發(fā)布時(shí)間：2018-11-28 16:12

【摘要】：目的:明確miR-34a負(fù)性調(diào)控YY1在氡致BEAS-2B細(xì)胞凋亡中的作用,為研究氡致支氣管上皮細(xì)胞凋亡的分子機(jī)制提供基礎(chǔ)資料。方法:對(duì)BEAS-2B細(xì)胞進(jìn)行常規(guī)培養(yǎng),胰酶消化后接種于Transwell培養(yǎng)皿,12h后將培養(yǎng)皿移至染毒裝置中進(jìn)行氡染毒,氡暴露濃度為20,000Bq/m3,染毒時(shí)間30min,連續(xù)染毒2次后作為染毒1代(Rn1)。分別染氡5代(Rn5)、10代(Rn10)、15代(Rn15)、20代(Rn20)后,流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡率,Real-time PCR檢測(cè)miRNA-34a基因的表達(dá)水平,Western blot檢測(cè)YY1的蛋白表達(dá)水平。人工合成人源性miR-34a mimic、miR-34a inhibitor,分別瞬時(shí)轉(zhuǎn)入BEAS-2B細(xì)胞中,觀察對(duì)細(xì)胞凋亡率、凋亡相關(guān)蛋白Bcl-2、PARP-1和Bax表達(dá)的影響。生物信息學(xué)分析發(fā)現(xiàn)轉(zhuǎn)錄因子YY1的3’-UTR存在與miR-34a互補(bǔ)結(jié)合的位點(diǎn),且符合miRNA靶基因的條件。在BEAS-2B細(xì)胞中分別瞬時(shí)轉(zhuǎn)入miR-34a mimic、miR-34a inhibitor,觀察YY1表達(dá)水平的改變。構(gòu)建YY1過(guò)表達(dá)載體和沉默載體,觀察對(duì)細(xì)胞凋亡、Bcl-2、PARP-1和Bax表達(dá)的影響。結(jié)果:(1)細(xì)胞凋亡率隨氡染毒劑量的增加而升高,miRNA-34a表達(dá)水平隨氡染毒劑量的增加而增加,而YY1表達(dá)水平隨氡染毒劑量的增加而降低;(2)轉(zhuǎn)染miR-34a mimic組細(xì)胞凋亡率增加,Bcl-2和PARP-1表達(dá)下調(diào),Bax表達(dá)上調(diào);(3)轉(zhuǎn)染YY1過(guò)表達(dá)載體組細(xì)胞凋亡率減少,Bcl-2和PARP-1表達(dá)上調(diào),Bax表達(dá)下調(diào)。結(jié)論:本次試驗(yàn)結(jié)果提示,在氡致支氣管上皮細(xì)胞凋亡的過(guò)程中,mi R-34a通過(guò)負(fù)性調(diào)控YY1的表達(dá),使Bax表達(dá)上調(diào),Bcl-2和PARP-1表達(dá)下調(diào),從而最終促進(jìn)細(xì)胞的凋亡。但其確切的分子生物學(xué)機(jī)制還需更多的實(shí)驗(yàn)加以證實(shí)。
[Abstract]:Aim: to investigate the role of miR-34a in the negative regulation of YY1 in radon induced apoptosis of BEAS-2B cells, and to provide basic information for studying the molecular mechanism of radon induced apoptosis of bronchial epithelial cells. Methods: BEAS-2B cells were cultured routinely, trypsin was digested and inoculated into Transwell petri dish. After 12 hours, the culture dish was transferred to the device for radon exposure. The exposure concentration of radon was 20000Bq / m3, and the exposure time was 30 min. After continuous exposure for 2 times, it was used as the first generation (Rn1). After radon exposure to radon for 5 (Rn5), 10 (Rn10), 15 (Rn15) and 20 (Rn20), the apoptosis rate was detected by flow cytometry, and the expression level of miRNA-34a gene was detected by Real-time PCR., Western blot was used to detect the expression of YY1 protein. Synthetic human miR-34a mimic,miR-34a inhibitor, was transiently transferred into BEAS-2B cells to observe the effects on apoptosis rate, Bcl-2,PARP-1 and Bax expression of apoptosis-related proteins. Bioinformatics analysis showed that the 3'-UTR of transcription factor YY1 had complementary binding sites with miR-34a, and it was in line with the condition of miRNA target gene. The changes of YY1 expression were observed by transient transfer into miR-34a mimic,miR-34a inhibitor, in BEAS-2B cells. YY1 overexpression vector and silencing vector were constructed to observe the effect on apoptosis, Bcl-2,PARP-1 and Bax expression. Results: (1) the cell apoptosis rate increased with the increase of radon exposure dose, the expression of miRNA-34a increased with the increase of radon exposure dose, while the expression level of YY1 decreased with the increase of radon exposure dose. (2) the rate of apoptosis increased, the expression of Bcl-2 and PARP-1 down-regulated and the expression of Bax up-regulated in miR-34a mimic transfection group, (3) the rate of apoptosis decreased, the expression of Bcl-2 and PARP-1 increased, and the expression of Bax decreased in the group transfected with YY1 overexpression vector. Conclusion: in the process of radon induced apoptosis of bronchial epithelial cells, mi R-34a can up-regulate the expression of YY1, up-regulate the expression of Bax, and down-regulate the expression of Bcl-2 and PARP-1, thus promoting the apoptosis of cells. But more experiments are needed to confirm its exact molecular biological mechanism.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R114

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本文編號(hào)：2363434