Background:Tetralogy of Fallot(TOF),the predominant cyanotic congenital heart defect,arisesfrom multifactorial gene-envirorment interactions disrup ting cardiac developmental networks.This studyinvestiga ted TOF-speci...Background:Tetralogy of Fallot(TOF),the predominant cyanotic congenital heart defect,arisesfrom multifactorial gene-envirorment interactions disrup ting cardiac developmental networks.This studyinvestiga ted TOF-specific transcriptional alterations and identified high-confidence candidate genes.Methods:Based on GSE36761 transcriptome data,a weighted gene co-exp ression network analysis(WGCNA)andprotein-protein interaction(PPI)network were conducted to identify TOF-related sub-netrwork and Hub genes.The potentialbiological functions among these genes were revealed by enrichment analysis.Genetic,epigeneticand transcriptional alteration in the Fub genes were analyzed with leveraged public resources:a methylationdataset(CSE62629)and two single-cell datasets(EGAS00001003996 and GSE126128),Results:Eight Hub geneswere identified using the WGCNA network and PPl network,and functional errichment analysis revealedthatGJA1,RUNX2,FTK7,PRICKLE1,and SPRP1 were involved in the morphogenesis of an epithelium,anddysregulation of the signaling were also found in the other two TOF datasets,Furthermore,the study foundthat the promoters of GJA1,RUNX2,FTK7,and PRICKLE1 genes were hypermethylated and that GJA1 andSFRP1 are highly expressed in mouse second heart field cells and neural crest cells,and the la tter is expressedin human embry onic outflow tract cells.Since RUNX2 was not expressed in human and mouse embryonichearts,GJA1,FTK7,PRICKLE1,and SPRP1 were ultimately identified as TOF candidate genes.Conclusion:Based on the WGCNA network and various bioinformatics analysis approaches,we screened 4 TOF candidatepathogenic genes,and found that the signaling pathways related to the morphogenesis of an epithelium maybe involved in the pathogenesis of TOF.展开更多
AIM:To investigate the prognostic impact of metastatic lymph node ratio(rN) on gastric cancer after curative distal gastrectomy.METHODS:A total of 634 gastric cancer patients who underwent curative resection(R0) of ly...AIM:To investigate the prognostic impact of metastatic lymph node ratio(rN) on gastric cancer after curative distal gastrectomy.METHODS:A total of 634 gastric cancer patients who underwent curative resection(R0) of lymph nodes at distal gastrectomy in 1995-2004.Correlations between positive nodes and retrieved nodes,between rN and retrieved nodes,and between rN and negative lymph node(LN) count were analyzed respectively.Prognostic factors were identif ied by univariate and multivariate analyses.Staging accuracy of the pN category(5th UICC/TNM system) and the rN category was compared according to the survival rates of patients.A linear regression model was used to identify the relation between rN and 5-year survival rate of the patients.RESULTS:The number of dissected LNs was related with metastatic LNs but not related with rN.Cox regression analysis showed that depth of invasion,pN and rN category were the independent predictors of survival(P < 0.05).There was a signif icant difference in survival between LN stages classif ied by the rN category or by the pN category(P < 0.05).However,no signif icant difference was found in survival rate between LN stages classif ied by the pN category or by the rN category(P > 0.05).Linear regression model showed a signif icant linear correlation between rN and the 5-year survival rate of gastric cancer patients(β = 0.862,P < 0.001).Pearson's correlation test revealed that negative LN count was negatively correlated with rN(P < 0.001).CONCLUSION:rN category is a better prognostic tool than the 5th UICC pN category for gastric cancer patients after curative distal gastrectomy.Increased negative LN count can reduce rN and improve the survival rate of gastric cancer patients.展开更多
BACKGROUND: This study was designed to probe the clinical value in assessing the degree of liver cirrhosis by using the arrival time of contrast agent in the right portal vein in contrast-enhanced ultrasonography, as ...BACKGROUND: This study was designed to probe the clinical value in assessing the degree of liver cirrhosis by using the arrival time of contrast agent in the right portal vein in contrast-enhanced ultrasonography, as well as the velocity and flow volume in the right portal vein using the color Doppler velocity profile technique. METHODS: Twenty-eight patients with HBV post-hepatic cirrhosis were grouped into compensated (13 patients) and decompensated cirrhosis (15); 30 patients without hepatic cirrhosis served as controls. Written informed consent was obtained from each patient. All the patients with hepatic cirrhosis were pathologically confirmed by percutaneous biopsy. SonoVue was injected to detect the arrival time in the right portal vein. The velocity and flow volume in the right portal vein were measured. The value of each parameter was compared for correlation analysis. RESULTS: The arrival time in the right portal vein in the cirrhosis group was much longer than that in the control group (24.92 +/- 1.34 vs. 20.81 +/- 0.55 sec, respectively, P<0.01). The mean velocity, maximal velocity and flow volume in the cirrhosis group were much lower than those in the control group (10.64 +/- 0.84 vs. 14.78 +/- 0.71 cm/sec, 13.68 +/- 1.02 vs. 17.30 +/- 0.68 cm/sec and 358.72 +/- 23.63 vs. 438.61 +/- 16.86 ml/min, respectively, P<0.01). With the development of cirrhosis, the arrival time in the right portal vein was longer (P<0.05), and the velocity and flow volume was lower (P<0.01). There was a negative correlation between arrival time and mean velocity, maximal velocity and flow volume in the right portal vein in the cirrhosis group (r=-0.547, P<0.01; r=-0.508, P<0.05; r=-0.471, P<0.05, respectively). CONCLUSIONS: With the development of liver cirrhosis, the arrival time of contrast agent in the right portal vein is gradually prolonged, whereas the velocity and flow volume in this vein decreases markedly, and there is a negative correlation between the results of the two methods.展开更多
