流式细胞仪碘化丙啶染色法测定类胰岛素生长因子Ⅰ对人胚胎原代及传代成肌细胞生长周期的作用

Effects of insulin-like growth factor-I on the growth cycle of primary and continuously subcultured human embryonic myoblasts by flow cytometry propidium iodide method

目的：大量研究表明,类胰岛素生长因子Ⅰ促成肌细胞增殖作用明显,低浓度水平就有活性,但是对于其作用机制目前还不太清楚。应用流式细胞仪碘化丙啶染色法（PⅠ法）检测成肌细胞生长周期,拟探讨类胰岛素生长因子Ⅰ对原代及传代人胚骨骼肌成肌细胞体外增殖的作用与机制。方法：实验于2004-06/12在四川大学华西医院生物治疗国家重点实验室、干细胞与组织工程研究室以及成都市第三人民医院检验科流式细胞室完成。在产妇知情同意的前提下,选取孕8-12周水囊引产胎儿,性别不限,切取部分小腿腓肠肌与股四头肌组织后,采用文献的方法进行成肌细胞分离、纯化与传代培养,实验经医院伦理委员会批准。选取原代、第2、4、6代成肌细胞以1×105/孔密度接种于24孔板,每3孔为1组计7组。1-7组细胞以无血清F12培养基同步化后分别予以含0,1,2,4,8,16,32μg/L类胰岛素生长因子Ⅰ的培养基,运用核素掺入法确定类胰岛素生长因子Ⅰ促增殖的适宜浓度用于实验。同步化后其中一板的生长培养基不加类胰岛素生长因子Ⅰ作为对照组;另一个加入适宜浓度的类胰岛素生长因子Ⅰ作为实验组。每组每天取3孔细胞计数,绘制生长曲线,推算成肌细胞的群体倍增时间,作为细胞增殖周期TC。运用流式细胞技术PI法分别测定细胞生长周期和各亚周期时相时间。结果：4-8μg/L类胰岛素生长因子Ⅰ具有良好促进成肌细胞增殖的能力。各代的实验组与对照组成肌细胞在24h后进入对数生长期,各实验组细胞生长曲线明显左移。6代以内成肌细胞的增殖周期、各亚周期细胞百分比、各亚周期所占时相以及各代细胞对类胰岛素生长因子Ⅰ的反应一致,生长模式与能力一致,细胞倍增时间平均从4.8d缩短到3.3d。结论：类胰岛素生长因子Ⅰ能促进成肌细胞体外增殖,成肌细胞的增殖指数明显提高。其促增殖作用是通过缩短DNA合成前期和合成期来实现的。

AIM： Insulin-like growth factor-Ⅰ （IGF-Ⅰ） can effectively promote the proliferation of sarcoblasts, even at a low concentration, but the mechanism is still unclear. Growth cycle of sarcoblasts is detected by flow cytometry propidium iodide method （PI）. This study investigated the effect and mechanism of IGF-Ⅰ on the growth of primary and continuously subcultured human embryonic myoblasts. METHODS： Experiments were performed at the State Key Laboratory of Biotherapy, Stem Cells and Tissue Engineering Laboratory, West China Hospital, Sichuan University and Flow cytometry Room, Department of Clinical Laboratory, Third People＇s Hospital from June to December 2004. Puerperants signed an informed consent. Fetus by induction of labor with water bag of pregnant 8-12 weeks, of either sex, were selected. Gastrocnemius and musculus quadriceps fexoris of cnemis were obtained. Sarcoblasts were isolated, purified and passaged by the method of references. The experimental procedures were approved by Hospital Ethics Committee. The primary, the 2nd, 4th and 6th passage of myoblasts were cultured in 24-well plate at 1 ～10/well in vitro. Three wells served as a group, totally 7 groups. Cells in the 1-7 groups were synchronized in serum-free F12 medium, and then incubated in medium containing 0,1, 2, 4, 8, 16, 32 μ g/L insulin-like growth factor Ⅰ （IGF-Ⅰ）. Suitable concentration of IGF-Ⅰ to promote proliferation was identified by incorporation assay. The control groups only received growth medium. The experimental groups received IGF-Ⅰ at suitable concentrations. Cells from 3 wells were counted and growth curves were observed in each group every day. Doubling time of myoblastic masses was calculated as cell generation cycle Tc. Cell cycle and time （h） of G0/G1 phase, S phase and G2M phase were detected by flow cytometry （PI method）. RESULTS： 4-8 g/L IGF-Ⅰ were chosen to stimulate myoblasts proliferation. Sarcoblasts in experimental groups and control groups entered logarithmic growth phase 24 hours later. Cell growth curve in each experimental group moved left. Reproductive cycle, cell percentage and time（h） of G0/G1 phase,S phase and G2M phase of 1-6 passage of myoblasts and the reaction to IGF-Ⅰ were similar, so did grow pattern and potential. Doubling time shortened from 4.8 days to 3.3 days averagely. CONCLUSION： IGF-Ⅰ can accelerate the proliferation of primary and continuously subcultured human embryonic myoblasts in vitro. IGF-Ⅰ can greatly shorten the time of G1 and S phase, so as to prompt the proliferation of myoblasts.