本文選題：ATP1A1 + RNA干擾　； 參考：《重慶醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的ATP1A1屬于Na+/K+ATPase酶家族之一,ATP1A1過(guò)表達(dá)已在多種癌癥中有所報(bào)道,包括食管癌、黑色素瘤、肝癌等。據(jù)報(bào)道它在與癌癥相關(guān)的各生物學(xué)過(guò)程起到關(guān)鍵作用,而沉默ATP1A1后對(duì)腫瘤的生長(zhǎng)、轉(zhuǎn)移抑制也有所報(bào)道,但尚未見在膠質(zhì)瘤的侵襲性方面有單獨(dú)報(bào)道。為了證明這一假想,我們借助RNAi(RNA干擾技術(shù)),定向沉默膠質(zhì)瘤U251細(xì)胞的ATP1A1基因,觀察細(xì)胞增殖、遷移、侵襲以及成瘤能力的改變,并探討MMP-2/9(基質(zhì)金屬蛋白酶2/9)表達(dá)的調(diào)節(jié)是否為其關(guān)鍵的作用靶點(diǎn)。方法構(gòu)建ATP1A1/sh RNA質(zhì)粒三對(duì)和陰性對(duì)照一對(duì),通過(guò)三質(zhì)粒系統(tǒng)包裝慢病毒并轉(zhuǎn)染U251細(xì)胞系從而抑制ATP1A1基因的表達(dá)。通過(guò)嘌呤霉素篩選,獲取嘌呤霉素抗性單克隆進(jìn)行培養(yǎng),擴(kuò)增后分別用免疫熒光技術(shù)觀察熒光量,RT-q PCR(實(shí)時(shí)定量PCR技術(shù))和Western blot(免役印跡技術(shù))各自檢測(cè)ATP1A1 m RNA和蛋白的表達(dá),從而驗(yàn)證獲得的穩(wěn)定轉(zhuǎn)染細(xì)胞株。后續(xù)實(shí)驗(yàn)分為空白組、對(duì)照組、實(shí)驗(yàn)組�？瞻捉M:未作任何干預(yù)的U251膠質(zhì)瘤細(xì)胞,對(duì)照組:空載質(zhì)粒轉(zhuǎn)染的U251膠質(zhì)瘤細(xì)胞,實(shí)驗(yàn)組:選出沉默效果最佳的一組。MTT法觀察細(xì)胞體外的增殖能力,細(xì)胞劃痕實(shí)驗(yàn)觀察細(xì)胞的遷移能力,Transwell小室觀察細(xì)胞的侵襲能力,裸鼠皮下成瘤觀察體內(nèi)成瘤能力,Western blot檢測(cè)MMP-2、MMP-9的表達(dá)。結(jié)果經(jīng)轉(zhuǎn)染和嘌呤霉素篩選后,熒光顯微鏡下見有明顯表達(dá)抑制ATP1A1基因的細(xì)胞,RT-q PCR、Western blot分別檢測(cè)穩(wěn)定株ATP1A1的m RNA和蛋白表達(dá),均顯著降低(P0.05),通過(guò)RNAi技術(shù)成功建立U251低表達(dá)ATP1A1的穩(wěn)定株。與對(duì)照組相比,實(shí)驗(yàn)組細(xì)胞的增殖和遷移、侵襲以及成瘤能力也顯著受抑(P0.05);MMP-2和MMP-9的表達(dá)也明顯降低(P0.05)。數(shù)據(jù)有統(tǒng)計(jì)學(xué)意義。結(jié)論靶向沉默ATP1A1基因能夠明顯抑制膠質(zhì)瘤U251細(xì)胞的體外增殖、遷移、侵襲及體內(nèi)成瘤性,其機(jī)制可能與MMP-2、MMP-9的下調(diào)相關(guān),ATP1A1可能作為膠質(zhì)瘤治療的一個(gè)潛在靶點(diǎn)。
[Abstract]:Objective the overexpression of ATP1A1 belongs to the Na / K ATPase family has been reported in many cancers, including esophageal cancer, melanoma, liver cancer and so on. It is reported that it plays a key role in various biological processes related to cancer, and the inhibition of tumor growth and metastasis after silencing ATP1A1 has been reported, but there has not been a separate report on the invasiveness of glioma. To prove this hypothesis, we used the RNAi(RNA interference technique to target the ATP1A1 gene of U251 glioma cells and observe the changes in cell proliferation, migration, invasion and tumorigenesis. And to investigate whether the regulation of MMP-2 / 9 (matrix metalloproteinase 2 / 9) expression is a key target. Methods three pairs of ATP1A1/sh RNA plasmids and a pair of negative controls were constructed. The lentivirus was packaged with three plasmids and transfected into U251 cell line to inhibit the expression of ATP1A1 gene. Purine mycin resistant monoclonal clone was obtained by purine mycin screening, and the expression of ATP1A1 m RNA and protein were detected by immunofluorescence technique using RT-q PCR (real-time quantitative PCR) and Western blot (no-service blotting), respectively. The stable transfection cell line was verified. The follow-up experiment was divided into blank group, control group and experimental group. Blank group: U251 glioma cells without any intervention, control group: U251 glioma cells transfected with empty plasmids, experimental group: select the best silencing effect group. MTT method to observe the proliferation of cells in vitro. The cell migration ability was observed by cell scratch assay and the invasion ability was observed by Transwell chamber, and the expression of MMP-2 and MMP-9 was detected by Western blot in nude mice by subcutaneous tumorigenesis. Results after transfection and purine mycin screening, the expression of m RNA and protein in the stable ATP1A1 strain was detected by RT-PCR blot. The stable strain of U251 with low expression of ATP1A1 was successfully established by RNAi. Compared with the control group, the proliferation and migration, invasion and tumorigenesis of the cells in the experimental group were also significantly inhibited by the expression of MMP-2 and MMP-9. The data are statistically significant. Conclusion silencing ATP1A1 gene can significantly inhibit the proliferation, migration, invasion and tumorigenesis of U251 glioma cells in vitro, and its mechanism may be related to the down-regulation of MMP-2 / MMP 9. It may be a potential target for the treatment of glioma.
【學(xué)位授予單位】：重慶醫(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)】：R739.41

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本文編號(hào)：1869443