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目的探索大鼠骨髓基质干细胞(MSCs)向肝细胞分化的能力,及肝细胞生长的微环境对其诱导分化的作用。方法采用梯度离心法,获取大鼠骨髓基质肝细胞;改良的两步法获取大鼠肝细胞。将鉴定的MSCs和肝细胞以半透膜相隔共同培养,以单独培养的MSCs作对照。在第1、3、7、14、21、28人, 18(CK-18)的基因和蛋白表达。结果在MSCs与肝细胞共同培养过程中,MSCs出现明显的细胞形态、体积和数量变化,可见双核或多核细胞,细胞轮廓较清晰。RT-PCR检测:共用培养的MSCs第7天即出现AFP基因表达,第14天表达增强,第21天表达减弱;第14天开始出现白蛋白、CK-18基因表达,并持续表达。单独培养的MSCs均无表达。共同培养的MSCs,于第7天进行免疫细胞化学检测,AFP即呈阳性; 第14天白蛋白和CK-18也呈阳性;单独培养的MSCs未见AFP、白蛋白及CK-18表达。结论大鼠骨髓基质肝细胞与肝细胞共同培养,可被诱导分化为肝细胞。
Objective To explore the ability of rat bone marrow stromal stem cells (MSCs) to differentiate into hepatocytes and the effect of microenvironment of hepatocyte growth on their differentiation. Methods Rat bone marrow stromal cells were obtained by gradient centrifugation. Rat liver cells were obtained by a modified two-step method. The identified MSCs and hepatocytes were co-cultured with semi-permeable membranes and MSCs cultured alone were used as controls. Genes and proteins were expressed on day 1, 3, 7, 14, 21, 28, and 18 (CK-18). Results In MSCs and hepatocytes co-culture process, MSCs showed obvious cell morphology, volume and number of changes, we can see binucleated or multinucleated cells, the cell outline more clearly. RT-PCR showed that AFP gene expression was observed on the 7th day in co-cultured MSCs, increased on the 14th day, and weakened on the 21st day. The albumin and CK-18 gene expression started to appear on the 14th day and was continuously expressed. MSCs cultured alone were not expressed. Co-cultured MSCs were positive for AFP on day 7 after immunocytochemistry, and positive for albumin and CK-18 on day 14; AFP, albumin and CK-18 were not expressed on MSCs cultured alone. Conclusion Rat bone marrow stromal hepatocytes co-cultured with hepatocytes can be induced to differentiate into hepatocytes.