發(fā)布時間：2018-11-22 09:31

【摘要】：目的構(gòu)建熱激啟動子(p HSP)驅(qū)動的靶向敲低Polo樣蛋白激酶1(PLK1)的真核表達(dá)質(zhì)粒,并觀察其對骨肉瘤U2OS細(xì)胞增殖的影響。方法首先合成熱休克蛋白HSP70的啟動子,克隆至帶有綠色熒光蛋白(GFP)標(biāo)簽的真核表達(dá)質(zhì)粒p EGFP-C1中,利用p HSP替換原有的CMV啟動子,構(gòu)建真核表達(dá)載體p HSP-GFP;其次,利用RNA干擾技術(shù)合成以plk1基因為靶向目標(biāo)的sh RNA,將其定向克隆至真核表達(dá)載體p HSP-GFP中形成重組質(zhì)粒p HSP-sh PLK1-GFP;另外設(shè)計一組陰性對照質(zhì)粒p HSP-NC-GFP,最后,利用脂質(zhì)體Lipo2000轉(zhuǎn)染的方法將質(zhì)粒轉(zhuǎn)染至U2OS細(xì)胞,非熱激組細(xì)胞正常培養(yǎng),熱激組及陰性對照組細(xì)胞42℃加熱2 h,通過細(xì)胞免疫熒光染色實驗檢測GFP的表達(dá),從而確定p HSP的驅(qū)動能力,采用Real-time PCR法檢測細(xì)胞內(nèi)plk1的m RNA表達(dá)水平,Western blot法檢測PLK1蛋白表達(dá)水平,MTT法檢測重組質(zhì)粒對U2OS細(xì)胞增殖的影響。結(jié)果成功構(gòu)建了p HSP驅(qū)動的真核表達(dá)質(zhì)粒p HSP-sh PLK1-GFP;細(xì)胞免疫熒光染色實驗結(jié)果顯示p HSP可正常驅(qū)動gfp基因的表達(dá),且GFP蛋白定位在胞質(zhì)中;Real-time PCR結(jié)果顯示熱激組細(xì)胞plk1基因表達(dá)量明顯降低,與非熱激組及陰性對照組相比差異有統(tǒng)計學(xué)意義(P0.05);Western blot結(jié)果顯示熱激組細(xì)胞PLK1蛋白表達(dá)量比非熱激組及陰性對照組低(P0.05);MTT結(jié)果顯示熱激組細(xì)胞的增殖活性被明顯抑制,與非熱激組及陰性對照組相比差異有統(tǒng)計學(xué)意義(P0.05)。結(jié)論 p HSP驅(qū)動的真核表達(dá)質(zhì)粒p HSP-sh PLK1-GFP能在U2OS細(xì)胞中表達(dá),在42℃加熱條件下,該重組質(zhì)粒呈現(xiàn)更強(qiáng)的下調(diào)plk1基因表達(dá)和抑制增殖作用。
[Abstract]:Objective to construct the eukaryotic expression plasmid of Polo like protein kinase 1 (PLK1) driven by heat shock promoter (p HSP) and to observe its effect on the proliferation of U2OS cells in osteosarcoma. Methods the promoter of heat shock protein HSP70 was synthesized and cloned into the eukaryotic expression plasmid p EGFP-C1 with green fluorescent protein (GFP) tag. The original CMV promoter was replaced by p HSP, and the eukaryotic expression vector p HSP-GFP; was constructed. Secondly, sh RNA, targeting plk1 was synthesized by RNA interference technique and cloned into eukaryotic expression vector p HSP-GFP to form recombinant plasmid p HSP-sh PLK1-GFP;. In addition, a group of negative control plasmids p HSP-NC-GFP, were designed. Finally, the plasmids were transfected into U2OS cells by liposome Lipo2000 transfection. The cells in non-heat shock group were cultured normally, and the cells in heat shock group and negative control group were heated at 42 鈩，

本文編號：2348876