雷帕霉素抑制膀胱癌细胞生长及转移的实验研究

目的：探讨新型免疫抑制剂雷帕霉素（RPM）对膀胱癌细胞生长及转移的影响,探究其在肾移植患者膀胱肿瘤治疗中的作用与应用前景。方法：将雷帕霉素分别用5ng/ml,10 ng/ml,15ng/ml 3个浓度梯度处理T24膀胱癌细胞,分别在药物处理后12h,24h,36h应用MTT法检测上述不同药物浓度对细胞增殖的影响。流式细胞仪检测不同浓度RPM对人T24膀胱癌细胞株凋亡和细胞周期的影响;应用RT-PCR检测经不同浓度雷帕霉素处理后血管内皮生长因子（VEGF）mRNA的表达以及划痕法检测不同浓度雷帕霉素对于T24膀胱癌细胞迁移率的影响。体内实验用14只4-6周龄Balb/c裸鼠种植转移性人膀胱癌细胞构建荷瘤模型,观察雷帕霉素对肿瘤生长及自发转移的影响。结果：体外实验显示3种浓度的雷帕霉素明显抑制T24细胞的增殖,随着浓度的增加和作用时间的增加对于肿瘤的抑制性明显加强,其中24h时各浓度作用明显强于12h时各浓度,且各浓度之间差异明显（P〈0.05）。雷帕霉素使细胞停留在G0/G1期,并促进细胞凋亡;同时下调VEGF的基因表达（P〈0.05）。在划痕实验中雷帕霉素明显抑制膀胱癌细胞的迁移（P〈0.01）。体内实验中,鼠成瘤率达85.7%（12/14）。治疗3周后,空白对照组较RPM组肿瘤大且裸鼠明显消瘦（P〈0.05）;探查肺部和肝脏转移灶空白组明显较RPM组多（P〈0.05）。结论：雷帕霉素明显抑制膀胱癌细胞的生长及迁移,以雷帕霉素为基础的免疫抑制剂在肾移植患者膀胱肿瘤治疗中有良好的临床应用前景。

Inhibitory effect of rapamycin on growth and metastasis of bladder tumor T24 cells

Objective：To investigate the effects of rapamycin（RPM） in inhibiting the growth and metastasis of bladder tumor.Methods：Human bladder tumor cells of the line T24 were divided into 3 groups to be cultured with different doses of RPM（5ng/ml,10 ng/ml,15ng/ml） for 12h,24h,36h and a control group with no-drug.MTT method was used to examine the effect of RMP on the proliferation of the T24 cells;Flow cytometry was used to examine the apoptosis and cell cycle;RT-PCR was used to detect the mRNA expression of vascular endothelial growth factor（VEGF）,and scarification was used for investigating the effect of RPM on the migration ability of T24 cells.In vivo experiment,14 only 4-6 weeks of age Balb/c nude mice were used to build bladder cancer model,which were randomly divided into RPM（2mg/kg） and control group（saline）,to observe the effect of immune inhibitor on tumor growth and metastasis.Results：RPM significantly blocked the proliferation,growth and migration of T24 cells and the cell cycle was arrested at the stage of G 0/G 1;MTT results：the growth inhibition rate（GIR） between the different doses of RPM was significant difference（P 0.01）;The results detected by flow cytometry for 5ng/ml,10 ng/ml,15ng/ml induced bladder tumor cell line T24 apoptosis,the apoptosis rate compare with each other was difference（P 0.05）,while at 36h there was no difference between 10 ng/ml and 15ng/ml groups;The results of RT-PCR：compare 5ng/ml group with 10 ng/ml and 15ng/ml groups,10 ng/ml and 15ng/ml groups were more stronger than 5ng/ml group（P 0.05）,as well as there was difference between 10 ng/ml group and 15ng/ml group（P 0.05）;The results of scarification：compare with each other with different doses was difference（P 0.05）.With the time going and the increased the doses the affection increased（P 0.05）.In vivo experiment,rat tumor formation rate was 85.7%（12/14）,3 weeks after treatment of nude mice were sacrificed,the weight of the nude mice in control group were significantly lighter than in RPM group,while contrary to the weight of tumors（P0.05）.Exploration the metastases of the lungs and liver in control group was significantly more than the RPM group（P0.05）.Conclusion：RPM significantly inhibits the growth and metastasis of bladder tumor,RPM based immunosuppressive regimen is valuable for bladder tumor patients received renal transplantation.