AIM： To transplant undifferentiated embryonic stem （ES） cells into the spleens of carbon tetrachloride （CCl4）-treated mice to determine their ability to differentiate into hepatocytes in the liver. METHODS： CCh,...AIM： To transplant undifferentiated embryonic stem （ES） cells into the spleens of carbon tetrachloride （CCl4）-treated mice to determine their ability to differentiate into hepatocytes in the liver. METHODS： CCh, 0.5 mL/kg body weight, was injected into the peritoneum of C57BL/6 mice twice a week for 5 wk. In group 1 （n = 12）, 1 × 10^5 undifferentiated ES cells （0.1 mL of 1 × 10^6/mL solution）, genetically labeled with GFP, were transplanted into the spleens 1 d after the second injection. Group 2 mice （n = 12） were injected with 0.2 mL of saline twice a week, instead of CCh, and the same amount of ES cells was transplanted into the spleens. Group 3 mice （n = 6） were treated with CCh and injected with 0.1 mL of saline into the spleen, instead of ES cells. Histochemical analyses of the livers were performed on post-transplantation d （PD） 10, 20, and 30. RESULTS： Considerable numbers of GFP-immunopositive cells were found in the periportal regions in group 1 mice （CCh-treated） on PD 10, however, not in those untreated with CCh （group 2）. The GFP-positive cells were also immunopositive for albumin （ALB）, alpha-1 antitrypsin, cytokeratin 18, and hepatocyte nuclear factor 4 alpha on PD 20. Interestingly, most of the GFP-positive cells were immunopositive for DLK, a hepatoblast marker, on PD 10. Although very few ES-derived cells were demonstrated immunohistologically in the livers of group 1 mice on PD 30, improvements in liver fibrosis were observed. Unexpectedly, liver tumor formation was not observed in any of the mice that received ES cell transplantation during the experimental period CONCLUSION： Undifferentiated ES cells developed into hepatocyte-like cells with appropriate integration into tissue, without uncontrolled cell growth.展开更多
BACKGROUND: We reported a case of malignant nonfunction islet cell tumor (10.0 cm in diameter) of the pancreas, with malignant histological features and splenic infiltration. The case is rare, and few reports have bee...BACKGROUND: We reported a case of malignant nonfunction islet cell tumor (10.0 cm in diameter) of the pancreas, with malignant histological features and splenic infiltration. The case is rare, and few reports have been published. METHODS: A 46-year-old woman with a vague pain in the left upper quadrant for 3 months was found to have a tumor in the spleen. Ultrasonography and computed tomography demonstrated a well-defined pancreatic tumor of 8.2×10.0 cm in size, her serum levels of pancreatic hormones were within normal limits. RESULTS: Splenectomy combined with pancreatectomy was performed for the tail of the pancreas. Resected specimens showed a malignant nonfunctioning islet cell tumor invading the spleen. CONCLUSIONS: The growth pattern of the tumor causes malignant features. Resection of the tumor should be performed by enucleation, pancreaticoduodenectomy or distal pancreatectomy.展开更多
In order to establish an animal model with hepatic metastasis intrasplenic inoculation of carcinoma cells from murine uterine cervical carcinoma (U14) was employed. Results showed a high incidence of hepatic metastasi...In order to establish an animal model with hepatic metastasis intrasplenic inoculation of carcinoma cells from murine uterine cervical carcinoma (U14) was employed. Results showed a high incidence of hepatic metastasis could be obtained through the intrasplenic inoculation of 1 × 106 carcinoma cells. Removal of the primary carcinoma through splenec-tomy at different intervals after intrasplenic inoculation proved that the hepatic metastatic mechanism was not due to mechanical pressure but occurred spontaneously. This experimental model provides a useful means for studying the mechanism and prevention of hepatic metastasis.展开更多
