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本研究旨在构建小鼠血管细胞粘附分子-1(vascular cell adhesion molecule-1,VCAM-1)的逆转录病毒载体,通过转染建立稳定高表达VCAM-1的间充质干细胞(mesenchymal stem cells,MSC),并初步探讨VCAM-1基因过表达对MSC免疫学特性的影响。以小鼠脾脏组织总RNA为模板,通过RT-PCR扩增小鼠VCAM-1基因cDNA,应用基因工程技术将扩增的cDNA片段插入PMSCVmigr-1逆转录病毒载体,构建重组逆转录病毒表达质粒PMSCVmigr-1-mVCAM-1,经限制性内切酶酶切分析及测序鉴定后,用脂质体转染技术转染293细胞,收获含有病毒颗粒的上清;用病毒上清感染间充质干细胞系(C3H10T 1/2)并检测其表达。采用3H-TdR掺入法测定高表达VCAM-1的MSC在淋巴母细胞转化实验中对淋巴细胞增殖的抑制作用。结果表明:酶切和测序鉴定证实,正确构建了小鼠VCAM-1的重组逆转录病毒表达质粒。逆转录病毒上清感染MSC后流式细胞术检测到目的基因VCAM-1在MSC表面高表达。高表达VCAM-1的MSC对刀豆素A诱导的淋巴细胞增殖具有显著抑制作用,且呈剂量效应关系。结论:成功构建了小鼠VCAM-1基因的重组逆转录病毒质粒并使其在MSC中稳定高表达。高表达VCAM-1的MSC对体外淋巴细胞增殖具有很强的抑制效果。本课题为进一步研究VCAM-1基因调控MSC干预免疫相关性疾病奠定了实验基础。
The purpose of this study was to construct retroviral vector of mouse vascular cell adhesion molecule-1 (VCAM-1) and to establish a mesenchymal stem cell cells, MSC). The effects of VCAM-1 gene overexpression on the immunological characteristics of MSC were also investigated. The mouse VCAM-1 cDNA was amplified by RT-PCR using total RNA of mouse spleen as a template. The amplified cDNA fragment was inserted into the PMSCVmigr-1 retrovirus vector to construct a recombinant retroviral expression plasmid PMSCVmigr-1-mVCAM-1 was digested with restriction endonucleases and identified by sequencing. The recombinant plasmid was transfected into 293 cells by lipofection technique to obtain the supernatant containing the virus particles. The supernatant was used to infect mesenchyme Stem cell line (C3H10T 1/2) and its expression detected. 3H-TdR incorporation was used to determine the inhibitory effect of VCAM-1-overexpressing MSCs on lymphocyte proliferation in lymphoblast transformation. The results showed that digestion and sequencing confirmed that recombinant retrovirus expression plasmid of mouse VCAM-1 was correctly constructed. The results of flow cytometry showed that the target gene VCAM-1 was highly expressed on the MSC surface after infected with retrovirus supernatant. MSC overexpressing VCAM-1 had a significant inhibitory effect on concanavalin A-induced lymphocyte proliferation with a dose-response relationship. Conclusion: The recombinant retrovirus plasmid of mouse VCAM-1 gene was successfully constructed and stably overexpressed in MSC. High expression of VCAM-1 of MSC on lymphocyte proliferation in vitro has a strong inhibitory effect. The topic for further study VCAM-1 gene regulation of MSC intervention immune-related diseases laid the foundation for the experiment.