The study of human PDGF-B gene transferred to cat corneal endothelial cells

作     者：Wen-Juan Luo, Chuan-Fu Wang 

作者机构：Department of Ophthalmology the Affiliated Hospital of Medical College Qingdao University Qingdao 266003 Shandong Province China School of Pharmacy Jiangxi Science and Technology Normal University Nan Chang 330013 Jiangxi Province China Department of Pathology the Affiliated Hospital of Medical College Qingdao University Qingdao 266003 Shandong Province China 

出 版 物：《International Journal of Ophthalmology(English edition)》 (国际眼科杂志（英文版）)

年 卷 期：2012年第5卷第1期

页      面：18-22页

核心收录：

学科分类：1002[医学-临床医学] 100212[医学-眼科学] 10[医学] 

基　　金：National Natural Science Foundation of China(No.30572011) Natural Science Foundation of Shandong Province,China(No.ZR2010HQ041) 

主　　题：platelet-derived growth factor corneal endothelial cell viability gene transfection. 

摘      要：AIM: To demonstrate that human platelet-derived growth factor-B (PDGF-B) cDNA could be Expressed in primary cultured cat corneal endothelia cells by using gene transfer techniques; to explore a useful tool for the further studies of the molecular mechanisms of corneal endothelium failure and provide a potential effective genetic therapy for the blind patients. METHODS: Human PDGF-B cDNA was isolated from human placent by RT-PCR and inserted into pcDNA(4) vector to construct recombinant eukaryotic expression plasmid pcDNA(4)-PDGF-B. The full length was confirmed by the DNA sequencing analysis. By tearing endothelium technique we obtained pure single layer of cat corneal endothelial cells. The pcDNA(4)-PDGF-B eukaryotic Expression vector was transferred into cat corneal endothelial cells by Effectene (TM) lipofectine. The transfection efficiency of Effectene (TM) lipofectine in pcDNA(4)-B was detected with pcDNA(4)-GFP. 5 days later, RT-PCR was used to check the PDGF-B expression. Cell viability was tested by modified tertrozalium salt (MU) method. Cell morphology was observed under inverted phase contrast microscope. RESULTS: The human PDGF-B cDNA was isolated successfully from healthy parturien placent tissue and the sequence was confirmed by computer automatic sequence and PCR analysis. Pure single layer cat corneal endothelial cells were successfully cultured by tearing endothelium technique. Effectene (TM) lipofectine transfection technique could be effectively used to transfer pcDNA(4)-PDGF-B into cat corneal endothelial cells in vitro, the transfection efficiency was 30%. RT-PCR result showed that human PDGF-B gene was highly expressed in transfected cat corneal endothelial cells. The expressed PDGF-BB protein promoted the viability of cat corneal endothelial cells. CONCLUSION: Human platelet-derived growth factor-B (PDGF-B) cDNA could be highly Expressed in cultured cat corneal endothelial cells by gene transfection techniques. Expressed PDGF-BB protein sign