【摘要】：目的：應(yīng)用焦磷酸測序技術(shù)對(duì)腎臟固有細(xì)胞表型轉(zhuǎn)化標(biāo)志性蛋白α-SMA的基因轉(zhuǎn)錄起始點(diǎn)附近序列的甲基化狀態(tài)進(jìn)行定位、定量檢測，分析比較腎小球系膜細(xì)胞（HBZY-1）及腎間質(zhì)成纖維細(xì)胞（NRK-49F）在表型轉(zhuǎn)化前后α-SMA基因的甲基化圖示及數(shù)量的差異，從表觀遺傳學(xué)水平闡述腎臟固有細(xì)胞發(fā)生表型轉(zhuǎn)化的可能機(jī)制，為腎臟纖維化的早期防治及尋找新的治療靶標(biāo)提供新的可行性研究思路。方法：（1）分別對(duì)HBZY-1及NRK-49F進(jìn)行體外培養(yǎng)，，同步化后兩株細(xì)胞均分為無血清培養(yǎng)的正常對(duì)照組和重組人TGF-β1（10ug/L，48h）誘導(dǎo)的誘導(dǎo)組各兩組。各組細(xì)胞分別進(jìn)行形態(tài)學(xué)觀察、細(xì)胞爬片及培養(yǎng)上清液收集，細(xì)胞爬片進(jìn)行免疫細(xì)胞化學(xué)染色觀察細(xì)胞胞漿內(nèi)α-SMA蛋白表達(dá)情況，ELISA檢測培養(yǎng)上清液中Ⅰ型膠原含量，分別比較兩株細(xì)胞各組實(shí)驗(yàn)結(jié)果，判定誘導(dǎo)條件誘導(dǎo)細(xì)胞發(fā)生表型轉(zhuǎn)化的結(jié)果。（2）根據(jù)實(shí)驗(yàn)前部分驗(yàn)證結(jié)果，采用同等條件誘導(dǎo)細(xì)胞表型轉(zhuǎn)化，收集各組新鮮細(xì)胞，提取全基因組DNA，應(yīng)用焦磷酸測序技術(shù)檢測α-SMA基因轉(zhuǎn)錄起始點(diǎn)附近甲基化狀態(tài)，比較兩株細(xì)胞各組甲基化情況。結(jié)果：（1）誘導(dǎo)后的兩株細(xì)胞均發(fā)生形態(tài)學(xué)改變，表現(xiàn)為細(xì)胞胞體增大，胞漿豐富，胞核增大，HBZY-1由梭形或不規(guī)則星形變?yōu)槎嘟切位驑渲π�，擴(kuò)張胞突連接成片，NRK-49F由長梭形變?yōu)槎趟笮位蚨嘟切危糠挚梢姲麅?nèi)空泡。（2）細(xì)胞免疫化學(xué)檢測顯示，正常HBZY-1胞漿內(nèi)僅有少量α-SMA蛋白呈弱陽性表達(dá)，誘導(dǎo)后胞漿內(nèi)α-SMA蛋白呈強(qiáng)陽性高表達(dá)（P＜0.05）；正常NRK-49F胞漿內(nèi)無α-SMA蛋白表達(dá)，誘導(dǎo)后胞漿α-SMA蛋白出現(xiàn)并呈強(qiáng)陽性高表達(dá)（P＜0.05）。（3）ELISA檢測顯示，正常HBZY-1及NRK-49F的培養(yǎng)上清液中均含有Ⅰ型膠原，誘導(dǎo)后兩株細(xì)胞培養(yǎng)上清液中Ⅰ型膠原含量均明顯增多（P＜0.05）。（4）正常HBZY-1及NRK-49F中α-SMA基因呈現(xiàn)不同程度甲基化，HBZY-1甲基化頻率在20%-50%之間呈低甲基化狀態(tài)，NRK-49F甲基化頻率＞50%呈高甲基化狀態(tài)，誘導(dǎo)后兩株細(xì)胞中α-SMA基因甲基化圖示及數(shù)量均無變化。結(jié)論：（1）TGF-β1可誘導(dǎo)HBZY-1及NRK-49F發(fā)生細(xì)胞表型轉(zhuǎn)化，轉(zhuǎn)變?yōu)楦弑磉_(dá)α-SMA蛋白、能分泌大量細(xì)胞外基質(zhì)的肌成纖維細(xì)胞，在腎臟纖維化形成中的發(fā)揮重要作用。（2）α-SMA基因轉(zhuǎn)錄起始點(diǎn)附近甲基化狀態(tài)在腎臟固有細(xì)胞表型轉(zhuǎn)化中作用尚不能完全明確，是否通過表觀遺傳修飾中DNA甲基化狀態(tài)的改變促進(jìn)腎臟纖維化的發(fā)展有待進(jìn)一步研究。
[Abstract]:Objective: to localize and quantitatively detect the methylated status of 偽-SMA gene near the starting point of gene transcription in renal intrinsic cell phenotypic transformation marker protein by pyrophosphate sequencing. The methylation patterns and quantity of 偽-SMA gene in Mesangial cells (HBZY-1) and interstitial fibroblasts (NRK-49F) before and after phenotypic transformation were analyzed and compared. This paper expounds the possible mechanism of phenotypic transformation of renal intrinsic cells from the level of epigenetics, and provides a new feasible research idea for the early prevention and treatment of renal fibrosis and the search for new therapeutic targets. Methods: (1) HBZY-1 and NRK-49F were cultured in vitro. After synchronization, the two cells were divided into two groups: normal control group without serum culture and recombinant human TGF- 尾 1 (10 ug 鈮

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