V/Ag multilayers with different periodic thicknesses were fabricated by magnetron sputtering deposition. The columnar structure and the orientation relationship of the multilayers were investigated by transmission ele...V/Ag multilayers with different periodic thicknesses were fabricated by magnetron sputtering deposition. The columnar structure and the orientation relationship of the multilayers were investigated by transmission electron microscopy, high resolution transmission electron microscopy, selected-area electron diffraction and X-ray diffraction. It was found that the multilayered structure became flatter as increasing individual layer thickness from 2 to 6 nm, and then became waved as the individual layer thickness increases to 8 nm. At the beginning of the growth, the morphology of the multilayers with small periodic thickness was influenced mainly by thermodynamic instabilities, and the morphology of the multilayers with larger periodic thickness was mainly influenced mainly by the columnar growth of V. When the waved interfaces were formed, the continuum growth of the multilayers was also influenced by the shadowing effect and the finite atomic size effect. All of these factors resulted in the columnar structure of the multilayers. Multilayers with small periodic thickness presented strong orientation relationship. Nano-hardness tests indicated that multilayers with flat sublayer morphology and clear interfaces exhibited larger hardness.展开更多
Objective:To explore the application effect of the path for improving humanistic literacy of operating room nursing students based on the ORTCC model,in order to make up for the deficiency of systematic humanistic edu...Objective:To explore the application effect of the path for improving humanistic literacy of operating room nursing students based on the ORTCC model,in order to make up for the deficiency of systematic humanistic education in the traditional teaching model.Methods:120 nursing students who practiced in the operating room from June 2023 to June 2024 were randomly divided into the experimental group and the control group,with 60 students in each group.The control group received routine teaching,while the experimental group applied the ORTCC model on the basis of routine and intervened through setting humanistic literacy goals,formulating behavioral norms,conducting diversified training,implementing two-way assessment,and creating departmental culture.The effect of the intervention was evaluated using the Nurse Humanistic Quality Scale,the Clinical Communication Ability Assessment Scale for Operating Room Nursing Students,and a self-made nursing satisfaction questionnaire.Results:After the intervention,the total score of humanistic literacy in the experimental group(4 weeks of intervention:161.43±10.06 points,12 weeks of intervention:165.71±9.45 points)was significantly higher than that in the control group(139.38±10.95 points,143.31±10.52 points)(all p<0.001);The total score of clinical communication ability in the experimental group(109.69±8.05 points for 4 weeks of intervention,116.90±7.21 points for 12 weeks of intervention)was also significantly better than that in the control group(99.14±9.23 points,103.03±8.78 points)(all p<0.001);The total score of patient care satisfaction in the experimental group(94.2±4.3)was significantly higher than that in the control group(82.6±5.7)(p<0.001).Conclusion:The ORTCC model can systematically enhance the humanistic literacy,clinical communication skills and patient satisfaction of operating room nursing students,providing an effective approach for optimizing clinical teaching in the operating room.展开更多
AIM: To determine the prognostic value of circulating indicators of cell death in acute-on-chronic liver failure (ACLF) patients with chronic hepatitis B virus (HBV) infection as the single etiology. METHODS: Full len...AIM: To determine the prognostic value of circulating indicators of cell death in acute-on-chronic liver failure (ACLF) patients with chronic hepatitis B virus (HBV) infection as the single etiology. METHODS: Full length and caspase cleaved cytokeratin 18 (detected as M65 and M30 antigens) represent circulating indicators of necrosis and apoptosis. M65 and M30 were identified by enzyme-linked immunosorbent assay in 169 subjects including healthy controls (n = 33), patients with chronic hepatitis B (CHB, n = 55) and patients with ACLF (n = 81). According to