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  本文選題：慢病毒 + RNA干擾��； 參考：《天津醫(yī)科大學(xué)》2008年碩士論文

【摘要】： 目的:應(yīng)用RNAi(RNA interference)技術(shù)探討靶向α1,3半乳糖基轉(zhuǎn)移酶(α1,3GT)的shRNA重組慢病毒載體抑制小鼠血管內(nèi)皮細(xì)胞α1,3GT和α1,3半乳糖殘基Galα(1,3)Gal表達(dá)的可行性。 方法:采用組織植塊法體外培養(yǎng)小鼠血管內(nèi)皮細(xì)胞;經(jīng)體外設(shè)計(jì)合成針對α1,3GTmRNA的序列特異性shRNA,并構(gòu)建攜帶α1,3GT shRNA的重組慢病毒載體,以慢病毒感染小鼠血管瘤內(nèi)皮細(xì)胞系EOMA;熒光實(shí)時定量PCR檢測轉(zhuǎn)染后α1,3GT mRNA表達(dá)水平的變化,免疫熒光檢測異種抗原Galα(1,3)Gal表達(dá)水平的變化;使用SPSS 13.0 for Windows進(jìn)行數(shù)據(jù)分析。 結(jié)果:體外組織植塊培養(yǎng)可獲得純度較高的小鼠血管內(nèi)皮細(xì)胞;成功構(gòu)建了重組慢病毒載體質(zhì)粒。經(jīng)病毒包裝后,得到了攜帶α1,3GT-shRNA的成熟的重組慢病毒顆粒;熒光實(shí)時定量PCR檢測顯示構(gòu)建的重組慢病毒轉(zhuǎn)染EOMA,能抑制α1,3GT的表達(dá),抑制率約為88%(P＜0.01)。空病毒載體對照組、陰性siRNA對照組與未處理細(xì)胞組的mRNA轉(zhuǎn)錄水平無顯著性差異(P＞0.05);免疫熒光檢測顯示α1,3GT-shRNA重組慢病毒轉(zhuǎn)染后細(xì)胞Galα(1,3)Gal抗原水平較對照組明顯降低(P＜0.01),空病毒載體對照組、陰性siRNA對照組與未處理細(xì)胞組間的Galα(1,3)Gal抗原表達(dá)無顯著性差異(P＞0.05)。 結(jié)論:通過組織植塊法可實(shí)現(xiàn)小鼠血管內(nèi)皮細(xì)胞的原代培養(yǎng)并傳代,但傳代的次數(shù)有限;本實(shí)驗(yàn)成功構(gòu)建了靶向α1,3GT基因的重組慢病毒載體,并利用該慢病毒載體成功地將α1,3GT-shRNA表達(dá)框架轉(zhuǎn)導(dǎo)入EOMA細(xì)胞,使其持續(xù)表達(dá),實(shí)現(xiàn)了靶向α1,3GT基因的RNA干擾。重組慢病毒載體有效地抑制了α1,3GTmRNA,并且抑制了其所催化的蛋白Galα(1,3)Gal的表達(dá)。通過實(shí)驗(yàn),初步說明慢病毒載體介導(dǎo)的RNAi技術(shù)對沉默α1,3GT基因從而下調(diào)異種抗原Galα(1,3)Gal表達(dá)的策略是可行的。從而為進(jìn)一步研究利用RNAi技術(shù)減輕異種移植超急性排斥反應(yīng)提供了實(shí)驗(yàn)依據(jù)。
[Abstract]:Aim: to investigate the feasibility of 偽 1N 3 galactosyltransferase (偽 1N 3 GTV) targeted shRNA recombinant lentivirus vector in inhibiting the expression of 偽 1N 3GT and 偽 1N 3 galactose residues Gal 偽 1 3G al in murine vascular endothelial cells (VECs) by RNAi(RNA interference technique. Methods: murine vascular endothelial cells were cultured in vitro by tissue grafting method, and sequence-specific shRNAs targeting 偽 1t3GT mRNA were designed and synthesized in vitro, and a recombinant lentivirus vector carrying 偽 1t3GT shRNA was constructed. Murine hemangioma endothelial cell line Eoma was infected with lentivirus, the expression level of 偽 1t3GT mRNA after transfection was detected by real-time quantitative PCR, and the expression level of heterologous antigen Gal 偽 1 tir 3G al was detected by immunofluorescence, and the data were analyzed by SPSS 13.0 for Windows. Results: high purity mouse vascular endothelial cells could be obtained by tissue graft culture in vitro, and recombinant lentivirus vector plasmid was successfully constructed. The recombinant lentivirus particles with 偽 1G T-shRNA were obtained by viral packaging, and the recombinant lentivirus was transfected into EOMA by real-time quantitative PCR assay, and the inhibition rate was about 88% (P < 0.01), and the recombinant lentivirus could inhibit the expression of 偽 1GT-shRNA (P < 0.01) after transfection of the recombinant lentivirus into EOMA.The recombinant lentivirus could inhibit the expression of 偽 1GT-shRNA. There was no significant difference in the level of mRNA transcription between the negative siRNA control group and the untreated cell group (P > 0.05), and the immunofluorescence assay showed that the level of the Gal 偽 -1GT-shRNA recombinant lentivirus-transfected lentivirus was significantly lower than that of the control group (P < 0.01), and the empty virus vector control group was significantly lower than that of the control group. There was no significant difference in the expression of Gal antigen between the negative siRNA control group and the untreated cell group (P > 0.05). Conclusion: the primary culture and passage of murine vascular endothelial cells can be achieved by tissue grafting method, but the number of passages is limited. The lentivirus vector was successfully used to transfer 偽 1GT-shRNA expression frame into EOMA cells, and the expression of 偽 1GT-shRNA was continuously expressed. The interference of RNA targeting 偽 1GT-GT gene was realized. The recombinant lentivirus vector effectively inhibited the expression of 偽 1t3 GTmRNAs and the expression of the protein Gal 偽 1 Gal catalyzed by the recombinant lentivirus vector. The results showed that the lentivirus vector-mediated RNAi technique was feasible for silencing the 偽 1t3GT gene and down-regulating the expression of the heterologous antigen Gal. The results provide experimental evidence for further study on the use of RNAi technique to alleviate hyperacute rejection in xenotransplantation.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R617;R346

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相關(guān)期刊論文 前1條

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本文編號：1880494