摘要
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。
This study was aimed to construct the mouse VCAM-1 expression vector,to establish the stably transfected MSC line and to investigate the effect of VCAM-1-modified mesenchymal stem cells （MSC） on the immunological characteristics of MSC.The cDNA of murine VCAM-1 gene was amplified by RT-PCR from the total RNA isolated from the mouse spleen; then the cDNA was inserted into the retrovirus vector PMSCVmigr-1 ; the recombinant plasmid was confirmed by restriction endonuclease experiments and sequencing,then designated as PMSCVmigr-1-mVCAM-1 ;the recombinant plasmid PMSCVmigr-1-mVCAM-1 was transfected into 293 cells by lipofecamin and the supernatant was collected to transfect MSC cell line （C3H10T1/2）.Moreover,VCAM-1 expression on MSC was evaluated by FACS.Furthermore,the inhibitory effect of VCAM-1-MSC on lymphocytic transformation was tested by 3H-TdR incorporation assay.The results indicated that the successful construction of recombinant retroviral expression plasmid of mouse VCAM-1 was confirmed by digesting and sequancing.After transfection of MSC with retroviral supernaptant,the high expression of VCAM-1 on MSC could be detected by flow cytometry.The MSC high expressing VCAM-1 could significantly inhibit the proliferation of Con A-inducing lymphocytes in dose-depentent marrer.It is concluded that recombinant retroviral encoding VCAM-1 （PMSCVmigr-1-mVCAM-1） has been successfully constructed and mouse VCAM-1 has been stably expressed in C3H10T1/2.MSC over-expressing VCAM-1 show more potent immunosuppressive effect on cellular immune reaction in vitro.Our data laid a foundation for the subsequent studying the effect of VCAM-1 transfecting into MSC on immune related disease study.
出处
《中国实验血液学杂志》
CAS
CSCD
北大核心
2014年第5期1396-1401,共6页
Journal of Experimental Hematology
基金
国家重点基础研究计划(973项目编号2010CB833600)
国家自然科学基金面上项目(编号31070996,31171084,81371945)
国家自然科学基金青年项目(编号81101342)
北京市自然科学基金面上项目(编号7132133)
