胰岛素样生长因子Ⅰ对大鼠纤维环细胞体外增殖活性影响的量效关系

Dose-effects of insulin-like growth factor-1 on proliferation of rat nucleus fibrosus cells in vitro

背景:近年来体外椎间盘髓核和纤维环组织细胞培养技术的建立,特别是软骨组织工程研究的不断深入及自体椎间盘细胞移植修复髓核缺损动物实验的初步成功,为退变椎间盘的形态结构与生理功能的完全再生修复带来了希望。目的:通过对椎间盘纤维环细胞的培养,观察胰岛素样生长因子Ⅰ对大鼠纤维环细胞体外增殖活性的影响及量效和时效关系。设计、时间及地点:单一样本观察,于2006-09/2007-01在山西医科大学寄生虫实验室完成。材料:清洁级1月龄Wistar系大鼠30只,雌雄不限。方法:体外分离培养大鼠纤维环细胞。剂量-效应实验:在培养的细胞中分别加入由含体积分数为0.01或0.1的小牛血清HAMF-12配制成的不同浓度的胰岛素样生长因子Ⅰ(0.1,1.0,10,100μg/L)。以不加生长因子做对照,培养72h。时间-效应实验:在培养的细胞中分别加入含有最佳效应浓度胰岛素样生长因子Ⅰ体积分数为0.1的小牛血清+F12培养液,分组为对照组和100μg/L的胰岛素样生长因子Ⅰ组,分别培养1,3,5,7d。主要观察指标:①苏木精-伊红、甲苯胺蓝、免疫细胞化学染色分析细胞的生物学特性。②噻唑蓝比色法观察胰岛素样生长因子Ⅰ在体积分数为0.01和0.1的血清浓度下对大鼠纤维环细胞体外增殖的调节作用及其剂量、时间与作用效果的关系。结果:苏木精-伊红染色细胞多为梭形,有伪足伸出,细胞核为圆形或椭圆形;甲苯胺蓝染色,胞浆为深蓝色;免疫细胞学方法检测表明纤维环细胞有Ⅰ型胶原表达;在体积分数为0.1的血清条件下,胰岛素样生长因子Ⅰ能提高细胞的增殖活性,并且在有效浓度范围内呈剂量效应关系。结论:胰岛素样生长因子Ⅰ能促进大鼠纤维环细胞的体外增殖,其效应在一定范围内与剂量和时间呈正相关。

BACKGROUND： Recently, in vitro nucleus gelatinosus and annulus fibrosus cell culture technology, especially cartilage tissue engineering development and primary success in autogenous intervertebral disc cell transplantation to repair nucleus pulposus defect, brings a hope for regeneration and reparation of degenerated intervertebral disc. OBJECTIVE： To explore the dose-dependent and time-dependent effects of insulin-like growth factor- Ⅰ （IGF- Ⅰ ） on proliferation of anulus fibrosus cells in vitro. DESIGN, TIME AND SETTING： Single-sample observation was performed at the Laboratory of Parasite, Shanxi Medical University between September 2006 and January 2007. MATERIALS： Thirty 1-month-old Wistar rats, irrespective of gender, were selected. METHODS： Anulus fibrosus cells were isolated and cultured in vitro. In dose-dependent test, IGF- Ⅰ （0.1, 1.0, 10, and 100 μ g/L）, prepared by volume fraction 0.01 or 0.1 calf serum HAMF-12, were added. Cells not with IGF- Ⅰ served as control. Cells were cultured for 72 hours. In time-dependent test, optimal dose of IGF- Ⅰ containing volume fraction 0.1 calf serum and F- 12 solution was added, and cells not with IGF- Ⅰ served as control. The cells were cultured for 1, 3, 5, and 7 days. MAIN OUTCOME MEASURES： Cell biological features were detected using HE staining and toluidine blue immunohistochemical staining; Dose-dependent and time-dependent effects were examined by MTT assay. RESULTS： HE stained cells were fusiform-shaped with pseudopodia, round or oval nucleus. The cytoplasm was blue after stained with the toluidine blue. The immunohistochemical test revealed that there existed positive expression of type Ⅰ collagen in the cells. In the presence of 10% calf serum, IGF- Ⅰ significantly improved cell proliferative activity in a dose-dependent manner within effective dose range. CONCLUSION： IGF- Ⅰ can stimulate cell proliferation of rat anulus fibrosus cells in vitro in a dose-dependent and time-dependent manner.