三种生长因子对人胚半月板细胞增殖及细胞表型的影响

目的研究碱性成纤维细胞生长因子（basic fibroblast growth factor,bFGF）、转化生长因子β1（transforming growth factorβ1,TGF-β1）、胰岛素样生长因子1（insulin-like growth factor1,IGF-1）单独或联合作用于人胚半月板细胞,探讨半月板组织工程种子细胞大量扩增最佳作用组合及浓度。方法无菌条件下取健康妇女意外流产并自愿捐献的4个月人胚半月板,体外分离、培养半月板纤维软骨细胞,用Ⅱ型胶原免疫组织化学染色和Aggrecan免疫荧光染色检测其性状。取第3代细胞贴壁后使用血清饥饿法同步化细胞,加入IGF-1（1、10、50、100μg/L）、TGF-β1（0.1、1.0、5.0、10.0、50.0μg/L）、bFGF（5、10、50、100、200μg/L）作用于半月板细胞,每样本设8个复孔和阴性对照组,分别于作用后48h和72h采用MTT法检测软骨细胞增殖情况,以确定各生长因子最佳效应浓度。同法设7个组,每组8个复孔,分别为：阴性对照组、bFGF最佳效应浓度组（50μg/L）、TGF-β1最佳效应浓度组（5μg/L）、IGF-1最佳效应浓度组（50μg/L）、bFGF和TGF-β1最佳效应浓度联用组、bFGF和IGF-1最佳效应浓度联用组、TGF-β1和IGF-1最佳效应浓度联用组,48h和72h用MTT比色法得出吸光度（A）值并分析软骨细胞的增殖效应。结果第3代半月板细胞扩增前后细胞均表达Ⅱ型胶原和Aggrecan蛋白。48h和72h50μg/L IGF-1,5μg/L TGF-β1及50μg/L bFGF浓度组与对照组相比均具有促细胞增殖作用,且差异有统计学意义（P〈0.05）;此即为各生长因子最佳效应浓度。生长因子联用：bFGF50μg/L与IGF-150μg/L联用、IGF-150μg/L与TGF-β15μg/L联用具有协同效应,差异有统计学意义（P〈0.05）;bFGF50μg/L与TGF-β15μg/L联用无协同效应,差异无统计学意义（P〉0.05）。结论bFGF、TGF-β1、IGF-1单独使用均可体外扩增人胚半月板细胞,最佳效应浓度联用时bFGF/IGF-1、IGF-1/TGF-β1的扩增效果优于各自单独使用,可用于体外大量扩增种子细胞。

EFFECT OF THREE GROWTH FACTORS ON PROLIFERATION AND CELL PHENOTYPE OF HUMAN FETAL MENISCAL CELLS

OBJECTIVE: To investigate the effects of the insulin-like growth factor 1 (IGF-1), the transforming growth factor beta1 (TGF-beta1), and the basic fibroblast growth factor (bFGF) on proliferation and cell phenotype of the human fetal meniscal cells, and to find out the best combination and concentration of the growth factors for the meniscus tissue engineering. METHODS: The fetus came from the healthy woman accidental abortion and the procedure had got her approval. The human fetal meniscal fibrochondrocytes were cultured in vitro. The cell phenotype was identified by the collagen type II immunohistochemistry and Aggrecan immunofluorescence. In the growth factor groups, the 3rd passage meniscal cells synchronized by the serum starvation method and were mixed with IGF-1 (1, 10, 50, 100 microg/L), TGF-beta1 (0.1, 1.0, 5.0, 10.0, 50.0 microg/L), and bFGF (5, 10, 50, 100, 200 microg/L), respectively, and in the combination groups, the combinations of bFGF and TGF-beta1, bFGF and IGF-1, TGF-beta1 and IGF-1 were established at their optimal effect concentrations. The control group was also established for comparison. The dose-response relationship was studied at 48 h and 72 h by the MTT colorimetric method. RESULTS: The 3rd passage meniscal cells could express collagen type II and Aggrecan before and after the addition of the three growth factors. The proliferating effects of the growth factors (IGF-1 50 microg/L, TGF-beta1 5 microg/L, bFGF 50 microg/L) on the 3rd passage cells at 48 h and 72 h were significantly better in the growth factor groups than in the control group (P < 0.05), and the combination groups of bFGF 50 microg/L and IGF-1 50 microg/L, IGF-1 50 microg/L and TGF-beta1 5 microg/L showed a significantly higher proliferating effect than that in the single growth factor group (P < 0.05). bFGF 50 microg/L and TGF-beta1 5 microg/L had no synergetic effect (P > 0.05). CONCLUSION: IGF-1, TGF-beta1 and bFGF can promote the proliferation of the human fetal meniscal cells, respectively, and the combinations of bFGF and IGF-1, IGF-1 and TGF-beta1 at their optimal concentrations can have better proliferating effects than the single growth factor. They can be used for the in vitro amplification of the meniscal seed cells.