钙三醇抑制肾间质成纤维细胞的活化

目的 通过观察钙三醇对肾间质成纤维细胞增殖和凋亡的影响，及其对转化生长因子（TGF）β1诱导的成纤维细胞转分化和细胞外基质(ECM)合成的干预作用，旨在探讨钙三醇抗肾间质纤维化的潜在效应及相关机制。 方法 体外培养大鼠肾间质成纤维细胞NRK-49F，分别以不同浓度TGF-β1（1、2、5和10 μg/L）和（或）不同浓度钙三醇（10－4、10－5、10－6、10－7、和10－8 mmol/L）进行干扰。MTT法观察细胞增殖情况；流式细胞术分析细胞周期和细胞凋亡改变；实时定量PCR和Western印迹法分别检测细胞内α平滑肌肌动蛋白（α-SMA）、结缔组织生成因子（CTGF）及纤连蛋白（FN）的mRNA和蛋白表达。 结果 钙三醇能明显抑制正常和TGF-β1（5 μg/L）诱导的NRK-49F细胞增殖（P < 0.01）。经不同浓度钙三醇（10－4、10－5、10－6、10－7、和10－8 mmol/L）作用48 h后，NRK-49F细胞G2/S期细胞比例较TGF-β1刺激组均明显减少（分别为25.88%、21.81%、21.73%、23.28%、23.61%比27.42%，均P < 0.05），但对细胞凋亡无明显影响。NRK-49F细胞经TGF-β1刺激后，其α-SMA、CTGF、FN mRNA表达量显著上升，并在TGF-β1 1~5 μg/L的范围内呈剂量依赖效应。钙三醇能明显抑制TGF-β1诱导的α-SMA、CTGF、FN mRNA表达上调（均P < 0.05），而不同钙三醇浓度干预组之间差异无统计学意义。钙三醇能显著降低TGF-β1诱导的α-SMA和FN蛋白表达（P < 0.05）。 结论 钙三醇可通过G1期停滞抑制大鼠肾间质成纤维细胞增殖，但不影响凋亡。钙三醇具有拮抗 TGF-β1致肾间质纤维化的潜在作用。

Effects of caleitriol on proliferation and extracellular matrix synthesis in renal interstitial fibroblasts

Objective To observe the effects of calcitriol on cellular proliferation and apoptosis,and on the expression of transforming growth factor-β1(TGF-β1)-induced α-SMA and extracellular matrix(ECM)synthesis in renal interstitial fibroblasts,so as to illustrate its potential roles and underlying mechanisms in preventing renal interstitial fibrosis. Methods In NRK-49F of cultured rats in vitro,cellular proliferation was detected by MTY.The cellular cycle and apoptosis were assessed by flow eytometry using annexin-V FITC/propidium iodide(PI).Real-time PCR was performed to detect the mRNA expression of α-SMA,CTGF and FN,and Western blot was performed to detect the protein expression of α-SMA and FN. Results Cellular proliferation was inhibited by calcitriol through G1-arrest (P＜0.05).But calcitriol had no effect on cell apoptosis(P＞0.05).Increased expression of α-SMA,CTGF and FN mRNA induced by TGF-β1were significantly suppressed by calcitriol as compared with TGF-β1 treated group (P＜0.05).However,there was no significant difference among the groups with various concentration of calcitrol (P＞0.05).The similar phenomenon of the level of α-SMA and FN protein expression was observed in all groups by Western blot. Conclusion Caleiniol inhibits cellular proliferation of renal interstitial fibroblast by G1-arrest,but has no effect on cell apoptosis,and can prevent renal interstitial fibrosis caused by TGF-β1.