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目的探讨胚胎肝组织滤液定向诱导大鼠骨髓间充质干细胞(BMSCs)分化为肝细胞的诱导条件,为肝组织工程提供新的种子细胞来源。方法在体外培养体系中加入胎肝滤液,模拟体内肝脏微环境,诱导BMSCs向肝细胞定向分化,以免疫细胞化学检测肝细胞标志物;PAS检测糖原表达;靛青绿染色检测转化细胞的分化程度;测定细胞培养上清液中丙氨酸氨基转移酶(ALT)、谷草转氨酶(AST)、碱性磷酸酶(ALP)的含量以检测其功能状态。结果BMSCs经胎肝滤液诱导14d时细胞呈现多角形、卵圆形或圆形细胞的特征性改变;甲胎蛋白(AFP)和白蛋白(ALB)免疫反应、PAS反应和吲哚靛青绿(ICG)摄入实验及上清液中ALT、AST、ALP等酶均在诱导的第7天开始出现,AFP和ALB免疫反应在21d时达高峰;PAS反应和ICG摄入实验随着时间的延长而增强;上清液中的各个酶在14d达高峰,之后呈下降趋势。结论胎肝滤液可诱导BMSCs形成具有肝细胞形态和功能特点的细胞。
OBJECTIVE: To investigate the induction of differentiation of rat bone marrow mesenchymal stem cells (BMSCs) into hepatocytes induced by the filtrate of embryonic liver tissue, and to provide a new source of seed cells for liver tissue engineering. Methods Fetal liver filtrate was added to in vitro culture system to simulate the in vivo liver microenvironment and to induce BMSCs to differentiate into hepatocytes. Immunocytochemistry was used to detect hepatocyte markers. PAS was used to detect glycogen expression. Indocyanine green staining was used to detect the differentiation of transformed cells The contents of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) in the cell culture supernatant were measured to test their functional status. Results The cells showed characteristic changes of polygonal, oval or round cells on the 14th day after being induced by fetal liver filtrate. Immunofluorescence of AFP and ALB, PAS and indocyanine green (ICG ) Intake experiments and supernatant ALT, AST, ALP and other enzymes began to appear on the 7th day induction, AFP and ALB immunoreactivity peaked at 21d; PAS response and ICG uptake experiments with time and Enhanced; each enzyme in the supernatant reached its peak on 14d, then showed a downward trend. Conclusion Fetal liver filtrate can induce BMSCs to form cells with morphological and functional characteristics of hepatocytes.