SCF对体外转染pEGFP-C1/Akt的骨髓间充质干细胞中c-kit、Akt及VEGF表达的影响  

Stem Cell Factor Enhances Expression of c-kit,Akt and VEGF in Mesenchymal Stem Cells Transfected by pEGFP-C1/Akt Gene in Vitro

作　　者：霍鑫[1] 唐海燕[2] 孔宏亮[3] 刘英[4] 张宏伟[1] 王欣[1] 胡新华[1] 张强[1] 

机构地区：[1]中国医科大学附属第一医院干诊科,沈阳110001 [2]大庆油田总医院内分泌科,大庆163000 [3]中国医科大学附属第一医院循环科,沈阳110001 [4]中国医科大学附属第一医院内分泌科,沈阳110001

出　　处：《中国普外基础与临床杂志》2008年第2期116-121,共6页Chinese Journal of Bases and Clinics In General Surgery

摘　　要：目的克隆构建绿色荧光蛋白(GFP)/Akt表达载体,观察其在鼠骨髓间充质干细胞(MSCs)中的表达和定位,并探讨干细胞因子(SCF)通过PI3-Akt途径对转染pEGFP-C1/Akt的MSCs中c-kit、Akt和VEGF mRNA及蛋白表达的影响。方法采用酶切法将Akt重组于GFP表达载体中,经酶切序列鉴定后,将该重组质粒GFP/Akt及GFP空质粒通过脂质体转染骨髓MSCs;荧光显微镜观察其转染及在细胞中的分布;分别采用Western blot和反转录多聚酶链反应(RT-PCR)方法检测pEGFP-C1和pEGFP-C1/Akt转染MSCs后对c-kit、Akt及VEGF mRNA和蛋白表达的影响,pEGFP-C1/Akt转染MSCs后不同浓度SCF对c-kit、Akt及VEGF mRNA和蛋白表达的影响。结果GFP/Akt基因表达载体经酶切鉴定和测序分析后确认构建成功,并在MSCs中表达。荧光显微镜下见pEGFP-C1转染后,绿色荧光均匀分布于整个细胞中;pEGFP-C1/Akt转染后,绿色荧光分布于细胞质中。与pEGFP-C1组相比较,pEGFP-C1/Akt转染组Akt及VEGF的mRNA和蛋白表达均明显增强(P<0.05),c-kit mRNA和蛋白表达则无明显变化(P>0.05)。加入SCF后其能增强实验组(SCF+pEGFP-C1/Akt)和对照组(SCF+pEGFP-C1)中c-kit、Akt及VEGF的mRNA和蛋白表达,并且随着SCF的浓度增高该作用则越明显(P<0.05);与对照组相比较,实验组c-kit mRNA和蛋白的表达无明显变化(P>0.05),而Akt和VEGF mRNA和蛋白表达则明显增强(P<0.01)。结论成功构建GFP/Akt融合基因表达载体在MSCs中表达,可诱导Akt及VEGF mRNA和蛋白的表达;SCF可能通过PI3/Akt途径刺激c-kit、Akt及VEGF mRNA和蛋白的表达。Objective To construct green fluorescent protein （GFP）/Akt fusion gene vector for observing the expression and localization of GFP/Akt in rats bone marrow-derived mesenchymal stem cells （MSCs）. Stem cell factor （SCF） effected expression of c-kit, Akt and VEGF mRNA and protein in MSCs transfected by pEGFP-C1/Akt through PI3-Akt pathway. Methods Akt recombined GFP vector by restriction enzymes, MSCs was transfeced by GFP/Akt and GFP through cationic liposomes, and then veritied by restriction endonuclease assay and sequence analysis. Transfection and localization of GFP were evaluated by fluorescene microscopy. The expressions of c-kit, Akt and VEGF mRNA and protein were examined by RT-PCR and Western blot after MSCs transfected by pEGFP- C1 and pEGFP-C1/Akt. SCF effected the expression of c-kit, Akt and VEGF mRNA and protein after MSCs transfected by pEGFP-C1 and pEGFP-C1/Akt. Results Restriction endonuclease assay and sequence analysis verified that the successful construction of the recombinant vector pEGFP-C1/Akt and efficient high expression of pEGFP- C1/Akt fusion protein in the MSCs of rats. Under fluorescent microscence, green flurescence was seen homogeneously distributed in the entire cell of the cells transfected by the recombinant vector pEGFP-C1, and diffusely in the cytoplasm of the cells transfected by the recombinant vector pEGFP-C1/Akt. The expression of Akt and VEGF mRNA and protein were significantly higher in MSCs transfected by pEGFP-C1/Akt （P〈0.05）. The expression of c-kit, Akt and VEGF mRNA and protein were significantly higher in experiment group （SCF＋ pEGFP-C1/Akt） and control group （SCF＋pEGFP-C1）, P〈0.05. In experiment group, SCF stimulation enhanced expression of Akt and VEGF mRNA and protein （P〈0.01）. Conclusion GFP/Akt fusion gene vector is successfully construted and the fusion protein expressed in the MSCs of rats induces the expression of Akt and VEGF mRNA and protein. SCF stimulation enhanced expression of c-kit, Akt and VEGF mRNA and protein th

关 键 词：骨髓间充质干细胞 AKT基因 干细胞因子 血管内皮生长因子 

分 类 号：Q78[生物学—分子生物学]