To investigate the feasibility and efficacy of liver gene therapy mediated by intrasplenic transplanta-tion of genetically modified hepatocytes, the normal mouse liver cell line BNL CL. 2 cells were introduced with Ne...To investigate the feasibility and efficacy of liver gene therapy mediated by intrasplenic transplanta-tion of genetically modified hepatocytes, the normal mouse liver cell line BNL CL. 2 cells were introduced with Neo-re-sistant (NeoR) gene or interleukin-2 (IL-2) gene in vitro, and transplanted intrasplenically into normal syngeneic mice (2 × 106 cell/mouse); subsequently, the expressions of the introduced genes in vivo were detected. The RT-PCR results showed that NeoR mRNA expressions were detectable in livers 24 h after transplantation and lasted over 11 weeks. Moreover, The NeoR mRNA was detected to be expressed temporarily in spleens (24 h- 1 week) and lungs (24-96 h) after transplantation. After intrasplenic transplantation of IL-2 gene-modified BNL CL.2 cells, the stable expressions of IL-2 mRNA in the livers of transplanted mice were detectable by RT-PCR (24 h-11 weeks), and certain levels of IL-2 (5-40 pg/mL) remained in the peripheral blood. When IL-2 gene-modified BNL CL. 2 cells were transplanted intrasplenically to treat the metastatic liver colon carcinoma-bearing mice, the survival time of the treated mice was significantly prolonged. The data indicate that intrasplenic transplantation of genetically modified hepatocytes could allow for oriental distribution in host livers and long-term survival of the transplanted liver cells, and effective expression nf exogenous genes in vivo suggesting that this can he a candidate approach to liver-directed gene therapy.展开更多
Orthotopic liver transplantation(OLT)is the only proven effective treatment for both end-stage and metabolic liver diseases.Hepatocyte transplantation is a promising alternative for OLT,but the lack of available donor...Orthotopic liver transplantation(OLT)is the only proven effective treatment for both end-stage and metabolic liver diseases.Hepatocyte transplantation is a promising alternative for OLT,but the lack of available donor livers has hampered its clinical application.Hepatocyte-like cells(HLCs)differentiated from many multi-potential stem cells can help repair damaged liver tissue.Yet almost suitable cells currently identified for human use are difficult to harvest and involve invasive procedures.Recently,a novel mesenchymal stem cell derived from human menstrual blood(MenSC)has been discovered and obtained easily and repeatedly.In this study,we examined whether the MenSCs are able to differentiate into functional HLCs in vitro.After three weeks of incubation in hepatogenic differentiation medium containing hepatocyte growth factor(HGF),fibroblast growth factor-4(FGF-4),and oncostain M(OSM),cuboidal HLCs were observed,and cells also expressed hepatocyte-specific marker genes including albumin(ALB),α-fetoprotein(AFP),cytokeratin 18/19(CK18/19),and cytochrome P450 1A1/3A4(CYP1A1/3A4).Differentiated cells further demonstrated in vitro mature hepatocyte functions such as urea synthesis,glycogen storage,and indocyanine green(ICG)uptake.After intrasplenic transplantation into mice with 2/3 partial hepatectomy,the MenSC-derived HLCs were detected in recipient livers and expressed human ALB protein.We also showed that MenSC-derived HLC transplantation could restore the serum ALB level and significantly suppressed transaminase activity of liver injury animals.In conclusion,MenSCs may serve as an ideal,easily accessible source of material for tissue engineering and cell therapy of liver tissues.展开更多
