【摘要】：目的：本研究采用siRNA技術(shù),構(gòu)建pSUPER-siCD46重組質(zhì)粒,與本室保存的pSUPER-siCD59重組質(zhì)粒,觀測(cè)CD46和CD59特異性沉默對(duì)T細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)的影響,旨在探討CD59與CD46在T細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)中的協(xié)同效應(yīng)。 方法：構(gòu)建pSUPER-siCD46重組質(zhì)粒,利用PCR、質(zhì)粒雙酶切及DNA測(cè)序?qū)ζ溥M(jìn)行鑒定。應(yīng)用陽離子脂質(zhì)體法轉(zhuǎn)染Jurkat細(xì)胞,將細(xì)胞分為未轉(zhuǎn)染的Jurkat細(xì)胞(Ⅰ組)、轉(zhuǎn)染pSUPER空質(zhì)粒Jurkat細(xì)胞(Ⅱ組)、轉(zhuǎn)染pSUPER-siCD59重組質(zhì)粒Jurkat細(xì)胞(Ⅲ組)和轉(zhuǎn)染pSUPER-siCD46重組質(zhì)粒Jurkat細(xì)胞(Ⅳ組),G418篩選穩(wěn)定表達(dá)的細(xì)胞克隆。采用RT-PCR、 Westernblot檢測(cè)各組細(xì)胞中CD46和CD59基因的表達(dá)。應(yīng)用CD59、CD46的單克隆抗體聯(lián)合作用于各組Jurkat細(xì)胞,MTT法測(cè)細(xì)胞增殖、Western Blot測(cè)ZAP-70磷酸化的水平。結(jié)果：經(jīng)PCR、限制性內(nèi)切酶雙酶切及測(cè)序鑒定,結(jié)果表明重組質(zhì)粒序列插入正確。重組質(zhì)粒pSUPER-siCD59和pSUPER-siCD46分別轉(zhuǎn)染的Jurkat細(xì)胞,可表達(dá)綠色熒光蛋白,經(jīng)G418篩選,得到陽性細(xì)胞克��；RT-PCR結(jié)果顯示：Ⅲ組CD59mRNA和Ⅳ組CD46mRNA的表達(dá)均被明顯抑制,與Ⅰ、Ⅱ組相比較(P0.05),差異有顯著性。CD59mAb、CD46mAb聯(lián)合作用于T細(xì)胞,Ⅰ組細(xì)胞的增殖能力,ZAP-70磷酸化條帶的灰度值明顯高于Ⅲ、Ⅳ組,差異有顯著性(P0.05),,其中Ⅱ組低于Ⅲ組(P0.05),差異有顯著性,但Ⅰ、Ⅱ組之間比較(P0.05),差異無統(tǒng)計(jì)學(xué)意義。 結(jié)論：成功構(gòu)建了重組質(zhì)粒PSUPER-siCD46;分別建立了穩(wěn)定轉(zhuǎn)染重組質(zhì)粒pSUPER-siCD59、 pSUPER-siCD46的Jurkat細(xì)胞真核表達(dá)系統(tǒng)；RT-PCR實(shí)驗(yàn)從mRNA水平證明重組質(zhì)粒PSUPER-siCD59和pSUPER-siCD46可分別特異性地沉默CD59基因和CD46基因；通過MTT、 Western Blot實(shí)驗(yàn)證實(shí),在CD59與CD46的共同作用下,Jurkat細(xì)胞的增殖、ZAP-70的磷酸化顯著增強(qiáng),由此證明CD59、CD46在T細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)過程中存在協(xié)同作用。該研究創(chuàng)新之處在于,為跨膜蛋白CD46在GPI錨固蛋白CD59的T細(xì)胞跨膜信號(hào)轉(zhuǎn)導(dǎo)中的作用提供了實(shí)驗(yàn)依據(jù),為T細(xì)胞白血病的基因靶向治療開辟了一條新的途徑,具有重要的理論意義及廣闊的臨床應(yīng)用前景。
[Abstract]:Aim: to construct pSUPER-siCD46 recombinant plasmid by using siRNA technique, and to observe the effect of CD46 and CD59 specific silencing on T cell signal transduction. The purpose of this study was to investigate the synergistic effect of CD59 and CD46 in T cell signal transduction. Methods: pSUPER-siCD46 recombinant plasmid was constructed and identified by double digestion of PCR, plasmid and DNA sequencing. Jurkat cells were transfected with cationic liposome. The cells were divided into untransfected Jurkat cells (group 鈪，

本文編號(hào)：2391023