the 3-mo survival period, ACLF patients were defined as having spontaneous recovery (n = 33) and non-spontaneous recovery which included deceased patients and those who required liver transplantation (n = 48). RESULTS: Both biomarker levels significantly increased gradually as liver disease progressed (for M65: P < 0.001 for all; for M30: control vs CHB, P = 0.072; others: P < 0.001 for all). In contrast, the M30/M65 ratio was significantly higher in controls compared with CHB patients (P = 0.010) or ACLF patients (P < 0.001). In addition, the area under receiver operating characteristic curve (AUC) analysis demonstrated that both biomarkers had diagnostic value (AUC >= 0.80) in identifying ACLF from CHB patients. Interestingly, it is worth noting that the M30/M65 ratio was significantly different between spontaneous and non-spontaneous recovery in ACLF patients (P = 0.032). The prognostic value of the M30/M65 ratio was compared with the Model for End-Stage Liver Disease (MELD) and Child-Pugh scores at the 3-mo survival period, the AUC of the M30/M65 ratio was 0.66 with a sensitivity of 52.9% and the highest specificity of 92.6% (MELD:AUC = 0.71; sensitivity, 79.4%; specificity, 63.0%; Child-Pugh: AUC = 0.77; sensitivity, 61.8%; specificity, 88.9%). CONCLUSION: M65 and M30 are strongly associated with liver disease severity. The M30/M65 ratio may be a potential prognostic marker for spontaneous recovery in patients with HBV-related ACLF. (C) 2014 Baishideng Publishing Group Co., Limited. All rights reserved.展开更多
AIM:To investigate the role of autophagy in the antiapoptotic effect of augmenter of liver regeneration(ALR).METHODS:Autophagy was induced through serum deprivation.An ALR-expressing plasmid was transfected into HepG2...AIM:To investigate the role of autophagy in the antiapoptotic effect of augmenter of liver regeneration(ALR).METHODS:Autophagy was induced through serum deprivation.An ALR-expressing plasmid was transfected into HepG2 cells,and autophagic flux was determined using fluorescence microscopy,electron microscopy,Western blot and quantitative polymerase chain reaction(q PCR) assays.After ALR-expressing plasmid transfection,an autophagy inhibitor [3-methyladenine(3-MA)] was added to HepG2 cells,and apoptosis was observed using fluorescence microscopy and flow cytometry.RESULTS:Autophagy was activated in HepG2 cells,peaking at 24 h after serum deprivation.Microtubuleassociated protein light chain three-II levels were higher in HepG2 cells treated with ALR than in control cells,fluorescence microscopy,electron microscopy and q PCR studies showed the similar trend,and p62 levels showed the opposite trend,which indicated that ALR may play an important role in increasing autophagy flux.The numbers of apoptotic cells were substantially higher in HepG2 cells treated with both ALR and 3-MA than in cells treated with ALR alone.Therefore,the protective effect of ALR was significantly attenuated or abolished when autophagy was inhibited,indicating that the anti-apoptotic effect of ALR may be related to autophagy.CONCLUSION:ALR protects cells from apoptosis partly through increased autophagy in HepG2 cells and may be valuable as a new therapeutic treatment for liver disease.展开更多
AIM: To investigate the relationship between serum vitamin D3 levels and liver fibrosis or inflammation in treatment-naive Chinese patients with chronic hepatitis C(CHC).METHODS: From July 2010 to June 2011, we enroll...AIM: To investigate the relationship between serum vitamin D3 levels and liver fibrosis or inflammation in treatment-naive Chinese patients with chronic hepatitis C(CHC).METHODS: From July 2010 to June 2011, we enrolled 122 CHC patients and 11 healthy controls from Dingxicity, Gansu Province, China. The patients were infected with Hepatitis C virus(HCV) during blood cell retransfusion following plasma donation in 1992-1995, and had never received antiviral treatment. At present, all the patients except two underwent liver biopsy with ultrasound guidance. The Scheuer Scoring System was used to evaluate hepatic inflammation and the Metavir Scoring System was used to evaluate hepatic fibrosis. Twelve-hour overnight fasting blood samples were collected in the morning of the day of biopsy. Serum levels of alanine aminotransferase, aspartate aminotransferase, total bilirubin, direct bilirubin, cholineste rase, prothrom binactivity, albumin, γ-glutamyl transpeptidase, hemoglobin, calcium