碱性成纤维细胞生长因子及胰岛素样生长因子-1对体外兔关节软骨细胞的生物学效应

目的：了解胰岛素样生长因子－1（IGF－1）及碱性成纤维细胞生长因子（bFGF)对兔关节软骨细胞分裂增殖及功能代谢的影响，为组织工程方法修复关节软骨缺损提供实验依据。方法：取生长良好的兔正常关节软骨细胞，在含体积分数为10％新生小牛血清的DMEM条件下体外单层培养，细胞贴壁后随机分组，并分别加入不同浓度的bFGF和（或）IGF－1；培养液不加任何因子为对照组，以四唑盐（MTT）法检测细胞相对数，二苯胺显色法测各瓶细胞DNA含量，咔唑硫酸法测基质中糖醛酸含量，以间接反映蛋白多糖含量。并采用流式细胞技术进行细胞周期亚时相分析。结果：在实验浓度范围内，两种因子均促进细胞增殖，DNA合成及增加胞外基质中葡萄糖醛酸含量，且呈剂量－效应依赖关系。当IGF－1浓度≥10ng/ml,bFGF浓度≥1ng/ml时，其促进效果较显著（P＜0．05，0．01）。bFGF的促细胞增殖作用更为明显，最大为对照组的1．32倍，而IGF－1对细胞外基质葡萄糖醛酸含量的影响更显著，最大为对照组的1．78倍，bFGF能明显缩短DNA合成前期（G1期）和分裂前期及分裂期（G2M期）时间；bFGF IGF-1作用后，同样缩短G1期和G2M期时间，显著缩短G1期时间；而IGF－1仅缩短G2M期时间。结论：在本实验条件下，bFGF及IGF－1均以剂量依赖性方式影响细胞，刺激细胞增殖及功能代谢，两种因子的协同作用仅表现在对细胞的增殖作用上，对细胞功能代谢没有明显影响。两种因子促进细胞增殖是通过缩短细胞周期不同亚时相而实现的。

Biological effects of basic fibroblast growth factor and insulin-like growth factor-1 on cultured articular chondrocyes in rabbits

Objective To study the effects of basic fibroblast growth factor (bFGF) and insulin-like growth factor-1 (IGF-1) on proliferation and metabolism of the rabbit articular chondrocytes in order to cater experimental base for rapair of articular chondrocytes by tissue engineering. Methods The monolayer articular chondrocytes from a healthy rabbit were cultured in DMEM containing 10% fetal calf serum and grouped randomly. Then,each group was given bFGF and/or IGF-1 with various concentrations. Pure culture fliud was set as control group. The relative number of chondrocytes was measured by methyl thiazolyl tetrazolium method, DNA content of chondrocytes by diphenylamine development process and the glucuronic acid (GlucA) content in the matrix by carbazole sulfate so as to reflect the content of proteoglycan. Subcycle of the chondrocytes was analyzed using flow cytometer. Results Both bFGF and IGF-1 promoted cell proliferation and DNA composition and increased the content of gluconate in the matrix in a dose-dependent fashion. Both of them showed more significant promotive effect when concentration of bFGF was over 1 ng/ml and that of IGF-1 over 10 ng/ml (P<0.05, P< 0.01). Compared with the control group, bFGF had more significant promotive effect on proliferation of the chondrocytes (1.32 times more that in the control group at the peak effect). While IGF-1 affected more significantly the content of gluconate in the matrix (1.78 times more than control group at the peak effect). bFGF could significantly shorten the period of pre-synthesis phase (Phase G 1), prophase of division and division phase (Phase G 2M). bFGF plus IGF-1 also could shorten the period of Phase G 1 and Phase G 2M, especially Phase G 1; but IGF-1 only shortened the period of Phase G 2M. Conclusions bFGF and IGF-1 stimulate cell proliferation and matrix metabolism in a dose-dependent fashion. The coordinating action of bFGF and IGF-1 is significant on cell proliferation rather than matrix metabolism. They promote cell proliferation through shortening the time of different subcycles of the chondrocytes.