人血管生成素1基因慢病毒表达载体的构建及其在脐带间充质干细胞的表达

doi:10.6040/j.issn.1671-7554.0.2016.730

山东大学学报（医学版） ›› 2016, Vol. 54 ›› Issue (12): 1-7.doi: 10.6040/j.issn.1671-7554.0.2016.730

• 基础医学 • 下一篇

人血管生成素1基因慢病毒表达载体的构建及其在脐带间充质干细胞的表达

刘宁¹,吕欣¹,李栋²,黄志伟³,刘毅¹,张乐玲³

1. 山东大学齐鲁儿童医院儿科医学研究所, 山东济南 250022;2.山东大学齐鲁医院低温医学研究室, 山东济南 250012;3. 山东大学齐鲁儿童医院血液肿瘤科, 山东济南 250022

收稿日期:2016-06-22 出版日期:2016-12-10 发布日期:2016-12-10
通讯作者: 张乐玲. E-mail: shzh870@yeah.net 刘毅. E-mail:liuyi-ly@126.com E-mail:shzh870@yeah.net
基金资助:
山东省自然科学基金(ZR2013HM001)

Construction and identification of lentiviral vector for angiopoietin-1 gene and its expression in human umbilical cord mesenchymal stem cells

LIU Ning¹, L(¨overU)Xin¹, LI Dong², HUNAG Zhiwei³, LIU Yi¹, ZHANG Leling³

1. Institute of Pediatric Research, Qilu Childrens Hospital of Shandong University, Jinan 250022, Shandong, China;
2. Cryomedicine Laboratory, Qilu Hospital of Shandong University, Jinan 250012, Shandong, China;
3. Department of Hematologyoncology, Qilu Childrens Hospital of Shandong University, Jinan 250022, Shandong, China

Received:2016-06-22 Online:2016-12-10 Published:2016-12-10

摘要/Abstract

摘要： 目的通过基因重组技术构建人血管生成素-1(Ang-1)基因慢病毒表达载体,并检测其在人脐带间充质干细胞(hUC-MSCs)的表达及对hUC-MSCs免疫抑制能力的影响。方法应用Trizol法从hUC-MSCs提取总RNA,反转录获取cDNA,PCR扩增获得编码Ang-1的序列克隆到GV287载体中。将重组GV287-Ang-1载体质粒和慢病毒包装质粒pHelper 1.0和pHelper 2.0共转染293T细胞,收集病毒上清,纯化浓缩测定病毒滴度。采用荧光显微镜观察转染效率,Western blotting法检测Ang-1蛋白表达,并通过CCK8试剂盒检测T淋巴细胞增殖活性。结果 Ang-1基因扩增PCR产物与预期大小一致。重组慢病毒GV287-Ang-1质粒经PCR和DNA测序分析显示,所得结果与目的基因序列一致且插入方向正确。包装慢病毒浓缩悬液滴度为2×10⁸TU/mL,最佳感染复数为8。GV287-Ang-1转染组细胞Ang-1表达显著高于未转染组和GV287转染组。过表达Ang-1的hUC-MSCs对T淋巴细胞的增殖抑制显著高于单纯的hUC-MSCs。结论成功构建携带Ang-1基因的慢病毒载体GV287-Ang-1,并可有效转染hUC-MSCs过表达Ang-1蛋白,且能显著提高hUC-MSCs的免疫抑制能力。

关键词: 血管生成素基因1, 293T细胞, 人脐带间充质干细胞, 过表达, 慢病毒载体

Abstract: Objective To construct the lentivirus vector carrying angiopoietin-1 gene(Ang-1), and to investigate its expression in human umbilical cord mesenchymal stem cells(hUC-MSCs)and the immunomodulatory effect on Ang-1-modified hUC-MSCs. Methods The cDNA encoding full-length human Ang-1 was amplified by RT-PCR from the total RNA of hUC-MSCs, and then subcloned to lentiviral vector backbone plasmid GV287. The recombinant plasmid GV287-Ang-1 was co-transfected with packaging and enveloping plasmids pHelper 1.0 and pHelper 2.0 into 293T cells. Vector titer was quantified using cell fluorescence. The transfection was detected by fluorescence microscopy, and protein expression of Ang-1 was detected by Western blotting. T lymphocytes proliferation was assessed by cell counting kit 8(CCK8). Results The recombinant lentiviral vector GV287-Ang-1 was verified correctly by PCR and DNA sequencing. The titer of concentrated virus was 2×10⁸ TU/mL, and the optimal MOI for transfecting hUC-MSCs was 8. Western blotting results showed that the Ang-1 expression of the GV287-Ang-1 transfected group was significantly higher 山东大学学报 (医学版)54卷12期 -刘宁,等.人血管生成素1基因慢病毒表达载体的构建及其在脐带间充质干细胞的表达 \=-than those of the non-transfected group and GV287 group. The suppression ratio of T lymphocytes in Ang-1-UC-MSCs group was significantly increased as compared to that in the UC-MSCs group. Conclusion The lentivirus vector carrying human Ang-1 was constructed successfully and overexpressed effectively in hUC-MSCs. Ang-1-modified hUC-MSCs can enhance the immunosuppressive capability, which suggests a potential clinical application.

Key words: Angiopoietin-1, 293T cells, Lentiviral vector, Overexpression, Human umbilical cord mesenchymal stem cells

中图分类号:

刘宁,吕欣,李栋,黄志伟,刘毅,张乐玲. 人血管生成素1基因慢病毒表达载体的构建及其在脐带间充质干细胞的表达[J]. 山东大学学报（医学版）, 2016, 54(12): 1-7.

LIU Ning, L(¨overU)Xin, LI Dong, HUNAG Zhiwei, LIU Yi, ZHANG Leling. Construction and identification of lentiviral vector for angiopoietin-1 gene and its expression in human umbilical cord mesenchymal stem cells[J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2016, 54(12): 1-7.

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人血管生成素1基因慢病毒表达载体的构建及其在脐带间充质干细胞的表达

Construction and identification of lentiviral vector for angiopoietin-1 gene and its expression in human umbilical cord mesenchymal stem cells

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