and phosphorus were determined. Serum HCV RNA levels were measured by real-time PCR. Serum levels of 25-hydroxyvitamin D3 [25(OH)D3] and 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] were measured by high-performance liquid chromatography tandem mass spectrometry.R E S U LT S : Serumlevels of 2 5(OH) D3 but not 24,25(OH)2D3 were significantly lower in CHC patients than in control subjects. Serum 25(OH)D3 levels did not correlate with liver fibrosis, inflammation, patient age, or levels of alanine aminotransferase, aspartate aminotransferase, total bilirubin, direct bilirubin, prothrombin activity, cholinesterase or HCV RNA. However, serum 25(OH)D3 levels did correlate with serum 24,25(OH)2D3 levels. Serum 25(OH)D3 and 24,25(OH)2D3 levels, and the 25(OH)D3/24,25(OH)2D3 ratio, have no difference among the fibrosis stages or inflammation grades.CONCLUSION: We found that serum levels of 25(OH)D3 and its degradation metabolite 24,25(OH)2D3 did not correlate with liver fibrosis in treatment-naive Chinese patient with CHC.展开更多
The ion implantation uniformity is of vital importance for an ion implanter.In this paper,we report the,uniformity measurement for a large current ion implanter(LC-16 type) by implanting of 190-keV Ar ions into Si to ...The ion implantation uniformity is of vital importance for an ion implanter.In this paper,we report the,uniformity measurement for a large current ion implanter(LC-16 type) by implanting of 190-keV Ar ions into Si to 3×1016 atoms/cm2,followed by Rutherford backscattering spectroscopy(RBS) and sheet resistance measurement providing quantitative information on spatial distribution of dopants.The implant doses obtained from RBS at selected points of the sample give a spatial uniformity of <5%,which are confirmed by the sheet resistance measurement.While sheet resistance is an indirect method for dose evaluation of ion-implanted samples,RBS provides a competent technique for calibration of the ion implantation system.And both measurements show that good uniformity can be achieved for the ion implanter by tuning of the scanning process.展开更多
In the alternative splicing, intron retention, of histamine H3 receptors in rats and mice, the short transcript isoforms that are excised alternatively spliced introns are easily detected in a very low level in rats a...In the alternative splicing, intron retention, of histamine H3 receptors in rats and mice, the short transcript isoforms that are excised alternatively spliced introns are easily detected in a very low level in rats and are undetectable in mice using the regular PCR protocol. The retained introns have common 5＇ splice site and different 3＇ splice sites. The detailed mechanism for the special alternative splicing remains largely unclear. In this study, we developed a minigene splicing system to recapitulate natural alternative splicing of the receptors and investigated the effects of 5＇ and 3＇ splice sites on intron retention in HeLa cells. Mutating weak 5＇ and 3＇ splice sites of the alternatively spliced introns toward the canonical consensus sequences promoted the splicing of the corresponding introns in rat and mouse minigenes. The effect of splice site strength was context-dependent and much more sigiaificant for the 3＇ splice site of the longer alternative intron than for the 3＇ splice site of the shorter alternative intron and the common 5＇ splice sites; it was also more significant in the rat minigene than in the mouse minigene. Mutating the 3＇ splice site of the longer alternative intron resulted in almost complete splicing of the intron and made the corresponding isoform to become the nearly exclusive transcript in the rat minigene.展开更多
AIM To prepare a Gpm6a/Reelin^(GFPCreERT2) construct with a rapid and reliable strategy using a bacterial artificial chromosome(BAC). METHODS Gpm6 a and Reelin BACs were purified and transformed into SW102 E. coli by ...AIM To prepare a Gpm6a/Reelin^(GFPCreERT2) construct with a rapid and reliable strategy using a bacterial artificial chromosome(BAC). METHODS Gpm6 a and Reelin BACs were purified and transformed into SW102 E. coli by electroporation. The GFPCreE RT2 fragment was prepared from a shuttle vector and transformed into SW102 E. coli carrying a BAC. Homologous recombination was induced in SW102 E. coli. Recombinant clones were screened and confirmed by PCR and restriction enzyme digestion. Recombinant clones were transformed into SW102 E. coli