發(fā)布時(shí)間：2019-03-05 10:59

【摘要】：目的明確低劑量重金屬鉻暴露后,對(duì)膀胱癌進(jìn)展產(chǎn)生的影響,并初步探討其對(duì)FLNA基因表達(dá)的影響及相關(guān)的細(xì)胞生物學(xué)行為。方法采用MTT方法篩選Cr(VI)暴露T24細(xì)胞的亞致死劑量,明確低劑量Cr(VI)對(duì)T24細(xì)胞亞致死濃度;目的是篩選出對(duì)細(xì)胞增殖效應(yīng)最為明顯的鉻濃度。在該濃度下的Cr(VI)處理T24細(xì)胞后分別進(jìn)行細(xì)胞劃痕實(shí)驗(yàn)、Transwell實(shí)驗(yàn)研究Cr(VI)暴露對(duì)細(xì)胞遷移能力、侵襲能力;采用免疫組化、qPCR免疫熒光和Western bolt等技術(shù)分析膀胱癌及其癌旁組織;膀胱癌細(xì)胞系中的表達(dá)水平,探討其與臨床分期的關(guān)系。利用shFLNA質(zhì)粒轉(zhuǎn)染T24細(xì)胞系后,利用流式細(xì)胞技術(shù)檢測(cè)敲低FLNA后的T24細(xì)胞凋亡影響。利用劃痕實(shí)驗(yàn)、Transwell實(shí)驗(yàn)檢測(cè)敲低前后的侵襲性、遷移性的改變。用Cr(VI)刺激T24細(xì)胞后,利用免疫熒光和Western bolt檢測(cè)FLNA的表達(dá)情況。結(jié)果Cr(VI)暴露可促進(jìn)T24細(xì)胞增殖,并發(fā)現(xiàn)在鉻暴露劑量為0.4uM時(shí),其促進(jìn)增殖效應(yīng)最為顯著(P0.05);Cr(VI)暴露T24細(xì)胞后,細(xì)胞遷移以及侵襲能力均顯著增加(P0.05);而免疫組化結(jié)果顯示,浸潤性尿路上皮癌組織中FLNA的表達(dá)呈強(qiáng)陽性,而非浸潤性尿路上皮癌的組織則為弱陽性,正常組織免疫組化結(jié)果為陰性,結(jié)果具有統(tǒng)計(jì)學(xué)意義。qPCR檢測(cè)膀胱癌組織及其癌旁組織FLNA mRNA表達(dá)量表明,肌層浸潤性尿路上皮癌FLNA的表達(dá)較非肌層浸潤性尿路上皮癌高,而癌旁組織的表達(dá)量最低。Western blot結(jié)果表明正常人臍靜脈細(xì)胞(HUVC)中FLNA的蛋白表達(dá)量低于T24細(xì)胞系qPCR結(jié)果也表明T24細(xì)胞系的FLNA表達(dá)高于HUVC細(xì)胞系。劃痕實(shí)驗(yàn)和Transwell實(shí)驗(yàn)的結(jié)果表明shFLNA腫細(xì)胞的遷移能力和侵襲能力較正常未處理對(duì)照組T24細(xì)胞低。而在低劑量鉻表露后Western blot結(jié)果表明FLNA的表達(dá)增高。而免疫熒光的結(jié)果同樣也表明FLNA在低劑量鉻暴露后在細(xì)胞質(zhì)中較未經(jīng)過鉻刺激的細(xì)胞呈高表達(dá)。結(jié)論Cr(VI)暴露可顯著影響膀胱細(xì)胞的生物學(xué)行為,促進(jìn)膀胱癌進(jìn)展。該作用機(jī)制是低劑量鉻調(diào)控FLNA及相關(guān)信號(hào)通路的表達(dá)發(fā)揮其促進(jìn)腫瘤進(jìn)展的作用。
[Abstract]:Objective To study the effect of low dose of heavy metal chromium on the progression of bladder cancer and to explore its effect on the expression of FLNA gene and the related cellular biological behavior. Methods The sublethal dose of Cr (VI) exposed to T24 cells was selected by MTT method, and the sublethal concentration of low dose of Cr (VI) on T24 cells was determined. The cell scratch test was carried out after the treatment of T24 cells with Cr (VI) at this concentration, and the cell migration ability and the invasion ability of Cr (VI) were studied by Transwell. The expression level of the bladder cancer cell line was analyzed by immunohistochemistry, qPCR immunofluorescence and Western blot. The relationship between the clinical stage and the clinical stage was discussed. After the T24 cell line was transfected with the shFLA plasmid, the apoptosis of T24 cells following the knockdown of FLNA was detected by flow cytometry. Using the scratch test, the transwell's experiment was used to detect the change of the invasion and migration before and after the knock. After T24 cells were stimulated with Cr (VI), the expression of FLNA was detected by immunofluorescence and Western blot. Results Cr (VI) exposure could promote the proliferation of T24 cells, and it was found that the effect of Cr (VI) on the proliferation of T24 cells was the most significant (P0.05). After the exposure of Cr (VI) to T24 cells, the cell migration and the invasion ability were significantly increased (P0.05). The expression of FLNA in the tissues of invasive urothelial carcinoma was strongly positive, while the non-invasive urothelial carcinoma was weakly positive, and the immunohistochemical results of normal tissues were negative and the results were of statistical significance. The expression of FLNA mRNA in bladder cancer tissues and its adjacent tissues was detected by qPCR, and the expression of FLNA in the invasive urothelial carcinoma of the myometrium was higher than that of the non-myometrial invasion urothelial carcinoma, while the expression of the adjacent tissue was the lowest. The results of Western blot show that the expression of FLNA in human umbilical vein cell (HUVC) is lower than that of T24 cell line, and the expression of FLNA in the T24 cell line is higher than that of HUVC cell line. The results of the scratch test and the Transwell experiment show that the ability and the invasion ability of the shFLA swollen cells are lower than that of the control group T24. The results showed that the expression of FLNA was increased after low dose of chromium. The results of immunofluorescence also show that FLNA is highly expressed in the cytoplasm, which is not stimulated by chromium, after low-dose chromium exposure. Conclusion The exposure of Cr (VI) can significantly affect the biological behavior of bladder cells and promote the progression of bladder cancer. The mechanism is the role of low-dose chromium control FLA and related signal pathways to play a role in promoting the progression of the tumor.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.14

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相關(guān)期刊論文 前1條

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本文編號(hào)：2434834