【作者】 许艳芳；

【导师】 万建新；

【作者基本信息】 福建医科大学 ， 内科学， 2008， 硕士

【摘要】 目的:观察骨形态发生蛋白-7(BMP-7)对转化生长因子-β1(TGF-β1)诱导人近端肾小管上皮细胞(HK-2)向间充质细胞转分化(EMT)及细胞外基质(ECM)分泌的影响,探讨其逆转肾间质纤维化的可能机制。方法:将HK-2细胞在体外与TGF-β1或TGF-β1+BMP-7共培养,分成对照组,不同浓度TGF-β1诱导组,TGF-β1不同时间诱导组,3ng/ml TGF-β1加不同浓度BMP-7组。相差显微镜观察细胞形态的改变;间接免疫荧光测定α-SMA、E-cadherin的表达; RT-PCR检测α-SMA、E-cadherin、ColⅠα1、FN、PAI-1 mRNA的表达;Western Blot检测α-SMA、E-cadherin蛋白的表达;ELISA方法测定细胞上清液ColⅠ、FN的表达。结果:(1)随着TGF-β1浓度的增加及作用时间的延长,HK-2细胞形态逐渐发生改变,由立方形铺路石样转变为梭形长条样;在3ng/ml TGF-β1诱导48h后再加入200ng/ml BMP-7组中,可见到已转变为梭形长条样的细胞大部分逆转回原来的形态。(2)免疫荧光检测显示,正常HK-2细胞表达E-cadherin、而无α-SMA表达,经3ng/ml TGF-β1刺激48h后,细胞浆中α-SMA表达明显增强,细胞质膜E-cadherin表达显著减弱;去除TGF-β1,再加入200ng/ml BMP-7干预48h,能明显抑制α-SMA的表达、恢复E-cadherin的表达。(3)TGF-β1在10ng/ml内呈剂量依赖性上调α-SMA mRNA的表达。在3ng/ml TGF-β1诱导下,α-SMA mRNA表达随作用时间的延长而增加,E-cadherin mRNA表达随作用时间的延长而逐渐减少。在3ng/ml TGF-β1与BMP-7共同干预下,α-SMA mRNA及蛋白的表达随着BMP-7(100 ng/ml ~400 ng/ml )的浓度增加而减少,400 ng/ml BMP-7即可显著减少α-SMA mRNA及蛋白的表达(P<0.01)。在3ng/ml TGF-β1的持续刺激下,E-cadherin mRNA及蛋白的表达随着BMP-7的浓度增加而逐渐增加,400 ng/ml BMP-7即可显著增加E-cadherin mRNA及蛋白的表达(P<0.01)。(4)在3ng/ml TGF-β1诱导下,随刺激时间的延长,ColⅠα1、FN mRNA表达逐渐增加; BMP-7呈剂量依赖性抑制ColⅠ、FN的表达, 400 ng/ml BMP-7即可显著抑制ColⅠ、FN的表达(P<0.01)。(5)在3ng/ml TGF-β1诱导下,PAI-1 mRNA表达明显增加(P<0.01);BMP-7呈剂量依赖性抑制PAI-1 mRNA表达,400ng/ml BMP-7可明显减少PAI-1 mRNA表达(P<0.01)。结论:(1)TGF-β1呈剂量和时间依赖性上调α-SMA的表达、抑制E-cadherin的表达,诱导HK-2细胞转分化,并促进ColⅠ、FN的合成(。2)BMP-7可呈剂量依赖性地抑制TGF-β1诱导下α-SMA mRNA和蛋白的表达,并通过再诱导E-cadherin mRNA和蛋白的表达抑制、逆转TGF-β1诱导的EMT。(3)BMP-7可呈剂量依赖性抑制TGF-β1诱导下HK-2细胞外基质成分ColⅠ、FN mRNA和蛋白的表达;抑制PAI-1 mRNA的表达,即BMP-7可抑制TGF-β1诱导下肾小管上皮细胞ECM的合成、促进ECM降解。更多还原

