中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (27): 5071-5074.doi: 10.3969/j.issn.1673-8225.2010.27.030

• 干细胞基础实验 basic experiments of stem cells • 上一篇    下一篇

人sTRAIL基因载体构建及其在真皮干细胞的表达

缪殿南1，梁宇佳1，闫国和2，董世武3，戴晓天1，熊  玮1   

  1. 1解放军第三军医大学西南医院呼吸内科，重庆市  400038；解放军第三军医大学，2军事预防医学院防原医学教研室，创伤、烧伤与复合伤国家重点实验室，3基础医学部解剖学教研室，国家教育部生物力学与组织工程重点实验室生物力学研究室，重庆市  400038
  • 出版日期:2010-07-02 发布日期:2010-07-02
  • 通讯作者: 熊玮，博士，副教授，解放军第三军医大学西南医院呼吸内科，重庆市 400038 xiongwei64@126.com
  • 作者简介:缪殿南★，男，江苏省南通市人，解放军第三军医大学在读硕士，主治医师，主要从事肿瘤发病机制及其治疗方面的研究。mdn7310@163.com
  • 基金资助:

    国家自然科学基金(30770929)。课题名称：受uPA激活的重组人sTRAIL对非小细胞肺癌抗瘤作用的实验研究。

Construction of human sTRAIL vector and its expression in dermis-derived mesenchymal stem cells 

Miao Dian-nan1, Liang Yu-jia1, Yan Guo-he2, Dong Shi-wu3, Dai Xiao-tian1, Xiong Wei1   

  1. 1 Department of Respiratory Diseases, Southwest Hospital, Third Military Medical University, Chongqing  400038, China; 2 State Key Laboratory of Trauma, Burns and Combined Injury, Department of Nuclear Prevention Medicine, College of Military Preventive Medicine, 3 Department of Anatomy, State Key Laboratory for Biomechanics & Tissue Engineering, Ministry of Education, Third Military Medical University, Chongqing  400038, China
  • Online:2010-07-02 Published:2010-07-02
  • Contact: Xiong Wei, Doctor, Associate professor, Department of Respiratory Diseases, Southwest Hospital, Third Military Medical University, Chongqing 400038, China xiongwei64@126.com
  • About author:Mao Dian-nan★, Studying for master’s degree, Attending physician, Department of Respiratory Diseases, Southwest Hospital, Third Military Medical University, Chongqing 400038, China mdn7310@163.com
  • Supported by:

    the National Natural Science Foundation of China, No. 30770929*

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摘要：

背景：基于肿瘤坏死因子(tumour necrosis factor，TNF)可溶性相关凋亡诱导配体(soluble related apoptosis inducing ligand, sTRAIL)抗瘤的生物特性，克隆sTRAIL基因，构建其真核表达载体，为肿瘤细胞凋亡的实验研究奠定基础。
目的：构建真核表达载体pEGFP-N1/sTRAIL，并转染真皮干细胞，以探究sTRAIL基因导入真皮干细胞的表达情况。
方法：采用RT-PCR二步法，以人胎盘组织总RNA为模板，扩增sTRAIL的cDNA序列，将其克隆入pMD18-T载体，随后亚克隆入载体pEGFP-N1中，构建其真核瞬时表达载体pEGFP-N1/sTRAIL，以Fugene 6转染技术，将sTRAIL基因导入真皮干细胞。倒置显微镜下观测转染后真皮干细胞生长变化及sTRAIL在其中的表达等情况。RT-PCR法对目的基因转染的真皮干细胞进行鉴定。
结果与结论：克隆到sTRAIL基因的cDNA序列，成功构建了其真核表达载体，该真核表达载体能携带sTRAIL基因在真皮干细胞中正常表达。倒置显微镜下观测到转染后真皮干细胞生长状态与未转染的真皮干细胞无差异。以经sTRAIL基因转染的真皮干细胞该基因组DNA为模板，用RT-PCR法能扩增到sTRAIL基因cDNA序列。

关键词: 人sTRAIL基因, 克隆, 真核表达载体, 真皮干细胞, 绿色荧光

Abstract:

BACKGROUND: Based on the antineoplastic features of tumour necrosis factor (TNF) soluble related apoptosis inducing ligand (sTRAIL), sTRAIL gene was cloned and its eukaryotic expression vector was constructed, which can provide a foundation for apoptotic study of tumour cells.
OBJECTIVE: To construct pEGFP-N1/sTRAIL eukaryotic expression vector and express it into dermis-derived mesenchymal stem cells (dMSCs).
METHODS: cDNA fragment encoding human sTRAIL gene were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) with the total mRNA isolated from human placenta tissue as template. The PCR amplified fragment of sTRAIL gene was first cloned into Pmd18-T vector, and then subcloned into the recombinant eukaryotic expression vector pEGFP-N1 to construct pEGFP-N1/sTRAIL plasmid after sequencing. Subsequently, plasmid DNA of pEGFP-N1/sTRAIL was transfected into dMSCs with the help of Fugene 6 transfection reagent. The growth of dMSCs and expression of transfected sTRAIL in dMSCs were observed under an inverted microscope. The dMSCs were identified by RT-PCR.
RESULTS AND CONCLUSION: cDNA sequence encoding human sTRAIL gene was successfully cloned, and recombinant eukaryotic expression vector pEGFP-N1/sTRAIL was constructed. The expression of sTRAIL in dMSCs was obtained with the transfecton of pEGFP-N1/sTRAIL plasmid. Under an inverted microscope, the transfected DMSCs could be seen grown well. cDNA sequence encoding human sTRAIL gene was successfully cloned into pEGFP-N1. The transient expression of sTRAIL gene in dMSCs has been realized.

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