BACKGROUND: Toll-like receptor 2 and 4 (TLR2/4) may play important roles in ischemia-reperfusion (I/R) injury, and N-acetylcysteine (NAC) can prevent the generation of reactive oxygen species (ROS) induced by I/R inju...BACKGROUND: Toll-like receptor 2 and 4 (TLR2/4) may play important roles in ischemia-reperfusion (I/R) injury, and N-acetylcysteine (NAC) can prevent the generation of reactive oxygen species (ROS) induced by I/R injury. This study aimed to investigate the changes in TLR2/4 gene expression in the liver and lung after I/R injury with or without NAC pretreatment. METHODS: BALB/c mice were used in a model of partial hepatic I/R injury and randomly assigned to a sham-operated control group (SH), a hepatic ischemia/reperfusion group (I/R) or a NAC pretreated, hepatic I/R group (I/R-NAC). The levels of TNF-alpha in the portal vein and plasma alanine aminotransferase (ALT) were measured at 1 and 3 hours after reperfusion. The lung wet-to-dry ratio was measured, and the expression of TLR2/4 mRNA and protein in the liver and lung were assessed with RT-PCR and Western blotting at the same time points. RESULTS: Compared with the I/R group, the expression of TLR2/4 mRNA and protein in the liver and lung in the I/R-NAC group was decreased at the same time point (P<0.05). The levels of portal vein TNF-a and plasma ALT increased continuously in the l/R group at I and 3 hours of reperfusion compared with the SH group; however, they declined significantly in the group pretreated with NAC (P<0.05). The extent of lung edema was relieved in the I/R-NAC group compared with the I/R group (P<0.05). CONCLUSIONS: TLR2/4 was activated in the liver and lung in the process of partial hepatic I/R injury. NAC inhibited the activation of TLR2/4 and the induction of TNF-alpha resulting from I/R injury via modulating the redox state, thus it may mitigate liver and lung injury following partial hepatic I/R in mice.展开更多
BACKGROUND: The pancreas has a strong regeneration potential in mammals. Previous studies suggested that pancreas regeneration is correlated with proliferation and differentiation of pancreatic stem cells, but the fie...BACKGROUND: The pancreas has a strong regeneration potential in mammals. Previous studies suggested that pancreas regeneration is correlated with proliferation and differentiation of pancreatic stem cells, but the field of pancreatic stem cells is still in its infancy. This study was undertaken to detect the expression of pancreas/duodenal homeobox-1 (PDX-1) and proliferation of pancreatic duct epithelial cells in remnant pancreas during regeneration after partial pancreatectomy in rats, and characterize the source of pancreatic stem cells. METHODS: Partial pancreatectomy (90%) was performed on four- to five-week-old Sprague-Dawley rats, and duct epithelial cells and acinar cells were detected by immunohistochemical staining and scored using the 5-bromo-2-deoxyuridine (BrdU) labelling index at various time points. Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to assess the expression of PDX-1 protein and mRNA, respectively. RESULTS: At 24 hours after partial pancreatectomy, proliferation started in the main, large and small duct cells, and persisted in small duct cells to day 5. The experimental and control groups were significantly different (P<0.001). BrdU-positive acinar cells were greatly increased and reached a peak on day 5. PDX-1 protein was only faintly detectable in pancreatic ductal cells on day I after partial pancreatectomy. On days 2 and 3, a 2-3 fold increase in PDX-1 protein was observed, corresponding to the characteristic 42 kD protein in Western blotting. The operated and sham-operated groups also differed significantly (P<0.05). PDX-1 protein expression on days 5 and 7 after operation did not differ from that of the control group. RT-PCR revealed that PDX-1 mRNA expression did not significantly differ between the operated group and the sham-operated group at various time points. CONCLUSIONS: Pancreatic stem cells in pancreatic ductal epithelial cells are involved in the regeneration of remnant pancreas and the expression of PDX-1 in ductal cells is due to posttranscriptional regulation.展开更多
BACKGROUND: Tissue inhibitor of metalloproteinases (TIMPs) can restrain the tumor growth by their protein property and matrix metalloproteinases (MMPs) inhibition. There are currently four known TIMP family members-TI...BACKGROUND: Tissue inhibitor of metalloproteinases (TIMPs) can restrain the tumor growth by their protein property and matrix metalloproteinases (MMPs) inhibition. There are currently four known TIMP family members-TIMP-1, 2, 3, 4. We determined whether increasing levels of TIMP-3 expression could suppress the malignant phenotype of human hepatocarcinoma cell line HCC-7721. METHODS: A recombinant expression vector, which contained full-length cDNA of human TIMP-3, was constructed and transfected into the human hepatocarcinoma cell line HCC-7721 by the lipofectamine technique. In vitro and in vivo tests such as Western blotting, immunohistochemistry as well as xenografting in nude mice were used to analyze expression levels of TIMP and MMP, and changes in malignant phenotype after the gene transfection. RESULTS: TIMP-3 expression in TIMP-3 gene-transfected HCC-7721 cells was upregulated as assessed by Western blotting. The ability of in vitro invasion through a Boyden chamber was significantly decreased compared to controls. Following subcutaneous injection into nude mice, the TIMP-3 transfected cells suppressed primary tumor growth, as characterized by reduced tumor weight, size and microvasculature as well as maintaining the extracellular matrix. CONCLUSION: The results suggest that upregulation of TIMP-3 expression in HCC-7721 cells inhibits invasion capacity in vitro as well as tumorigenic and metastatic potential in nude mice.展开更多
BACKGROUND: Focal nodular hyperplasia (FNH) is increasingly diagnosed as a result of the advances in imaging studies such as contrast-enhanced ultrasonography (CEUS), enhanced computed tomography and magnetic resonanc...BACKGROUND: Focal nodular hyperplasia (FNH) is increasingly diagnosed as a result of the advances in imaging studies such as contrast-enhanced ultrasonography (CEUS), enhanced computed tomography and magnetic resonance imaging. However, FNH with atypical features can be difficult to differentiate from other benign and malignant tumors. The aim of this study was to investigate the influence of fatty liver background on the CEUS characteristics of FNH. METHODS: Twenty-six patients with FNH were divided into two groups: group A included 14 patients with fatty liver and group B included 12 patients with normal liver background. Conventional two-dimensional ultrasonography and color Doppler flow imaging (CDFI) were conducted and followed by real-time dual-frame CEUS. RESULTS: On two-dimensional ultrasonography, hypoechoic nodules were present in most of the patients in group A (12/14) and hyperechoic nodules in most of those in group B (7/12). The difference in the nodule echotextures between the two groups was statistically significant (P<0.05). Nodules with centrifugal blood flow signals on CDFI were found in 6 of the 14 patients in group A and 5 of the 12 in group B (P>0.05). On CEUS, nodules with a central spoked-wheel-like enhancement pattern in the early arterial phase were observed in 8 patients in group A and those with an eccentric enhancement pattern in the remaining 6 patients. In this group, 3 patients had hypoechoic nodules in the delayed phase. Eleven of the 14 patients in this group were diagnosed accurately with CEUS. In group B, nodules with a rapid central spoked-wheel-like enhancement pattern in the early arterial phase were found in 8 patients by CEUS and those with rapid an eccentric enhancement pattern in 4. The nodules were found to be continuously enhanced in the delayed phase. All of the patients in group B were accurately diagnosed with CEUS. CONCLUSIONS: A FNH nodule on a background of fatty liver may present a hypoechoic pattern on two-dimensional ultrasonography and a hypoechoic wash-out pattern in the delayed phase on CEUS. At this time, punch biopsy is needed for the diagnosis or differential diagnosis of FNH.展开更多