【Abstract】 Objective: To investigate the effects of bone morphogenic protein(BMP)-7 on epithelial-to-mesenchymal transition(EMT) and the expression of fibronectin(FN) and type collagenⅠ(ColⅠ) in human renal proximal tubular cells(HK-2) induced by transforming growth factor-β1(TGF-β1),and to explore the possible mechanisms of BMP-7 for the inhibition and reversal of renal interstitial fibrosis.Methods: HK-2 cells were treated with TGF-β1 or a combination of TGF-β1 and BMP-7. HK-2 cells divided into the following groups according to different factors: Control group, TGF-β1(different time and concentration) group, 3ng/ml TGF-β1+ BMP-7 (different concentration) group. Morphological changes were assessed by phase contrast microscopy.The expression ofα-SMA and E-cadherin were analysed by indirect immunofluorescence ,RT-PCR and Western Blot respectively.The mRNA levels of extracelluar matrix components(ColⅠα1 and FN) and plasminogen activator inhibitior(PAI)-1 were measured by RT-PCR..The secretion of ColⅠand FN protein was quantitated by ELISA.Results: (1) TGF-β1 treatment of confluent HK-2 cells resulted in distinct morphological changes in a time and dose-dependent manner.The control cells displayed typical cobblestone morphology of epithelial cells. 3ng/ml TGF-β1 induced profound morphologic changes after 48h, with cells becoming elongated in shape, but addition of 200ng/ml BMP-7 for 48h restored the epithelial morphology of the HK-2 cells.(2)Indirect immunofluorescence staining showed that expression ofα-SMA could not be seen in control cells but upregulated and enhanced by 3ng/ml TGF-β1 after 48h.Incubation with 200ng/ml BMP-7 for 48h dramatically abrogated TGF-β1-inducedα-SMA expression. Treatment of 3ng/ml TGF-β1 resulted in distinct loss of Ecadherin staining in the plasma membrane of HK-2 cells but subsequent treatment with 200ng/ml BMP-7 largely restored the E-cadherin protein staining. (3)The expression ofα-SMA mRNA were upregulated by TGF-β1 in a dose-dependent manner maximally at the concentration of 10ng/ml TGF-β1. The 3ng/ml TGF-β1-induced levels ofα-SMA mRNA and protein were increased while E-cadherin decreased in a time-dependent manner. BMP-7 dramatically supressed TGF-β1-inducedα-SMA mRNA and protein expression in a dose-dependent manner. 400 ng/ml BMP-7 could significantly block the expression ofα-SMA mRNA and protein induced by 3ng/ml TGF-β1(P<0.01).Under the continuous influence of 3ng/ml TGF-β1,increasing doses of BMP-7(100～400ng/ml) substantially restored the expression of E-cadherin mRNA and protein (P<0.01, 400 ng/ml BMP-7+ TGF-β1VS. TGF-β1 alone). (4)3ng/ml TGF-β1 could increase the mRNA expression levels of FN and ColⅠα1 in a time-depedent manner.BMP-7 could inhibit the expression of ECM components(FN, ColⅠ) induced by TGF-β1 in a dose-depedent manner:400ng/ml BMP-7 could supress the expression of FN and ColⅠsignificantly(P<0.01). (5) 3ng/ml TGF-β1 could increase the expression of PAI-1 mRNA(P<0.01 VS. control).BMP-7 could inhibit the expression of PAI-1 mRNA induced by TGF-β1 in a dose-dependent manner.400ng/ml BMP-7 could inhibit the expression of PAI-1 mRNA significantly(P<0.01).Conclusions: (1) TGF-β1 can induce EMT and produce ECM components in association with an inhibition of E-cadherin expression, increase ofα-SMA, ColⅠand FN expression and loss of epithelial cell morphology.(2)BMP-7 can block and reverse TGF-β1-induced EMT by supressing the expression ofα-SMA and restoring the expression of E-cadherin in a dose-depedent manner.(3)BMP-7 can prevent accumulation of ECM components and enhance degradation of ECM components induced by TGF-β1 through reducing the expression of ColⅠ,FN and PAI-1.更多还原