补体C5a 及受体对人肾间质成纤维细胞增殖的影响及机制研究

目的 探讨补体C5a 及受体(C5aR)对人肾间质成纤维细胞(human renal interstitial fibroblasts，hRIFs)增殖及纤维化相关因子表达的影响。方法 原代培养hRIFs 细胞，对细胞进行鉴定后，检测C5aR 表达情况；同用C5a和C5aR 拮抗剂分别对hRIFs 处理后检测细胞增殖、细胞外基质蛋白(fibronectin，Collagen-I)以及转化生长因子-β1(TGF-β1)的蛋白表达情况。结果 hRIFs 细胞vimentin 蛋白表达阳性，desmin 蛋白表达阴性，C5aR 表达于hRIFs细胞表面。MTS 实验结果显示:与对照组相比，C5a 纯品(浓度分别为10，15，20nmol/L)刺激后第2h，8h 的A 值差异无统计学意义 (P>0.05)，刺激后第24h 的A 值(0.217±0.037 vs 0.613±0.016, 0.793±0.041, 0.887±0.039) 显著升高，差异有统计学意义(t=4.891，4.211，8.408，均P ＜ 0.05)。与C5a 纯品组相比，C5a 纯品+C5aR 拮抗剂组的A 值明显降低(0.887±0.039 vs 0.424±0.016)，差异有统计学意义(t=8.657，P ＜ 0.05)。Western-Blot 结果显示，与对照组相比，补体C5a(20nmol/L)刺激后第24h，fibronectin(26 091±128 vs 15 400±105), Collagen-I(31 229±129 vs 24 823±136)以及TGF-β1(27 855±161 vs 20 326±152) 蛋白表达水平都有明显增加，差异具有统计学意义(t=4.891，5.820, 2.311,均P ＜ 0.05)。与C5a 纯品组相比，C5a 纯品+C5aR 拮抗剂组fibronectin(22 188±132 vs 26 091±128) ,Collagen-I(27181±113 vs 31 229±129) 以及TGF-β1(25 013±139 vs 27 855±161) 蛋白表达水平显著降低，差异具有统计学意义(t=2.349，1.618，3.774，均P ＜ 0.05)。结论 补体C5a-C5aR 相互作用可导致hRIFs 细胞增殖、促纤因子TGF-β1表达升高及细胞外基质蛋白生成增加，对肾间质纤维化具有重要意义。

Effect of Complement C5a on the Proliferation of Human Renal Mesenchymal Fibroblasts and Expression of Fibrosis-related Factors

Objective To investigate the effect of complement C5a-C5aR on the proliferation and expression of fibrosis-related factors in human renal mesenchymal fibroblasts (HhRIFs) cells. Methods After primary culture of hRIFs cells, the cells were identified and C5aR expression was detected. HRIFs were treated with C5a and C5aR antagonists to detect cell proliferation, extracellular matrix protein (fibronectin, Collagen-I) and transforming growth factor-β1(TGF-β1) protein expression, respectively. Results The results of the MTS experiment showed that the A values of C5a pure (10, 15, 20nmol/L) were not statistically significant (P ＞ 0.05) at the 2h and 8h after stimulation compared with the control group. The A values at the 24h after stimulation (0.217±0.037 vs 0.613±0.016, 0.793±0.041, 0.887±0.039)were significantly higher, the differences were statistically significant(t=4.891, 4.211, 8.408, all ＜ 0.05). The A values of C5a pure + C5aR antagonist group at 24h after stimulation(0.887±0.039 vs 0.424±0.016)were significantly lower compared to C5a pure group, and the difference was statistically significant (t=8.657, P ＜ 0.05). Western-Blot results showed that compared to the control group, at the 24h after stimulation with complement C5a (20nmol/L) fibronectin (26 091±128 vs 15 400±105), Collagen-I (31 229±129 vs 24 823±136) and TGF-β1 (27 855±161 vs 20 326±152) protein expression levels were significantly increased compared with the control group, and the results were statistically significant (t=4.891, 5.820, 2.311, all P ＜ 0.05). Compared with the C5a-only group, the C5a-only + C5aR antagonist groups fibronectin (22 188±132 vs 26 091±128), Collagen-I (27 181±113 vs 31 229±129), and TGF-β1 (25 013±139 vs 27 855±161) protein expression levels were significantly reduced and the results were statistically significant (t=2.349, 1.618, 3.774, all P ＜ 0.05). Conclusion Complement C5a-C5aR interaction could leads to proliferation of hRIFs cells, elevated expression of pro-fibrotic factor TGF-β1 and increased production of extracellular matrix proteins, which are important for renal interstitial fibrosis.