胰岛素样生长因子Ⅰ对组织工程肌腱细胞增殖影响的量效关系

Dose-effect relationship of insulin-like growth factor Ⅰand the proliferation of tenocytes in tissue engineering

目的:胰岛素样生长因子Ⅰ是一种潜在的促有丝分裂剂,对肌腱细胞有促进分裂增殖的作用。实验将不同剂量胰岛素样生长因子Ⅰ作用于第2代肌腱细胞,进一步验证其对细胞增殖的影响并探讨其量效关系。方法:实验于2004-11/2005-04在天津医院实验室完成。①实验材料:天津医院实验室提供的精选法国罗曼鸡受精鸡蛋200只,在孵育19d时,随机取出10只鸡胚肌腱。②实验过程及分组:分离培养肌腱细胞,观察肌腱细胞形态及生长规律。取第2代肌腱细胞接种于六孔板,分别给予不同浓度的胎牛血清,观察血清浓度对肌腱贴壁及生长的影响。取第2代肌腱细胞接种于96孔板,分为7组。前5组分别加入含不同剂量胰岛素样生长因子Ⅰ(1,5,10,50,200μg/L)的体积分数为0.02的胎牛血清培养液;第6组加入体积分数为0.05的胎牛血清培养液做为阳性对照,第7组加入体积分数为0.02的胎牛血清培养液做为阴性对照。③实验评估:采用四甲基偶氮唑盐法及瑞士-姬姆萨染色观察不同剂量的胰岛素样生长因子Ⅰ对细胞增殖的影响。结果:①肌腱细胞贴壁后生长很快,原代细胞1周左右即可传代,增殖速度与营养液中胎牛血清浓度呈正相关。②肌腱细胞增殖速度随胰岛素样生长因子Ⅰ剂量加大而有增加趋势。第2天、第4天,胰岛素样生长因子Ⅰ1μg/L组、5μg/L组和阴性对照组之间差异无显著性(P>0.05);胰岛素样生长因子Ⅰ10μg/L组、50μg/L组和200μg/L组之间差异无显著性(P>0.05)。②第2天,阳性对照组增殖高于胰岛素样生长因子Ⅰ1μg/L组与5μg/L组,低于10μg/L组、50μg/L组和200μg/L组,差异有显著性(P<0.000或<0.006);第4天,阳性对照组增殖高于胰岛素样生长因子Ⅰ各剂量组和阴性对照组,差异有显著性(P<0.000或<0.006)。③第2天、第4天,胰岛素样生长因子Ⅰ1μg/L组、5μg/L组和阴性对照组低于10μg/L组,50μg/L组,200μg/L组,差异有显著性(P<0.000);阳性对照组增殖高于阴性对照组,统计分析差异有显著性(P<0.000)。结论:胰岛素样生长因子Ⅰ对肌腱细胞增殖的促进作用,随胰岛素样生长因子Ⅰ浓度增高而有增高趋势,10μg/L为其较适合浓度,同时发现培养血清浓度对肌腱细胞有其特殊作用,浓度越高,肌腱细胞贴壁越快,并对增殖有明显促进作用。

AIM： Insulin-like growth factor Ⅰ（IGF- Ⅰ ） is a potent mitogen and powerful stimulator for the division and growth of tenocytes. In this study, we cultured the tenocytes of the second generation with IGF- Ⅰ at different doses to verify the effects of IGF- Ⅰ on cell proliferation and the dose effect. METHODS： The experiment was performed in the laboratory of Tianjin Hospital from November 2004 to April 2005. （1）200 France Lohmann fertilized eggs were provided by the laboratory of Tianjin Hospital. On day 19, the tendons of 10 embryo chickens were obtained. （2）Tenocytes were isolated and cultured. The cell morphous and growth rules were observed. The second generation cells were seeded onto 6-well culture plate and added with fetal calf serum （FCS） of different concentrations. The effects of serum concentration on cell attachment and growth were observed. The second generation tenocytes were seeded onto 96-well culture plate and divided into 7 groups. The former 5 groups were cultured in 0.02 volume fraction FCS culture medium containing IGF- Ⅰ at doses of 1, 5, 10, 50, 200 μ g/L; the sixth group was cultured in 0.05 volume fraction FCS culture medium as positive control, and the seventh group was cultured in 0.02 volume fraction FCS culture medium as negative control. （3）The effects of IGF- Ⅰ on cell proliferation were observed with MTT test and Swiss-gimmsa taint test. RESULTS： （1）The tenocytes grew very fast after attaching to the wall. The primary cells started to passage in about 1 week. The proliferative rate of tenocytes was positively correlated with the concentration of FCS in culture medium. （2）The proliferative rate of tenocytes was accelerated with the increase in IGF- Ⅰ dose. There was no difference among the 1μ g/L and 50μ g/L IGF-Ⅰ groups and negative control group on days 2 and 4 （P 〉 0.05）; There was no difference among the 10μ g/L, 50μ g/L and 200μ g/L IGF-Ⅰ groups （P 〉 0.05）. （3）On the second day, the proliferative rate of tenocytes in the positive control group was significantly higher than that in 1 μ g/L and 5 μ g/L IGF-Ⅰ groups but lower than in 10 μ g/L, 50 μ g/L and 200 μ g/L IGF-Ⅰ groups （P 〈 0.000 or 〈 0.006）. On the fourth day, the proliferative rate of tenocytes in the positive control group was significantly higher than that in each IGF-Ⅰ group and negative control group （P 〈 0.000 or 〈 0.006）. （On days 2 and 4, the proliferative rate of tenocytes in 11μ g/L and 51μ g/L IGF-Ⅰ groups was significantly lower than that in 10μ g/L, 50μ g/L and 200μ g/L IGF-Ⅰ groups （P 〈 0.000）; the positive control group was remarkably higher than the negative control group （P 〈 0.000）. CONCLUSION： IGF-Ⅰ promotes the proliferation of tenocytes in a dose-dependent manner. 10 μ g/L is found to be the most effective concentration. Meanwhile, the proliferative rate of tenocytes is accelerated when the cells are cultured in higher concentration of FCS.