Objective： Hepatic progenitor cell transplantation has shed light on the treatment of liver failure. The present study was designed to evaluate whether xenogeneic liver epithelial progenitor cells （LEPCs） transplan...Objective： Hepatic progenitor cell transplantation has shed light on the treatment of liver failure. The present study was designed to evaluate whether xenogeneic liver epithelial progenitor cells （LEPCs） transplantation could promote liver recovery in a rat model of acute liver failure. The engraftment and hepatocytic differentiation of transplanted hepatic progenitor cells in the rat spleen was also investigated. Methods： LEPCs were propagated in vitro for long and transduced with lentiviral vector carrying mCherry gene. Intraperitoneal injection of CC14 followed by 2/3 partial hepatectomy three days later were used to establish rat models of acute liver failure. Rats were intrasplenically injected with mCherry modified LEPCs （n=20, 1× 107 cells/0.5 mL） or the same volume of medium （n=20）. Serum liver enzymes （ALT, AST） and liver histology were evaluated for 21 days after transplantation. The engraftment of transplanted LEPCs in the spleens was tested by polymerase chain reaction （PCR） amplification targeting mCherry gene. The differentiation into hepatocytic lineage of transplanted LEPCs was investigated usingimmunohistochemistry staining against Alb. Results： LEPCs were effectively transduced with lentiviral vector showing a transduction efficiency of 90%. Compared with control, cell-injected group displayed significantly lower levels of ALT and AST （P〈0.05） and better histological features including less swelling change and hepatocyte death. PCR amplification of mCherry sequences confirmed the engraftment of LEPCs in the spleens. Alb-positive cells first appeared 5 days after cell transplantation and the number of Alb-positive cells increased substantially （P〈0.05）, which revealed the hepatocytic differentiation process Conclusion： Xenogeneic hepatic progenitor cells can engraft and differentiate into hepatocytes in the splenic parenchyma. Intrasplenic delivery of hepatic progenitor cells ameliorates CCh/partial hepatectomy-induced liver injury in rats展开更多
Inoculation with purified specific protein is usually the first step for preparation of monoclonal antibody (mAb). But it is quite difficult to obtain pure proteins especially with natural structures.Here we attempt t...Inoculation with purified specific protein is usually the first step for preparation of monoclonal antibody (mAb). But it is quite difficult to obtain pure proteins especially with natural structures.Here we attempt to replace the protein inoculation with DNA immunization in the preparation of mAb.The eukaryotic expression vectors pcDNA3-PreS2/S and pVAX-PreS2/S encoding the HBV M protein were constructed and prepared for DNA immunization.Female BALB/c mice developed a well antibody response to the target antigen after muscle injection with corresponding plasmids.The mice with effective antibodies induced were used for preparation of mAb.We found the mice immunized with three administrations of pcDNA3-PreS2/S and boosted by intrasplenic injection with the same plasmid could be exploited for preparation of mAb.And positive hybridoma cell 2D3 that can secrete specific mAb was cloned and analyzed.Our studies demonstrate that gene immunization may provide a convenient and efficient way to prepare mAbs.Cellular & Molecular Immunology. 2004;1(4):295-299.展开更多
Objective To investigate the antitumor effects of intrasplenically transplanted interleukin-18 (IL-18) gene-modified hepatocytes on murine implanted liver carcinoma. Methods Embryonic murine hepatocyte cell line (BNL-...Objective To investigate the antitumor effects of intrasplenically transplanted interleukin-18 (IL-18) gene-modified hepatocytes on murine implanted liver carcinoma. Methods Embryonic murine hepatocyte cell line (BNL-CL2) was transfected with a recombinant adenovirus encoding IL-18 and used as delivery cells for IL-18 gene transfer. Two cell lines,BNL-LacZ and BNL-CL2, were used as controls. One week after intrasplenic injection of C26 cells (colon carcinoma line),tumor-bearing syngeneic mice underwent the intrasplenic transplantation of IL-18 gene-modified hepatocyte cell line and were divided into treatment group (BNL IL-18) and control groups (BNL-LacZ and BNL-CL2 ). Two weeks later,the serum levels of IL-18,interferon-γ (IFN-γ),tumor necrosis factor-α (TNF-α) and nitric oxide (NO) in the implanted liver carcinoma-bearing mice were assayed,the cytotoxicity of murine splenic cytotoxic T-lymphocytes (CTLs) was measured,and the morphology of the hepatic tumors