PSA启动子介导的TAT-p21^(WAF1/CIP1)融合蛋白在前列腺癌细胞中的特异表达及活性初探  

Targeting expression of TAT-p21^(WAF1/CIP1) fusion protein driven by PSA promoter in prostate cancer cells

作　　者：蔡捷[1] 苑辉卿[1] 孔峰[1] 任凯[1] 王小玲[1] 吉恺[1] 胡晓燕[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教研室,山东济南250012

出　　处：《中国病理生理杂志》2008年第3期498-503,共6页Chinese Journal of Pathophysiology

基　　金：山东省卫生厅重点资助项目(No.HZ023);山东大学大学生科技创新基金

摘　　要：目的:设计并构建含有前列腺组织特异性抗原(PSA)启动子的TAT-p21WAF1/CIP1融合蛋白真核表达载体(pPSA-TAT-p21),使由TAT蛋白转导结构域(TAT)介导的抑癌基因蛋白p21WAF1/CIP1(p21)能够在前列腺癌细胞中实现特异性的跨膜转运,增强p21对前列腺癌的抑制作用。方法:利用PCR技术构建含有PSA启动子、TAT蛋白转导序列和p21读码框架的真核表达载体pPSA-TAT-p21,并进行酶切、测序鉴定。脂质体法在前列腺癌LNCaP细胞和大肠癌HT-29细胞中转染上述表达载体及对照质粒后进行反转录PCR(RT-PCR),检测p21的mRNA以及蛋白在不同细胞中的表达情况。MTT法检测转染pPSA-TAT-p21后LNCaP细胞的增殖情况。结果:成功构建pPSA-TAT-p21真核表达载体。RT-PCR和Western blotting结果显示PSA-TAT-p21在前列腺癌细胞中有明显表达,在大肠癌细胞中基本不表达,呈现特异性的表达,而由CMV启动子介导的p21(pCMV-21)对照组在两个细胞株中均有表达。细胞增殖实验结果显示,含有TAT蛋白转导结构域的PSA-TAT-p21的融合蛋白对前列腺癌细胞增殖的抑制作用明显高于不含TAT的PSA-p21蛋白。结论:pPSA-TAT-p21能够在前列腺癌细胞中特异性地高效表达,为前列腺癌基因的靶向治疗提供了实验依据。AIM： A eukaryotic expression vector containing p21^WAF1/CIP1 （p21） gene fused to TAT transduction domain under the control of prostate - specific antigen promoter was constructed. Targeting expression of TAT - p21 fusion protein was examined in prostate cancer cells and colonic cancer cells. METHODS： The eukaryotic expression vector pPSA -TAT- p21 was produced by using the polymerase chain reaction （PCR）. Transient transfections of pPSA -TAT- p21, pPSA - p21 or pCMV - p21 into the prostate cancer cell LNCaP and colonic cancer cell HT - 29 were conducted by using the lipofectamine 2000. Reverse transcription PCR （ RT - PCR） was used to detect the expression of p21 in the level of mRNA. Prostate cancer cell growth was analyzed by the method of MTT. RESULTS： The vector pPSA -TAT -p21 was constructed and the entire coding region was confirmed by sequencing. RT - PCR results indicated that expressions of pPSA -TAT- p21 and pPSA -p21 were shown in prostate cancer cells and no detectable band was observed in colonic cancer cells, whereas pCMV -p21 expressed in both the cell lines. Inhibitory effect of p21 on cell growth was found from the resuits of MTT assay, indicating that pPSA - TAT - p21 had higher inhibitory effect when compared to pPSA - p21 group. CONCLUSION： The vector pPSA- TAT- p21 targeting expresses in prostate cancer cells and inhibits cell proliferation.

关 键 词：前列腺特异抗原 TAT—p21^WAF1/CIP1融合蛋白 前列腺肿瘤 LNCAP细胞 HT-29细胞 

分 类 号：R363[医药卫生—病理学]