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目的:构建含人白介素10(hIL-10)的腺相关病毒血清型2/1杂合载体(AAV2/1),并观察其在供肝移植物中的表达.方法:采用RT-PCR方法从人外周血淋巴细胞中扩增hIL-10cDNA,将其克隆对pMD18-T载体中,测序后,再经酶切定向插入真核表达载体pSNAV,用脂质体介导转染BHK21细胞,经G418筛选得到含ITR—hIL-10-ITR的BHK21细胞株,用携带rep2-cap1基因的辅助病毒(rHSV/r2c1)感染该细胞株.通过细胞孵育、裂解和病毒纯化,得到含hIL-10的AAV2/1载体.在供肝灌洗、保存中应用该重组病毒载体转染供肝移植物,移植后检测其在移植物中的表达.结果:RT-PCR扩增的hIL-10cDNA序列与GenBank中hIL-10cDNA序列一致,构建了rAAV2/1-hIL-10表达载体.转染供肝移植物后可检测到hIL-10,持续24wk表达.移植后24wk,实验组供肝中hIL-10的表达比空白对照组和rAAV2/1-GFP对照组显著增高(219.15±45.83ng/Lvs40.02,38.64ng/L,P<0.05).结论:成功构建了rAAV2/1-hIL-10表达载体系统,并在大鼠供肝中持续表达hIL-10,为IL-10在器官移植中的生物学作用机制及临床应用的深入研究奠定基础.
OBJECTIVE: To construct AAV2 / 1 adenovirus vector containing human interleukin 10 (hIL-10) and observe its expression in donor liver grafts.Methods: RT-PCR was used to detect the expression of hAV- The hIL-10 cDNA was amplified from human peripheral blood lymphocytes and cloned into pMD18-T vector. After sequencing, the recombinant plasmid pSNAV was inserted into the eukaryotic expression vector pSNAV by lipofectamine. BHK21 cells were transfected by lipofectamine and transfected into BHK21 cells by G418 The BHK21 cell line with ITR-hIL-10-ITR was screened and infected with the helper virus carrying the rep2-cap1 gene (rHSV / r2c1) .The cell lines were incubated, lysed and purified by virus to obtain hIL-10 containing AAV2 / 1 vector.The donor liver graft was transfected with the recombinant virus vector during the lavage and preservation of the liver and the expression of the hIL-10 cDNA in the graft was detected after transplantation.Results: The hIL-10 cDNA sequence was consistent and the rAAV2 / 1-hIL-10 expression vector was constructed.The expression of hIL-10 in donor liver was detected 24 h after transplantation (219.15 ± 45.83ng / L vs 40.02, 38.64ng / L, P <0.05) .Conclusion: The rAAV2 / 1-hIL-10 Expression vector system, and persistent expression in rat liver hIL-10, lay the foundation for further study of the biological mechanisms of action and clinical application of IL-10 in organ transplantation.