转化生长因子β1和胰岛素样生长因子1对体外培养人退变髓核细胞生物学活性的影响

Effect of transforming growth factor-β1 and insulin-like growth factor-1 on the biological potential of the human degenerative nucleus pulposus cells in vitro

目的:观察在体外培养条件下转化生长因子β1(TGF-β1)和胰岛素样生长因子1(IGF-1)对人退变髓核细胞生物学活性的影响。方法:体外培养人退变髓核细胞,台盼蓝染色法测定活细胞率。将每份髓核细胞样本分为对照组、TGF-β1组和TGF-β1+IGF-1组。采用考马斯亮蓝法测定TGF-β1和IGF-1干预后第0、2、4、6天细胞内总蛋白含量。采用免疫组织化学和免疫荧光化学染色方法测定细胞在TGF-β1和IGF-1干预后0～6d合成Ⅱ型胶原的情况。结果:体外培养人退变髓核细胞的活细胞率为90%～95%。干预后第4、6天TGF-β1组和TGF-β1+IGF-1组与对照组比较、TGF-β1+IGF-1组与TGF-β1组比较细胞内总蛋白含量均显著增加(P<0.05),TGF-β1+IGF-1组Ⅱ型胶原的合成较对照组显著增加(P<0.05);干预后第6天TGF-β1组和TGF-β1+IGF-1组与对照组比较、TGF-β1+IGF-1组与TGF-β1组比较Ⅱ型胶原均显著增加(P<0.05)。结论:一定浓度的TGF-β1能够促进人退变髓核细胞合成Ⅱ型胶原,改善其生物学活性,合用TGF-β1和IGF-1上述作用更加明显。

Objective：To stimulate the human degenerative nucleus pulpesus （NP） cells by transforming growth factor-β1 （TGF-β1） and insulin-like growth factor-1 （IGF-1） in vitro and to observe the changes of biological potential of the cells,Method：Human degenerative nucleus pulposus cells were isolated from degenerated disc and cultured in DMEM/F12 in vitro.The viability of cells were determined by trypan blue exclusion test.They were then treated separately with serum-free medium,TGF-β1,TGF-β1 plus IGF-1.The immunocytochemistry and Coomassie brilliant blue method were used to detecte type Ⅱ collagen that were secreted by NP cell and total protein content.Result：The viability of primary cell after isolation was about 90% -95%.The total protein content in NP cells of TGF-β1 group and TGF-β1＋IGF-1 group increased significantly compared with the control group （P〈0.05）,and TGF-β1＋IGF-1 group cause a dramatic increase compared with TGF-β1 group （P〈0.05）.Immunocytochemical analysis demonstrated that after combination with TGF-β1 plus IGF-1 for 4d and 6d there was statistically significant increasing expression of the collagen type Ⅱ than the control group （P〈0.05）.The indirect-1 results revealed that compared with the control group the expression of the collagen type H in NP cells increased significantly after treated with TGF-β1 or with TGF-β1 plus IGF-1 for 6d （P〈0.05）.Furthermore,the expression of the collagen type Ⅱ in the TGF-β1＋ IGF-1 group increased markedly than the TGF-β1 group（P〈0.05）.Couclusion：TGF-β1 can significantly induce the expression of the collagen type H in the human degenerative NP cells and enhance the biological potential of NP cells.And combined use of TGF-β1 and IGF-1 have better effect.