蛙皮素对雄激素非依赖型前列腺癌PC-3细胞系的作用

Effect of bombesin on androgen-independent prostate cancer PC-3 cells line

目的观察蛙皮素(bombesin,BBS)对雄激素非依赖型前列腺癌PC-3细胞系的作用;检测PC-3细胞BBS受体及其 mRNA的表达。方法①MTT法检测BBS对PC-3细胞增殖的影响;②检测BBS处理后PC-3细胞贴壁率;③Millcell小室实验检测BBS对PC-3细胞播散能力的影响;④免疫荧光组织化学结合激光扫描共聚焦显微镜检测BBS处理的PC-3细胞角蛋白(CK)的表达[1];⑤用Fluo-3/AM荧光标记技术检测不同浓度BBS处理后PC-3细胞[Ca2+]i浓度[2]。⑥免疫组化方法检测PC-3细胞中蛙皮素受体(BBS-R)蛋白表达;⑦利用逆转录聚合酶链反应(Rt-PCR)观察PC-3细胞BBS-R mRNA的表达。结果经BBS处理的PC-3细胞吸光度A值增高,细胞贴壁率增高,细胞播散能力提高,并呈现一定浓度依赖性;10-5mol/L浓度的BBS可促进PC-3细胞CK表达;BBS提高PC-3细胞[Ca2+]i浓度;PC-3细胞中BBS-R蛋白表达呈阳性;Rt-PCR产物与预期的BBS-R的cDNA分子量完全相符。结论BBS可通过特异性受体介导致PC-3细胞系增殖、粘附、播散、伪足形成。一定浓度的BBS可明显提高PC-3细胞[Ca2+]i浓度以及CK表达进而影响细胞骨架形态。

Objective To observe the effect of BBS on human androgen-independent prostate cancer PC-3 cells line.To observe the expression of bombesin receptor mRNA and its protein in PC-3 Cell. Methods ①The proliferation effects of BBS were detected on PC-3 cells by MTT method;②The effects of BBS on percentage of cell adhesion were observed;③Millcell test was down to observe the effect of BBS on cell spreading;④The immunofluorescent histochemistry(IH) combined with laser scanning confocal microscopy(LSCM) was used to examine the expression of cytokeratin(CK) in PC-3 cell treated with BBS;⑤Fluo-3/AM fluorescence technique and LSCM were used to measure the [Ca2+]i concentration after different concentrations BBS were added in PC-3 cell. ⑥ Bombesin receptor protein expression in PC-3 cell were detected by immunohistochemical method staining;⑦RT-PCR were performed for detection of BBS-R mRNA in PC-3 Cell. Results Proliferation of PC-3 cell was observed after treated with BBS.BBS can stimulate cell adhesion , spreading and expression of CK in PC-3 cell,and markedly increase the formation of lamellipodium.[Ca2+]i concentration was rised treated with BBS,and showed concentration dependence.The positive expression rates of bombesin receptor protein were significantly higher in PC-3 Cell.It is shown that the molecular weight of the cDNA observed by RT-PCR was consistent with that of expect. Conclusions These results suggest that the effect of proliferation,adhesion, spreading ,lamellipodia formation by BBS can be mediated through BBS receptor in PC-3 cells line. These results clearly indicate that definite concentrations BBS exerts morphological and [Ca2+]i concentration on PC-3 cell.By using immunohistochemical method and RT-PCR, it was demonstrated that PC-3 Cell can express specific bombesin receptor protein.