BACKGROUND: Pancreatic cancer is one of the most aggressive malignancies, and has a poor prognosis. Despite efforts made in multiple fields, there has been little success in improving the disease-free survival rate of...BACKGROUND: Pancreatic cancer is one of the most aggressive malignancies, and has a poor prognosis. Despite efforts made in multiple fields, there has been little success in improving the disease-free survival rate of patients. This study was undertaken to investigate the effectiveness and feasibility of using intra-tumoral injection of ricin-loaded thermosensitive hydrogel for treatment of pancreatic cancer xenografts, attempting to develop a new treatment for human pancreatic cancer. METHODS: BALB/c-(nu/nu) nude mice were inoculated subcutaneously in the right flank with the human pancreatic cancer cells, SW1990. Fourteen days after inoculation, 32 mice, bearing tumors of volume 1.5-2.0 cm(3), were randomly assigned to one of four groups, and given an intra-tumoral injection of: (1) saline; (2) 23% w/w thermosensitive hydrogel alone; (3) ricin, 10 mu g/kg; or (4) 10 mu g/kg ricin loaded in thermosensitive hydrogel. On day 14 after administration, the tumors were excised to calculate the inhibition rate of tumor growth and perform histopathological examination. Tumor cell apoptosis was detected by flow cytometry, and RT-PCR was performed to evaluate the mRNA expression levels of Bc12 and Bax. RESULTS: Intra-tumoral injection of ricin-loaded thermosensitive hydrogel resulted in remarkable control of tumor growth. The tumor became necrotic by day 14 after administration. The histological results clearly confirmed that the tumor cells were lysed. The percentage of apoptotic cells detected by flow cytometry was higher in the ricin hydrogel group than in the other groups. Semi-quantitative RT-PCR revealed that the mRNA expression level of Bc12 was down-regulated whereas Bax was upregulated. CONCLUSIONS: Intra-tumoral injection of ricin-loaded thermosensitive hydrogel may provide an effective approach for interstitial chemotherapy in pancreatic cancer. Inducing apoptosis by downregulating Bcl2 expression and upregulating Bax expression may be a key molecular mechanism.展开更多
BACKGROUND: Inflammatory pseudotumor of the biliary tract is a benign disease, and is extremely rare. Its diagnosis often depends on pathological examination after operation. The histopathological examination shows in...BACKGROUND: Inflammatory pseudotumor of the biliary tract is a benign disease, and is extremely rare. Its diagnosis often depends on pathological examination after operation. The histopathological examination shows inflammatory lesions with a polymorphous infiltration and variable amounts of fibrous tissue. This study was undertaken to elucidate that an inflammatory pseudotumor in the right hepatic duct is especially difficult to distinguish from hilar cholangiocarcinoma. METHOD: The clinical data of one patient with inflammatory pseudotumor of the right hepatic duct were analyzed. RESULTS: An occupying lesion of the right hepatic duct was revealed by abdominal ultrasound and magnetic resonance cholangiopancreatography. The right hepatic duct inflammatory pseudotumor was not identified during the operation but was confirmed by postoperative histopathological analysis. The patient recovered well without any serious complication. CONCLUSIONS: The preoperative evaluation for optimizing surgical management is important to the diagnosis of hepatobiliary occupying lesions. The evaluation involves clinical manifestations, imaging appearance and tumor markers. Malignant tumors and possible benign lesions should be considered to avoid aggressive surgical treatment. (Hepatobiliary Pancreat Dis Int 2010; 9: 219-221)展开更多
BACKGROUND: Currently, one of the tough problems for the application of bioartificial liver (BAL) is the shortage of suitable hepatocytes. There are reports on different types of BAL assistance developed with porcine ...BACKGROUND: Currently, one of the tough problems for the application of bioartificial liver (BAL) is the shortage of suitable hepatocytes. There are reports on different types of BAL assistance developed with porcine hepatocytes and HepG2 C3A cells, but their defects are obvious. In recent years, some studies focus more on liver cells with features of human origin and improved detoxification. In this study, a hepatocyte line with high expression of human glutamine synthetase (hGS) was raised and its capacity for ammonia metabolism was investigated. METHODS: hGS cDNA and alpha-fetoprotein transcription regulatory element (AFP-TRE) were cloned with the designed primers. The eukaryotic expression vectors, pLNChGS and pLNAFhGS, were constructed and transfected into PA317 cells. Recombinant retroviruses (Retro-hGS and Retro-AFhGS) were produced and then infected into HepG2 cells. G418-resistant cell clones, HepG2/pLNChGS and HepG2/pLNAFhGS, were selected and amplified. Then hGS mRNA was measured by semi-quantitative RT-PCR; hGS enzymatic activity and ammonia metabolism analysis in different concentration of NH(4)(+) were detected with a quantitative biochemistry kit. The cell proliferation was also detected by MTT chromatometry. RESULTS: The expression of hGS mRNA in HepG2/pLNChGS cells (8.306+/-0.336) and HepG2/pLNAFhGS cells (21.358+/-1.716) was much stronger than in control cells (P<0.05), and that in HepG2/pLNAFhGS cells was markedly stronger than in HepG2/pLNChGS cells (P<0.05). The hGS enzymatic activities of HepG2/pLNChGS cells (3.279+/-0.328 U/mg prot) and HepG2/pLNAFhGS cells (4.557+/-0.253 U/mg prot) were higher than those of control cells (P<0.05), and those of HepG2/ pLNAFhGS cells were also higher than the activities of HepG2/pLNChGS cells (P<0.05). In addition, the effect of hGS introduction on HepG2 cell proliferation was not significant. The amount of glutamine synthesis in HepG2/pLNChGS or HepG2/pLNAFhGS cells in three different concentrations of NH(4)(+) was higher than in the two control cells (P<0.05). The amount of glutamine synthesis and cell proliferation in the higher concentrations of NH(4)(+) (5 or 10 mmol/L) in HepG2/pLNAFhGS cells increased more than those in HepG2/pLNChGS cells (P<0.05). NH(4)(+) at a high concentration (10 mmol/L) was toxic to HepG2 and HepG2/pLNCX cells, but less toxic to HepG2/pLNChGS and HepG2/pLNAFhGS cells. CONCLUSION: The constructed hepatocytes (HepG2 cells) with specific high-expression of hGS have a powerful ability to degrade ammonia in vitro, and provide necessary experimental data for the selection of biomaterials in BAL.展开更多