was studied to evaluate the antitumor effects of the approach. Results In the treatment group,the serum levels of IL-18,IFN-γ,TNF-α and NO increased significantly. The splenic CTL activity increased markedly ( P <0.01) ,accompanied by a substantial decrease in tumor volume and the percentage of tumor area and prolonged survival of liver carcinomo-being mice. Conclusions In vivo IL-18 expression by ex vivo manipulated cells with IL-18 recombinant adenovirus is able to exert potent antitumor effects by inducing a predominantly T-cell-helper type 1 (Th1) immune response. Intrasplenic transplantation of adenovirus-mediated IL-18 gene-modified hepatocytes could be used as a targeting treatment for implanted liver carcinoma.展开更多
文摘AIM： To transplant undifferentiated embryonic stem （ES） cells into the spleens of carbon tetrachloride （CCl4）-treated mice to determine their ability to differentiate into hepatocytes in the liver. METHODS： CCh, 0.5 mL/kg body weight, was injected into the peritoneum of C57BL/6 mice twice a week for 5 wk. In group 1 （n = 12）, 1 × 10^5 undifferentiated ES cells （0.1 mL of 1 × 10^6/mL solution）, genetically labeled with GFP, were transplanted into the spleens 1 d after the second injection. Group 2 mice （n = 12） were injected with 0.2 mL of saline twice a week, instead of CCh, and the same amount of ES cells was transplanted into the spleens. Group 3 mice （n = 6） were treated with CCh and injected with 0.1 mL of saline into the spleen, instead of ES cells. Histochemical analyses of the livers were performed on post-transplantation d （PD） 10, 20, and 30. RESULTS： Considerable numbers of GFP-immunopositive cells were found in the periportal regions in group 1 mice （CCh-treated） on PD 10, however, not in those untreated with CCh （group 2）. The GFP-positive cells were also immunopositive for albumin （ALB）, alpha-1 antitrypsin, cytokeratin 18, and hepatocyte nuclear factor 4 alpha on PD 20. Interestingly, most of the GFP-positive cells were immunopositive for DLK, a hepatoblast marker, on PD 10. Although very few ES-derived cells were demonstrated immunohistologically in the livers of group 1 mice on PD 30, improvements in liver fibrosis were observed. Unexpectedly, liver tumor formation was not observed in any of the mice that received ES cell transplantation during the experimental period CONCLUSION： Undifferentiated ES cells developed into hepatocyte-like cells with appropriate integration into tissue, without uncontrolled cell growth.
文摘BACKGROUND: We reported a case of malignant nonfunction islet cell tumor (10.0 cm in diameter) of the pancreas, with malignant histological features and splenic infiltration. The case is rare, and few reports have been published. METHODS: A 46-year-old woman with a vague pain in the left upper quadrant for 3 months was found to have a tumor in the spleen. Ultrasonography and computed tomography demonstrated a well-defined pancreatic tumor of 8.2×10.0 cm in size, her serum levels of pancreatic hormones were within normal limits. RESULTS: Splenectomy combined with pancreatectomy was performed for the tail of the pancreas. Resected specimens showed a malignant nonfunctioning islet cell tumor invading the spleen. CONCLUSIONS: The growth pattern of the tumor causes malignant features. Resection of the tumor should be performed by enucleation, pancreaticoduodenectomy or distal pancreatectomy.
文摘In order to establish an animal model with hepatic metastasis intrasplenic inoculation of carcinoma cells from murine uterine cervical carcinoma (U14) was employed. Results showed a high incidence of hepatic metastasis could be obtained through the intrasplenic inoculation of 1 × 106 carcinoma cells. Removal of the primary carcinoma through splenec-tomy at different intervals after intrasplenic inoculation proved that the hepatic metastatic mechanism was not due to mechanical pressure but occurred spontaneously. This experimental model provides a useful means for studying the mechanism and prevention of hepatic metastasis.
基金Project supported by the National Natural Science Foundation of China.