to remove the kanamycin unit.RESULTS A complete BAC was successfully transformed into SW102 E. coli by electroporation because BAC purified from SW102 E. coli showed the same pattern as the original BAC with Bam H I digestion. The GFPCre ERT2 fragment was deemed to have been prepared successfully because we obtained the same size fragment as expected. Homologous recombination was induced, and GFPCre ERT2 was deemed to have been inserted into the correct site of the BAC because we found the band change was the same as the expected pattern after restriction enzyme digestion. The kanamycin unit was deemed to have been removed successfully because we obtained different sizes of bands that were consistent with the results expected by PCR with different primers. CONCLUSION The construct of Gpm6 a^(GFPCreERT2) or Reelin^(GFPCreERT2) was prepared successfully, which will establish a foundation for tracing the hepatic stellate cell lineage and studying its function.展开更多
基金the National Natural Science Foundation of China(Nos.91026014 and 11175133)the Foundations from Chinese Ministry of Education(Nos.2011014113004 and NCET-13-0438)the Hubei Provincial Natural Science Foundation(No.2012FFA042) for financial support
文摘V/Ag multilayers with different periodic thicknesses were fabricated by magnetron sputtering deposition. The columnar structure and the orientation relationship of the multilayers were investigated by transmission electron microscopy, high resolution transmission electron microscopy, selected-area electron diffraction and X-ray diffraction. It was found that the multilayered structure became flatter as increasing individual layer thickness from 2 to 6 nm, and then became waved as the individual layer thickness increases to 8 nm. At the beginning of the growth, the morphology of the multilayers with small periodic thickness was influenced mainly by thermodynamic instabilities, and the morphology of the multilayers with larger periodic thickness was mainly influenced mainly by the columnar growth of V. When the waved interfaces were formed, the continuum growth of the multilayers was also influenced by the shadowing effect and the finite atomic size effect. All of these factors resulted in the columnar structure of the multilayers. Multilayers with small periodic thickness presented strong orientation relationship. Nano-hardness tests indicated that multilayers with flat sublayer morphology and clear interfaces exhibited larger hardness.
文摘Objective:To explore the application effect of the path for improving humanistic literacy of operating room nursing students based on the ORTCC model,in order to make up for the deficiency of systematic humanistic education in the traditional teaching model.Methods:120 nursing students who practiced in the operating room from June 2023 to June 2024 were randomly divided into the experimental group and the control group,with 60 students in each group.The control group received routine teaching,while the experimental group applied the ORTCC model on the basis of routine and intervened through setting humanistic literacy goals,formulating behavioral norms,conducting diversified training,implementing two-way assessment,and creating departmental culture.The effect of the intervention was evaluated using the Nurse Humanistic Quality Scale,the Clinical Communication Ability Assessment Scale for Operating Room Nursing Students,and a self-made nursing satisfaction questionnaire.Results:After the intervention,the total score of humanistic literacy in the experimental group(4 weeks of intervention:161.43±10.06 points,12 weeks of intervention:165.71±9.45 points)was significantly higher than that in the control group(139.38±10.95 points,143.31±10.52 points)(all p<0.001);The total score of clinical communication ability in the experimental group(109.69±8.05 points for 4 weeks of intervention,116.90±7.21 points for 12 weeks of intervention)was also significantly better than that in the control group(99.14±9.23 points,103.03±8.78 points)(all p<0.001);The total score of patient care satisfaction in the experimental group(94.2±4.3)was significantly higher than that in the control group(82.6±5.7)(p<0.001).Conclusion:The ORTCC model can systematically enhance the humanistic literacy,clinical communication skills and patient satisfaction of operating room nursing students,providing an effective approach for optimizing clinical teaching in the operating room.