BACKGROUND: Hepatic fungal infection is difficult to diagnose early because of the lack of related clinical knowledge. In this study, we analyzed the ultrasonic manifestations of hepatic fungal infection, in order to ...BACKGROUND: Hepatic fungal infection is difficult to diagnose early because of the lack of related clinical knowledge. In this study, we analyzed the ultrasonic manifestations of hepatic fungal infection, in order to determine the diagnostic value of ultrasonography. METHODS: In 8 patients with hepatic fungal infection, the results of color Doppler flow images (CDFI) and contrast-enhanced ultrasonography (CEUS) were compared with those of ultrasound-guided biopsy. RESULTS: In the 8 patients with pathologically proven hepatic fungal infection, 5 showed multiple hypoechoic lesions with unequal sizes in the liver, commonly less than 2 cm in diameter. Some of the hypoechoic lesions showed a hyperechoic strip in the periphery, while a minority showed a 'bull's eye' pattern, which is considered a typical manifestation of metastatic hepatic carcinoma. Solitary lesions in the right lobe of the liver were found in the other 3 patients. No blood flow signals were found in any lesions with CDFI. In the lesions a 'filling defect' pattern was shown by CEUS, indicating no enhancement in the arterial phase, the portal vein phase and the delayed phase, except for 3 patients, in whom the lesions were slightly enhanced in the periphery during the early arterial phase. CONCLUSIONS: Hepatic fungal infection is characterized by an ultrasonic manifestation of multiple hypoechoic lesions with inequal size. A small number of lesions may show a characteristic 'bull's eye' appearance. Moreover, a 'filling defect' pattern on CEUS is helpful to make a correct diagnosis. In addition, ultrasound-guided biopsy is an important method for accurate diagnosis of hepatic fungal infection.展开更多
Objective Evaluated NOS expression in bladder tissue from the patients with transitional cell carcinoma (TCC) of the bladder and studied its relationship with angiogenesis. Methods Bladder carcinoma tissue specimens w...Objective Evaluated NOS expression in bladder tissue from the patients with transitional cell carcinoma (TCC) of the bladder and studied its relationship with angiogenesis. Methods Bladder carcinoma tissue specimens were procured from 58 patients with TCC and 14 cases of benign bladder tissue as contrast group. NOS immunohistochemistry was performed on all tissue specimens and microvessal density (MVD) was counted by endothelial cells immunostained. Results Inducible NOS specific proteins were found in 47 of 58 bladder cancer specimens but not in control bladder tissue. The malignant cells and inflammatory cells within the carcinomas were highly iNOS positive whereas specimens of bladder mucosa outside the malignant regions showed only a weak positive iNOS immunostaining. The endothelial cells in both normal urothlium and tumor tissue showed a highly positive eNOS immunotaining but its immunoreaction was not detected in either malignant or benign epithelium. MVD was (39. 3 ± 19. 5)/HP and (29.3 ± 10.展开更多
Objective To investigate the expression of bradykinin as a substrate of CD26 /DPP IV in rats with ischemia/reperfusion injury following lung transplantation ( LTx) . Methods Thirty - six syngeneic male SD rats were ra...Objective To investigate the expression of bradykinin as a substrate of CD26 /DPP IV in rats with ischemia/reperfusion injury following lung transplantation ( LTx) . Methods Thirty - six syngeneic male SD rats were randomly allocated into control group and experimental group ( n = 18 each) ,and 36 rats served as do-展开更多
Objective To investigate the effect of the implant composite of poly lactide-co-glycolide(PLGA)and bone mesenchymal stem cells (BMSCs) modified by basic fibroblast growth factor (bFGF) on injured spinal cord in rats.M...Objective To investigate the effect of the implant composite of poly lactide-co-glycolide(PLGA)and bone mesenchymal stem cells (BMSCs) modified by basic fibroblast growth factor (bFGF) on injured spinal cord in rats.Methods Two hundred and展开更多
Objective To investigate the effect of ifenprodil pretreatment on the expression of NR2B subunit protein of NMDA receptor in the dorsal horn of spinal cord in a rat model of neuropathic pain. Methods Fifty male SD rat...Objective To investigate the effect of ifenprodil pretreatment on the expression of NR2B subunit protein of NMDA receptor in the dorsal horn of spinal cord in a rat model of neuropathic pain. Methods Fifty male SD rats weighing 180-200 g were used in this study. Neuropathic pain was produced by chronic constrietive injury （CCI）. The left sciatic nerve was exposed and 4 loose ligatures were placed on the sciatic nerve at 1-2 mm intervals with 4-0 chromic catgut under pentobarbital anesthesia.展开更多
Objective To study the mechanism of the neuronal apoptosis induced by NOS, p38 MAPK and Caspase-3 after hypoxia injury. Methods The apoptosis of PC12 cells intoxicated by hypoxia was determined by using MTT assay,fluo...Objective To study the mechanism of the neuronal apoptosis induced by NOS, p38 MAPK and Caspase-3 after hypoxia injury. Methods The apoptosis of PC12 cells intoxicated by hypoxia was determined by using MTT assay,fluorescent staining, and FCM. RT-PCR was used to detect the pro-portion of nNOS, iNOS, p38 MAPK and Caspase-3 mRMA expression. Results MTT assay indicated that hypoxia-induced suppression of growth of PC12 cells was negatively related to hypoxia time.展开更多
Objective To investigate the transfection and expression of vascular endothelial growth factor in primary cultured rat skeletal moblasts.Methods VEGF121 gene was transfected to primary cultured rat skeletal myoblasts ...Objective To investigate the transfection and expression of vascular endothelial growth factor in primary cultured rat skeletal moblasts.Methods VEGF121 gene was transfected to primary cultured rat skeletal myoblasts by lipofectamine in vitro.Gene expression of transfected myoblasts was detected by RT-PCR,Western-Blot and immunofluorescent staining.展开更多
Objective To investigate the effects of gel and coarse polysaccharide extracted from aloe barbadensis in tissue microvascular formation in burned rats. Methods Forty-eight male Wistar rats were randomly divided into e...Objective To investigate the effects of gel and coarse polysaccharide extracted from aloe barbadensis in tissue microvascular formation in burned rats. Methods Forty-eight male Wistar rats were randomly divided into eight groups （six rats in each group） ,each of which was given four deep degree Ⅱ burn wound with 3 cm in diameter on the back. After 5 % aloe coarse polysaccharide, 10% aloe vera gel, 1% sulfanilamide pyridine silver （SD-Ag） cream,展开更多
