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

doi:10.3969/j.issn.2095-4344.2016.36.003

Abstract

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

CLC Number:

R394.2

Liu Jing-wen, Ren Jing, Chen Sheng, Li Bai-jun, Liu Shen-wen, Qin Bo. Construction of a lentivirus vector containing Pax6 and its transfection of human bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(36): 5338-5344.

Figures/Tables 2

2.1 目的基因片段Pax6的获取设计扩增引物，并在上下游引物分别加上COZAK增强子序列、XbaⅠ和BamHⅠ酶切位点，从人Pax6 cDNA基因克隆载体pGEM-PAX6上扩增Pax6基因片段，用XbaⅠ和BamHⅠ双酶切，电泳结果显示酶切后1 269 bp处有明亮特异的目的条带，与预期相符，同时PHIV-EGFP质粒也用XbaⅠ和BamHⅠ双酶切，切胶纯化，见图1。 2.2 慢病毒载体Pax6-EGFP的鉴定及测序结果分析重组质粒连接后转化到DH5α感受态，挑选10个克隆，经菌液PCR筛选后可见3个阳性克隆(图2A)，抽提3个阳性克隆的质粒，继续做PCR，均可见长度为1 269 bp左右基因片段(图2B)，与目的基因Pax6大小基本一致。采用通用引物检测重组载体PHIV-EGFP-Pax6中的Pax6序列信息，经测序分析并与GeneBank中基因序列比对(图3)，发现其相似性为100%，表明成功构建载体。 2.3 显微镜下观察细胞形态及绿色荧光表达经MOI=10，20，40的Pax6-EGFP慢病毒转染骨髓间充质干细胞24 h，均可见绿色荧光蛋白阳性细胞但数量较少，且胞浆内荧光较弱，间隔1 d重复感染，72 h后见绿色荧光蛋白阳性细胞增多，荧光增强，其中MOI=20时阳性细胞数量及荧光强度最优。以相同MOI值及相同步骤对空白组进行pHIV-EGFP转染，两组分别置于普通显微镜(图4)、荧光显微镜(图5)下观察。普通显微镜下两组细胞生长状态良好，细胞形态均未见明显改变，多数细胞呈长梭形。荧光显微镜下两组均能观察到绿色荧光蛋白表达，感染效率约为30%，并且在转染后第10天荧光依旧可见。 2.4 RT-PCR及Real time PCR检测各组细胞内Pax6的表达 168 bp的GAPDH作为内参，1%琼脂糖凝胶电泳于相应位置可见目的条带。取转染72 h后的两组骨髓间充质干细胞分别行RT-PCR，以设计的Pax6 Real time PCR引物作为RT-PCR的引物，扩增产物为95 bp，经电泳40 min后凝胶成像仪观察结果见图6，可见实验组在95 bp左右出现阳性条带，对照组则未见相应条带。利用2-??Ct方法，以对照组作为参照标准，即表达水平为1，计算对照组与实验组骨髓间充质干细胞中Pax6基因相对表达水平，可见实验组表达水平明显高于对照组，差异有显著性意义(图7)。"

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