【作者】 孙海晨； 于丁宁； 张淑文； 刘爽； 崔叶青； 李非； 孙家邦； 李铎；

【机构】 首都医科大学宣武医院外科实验室；

【摘要】 目的:探讨人胎肝细胞分离、培养的简单方法并证实其具有近似正常肝细胞功能。方法:取水囊引产中期(4～6月龄)妊娠死胎,用两步灌注法分离胎肝细胞,观察细胞形态及存活情况,并以正常成人肝组织作正常对照,检测胎肝细胞五种基因的表达,进行光密度分析。结果:经台盼蓝染色、FDA 荧光染色观察,细胞培养后11天内均存活良好,最长可存活18天:新鲜分离、培养3、5、7、10天组胎肝细胞均可表达 GAPDH(Glyceraldehyde Phosphate Dehydrogenase 磷酸甘油醛脱氢酶)、ALB(Aibumin 白蛋白)、 GS(Glutamine Synthetase 谷氨酰胺合成酶)、CK(Cytokeratin 细胞角蛋白),经光密度分析证实,上述四种基因在培养肝细胞和正常成人肝细胞中的表达无明显统计学差异;AFP 基因表达明显强于正常肝细胞,并且于培养5天后呈显著的减弱。结论:经分离、培养的人胎肝细胞存活良好,具有和正常成人肝细胞类似的基因表达。更多还原

【Abstract】 Objective:To investigate a simple method of hunman fetal hepatocytes separation and culture,and prove their normal function similarly with adult hepatocytes.Methods:Liver cells were isolated from abortion fetus(4～6 month old)with collagenase perfusing in situ via umbilical vein.Then the cells were inoculated in culture flask and observed in microscope every 2 or 3 days to exam the survival rate.The cells were divided into four groups according to culture time with adult normal hepatocytes in control.NTCP,GAPDH,ALB,GS,CK and AFP gene express were examined by RT-PCR and the optic density of bands on the electrophotograms were analysed by PhotoShop 7.0.RESULTS:The cultured Cells represent normal hepatoce-llular morphology and normal GAPDH,ALB,GS and CK gene expression.AFP gene expression,was more stronger than normal cells and began attenuating after 5 days culture.CONCLUSIONS:Fetal hepatocytes cultured in vitro demonstrated some normal adult hepatocellular functions.更多还原