Objective To study the effects of combination of bone morphogenetic protein-2 (BMP-2) and strontium chloride on proliferation and osteogenic differentiation of human umbilical cord mesenchymal stem cells(hUCM-SCs)in v...Objective To study the effects of combination of bone morphogenetic protein-2 (BMP-2) and strontium chloride on proliferation and osteogenic differentiation of human umbilical cord mesenchymal stem cells(hUCM-SCs)in vitro culture.展开更多
Objective To investigate immunomodulatory effects of adipose-derived mesenchymal stem ceils in a rat orthotopic liver transplantation model. Methods Mesenchymal stem ceils were isolated from adipose tissue of SD rats....Objective To investigate immunomodulatory effects of adipose-derived mesenchymal stem ceils in a rat orthotopic liver transplantation model. Methods Mesenchymal stem ceils were isolated from adipose tissue of SD rats. In in vitro mixed lymphocyte culture system by using Wistar T cells as responders and SD spleen cells as stimulators,展开更多
基金supported by the National Natural Science Found ation of China(No.8230045i for Zhen Wang,82302230 for jiawei Shi,82202194 for Jing Wang and 82171961 for Haiyan Cao).
文摘Background:Tetralogy of Fallot(TOF),the predominant cyanotic congenital heart defect,arisesfrom multifactorial gene-envirorment interactions disrup ting cardiac developmental networks.This studyinvestiga ted TOF-specific transcriptional alterations and identified high-confidence candidate genes.Methods:Based on GSE36761 transcriptome data,a weighted gene co-exp ression network analysis(WGCNA)andprotein-protein interaction(PPI)network were conducted to identify TOF-related sub-netrwork and Hub genes.The potentialbiological functions among these genes were revealed by enrichment analysis.Genetic,epigeneticand transcriptional alteration in the Fub genes were analyzed with leveraged public resources:a methylationdataset(CSE62629)and two single-cell datasets(EGAS00001003996 and GSE126128),Results:Eight Hub geneswere identified using the WGCNA network and PPl network,and functional errichment analysis revealedthatGJA1,RUNX2,FTK7,PRICKLE1,and SPRP1 were involved in the morphogenesis of an epithelium,anddysregulation of the signaling were also found in the other two TOF datasets,Furthermore,the study foundthat the promoters of GJA1,RUNX2,FTK7,and PRICKLE1 genes were hypermethylated and that GJA1 andSFRP1 are highly expressed in mouse second heart field cells and neural crest cells,and the la tter is expressedin human embry onic outflow tract cells.Since RUNX2 was not expressed in human and mouse embryonichearts,GJA1,FTK7,PRICKLE1,and SPRP1 were ultimately identified as TOF candidate genes.Conclusion:Based on the WGCNA network and various bioinformatics analysis approaches,we screened 4 TOF candidatepathogenic genes,and found that the signaling pathways related to the morphogenesis of an epithelium maybe involved in the pathogenesis of TOF.
文摘AIM:To investigate the prognostic impact of metastatic lymph node ratio(rN) on gastric cancer after curative distal gastrectomy.METHODS:A total of 634 gastric cancer patients who underwent curative resection(R0) of lymph nodes at distal gastrectomy in 1995-2004.Correlations between positive nodes and retrieved nodes,between rN and retrieved nodes,and between rN and negative lymph node(LN) count were analyzed respectively.Prognostic factors were identif ied by univariate and multivariate analyses.Staging accuracy of the pN category(5th UICC/TNM system) and the rN category was compared according to the survival rates of patients.A linear regression model was used to identify the relation between rN and 5-year survival rate of the patients.RESULTS:The number of dissected LNs was related with metastatic LNs but not related with rN.Cox regression analysis showed that depth of invasion,pN and rN category were the independent predictors of survival(P < 0.05).There was a signif icant difference in survival between LN stages classif ied by the rN category or by the pN category(P < 0.05).However,no signif icant difference was found in survival rate between LN stages classif ied by the pN category or by the rN category(P > 0.05).Linear regression model showed a signif icant linear correlation between rN and the 5-year survival rate of gastric cancer patients(β = 0.862,P < 0.001).Pearson's correlation test revealed that negative LN count was negatively correlated with rN(P < 0.001).CONCLUSION:rN category is a better prognostic tool than the 5th UICC pN category for gastric cancer patients after curative distal gastrectomy.Increased negative LN count can reduce rN and improve the survival rate of gastric cancer patients.
基金a grant from the Science Foundation of Fujian Province (No. 2000Z138).
文摘BACKGROUND: This study was designed to probe the clinical value in assessing the degree of liver cirrhosis by using the arrival time of contrast agent in the right portal vein in contrast-enhanced ultrasonography, as well as the velocity and flow volume in the right portal vein using the color Doppler velocity profile technique. METHODS: Twenty-eight patients with HBV post-hepatic cirrhosis were grouped into compensated (13 patients) and decompensated cirrhosis (15); 30 patients without hepatic cirrhosis served as controls. Written informed consent was obtained from each patient. All the patients with hepatic cirrhosis were pathologically confirmed by percutaneous biopsy. SonoVue was injected to detect the arrival time in the right portal vein. The velocity and flow volume in the right portal vein were measured. The value of each parameter was compared for correlation analysis. RESULTS: The arrival time in the right portal vein in the cirrhosis group was much longer than that in the control group (24.92 +/- 1.34 vs. 20.81 +/- 0.55 sec, respectively, P<0.01). The mean velocity, maximal velocity and flow volume in the cirrhosis group were much lower than those in the control group (10.64 +/- 0.84 vs. 14.78 +/- 0.71 cm/sec, 13.68 +/- 1.02 vs. 17.30 +/- 0.68 cm/sec and 358.72 +/- 23.63 vs. 438.61 +/- 16.86 ml/min, respectively, P<0.01). With the development of cirrhosis, the arrival time in the right portal vein was longer (P<0.05), and the velocity and flow volume was lower (P<0.01). There was a negative correlation between arrival time and mean velocity, maximal velocity and flow volume in the right portal vein in the cirrhosis group (r=-0.547, P<0.01; r=-0.508, P<0.05; r=-0.471, P<0.05, respectively). CONCLUSIONS: With the development of liver cirrhosis, the arrival time of contrast agent in the right portal vein is gradually prolonged, whereas the velocity and flow volume in this vein decreases markedly, and there is a negative correlation between the results of the two methods.
