骨髓间质干细胞通过影响PI3K／Akt信号通路抑制肝星状细胞的增殖

Bone marrow mesenchymal stem cells can inhibit hepatic stellate cells proliferation by influencing PI3K/Akt singal pathway

目的探讨在体外非接触共培养条件下，大鼠骨髓间质干细胞（MSCs）如何调控肝星状细胞（HSCs）的增殖。方法实验组将MSCs／HSCs按2×10^4／2×10^4（细膨孔）的比例接种于TransweH共培养板上下层，建立非接触共培养体系；对照组为HSCs（2×10^4细胞／孔）单独培养；阳性对照组为加入抑制剂LY294002（20μmol／m1）观察抑制效果。共培养24、48、72h后应用流式细胞仪分析细胞周期，Western blot检测HSCs的P—Akt及Akt蛋白的表达。结果共培养2，4、48、72h后HSCs的S期细胞与对照组比较明显减少，且抑制程度呈时间依赖性（11．24±0．34〈15．73±0．76〈19．14±0．91〈23．16±1．80，P〈0．05）；加入LY294002后可明显增加共培养组抑制效果，与单独使用抑制剂比较s期细胞减少更加明显（8．2±0．8〈11．7±1．6，P〈0．05）；共培养组及共培养＋抑制剂组p-Akt蛋白表达较单独培养组均显著下调，而Akt蛋白表达差异无统计学意义（P〉0．05）；共培养＋抑制剂组较共培养组p-Akt蛋白表达下调。结论MSCs可明显抑制HSCs的增殖，可能是通过影响P13K／Akt信号通路达到这一作用，磷酸化的Akt蛋白在其中起关键作用。

Objective To investigate the mechanism of bone marrow mesenchymal stem cells （MSCs） regulating the proliferation of hepatic stellate cells （HSCs） under non-contact co-culture in vitro. Methods Rat MSCs/Rats HSCs were seeded in proportion （2× 10^4/2×10^4 cells/well） in the Transwell co-culture plate as the experimental group to establish the upper and lower double-cell co-culture system. HSCs were separately cultured （2 ×10^4 cells/well） in a well as control group. LY294002 as the inhibitor of PI3K/Akt signal pathway was added to co-culture group and control group respectively （20μmol/ml）, which served as the positive control group. Cell cycle was determined at different co-culture time points （24, 48, 72 h） by using flow cytometry. The p-Akt and Akt protein expression in HSCs was detected by using Western blotting. Results HSCs co-cultured with MSCs at different time points （72, 48, 24 h） significantly inhibi- ted HSCs proliferation as compared with the mono-cuhure groups, and the tendency become obvious with the passage of time （ 11.24 ± 0. 34 〈 15.73 ± 0. 76 〈 19. 14± 0. 91 〈 23.16 ± 1.80, P 〈 0. 05 ）. The inhibitory effect was become larger after adding the LY294002 into the co-culture groups than the co-culture groups （8. 2±0. 8 〈 11.7± 1.6,P 〈0. 05）. After HSCs co-cultured with MSCs for 24 h, the expression of p-Akt protein was reduced, and significantly reduced in the co-culture group added with the LY294002. No sig- nificant difference was found in Akt protein in any group. Conclusion MSCs can significantly inhibit HSCs proliferation through influencing the PI3K/Akt signal pathway, and the p-Akt is the key point.