鞘脂激活肽真核表达载体的构建及其对前列腺癌细胞增殖的影响  

Effect of ectopic expressed Neurotrophic Peptide on proliferation of human prostate cancer cells

作　　者：任凯[1] 苑辉卿[1] 孔峰[1] 蔡捷[1] 王小玲[1] 胡晓燕[1] 徐霞[1] 张建业[1] 

机构地区：[1]山东大学医学院生物化学与分子生物学研究所,山东济南250012

出　　处：《山东大学学报（医学版）》2007年第1期1-5,9,共6页Journal of Shandong University:Health Sciences

基　　金：教育部科学技术研究重点项目(106101);山东省卫生厅重点项目(HZ023)

摘　　要：目的:构建含有鞘脂激活蛋白原神经营养序列短肽-鞘脂激活肽NP(neurotrophic peptide,NP)的真核表达载体以及NP与TAT的融合蛋白真核表达载体(TAT-NP),探讨NP在前列腺癌细胞中的作用。方法:根据NP和TAT的氨基酸序列合成其DNA序列,利用基因克隆技术构建含有TAT和NP读码框架的真核表达载体pcDNA-TAT-NP和pcDNA-NP。在前列腺癌PC3、LNCaP细胞中转染上述载体后通过RT-PCR、MTT和流式细胞术,分别检测TAT-NP、NP的mRNA的表达,NP对前列腺癌细胞的增殖情况以及对细胞周期的影响。结果:经测序鉴定,成功构建pcDNA-TAT-NP和pcDNA-NP真核表达载体。RT-PCR结果显示,TAT-NP、NP能够在前列腺癌细胞中表达。MTT结果表明,TAT-NP、NP均对前列腺癌细胞增殖具有促进作用。流式细胞结果显示,TAT-NP、NP具有促进细胞进入S和G2/M期的作用。结论:外源表达的鞘脂激活肽NP能够促进前列腺癌细胞的增殖,并能影响细胞周期各时相的变化。Objective： Based on neurotrophic domain sequence （NP） of saposin C, eukaryotic expression vectors containing NP sequence and NP fused to TAT transduction domain were respectively constructed. The TAT trans- membrane signal was expected to help NP go through cell membranes and have effects on prostate cancer cells, Methods： The eukaryotic expression vectors pcDNA-TAT-NP and pcDNA-NP were constructed by using gene cloning technology. After transient transfection of pcDNA-TAT-NP and pcDNA-NP into the prostate cancer cells PC3 and LNCaP, RT- PCR, MTT and flow cytometry were used to respectively detect the expression of TAT-NP and NP in the level of mRNA, prostate cancer cell growth and the cell cycle changes. Results： The eukaryotic expression vectors pcDNA-TAT-NP and pcDNA-NP were constructed and confirmed by sequencing. The result obtained from RT-PCR indicated both pcDNA- TAT-NP and pcDNA-NP were expressed in prostate cancer cells. MTT analysis showed that expressions of TAT-NP and NP had a stimulating effect on prostate cancer cell proliferation, and promoted the cells to enter S and G2/M phases. Conclusion： Ectopic expression of NP could enhance the prostate cancer cell growth and affect the cell cycle.

关 键 词：鞘脂激活蛋白原 鞘脂激活肽 TAT蛋白转导结构域 流式细胞术 前列腺肿瘤 细胞株 

分 类 号：R737.25[医药卫生—肿瘤]