基金This work was supported by a grant from theNational Natural Science Foundation of China(No.30200272).
文摘BACKGROUND: Toll-like receptor 2 and 4 (TLR2/4) may play important roles in ischemia-reperfusion (I/R) injury, and N-acetylcysteine (NAC) can prevent the generation of reactive oxygen species (ROS) induced by I/R injury. This study aimed to investigate the changes in TLR2/4 gene expression in the liver and lung after I/R injury with or without NAC pretreatment. METHODS: BALB/c mice were used in a model of partial hepatic I/R injury and randomly assigned to a sham-operated control group (SH), a hepatic ischemia/reperfusion group (I/R) or a NAC pretreated, hepatic I/R group (I/R-NAC). The levels of TNF-alpha in the portal vein and plasma alanine aminotransferase (ALT) were measured at 1 and 3 hours after reperfusion. The lung wet-to-dry ratio was measured, and the expression of TLR2/4 mRNA and protein in the liver and lung were assessed with RT-PCR and Western blotting at the same time points. RESULTS: Compared with the I/R group, the expression of TLR2/4 mRNA and protein in the liver and lung in the I/R-NAC group was decreased at the same time point (P<0.05). The levels of portal vein TNF-a and plasma ALT increased continuously in the l/R group at I and 3 hours of reperfusion compared with the SH group; however, they declined significantly in the group pretreated with NAC (P<0.05). The extent of lung edema was relieved in the I/R-NAC group compared with the I/R group (P<0.05). CONCLUSIONS: TLR2/4 was activated in the liver and lung in the process of partial hepatic I/R injury. NAC inhibited the activation of TLR2/4 and the induction of TNF-alpha resulting from I/R injury via modulating the redox state, thus it may mitigate liver and lung injury following partial hepatic I/R in mice.
基金This study was supported by a grant from the National Natural Sciences Foundation of China (No. 30571817).
文摘BACKGROUND: The pancreas has a strong regeneration potential in mammals. Previous studies suggested that pancreas regeneration is correlated with proliferation and differentiation of pancreatic stem cells, but the field of pancreatic stem cells is still in its infancy. This study was undertaken to detect the expression of pancreas/duodenal homeobox-1 (PDX-1) and proliferation of pancreatic duct epithelial cells in remnant pancreas during regeneration after partial pancreatectomy in rats, and characterize the source of pancreatic stem cells. METHODS: Partial pancreatectomy (90%) was performed on four- to five-week-old Sprague-Dawley rats, and duct epithelial cells and acinar cells were detected by immunohistochemical staining and scored using the 5-bromo-2-deoxyuridine (BrdU) labelling index at various time points. Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to assess the expression of PDX-1 protein and mRNA, respectively. RESULTS: At 24 hours after partial pancreatectomy, proliferation started in the main, large and small duct cells, and persisted in small duct cells to day 5. The experimental and control groups were significantly different (P<0.001). BrdU-positive acinar cells were greatly increased and reached a peak on day 5. PDX-1 protein was only faintly detectable in pancreatic ductal cells on day I after partial pancreatectomy. On days 2 and 3, a 2-3 fold increase in PDX-1 protein was observed, corresponding to the characteristic 42 kD protein in Western blotting. The operated and sham-operated groups also differed significantly (P<0.05). PDX-1 protein expression on days 5 and 7 after operation did not differ from that of the control group. RT-PCR revealed that PDX-1 mRNA expression did not significantly differ between the operated group and the sham-operated group at various time points. CONCLUSIONS: Pancreatic stem cells in pancreatic ductal epithelial cells are involved in the regeneration of remnant pancreas and the expression of PDX-1 in ductal cells is due to posttranscriptional regulation.
文摘BACKGROUND: Tissue inhibitor of metalloproteinases (TIMPs) can restrain the tumor growth by their protein property and matrix metalloproteinases (MMPs) inhibition. There are currently four known TIMP family members-TIMP-1, 2, 3, 4. We determined whether increasing levels of TIMP-3 expression could suppress the malignant phenotype of human hepatocarcinoma cell line HCC-7721. METHODS: A recombinant expression vector, which contained full-length cDNA of human TIMP-3, was constructed and transfected into the human hepatocarcinoma cell line HCC-7721 by the lipofectamine technique. In vitro and in vivo tests such as Western blotting, immunohistochemistry as well as xenografting in nude mice were used to analyze expression levels of TIMP and MMP, and changes in malignant phenotype after the gene transfection. RESULTS: TIMP-3 expression in TIMP-3 gene-transfected HCC-7721 cells was upregulated as assessed by Western blotting. The ability of in vitro invasion through a Boyden chamber was significantly decreased compared to controls. Following subcutaneous injection into nude mice, the TIMP-3 transfected cells suppressed primary tumor growth, as characterized by reduced tumor weight, size and microvasculature as well as maintaining the extracellular matrix. CONCLUSION: The results suggest that upregulation of TIMP-3 expression in HCC-7721 cells inhibits invasion capacity in vitro as well as tumorigenic and metastatic potential in nude mice.