基金Supported by National Science and Technology Key Project of China on"Major Infectious Diseases",No.2012ZX10002004-006,No.2012ZX10004904-003-001,No.2013ZX10002002-006-001Beijing Municipal Science and Technology Commission,No.Z131107002213019,No.Z131100004613030+2 种基金High Technical Personnel Training Program in Beijing Health System,No.2011-3-083,No.2013-3-071Special Scientific Research Fund for Beijing Health Development,No.2011-2018-04National Natural Science Foundation of China,No.30800979,No.30800517
文摘AIM: To determine the prognostic value of circulating indicators of cell death in acute-on-chronic liver failure (ACLF) patients with chronic hepatitis B virus (HBV) infection as the single etiology. METHODS: Full length and caspase cleaved cytokeratin 18 (detected as M65 and M30 antigens) represent circulating indicators of necrosis and apoptosis. M65 and M30 were identified by enzyme-linked immunosorbent assay in 169 subjects including healthy controls (n = 33), patients with chronic hepatitis B (CHB, n = 55) and patients with ACLF (n = 81). According to the 3-mo survival period, ACLF patients were defined as having spontaneous recovery (n = 33) and non-spontaneous recovery which included deceased patients and those who required liver transplantation (n = 48). RESULTS: Both biomarker levels significantly increased gradually as liver disease progressed (for M65: P < 0.001 for all; for M30: control vs CHB, P = 0.072; others: P < 0.001 for all). In contrast, the M30/M65 ratio was significantly higher in controls compared with CHB patients (P = 0.010) or ACLF patients (P < 0.001). In addition, the area under receiver operating characteristic curve (AUC) analysis demonstrated that both biomarkers had diagnostic value (AUC >= 0.80) in identifying ACLF from CHB patients. Interestingly, it is worth noting that the M30/M65 ratio was significantly different between spontaneous and non-spontaneous recovery in ACLF patients (P = 0.032). The prognostic value of the M30/M65 ratio was compared with the Model for End-Stage Liver Disease (MELD) and Child-Pugh scores at the 3-mo survival period, the AUC of the M30/M65 ratio was 0.66 with a sensitivity of 52.9% and the highest specificity of 92.6% (MELD:AUC = 0.71; sensitivity, 79.4%; specificity, 63.0%; Child-Pugh: AUC = 0.77; sensitivity, 61.8%; specificity, 88.9%). CONCLUSION: M65 and M30 are strongly associated with liver disease severity. The M30/M65 ratio may be a potential prognostic marker for spontaneous recovery in patients with HBV-related ACLF. (C) 2014 Baishideng Publishing Group Co., Limited. All rights reserved.
