高糖应激致人PRKCD基因过表达内皮细胞模型构建及鉴定

目的:构建高糖应激下人PRKCD基因过表达内皮细胞模型并鉴定。方法:设计含AgeⅠ和NheⅠ酶切位点的PRKCD基因上下游引物,以含PRKCD基因的原始质粒为模版,PCR扩增获得PRKCD全部序列,经AgeⅠ和NheⅠ酶切后与同样酶切后的真核表达载体pDC316-LacZα重组获得穿梭质粒pDC316-PRKCD,经PCR及酶切、基因测序鉴定后,与腺病毒骨架质粒pB-HGlox△E1,3Cre共转染293细胞获得重组腺病毒Ad5-PRKCD,行PCR鉴定并反复纯化扩增后用TCID50法测定病毒滴度。分组培养人脐静脉内皮细胞,转染重组腺病毒后于高糖(25mmol/L)负荷,并设立空载体对照及渗透压对照组,以免疫荧光法检测PKCδ在细胞中的表达。结果:目的基因成功插入穿梭质粒,基因测序结果与GenBank公布序列一致,重组腺病毒Ad5-PRKCD经PCR鉴定及免疫荧光鉴定成功。测得病毒滴度为1.0×1010IU/ml。激光共焦聚观察高糖负荷下,胞内PRKCD翻译产物PKCδ荧光表达强度明显增强,为正常对照组的1.5倍(P0.05),高糖负荷下内皮细胞感染重组腺病毒后PKCδ荧光强度明显增加,浆/核荧光强度比值较高糖组进一步降低了35%(p0.05),提示核转位明显。结论:成功构建了人重组腺病毒Ad5-PRKCD并有效转染人脐静脉内皮细胞,高糖负荷使PKCδ表达上调并发生核转位激活,为筛选稳定表达PKCδ的内皮细胞株及其蛋白复合体奠定了基础。

Construction and Identification of Endothelial Cell Model with Human PRKCD Gene Overexpression under High Glucose Stress

Objective:To construct endothelial cell model with hPRKCD gene overexpression under high glucose stress, and to investigate the expression of PKCδ in human umbilical vein endothelial cell (HUVEC) transfected with Ad5 - PRKCD. Method: Full length PRKCD cDNA was obtained from p rimary plasmid by PCR amplification. The PCR product and eukaryotic expression vector pDC316 - lacZα were digested by restriction endonucleases Age Ⅰ and Nhe Ⅰ and constructed the recombinant shuttle plasmid pDC316 - PRKCD. Gene sequencing was performed to identify the recombinant shuttle plasmid. The adenovirus genomic plasmid pBHGloxΔE1 ,3Cre co - transfected with shuttle plasmid into 293 cells to construct recombinant adenovirus Ad5 - PRKCD. The virus titer was calculated by TCID50. Ad5 -PRKCD was then used to transfect into HUVEC under high glucose stress (25 mmol/L) , and the expression of PKCδ was investigated by immunofluorescence assay. Result: The recombinant shuttle plasmid was successfully identified by gene sequencing as repoted in Gen-Bank, the recombinant adenovirus Ad5 - PRKCD was successfully constructed with high titer ( 1. 0 × 10~(10) IU/ml). under high glucose stress, the expression of PKCδ was increased by 1. 5 fold compared with the normal control(P <0.05) ,thus after transfected with Ad5 -PKCδ, it is even more increased under high glucose stress, and the fluorescence of cytosol - to - nuclei is decreased by 35% (p < 0.05 ) ,indicating obvious nuclear translocation. Conclusion: The recombinant adenovirus vector Ad5 -PRKCD was successfully constructed and effectively transfected into HUVEC, PKCδ translocated to nucleus under high glucose stress, It is a potential tool for screening HUVEC stably expressing PKCδ. And isolating protein complex.