CD44过表达慢病毒载体的构建及其在前列腺癌LNCaP细胞中的表达  

Construction of CD44 gene overexpression lentiviral vector and its expression in LNCaP cells

作　　者：潜力[1] 符翠莉[1] 李伟[2] 

机构地区：[1]广西壮族自治区人民医院药学部,南宁530021 [2]广西壮族自治区人民医院泌尿外科,南宁530021

出　　处：《中华老年医学杂志》2017年第9期1015-1018,共4页Chinese Journal of Geriatrics

基　　金：国家自然科学基金（81460387）;广西卫生和计划生育委员会自筹基金（Z2016586）

摘　　要：目的构建CD44基因过表达慢病毒载体并建立稳定过表达CD44的雄激素依赖前列腺癌LNCaP细胞株。方法聚合酶链反应（PCR）法获得CD44基因序列，与经NotI和NsiI双酶切的LV5载体连接获得CD44过表达重组慢病毒载体，重组质粒经双酶切鉴定和测序验证无误后，进行高纯度抽提，共转染293T细胞，收集富含慢病毒颗粒的细胞上清液，对其浓缩后得到高滴度的慢病毒浓缩液，倍比稀释法检测病毒滴度。用病毒颗粒感染LNCaP细胞，荧光显微镜检测CD44荧光水平，定量反转录聚合酶链反应（QRT—PcR）检测CD44基因的表达，免疫印迹检测CD44蛋白的表达。结果成功构建CD44基因过表达慢病毒载体，并在293T细胞中包装获得病毒。重组病毒感染LNCaP细胞后，荧光显微镜检测CD44基因过表达细胞荧光水平增加；QRT—PCR和免疫印迹检测显示，CD44在LNCaP细胞中表达显著升高（均P〈O．05）。结论成功构建CD44基因过表达慢病毒载体，并建立CD44过表达LNCaP细胞株。Objective To construct an overexpression lentiviral vector of CD44 and obtain an androgen-dependent prostate cancer LNCaP cell line with CD44 overexpression. Methods CD44 gene sequence was obtained by PCR amplification. The synthesized oligonucleotides were cloned in the lentiviral vector LV5 after digested with NotI and NsiI enzyme. Recombinant plasmids were verified by enzyme digestion analysis and sequencing, and were transfected into 293T package cells. Supernatant was collected and concentrated to obtain lentivirus solution at a high titer. The titer of virus was identified by multiple proportion dilution methods. Then the recombinant viruses were transfected into LNCaP cells. Results Fluorescence intensity in infected LNCaP cells was increased,detected by a fluorescence microscope. And QRT-PCR and Western blot results showed that the expression of CD44 was significantly increased（P〈0.05）. Conclusions The overexpression lentiviral vector of CD44 is successfully constructed,and the LNCaP cell line with CD44 overexpression is obtained.

关 键 词：前列腺肿瘤 抗原 CD44 

分 类 号：R737.25[医药卫生—肿瘤]