文摘To investigate the feasibility and efficacy of liver gene therapy mediated by intrasplenic transplanta-tion of genetically modified hepatocytes, the normal mouse liver cell line BNL CL. 2 cells were introduced with Neo-re-sistant (NeoR) gene or interleukin-2 (IL-2) gene in vitro, and transplanted intrasplenically into normal syngeneic mice (2 × 106 cell/mouse); subsequently, the expressions of the introduced genes in vivo were detected. The RT-PCR results showed that NeoR mRNA expressions were detectable in livers 24 h after transplantation and lasted over 11 weeks. Moreover, The NeoR mRNA was detected to be expressed temporarily in spleens (24 h- 1 week) and lungs (24-96 h) after transplantation. After intrasplenic transplantation of IL-2 gene-modified BNL CL.2 cells, the stable expressions of IL-2 mRNA in the livers of transplanted mice were detectable by RT-PCR (24 h-11 weeks), and certain levels of IL-2 (5-40 pg/mL) remained in the peripheral blood. When IL-2 gene-modified BNL CL. 2 cells were transplanted intrasplenically to treat the metastatic liver colon carcinoma-bearing mice, the survival time of the treated mice was significantly prolonged. The data indicate that intrasplenic transplantation of genetically modified hepatocytes could allow for oriental distribution in host livers and long-term survival of the transplanted liver cells, and effective expression nf exogenous genes in vivo suggesting that this can he a candidate approach to liver-directed gene therapy.
基金Project supported by the National High-Tech R&D Program(863) of China(Nos.2011AA020102 and 2012AA020905)the Key Technologies R&D Program of Zhejiang Province(Nos.2012C13015-2and 2011C13029-1)+1 种基金the Hangzhou Key Technologies R&D Program(No.20122513A49)the National Natural Science Foundation of China(Nos.81201783 and 81201089)
文摘Orthotopic liver transplantation(OLT)is the only proven effective treatment for both end-stage and metabolic liver diseases.Hepatocyte transplantation is a promising alternative for OLT,but the lack of available donor livers has hampered its clinical application.Hepatocyte-like cells(HLCs)differentiated from many multi-potential stem cells can help repair damaged liver tissue.Yet almost suitable cells currently identified for human use are difficult to harvest and involve invasive procedures.Recently,a novel mesenchymal stem cell derived from human menstrual blood(MenSC)has been discovered and obtained easily and repeatedly.In this study,we examined whether the MenSCs are able to differentiate into functional HLCs in vitro.After three weeks of incubation in hepatogenic differentiation medium containing hepatocyte growth factor(HGF),fibroblast growth factor-4(FGF-4),and oncostain M(OSM),cuboidal HLCs were observed,and cells also expressed hepatocyte-specific marker genes including albumin(ALB),α-fetoprotein(AFP),cytokeratin 18/19(CK18/19),and cytochrome P450 1A1/3A4(CYP1A1/3A4).Differentiated cells further demonstrated in vitro mature hepatocyte functions such as urea synthesis,glycogen storage,and indocyanine green(ICG)uptake.After intrasplenic transplantation into mice with 2/3 partial hepatectomy,the MenSC-derived HLCs were detected in recipient livers and expressed human ALB protein.We also showed that MenSC-derived HLC transplantation could restore the serum ALB level and significantly suppressed transaminase activity of liver injury animals.In conclusion,MenSCs may serve as an ideal,easily accessible source of material for tissue engineering and cell therapy of liver tissues.
