慢病毒介导的RNAi抑制小鼠血管内皮细胞α1，3GT表达的研究

发布时间：2018-05-12 22:48

本文选题：慢病毒 + RNA干扰　；参考：《天津医科大学》2008年硕士论文

【摘要】： 目的:应用RNAi(RNA interference)技术探讨靶向α1,3半乳糖基转移酶(α1,3GT)的shRNA重组慢病毒载体抑制小鼠血管内皮细胞α1,3GT和α1,3半乳糖残基Galα(1,3)Gal表达的可行性。方法:采用组织植块法体外培养小鼠血管内皮细胞;经体外设计合成针对α1,3GTmRNA的序列特异性shRNA,并构建携带α1,3GT shRNA的重组慢病毒载体,以慢病毒感染小鼠血管瘤内皮细胞系EOMA;荧光实时定量PCR检测转染后α1,3GT mRNA表达水平的变化,免疫荧光检测异种抗原Galα(1,3)Gal表达水平的变化;使用SPSS 13.0 for Windows进行数据分析。结果:体外组织植块培养可获得纯度较高的小鼠血管内皮细胞;成功构建了重组慢病毒载体质粒。经病毒包装后,得到了携带α1,3GT-shRNA的成熟的重组慢病毒颗粒;荧光实时定量PCR检测显示构建的重组慢病毒转染EOMA,能抑制α1,3GT的表达,抑制率约为88%(P＜0.01)。空病毒载体对照组、阴性siRNA对照组与未处理细胞组的mRNA转录水平无显著性差异(P＞0.05);免疫荧光检测显示α1,3GT-shRNA重组慢病毒转染后细胞Galα(1,3)Gal抗原水平较对照组明显降低(P＜0.01),空病毒载体对照组、阴性siRNA对照组与未处理细胞组间的Galα(1,3)Gal抗原表达无显著性差异(P＞0.05)。结论:通过组织植块法可实现小鼠血管内皮细胞的原代培养并传代,但传代的次数有限;本实验成功构建了靶向α1,3GT基因的重组慢病毒载体,并利用该慢病毒载体成功地将α1,3GT-shRNA表达框架转导入EOMA细胞,使其持续表达,实现了靶向α1,3GT基因的RNA干扰。重组慢病毒载体有效地抑制了α1,3GTmRNA,并且抑制了其所催化的蛋白Galα(1,3)Gal的表达。通过实验,初步说明慢病毒载体介导的RNAi技术对沉默α1,3GT基因从而下调异种抗原Galα(1,3)Gal表达的策略是可行的。从而为进一步研究利用RNAi技术减轻异种移植超急性排斥反应提供了实验依据。
[Abstract]:Aim: to investigate the feasibility of 伪 1N 3 galactosyltransferase (伪 1N 3 GTV) targeted shRNA recombinant lentivirus vector in inhibiting the expression of 伪 1N 3GT and 伪 1N 3 galactose residues Gal 伪 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 伪 1t3GT mRNA were designed and synthesized in vitro, and a recombinant lentivirus vector carrying 伪 1t3GT shRNA was constructed. Murine hemangioma endothelial cell line Eoma was infected with lentivirus, the expression level of 伪 1t3GT mRNA after transfection was detected by real-time quantitative PCR, and the expression level of heterologous antigen Gal 伪 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 伪 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 伪 1GT-shRNA (P < 0.01) after transfection of the recombinant lentivirus into EOMA.The recombinant lentivirus could inhibit the expression of 伪 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 伪 -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 伪 1GT-shRNA expression frame into EOMA cells, and the expression of 伪 1GT-shRNA was continuously expressed. The interference of RNA targeting 伪 1GT-GT gene was realized. The recombinant lentivirus vector effectively inhibited the expression of 伪 1t3 GTmRNAs and the expression of the protein Gal 伪 1 Gal catalyzed by the recombinant lentivirus vector. The results showed that the lentivirus vector-mediated RNAi technique was feasible for silencing the 伪 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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R617;R346

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