大鼠肝部分切除术后mTOR信号通路对肝再生的作用

Effects of Rapamycin on the Liver Regeneration after Partial Hepatectomy in Rats

目的:探讨mTOR信号通路对大鼠肝部分切除术后肝细胞蛋白质合成功能及肝细胞大小的影响。方法:采用Sprague-Dawley雄性大鼠70％肝切除模型和肝细胞分离与培养方法,在术后不同的时相点分离残肝的肝细胞,进行培养。实验分组:分离的残肝肝细胞分两组:对照组(Control组、C组)、雷帕霉素组(Rapamycin组、R组)。采用Western blot方法检测磷酸化mTOR蛋白在不同时相点的变化;采用3H-亮氨酸(3H-Leucine)掺入法测定肝细胞蛋白质合成;扫描电镜(AMRAY 1000B,US)获取肝细胞图像,病理图像分析系统(北航CM2000B)测定细胞面积。结果:1)C组中,磷酸化mTOR蛋白的含量由0h开始升高,6h达到高峰,以后即降低,而R组明显较C组含量降低;2)在术后的各时相点,C组的肝细胞的蛋白质合成率较R组显著升高(P<0．05),而且随时间点的延长,蛋白质合成率呈上升趋势;3)在2h和6h时相点C组肝细胞面积较R组显著增大(P<0．05)。但是,在C组肝细胞24h时相点面积较2h和6h减小(P<0．05)。结论:体外实验证实,肝部分切除术后mTOR信号通路即活化,促进肝细胞的蛋白质合成和细胞生长。因此,我们推测mTOR信号通路在肝再生过程中发挥重要作用。

Objective： To explore the role of the mTOR-signalling pathway on the liver regenetation after partial hepatectomy in normal rats. Methods： A method to separate and culture hepatocytes was established and the hepatocytes were separated and cultured at different time points after partial hepaectomy in normal rats.The cultivation of separated hepatocytes were divided into two groups ： control group （C） and rapamycin group （R）. The protein expression of phosphor-mTOR was determined by Western blotting. The level of synthesis of protein in regenerative hepatocytes was assayed by 3H-Leucine incorporation, and the cross-sectional area of hepatocytes was assayed by scanning electron microscope （SEM）and computer-assisted inage analysis system（BEIHANG CM2000B）. Results： The protein phosphor-roTOR expression was significantly enhanced at 2h and at 6h with a peak at 6h after hepatectomy, then decreased gradually,but the protein phosphor-mTOR expression was suppressed by rapamycin in group R. The level of protein synthesis in regenerating hepatocytes cultured in group C was higger than that in group R after partial hepatectomy at all time points （P〈0.01）; and the cross-sectional area of hepatocytes growing on glass coverslips cultured in group C increased more significantly than that in group R after partial hepatectomy in 2h and 6h （357.02±166.02vs294.37±157.94 P〈0.05; 362.26±140.43 vs278.15±93.24 P〈0.01 ）. Conclusion： The experiment in vitro demonstrate that the mTOR-signalling pathway was activated immediately after partial hepatectomy, which could promote the synthesis of protein and cell growth in hepatocytes. The roTOR-signalling pathway must have played a pivotal role in liver regenatation.