中国组织工程研究

中国组织工程研究 ›› 2016, Vol. 20 ›› Issue (36): 5338-5344.doi: 10.3969/j.issn.2095-4344.2016.36.003

• 骨髓干细胞 bone marrow stem cells • 上一篇    下一篇

慢病毒载体Pax6-EGFP构建及转染人骨髓间充质干细胞

刘静雯1，2，任  静1，2，陈  胜1，李柏军1，刘身文1，秦  波1   

  1. 1深圳市眼科医院眼外伤科，暨南大学附属深圳眼科医院，深圳大学眼视光学院，深圳眼科学重点实验室，广东省深圳市  518000
    2暨南大学，广东省广州市  510632
  • 修回日期:2016-07-08 出版日期:2016-09-02 发布日期:2016-09-02
  • 通讯作者: 秦波，博士，教授，主任医师，深圳市眼科医院眼外伤科，暨南大学附属深圳眼科医院，深圳大学眼视光学院，深圳眼科学重点实验室，广东省深圳市 518000
  • 作者简介:刘静雯，女，1989年生，广东省清远市人，汉族，2016年暨南大学毕业，硕士，医师，主要从事眼外伤、眼底病研究。
  • 基金资助:

    深圳市知识创新计划基础研究计划项目(JCYJ20130401152829828)

Construction of a lentivirus vector containing Pax6 and its transfection of human bone marrow mesenchymal stem cells

Liu Jing-wen1, 2, Ren Jing1, 2, Chen Sheng1, Li Bai-jun1, Liu Shen-wen1, Qin Bo1   

  1. 1Ocular Trauma Department of Shenzhen Eye Hospital, Affiliated Shenzhen Eye Hospital of Jinan University, Joint College of Optometry of Shenzhen University, Shenzhen Key Laboratory of Ophthalmology, Shenzhen 518000, Guangdong Province, China
    2Jinan University, Guangzhou 510632, Guangdong Province, China
  • Revised:2016-07-08 Online:2016-09-02 Published:2016-09-02
  • Contact: Qin Bo, M.D., Professor, Chief physician, Ocular Trauma Department of Shenzhen Eye Hospital, Affiliated Shenzhen Eye Hospital of Jinan University, Joint College of Optometry of Shenzhen University, Shenzhen Key Laboratory of Ophthalmology, Shenzhen 518000, Guangdong Province, China
  • About author:Liu Jing-wen, Master, Physician, Ocular Trauma Department of Shenzhen Eye Hospital, Affiliated Shenzhen Eye Hospital of Jinan University, Joint College of Optometry of Shenzhen University, Shenzhen Key Laboratory of Ophthalmology, Shenzhen 518000, Guangdong Province, China; Jinan University, Guangzhou 510632, Guangdong Province, China
  • Supported by:

    Basic Research Project of Knowledge Innovation Program in Shenzhen, China, No. JCYJ20130401152829828

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摘要：

文章快速阅读：

文题释义：
慢病毒载体：是以HIV-1(人类免疫缺陷1型病毒)为基础发展起来的基因治疗载体，它能将外源基因整合到宿主细胞染色体上并长期表达目的基因及蛋白。四质粒系统是目前最新、应用最广的慢病毒载体系统，分别由pRev、pGag/Pol、pVSV-G和一个携带目的基因序列的载体构成，由于其包装序列不能整合到基因组中，病毒感染宿主后无法复制，也不能产生新的病毒颗粒，因此安全性得到保证。
感染复数：指的是有感染力的病毒数量与待感染的细胞数量的比值，通常以不同浓度梯度的病毒液感染靶细胞并观察转染效果，以确定该病毒载体的最适感染复数。

 

摘要
背景：Pax6基因是影响眼发育、分化的关键基因，获得过表达Pax6基因的稳定细胞株是研究其对骨髓间充质干细胞分化作用的首要任务，也是干细胞替代治疗的研究基础。
目的：构建重组慢病毒载体Pax6-EGFP，体外转染人骨髓间充质干细胞，检测Pax6基因在人骨髓间充质干细胞中的表达情况。
方法：利用PCR法从人Pax6 cDNA基因克隆载体pGEM-PAX6上扩增Pax6目的片段，将其与XbaⅠ和BamHⅠ双酶切的慢病毒载体pHIV-EGFP连接，转化感受态细胞DH5α，挑取阳性克隆提取质粒，测序鉴定。经293T细胞包装后，收集含病毒颗粒的上清液，体外转染人骨髓间充质干细胞，同时设立阴性对照组(pHIV-EGFP空载体)。荧光显微镜下观察转染是否成功，Real time PCR检测转染细胞Pax6基因的表达。
结果与结论：①酶切电泳及基因测序证实携带Pax6基因的慢病毒载体构建成功，包装后获得滴度为3×109 pfu/L的Pax6-EGFP重组载体；②转染人骨髓间充质干细胞后，在荧光显微镜下可以发出绿色荧光，且荧光强度不随培养时间延长而衰退；③经Real time PCR检测，被转染细胞内有Pax6基因表达，表明慢病毒转染是一种安全高效的基因修饰手段，能够使外源基因整合到宿主细胞基因组中。

 

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程
ORCID:0000-0003-2533-1378(刘静雯)

关键词: 干细胞, 骨髓干细胞, 骨髓间充质干细胞, 过表达, Pax6基因, 慢病毒, 基因转染, 绿色荧光蛋白

Abstract:

BACKGROUND: Pax6 gene plays an important role in eye development and differentiation, and to study how it regulates the differentiation of human bone marrow mesenchymal stem cells (BMSCs), gaining the BMSCs stably over-expressing Pax6 is crucial, which is also the basis of stem cell replacement therapy.
OBJECTIVE: To construct a lentivirus vector containing Pax6 and detect the expression of Pax6 in transfected human BMSCs.
METHODS: Pax6 gene was extracted using PCR. After its connection with lentivirus vector pHIV-EGFP, it was then packaged by 293T cells. The human BMSCs were transfected with recombinant lentivirus Pax6-EGFP as well as lentivirus vector pHIV-EGFP, which was considered negative control group. The cellular morphology was observed by a fluorescence microscope, and the mRNA expression of Pax6 was detected by real-time PCR.
RESULTS AND CONCLUSION: The recombinant lentivirus Pax6-EGFP was constructed successfully with a titer of 3×109 pfu/L. After the transfection, both the green fluorescent protein and Pax6 gene were expressed detected using fluorescence microscope and real-time PCR, showing that the method of lentiviral transfection is a safe and effective way to modify BMSCs.

 

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Bone Marrow, Mesenchymal Stem Cells, Paired Box Transcription Factors, Lentivirus, Transfection, Green Fluorescent Proteins, Tissue Engineering

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