重组分泌型肿瘤坏死因子相关凋亡诱导配体腺病毒对前列腺癌细胞增殖的影响  

Construction of recombinant secreting tumor necrosis factor related apoptosis inducing llgand ade- noviral vector and the effects of secreting - trail adenovirns on the prostate cancer PC - 3 cells＇ proliferation

作　　者：高磊[1] 潘铁军[1] 蒋渊[1] 沈国球[1] 谢森[1] 文瀚东[1] 

机构地区：[1]广州军区武汉总医院泌尿外科,武汉430070

出　　处：《中华实验外科杂志》2016年第12期2632-2634,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金（81502210）;湖北省自然科学基金（2015CFC863）

摘　　要：目的构建表达分泌型肿瘤坏死因子相关凋亡诱导配体（TRAIL）基因的腺病毒载体，观察TRAIL基因对前列腺癌细胞株PC-3增殖的影响。方法采用基因合成的方法获得含有分泌型信号肽的TRAIL基因，连接人GV315载体，在293A细胞中进行重组腺病毒的包装及扩增。以构建好的腺病毒载体感染体外培养的前列腺癌细胞株PC-3。采用间接免疫荧光法检测腺病毒感染后PC-3细胞中TRAIL表达的变化。Westernblot检测半胱氨酰天冬氨酸特异性蛋白酶（Caspase）-3和Caspase-8表达的变化，通过平板克隆实验、细胞生长实验检测TRAIL对PC-3细胞增殖能力的影响。结果成功构建重组分泌型腺病毒载体Ad-sTRAIL。PC-3细胞经Ad-sTRAIL感染后TRAIL蛋白表达水平明显增加，并且active-Caspase-3和active．Caspase-8的表达明显增加。平板克隆实验结果显示Ad-sTRAIL组克隆形成率[（30．60±2．10）％]明显低于Control组[（46．60±4．86）％，q=7．56，P〈0．01]及Ad-LaeZ组[（45．90±3．56）％，q=7．19，P〈0．01]。噻唑蓝（MTT）实验结果显示Ad-sTRAIL对Pc-3细胞的抑制作用自48h后（20．15％）差异有统计学意义（q=6．82，P〈0．01）。结论通过重组分泌型TRAIL腺病毒载体Ad-sTRAIL使PC-3细胞表达TRAIL基因，并可增加active-Caspase-3和active-Caspase-8的表达，从而抑制前列腺癌细胞的增殖。Objective To construct a recombinant secreting- trail adenoviral vector and to inves- tigate the effect of tumor necrosis factor related apoptosis inducing ligand （TRAIL） gene on the prostate cancer PC - 3 cells＇ proliferation. Methods The TRAIL gene with a secreting signal peptide was ob- tained by gene synthesis, and cloned into GV315 plasmid vector. Then plasmids were constructed and am- plified in 293A cells. The adenoviral vector were infected into prostate cancer cell line PC - 3 in vitro. The up -regulation of TRAIL protein was determined by indirect immunofluorescence assay. The protein ex- pressions of cysteinyl aspartate - specific protease （Caspase） - 3 and Caspase - 8 in the cells infected with Ad -sTRAIL and Ad- LacZ were determined by Western blotting respectively. Methyl thiazol tetrazolium （MTT） assay and plate colony formation were used to determine the effect of cells proliferation. Results Construct the Ad - sTRAIL successfully. After infection, it had been verified that the protein expression of TRAIL in the cells by indirect immunofluorescence assay. Through the Western - blot, the expressions of ac- tive- Caspase- 3 and active -Caspase -8 in Ad-sTRAIL infected PC -3 cells were significantly higher than the Control group and Ad - LacZ group. The colony forrrmtion rate of the PC - 3 cells was [ （30. 6 ± 2. 10） % ] in Ad - sTRAIL group significantly lower than （46. 6 ± 4. 86） % in Control group （ q = 7. 56, P 〈 0. 01 ） and （45.9± 3.56） % in Ad - LacZ group （ q = 7.19, P 〈 0.01 ）. MTT assay showed that Ad - sTRAIL significantly inhibited the growth of the PC - 3 cells （ 20. 15% ） at 48 h （ q = 6. 82, P 〈 0. 01）. Conclusion The secreting- trail adenoviral vector could up regulate the expression of TRAIL, active - Caspase - 3 and active - Caspase - 8, furthermore it could inhibit the proliferation of PC - 3.

关 键 词：前列腺癌 肿瘤坏死因子相关凋亡诱导配体基因 细胞增殖 分泌型腺病毒 载体 

分 类 号：R737.25[医药卫生—肿瘤]