两种肝组织匀浆上清液诱导人脐带间充质干细胞向肝细胞分化的比较  

Differentiation efficiency of human umbilical cord mesenchymal stem cells into hepatocytes under two kinds of liver homogenate supernatants:a comparative study

作　　者：闫成[1,2] 薛改[2] 吴丽颖[2] 刘建芳[2] 侯艳宁[2] 

机构地区：[1]解放军医学院,北京市100853 [2]解放军白求恩国际和平医院药剂科,河北省石家庄市050082

出　　处：《中国组织工程研究》2015年第19期2993-2998,共6页Chinese Journal of Tissue Engineering Research

基　　金：军队"十二五"重点课题(BWS11J002);课题名称:成体间充质干细胞移植修复战创伤重要器官功能损害的前沿技术研究~~

摘　　要：背景:前期研究证明正常大鼠肝组织匀浆上清液可诱导人脐带间充质干细胞定向分化为具有部分肝细胞功能的肝样细胞,而肝纤维化大鼠肝组织匀浆上清液的诱导可行性及与前者诱导效果的比较尚不明确。目的:比较正常肝组织及纤维化肝组织微环境诱导人脐带间充质干细胞向肝细胞分化的能力。方法:采用腹腔注射3%硫代乙酰胺生理盐水溶液(200 mg/kg,2次/周,共4周)的方法制备SD大鼠肝纤维化模型,取肝纤维化大鼠及正常大鼠肝脏制备肝组织匀浆上清。将第3代人脐带间充质干细胞进行分组诱导培养:1标准对照组:加入含体积分数为10%胎牛血清的DMEM/F12培养基。2纤维化肝组织匀浆组:加入含体积分数为10%胎牛血清及50 g/L纤维化肝组织匀浆的DMEM/F12培养基。3正常肝组织匀浆组:加入含体积分数为10%胎牛血清及100 g/L正常肝组织匀浆的DMEM/F12培养基。诱导开始后于倒置显微镜下观察各组细胞形态学变化;取标准对照组及诱导7 d的匀浆组细胞采用Western Blot法检测CK18、AFP、CYP3A4、CYP2E1、CYP2D6、TPH2的表达情况,采用ELISA法测定各组细胞培养液中白蛋白水平。结果与结论:与标准对照组长梭形成纤维样细胞相比,诱导7 d时,肝纤维化匀浆组及正常匀浆组细胞形态变化明显,呈类圆形,胞体丰满。与标准对照组相比,两匀浆组细胞均可表达肝细胞标志物CK18和AFP。标准对照组细胞可表达少量CYP2E1,两匀浆组细胞表达CYP3A4、CYP2E1、CYP2D6、TPH2等肝细胞功能蛋白,且CYP2E1的表达量较标准对照组明显增加(P<0.01),而两匀浆组间上述蛋白的相对表达量差异无显著性意义(P>0.05)。同时,两匀浆组细胞分泌白蛋白的能力较标准对照组亦明显增强(P<0.01),两匀浆组相比差异无显著性意义(P>0.05)。实验结果显示正常肝组织和纤维化肝组织微环境均可诱导人脐带间充质干细胞向肝细胞分化,在达到相同诱BACKGROUND： Previous studies have demonstrated that normal rat liver homogenate supernatant can induce human umbilical cord mesenchymal stem cells to differentiate into hepatocyte-like cells with partial hepatocytefunctions. However, whether fibrotic liver homogenate supematant can work or how the inducing effect is remains unclear. OBJECTIVE： To investigate the differentiation potential of human umbilical cord mesenchymal stem cells into hepatocytes under the normal liver and fibretic liver micreenvirenment in vitro. METHODS： Liver fibrosis was induced in the SD rats by repeated intraperitoneal injectiOns of 3% thioacetamide at a dose of 200 mg/kg body mass, twice a week for 4 weeks, and then fibrotic liver tissues and normal liver tissues were used to prepare liver homogenate supernatants. Passage 3 human umbilical cord mesenchymal stem cells were used and divided into standard control group （cells were cultured in DMEM/F12 with 10% fetal bovine serum）, fibretic liver homogenate supernatants group （cells were cultured in DMEM/F12 with 10% fetal bovine serum and 50 g/L fibrotic liver homogenate supernatants）, normal liver homogenate supernatants group （cells were cultured in DMEM/F12 with 10% fetal bovine serum and 100 g/L normal liver homogenate supernatants）. The morphological changes of the cells in each group were recorded under inverted microscope; the protein levels of CK18, AFP, CYP3A4, CYP2E1, CYP2D6 and TPH2 were evaluated using western blot assay. Furthermore, the concentration of albumin in the cells was measured. RESULTS AND CONCLUSION： Aftelr a 7-day inducement, the stem cells in liver homogenate supernatants groups lost their fusiform shape and changed into hepatocyte-like cells with the morphous of round shape. Compared with the standard control group, the hepatocyte-like cells in the two liver homogenate supernatants groups exhibited human hepatocyte biomarkers, CK18 and AFP. The standard control group cells could express a little amount of CYP2E1, while cells in the two liver

关 键 词：肝硬化 脐带 间质干细胞 肝细胞 细胞分化 干细胞 脐带脐血干细胞 肝纤维化 肝组织匀浆上清液 微环境 人脐带间充质干细胞 诱导分化 细胞色素P450酶 白蛋白 

分 类 号：R394.2[医药卫生—医学遗传学]