DAB2IP表达载体的构建及其对前列腺癌细胞LNCaP DNA合成及凋亡的影响  

Construction of DAB2IP expression vector and its influence on the apoptosis and DNA synthesis of prostate cancer cell LNCaP

作　　者：肖黎[1] 佟明[1] 金艳阳[1] 秦盛斐[1] 苏雷[1] 

机构地区：[1]辽宁医学院附属第一医院泌尿外科,辽宁锦州121001

出　　处：《中国医学工程》2012年第10期7-8,10,共3页China Medical Engineering

摘　　要：目的构建表达载体pEGFP-N1-DAB2IP,观察DAB2IP在前列腺癌细胞LNCaP中高表达后对细胞凋亡及DNA合成的影响。方法 PCR扩增DAB2IP基因全长,经EcoRⅠ和SalⅠ双酶切后T4 DNA连接酶连接,DH5α转化,用酶切及测序签定来确定构建的质粒正确。用对照载体pEGFP-N1和pEGFP-N1-DAB2IP转染前列腺癌细胞LNCaP,荧光显微镜下观察转染效率,Western blot检测DAB2IP蛋白的表达,应用BrdU/PI掺入法检测DAB2IP对细胞DNA合成的影响,应用AnnexinⅤ-FITC/PI双染法检测两组细胞凋亡情况。结果 pEGFP-N1-DAB2IP表达载体经酶切和测序鉴定正确,并成功转染LNCaP细胞,Western blot显示转染后DAB2IP的蛋白表达水平明显升高。BrdU/PI掺入法检测:pEGFP-N1组BrdU+细胞比例:0.66±6%,pEGFP-N1-DAB2IP组BrdU+细胞比例:0.45±3%,两组比较差异有统计学意义(P<0.05),提示DAB2IP能抑制LNCaP细胞的DNA合成。细胞凋亡检测:pEGFP-N1组凋亡细胞比例:7.44±0.68%,pEGFP-N1-DAB2IP组:17.98±1.5%,两组比较差异有统计学意义(P<0.05),提示DAB2IP能诱导前列腺癌细胞LNCaP的凋亡。结论成功构建了表达载体pEGFP-N1-DAB2IP,并在前列腺癌细胞LNCaP中成功高表达DAB2IP,DAB2IP高表达后能抑制LNCaP DNA合成并诱导细胞的凋亡。【Objective】 To construct the recombinant plasmid pEGFP-N1-DAB2IP and observe its effect on apoptosis and DNA synthesis in human prostate cancer cell LNCaP.【Methods】The full length of DAB2IP was amplified by PCR.After digestion by EcoRⅠand SalⅠ,ligation with pEGFP by T4 DNA ligase,and transformation into DH5α,the recombinant plasmid pEGFP-N1-DAB2IP was identified by enzyme digestion and DNA sequencing.LNCaP cells were transfected with pEGFP-N1-DAB2IP and the control plasmid pEGF-N1 with liposome.Transfection efficiency was observed by fluorescence microscopy.The protein expression of DAB2IP was examined by Western blot.Brdu/PI incorporation was used to determine DNA synthesis of LNCaP cells.Annexin V-FITC/PI double staining were used to detect apoptosis.【Resluts】The recombinant plasmid pEGFP-N1-DAB2IP was successfully constructed.The expression of DAB2IP was significantly increased in the LNCaP cells transfected with the DAB2IP plasmid compared to the control cells.Percentages of apoptotic cells for the pEGFP-N1 group and the pEGFP-N1-DAB2IP group are 7.44±0.68%,and 17.98±1.5% respectively.This result suggested that DAB2IP can induce the cell apoptosis in LNCaP cells.Percentages of Brdu＋ cellsfor the pEGFP-N1 group and pEGFP-N1-DAB2IP group are 0.66±6%,and 0.45±3% respectively..This result suggested that DAB2IP can inhibit the DNA synthesis of LNCaP cells.【Conclusion】The expression vector pEGFP-N1-DAB2IP was successfully constructed.Overexpression of DAB2IP in human prostate cancer cells LNCaP could inhibit DNA synthesis and induce apoptosis.

关 键 词：DAB2IP 前列腺癌 LNCAP DNA 细胞凋亡 

分 类 号：R737.25[医药卫生—肿瘤]