【作者】 张志华；

【导师】 郭杰；

【作者基本信息】 山东大学 ， 口腔临床医学， 2015， 硕士

【摘要】 目的：增殖和迁移是细胞参与各种细胞反应的关键行为,细胞生长因子发挥其生理功能的主要方式就是通过调节效应细胞的增殖和迁移。胰岛素样生长因子I(insulin-like growth factor I, IGF-I)是影响肌肉卫星细胞功能状态的一个重要细胞因子,因此,对IGF-I促进肌肉卫星细胞增殖、迁移机制的研究具有现实意义。mTOR/STAT3信号通路是细胞代谢和生长的中央控制器,对细胞的各项生命活动起着重要的调控作用,而本课题组前期实验发现STAT3蛋白在IGF-I介导的成肌细胞增殖过程中发挥着重要作用。基于以上背景本文拟探讨mTOR/STAT3信号通路在IGF-I诱导的成肌细胞增殖、迁移中的作用。方法：第一部分：应用MTT法检测10ng/ml、50ng/ml、100ng/ml、200ng/ml的IGF-I与2%FBS分别作用于L6成肌细胞24h、48h、72h的OD值；根据MTT结果选出适宜的IGF-I浓度后,实时细胞分析系统(Real Time Cell Analyzer, RTCA)连续监测IGF-I对L6成肌细胞增殖的影响；用RTCA法检测以上各组细胞在12h的穿膜细胞数,从而探讨IGF-I是否可以促进成肌细胞迁移,并筛选其促进成肌细胞增殖、迁移的最佳浓度。第二部分：根据第一部分实验结果,选取100ng/ml IGF-I作用于L6成肌细胞,以2%FBS作对照,在Omin,20min,30min, 1h,2h,4h,12h,24h,48h后提取细胞总蛋白,利用免疫印迹技术(Western Blot)检测IGF-I刺激成肌细胞后mTOR/STAT3信号通路活化水平,从而探讨mTOR/STAT3信号通路是否被激活。第三部分：将细胞分为2%FBS对照组、50μmol/L雷帕霉素(rapamycin, RAPA)+100ng/mlIGF-I处理组、100ng/mlIGF-I处理组三组,用Western Blot观察各组目的蛋白磷酸化水平的变化,应用MTT法检测24h、48h、72h成肌细胞增殖的变化；用RTCA法检测12h成肌细胞迁移能力的变化,验证IGF-1促进成肌细胞增殖、迁移过程中mTOR/STAT3信号通路的作用。结果：第一部分：在同一时间,高浓度(50ng/ml, 100ng/ml,200ng/ml)的IGF-I具有促进L6成肌细胞增殖的作用(p<0.05),以100ng/ml,200ng/ml的IGF-I促进作用更为显著(p<0.01)；10ng/ml则无明显促进作用(p>0.05)。100ng/ml, 200ng/ml的IGF-I能有效促进L6成肌细胞迁移(p<0.01),而较低浓度(Ong/ml, lOng/ml,50ng/ml)的IGF-I则无明显促迁移作用(p>0.05)第二部分：实验组细胞各时间点之间哺乳动物雷帕霉素靶蛋白(the mammalian target of rapamycin, mTOR)及信号转导和转录激活因子3 (signal transducers and activators of transcription 3, STAT3)总蛋白表达量无明显差异,然而mTOR及STAT3蛋白活化水平(p-mTOR、p-STAT3)均随IGF-I作用时间延长而升高,在4h达到峰值,随后降低,然而在48h其蛋白磷酸化水平仍显著高于IGF-I作用起始,差异有统计学意义(p<0.05),即IGF-I作用下,mTOR/STAT3信号通路被激活。第三部分：RAPA显著抑制了IGF-I激活的目的蛋白磷酸化水平,且该通路抑制剂RAPA的加入显著降低了IGF-I诱导的L6成肌细胞增殖及迁移能力,说明IGF-I通过mTOR/STAT3信号通路促进了L6成肌细胞的增殖及迁移。结论：适宜浓度的IGF-I对成肌细胞增殖、迁移有促进作用,这种促进作用与mTOR/STAT3信号通路的激活有关,但这并不是唯一的激活通路。更多还原

