【作者】 杨丽霞；

【导师】 刘铜华；

【作者基本信息】 北京中医药大学 ， 中医内科学， 2009， 博士

【摘要】 【目的】本课题采用转化生长因子-β1（TGF-β1）诱导体外培养人肾小管上皮细胞（HK-2）转分化构建肾间质纤维化细胞模型,通过姜黄素（Cur）干预TGF-β1诱导人肾小管上皮细胞增殖、表型转化、细胞外基质成分合成以及TGF-β/Smad信号转导途径的实验研究,探讨姜黄素干预肾间质纤维化的作用及机制,为其防治肾间质纤维化的临床应用提供实验依据。【方法】HK-2细胞用含10%胎牛血清、100 U/mL青霉素、100μg/mL链霉素DMEM/F12（1:1）完全培养基,于37℃、5%CO₂细胞培养箱中培养。每隔2-3天换新鲜培养基一次,培养融合至80%以上,用含0.25%胰蛋白酶、0.05%EDTA的消化液消化,用完全培养基终止消化后,离心5 min（1000g/min）。用新鲜培养基将细胞重悬后进行传代培养。实验前先用无血清培养基培养24h使细胞同步化。实验分为以下5组:空白对照组（DMEM/F12培养液）、单纯TGF-β刺激组（DMEM/F12培养液+10 ng/mL TGF-β₁）、低浓度药物处理组（DMEM/F12培养液+10 ng/mL TGF-β1+1μmoL/L Cur）、中浓度药物处理组（DMEM/F12培养液+10ng/mL TGF-β1+5μmoL/L Cur）、高浓度药物处理组（DMEM/F12培养液+10 ng/mLTGF-β1+10μmoL/L Cur）。然后进行下列实验研究:（1）通过倒置显微镜下观察细胞形态、MTT法检测细胞增殖,探讨姜黄素对细胞增殖的干预作用;（2）通过免疫细胞化学法检测α-SMA、E-cadherin的表达,探讨姜黄素对细胞表型转化的干预作用;（3）通过ELISA法检测细胞培养上清ColⅠ、ColⅢ、FN的含量,以及RT-PCR法检测细胞ColⅠ、ColⅢmRNA的表达,探讨姜黄素对细胞外基质成分合成的干预作用;（4）通过Western blot法检测TβRⅠ、TβRⅡ、smad2、Smad 3、P-Smad 2/3以及smad 7蛋白表达,探讨姜黄素对TGF-β/Smad信号转导途径的干预作用。【结果】（1）姜黄素对细胞增殖的干预作用倒置显微镜下观察发现,正常HK-2细胞呈立方形铺路石样贴壁生长,当培养液中加入TGF-β₁作用24h后,细胞拉长增大呈梭形,细胞间隙加大,具有纤维样特征,与姜黄素共同作用后,细胞形态随着姜黄素浓度的增加在一定程度上趋向于正常形态。MTT比色结果显示:TGF-β₁能够诱导HK-2细胞增殖,与空白对照组相比有显著性差异（P＜0.05）,但和姜黄素共同作用后,其促细胞增殖作用受到显著抑制（P＜0.05）。（2）姜黄素对细胞表型转化的干预作用免疫细胞化学实验结果显示:正常HK-2细胞胞膜强烈表达E-cadherin,几乎没有α-SMA表达,经TGF-β₁诱导后胞浆强烈表达α-SMA,E-cadherin表达几乎消失,但与姜黄素共同作用后α-SMA表达明显减弱,E-cadherin表达显著增强。（3）姜黄素对细胞外基质成分合成的干预作用ELISA实验结果显示:HK-2细胞经TGF-β₁处理24h、48h、72h后,时间依赖性地促进ColⅠ、ColⅢ、FN的分泌（p＜0.05）,但经姜黄素干预后ColⅠ、ColⅢ、FN的分泌均受到显著抑制（p＜0.05）。并且RT-PCR实验结果显示,姜黄素也能抑制ColⅠ、ColⅢmRNA的表达。（4）姜黄素对TGF-β/Smad信号转导途径的干预作用Western blot实验结果显示:HK-2细胞经TGF-β₁作用24h后,TβRⅠ、TβRⅡ、smad2、Smad 3、P-Smad 2/3蛋白表达显著增强,smad7蛋白表达显著减弱,与空白对照组相比有显著性差异（p＜0.05）,但和姜黄素共同作用后,TβRⅠ、TβRⅡ、smad2、Smad 3、P-Smad 2/3蛋白表达受到抑制,而smad7蛋白表达增强,与单纯TGF-β1作用相比有显著性差异（P＜0.05）。【结论】姜黄素能够抑制肾小管上皮细胞增殖,阻止肾小管上皮细胞向肌成纤维细胞转分化,并能减少细胞外基质成分的合成,阻断TGF-β/Smad信号转导途径,具有干预肾间质纤维化的作用,为肾间质纤维化的临床治疗提供了实验依据。更多还原

