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目的:通过同源重组构建CTLA4Ig腺病毒载体,研究其生物学活性,并用于基因治疗诱导心脏移植耐受。方法:通过基因重组技术,将CTLA4Ig基因克隆至腺病毒穿梭质粒pCA13中。然后将该质粒与腺病毒辅助质粒同源重组,经过293细胞包装,构建CTLA4Ig腺病毒载体。用RT-PcR、SDS-PAGE及Western blot等技术,检测包装的病毒感染293细胞后CTLA4Ig蛋白的表达及分泌,通过体外实验,观察病毒感染的293细胞上清对混合淋巴细胞反应(MLR)的抑制作用。通过大鼠体内实验,检测用CTLA4Ig腺病毒载体基因治疗后,CTLA4Ig在体内的表达。结果:CTLA4Ig腺病毒载体构建成功。体外实验证实,CTLA4Ig腺病毒感染的293细胞上清液,能够抑制异基因脾细胞的单向MLR。体内实验表明,经过静脉途径给予受体大鼠CTLA4Ig腺病毒载体,能够诱导移植耐受,延长心脏移植物的存活。结论:构建的CTLA4Ig腺病毒载体,体外感染293细胞能够分泌CTLA4Ig蛋白。该蛋白可抑制T细胞的活化,CTLA4Ig腺病毒载体可用于体内基因治疗诱导移植耐受。
OBJECTIVE: To construct CTLA4Ig adenovirus vector by homologous recombination and study its biological activity, and to use it in gene therapy to induce cardiac transplantation tolerance. Methods: CTLA4Ig gene was cloned into adenoviral shuttle plasmid pCA13 by gene recombination technology. The plasmid was then homologously recombined with the adenovirus helper plasmid and packaged in 293 cells to construct CTLA4Ig adenoviral vector. The expression and secretion of CTLA4Ig protein were detected by RT-PCR, SDS-PAGE and Western blot. The effect of virus-infected 293 cells supernatant on mixed lymphocyte reaction (MLR) Inhibition. In vivo experiments in rats, CTLA4Ig adenovirus vector gene therapy, CTLA4Ig expression in vivo. Results: CTLA4Ig adenovirus vector was constructed successfully. In vitro experiments confirmed that, CTLA4Ig adenovirus-infected 293 cell supernatant, allogeneic spleen cells can inhibit one-way MLR. In vivo experiments show that, after intravenous administration of the recipient rat CTLA4Ig adenovirus vector, can induce graft tolerance, prolong cardiac graft survival. Conclusion: CTLA4Ig adenovirus vector constructed in vitro can infect 293 cells to secrete CTLA4Ig protein. The protein inhibits T cell activation and the CTLA4Ig adenoviral vector can be used for in vivo gene therapy to induce transplant tolerance.