重组质粒rAd-EGF构建并转染hDPSCs对其增殖的影响  

Proliferation of human dental pulp stem cells transfected by recombinant adenovirus mediated epidermal growth factor in vitro

作　　者：付广丽[1] 鲍艳[1] 吴桂堂[1] 刘兴容[1] 

机构地区：[1]泸州医学院附属口腔医院预防保健科,四川泸州646000

出　　处：《泸州医学院学报》2015年第1期27-31,共5页Journal of Luzhou Medical College

基　　金：四川省卫生厅基金项目(060051)

摘　　要：目的:重组腺病毒介导的表皮生长因子(recombinant adenovirus mediated epidermal growth factor,r Ad-EGF)质粒的构建,及体外转染人牙髓干细胞(human dental pulp stem cells,h DPSCs)后对其增殖的影响。方法:采用目的载体PYr-adshuttle-1和EGF基因(PCR4-TOPO-EGF)构建PYr-ads-1-EGF,在体外重组到PAd/PL-DEST构建r Ad-EGF,并将其体外转染到h DPSCs。将h DPSCs分为3组:r Ad-EGF转染组(实验组)、r Ad-EGFP阴性对照组和空白对照组。当细胞密度达到70%时,用MOI=100的r Ad-EGF或r Ad-EGFP转染各组细胞,在37℃,5%CO2的条件下,用胰酶消化培养48 h后的各组细胞并收集,采用Western blotting检测转染后各组细胞EGF蛋白的表达,采用MTT比色法检测细胞增殖情况。结果:经PCR鉴定,r AdEGF包装成功。在一定时间内,r Ad-EGF转染入h DPSCs,实验组h DPSCs的EGF蛋白表达水平显著高于其他两组(F=339.795,P<0.001),其细胞增殖能力明显升高(F=23.937,P<0.001)。结论 :r Ad-EGF质粒构建成功,r Ad-EGF转染h DPSCs后,其EGF蛋白明显升高,对其增殖具有促进作用。Objective： To research the proliferation of human dental pulp stem cells（h DPSCs） transfected by recombinant adenovirus mediated human epidermal growth factor（r Ad-EGF） in vitro. Methods： The human dental pulp stem cells were divided into 3 groups：the experimental group transfected by r Ad-EGF, the control group transfected by r Ad-EGFP and the blank. When the cell density reached to 70%, the experimental and control group were transfected by 100 MOI r Ad-EGF or r Ad- EGFP, and the same amount DMEM was added into blank group. After cultured at 37 ℃, 5% CO2 for 48 h, the cells were digested and collected. The expression of EGF proteins and m RNA was detected by Western-blot and RT-PCR in each group, and the proliferation of h DPSCs was measured by MTT. Results： The r Ad-EGF was constructed successfully.The expression of EGF protein and m RNA in the r Ad- EGF group was significantly higher than that of other two groups（F = 442.797, P〈0.001）,and the proliferation of h DPSCs increased（F = 23.937, P〈0.001）. Conclusion： The expression of EGF protein and m RNA could be increased obviously after the r Ad-EGF was tranfected into human dental pulp stem cell,which could promote the proliferation of h DPSCs.

关 键 词：腺病毒 表皮生长因子 人牙髓干细胞 转染 增殖 

分 类 号：R789[医药卫生—口腔医学]