摘要
背景:生物型人工肝的构建有望成为治疗急性肝衰竭的有效方法,但构建人工肝的种子细胞来源、培养模式、营养获取等方面仍存在较多难题,制约血液净化-人工肝的发展与临床应用。目的:探讨在不添加外源血清等异种来源物质的条件下将人诱导性多能干细胞诱导分化为肝细胞样细胞的可行性。方法:应用含Transwell小室的6孔板构建3D培养体系,外源添加丙戊酸和尼克酰胺对人诱导性多能干细胞进行诱导分化,并将分化的肝细胞样细胞与生物补片共培养。实验分为5组:人诱导性多能干细胞为A组、正常肝细胞为B组、不添加尼克酰胺诱导分化的肝细胞样细胞为C组、添加尼克酰胺诱导分化的肝细胞样细胞为D组、在生物外科补片上培养的肝细胞样细胞为E组。倒置相差显微镜下观察D组肝细胞样细胞的形态;免疫荧光检测D组细胞中肝细胞核因子4α和甲胎蛋白的表达;荧光实时定量PCR和Western blot检测A、B、C、D组细胞中甲胎蛋白与白蛋白的mRNA和蛋白表达;流式细胞术检测A、C、D组细胞的分化效率;免疫细胞化学检测B组和E组细胞中胆盐输出泵蛋白的表达;ELISA检测D组和E组上清液中乳酸脱氢酶活性、白蛋白和尿素氮含量。结果与结论:(1)D组细胞由梭形逐渐转变成多边形;(2)D组细胞中肝细胞核因子4α和甲胎蛋白呈阳性表达;(3)D组细胞中甲胎蛋白、白蛋白的基因和蛋白表达明显高于A组和C组(P<0.01);(4)B组和E组细胞中胆盐输出泵蛋白呈明显阳性表达;(5)E组细胞乳酸脱氢酶活性、白蛋白和尿素氮的含量明显高于D组(P<0.01);(6)结果表明,外源添加小分子化合物的三维培养体系和生物外科补片联合应用有助于促进诱导性多能干细胞在体外诱导分化为功能性肝细胞样细胞。
BACKGROUND:The construction of bioartificial liver is expected to be an effective method for the treatment of acute liver failure.However,there are still many problems in seed cell source,culture mode and nutrient acquisition,which restrict the development and clinical application of blood purification-artificial liver.OBJECTIVE:To investigate the feasibility of inducing the differentiation of induced pluripotent stem cells from human skin into hepatocyte-like cells without addition of exogenous sources such as exogenous serum.METHODS:A 3D culture system was constructed by using a three-dimensional culture system containing 6-well plates of the Transwell chamber.The induction and differentiation of human induced pluripotent stem cells were performed by addition of valproic acid and nicotinamide.The differentiated hepatocyte-like cells were co-cultured with biological patches.There were five groups in the experiment:human induced pluripotent stem cells were set as group A,normal hepatocytes as group B,hepatocyte-like cells without induction by nicotinamide as group C,hepatocyte-like cells with induction by nicotinamide as group D,and hepatocyte-like cells cultured on the biological patch as group E.The morphology of hepatocyte-like cells in group D was observed under inverted phase contrast microscope.The expression of nuclear factor 4αand alpha-fetoprotein in hepatocytes of group D was detected by immunofluorescence.Real-time quantitative PCR and western blot were used to detect mRNA and protein expressions of alpha-fetoprotein and albumin in the cells of groups A,B,C and D.Flow cytometry was used to detect the differentiation efficiency of cells in groups A,C and D.Immunocytochemistry detection was used to detect the protein expression of bile salt export pump in groups B and E.ELISA assay was used to detect lactate dehydrogenase activity,albumin and urea nitrogen contents in the supernatants of the groups D and E.RESULTS AND CONCLUSION:(1)The cells of group D changed from fusiform to polygon in shape.(2)Positive expression of hepatocyte nuclear factor 4 alpha and alpha fetoprotein was found in the cells of group D.(3)The gene and protein expressions of alpha fetoprotein and albumin in group D was significantly higher than those in groups A and C(P<0.01).(4)The protein expression of bile salt export pump in the cells was remarkably positive in groups B and E.(4)The activity of lactate dehydrogenase and the contents of albumin and urea nitrogen in cultured cells of group E were significantly higher than those in group D(P<0.01).To conclude,the combination of 3D culture system with exogenous small molecules and biological surgical patch helps to induce induced pluripotent stem cells to differentiate into functional hepatocyte-like cells in vitro.
作者
李嘉晋
王荣丽
李婷婷
何东
石伟
Li Jia-jin;Wang Rong-li;Li Ting-ting;He Dong;Shi Wei(School of Clinical Medicine,Southwest Medical University,Luzhou 646000,Sichuan Province,China;Department of Critical Care Medicine,Mianyang People’s Hospital,Mianyang 621000,Sichuan Province,China;Department of Nephropathy,Mianyang People’s Hospital,Mianyang 621000,Sichuan Province,China)
出处
《中国组织工程研究》
CAS
北大核心
2018年第29期4681-4686,共6页
Chinese Journal of Tissue Engineering Research
