發(fā)布時(shí)間：2018-04-25 21:15

  本文選題：慢病毒屬 + RNA干擾��； 參考：《福建醫(yī)科大學(xué)學(xué)報(bào)》2017年03期

【摘要】：目的構(gòu)建靶向YWHAE基因的shRNA慢病毒表達(dá)質(zhì)粒,并驗(yàn)證其對(duì)AGS胃癌細(xì)胞增殖的影響。方法設(shè)計(jì)并合成5對(duì)針對(duì)人YWHAE基因的shRNA序列,分別克隆入pLentiLox3.7(pLL3.7)慢病毒表達(dá)質(zhì)粒,接著將重組的慢病毒表達(dá)質(zhì)粒和包裝質(zhì)粒PHR、包膜質(zhì)粒VSVG一起采用磷酸鈣法轉(zhuǎn)染293T細(xì)胞包裝慢病毒,收集制備的慢病毒感染AGS細(xì)胞,用抗生素Puromycine進(jìn)行篩選。Western-blot檢測(cè)感染細(xì)胞YWHAE蛋白的表達(dá)。選取干擾效率最高的慢病毒表達(dá)質(zhì)粒,針對(duì)性設(shè)計(jì)siRNA的點(diǎn)突變引物,重疊延伸PCR法擴(kuò)增獲得點(diǎn)突變的YWHAE基因,克隆入pcDNA3.1/myc-His(-)A載體,構(gòu)建YWHAE點(diǎn)突變的表達(dá)質(zhì)粒,轉(zhuǎn)染YWHAE沉默效果最好的AGS細(xì)胞株,Western-blot檢測(cè)轉(zhuǎn)染細(xì)胞YWHAE蛋白的回復(fù)表達(dá)。采用MTS法檢測(cè)YWHAEshRNA慢病毒對(duì)AGS細(xì)胞增殖的影響。結(jié)果5對(duì)針對(duì)人YWHAE基因的shRNA序列構(gòu)建的慢病毒中,pLL3.7-siYWHAE-5包裝成的慢病毒抑制AGS細(xì)胞的YWHAE蛋白表達(dá)的效果最明顯,僅為對(duì)照組相對(duì)表達(dá)量的(0.269±0.083)倍;pLL3.7-siYWHAE-5包裝的慢病毒,其干擾效應(yīng)可經(jīng)由YWHAE點(diǎn)突變表達(dá)質(zhì)�；貜�(fù)。與對(duì)照組比較,YWHAE-shRNA組AGS細(xì)胞增殖能力明顯下降。結(jié)論成功構(gòu)建了靶向YWHAE基因的shRNA慢病毒表達(dá)質(zhì)粒,獲得YWHAE基因表達(dá)顯著下調(diào)的AGS細(xì)胞株;探明YWHAE表達(dá)的下調(diào)可有效抑制AGS細(xì)胞的增殖,提示YWHAE在胃癌中的致癌潛能。
[Abstract]:Objective to construct the shRNA lentivirus expression plasmid targeting YWHAE gene and verify its effect on the proliferation of AGS gastric cancer cells. Methods five pairs of shRNA sequences targeting human YWHAE gene were designed and synthesized and cloned into lentivirus expression plasmids pLL3.7pL3.7pL3.7pL3.7pL3.7. the recombinant lentiviral expression plasmids and packaging plasmids were then transfected into 293T cells by calcium phosphate method with encapsulated plasmid VSVG. The lentivirus was collected and infected with AGS cells. The expression of YWHAE protein in infected cells was detected by Puromycine screening. The point mutation primers of siRNA were designed by using the lentivirus expression plasmid with the highest interference efficiency. The point mutant YWHAE gene was amplified by overlapping extension PCR and cloned into the pcDNA3.1/myc-His(-)A vector to construct the point mutation expression plasmid of YWHAE. Western-blot was used to detect the expression of YWHAE protein in transfected AGS cells. The effect of YWHAEshRNA lentivirus on the proliferation of AGS cells was detected by MTS assay. Results 5 lentiviruses packaged with lentivirus pL3.7-siYWHAE-5 targeting the shRNA sequence of human YWHAE gene had the most obvious effect on inhibiting the expression of YWHAE protein in AGS cells, only 0.269 鹵0.083 times of the relative expression of pLL3.7-siYWHAE-5 in the control group. Its interference effect can be recovered by YWHAE point mutation expression plasmid. Compared with the control group, the proliferation ability of AGS cells in YWHAE-shRNA group was significantly decreased. Conclusion the expression plasmid of shRNA lentivirus targeting YWHAE gene was successfully constructed and the AGS cell line with significantly down-regulated expression of YWHAE gene was obtained. It was found that the down-regulation of YWHAE expression could effectively inhibit the proliferation of AGS cells and suggest the carcinogenic potential of YWHAE in gastric cancer.
【作者單位】： 福建醫(yī)科大學(xué)消化道惡性腫瘤教育部重點(diǎn)實(shí)驗(yàn)室福建省腫瘤微生物重點(diǎn)實(shí)驗(yàn)室;
【基金】：國家自然科學(xué)基金(81271784) 福建省教育廳項(xiàng)目(JA12150) 福建醫(yī)科大學(xué)重大科研項(xiàng)目基金(09ZD018);福建醫(yī)科大學(xué)苗圃科研基金(2010MP029)
【分類號(hào)】：R735.2
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本文編號(hào)：1802995