KDR启动子／增强子靶向干扰磷脂酰肌醇3激酶信号通路对大鼠血管内皮细胞增殖和凋亡的影响  

RNA interference of phosphatidylinositol 3-kinase signaling pathway by kinase insert domain-con- taining receptor promoter/enhancer influences the proliferation and apoptosis of rat vascular endo- thelial cells

作　　者：王倩[1] 邓勇志[1] 徐俊文[1] 陈丽[1] 杨雪峰[1] 

机构地区：[1]山西医科大学第二医院心胸外科,太原030001

出　　处：《中华实验外科杂志》2012年第8期1440-1442,共3页Chinese Journal of Experimental Surgery

基　　金：山西省回国留学人员科研资助项目（2011-107）;太原市技术创新与人才扶持计划人才扶持专项（11014926）

摘　　要：目的针对大鼠血管内皮细胞（VEC）的磷脂酰肌醇3激酶p亚单位（Pik3cb），构建KDR特异性启动子／增强子真核表达载体，观察其对VEC增殖和凋亡的影响。方法构建KDR特异性启动子／增强子真核表达载体并转染大鼠VEC。实验分5组，A组：正常VEC；B组：转染特异性KDR质粒，浓度2．0mg／L；C组：转染非特异性巨细胞病毒（CMV）质粒，浓度2．0mg／L；D组：转染空质粒，浓度2．0mg／L；E组：阳性对照渥曼青霉素，浓度50nmol／L。分别于转染24、48、72h后，实时荧光定量逆转录．聚合酶链反应（RT．qPCR）法检测各组细胞Pik3cbmRNA相对表达量，细胞计数试剂盒（CCK_8）和流式细胞仪检测各组细胞增殖抑制率和凋亡率。结果转染24、48、72h后，RT—qPCR法测得KDR组Pik3cbmRNA相对表达量分别为（54．82±2．77）％、（50．54±3．98）％和（35．47±4．83）％，均明显低于正常对照组（P〈0．05）；CCK_8法测得KDR组细胞增殖抑制率分别为（21．98±2．25）％、（24．32±3．04）％和（26．38±5．06）％；流式细胞仪测得KDR组细胞凋亡率分别为（9．9±1．3）％、（31．0±7．4）％和（44．5±8．3）％，细胞增殖抑制率和凋亡率均明显高于正常对照组（P〈0．05）。结论KDR特异性启动子／增强子真核表达载体可通过下调磷脂酰肌醇3激酶（P13K）信号通路特异性抑制VEC增殖并促使其凋亡。Objective To construct the kinase insert domain-containing receptor （KDR） specific protnoter/enhancer eukaryon expression vector for RNA interference of phosphatidylinositol 3-kinase, cata- lytic, beta polypeptide （Pik3cb） gene in rat vascular endothelial cells （VECs） and evaluate the effects on the proliferation and apoptosis of VECs. Methods Both the specific KDR promoter/enhancer eukaryon ex- pression vector and non-specific cytomegalovirus （CMV） eukaryon expression vector were constructed and transfected into VECs, respectively. The samples were divided into 5 groups ： group A, normal VECs with- out any treatment ; group B, VECs transfected with 2. 0 mg/L KDR plasmid ; group C, VECs transfected with 2. 0 mg/L CMV plasmid ; group D, VECs transfected with 2. 0 mg/L empty plasmid ; group E ： VECs treated with 50 nmol/L wortmannin. At 24, 48, and 72 h after treatments, the expression level of Pik3cb mRNA was detected by using real-time quantitative reverse transcription-polymerase chain reaction （RT- qPCR） , and proliferation and apoptosis of VECs were analyzed by cell counting Kit-8 （ CCK-8 ） and flow cytometry, respectively. Results At 24, 48 and 72 h after treatments, the real-time RT-PCR revealed that the relative expression of PikScb mRNA in group B was （54. 82 ±2.77）% , （50. 54 ±3.98）% and （35.47 ± 4. 83 ）% respectively, significantly lower than in groups A and D （P 〈 0. 05 ） ; The CCK-8 anal- ysis showed the inhibition rate of VECs proliferation in group B was （ 21.98± 2. 25 ） %, （ 24. 32 ± 3.04） % and （ 26. 38 ± 5.06 ） % respectively, significantly higher than in groups A and D （ P 〈 0. 05 ） ; the results of flow cytometry indicated that the apoptosis rate in group B was （9.9 ± 1.3）% , （31.0 ±7.4 ） % and （44. 5 ± 8.3 ） % respectively, significantly higher than in group A （ P 〈 0. 05 ）. Conclusion The inhibition of P13K signaling pathway by KDR specific promoter/enhancer eukaryon expression vector in rat VECs down-re

关 键 词：RNA干扰 血管内皮细胞 磷脂酰肌醇3激酶 

分 类 号：R654.3[医药卫生—外科学]