脐带间充质干细胞通过旁分泌机制维持原代小鼠肝细胞的体外生长  

Umbilical cord mesenchymal stem cells support the in vitro growth of primary mouse hepatocytes by a paracrine mechanism

作　　者：吴源泉 吴建华 王梁 林洋 Wu Yuanquan;Wu Jianhua;Wang Liang;Lin Yang(Kashgar Region First Hospital, Kashgar 844000, XinjiangUygur Autonomous Region, China)

机构地区：[1]喀什地区第一医院

出　　处：《中国组织工程研究》2019年第25期4031-4036,共6页Chinese Journal of Tissue Engineering Research

基　　金：新疆维吾尔自治区自然科学基金(2016D01C025)项目负责人:吴源泉~~

摘　　要：背景:肝细胞移植对于晚期肝功能衰竭患者是一种非常有效的替代治疗方法,体外条件下扩增培养肝细胞具有非常重要的临床意义。目的:体外实验探讨人脐带间充质干细胞对原代小鼠肝细胞功能的调控作用。方法:实验分3组:①人脐带间充质干细胞与原代小鼠肝细胞非接触共培养(非接触共培养组);②人脐带间充质干细胞培养液上清与原代小鼠肝细胞共培养(上清组);③原代小鼠肝细胞用含体积分数为10%胎牛血清的低糖DMEM培养(常规培养组)。培养48,72,96 h,采用MTT法检测3组肝细胞增殖情况,吲哚菁绿检测肝细胞相关功能,ELISA法检测白蛋白及尿素分泌情况,RT-PCR检测肝细胞相关基因表达。该研究的实施符合喀什地区第一人民医院的相关伦理要求(伦理批件号:01号)。结果与结论:①与常规培养组相比,培养48,72,96 h时非接触共培养组和上清组都可以显著促进小鼠肝细胞增殖,差异有显著性意义(P <0.05);②与常规培养组相比,培养96 h时非接触共培养组和上清组都可以维持小鼠肝细胞的摄取功能活性,差异有显著性意义(P <0.05);③与常规培养组相比,在48 h和96 h时非接触共培养组和上清组小鼠肝细胞分泌白蛋白及尿素水平显著升高,差异有显著性意义(P <0.05);④与常规培养组相比,非接触共培养组和上清组HGF、b FGF以及ALB mRNA表达显著升高,差异有显著性意义(P <0.05);但细胞色素基因CYP2B9的表达在3组细胞内差异无显著性意义(P> 0.05);⑤结果表明,脐带间充质干细胞可通过旁分泌机制在体外维持原代小鼠肝细胞功能,并促进其增殖,是体外条件下人工扩增肝细胞的一种新的应用策略。BACKGROUND: Hepatocyte transplantation is a very effective alternative treatment for patients with advanced liver failure. It is of very important clinical significance to expand and culture hepatocytes in vitro.OBJECTIVE: To explore the regulatory effect of human umbilical cord mesenchymal stem cells on the function of primary mouse hepatocytes under in vitro conditions.METHODS: Umbilical cord mesenchymal stem cells were co-cultured with primary mouse hepatocytes in a non-contacted way(co-culture group). Umbilical cord mesenchymal stem cell supernatant was collected and co-cultured with primary mouse hepatocytes(conditioned medium). Primary mouse hepatocytes were cultured in conventional culture medium containiing low-dose DMEM+10% fetal bovine serum as control group. Cell counting kit-8 method was used to detect the proliferation of three groups of hepatocytes. RT-PCR was used to detect the expression of hepatocyte-related genes. Indocyanine green detection was used to detect the function of hepatocytes. ELISA method was used to detect the secretion of albumin and urea. The implementation of the study was in line with the relevant ethical requirements of Kashgar Region First Hospital(ethical approval No. 01)RESULTS AND CONCLUSION: Compared with the control group, umbilical cord mesenchymal stem cells could significantly retain the uptake ability of mouse hepatocytes(96 hours of culture) and promote their proliferation(48, 72, and 96 hours of culture) by non-contact co-culture(P < 0.05) or supernatant culture(P < 0.05). Compared with the control group, the primary mouse hepatocytes in the co-culture group and the conditioned medium group had significantly stronger albumin and urea secretion ability(P < 0.05). RT-PCR detection of related genes revealed higher expression of hepatocyte growth factor, basic fibroblast growth factor and album mRNAs in the co-culture group and conditioned medium group than the control group(P < 0.05). However, we observed similar results in the expression of the cytochrome gene Cyp2 b

关 键 词：人脐带间充质干细胞 肝细胞 共培养 旁分泌 肝细胞生长因子 肝功能 新疆维吾尔自治区自然科学基金 

分 类 号：R459.9[医药卫生—治疗学] R394.2[医药卫生—临床医学]