基金Supported by the National Natural Science Foundation of China(30600575,30830099)
文摘Objective： Hepatic progenitor cell transplantation has shed light on the treatment of liver failure. The present study was designed to evaluate whether xenogeneic liver epithelial progenitor cells （LEPCs） transplantation could promote liver recovery in a rat model of acute liver failure. The engraftment and hepatocytic differentiation of transplanted hepatic progenitor cells in the rat spleen was also investigated. Methods： LEPCs were propagated in vitro for long and transduced with lentiviral vector carrying mCherry gene. Intraperitoneal injection of CC14 followed by 2/3 partial hepatectomy three days later were used to establish rat models of acute liver failure. Rats were intrasplenically injected with mCherry modified LEPCs （n=20, 1× 107 cells/0.5 mL） or the same volume of medium （n=20）. Serum liver enzymes （ALT, AST） and liver histology were evaluated for 21 days after transplantation. The engraftment of transplanted LEPCs in the spleens was tested by polymerase chain reaction （PCR） amplification targeting mCherry gene. The differentiation into hepatocytic lineage of transplanted LEPCs was investigated usingimmunohistochemistry staining against Alb. Results： LEPCs were effectively transduced with lentiviral vector showing a transduction efficiency of 90%. Compared with control, cell-injected group displayed significantly lower levels of ALT and AST （P〈0.05） and better histological features including less swelling change and hepatocyte death. PCR amplification of mCherry sequences confirmed the engraftment of LEPCs in the spleens. Alb-positive cells first appeared 5 days after cell transplantation and the number of Alb-positive cells increased substantially （P〈0.05）, which revealed the hepatocytic differentiation process Conclusion： Xenogeneic hepatic progenitor cells can engraft and differentiate into hepatocytes in the splenic parenchyma. Intrasplenic delivery of hepatic progenitor cells ameliorates CCh/partial hepatectomy-induced liver injury in rats
基金supported by National High Technology Research and Development Program of China(863 program 2004AA215242)National Science Found for Distinguished Young Scholars from NSFC(No.39925031)Science and Technology commission of Shanghai municipality(024319112).
文摘Inoculation with purified specific protein is usually the first step for preparation of monoclonal antibody (mAb). But it is quite difficult to obtain pure proteins especially with natural structures.Here we attempt to replace the protein inoculation with DNA immunization in the preparation of mAb.The eukaryotic expression vectors pcDNA3-PreS2/S and pVAX-PreS2/S encoding the HBV M protein were constructed and prepared for DNA immunization.Female BALB/c mice developed a well antibody response to the target antigen after muscle injection with corresponding plasmids.The mice with effective antibodies induced were used for preparation of mAb.We found the mice immunized with three administrations of pcDNA3-PreS2/S and boosted by intrasplenic injection with the same plasmid could be exploited for preparation of mAb.And positive hybridoma cell 2D3 that can secrete specific mAb was cloned and analyzed.Our studies demonstrate that gene immunization may provide a convenient and efficient way to prepare mAbs.Cellular & Molecular Immunology. 2004;1(4):295-299.
基金Thisstudywassupportedby grantsfromtheNationalNaturalScienceFoundationofChina (No 3 0 12 10 0 2 )andNationalKeyBasicResearchProgramofChina (No .2 0 0 1CB5 10 0 0 2 )
文摘Objective To investigate the antitumor effects of intrasplenically transplanted interleukin-18 (IL-18) gene-modified hepatocytes on murine implanted liver carcinoma. Methods Embryonic murine hepatocyte cell line (BNL-CL2) was transfected with a recombinant adenovirus encoding IL-18 and used as delivery cells for IL-18 gene transfer. Two cell lines,BNL-LacZ and BNL-CL2, were used as controls. One week after intrasplenic injection of C26 cells (colon carcinoma line),tumor-bearing syngeneic mice underwent the intrasplenic transplantation of IL-18 gene-modified hepatocyte cell line and were divided into treatment group (BNL IL-18) and control groups (BNL-LacZ and BNL-CL2 ). Two weeks later,the serum levels of IL-18,interferon-γ (IFN-γ),tumor necrosis factor-α (TNF-α) and nitric oxide (NO) in the implanted liver carcinoma-bearing mice were assayed,the cytotoxicity of murine splenic cytotoxic T-lymphocytes (CTLs) was measured,and the morphology of the hepatic tumors was studied to evaluate the antitumor effects of the approach. Results In the treatment group,the serum levels of IL-18,IFN-γ,TNF-α and NO increased significantly. The splenic CTL activity increased markedly ( P <0.01) ,accompanied by a substantial decrease in tumor volume and the percentage of tumor area and prolonged survival of liver carcinomo-being mice. Conclusions In vivo IL-18 expression by ex vivo manipulated cells with IL-18 recombinant adenovirus is able to exert potent antitumor effects by inducing a predominantly T-cell-helper type 1 (Th1) immune response. Intrasplenic transplantation of adenovirus-mediated IL-18 gene-modified hepatocytes could be used as a targeting treatment for implanted liver carcinoma.