基金National Natural Science Foundation of China,No.81300349 and No.81270532Beijing Natural Science Foundation,No.7144216+2 种基金Beijing Nova Program,No.Z131107000413016Project of Science and Technology Activities of Preferred Overseas Personnel of Beijing(2014)Project of Cultivation of High Level Medical Technical Personnel in Health System of Beijing
文摘AIM:To investigate the role of autophagy in the antiapoptotic effect of augmenter of liver regeneration(ALR).METHODS:Autophagy was induced through serum deprivation.An ALR-expressing plasmid was transfected into HepG2 cells,and autophagic flux was determined using fluorescence microscopy,electron microscopy,Western blot and quantitative polymerase chain reaction(q PCR) assays.After ALR-expressing plasmid transfection,an autophagy inhibitor [3-methyladenine(3-MA)] was added to HepG2 cells,and apoptosis was observed using fluorescence microscopy and flow cytometry.RESULTS:Autophagy was activated in HepG2 cells,peaking at 24 h after serum deprivation.Microtubuleassociated protein light chain three-II levels were higher in HepG2 cells treated with ALR than in control cells,fluorescence microscopy,electron microscopy and q PCR studies showed the similar trend,and p62 levels showed the opposite trend,which indicated that ALR may play an important role in increasing autophagy flux.The numbers of apoptotic cells were substantially higher in HepG2 cells treated with both ALR and 3-MA than in cells treated with ALR alone.Therefore,the protective effect of ALR was significantly attenuated or abolished when autophagy was inhibited,indicating that the anti-apoptotic effect of ALR may be related to autophagy.CONCLUSION:ALR protects cells from apoptosis partly through increased autophagy in HepG2 cells and may be valuable as a new therapeutic treatment for liver disease.
基金Grants from High technical personnel training item from Beijing Health System,No.2011-3-083,No.2013-3-071Beijing Municipal Science&Technology Commission,No.Z131107002213019,No.Z151100004015066+4 种基金Base to Clinical Scientific Research Cooperation fund of Capital Medical University No.15JL67National Science and Technology Key Project on"Major Infectious Diseases such as HIV/AIDS,Viral Hepatitis Prevention and Treatment",No.2012ZX10002004-006,No.2012ZX10004904-003-001,and No.2013ZX10002002-006Beijing Municipal Administration of Hospitals Clinical medicine Development of special funding support,No.XM201308National Key Subject Construction Project,NO.WJWYA-2014-002Scientific research base construction-the major infectious diseases prevention and control of collaborative innovation center,No.115215
文摘AIM: To investigate the relationship between serum vitamin D3 levels and liver fibrosis or inflammation in treatment-naive Chinese patients with chronic hepatitis C(CHC).METHODS: From July 2010 to June 2011, we enrolled 122 CHC patients and 11 healthy controls from Dingxicity, Gansu Province, China. The patients were infected with Hepatitis C virus(HCV) during blood cell retransfusion following plasma donation in 1992-1995, and had never received antiviral treatment. At present, all the patients except two underwent liver biopsy with ultrasound guidance. The Scheuer Scoring System was used to evaluate hepatic inflammation and the Metavir Scoring System was used to evaluate hepatic fibrosis. Twelve-hour overnight fasting blood samples were collected in the morning of the day of biopsy. Serum levels of alanine aminotransferase, aspartate aminotransferase, total bilirubin, direct bilirubin, cholineste rase, prothrom binactivity, albumin, γ-glutamyl transpeptidase, hemoglobin, calcium and phosphorus were determined. Serum HCV RNA levels were measured by real-time PCR. Serum levels of 25-hydroxyvitamin D3 [25(OH)D3] and 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] were measured by high-performance liquid chromatography tandem mass spectrometry.R E S U LT S : Serumlevels of 2 5(OH) D3 but not 24,25(OH)2D3 were significantly lower in CHC patients than in control subjects. Serum 25(OH)D3 levels did not correlate with liver fibrosis, inflammation, patient age, or levels of alanine aminotransferase, aspartate aminotransferase, total bilirubin, direct bilirubin, prothrombin activity, cholinesterase or HCV RNA. However, serum 25(OH)D3 levels did correlate with serum 24,25(OH)2D3 levels. Serum 25(OH)D3 and 24,25(OH)2D3 levels, and the 25(OH)D3/24,25(OH)2D3 ratio, have no difference among the fibrosis stages or inflammation grades.CONCLUSION: We found that serum levels of 25(OH)D3 and its degradation metabolite 24,25(OH)2D3 did not correlate with liver fibrosis in treatment-naive Chinese patient with CHC.
