抗内皮细胞表面主要组织相容性复合物Ⅱ类分子转录激活因子锤头状核酶的克隆及活性表达

The biological activity of anti-CⅡTA hammerhead ribozyme: A novel approach for graft vascular disease treatment

目的 :拟克隆抗内皮细胞表面主要组织相容性复合物Ⅱ类分子转录激活因子 (CⅡTA)锤头状核酶 ,抑制血管内皮细胞表面主要组织相容性复合物 (MHC Ⅱ )分子的表达。方法 :设计并合成针对人类CⅡTA第 134、2 18、4 6 4位点的一组锤头状核酶 (Rz134、Rz2 18、Rz4 6 4 ) ,及其对应的CⅡTA靶基因m5 2 3。核酶体外切割实验鉴定Rz4 6 4活性最高 ,将Rz4 6 4克隆到真核表达载体pIRES2 EGFP(pIRES2 Rz4 6 4 ) ,并通过纳米载体介导其稳定转染人类脐静脉内皮细胞系 ,流式细胞术检测HLA DR、DP、DQ类抗原表达 ,RT PCR检测CⅡTAmRNA水平。结果 :pIRES2 Rz4 6 4阳性内皮细胞与对照组比较 ,HLA DR、DP、DQ抗原表达分别降低了 76 .2 6 %、87.75 %及 78.99% ,同时CⅡTAmRNA含量亦下降了 6 9%。结论 :Rz4 6 4抑制了CⅡTAmRNA含量 。

Objective:To investigate the inhibiting effect of ribozymes against MHCⅡ transactivator ( CⅡTA) on the expression of MHCⅡ on vascular endothelium.Methods:Three hammerhead ribozymes recognizing the 134,218,464 site of CⅡTA respectively were synthesized(Rz134,Rz218,Rz464). CⅡTA target gene(m523) was obtained from Raji cell by RT-PCR. Rz134,Rz218,Rz464 and their target RNA m523 were transcribed and then incubated in cell-free conditions. It showed that Rz464 could exclusively cleave target RNA, then it was cloned into the pIRES2-EGFP vector (pIRES2-Rz464, pRz464). Stable transfectants of umbilical venous endothelial cell line with pRz464 were analyzed for classic MHCⅡ(HLA-DR, DP, DQ) expression by flow cytometry. mRNA of CⅡTA was detected by RT-PCR.Results:The expression of HLA-DR,-DP ,-DQ on pRz464 positive endothelium was reduced 76.26%, 87.75% and 78.99% respectively. So did the mRNA contents of CⅡTA (69%).Conclusion:pRz464 inhibit CⅡTA and thus the family of MHCⅡmolecules are regulated by CⅡTA.