卡波氏肉瘤病毒编码的miR-K7-3p重组慢病毒载体构建及其对血管内皮细胞增殖的影响  

Construction of overexpression plasmid carrying KSHV-encoded miR-K7-3p and identification of its role in cell proliferation

作　　者：朱凯翔[1] 卢春[1] 

机构地区：[1]南京医科大学病原生物学系,江苏南京211166

出　　处：《江苏大学学报（医学版）》2017年第1期18-23,共6页Journal of Jiangsu University：Medicine Edition

摘　　要：目的:构建卡波氏肉瘤病毒(KSHV)编码的miR-K7-3p的重组慢病毒载体,并检测其对人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)增殖能力的影响。方法:将miR-K7-3p的序列及互补序列插入miR-30茎环结构,以此为模板扩增出目的片段,插入慢病毒载体mp CDH-CMV-MCS-EF1-tRFP(简写为mp CDH,tRFP基因可表达红色荧光蛋白)中,构建重组慢病毒载体mp CDH-miR-K7-3p。将构建的目的质粒mp CDH-K7-3p与包装质粒ps PAX2、包膜质粒p MD2.G共同转染人胚肾上皮细胞(293 T),包装表达miR-K7-3p的慢病毒。采用病毒梯度稀释法测定病毒滴度。用包装成功的miR-K7-3p慢病毒感染HUVECs,荧光实时定量PCR(qRT-PCR)检测miR-K7-3p的表达,CCK-8及平板克隆实验检测细胞增殖力。结果:双酶切鉴定、核酸序列测定与比对结果证实重组慢病毒质粒mp CDH-miR-K7-3p构建成功。qRT-PCR检测到重组慢病毒感染的HUVECs中miR-K7-3p的表达水平升高。增殖实验结果显示,miR-K7-3p慢病毒感染后的HUVECs增殖能力明显强于对照组。结论:miR-K7-3p可以明显促进HUVECs的增殖。Objective： To construct the recombinant lentivirus expressing vector carrying Kaposi＇s sarcoma-associated herpesvirus（ KSHV）-encoded miR-KT-3p, and identify its function in regulating the proliferation of endothelial cells. Methods： miR-30-stem-loop inserted with miR-K7-3p sequence and its complementary sequence was used as template for polymerase chain reaction（PCR）. Sequences were then inserted into a lentiviral vector mpCDH-CMV-MCS-EFI-tRFP to construct the recombinant lentivirus vector mpCDH- miR-K7-3p. The recombinant plasmid mpCDH-miR-K7-3p, package plasmid psPAX2 and envelope plas- mid pMD2. G were co-transfected into the 293 T cells, and filtered culture media was collected. Viral titer was determined by virus gradient dilution, together with observing the expression of red fluorescent protein （RFP）. The human umbilical vein endothelial cells（HUVECs） were infected with lentivirus-miR-K7-3p and lentivirus-mpCDH in the same multiplicity of infection （MOI） and efficiency of overexpression was detected by qRT-PCR. At last, cell proliferation was detected by Cell Counting Kit-8 （CCK-8） assays. Results： Double digestion of the plasmid and nucleic acid sequencing identified that the recombinant plasmid carrying miR-KT-3p were successfully constructed, qRT-PCR showed the elevated level of miR-K7-3p mR-NA in virus infected HUVECs. Further, the CCK-8 assay and tablet clone forming experiment offered efficient evidence that miR-K7-3p can enhance cell proliferation. Conclusion： The recombinant lentivirus vector of miR-K7-3p was successfully constructed and can effectively enhance cell proliferation.

关 键 词：卡波氏肉瘤病毒 miR-K7-3p 慢病毒 人脐静脉内皮细胞 细胞增殖 平板克隆 

分 类 号：R73-36[医药卫生—肿瘤]