文摘BACKGROUND: Focal nodular hyperplasia (FNH) is increasingly diagnosed as a result of the advances in imaging studies such as contrast-enhanced ultrasonography (CEUS), enhanced computed tomography and magnetic resonance imaging. However, FNH with atypical features can be difficult to differentiate from other benign and malignant tumors. The aim of this study was to investigate the influence of fatty liver background on the CEUS characteristics of FNH. METHODS: Twenty-six patients with FNH were divided into two groups: group A included 14 patients with fatty liver and group B included 12 patients with normal liver background. Conventional two-dimensional ultrasonography and color Doppler flow imaging (CDFI) were conducted and followed by real-time dual-frame CEUS. RESULTS: On two-dimensional ultrasonography, hypoechoic nodules were present in most of the patients in group A (12/14) and hyperechoic nodules in most of those in group B (7/12). The difference in the nodule echotextures between the two groups was statistically significant (P<0.05). Nodules with centrifugal blood flow signals on CDFI were found in 6 of the 14 patients in group A and 5 of the 12 in group B (P>0.05). On CEUS, nodules with a central spoked-wheel-like enhancement pattern in the early arterial phase were observed in 8 patients in group A and those with an eccentric enhancement pattern in the remaining 6 patients. In this group, 3 patients had hypoechoic nodules in the delayed phase. Eleven of the 14 patients in this group were diagnosed accurately with CEUS. In group B, nodules with a rapid central spoked-wheel-like enhancement pattern in the early arterial phase were found in 8 patients by CEUS and those with rapid an eccentric enhancement pattern in 4. The nodules were found to be continuously enhanced in the delayed phase. All of the patients in group B were accurately diagnosed with CEUS. CONCLUSIONS: A FNH nodule on a background of fatty liver may present a hypoechoic pattern on two-dimensional ultrasonography and a hypoechoic wash-out pattern in the delayed phase on CEUS. At this time, punch biopsy is needed for the diagnosis or differential diagnosis of FNH.
文摘BACKGROUND: Pancreatic cancer is one of the most aggressive malignancies, and has a poor prognosis. Despite efforts made in multiple fields, there has been little success in improving the disease-free survival rate of patients. This study was undertaken to investigate the effectiveness and feasibility of using intra-tumoral injection of ricin-loaded thermosensitive hydrogel for treatment of pancreatic cancer xenografts, attempting to develop a new treatment for human pancreatic cancer. METHODS: BALB/c-(nu/nu) nude mice were inoculated subcutaneously in the right flank with the human pancreatic cancer cells, SW1990. Fourteen days after inoculation, 32 mice, bearing tumors of volume 1.5-2.0 cm(3), were randomly assigned to one of four groups, and given an intra-tumoral injection of: (1) saline; (2) 23% w/w thermosensitive hydrogel alone; (3) ricin, 10 mu g/kg; or (4) 10 mu g/kg ricin loaded in thermosensitive hydrogel. On day 14 after administration, the tumors were excised to calculate the inhibition rate of tumor growth and perform histopathological examination. Tumor cell apoptosis was detected by flow cytometry, and RT-PCR was performed to evaluate the mRNA expression levels of Bc12 and Bax. RESULTS: Intra-tumoral injection of ricin-loaded thermosensitive hydrogel resulted in remarkable control of tumor growth. The tumor became necrotic by day 14 after administration. The histological results clearly confirmed that the tumor cells were lysed. The percentage of apoptotic cells detected by flow cytometry was higher in the ricin hydrogel group than in the other groups. Semi-quantitative RT-PCR revealed that the mRNA expression level of Bc12 was down-regulated whereas Bax was upregulated. CONCLUSIONS: Intra-tumoral injection of ricin-loaded thermosensitive hydrogel may provide an effective approach for interstitial chemotherapy in pancreatic cancer. Inducing apoptosis by downregulating Bcl2 expression and upregulating Bax expression may be a key molecular mechanism.
文摘BACKGROUND: Inflammatory pseudotumor of the biliary tract is a benign disease, and is extremely rare. Its diagnosis often depends on pathological examination after operation. The histopathological examination shows inflammatory lesions with a polymorphous infiltration and variable amounts of fibrous tissue. This study was undertaken to elucidate that an inflammatory pseudotumor in the right hepatic duct is especially difficult to distinguish from hilar cholangiocarcinoma. METHOD: The clinical data of one patient with inflammatory pseudotumor of the right hepatic duct were analyzed. RESULTS: An occupying lesion of the right hepatic duct was revealed by abdominal ultrasound and magnetic resonance cholangiopancreatography. The right hepatic duct inflammatory pseudotumor was not identified during the operation but was confirmed by postoperative histopathological analysis. The patient recovered well without any serious complication. CONCLUSIONS: The preoperative evaluation for optimizing surgical management is important to the diagnosis of hepatobiliary occupying lesions. The evaluation involves clinical manifestations, imaging appearance and tumor markers. Malignant tumors and possible benign lesions should be considered to avoid aggressive surgical treatment. (Hepatobiliary Pancreat Dis Int 2010; 9: 219-221)
文摘BACKGROUND: Currently, one of the tough problems for the application of bioartificial liver (BAL) is the shortage of suitable hepatocytes. There are reports on different types of BAL assistance developed with porcine hepatocytes and HepG2 C3A cells, but their defects are obvious. In recent years, some studies focus more on liver cells with features of human origin and improved detoxification. In this study, a hepatocyte line with high expression of human glutamine synthetase (hGS) was raised and its capacity for ammonia metabolism was investigated. METHODS: hGS cDNA and alpha-fetoprotein transcription regulatory element (AFP-TRE) were cloned with the designed primers. The eukaryotic expression vectors, pLNChGS and pLNAFhGS, were constructed and transfected into PA317 cells. Recombinant retroviruses (Retro-hGS and Retro-AFhGS) were produced and then infected into HepG2 cells. G418-resistant cell clones, HepG2/pLNChGS and HepG2/pLNAFhGS, were selected and amplified. Then hGS mRNA was measured by semi-quantitative RT-PCR; hGS enzymatic activity and ammonia metabolism analysis in different concentration of NH(4)(+) were detected with a quantitative biochemistry kit. The cell proliferation was also detected by MTT chromatometry. RESULTS: The expression of hGS mRNA in HepG2/pLNChGS cells (8.306+/-0.336) and HepG2/pLNAFhGS cells (21.358+/-1.716) was much stronger than in control cells (P<0.05), and that in HepG2/pLNAFhGS cells was markedly stronger than in HepG2/pLNChGS cells (P<0.05). The hGS enzymatic activities of HepG2/pLNChGS cells (3.279+/-0.328 U/mg prot) and HepG2/pLNAFhGS cells (4.557+/-0.253 U/mg prot) were higher than those of control cells (P<0.05), and those of HepG2/ pLNAFhGS cells were also higher than the activities of HepG2/pLNChGS cells (P<0.05). In addition, the effect of hGS introduction on HepG2 cell proliferation was not significant. The amount of glutamine synthesis in HepG2/pLNChGS or HepG2/pLNAFhGS cells in three different concentrations of NH(4)(+) was higher than in the two control cells (P<0.05). The amount of glutamine synthesis and cell proliferation in the higher concentrations of NH(4)(+) (5 or 10 mmol/L) in HepG2/pLNAFhGS cells increased more than those in HepG2/pLNChGS cells (P<0.05). NH(4)(+) at a high concentration (10 mmol/L) was toxic to HepG2 and HepG2/pLNCX cells, but less toxic to HepG2/pLNChGS and HepG2/pLNAFhGS cells. CONCLUSION: The constructed hepatocytes (HepG2 cells) with specific high-expression of hGS have a powerful ability to degrade ammonia in vitro, and provide necessary experimental data for the selection of biomaterials in BAL.
