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目的:针对同种异体细胞移植的免疫反应主要由HLA-Ⅱ类抗原介导,而CⅡTA基因对HLA-Ⅱ分子的表达起严格且专一性的调控作用,拟通过CⅡTA的反义cDNA抑制细胞表面HLAⅡ分子的表达。方法:以RT-PCR从HLAⅡ抗原组成型表达的Raji细胞株克隆CⅡTA反义片段(arⅡ),插入腺相关病毒载体psNAV中(pAarⅡ)。将该质粒通过脂质体稳定转染Heda细胞(pAar Ⅱ H),检测其表面HLAⅡ分子表达,并以RT-PCR检测其CⅡTA、HLA-Ⅱ(DR、DP及DQ)及Ii分子的mRNA水平。结果:pAarⅡH在重组人IFN-γ诱导下,DR、DP及DQ抗原表达分别降低了79±12%、90±15%及40±8%;同时HLA-Ⅱ及Ii分子的mRNA被明显抑制。结论:提示CⅡTA反义基因片段抑制了自身组成型及诱导型表达量,并相应阻止了CⅡTA所调控的基因(HLA-Ⅱ及Ii)的表达,从而为进一步探讨免疫耐受形成及其在组织工程中的应用奠定了基础。
OBJECTIVE: The immune response to allogeneic cell transplantation is mainly mediated by HLA-Ⅱ antigens, while CⅡTA gene plays a strict and specific regulatory role on the expression of HLA-Ⅱ molecules. The anti-sense cDNA of CⅡTA suppresses cells Surface HLA Ⅱ molecule expression. Methods: The C Ⅱ TA antisense fragment (arⅡ) was cloned from Raji cells constitutively expressed by HLA Ⅱ antigen by RT-PCR and inserted into the adeno-associated virus vector psNAV (pAarⅡ). The plasmid was stably transfected into Heda cells (pAar Ⅱ H) by liposome, and the expression of HLA Ⅱ molecules on the surface of Heda cells was detected by RT-PCR. The mRNA levels of CⅡTA, HLA-Ⅱ (DR, DP and DQ) and Ii . Results: The expression of DR, DP and DQ antigen in pAarⅡH was reduced by 79 ± 12%, 90 ± 15% and 40 ± 8% respectively under the induction of recombinant human IFN-γ. At the same time, mRNA of HLA-Ⅱ and Ii was significantly inhibited. CONCLUSION: The antisense gene fragment of CⅡTA inhibits the constitutive and inducible expression of CⅡTA, and accordingly blocks the expression of CⅡTA-regulated genes (HLA-Ⅱ and Ii), so as to further explore the formation of immune tolerance and its role in tissue Engineering application laid the foundation.