摘要
目的探讨阿霉素与肿瘤坏死因子相关凋亡诱导配体(TRAIL)真核表达载体诱导前列腺癌细胞凋亡的协同作用。方法RT-PCR方法克隆人TRAIL全长基因,并插入真核表达载体pLX IN多克隆位点,经酶切和测序鉴定。将pLX IN-TRAIL质粒转染PC3-M细胞,RT-PCR方法检测外源性TRAIL表达率,TUNEL染色和流式细胞术比较单独表达TRAIL和联合应用阿霉素对PC3-M细胞凋亡率的影响,W estern b lot法检测经阿霉素诱导前后PC-3M细胞死亡受体-5(DR5)的表达变化。结果成功构建pLX IN-TRAIL真核表达载体,外源性TRAIL表达可以诱导PC-3M细胞凋亡,阿霉素能够增强TRAIL的凋亡诱导效应,6μg pLX IN-TRAIL质粒DNA组,加入1 mg/L阿霉素前后平均凋亡率分别为11.8%和20.7%,同时可提高PC-3M细胞DR5的表达。结论TRAIL真核表达载体介导的细胞凋亡是治疗前列腺癌的新途径,阿霉素可以通过提高DR5表达而增强PC-3M细胞对TRAIL诱导的凋亡敏感性。
Objective To construct tumor necrosis factor-related apoptosis-inducing ligand （TRAIL） eukaryotic expression vector, and to investigate the synergistic effects of doxorubicin （DOX） and TRAIL on induction of apoptosis in androgen-independent prostate cancer cell line PC-3M. Methods The full-length cDNA sequence of human TRAIL gene was cloned by RT-PCR from the total mRNA of HL-60 cells. The TRAIL gene was sub-cloned into plasmid vector pLXIN. The vector was analyzed by enzyme digestion and cDNA sequencing. The pLXIN-TRAIL plasmids were transfected into prostate cancer line PC-3M cells and the cells were incubated with DOX simultaneously. The expressing of exogenous TRAIL was detected with RT-PCR. Apoptosis of PC-3M was analyzed with TUNEL staining and flow cytometry. The expression of PC-3M cell death receptor 5 （ DR5 ） was detected by Western blot analysis. Results The full-length cDNA of human TRAIL gene was inserted into the plasmid vector successfully. TUNEL and flow cytometry analysis confirmed apoptosis induction of exogenous expressing-TRAIL. DOX could enhance the apoptosis-inducing effect. In 6μg pLXIN-TRAIL plasmids DNA group,the mean apoptosis rates before and after addition of 1 mg/L DOX were 11.8% and 20.7% ,respectively. In addition,DOX could elevate the expression of DR5 in a dose-dependent manner. Conclusions The combination of exogenous TRAIL with DOX provide a novel and possible therapeutic method for treating prostate cancer. The increasing expression of DR5 in prostate cancer cells induced by DOX may be the important molecular mechanism.
出处
《中华泌尿外科杂志》
CAS
CSCD
北大核心
2005年第12期799-802,共4页
Chinese Journal of Urology
基金
国家自然科学基金资助项目(30070756)
关键词
前列腺肿瘤
癌
阿霉素
凋亡
Prostatic neoplasms
Carcinoma
Doxorubicin
Apoptosis
