人骨生成诱导因子基因慢病毒载体构建及在人主动脉平滑肌细胞中稳定过表达

张惠洁; 刘卉芳; 张晓娜; 陈凤玲

人骨生成诱导因子基因慢病毒载体构建及在人主动脉平滑肌细胞中稳定过表达

1.
1. 上海交通大学医学院附属第三人民医院内分泌科, 上海 201900;
2.
2. 上海交通大学医学院附属瑞金医院, 上海市内分泌代谢病研究所, 上海 200025

作者简介: 张惠洁(1987- ), 女, 硕士研究生.

通讯作者: 陈凤玲, CFL1993@126.com

基金项目:
国家自然科学基金面上项目(30870954)

上海交通大学校基金资助项目(YZ1054)

上海交通大学新百人计划(09XBR01)

上海交通大学医学院附属第三人民医院院基金资助项目(syz2010-02)

Construction of human osteoinductive factor gene lentivirus vector and stable over-expression in human aortic smooth muscle cells

1.
1. Department of Endocrinology, The Third People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 201900;
2.
2. Shanghai Institution of Endocrinology, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China

Corresponding author: CHEN Feng-ling, CFL1993@126.com

摘要: 目的:构建人骨生成诱导因子基因(osteoinductive factor, OIF)慢病毒载体, 并在人主动脉平滑肌细胞(human aorticsmooth muscle cell, HASMC)中稳定过表达OIF。方法:PCR扩增OIF-3FLAG, 并克隆到慢病毒载体pMSCV PIG中, 构建慢病毒表达载体pMSCV PIG-OIF-3FLAG, 并测序鉴定。应用脂质体将pMSCV PIG-OIF-3FLAG或pMSCV PIG与VSVG、GAG-POL共转染293T细胞, 48 h后收集细胞上清, 感染HASMC, 48 h后加入嘌呤霉素4 d, 即筛选出稳定过表达pMSCV PIG-OIF-3FLAG的HASMC细胞株, 采用real-time PCR和Western blot检测OIF mRNA和蛋白表达。结果:构建OIF基因慢病毒表达载体pMSCV PIG-OIF-3FLAG, 经酶切及测序后证实目的基因的插入位点和读码框正确;包装病毒感染HASMC, 经real-time PCR和Western blot检测证实OIF mRNA和蛋白水平在该细胞株中高表达。结论:成功构建人OIF基因的慢病毒载体, 并获得稳定过表达该基因的HASMC。
- 人骨生成诱导因子 /
- 慢病毒载体 /
- 过表达 /
- 人主动脉平滑肌细胞
Abstract: Objective: To construct human osteoinductive factor(OIF) recombinant lentivirus vector and over-express OIF stably in human aortic smooth muscle cells(HASMCs) . Methods: OIF-3FLAG was amplified by PCR and cloned into vector(pMSCV PIG) . The lentivirus vector pMSCV PIG-OIF-3FLAG was constructed and indentified by sequencing. pMSCV PIG-OIF-3FLAG or pMSCV PIG with helper vectors of VSVG and GAG-POL were co-transfected into 293T cells. Infectious lentivirus was harvested at 48 h post-transfection. The lentivirus was added in HASMCs. After 48 h, puromycin was added for 4 d. When the cells reached 90%, HASMCs over-expressed pMSCV PIG-OIF-3FLAG stably. Human OIF expression was verified by quantitative real-time PCR and western blot analysis. Results: The lentivirus vector pMSCV PIG-OIF-3FLAG was successfully constructed and confirmed by enzyme digestion and sequencing. HASMCs were infected by lentivirus supernatant. Over-expression of OIF in HASMCs was revealed by real-time PCR and western blot. Conclusions: Human OIF gene recombined lentivirus vector and a cell model which over-expessing OIF stably in HASMCs were successfully established.
- human osteoinductive factor /
- lentivirus vector /
- over-expression /
- human aortic smooth muscle cell

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人骨生成诱导因子基因慢病毒载体构建及在人主动脉平滑肌细胞中稳定过表达

作者简介: 张惠洁(1987- ), 女, 硕士研究生.

通讯作者: 陈凤玲, CFL1993@126.com

Construction of human osteoinductive factor gene lentivirus vector and stable over-expression in human aortic smooth muscle cells

Corresponding author: CHEN Feng-ling, CFL1993@126.com

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人骨生成诱导因子基因慢病毒载体构建及在人主动脉平滑肌细胞中稳定过表达

通讯作者: 陈凤玲, CFL1993@126.com

作者简介: 张惠洁(1987- ), 女, 硕士研究生.

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Construction of human osteoinductive factor gene lentivirus vector and stable over-expression in human aortic smooth muscle cells

Corresponding author: CHEN Feng-ling, CFL1993@126.com

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人骨生成诱导因子基因慢病毒载体构建及在人主动脉平滑肌细胞中稳定过表达

作者简介: 张惠洁(1987- ), 女, 硕士研究生.

通讯作者: 陈凤玲, CFL1993@126.com

Construction of human osteoinductive factor gene lentivirus vector and stable over-expression in human aortic smooth muscle cells

Corresponding author: CHEN Feng-ling, CFL1993@126.com

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人骨生成诱导因子基因慢病毒载体构建及在人主动脉平滑肌细胞中稳定过表达

通讯作者: 陈凤玲, CFL1993@126.com

作者简介: 张惠洁(1987- ), 女, 硕士研究生.

English Abstract

Construction of human osteoinductive factor gene lentivirus vector and stable over-expression in human aortic smooth muscle cells

Corresponding author: CHEN Feng-ling, CFL1993@126.com

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