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目的 研究抗组织相容性复合物(MHC)Ⅱ类分子转录激活因子(CⅡTA)核糖核酸酶P(RNaseP)抑制肝细胞表面MHC Ⅱ分子的表达。方法 M1—RNA是RNaseP的催化活性单位,设计并克隆针对C Ⅱ TA第629位点的M1-RNA(M1—629—GS)及其对应的C Ⅱ TA靶序列,分别插入腺相关病毒载体psNAV(psNAV—M1—629-GS)及pGEM—7zf(+)载体(pGEM—629),进行体外转录和切割反应鉴定其活性。通过纳米载体介导psNAV—M1—629—GS稳定转染人胎肝细胞,流式细胞术检测MHC Ⅱ类抗原表达,逆转录聚合酶链反应检测CⅡTA mRNA水平。结果 M1—629—GS与pGEM—800体外切割产物电泳见预期切割条带(553 nt和176 nt)。psNAV—M1—629—GS~+肝细胞表面人白细胞抗原(HLA)—DR、DP、DQ的诱导型表达分别为(1.01±0.51)％、(4.37±1.28)％、(1.98±0.42)％,对照组psNAV-M1—452-GS~+分别为(10.81±3.09)％、(40.12±2.60)％、(5.71±0.11)％,两组比较分别下调90.65％、89.11％及65.32％;psNAV—M1-629一GS~+克隆诱导型CⅡTA mRNA与β-actin比值为0.94±0.25,空载体组比值为2.30±0.49,M1—629—GS可明显下调C Ⅱ TA mRNA含量(t=5.56,P<0.01)。结论 抗C Ⅱ TA RNasep-M1—629—GS可发展为新一代抗肝移植排斥的核酸药物。
Objective To study the inhibitory effect of RNase P (MHC Ⅱ) on the expression of MHC Ⅱ on the surface of hepatocytes. Methods M1-RNA was the catalytic unit of RNaseP. M1-RNA (M1-629-GS) and its corresponding C Ⅱ TA target sequence at the 629th site of C Ⅱ TA were designed and cloned, and inserted into adeno-associated virus vector psNAV (psNAV-M1-629-GS) and pGEM-7zf (+) vector (pGEM-629). The in vitro transcription and cleavage reactions were used to identify the activity. Human fetal hepatocytes were stably transfected with psNAV-M1-629-GS through nanocarriers. The expression of MHC class II antigen was detected by flow cytometry. The level of CⅡTA mRNA was detected by reverse transcription-polymerase chain reaction. Results The expected cleavage bands of M1-629-GS and pGEM-800 in vitro cleavage products (553 nt and 176 nt) were observed. The induced expressions of HLA-DR, DP and DQ on psNAV-M1-629-GS ~ + hepatocytes were (1.01 ± 0.51)%, (4.37 ± 1.28)% and (1.98 ± 0.42)% (10.81 ± 3.09)%, (40.12 ± 2.60)% and (5.71 ± 0.11)% respectively in the control group, and were decreased by 90.65%, 89.11% and 65.32% respectively in the control group. The levels of psNAV-M1-452- The ratio of C Ⅱ TA mRNA to β-actin in M1-629-GS1 + clone was 0.94 ± 0.25, and the ratio of empty vector group was 2.30 ± 0.49. M1-629-GS significantly decreased the content of C Ⅱ TA mRNA (t = 5.56, P <0.01). Conclusion Anti-C Ⅱ TA RNasep-M1-629-GS can be developed as a new generation of nucleic acid drugs against liver transplantation rejection.