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目的 针对同种异体细胞移植的免疫反应主要是由主要组织相容性复合物Ⅱ (majorhistocompatibilitycomplex ,MHC Ⅱ ,人类中亦称HLA Ⅱ )类抗原介导 ,而MHCⅡ类分子转录激活因子 (MHCclassⅡtransactivator ,CⅡTA)对MHCⅡ分子的表达起严格且专一性的调控作用 ,拟通过抗CⅡTA锤头状核酶 (ribozyme,Rz)抑制细胞表面MHCⅡ分子的表达。 方法 设计并合成针对人类CⅡTA基因第 134、2 18、4 6 4位点的一组核酶 ,分别命名为Rz134、Rz2 18、Rz4 6 4。通过体外制备和活性鉴定 ,筛选出活性较高的核酶———Rz4 6 4。将Rz4 6 4克隆到含有核糖体内进入位点及增强型绿色荧光蛋白的表达载体 (internalribosomeentrysite enhancedgreenfluorescentprotein ,pIRES2 EGFP) ,简称为 pRz4 6 4 ,并稳定转染Jukart细胞株 (pRz4 6 4 J) ,流式细胞术检测经典的MHCⅡ (HLA DR、DP、DQ)抗原表达 ,逆转录 聚合酶链反应 (RT PCR)检测CⅡTAmRNA水平。结果 pRz4 6 4 J与无关核酶组比较 ,HLA DR、DP、DQ抗原诱导型表达分别降低了 73 2 7%、88 93%、5 8 82 % ;同时CⅡTA的诱导性mRNA含量明显减少 (P <0 0 1)。结论 抗CⅡTA锤头状核酶可抑制CⅡ ATmRNA的表达 ,从而阻止其调控的相应基因———MHCⅡ分子的表达 ,为进一步探讨免疫?
Objective The immune response induced by allogeneic cell transplantation is mainly mediated by major histocompatibility complex Ⅱ (MHC Ⅱ, human also known as HLA Ⅱ) antigen, and MHC class Ⅱ transcription activator (MHC class Ⅱ transactivator, C Ⅱ TA ) Strictly and specifically regulate the expression of MHC II molecules, and it is proposed that the expression of MHC II molecules on the cell surface is inhibited by ribozyme (Rz) against CⅡTA. Methods A series of ribozymes targeting at positions 134, 2 18 and 4 6 4 of human C Ⅱ TA gene were designed and synthesized, which were named Rz134, Rz2 18 and Rz4 6 4, respectively. Through in vitro preparation and activity identification, the ribozyme Rz4 6 4 with high activity was screened out. Rz4 6 4 was cloned into the expression vector pRES4 EGFP (pIRES2 EGFP) containing ribosomal entry sites and enhanced green fluorescent protein (pIRES2 EGFP) for short, and was stably transfected into the Jukart cell line (pRz4 6 4 J) The classical MHCⅡ (HLA DR, DP, DQ) antigen expression was detected by cytometry, and the level of CⅡTA mRNA was detected by reverse transcription polymerase chain reaction (RT PCR). Results Compared with the unrelated ribozyme group, the induced expression of HLA DR, DP and DQ antigen was decreased by 73.27%, 88.93% and 58.282%, respectively. Meanwhile, the induced mRNA level of CⅡTA was significantly decreased (P <0 0 1). Conclusion Anti-CⅡTA hammerhead ribozyme can inhibit the expression of CⅡ AT mRNA, thus preventing the expression of MHCⅡ gene, which is its regulated gene.