小剂量环磷酰胺治愈荷膀胱癌小鼠动物模型的建立

[目的]建立单一剂量环磷酰胺（cyclophosphamide,CTX）治愈荷膀胱癌小鼠的动物模型。[方法]T739小鼠皮下接种小鼠可移植性膀胱移行细胞癌组织建立荷瘤小鼠模型。肿瘤接种后第7天,不同剂量的CTX单次腹腔内注射,观察荷瘤小鼠用药后体重和肿瘤结节直径的变化,找出CTX可治愈多数荷瘤小鼠的最低有效剂量。然后再取12只荷瘤T739小鼠随机分成两组,最低有效剂量的CTX单次腹腔内注射,等量生理盐水作对照,分别在治疗后第4、14天内眦静脉取血进行白细胞计数。[结果]接种2 mm^3的小鼠膀胱癌肿瘤组织可使T739小鼠肿瘤进行性生长,接种后第7天肿瘤直径约为2-4 mm。15-400 mg/kg的CTX单次腹腔内注射后1周内,荷瘤小鼠肿瘤结节缩小的速率和毒性反应均与所用CTX的剂量呈正相关。CTX治疗后2月,100-200 mg/kg CTX治疗组的荷瘤小鼠全部死亡;40 mg/kg CTX治疗后20%的荷瘤小鼠存活;15 mg/kg CTX治疗组60%的荷瘤小鼠存活,与对照组比较差异具有统计学意义（P〈0.01）。15 mg/kg CTX治疗后第4天荷瘤小鼠外周血WBC为（11.36±0.30）×10^9/L,对照组为（11.14±2.14）×10^9/L,两组数据比较差异无统计学意义（P〉0.05）;治疗后第14天,治疗组小鼠WBC计数降为（8.46±0.76）×10^9/L,与CTX治疗后第4天WBC计数比较差异具有统计学意义（P〈0.05）,但与对照组（9.86±3.40）×10^9/L比较差异无统计学意义（P〉0.05）。[结论]15 mg/kg CTX单次腹腔内注射后荷瘤小鼠无明显毒性反应,并可使多数荷瘤小鼠肿瘤治愈,因此,小剂量CTX治愈荷膀胱癌小鼠模型成功建立。

依维莫司负调控mTOR信号通路对膀胱癌BTT739细胞增殖和迁移的抑制作用及机制研究

目的:探索mTOR抑制剂依维莫司(RAD001)对膀胱癌BTT739细胞在体外和小鼠体内增殖和迁移的抑制作用及其作用机制,为依维莫司在临床上治疗膀胱癌提供理论依据。方法:体外培养小鼠膀胱癌BTT739细胞,待其生长至对数期后用梯度浓度的RAD001(0 nmol/L、5 nmol/L、10 nmol/L和20 nmol/L)干预24 h,用MTT法检测细胞增殖的变化;Tranwell实验检测RAD001对BTT739细胞迁移的影响;ELISA试剂盒检测细胞分泌蛋白E-钙黏蛋白表达的变化。Western blot检测Akt、mTOR、4EBP和PTEN蛋白的表达,RTPCR检测mTOR mRNA的表达。将BTT739细胞接种于昆明鼠皮下,待成瘤后,用依维莫司干预14天,取小鼠肿瘤组织检测E-钙黏蛋白及AKT、mTOR、4EBP和PTEN蛋白表达的情况。结果:体外实验显示RAD001可以抑制BTT739细胞的增殖和迁移并与剂量有关(P<0.05)。还可以增加E-钙黏蛋白的含量,抑制癌细胞的转移。实验证明RAD001在体外抑制Akt、mTOR、4 EBP的表达,促进肿瘤抑制因子PTEN的表达(P<0.05)。将BTT739细胞接种于小鼠皮下后发现,RAD001在小鼠体内亦可以抑制病变情况,促进PTEN和E-钙黏蛋白的表达,抑制Akt、mTOR和4EBP的表达。结论:mTOR抑制剂依维莫司通过抑制mTOR信号通路抑制膀胱癌细胞BTT739的增殖、迁移和侵袭。

Effects of dendritic cell vaccines on hematogenous micrometastasis of bladder cancer carrying for PBL-SCID mice

Objective： To study the effect of dendritic cells loaded with whole tumor antigen on hematogenous micrometastasis of bladder cancer model in hu-PBL-SCID mice. Methods： T24-3 ceil subset was selected from human bladder transitional cell carcinoma T24 cell line by Boyden chamber system. The SCID mice intraperitoneally injected with 4 × 10^7 hu-PBL and subcutaneously injected with 3 × 10^6 T24-3 cells were named hu-PBL-T24-3-SCID model. Human IgG level in the blood plasma of mice was detected by ELISA, and human CD3^＋, CD4^＋, CD8^＋ T cells in blood and spleen cells of mice were detected by FCM analysis for human immune reconstruction study. Human CK20 mRNA expression in mice peripheral blood was detected by RT-PCR to investigate metastasis of tumor cells. The PBMCs were isolated from human peripheral blood, and were induced into DCs by co-culture with rhGM-CSF and rhlL-4 in vitro. The DC vaccines were produced by co-culturing with whole tumor antigen which was purified through freezing and melting T24-3 cell subset. After T24-3 cells injected into SCID mice for 5 weeks, the mice were treated with DC vaccines. Results： All mice were initially treated at 5th week. The expression of CK20 mRNA in peripheral blood of DC vaccines treated mice was the lowest. There was 2 mice showing CK20 mRNA expression and 3 mice with metastasis tumor in PBS group. MMP-7 mRNA expression in tumor tissues of DC vaccines treated mice was statistically lower than that of PBS group （P 〈 0.01）. Conclusion： DC vaccines have a good effect on hu-PBL-SCID mice bladder cancer model by reducing hematogenous micrometastasis.