Yan Cheng, Xue Gai, Wu Liying, Liu Jianfang, Hou Yanning
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2015 Jul;29(7):878-83.
To investigate the differentiation potential of human umbilical cord mesenchymal stem cells (HUCMSCs) into hepatocytes induced by rat fibrotic liver tissue extracts.
Liver fibrosis was induced in the Sprague Dawley rats (weighting, 180-220 g) by repeated intraperitoneal injections of 3% thioacetamide-saline at a dose of 200 mg/kg twice a week for 4 weeks; fibrotic liver tissues were used to prepare liver homogenate supernatants. The HUCMSCs at passage 3 were cultured in DMEM/F12 with 10% fetal bovine serum (FBS) (control group) and in DMEM/F12 with 10% FBS and 50 g/L liver homogenate supernatants (experimental group) for 7 days. The morphological changes of the cells were recorded; the protein levels of cytokeratin 18 (CK18), alpha fetoprotein (AFP), and CYP3A4 were measured using Western blot. The glycogen storing ability of the cells was detected by periodic acid-schiff (PAS) staining. Furthermore, the synthesis of albumin (ALB) and blood urea nitrogen (BUN) was measured.
In experimental group, after 1 day of induction, the stem cells of fusiform shape began to lose sharp edges and progressively shrunk, and then they changed into hepatocyte-like cells with round and irregular shape at 7 days. Positive expressions of AFP, CK18, and CYP3A4 were observed in the experimental group, but negative expression in the control group. The concentrations of BUN and ALB were (0.43 ± 0.07) mmol/L and (8.08 ± 0.41) µg/mL in the control group and were (2.52 ± 0.20) mmol/L and (41.48 ± 4.11) µg/mL in the experimental group, showing significant differences (t=24.160, P = 0.000; t = 19.810, P = 0.000). PAS staining results showed navy blue nucleus and lavender cytoplasm in the control group, but dark purple cell body and visible nucleus in the experimental group.
HUCMSCs could differentiate into hepatocyte-like cells induced by rat fibrotic liver tissue extracts, which have hepatocyte biomarkers (AFP, CK18, and CYP3A4) and hepatocyte-specific functions of glycogen storage, urea production and ALB secretion, so they could partially replace the function of hepatocytes, that may be one of the therapeutic mechanisms of stem cell transplantation.
研究大鼠纤维化肝组织提取物诱导人脐带间充质干细胞（HUCMSCs）向肝细胞分化的潜能。
采用体重180 - 220 g的Sprague Dawley大鼠，通过每周两次腹腔注射200 mg/kg的3%硫代乙酰胺生理盐水溶液，连续注射4周诱导肝纤维化；用纤维化肝组织制备肝匀浆上清液。将第3代HUCMSCs在含10%胎牛血清（FBS）的DMEM/F12培养基中培养（对照组），以及在含10% FBS和50 g/L肝匀浆上清液的DMEM/F12培养基中培养（实验组）7天。记录细胞的形态变化；采用蛋白质印迹法检测细胞角蛋白18（CK18）、甲胎蛋白（AFP）和细胞色素P450 3A4（CYP3A4）的蛋白水平。通过过碘酸 - 希夫（PAS）染色检测细胞的糖原储存能力。此外，检测白蛋白（ALB）和血尿素氮（BUN）的合成。
实验组中，诱导1天后，梭形干细胞开始失去锐利边缘并逐渐收缩，7天时转变为圆形和不规则形的类肝细胞。实验组中观察到AFP、CK18和CYP3A4的阳性表达，而对照组为阴性表达。对照组中BUN和ALB的浓度分别为（0.43 ± 0.07）mmol/L和（8.08 ± 0.41）μg/mL，实验组中分别为（2.52 ± 0.20）mmol/L和（41.48 ± 4.11）μg/mL，差异有统计学意义（t = 24.160，P = 0.000；t = 19.810，P = 0.000）。PAS染色结果显示对照组细胞核呈深蓝色，细胞质呈淡紫色，而实验组细胞体呈深紫色且可见细胞核。
大鼠纤维化肝组织提取物可诱导HUCMSCs分化为类肝细胞，这些细胞具有肝细胞生物标志物（AFP、CK18和CYP3A4）以及糖原储存、尿素生成和ALB分泌等肝细胞特异性功能，因此它们可部分替代肝细胞的功能，这可能是干细胞移植的治疗机制之一。
