Wilson S E, Lloyd S A, He Y G, McCash C S
Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas 75235-9057.
Invest Ophthalmol Vis Sci. 1993 May;34(6):2112-23.
To transfect human corneal endothelial cells with a plasmid vector coding for the SV40 large T antigen to extend the life of the cells in culture.
Human corneal endothelial cells were transfected with the SV40 large T antigen-coding plasmid pSV3neo using the electroporation method. Transfected and control cells were propagated in culture until senescence. Polymerase chain reaction and immunofluorescence were used to demonstrate messenger RNA and protein, respectively, for the Simian virus 40 large T antigen in the transfected cells. Polymerase chain reaction and hot blotting were used to demonstrate messenger RNA coding for several growth factors and receptors in transfected and control cells.
The transfected cells continued to proliferate to 38 passages (more than 120 population doublings) in culture (control cells, 8 population doublings). Transfected cells, but not control cells, expressed messenger RNA coding for the Simian virus 40 large T antigen. Similarly, immunofluorescent staining with monoclonal antibodies demonstrated that the Simian virus 40 large T antigen protein was present in the nucleus of the transfected cells. Transfected cells were shown to produce messenger RNA coding for epidermal growth factor, epidermal growth factor receptor, basic fibroblast growth factor, fibroblast growth factor receptor-1, interleukin-1 alpha, the interleukin-1 receptor, transforming growth factor beta-1, and the glucocorticoid receptor. Qualitative expression of the messenger RNA coding for each of these modulators was similar in proliferating primary corneal endothelial cells and proliferating or confluent transfected corneal endothelial cells.
In culture, the life of human corneal endothelial cells transfected with a plasmid vector coding for the Simian virus 40 large T antigen is extended. This study suggests that human corneal endothelial cells have the capacity for extensive proliferation, but the proliferation of untransfected cells is regulated through mechanisms that have not yet been characterized.
用编码SV40大T抗原的质粒载体转染人角膜内皮细胞，以延长其在培养中的寿命。
采用电穿孔法用编码SV40大T抗原的质粒pSV3neo转染人角膜内皮细胞。转染细胞和对照细胞在培养中传代直至衰老。分别用聚合酶链反应和免疫荧光法检测转染细胞中猿猴病毒40大T抗原的信使核糖核酸和蛋白质。用聚合酶链反应和热印迹法检测转染细胞和对照细胞中几种生长因子和受体的信使核糖核酸编码情况。
转染细胞在培养中持续增殖至38代（超过120次群体倍增）（对照细胞为8次群体倍增）。转染细胞而非对照细胞表达编码猿猴病毒40大T抗原的信使核糖核酸。同样，用单克隆抗体进行免疫荧光染色显示，猿猴病毒40大T抗原蛋白存在于转染细胞的细胞核中。转染细胞被证明可产生编码表皮生长因子、表皮生长因子受体、碱性成纤维细胞生长因子、成纤维细胞生长因子受体-1、白细胞介素-1α、白细胞介素-1受体、转化生长因子β-1和糖皮质激素受体的信使核糖核酸。这些调节因子的信使核糖核酸编码在增殖的原代角膜内皮细胞以及增殖或汇合的转染角膜内皮细胞中的定性表达相似。
在培养中，用编码猿猴病毒40大T抗原的质粒载体转染的人角膜内皮细胞寿命延长。本研究表明，人角膜内皮细胞具有广泛增殖的能力，但未转染细胞的增殖通过尚未明确的机制受到调控。
