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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。
OBJECTIVE: To investigate the biological behavior of mouse vascular smooth muscle cells (VSMCs) transfected with H2-Bl gene and observe their biological behaviors such as apoptosis, proliferation and anti-PBMC cytotoxicity Mechanism of immune rejection after heart transplantation. Methods: After transfection of mouse vascular smooth muscle cells with 0.5mg / L empty plasmid, 0.5mg / LH2-B1 plasmid and 1.0mg / LH2-B1 plasmid, real-time fluorescence quantitative PCR, flow cytometry and microplate reader were used to detect Plasmid transfection efficiency, H2-Bl gene mRNA expression, apoptosis and proliferation of transfected VSMC and PBMC cytotoxicity on target cells. Results: Fluorescent quantitative PCR results showed that the mRNA expression of H2-B1 gene in the genome was significantly higher than that in the control group (P <0.001). H2-B1 gene could promote the apoptosis of VSMC, the difference was statistically significant (P <0.05, P <0.01) compared with the blank control group after 0.5mg / LH2-Bl plasmid group and 1.0mg / LH2- . The H2-B1 gene inhibited the proliferation of VSMC. The difference was statistically significant (P <0.05, P <0.01) between 0.5mg / LH2-B1 plasmid group and 1.0mg / LH2-B1 plasmid group at 24h and 48h after transfection. . Cytotoxicity assay Cytotoxicity of 0.5mg / LH2-B1 plasmid group and 1.0mg / LH2-B1 plasmid group was significantly lower than that of the blank control group after transfection 24h (P <0.05, P <0.01). CONCLUSION: The pEGFP-N1-H2B1 plasmid vector transfected mouse VSMC can effectively promote its apoptosis, inhibit its proliferation, reduce the toxic effect of PBMC on target cells and induce immune tolerance.