基金supported by the National Natural Science Foundation of China(Nos.11405117 and 11205116)International Cooperation Program of the Ministry of Science and Technology of China(No.2015DFR00720)
文摘The ion implantation uniformity is of vital importance for an ion implanter.In this paper,we report the,uniformity measurement for a large current ion implanter(LC-16 type) by implanting of 190-keV Ar ions into Si to 3×1016 atoms/cm2,followed by Rutherford backscattering spectroscopy(RBS) and sheet resistance measurement providing quantitative information on spatial distribution of dopants.The implant doses obtained from RBS at selected points of the sample give a spatial uniformity of <5%,which are confirmed by the sheet resistance measurement.While sheet resistance is an indirect method for dose evaluation of ion-implanted samples,RBS provides a competent technique for calibration of the ion implantation system.And both measurements show that good uniformity can be achieved for the ion implanter by tuning of the scanning process.
基金supported by the National Basic Research Program of China (973 Program) (No.2006CB943601)
文摘In the alternative splicing, intron retention, of histamine H3 receptors in rats and mice, the short transcript isoforms that are excised alternatively spliced introns are easily detected in a very low level in rats and are undetectable in mice using the regular PCR protocol. The retained introns have common 5＇ splice site and different 3＇ splice sites. The detailed mechanism for the special alternative splicing remains largely unclear. In this study, we developed a minigene splicing system to recapitulate natural alternative splicing of the receptors and investigated the effects of 5＇ and 3＇ splice sites on intron retention in HeLa cells. Mutating weak 5＇ and 3＇ splice sites of the alternatively spliced introns toward the canonical consensus sequences promoted the splicing of the corresponding introns in rat and mouse minigenes. The effect of splice site strength was context-dependent and much more sigiaificant for the 3＇ splice site of the longer alternative intron than for the 3＇ splice site of the shorter alternative intron and the common 5＇ splice sites; it was also more significant in the rat minigene than in the mouse minigene. Mutating the 3＇ splice site of the longer alternative intron resulted in almost complete splicing of the intron and made the corresponding isoform to become the nearly exclusive transcript in the rat minigene.
基金Supported by National Natural Science Foundation of China,No.81300349 and No.81270532the Beijing Natural Science Foundation,No.7144216+3 种基金the Beijing Nova Program,No.Z131107000413016the Project of Science and Technology Activities of Preferred Overseas Personnel of Beijing(2014)the Project of Cultivation of High Level Medical Technical Personnel in the Health System of Beijing,No.2014-3-090 and No.2013-3-071Beijing Municipal Institute of public medical research development and reform pilot project,No.2016-2
文摘AIM To prepare a Gpm6a/Reelin^(GFPCreERT2) construct with a rapid and reliable strategy using a bacterial artificial chromosome(BAC). METHODS Gpm6 a and Reelin BACs were purified and transformed into SW102 E. coli by electroporation. The GFPCreE RT2 fragment was prepared from a shuttle vector and transformed into SW102 E. coli carrying a BAC. Homologous recombination was induced in SW102 E. coli. Recombinant clones were screened and confirmed by PCR and restriction enzyme digestion. Recombinant clones were transformed into SW102 E. coli to remove the kanamycin unit.RESULTS A complete BAC was successfully transformed into SW102 E. coli by electroporation because BAC purified from SW102 E. coli showed the same pattern as the original BAC with Bam H I digestion. The GFPCre ERT2 fragment was deemed to have been prepared successfully because we obtained the same size fragment as expected. Homologous recombination was induced, and GFPCre ERT2 was deemed to have been inserted into the correct site of the BAC because we found the band change was the same as the expected pattern after restriction enzyme digestion. The kanamycin unit was deemed to have been removed successfully because we obtained different sizes of bands that were consistent with the results expected by PCR with different primers. CONCLUSION The construct of Gpm6 a^(GFPCreERT2) or Reelin^(GFPCreERT2) was prepared successfully, which will establish a foundation for tracing the hepatic stellate cell lineage and studying its function.
