过表达前列腺凋亡反应因子-4对NIT-1细胞的影响  

Effects of overexpression of prostate apoptosis response-4 on the NIT-1 cell

作　　者：吴绮楠[1] 甘霞光 邓武权[1] 张玲 陈兵[1] 

机构地区：[1]第三军医大学第一附属医院内分泌科,重庆400038 [2]第三军医大学第一附属医院门诊部,重庆400038

出　　处：《重庆医科大学学报》2015年第7期931-934,共4页Journal of Chongqing Medical University

基　　金：国家自然科学基金资助项目(编号:81370885);重庆市教委研究生科研创新资助项目(编号:CYB14097);第三军医大学青年人才基金资助项目(编号:SWH2013QN02)

摘　　要：目的:构建携带前列腺凋亡反应因子-4(prostate apoptosis response-4,PAR-4)基因PRKC apoptosis WT1 regulator(pawr)表达的重组腺病毒载体,研究PAR-4对胰岛β细胞株NIT-1的影响。方法:利用Oligo Designer3.0设计Pawr模版引物,通过RT-PCR扩增出pawr基因片段,使用双酶切克隆至腺病毒载体ADV1,构建重组载体质粒p Ad Track-pawr-CMV。线性化p Ad Track-pawr-CMV和p Ad-Easy-1共同转化到感受态大肠杆菌构建重组腺病毒载体质粒p Ad Track-pawr。将NIT-1细胞分为4组:低糖组、高糖对照组、低糖+PAR-4过表达组以及高糖+PAR-4过表达组,Western blot检测各组PAR-4蛋白表达,MTT法检测各组细胞增殖能力,ELISA检测经葡萄糖刺激后的各组细胞胰岛素分泌功能。结果:克隆的pawr基因片段测序正确。高纯度质粒中量抽提试剂盒抽提重组腺病毒载体质粒成功,单酶切鉴定,质粒转染HEK293细胞后有GFP表达,扩增48 h后出现细胞病变效应,病毒滴度测定为1×109 pfu/ml,低糖和高糖过表达PAR-4后均较相应对照组PAR-4表达明显升高,高糖培养+过表达PAR-4组细胞增殖能力均较低糖(P=0.000)和高糖对照组细胞(P=0.000)明显下降,较低糖(P=0.003)和高糖对照组细胞(P=0.001)胰岛素分泌明显下降。结论:成功构建高滴度pawr基因表达的重组腺病毒载体并转染NIT-1细胞,转染PAR-4后高糖培养的NIT-1细胞增殖能力和胰岛素分泌功能明显下降。Objective：To construct recombinant adenovirus vector containing PAR-4 gene,so as to lay a foundation for further study of the function of PAR-4 and to illuminate the effect of PAR-4 on islet β cell line-NIT-1. Methods：Oligo Designer 3.0 was used to design the PCR primers：PRKC apoptosis WT1 regulator（pawr）. Then,the gene fragments of pawr were obtained by RT-PCR and inserted into the ADV1 vector by restriction enzyme digestion.The recombinant vector plasmid p Adtrack-pawr-CMV was constructed,and then,the recombinant p Ad Track-pawr-CMV was linearized and translated into the competent bacteria with p Ad-Easy-1 for homologous recombination to obtain p Ad Track-PAR-4. NIT-1 cell was divided into 4 groups：low glucose control,low glucose＋overexpression of PAR-4,high glucose control,high glucose ＋ overexpression of PAR-4,and protein expression level was detected by Western blot,the proliferation ability by MTT,the secretion ability after glucose stimulation by ELISA. Results：The pawr genes were verified by gene sequencing. The recombinant plasmid was successfully extracted by high purity plasmid extraction median kit. The recombinant adenovirus vector plasmid with mono-restriction endonuclease enzyme was evaluated. The earliest expression of GFP was observed in HEK293 cells and after 48 h of amplification the CPE was observed and the final viral titer was 1×109pfu/ml. The protein expression level of PAR-4 was significantly increased in low glucose＋overexpression of PAR-4 group and high glucose＋overexpression of PAR-4 group than in its control groups. Proliferation ability and insulin secretion were decreased in high glucose＋overexpression of PAR-4 group than in low glucose ＋overexpression of PAR-4 group and high glucose control group. Conclusion：The over-expression of PAR-4 group has higher expression of PAR-4. After transfection and culture by high glucose concentration,the proliferation ability of high-glucose cultured NIT-1 is decreased.

关 键 词：前列腺凋亡反应因子-4 腺病毒载体 2型糖尿病 

分 类 号：R58[医药卫生—内分泌]