發(fā)布時(shí)間：2018-03-09 19:07

  本文選題：Argonaute　切入點(diǎn)：2　出處：《重慶醫(yī)科大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：目的: 我們在前期研究中通過sertoli細(xì)胞與生精細(xì)胞共培養(yǎng)發(fā)現(xiàn)Argonaute 2基因可能與血睪屏障相關(guān)基因claudin-11表達(dá)有關(guān)。為了深入研究相關(guān)機(jī)制,本課題設(shè)計(jì)構(gòu)建Ago2基因miRNA RNAi慢病毒表達(dá)載體與pEGFP-C1-Ago2表達(dá)載體,為研究其對血睪屏障相關(guān)基因claudin-11表達(dá)的影響奠定基礎(chǔ)。 方法: 提取小鼠睪丸組織中總RNA,采用RT-PCR的方法擴(kuò)增Ago2基因編碼區(qū)全長,擴(kuò)增產(chǎn)物克隆入pEGFP-C1載體EcoRI、Sa1I位點(diǎn),獲得重組表達(dá)質(zhì)粒,并對重組質(zhì)粒進(jìn)行PCR、酶切及測序鑒定。應(yīng)用LipofectamineTM 2000將重組質(zhì)粒轉(zhuǎn)染15P-1細(xì)胞,并通過RT-PCR及Western blotting法分別檢測Ago2基因及血睪屏障相關(guān)基因claudin-11基因和蛋白表達(dá)水平。以Ago2為靶向基因,利用www.rnaidesigner.invitrogen.com/rna1iexpress在線設(shè)計(jì)網(wǎng)站設(shè)計(jì)2對Ago2 miR RNAi序列,并將其克隆至pcDNA?6.2-GW/EmGFP-miR載體中,經(jīng)測序驗(yàn)證后,用Lipofectamine? 2000瞬時(shí)轉(zhuǎn)染15P-1細(xì)胞,通過RT-PCR法檢測Ago2表達(dá)情況,選擇干擾效果較強(qiáng)的質(zhì)�？寺≈罛LOCK-iTTM HiPerformTM Lentiviral PolⅡmiR RNAi Expression System,經(jīng)過氨芐青霉素和氯霉素篩選后,轉(zhuǎn)染293FT細(xì)胞中包裝慢病毒,轉(zhuǎn)染15P-1支持細(xì)胞,殺稻瘟菌素篩選獲得穩(wěn)定轉(zhuǎn)染后,通過RT-PCR及Western blotting法分別檢測Ago2及血睪屏障相關(guān)基因claudin-11基因和蛋白表達(dá)水平。 結(jié)果: 1.成功構(gòu)建了pEGFP-C1-Ago2表達(dá)載體,可有效上調(diào)15P-1 sertoli細(xì)胞Ago2基因表達(dá);成功構(gòu)建了Ago2基因miRNA RNAi慢病毒表達(dá)載體,通過殺稻瘟菌素篩選,獲得15P-1 Ago2-miRNA-RNAi穩(wěn)定株,可顯著下調(diào)Ago2基因表達(dá)。 2.RT-PCR與western blotting結(jié)果顯示,轉(zhuǎn)染pEGFP-C1-Ago2表達(dá)載體上調(diào)claudin-11基因表達(dá)。相反,轉(zhuǎn)染Ago2-miRNA RNAi慢病毒下調(diào)claudin-11基因表達(dá)。 結(jié)論: 1.成功構(gòu)建了Ago2基因pEGFP-C1-Ago2表達(dá)載體與miRNA RNAi慢病毒表達(dá)載體,為進(jìn)一步研究Ago2基因功能奠定了基礎(chǔ)。 2.Ago2基因正性調(diào)節(jié)claudin-11表達(dá)。
[Abstract]:Objective:. In our previous study, we found that the Argonaute 2 gene may be related to the expression of the blood-testis barrier gene claudin-11 through co-culture of sertoli cells and spermatogenic cells. In this study, we designed and constructed miRNA RNAi lentivirus expression vector and pEGFP-C1-Ago2 expression vector of Ago2 gene, which laid a foundation for the study of its effect on claudin-11 expression of blood-testis barrier related gene. Methods:. Total RNAs were extracted from mouse testis. The full length of Ago2 gene coding region was amplified by RT-PCR. The amplified product was cloned into the pEGFP-C1 vector Ecor RIN Sa1I site, and the recombinant expression plasmid was obtained. LipofectamineTM 2000 was used to transfect the recombinant plasmid into 15P-1 cells. The expression levels of Ago2 gene and claudin-11 gene and protein related to blood testis barrier were detected by RT-PCR and Western blotting, respectively. Ago2 was used as target gene. Design 2 pairs of Ago2 miR RNAi sequences using www.rnaidesigner.invitrogen.com/rna1iexpress online design site and clone them into pcDNAs? In 6.2-GW / EmGFP-miR vector, after sequencing, Lipofectamine? After transient transfection of 15P-1 cells in 2000, the expression of Ago2 was detected by RT-PCR method. The plasmid with strong interfering effect was cloned into BLOCK-iTTM HiPerformTM Lentiviral Pol 鈪，

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