Experimental study on establishing eukaryotic expression vector pcDNA3.1(+) TPA and its expressing activity in human skin fibroblists.

Q3 Medicine 中南大学学报(医学版) Pub Date : 2005-08-01

Hong-bo Deng, Yu Yang, Jin-fu Yang, Tao Tang, Wen-wu Zhou, Jian Liu

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Abstract

Objective: To establish a cell line stably expressing the tissue plasminogen activator (TPA) in human skin fibroblasts so as to develop the function analysis and gene therapy of TPA in ischemic heart diseases.

Methods: Eukaryotic expression vector pcDNA3.1(+) TPA was constructed and transferred into human skin fibroblasts. After G418 selection, the exogenous expression and activity of TPA were observed subsequently by reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and chromogenic substrate assay.

Results: Eukaryotic expression vector pcDNA3.1(+) TPA was expressed effectively in human skin fibroblasts. Quantitative ELISA showed that the expression of TPA protein of the experiment group was much higher than that of the control group (643.5 ng/10(6) cells per 24 hours vs. 19.2 ng/10(6) cells per 24 hours). And the chromogenic substrate assay showed that the exogenous TPA activity of the experimental group was also much higher than that of the control group (122.6 U/10(6) cells per 24 hours vs. 5.8 U/10(6) cells per 24 hours).

Conclusion: The exogenous TPA gene can be expressed effectively after pcDNA3.1(+)TPA was transferred into human skin fibroblasts, suggesting that the cell model will become an important tool in the further study of TPA function and gene therapy in ischemic heart diseases.

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真核表达载体pcDNA3.1(+) TPA的建立及其在人皮肤纤维细胞中的表达活性的实验研究。

目的:建立在人皮肤成纤维细胞中稳定表达组织纤溶酶原激活物(TPA)的细胞系，开展TPA在缺血性心脏病中的功能分析和基因治疗。方法:构建真核表达载体pcDNA3.1(+) TPA，转染人皮肤成纤维细胞。选择G418后，通过逆转录聚合酶链反应(RT-PCR)、酶联免疫吸附试验(ELISA)和显色底物试验观察外源TPA的表达和活性。结果:真核表达载体pcDNA3.1(+) TPA在人皮肤成纤维细胞中有效表达。定量ELISA结果显示，实验组TPA蛋白表达量明显高于对照组(643.5 ng/10(6)个细胞/ 24小时比19.2 ng/10(6)个细胞/ 24小时)。显色底物实验显示，实验组外源TPA活性也远高于对照组(122.6 U/10(6)个细胞/ 24小时vs. 5.8 U/10(6)个细胞/ 24小时)。结论:pcDNA3.1(+)TPA转染人皮肤成纤维细胞后可有效表达外源性TPA基因，提示该细胞模型将成为进一步研究TPA功能及缺血性心脏病基因治疗的重要工具。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

中南大学学报(医学版) Medicine-Medicine (all)

CiteScore

1.00

自引率

0.00%

发文量

8237

期刊介绍： Journal of Central South University (Medical Sciences), founded in 1958, is a comprehensive academic journal of medicine and health sponsored by the Ministry of Education and Central South University. The journal has been included in many important databases and authoritative abstract journals at home and abroad, such as the American Medline, Pubmed and its Index Medicus (IM), the Netherlands Medical Abstracts (EM), the American Chemical Abstracts (CA), the WHO Western Pacific Region Medical Index (WPRIM), and the Chinese Science Citation Database (Core Database) (CSCD); it is a statistical source journal of Chinese scientific and technological papers, a Chinese core journal, and a "double-effect" journal of the Chinese Journal Matrix; it is the "2nd, 3rd, and 4th China University Excellent Science and Technology Journal", "2008 China Excellent Science and Technology Journal", "RCCSE China Authoritative Academic Journal (A+)" and Hunan Province's "Top Ten Science and Technology Journals". The purpose of the journal is to reflect the new achievements, new technologies, and new experiences in medical research, medical treatment, and teaching, report new medical trends at home and abroad, promote academic exchanges, improve academic standards, and promote scientific and technological progress.