【Abstract】 Objective:Proliferation and migration play a crucial role in cellsreaction, the main way through which cell growth factor plays its physiological function is to regulate the proliferation and migration of effector cell. Insulin-like factor I(IGF-I) is an important cytokine which can impact the function of muscle satelite cells. Therefore, it is of practical significance to study the mechanism of IGF-I stimulation on proliferation and migration in muscle satellite cells. mT0R/STAT3 signal pathway is a central controller of cell metabolism and growth, which plays an important role in cellular biotic activities. Also, our early experiments found that STAT3 protein play an important role in IGF-I-stimulated myoblast proliferation. Accordingly, the following experiments attempt to discuss the role of mTOR/STAT3 pathway in IGF-I-stimulated myoblast proliferation and migration.Methods:Part Ⅰ:MTT method was used to examine the OD value of L6 myoblast at 24h, 48h and 72h, which are incubated with 2%FBS, 10ng/mL,50ng/mL, 100ng/mL or 200ng/mL IGF-I respectively. After selecting the proper IGF-I concentration according to the MTT results, RTCA was used to monitor the effect of IGF-I on proliferation of L6 myoblasts. Then the number of transmembrane cell was detected in above groups in 12h to investigate whether IGF-I could promote the migration of myoblast and conclude the optimum concentration for cell proliferation and migration.Part Ⅱ:Refer to the result of Part I, we chose 100ng/mL IGF-I to incubate L6 myoblast (2%FBS as control) and total cellular protein was collected after min, 20min,30min, 1h,2h,4h,12h,24h and 48h after incubation. Then analyzed mTOR/STAT3 pathway activation levels using Western blot assay to find out whether this signaling pathway could be activated after stimulation.Part Ⅲ:Divided cells into three groups:2% FBS group (control),50μmol/L RAPA + 100ng/mL IGF-I group and 100ng/mL IGF-I group. Western blot was used to detect the varies phosphorylation levels of target proteins in each group; MTT assay was used to test changes of myoblast proliferation after 24h,48h and 72h stimulation and RTCA was used to monitor changes of migration ability of myoblast at 12h. Thus we could determine the role of mTOR/STAT3 pathway in IGF-I-stimulated myoblast proliferation and migration.Results:Part Ⅰ:At the same time point, IGF-I of high concentration (50ng/mL, 100ng/mL,200ng/mL) could stimulate L6 myoblast proliferation (p<0.05), among which 100ng/mL and 200 ng/mL IGF-I had a more dramatic effect, while 10ng/mL IGF-I had no significant function (p>0.05). 100ng/mL and 200ng/mL IGF-I could significantly increase L6 myoblast migration (p<0.01), while IGF-I of low concentration (Ong/mL, 10ng/mL and 50ng/mL) has little impact on this.Part Ⅱ:There was no significant differences in total mTOR, STAT3 protein expression between the cells in the experimental group at each time point, while the phosphorylation protein levels (both p-mTOR and p-STAT3) increased along with the the prolongation of action time of IGF-I, which peaked at 4h and then decreased afterwards. However, the phosphorylation level at 48h was still higher than the initial value, which showed statistical significance. Conclusively, mTOR/STAT3 pathway was activated under IGF-I.Part Ⅲ:RAPA, an inhibitor of mTOR/STAT3 signaling pathway, significantly inhibited the IGF-I-stimulated phosphorylation levels of mTOR and STAT3 protein. With the addition of RAPA, the IGF-stimulated L6 myoblast migration was dramatically suppressed, suggesting that IGF-I stimulated proliferation and migration of L6 myoblast through mTOR/STAT3 pathway.Conclusion:IGF-I can promote the proliferation and migration of myoblasts to some extent, this promoting role is related to the activation of the mTOR/STAT3 signaling pathway, but is not the only pathway of activation.更多还原