雷帕霉素对小鼠H_(22)肝癌细胞生长增殖的影响

目的 研究雷帕霉素(rapamycin, RPM)对H_(22)肝癌细胞生长增殖的影响.方法 体外培养小鼠H_(22)肝细胞癌株,分别与RPM、CsA、FK506和5-Fu共同孵育48 h,进行MTT实验.流式细胞仪测定各组H_(22)细胞的细胞周期变化,ELISA法测定各组上清液中VEGF含量.体内实验建立H_(22)肝细胞癌皮下移植瘤模型,同时完成C57BL/6→BALB/c小鼠异体皮肤移植模型.给予RPM、CsA、FK506和5-Fu灌胃,观察皮片存活情况.获取实验小鼠血清及肿瘤组织.计算各组肿瘤体积,通过CD34免疫组化染色测定各组肿瘤组织的微血管密度(MVD).结果 剂量为0.01、0.1、1 mg/L的RPM对对数生长的H_(22)肝细胞具有细胞毒性,抑制H_(22)小鼠肝癌细胞增殖,培养上清液中的VEGF浓度较对照组显著降低(P<0.05),细胞周期分析显示S期细胞数较其他免疫抑制剂组显著减少(P<0.05).体内实验显示给予1.5 mg/(kg·d)和4.5 mg/(kg·d)的RPM与5 mg/(kg·d) FK506、20 mg/(kg·d) CsA的皮片存活时间相等,而肿瘤体积显著减小(P<0.05).与对照组相比,实验剂量RPM显著降低了荷瘤小鼠血清中的VEGF含量(P<0.05),同时瘤组织内的MVD显著减少(P<0.05).结论 体外实验研究和动物实验证实,RPM具有抗免疫排斥和抗肿瘤增殖的特点,可能在肝移植治疗肝脏恶性肿瘤方面发挥优势.

Inhibitory effect of rapamycin on proliferation of H_(22) hepatic cancer in mice

Objective To explore rapamycin's inhibitory effect on proliferation of H_(22) hepatic cancer in mice. Methods In vitro study: H_(22) hepatic cancer cell lines were cultured with rapamycin, CsA, FK506, and proliferation was determined through MTT. The influences of different agents on the H_(22) hepatic cancer cell cycle were observed by flow cytometry. The vascular endothelial growth factor (VEGF) concentration of the supernatant fluid of the cultured H_(22) hepatic cancer cell was detected by ELISA. In vivo study: C57BL/6 to Balb/c mice allogenic skin transplant was established, and the H_(22) hepatic cancer cell was implanted under skin. Rapamycin, CsA, FK506 and 5-FU were fed to the mice, respectively. The effect of different immunosuppressors on the survival of skin graft was observed while the proliferation of the transplant tumor was investigated. VEGF concentration of treated mice serum was examined by ELISA. The microvessel density of the transplanted tumor was observed through immunohistochemistry staining of CD34. Results The proliferation of the H_(22) hepatic cancer cells was inhibited by rapamycin at the concentration different dose of rapamycin, the VEGF concentration of the supernatant fluid decreased significantly (P<0.05). The number of S phase cells decreased significantly compared to that of other agents (P<0.05). When rapamycin, the lengthened survival time of the skin grafts was similar to that in CsA and FK506 groups. But the tumor volume was smaller than that in CsA and FK506 groups (P<0.05). Compared to that in the control group, the VEGF concentration of mice serum decreased in rapamycin group (P<0.05), and the microvessel density of the transplant tumor was reduced greatly (P<0.05). Conclusion Rapamycin, as an immunosuppressor, significantly resists immunologic rejection and inhibits the proliferation of H_(22) hepatic cancer, thus having its advantage in treating malignant hepatic cancer with liver transplantation.