文摘BACKGROUND: Hepatic fungal infection is difficult to diagnose early because of the lack of related clinical knowledge. In this study, we analyzed the ultrasonic manifestations of hepatic fungal infection, in order to determine the diagnostic value of ultrasonography. METHODS: In 8 patients with hepatic fungal infection, the results of color Doppler flow images (CDFI) and contrast-enhanced ultrasonography (CEUS) were compared with those of ultrasound-guided biopsy. RESULTS: In the 8 patients with pathologically proven hepatic fungal infection, 5 showed multiple hypoechoic lesions with unequal sizes in the liver, commonly less than 2 cm in diameter. Some of the hypoechoic lesions showed a hyperechoic strip in the periphery, while a minority showed a 'bull's eye' pattern, which is considered a typical manifestation of metastatic hepatic carcinoma. Solitary lesions in the right lobe of the liver were found in the other 3 patients. No blood flow signals were found in any lesions with CDFI. In the lesions a 'filling defect' pattern was shown by CEUS, indicating no enhancement in the arterial phase, the portal vein phase and the delayed phase, except for 3 patients, in whom the lesions were slightly enhanced in the periphery during the early arterial phase. CONCLUSIONS: Hepatic fungal infection is characterized by an ultrasonic manifestation of multiple hypoechoic lesions with inequal size. A small number of lesions may show a characteristic 'bull's eye' appearance. Moreover, a 'filling defect' pattern on CEUS is helpful to make a correct diagnosis. In addition, ultrasound-guided biopsy is an important method for accurate diagnosis of hepatic fungal infection.
文摘Objective Evaluated NOS expression in bladder tissue from the patients with transitional cell carcinoma (TCC) of the bladder and studied its relationship with angiogenesis. Methods Bladder carcinoma tissue specimens were procured from 58 patients with TCC and 14 cases of benign bladder tissue as contrast group. NOS immunohistochemistry was performed on all tissue specimens and microvessal density (MVD) was counted by endothelial cells immunostained. Results Inducible NOS specific proteins were found in 47 of 58 bladder cancer specimens but not in control bladder tissue. The malignant cells and inflammatory cells within the carcinomas were highly iNOS positive whereas specimens of bladder mucosa outside the malignant regions showed only a weak positive iNOS immunostaining. The endothelial cells in both normal urothlium and tumor tissue showed a highly positive eNOS immunotaining but its immunoreaction was not detected in either malignant or benign epithelium. MVD was (39. 3 ± 19. 5)/HP and (29.3 ± 10.
文摘Objective To investigate the expression of bradykinin as a substrate of CD26 /DPP IV in rats with ischemia/reperfusion injury following lung transplantation ( LTx) . Methods Thirty - six syngeneic male SD rats were randomly allocated into control group and experimental group ( n = 18 each) ,and 36 rats served as do-
文摘Objective To investigate the effect of the implant composite of poly lactide-co-glycolide(PLGA)and bone mesenchymal stem cells (BMSCs) modified by basic fibroblast growth factor (bFGF) on injured spinal cord in rats.Methods Two hundred and
文摘Objective To investigate the effect of ifenprodil pretreatment on the expression of NR2B subunit protein of NMDA receptor in the dorsal horn of spinal cord in a rat model of neuropathic pain. Methods Fifty male SD rats weighing 180-200 g were used in this study. Neuropathic pain was produced by chronic constrietive injury （CCI）. The left sciatic nerve was exposed and 4 loose ligatures were placed on the sciatic nerve at 1-2 mm intervals with 4-0 chromic catgut under pentobarbital anesthesia.
文摘Objective To study the mechanism of the neuronal apoptosis induced by NOS, p38 MAPK and Caspase-3 after hypoxia injury. Methods The apoptosis of PC12 cells intoxicated by hypoxia was determined by using MTT assay,fluorescent staining, and FCM. RT-PCR was used to detect the pro-portion of nNOS, iNOS, p38 MAPK and Caspase-3 mRMA expression. Results MTT assay indicated that hypoxia-induced suppression of growth of PC12 cells was negatively related to hypoxia time.
文摘Objective To investigate the transfection and expression of vascular endothelial growth factor in primary cultured rat skeletal moblasts.Methods VEGF121 gene was transfected to primary cultured rat skeletal myoblasts by lipofectamine in vitro.Gene expression of transfected myoblasts was detected by RT-PCR,Western-Blot and immunofluorescent staining.
文摘Objective To investigate the effects of gel and coarse polysaccharide extracted from aloe barbadensis in tissue microvascular formation in burned rats. Methods Forty-eight male Wistar rats were randomly divided into eight groups （six rats in each group） ,each of which was given four deep degree Ⅱ burn wound with 3 cm in diameter on the back. After 5 % aloe coarse polysaccharide, 10% aloe vera gel, 1% sulfanilamide pyridine silver （SD-Ag） cream,
文摘Objective To study the effects of combination of bone morphogenetic protein-2 (BMP-2) and strontium chloride on proliferation and osteogenic differentiation of human umbilical cord mesenchymal stem cells(hUCM-SCs)in vitro culture.
文摘Objective To investigate immunomodulatory effects of adipose-derived mesenchymal stem ceils in a rat orthotopic liver transplantation model. Methods Mesenchymal stem ceils were isolated from adipose tissue of SD rats. In in vitro mixed lymphocyte culture system by using Wistar T cells as responders and SD spleen cells as stimulators,
