【摘要】：目的:探討膠質(zhì)瘤組織細(xì)胞中GDNF基因在轉(zhuǎn)錄過(guò)程中發(fā)生的選擇性剪接異常變化,并揭露這種變化對(duì)膠質(zhì)瘤遷移能力可能存在的影響。方法:針對(duì)高度同源的GDNF剪接變異體cDNA的高效引物,應(yīng)用Real-time PCR檢測(cè)膠質(zhì)瘤組織細(xì)胞中GDNF選擇性剪接體α-pro-GDNF和β-pro-GDNF的mRNA表達(dá)水平;利用western blot檢測(cè)細(xì)胞中α-pro-GDNF和β-pro-GDNF蛋白的表達(dá)情況,同時(shí)選擇免疫熒光染色的方法檢測(cè)膠質(zhì)瘤細(xì)胞中蛋白的表達(dá)分布及表達(dá)水平。構(gòu)建過(guò)表達(dá)和干擾α-pro-GDNF蛋白的慢病毒質(zhì)粒并轉(zhuǎn)染膠質(zhì)瘤細(xì)胞U251,使α-pro-GDNF蛋白的表達(dá)上調(diào)或下降,隨后利用劃痕實(shí)驗(yàn)檢測(cè)轉(zhuǎn)染后的U251膠質(zhì)瘤細(xì)胞遷移能力的變化。結(jié)果:Real-time PCR和western blot結(jié)果均表明膠質(zhì)瘤細(xì)胞中GDNF基因存在選擇性剪接體,且隨著膠質(zhì)瘤WHO級(jí)別升高,α-pro-GDNF和β-pro-GDNF的mRNA和蛋白的表達(dá)水平均呈逐漸升高趨勢(shì),且α-pro-GDNF的mRNA和蛋白的升高趨勢(shì)較β-pro-GDNF尤為明顯,呈優(yōu)勢(shì)表達(dá);免疫熒光染色表明α-pro-GDNF和β-pro-GDNF蛋白主要表達(dá)于細(xì)胞胞質(zhì)中,熒光強(qiáng)度定量分析進(jìn)一步提示膠質(zhì)瘤細(xì)胞中蛋白表達(dá)量較正常細(xì)胞多;慢病毒轉(zhuǎn)染成功后,過(guò)表達(dá)組α-pro-GDNF蛋白升高,干擾組α-pro-GDNF蛋白降低,劃痕實(shí)驗(yàn)初步表明α-pro-GDNF蛋白表達(dá)的變化會(huì)影響膠質(zhì)瘤細(xì)胞的遷移能力。結(jié)論:膠質(zhì)瘤細(xì)胞中GDNF基因發(fā)生異常選擇性剪接,其中α-pro-GDNF優(yōu)先表達(dá),并與膠質(zhì)瘤遷移能力密切相關(guān),為膠質(zhì)瘤的治療提供新的作用靶點(diǎn)。
[Abstract]:Aim: to investigate the selective splicing abnormal changes of GDNF gene in glioma tissue cells during transcription, and to explore the possible effect of this change on glioma migration ability. Methods: Real-time PCR was used to detect the expression of 偽 -pro-GDNF and 尾 -pro-GDNF in glioma cells, and the expression of 偽 -pro-GDNF and 尾 -pro-GDNF in glioma cells was detected by western blot. At the same time, immunofluorescence staining was used to detect the distribution and level of protein expression in glioma cells. The lentivirus plasmid which overexpressed and interfered with 偽 -pro-GDNF protein was constructed and transfected into glioma cell U251. The expression of 偽 -pro-GDNF protein was up-regulated or down-regulated. The migration ability of transfected U251 glioma cells was detected by scratch test. Results the results of western blot and real-time PCR showed that there was a selective splicing of GDNF gene in glioma cells, and the mRNA and protein levels of 偽 -pro-GDNF and 尾 -pro-GDNF increased gradually with the increase of WHO grade of glioma. The expression of 偽 -pro-GDNF mRNA and protein was more obvious than that of 尾 -pro-GDNF, and immunofluorescence staining showed that 偽 -pro-GDNF and 尾 -pro-GDNF mainly expressed in cytoplasm. The quantitative analysis of fluorescence intensity further indicated that the expression of 偽 -pro-GDNF protein in glioma cells was higher than that in normal cells, and that the 偽 -pro-GDNF protein increased in the overexpression group and decreased in the interference group after lentivirus transfection. Scratch test showed that the expression of 偽 -pro-GDNF protein affected the migration ability of glioma cells. Conclusion: there is abnormal selective splicing of GDNF gene in glioma cells, in which 偽 -pro-GDNF is preferentially expressed and closely related to glioma migration, which provides a new target for the treatment of glioma.
【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】：2016
【分類(lèi)號(hào)】：R739.4

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