【摘要】 目的构建过表达HLA-G5的慢病毒载体,感染人羊膜间充质干细胞(human amnion drived mesenchymal stem cells,h AD-MSCs),为后续探索HLA-G5在h AD-MSCs诱导免疫耐受中的作用和机制,并为探索以其作为生物反应器生产HLA-G5提供前期实验基础。方法人工合成HLA-G5全长序列,定向接入真核表达载体p CDH-CMV-MCS-EF1-cop GFP,转化入大肠杆菌,酶切鉴定和测序正确后进行慢病毒包装。按照慢病毒滴度梯度感染h AD-MSCs,DAPI复染细胞核,荧光显微镜下观察感染效率。MTS检测连续感染24 h后慢病毒对h AD-MSCs的毒性。ELISA法检测感染后细胞上清中的可溶性HLA-G5表达。结果 (1)酶切鉴定和测序结果显示,HLA-G5表达质粒质量合格,序列比对与设计序列符合率100%;(2)感染h AD-MSCs后有较高的感染效率(接近100%);(3)构建的慢病毒毒性较小;(4)感染后h AD-MSCs分泌可溶性HLA-G5增高。结论本研究中构建的HLA-G5慢病毒过表达载体能够安全高效的感染h AD-MSCs,为进一步研究其体内外诱导免疫耐受的作用和机制,并将其作为细胞反应器生产HLA-G5的前期工艺奠定了基础。更多还原

【Abstract】 Objective To construct a lentiviral vector overexpressing HLA-G5 and infect human amnion drived mesenchymal stem cells( h AD-MSCs),so as to explore the role and mechanisms of HLA-G5 in h AD-MSCs for immune tolerance,and further provide a preliminary experimental basis for the use of h AD-MSCs as a bioreactor producing HLA-G5. Methods HLA-G5 full-length sequence was synthesized artificially and p CDH-CMV-MCS-EF1-COP GFP was inserted into Eukaryotic expression vector,transformed into E coli,and then lentivirus packaging was performed after the sequence was identified. The h AD-MSCs were infected according to the lentivirus titer gradient,DAPI stained the nucleus again,and the infection efficiency was observed under fluorescence microscope. MTS was used to detect the toxicity of lentivirus on h AD-MSCs after 24 hours of continuous infection. The expression of soluble HLA-G5 in the supernatant of infected cells was detected by ELISA.Results(1) The results of restriction endonuclease identification and sequencing showed that the quality of HLA-G5 expression plasmid was qualified,and the coincidence rate of sequence alignment and design sequence was100%.(2) The infection efficiency of h AD-MSCs was high( close to 100%).(3) The constructed lentivirus had less toxicity.(4) The secretion of soluble HLA-G5 was increased in the supernatant of h AD-MSCs after infection. Conclusion The HLA-G5 lentivirus overexpression vector constructed in this study could infect h AD-MSCs safely and efficiently,which lays a foundation for further study on the role and mechanisms of HLA-G5 in h AD-MSCs for immune tolerance,and to provide a preliminary experimental basis for exploring the use of h AD-MSCs as a bioreactor producing HLA-G5.更多还原