过表达肝细胞核因子4α脐带间充质干细胞促进小鼠大部肝切后肝再生

Overexpression of hepatocyte nuclear factor 4α in umbilical cord mesenchymal stem cells promotes liver regeneration after subtotal hepatectomy in mice

摘要:

目的 探讨人源性脐带间充质干细胞（UC-MSC）以及过表达肝细胞核因子4α（HNF4α）的UC-MSC能否促进小鼠大部肝切后肝再生。方法 体外分离、培养、鉴定人UC-MSC，慢病毒转染过表达HNF4α。体外收集细胞培养上清液，将L02与上清液共培养，通过细胞增殖实验试剂盒（CCK8）检测细胞增殖活性。体内实验建立肝大部切除模型（约70%），分别经尾静脉向肝切除小鼠移植0.9%生理盐水（NS）、MSC、MSC-HNF4α。48h后比较3组肝切后肝再生的变化，通过免疫组化来观察肝标本Ki67的表达。结果 成功分离鉴定UC-MSC，并且成功建立稳定过表达HNF4α的MSC。体外实验MSC组和MSC-HNF4α组中L02的增殖能力都明显高于NS组（P＜0.01），MSC组高于MSC-HNF4α组（P＜0.05）。同样体内实验相对于NS组，经MSC或MSC-HNF4α细胞处理的肝脏，其Ki67的表达明显高于NS组（P＜0.01），同样MSC组高于MSC-HNF4α组（P＜0.05）。结论 UC-MSC和过表达HNF4α的MSC都分泌一系列因子促进肝再生。

Abstract:

Objective To investigate whether human umbilical cord mesenchymal stem cells (UC-MSC) and the cells with overexpression of hepatocyte nuclear factor 4α (HNF4α) can promote liver regeneration after subtotal hepatectomy in mice. Methods After human UC-MSC were isolated, cultured and identified, the cells were transfected with lentiviral vector LV-HNF4α. Then the cell supernatant was collected and cultured with L02 cells, and the L02 cell viability was detected by cell counting kit-8 (CCK8) assay. In the in vivo study, a total of 18 mice were randomly divided into 3 groups (n=6 for each group): normal saline (NS) group, MSC group and MSC-HNF4α group, and received an injection of NS, MSC and MSC-HNF4α (5×106/ml), respectively via tail vein in 2 h before the establishment of subtotal hepatectomy model (70%). In 48 h later, the expression of Ki67 in the liver tissues was detected by immunohistochemical assay, and the results were compared among 3 groups. Results UC-MSC was successfully isolated from human umbilical cord, and the UC-MSC with stable overexpression of HNF4α were established. In vitro study indicated that the L02 cells showed stronger proliferative ability when cocultured with MSC and MSC-HNF4α than cultured alone (P＜0.01), and those cocultured with MSC-HNF4α stronger than the cells with MSC (P＜0.05). In vivo study showed similar findings: the expression of Ki67 was stronger in the mice treated with MSC or MSC-HNF4α than those with NS (P＜0.01), and also overexpression of HNF4α resulted in more significant expression of Ki67 (P＜0.05). Conclusion Both MSC and MSC-HNF4α secret some cytokines to promote liver regeneration.