本文選題：腎間質(zhì)纖維化 + 蛋白磷酸酶2A　； 參考：《中南大學(xué)》2014年碩士論文

【摘要】：目的 通過TGF-β1體外刺激人腎小管上皮細(xì)胞(HK-2細(xì)胞)建立腎間質(zhì)纖維化細(xì)胞模型,探討：(1)NCTD是否通過靶向抑制PP2Ac發(fā)揮抗腎間質(zhì)纖維化作用；(2)NCTD抗腎間質(zhì)纖維化是否與其抑制PP2Ac介導(dǎo)Smad3-L區(qū)去磷酸化有關(guān)。 方法 1、常規(guī)培養(yǎng)HK-2細(xì)胞,分別通過過表達(dá)PP2Ac和小干擾RNA敲低PP2Ac表達(dá)后,應(yīng)用TGF-β1(5ng/ml)刺激和NCTD(2.5μg/ml)進(jìn)行干預(yù)24h。采用RT-PCR和Western印跡法檢測(cè)PP2Ac、FN、 Col-I、α-SMA和E-cadherin mRNA及蛋白表達(dá)水平。 2、常規(guī)培養(yǎng)HK-2細(xì)胞,應(yīng)用NCTD (2.5μg/ml)進(jìn)行預(yù)干預(yù)24h和TGF-β1(5ng/ml)刺激1h。采用間接細(xì)胞免疫熒光法檢測(cè)pSmad3-L(Ser204)、pSmad3-L(Ser208)在細(xì)胞的分布,Western印跡法檢測(cè)總蛋白中PP2Ac和核蛋白中pSmad3-L(Ser204)、pSmad3-L(Ser208)的表達(dá)。 結(jié)果 1、TGF-β1刺激HK-2細(xì)胞24h致PP2Ac表達(dá)上調(diào)的同時(shí),FN、 Col-I和α-SMA表達(dá)上調(diào),E-cadherin表達(dá)下調(diào),HK-2細(xì)胞纖維化加重;而轉(zhuǎn)染PP2Ac過表達(dá)質(zhì)粒后再用TGF-β1刺激,則PP2Ac表達(dá)上調(diào)更為明顯,HK-2細(xì)胞纖維化加重更顯著；NCTD干預(yù)使PP2Ac表達(dá)下調(diào),同時(shí)FN、Col-I和α-SMA表達(dá)下調(diào),E-cadherin表達(dá)上調(diào),HK-2細(xì)胞纖維化緩解； 2、小干擾RNA敲低PP2Ac表達(dá)后,FN、Col-I和α-SMA表達(dá)下調(diào),E-cadherin表達(dá)上調(diào),HK-2細(xì)胞纖維化緩解；而NCTD與小干擾RNA共同抑制PP2Ac表達(dá)后,對(duì)HK-2細(xì)胞纖維化的緩解程度同單純PP2Ac小干擾RNA干預(yù)組比較無明顯差異； 3、免疫熒光結(jié)果顯示,空白對(duì)照組中pSmad3-L(Ser204)、 pSmad3-L(Ser208)基本無表達(dá),TGF-β1刺激HK-2細(xì)胞1h后pSmad3-L(Ser204)、pSmad3-L(Ser208)在細(xì)胞核內(nèi)表達(dá)明顯增多,NCTD干預(yù)使pSmad3-L(Ser204)、pSmad3-L(Ser208)在細(xì)胞核內(nèi)表達(dá)進(jìn)一步增多。Western印跡結(jié)果顯示,TGF-β1刺激1h使得PP2Ac蛋白表達(dá)上調(diào)的同時(shí),細(xì)胞核內(nèi)pSmad3-L(Ser204)、pSmad3-L(Ser208)蛋白表達(dá)均增多,NCTD干預(yù)使PP2Ac表達(dá)下調(diào)的同時(shí),細(xì)胞核內(nèi)pSmad3-L(Ser204)、pSmad3-L(Ser208)蛋白表達(dá)進(jìn)一步增多。 結(jié)論 1.NCTD靶向抑制PP2Ac發(fā)揮抗腎間質(zhì)纖維化作用； 2.NCTD可能通過抑制PP2Ac介導(dǎo)Smad3-L區(qū)去磷酸化,即促進(jìn)Smad3-L區(qū)磷酸化發(fā)揮抗腎間質(zhì)纖維化作用。
[Abstract]:Objective to establish a renal interstitial fibrosis cell model by stimulating human renal tubular epithelial cells (HK-2 cells) with TGF- 尾 1 in vitro, and to explore whether NCTD can inhibit renal interstitial fibrosis by targeting PP2Ac; (2) whether NCTD inhibits renal interstitial fibrosis by inhibiting PP2Ac mediated Smad3-L dephosphorylation. Methods 1. HK-2 cells were treated with TGF- 尾 1 (5ng/ml) stimulation and NCTD (2.5 渭 g/ml) for 24 h after overexpression of PP2Ac and small interfering RNA knockout PP2Ac. The mRNA and protein expressions of PP2ActFN, Col-I, 偽 -SMA and E-cadherin were detected by RT-PCR and Western blot. HK-2 cells were cultured routinely. NCTD (2.5 渭 g/ml) was pretreated for 24 h and stimulated by TGF- 尾 1 (5ng/ml) for 1 h. The expression of pSmad3-L (Ser204) pSmad3-L (Ser208) in the total protein and pSmad3-L (Ser204) pSmad3-L (Ser208) in the total protein was detected by Western blot. Results 1 the expression of PP2Ac was up-regulated in HK-2 cells induced by TGF- 尾 1 for 24 h, while the expression of FN, Col-I and 偽 -SMA was down-regulated and the fibrosis of HK-2 cells was aggravated after transfection of PP2Ac overexpression plasmid, and then stimulated by TGF- 尾 1. The expression of PP2Ac was up-regulated and the fibrosis of HK-2 cells was aggravated significantly. The expression of PP2Ac was down-regulated by NCTD, and the expression of E-cadherin was up-regulated by FN-1 Col-I and 偽 -SMA, and the fibrosis of HK-2 cells was alleviated. 2After the expression of PP2Ac was reduced by small interfering RNA knockout, the expression of PP2Ac was down-regulated and the expression of E-cadherin was up-regulated, while the expression of PP2Ac was inhibited by NCTD and small interfering RNA, and the expression of PP2Ac was inhibited by NCTD and small interfering RNA. The remission degree of HK-2 cells fibrosis was not significantly different from that of PP2Ac small interfering RNA intervention group. The expression of pSmad3-L (Ser204), pSmad3-L (Ser208) and pSmad3-L (Ser208) in the nucleus of HK-2 cells was significantly increased 1 h after stimulation of pSmad3-L (Ser204) and pSmad3-L (Ser208). Western blot showed that the expression of pSmad3-L (Ser204) pSmad3-L (Ser208) increased in the nucleus of HK-2 cells. Western blotting showed that the expression of PP2Ac protein was up-regulated by TGF- 尾 1 stimulation for 1 h, while the expression of pSmad3-L (Ser204) pSmad3-L (Ser208) increased significantly. The expression of pSmad3-L (Ser204) pSmad3-L (Ser208) in the nucleus was increased. The expression of pSmad3-L (Ser204) pSmad3-L (Ser208) in the nucleus was further increased while the expression of PP2Ac was down-regulated by NCTD. 2. NCTD could inhibit the dephosphorylation of Smad3-L region by inhibiting PP2Ac phosphorylation, that is, promote the phosphorylation of Smad3-L region and play an anti-interstitial role in renal interstitial fibrosis. 2.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R692

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