肝硬化患者骨髓间充质干细胞诱导分化为肝样细胞的实验

Differentiation of mesenchymal stem cells isolated from patients with hepatocirrhosis into hepatocyte-like cells

目的:探讨肝硬化患者骨髓间充质干细胞向肝样细胞的诱导分化,为患者自体骨髓间充质干细胞移植修复肝损伤提供实验依据。方法:实验于2005-05/06在解放军北京军区总医院肝病中心完成。骨髓来自北京军区总医院肝病中心一例肝硬化患者,应用Ficoll分离液分离培养其骨髓间充质干细胞,应用人肝细胞生长因子对骨髓间充质干细胞进行定向诱导分化,采用倒置显微镜形态观察、反转录聚合酶链反应及免疫组织化学染色检测诱导前后不同阶段骨髓间充质干细胞中白蛋白和角蛋白18核糖核酸和蛋白的表达,通过细胞形态及其白蛋白和角蛋白18的表达等指标确定骨髓间充质干细胞向肝样细胞的分化。结果:①经Ficoll分离液分离得到的人骨髓间充质干细胞体外培养20d左右细胞生长达80%～90%汇合,其形态呈较均一的长梭形。②人骨髓间充质干细胞经肝细胞生长因子诱导培养3周后,诱导组中出现圆形细胞,呈克隆样生长。③人骨髓间充质干细胞经肝细胞生长因子诱导2周时,表达少量白蛋白核糖核酸,于诱导3周时可见白蛋白核糖核酸的表达明显增多。④应用肝细胞生长因子诱导3周后人骨髓间充质干细胞胞浆中白蛋白及角蛋白18染色均呈阳性。结论:肝硬化患者骨髓间充质干细胞在肝细胞生长因子诱导下能定向分化为肝样细胞。

AIM： To investigate the differentiation of mesenchymal stem cells （MSCs） isolated from the patient with hepatoeirrhosis into hepatoeyte-like cells, so to make a foundation for the use of MSCs transplantation to repair the hepatic injury. METHODS： The experiment was carried out in the Therapeutic Center of Liver Diseases. General Hospital of Beijing Military Area Command of Chinese PLA, Beijing 100700, China from May to June in 2005. Bone marrow was obtained from a patient with hepatocirrhosis. MSCs were isolated by Fieoll-Pague density gradient centrifugation and then were induced by hepatocyte growth factor （HGF）. The morphological changes of MSCs and the expression of albnmin and keratin 18 in MSCs were detected bv means of inversion microscope observation, transcription-polymerase chain reaction （RT-PCR） and immunohistochemical analysis. The morphological changes and the expression of albumin and keratin 18were used as the markers to show whether the MSCs had differentiated into hepatocyte-like cells. RESULTS： ① MSCs isolated by Ficoll were shuttle shape and 80-90% confluent after 20 days. ② Three weeks after induced by HGF, some MSCs became round and grew in clones. ③ Two weeks after induced by HGF, MSCs expressed a little albumin mRNA, then 3 weeks after induced by HGF, the expression of albumin mRNA in MSCs increased significantly. ④Three weeks after induced by HGF, MSCs expressed positive albumin and keratin 18. CONCLUSION： The MSCs isolated from the patients with hepatocirrhosis can be induced into hepatocyte-like cells by HGF.