【Abstract】 Objective To study the effect of curcumin on proliferation,tubular epithelial-myofibroblast transdifferentiation,secretion of extracellular material,and TGF-β/Smad signal transduction pathway of human renal tubular epithelial cells HK-2 induced by transforming growth factor-β₁（TGF-β₁）,and further to explore the mechanism of curcumin on the prevention and treatment of renal interstitial fibrosis.Methods The HK -2 cells were cultured by DMEM/F12（1:1） with 10%fetal bovine serum in 37℃and 5%CO₂.After being cultured 24 hours by free serm,cells were divided into the control group,TGF-β₁ group（TGF-β₁ 10 ng/mL）,curcumin low dose therapy group （TGF-β₁ 10 ng/mL +Cur 1μmol/L）,curcumin median dose therapy group（TGF-β₁ 10 ng/mL +Cur 5μmol/L）,and curcumin high dose therapy group（TGF-β₁ 10 ng/mL +Cur 10μmol/L）. Then the HK-2 cell morphology were observed through the microscope.The cell proliferation were tested by MTT assay after being treated for 24 hours,48 hours and 72 hours.The expression ofα-SMA and E-cadherin of HK-2 were tested by indirect immunohistochemical staining after being treated for 24 hours.And the concentration of typeⅠcollagen,typeⅢcollagen and FN in culture medium supernatant were detected by ELISA after being treated for 24 hours,48 hours and 72 hours.And the expression of mRNA of typeⅠcollagen,typeⅢcollagen were tested after being treated for 24 hours.And the expression of protein of TβRⅠ,TβRⅡ,smad2,Smad 3,P-Smad 2/3 and smad7 were detected after being treated for 24 hours.Results We found that TGF-β₁ could stimulate HK-2 cells proliferation time-dependently compared with the control group,and there was significant difference（P＜0.05）.But the cell proliferation was inhibited after treated by curcumin（P＜0.05）,and the cells morphology could be maintained by curcumin.Secondly,we found that the expression ofα-SMA of HK-2 cultured with TGF-β₁ was much notable than the control group,but significantly attenuated after treated by curcumin（P＜0.05）.And the expression of E-cadherin was markedly decreased after treated by TGF-β₁ alone,but recovered after treated by curcumin（P＜0.05）.Thirdly,we also found that the secretion of typeⅠcollagen,typeⅢcollagen and FN were markedly inhibited in the culture medium supernatant treated by curcumin（P＜0.05）.At the same time the expression of mRNA of typeⅠcollagen,typeⅢcollagen also attenuated.Finally,we found that TGF-β₁ could enhance the expression of TβRⅠ,TβRⅡ,smad2,Smad3,P-Smad 2/3,and attenuate the expression of smad7,but after treated by curcumin,the expression of TβRⅠ,TβRⅡ,smad2,Smad3,P-Smad 2/3 were down-regulated obviously,and the expression of smad7 were up-regulated（P＜0.05）.Conclusion We obtained that TGF-β₁ could induce tubular epithelial cells proliferation, lead to tubular epithelial-myofibroblast transdifferentiation,and increase the amount of extracellular matrix,such as typeⅠcollagen,typeⅢcollagen and FN.In addition,it could activate TGF-β/Smad signal transduction pathway.All these results confirmed that TGF-β₁ is the key factor in development of renal interstitial fibrosis,and this is consistent with previous reports.At the same time,we also obtained that curcumin at given concentrations could inhibit the tubular epithelial cells proliferation,and inhibit tubular epithelial-myofibroblast transdifferentiation,and reduce synthesis and secretion extracellular matrix.In addition, curcumin could obstruct the TGF-β/Smad signal transduction pathway.So we concluded that curcumin could prevent the development of renal interstitial fibrosis to a certain extent.And this would provide evidence for later clinical application.更多还原