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目的探讨姜黄素抗肾纤维化的分子生物学机制,为临床治疗提供可靠的实验依据。方法以人肾小管上皮细胞(HKC)为研究对象,采用不同浓度的姜黄素(0、0.780、1.563、3.125、6.250、12.500、25.000、50.000和100.000μmol/L)刺激HKC 72h,倒置相差显微镜下观察细胞形态变化,并应用MTT比色法检测姜黄素对HKC生长的影响;应用不同浓度TGF-β1(0、1、5、10ng/mL)单独或联合姜黄素(6.250、12.500、25.000、50.000μmol/L和100.000μmol/L)共同刺激HKC细胞72h,观察细胞形态变化,应用逆转录PCR(RT-PCR)检测TGF-β1、骨形态发生蛋白-7(BMP-7)和Ⅰ型胶原基因表达变化。结果 HKC贴壁生长,呈铺路石样外观。姜黄素刺激对细胞形态无影响;TGF-β1刺激能明显促进细胞由上皮样形态向纤维样细胞形态转化,且发生形态转变的细胞数量和TGF-β1基因表达呈TGF-β1浓度依赖性增加(P<0.05,P<0.01);MTT结果显示:姜黄素在3.125~25.000μmol/L浓度范围内,能显著促进HKC增殖(P<0.05,P<0.01);姜黄素能对抗TGF-β1诱导的细胞形态转变,以12.500~50.000μmol/L的姜黄素作用最明显,同时,下调TGF-β1基因和蛋白的表达,相反上调BMP-7基因和蛋白表达,并伴随着Ⅰ型胶原基因和蛋白的表达下降(P<0.05,P<0.01)。结论姜黄素能促进HKC增殖并维持其表型,能对抗TGF-β1诱导的HKC向梭形细胞转化,抑制细胞上皮TGF-β1和Ⅰ型胶原的表达,而促进BMP-7表达。
Objective To explore the molecular biological mechanism of curcumin against renal fibrosis and provide a reliable experimental basis for clinical treatment. Methods Human renal tubular epithelial cells (HKC) were cultured in HKC for 72h under different concentrations of curcumin (0, 0.780, 1.563, 3.125, 6.250, 12.500, 25.000, 50.000 and 100.000 μmol / L) The morphological changes of the cells were observed. The effect of curcumin on the growth of HKCs was detected by MTT colorimetric assay. The effects of curcumin (6.250,12.500,25.000,50.000) and different concentrations of TGF-β1 (0,1,5,10ng / mL) HKC cells were stimulated with different concentrations of TGF-β1, BMP-7 and type Ⅰ collagen mRNA by reverse transcription polymerase chain reaction (RT-PCR) Change of expression Results HKC adherent growth, paving stone-like appearance. Curcumin had no effect on cell morphology. TGF-β1 stimulation could significantly promote the transformation from epithelial to fibroblast-like cells, and the number of TGF-β1-producing cells and TGF-β1 gene expression increased in a concentration-dependent manner (P <0.05, P <0.01). The results of MTT assay showed that curcumin can significantly promote the proliferation of HKC in the concentration range of 3.125 ~ 25.000 μmol / L (P <0.05, P <0.01) The morphological changes of cells showed the most obvious effect of 12.500 ~ 50.000μmol / L curcumin. At the same time, the expression of TGF-β1 gene and protein was down-regulated while the expression of BMP-7 gene and protein was up-regulated, accompanied by the type Ⅰ collagen gene and protein (P <0.05, P <0.01). Conclusion Curcumin can promote HKC proliferation and maintain its phenotype. It can antagonize the transformation of HKC to spindle cells induced by TGF-β1 and inhibit the expression of TGF-β1 and type Ⅰ collagen in the epithelial cells of the HKCs, and promote the expression of BMP-7.