摘要
目的构建共同表达人吲哚胺2,3-过氧化酶(IDO)基因和增强型绿色荧光蛋白(EGFP)基因的慢病毒表达载体,探讨其在人脐带间充质干细胞(hUCMSCs)中的表达,为下一步利用高表达IDO的hUCMSCs移植治疗再生障碍性贫血奠定实验基础。方法将IDO基因克隆至慢病毒pGC-FU-EGFP表达载体中,通过PCR和测序鉴定获得连接正确的克隆。重组慢病毒表达载体质粒及辅助包装质粒共转染293T细胞,荧光显微镜下观察绿色荧光蛋白(GFP)的表达情况;制备携带IDO基因的慢病毒Lentivirus-IDO,并测定重组慢病毒的滴度。用重组慢病毒以最佳MOI值感染hUCM-SCs作为实验组,以空载体转染的hUCMSCs(空载体组)及未转染的hUCMSCs(未转染组)作为对照,应用RT-PCR及Western blot法分别检测细胞中IDO mRNA及IDO蛋白水平的表达情况。结果经PCR及基因测序鉴定pGC-FU-EGFP-IDO重组慢病毒表达载体构建成功,包装慢病毒Lentivirus-IDO滴度为2×108 TU/mL。通过EGFP表达的阳性细胞数筛选其最适MOI为30,此时对细胞的转染效率可达到80%以上。RT-PCR检测显示实验组IDO mRNA表达阳性,空载体组和未转染组均为阴性;Western blot检测示实验组细胞内IDO蛋白表达阳性,空载体组和未转染组均为阴性。结论成功构建了人IDO基因的重组慢病毒表达载体,慢病毒Lentivirus-IDO能有效感染hUCMSCs,IDO蛋白可在hUCMSCs中长期、稳定表达。
Objective To construct the lentiviral vector co-expressing enhanced human indoleamine 2,3-dioxygenase（IDO） gene and green fluorescent protein（EGFP）gene,and to explore its transfection efficiency in human umbilical cord mesenchymal stem cells（hUCMSCs）in order to lay an experiment foundation for transplantation of IDO-expressing hUCMSCs to treat aplastic anemia.Methods The IDO gene was cloned into the lentiviral expression vector（pGC-FU-EGFP）,which was verified by PCR and sequencing analysis.The recombinant lentiviral vector and packaging plasmids were co-transfected into 293Tcells.The expression of green fluorescent protein（GFP）was observed to evaluate gene delivery efficiency under the fluorescence microscope.The IDO-expressing recombinant lentiviral vectors（Lentivirus-IDO）was prepared and harvested from 293Tcells,and the virus titer was determined.hUCMSCs infected with recombinant lentiviruses at the best MOI was designated as the experiment group,and those transfected with empty vector（empty vector group）and non-transfected hUCMSCs（non-transfection group） as control groups.RT-PCR and Western blot were used to detect IDO mRNA and protein expression levels in cells.Results Recombinant lentiviral vectors that expressed human IDO gene were successfully constructed and verified by PCR and gene sequencing.The Lentivirus-IDO at a titer of 2×108 TU/mL was successfully packaged.The best MOI was 30and the transfection efficiency was up to 80%.RT-PCR and Western blot results showed that IDO was positively expressed at mRNA and protein levels in hUCMSCs infected with recombinant lentiviruses,while it was not expressed in the empty vector-transfected and nontransfected hUCMSCs.Conclusion The Lentivirus-IDO vectors were successfully constructed and they could effectively infect hUCMSCs to allow the long-term and stable expression of IDO in the cells.
出处
《华中科技大学学报（医学版）》
CAS
CSCD
北大核心
2013年第4期395-400,423,共7页
Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
基金
安徽省高等学校省级自然科学研究项目(No.Kj2010B118)
安徽省高等学校省级自然科学研究重点项目(No.Kj2012A198)
安徽省蚌埠市科技计划项目(No.蚌科[2011]33)
作者简介
李玉云，女，1964年生，副教授，E—mail：bbmcliyuyun@163．com
通讯作者，corresponding author，E-mail：bbyxyzq@sina．com
