H2-Bl基因转染对小鼠血管平滑肌细胞生物学行为的影响

Effects of H2-Bl gene transfection on biological behaviour of mouse vascular smooth muscle cell

目的:H2-Bl基因转染小鼠血管平滑肌细胞(VSMC),观察其凋亡、增殖和抗外周血单个核细胞(PBMC)杀伤性等生物学行为的变化,探讨借鉴母胎免疫耐受模型防治心脏移植术后免疫排斥反应的机制。方法:0.5mg/L空质粒、0.5mg/LH2-Bl质粒、1.0mg/LH2-Bl质粒转染小鼠血管平滑肌细胞后,采用实时荧光定量PCR、流式细胞术、酶标仪检测不同时间质粒的转染效率、H2-Bl基因mRNA的表达量、转染VSMC的凋亡与增殖情况以及PBMC对靶细胞的毒性作用。结果:荧光定量PCR结果显示,基因组的H2-Bl基因mRNA表达量明显高于对照组(P<0.001)。H2-Bl基因促进VSMC凋亡,转染24h后0.5mg/LH2-Bl质粒组、1.0mg/LH2-Bl质粒组与空白对照比较,差异均有统计学意义(P<0.05,P<0.01)。H2-Bl基因抑制VSMC增殖,转染24h和48h后0.5mg/LH2-Bl质粒组、1.0mg/LH2-Bl质粒组与对照组比较差异均有统计学意义(P<0.05,P<0.01)。细胞毒性试验在转染24h后,0.5mg/LH2-Bl质粒组、1.0mg/LH2-Bl质粒组细胞毒性均较空白对照明显降低(P<0.05,P<0.01)。结论:pEGFP-N1-H2B1质粒载体转染小鼠VSMC后,能够有效促进其凋亡,抑制其增殖,降低PBMC对靶细胞的毒性作用,诱导免疫耐受。

AIM： To investigate the effects of H2-Bl gene on biological behaviour of mouse vascular smooth muscle cells （VSMCs） including apoptosis, proliferation and anti-cytolysis to peripheral blood mononuclear cells （PBMCs）, then speculate the possible mechanism of immunological rejection after heart transplantation in maternalfetal immune tolerance. METHODS： The mouse VSMC were transfected with 0.5 mg/L of pEGFP-N1 plasmid vector, 0.5 mg/L of pEGFP-N1-H2B1 plasmid vector and 1.0 mg/L of pEGFP-NI-H2B1 plasmid vector, respectively. The efficiency of transfection was detected, and the H2-B1 mRNA level, the apoptosis, the proliferation and cytotoxicity of VSMCs were also investigated at 24 h, 48 h and 72 h, respectively. RESULTS： Expression of the H2-BI gene was determined by real time quantitative PCR. Expressions of the H2-B1 in experimental groups were higher than that of the control group （ P 〈 0. 001 ）. VSMC apoptosis was observed after 24 h in the 0.5 mg/L and 1.0 mg/L H2-B1 experimental groups compared with control group （P 〈 0.05, P 〈 0.001, respectively）. VSMC proliferation was also inhibited in the 0. ,5 mg/L and 1. 0 mg/L H2-B1 gene groups at 24 h and 72 h （P〈0.05, P〈0.0I, respectively）. The cytotoxicity of PBMC in the 0.5 mg/L and 1.0 mg/L H2-B1 gene groups at 24 h was lower than the control group （ P 〈 0.005,P 〈 0. OOt, respectively）. CONCLUSION： Transfection of pEGFP-N1-H2B1 plasmid to mouse VSMC causes the over expression of H2-B1 mRNA, induces the apoptosis, inhibits the proliferation and attenuates the cytotoxicity of PBMC, leading to immune tolerance.