Construction and identification of endothelial cell model with overexpressed human protein kinase Cβ<sub>2</sub> induced by high glucose

段炼; 林雪波; 周波

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Construction and identification of endothelial cell model with overexpressed human protein kinase Cβ₂ induced by high glucose

更新时间：2026-01-09

- Construction and identification of endothelial cell model with overexpressed human protein kinase Cβ₂ induced by high glucose
- Journal of Army Medical University Vol. 31, Issue 20, Pages: 1993-1996(2009)
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  CLC： R394-33;R394.3;R587.102
- Published：2009
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段炼,林雪波,周波.高糖负荷下人蛋白激酶Cβ2基因过表达内皮细胞模型的构建及鉴定[J].陆军军医大学学报(原第三军医大学学报),2009,31(20):1993-1996.

　DUAN Lian,LIN Xue-bo,ZHOU Bo.Construction and identification of endothelial cell model with overexpressed human protein kinase Cβ2 induced by high glucose[J].J Amry Med Univ (J Third Mil Med Univ),2009,31(20):1993-1996.
段炼,林雪波,周波.高糖负荷下人蛋白激酶Cβ2基因过表达内皮细胞模型的构建及鉴定[J].陆军军医大学学报(原第三军医大学学报),2009,31(20):1993-1996. DOI：

　DUAN Lian,LIN Xue-bo,ZHOU Bo.Construction and identification of endothelial cell model with overexpressed human protein kinase Cβ2 induced by high glucose[J].J Amry Med Univ (J Third Mil Med Univ),2009,31(20):1993-1996. DOI：

摘要

目的应用AdMax系统构建含人蛋白激酶Cβ

(protein kinase C β

PKCβ

)基因的重组腺病毒载体，转染人脐静脉内皮细胞(HUVEC)，高糖刺激下观察蛋白量的表达，构建进一步功能研究的细胞模型。方法从pMD18-T-PRKCB1质粒中，扩增出人PRKCB1。所得片段定向克隆至穿梭质粒pDC315上，后与骨架质粒pBHGlox△E1，3Cre共转染293细胞，经同源重组构建腺病毒Ad-PRKCB1，TCID50测定重组病毒的滴度，免疫细胞化学和RT-PCR方法进行鉴定。构建成功后转染HUVEC，激光共聚焦显微镜(LSCM)下，观察高糖刺激下荧光蛋白转位及表达量

鉴定细胞模型构建。结果通过酶切鉴定目的片段正确插入穿梭质粒。倒置显微镜下观察到细胞的病变效应。测得病毒滴度为7.9×10

IU/ml，免疫细胞化学和RT-PCR验证了目的蛋白表达。LSCM下观察到高糖负荷下荧光蛋白转位及过表达。结论成功构建了含PRKCB1基因的重组腺病毒载体并在高葡萄糖负荷下观察到其活化及蛋白过表达，为下一步信号蛋白组学研究提供分子工具。

Abstract

Objective To construct the endothelial cell model with overexpressed human protein kinase C β

(PKCβ

) after high glucose inducement in order to study the function of human PKCβ

. Methods The PRKCB1 gene was amplified from pMD18-T-PRKCB1 plasmid and then directly cloned into shuttle plasmid pDC315 to construct shuttle plasmid. Then the recombinant shuttle plasmid and adenovirus genomic plasmid pBHGlox△E1

3Cre were co-transfected into 293

cells to construct recombinant adenovirus Ad-PRKCB1. The virus titer was calculated by TCID50. The Ad-PRKCB1 was verified by immunocytochemistry and RT-PCR. Ad-PRKCB1 was transfected into human umbilical vein endothelial cells (HUVECs) followed by the treatment of high glucose (25 mmol/L) for 96 h

and then the cells were observed by laser scanning confocal microscopy (LSCM). Results Restrictive endonuclease digestion and PCR result showed that the target gene was correctly cloned into shuttle plasmid. Cytopathic effect (CPE) was observed under inverted microscope through homologous recombination. The virus titer was 7.9×10

IU/ml. Immunocytochemical staining and RT-PCR indicated the expression of human PKCβ

in the transfected HUVECs. The translocation was observed under LSCM. Conclusion A recombinant adenovirus vector with human PKCβ

and the endothelial cell model with human PKCβ

overexpression by high glucose are constructed successfully.

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Construction and identification of endothelial cell model with overexpressed human protein kinase Cβ2 induced by high glucose

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摘要

Abstract

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references

Construction and identification of endothelial cell model with overexpressed human protein kinase Cβ₂ induced by high glucose