共同培养诱导骨髓基质干细胞向肝细胞分化的研究

A study of rat mesenchymal stem cells (MSCs) differentiating into liver cells when co-cultured with rat hepatocytes

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。

Objectives To explore whether rat mesenchymal stem cells （MSCs） can be induced to develop into hepatocytes and the role of the microenvironment of hepatocytes growth in inducing MSCs differentiating into hepatocytes in vitro. Methods Mesenchymal stem cells were collected from the aspirates from femurs of SD rats by density gradient centrifugation and identified by flow cytometric analysis and alkaline phosphatase （ALP） staining. Rat hepatocytes were isolated by the modified two-step method described by Seglen. Two 6-well culture plates were piled up after the chambers＇ bottoms of the upper plate was removed. Then the upper and lower chambers were separated by a semipermeable membrane. MSCs and hepatocytes of rats were plated separately in the upper and lower chambers of the two 6-well culture plates for co-culturing. MSCs cultured alone without co-culturing with hepatocytes served as controls. On days 1, 3, 7, 14, 21 and 28, mRNA of cytokeratin 18 （CK-18）, alpha-fetoprotein （AFP） and albumin were detected by reverse transcriptase-polymerase chain reaction （RT-PCR）, and immunocytochemistry staining of CK-18 AFP and albumin were also examined. Results The shapes of MSCs co-cultured with hepatocytes changed and their sizes and uumbers increased in the course of the culturing. When MSCs were co-cultured with hepatocytes for 2 weeks, colonies composed of polygonal cells resembling mature hepatocytes were found. In the controls, shapes of cells also changed and their sizes and numbers increased, but colonies composed of polygonal cells resembling mature hepatocytes were not found. Of the MSCs co-cultured with hepatocytes, on day 7, the mRNA of AFP was detected by RT-PCR, and it increased on day 14, and then decreased on day 21. mRNA of albumin and CK-18were detected by RT-PCR from day 14 to day 28in the co-cultured cells, but mRNA of AFP and CK-18 and albumin were not detected in the controls in the course of the culturing. Immunocytochemical analysis for CK-18, albumin, and AFP, showed positive staining reaction for AFP on day 7, for CK-18 and AFP on day 14 in the co-cultured cells but not in the controls. Conclusion Rat MSCs co-cultured with hepatocytes can differentiate into hepatocytes.