Chen Ping, Yan Hongtao, Chen Yin, He Zhonglin
Department of Hepatobiliary Surgery, Daping Hospital, the Third Military Medical University, Chongqing, China.
Exp Mol Pathol. 2009 Apr;86(2):101-7. doi: 10.1016/j.yexmp.2009.01.013.
The aim of this study was to investigate the role and regulatory mechanisms of Akt/TSC1-TSC2/mTOR signal pathway on the hepatocyte growth and proliferation after partial hepatectomy in rats.
We used the animal model of 70% hepatectomy, separated and cultivated hepatocytes. According to the different time points after partial hepatectomy, it could be grouped into 0 h, 2 h, 6 h, 24 h and 72 h. According to the different kinds of specific inhibitor in the nutritive medium after the separation of hepatocytes, it could be grouped into Triciribine (TR), Rapamycin (RA) and Control (CO). We investigated (3)H-Leucine incorporation into protein, the cross section areas of hepatocytes, and detected cell cycle through FCM. The expressions of phosphorylated protein TSC2 and mTOR were observed.
(1) The content of phosphorylated protein TSC2 in group CO began to increase at 2 h and got to the peak at 6 h but declined at 24 h. The content of phosphorylated protein TSC2 in group RA had the same variation with that of phosphorylated protein TSC2 in group CO. (2) At the time point of 0 h, 2 h, 6 h and 24 h after operation, the incorporation efficiency of (3)H-Leucine in groups RA and TR was different from that in group CO in statistics (P<0.01). (3) It could be seen that the cross section areas of hepatocytes in groups RA and TR were different from that in group CO in statistics at 2 h and 6 h after operation (P<0.05). (4) Comparing with the other two inhibitor groups (TR and RA), the number of cells during the period of G0/G1 in group CO became fewer, while the number of cells during the period of S and G2/M grew obviously (referring to Fig. 8). After operation, each time point was different from the inhibitor groups obviously (P<0.05 or P<0.01). The peak declined greatly at 24 h and 72 h after operation.
These data strongly suggest the effects of Akt/TSC1-TSC2/mTOR signal pathway on hepatocyte growth, protein synthesis and cell cycle, and prove its contribution to liver regeneration.
本研究旨在探讨Akt/TSC1-TSC2/mTOR信号通路在大鼠部分肝切除术后肝细胞生长和增殖中的作用及调控机制。
采用70%肝切除动物模型，分离培养肝细胞。根据部分肝切除术后不同时间点，分为0 h、2 h、6 h、24 h和72 h组。根据肝细胞分离后营养培养基中不同种类的特异性抑制剂，分为三氮脒（TR）、雷帕霉素（RA）和对照组（CO）。我们检测了（3）H-亮氨酸掺入蛋白质的情况、肝细胞的横截面积，并通过流式细胞术检测细胞周期。观察磷酸化蛋白TSC2和mTOR的表达。
（1）CO组磷酸化蛋白TSC2含量在2 h开始升高，6 h达到峰值，24 h下降。RA组磷酸化蛋白TSC2含量变化与CO组磷酸化蛋白TSC2相同。（2）术后0 h, 2 h, 6 h和24 h时间点，RA组和TR组（3）H-亮氨酸掺入效率与CO组相比有统计学差异（P<0.01）。（3）术后2 h和6 h可见，RA组和TR组肝细胞横截面积与CO组相比有统计学差异（P<0.05）。（4）与其他两个抑制剂组（TR和RA）相比，CO组G0/G1期细胞数量减少，而S期和G2/M期细胞数量明显增加（见图8）。术后各时间点与抑制剂组相比差异明显（P<0.05或P<0.01）。术后24 h和72 h峰值大幅下降。
这些数据有力地表明了Akt/TSC1-TSC2/mTOR信号通路对肝细胞生长、蛋白质合成和细胞周期的影响，并证明了其对肝脏再生的作用。
