Title

Experimental Study on Establishing Eukaryotic Expression Vector pcDNA3.1(+) TPA and Its Expressing Activity in Human Skin Fibroblasts

Translated Titles

真核表達載體pcDNA3.1(+) TPA的建立及其在人皮膚成纖維細胞的表達活性

Authors

鄧洪波(Hong-Bo Deng);楊宇(Yu Yang);楊進福(Jin-Fu Yang);唐滔(Tao Tang);周文武(Wen-Wu Zhou);劉劍(Jian Liu)

Key Words

組織型纖溶酶原激活因子 ; 基因治療 ; pcDNA3.1 ; 皮膚成纖維細胞 ; 缺血性心臟病 ; tissue plasminogen activator ; gene therapy ; pcDNA3.1(+) ; skin fibroblast ; ischemic heart diseases

PublicationName

中南大學學報(醫學版)

Volume or Term/Year and Month of Publication

30卷4期(2005 / 08 / 01)

Page #

373 - 378

Content Language

英文

Chinese Abstract

目的:建立在人皮膚成纖維細胞中穩定表達的細胞株,為組織型纖溶酶原激活因子(TPA)功能分析和對缺血性心臟疾病基因治療提供理論依據。方法:構建真核表達載體pcDNA3.1(+) TPA,然後將其轉入人皮膚成纖維細胞,經G418篩選後,用RT-PCR, ELISA和發色底物法觀察外源性TPA表達情況。結果:真核表達載體pcDNA3.l(+) TPA在人皮膚成纖維細胞中獲有效表達,酶聯免疫吸附法TPA蛋白表達定量檢測結果為每24小時643.5ng/10^6個細胞,高於未轉pcDNA3.l(+) TPA的皮膚成纖維細胞每24小時19.2ng/10^6個細胞;發色底物法測得外源性TPA活性為每24小時122.6U/l0^6個細胞,亦高於未轉pcDNA3.1(+) TPA的皮膚成纖維細胞每24小時5.8U/10^6個細胞。結論:pcDNA3.1(+) TPA轉入人皮膚成纖維細胞後,外源性TPA基因獲有效表達,該細胞模型將成為TPA功能研究和缺血性心臟病基因治療的重要方法。

English 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 (643.5ng/l0^6 cells per 24 hours vs. 19.2ng/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 (122.6U/10^6 cells per 24 hours vs. 5.8U/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.

Topic Category 醫藥衛生 > 醫藥衛生綜合
醫藥衛生 > 預防保健與衛生學
醫藥衛生 > 中醫藥學
醫藥衛生 > 藥理醫學
醫藥衛生 > 社會醫學