雷帕霉素抑制肝癌细胞生长及转移的实验研究

Inhibition of growth and metastasis of hepatocellular carcinoma by rapamycin:experiment with mice

目的探讨具有抗肿瘤特性的新型免疫抑制剂雷帕霉素(RPM)对肝癌细胞生长及转移的作用。方法应用流式细胞仪检测RPM(10 ng/m l)、环孢素(CsA)(100 ng/m l)、以及两者合用对人肝癌高转移细胞株MHCC97H细胞凋亡及细胞周期的影响;应用MTT法检测上述药物对细胞增殖的影响。用实时定量聚合酶链反应(PCR)法检测RPM处理后血管内皮细胞生长因子(VEGF)mRNA、碱性成纤维细胞生长因子(bFGF)mRNA、缺氧诱导因子-1α(H IF-1α)mRNA、转化生长因子b(TGFb)的表达。ELISA法检测MHCC97H培养液上清VEGF蛋白水平的变化。28只原位种植高转移性人肝癌模型LC I-D20裸鼠,随机分为CsA(25 mg/kg)组、RPM(2 mg/kg)组、CsA+RPM组及对照组(生理盐水),每组7只;探讨免疫抑制剂对肿瘤生长及自发肺转移的影响。结果体外实验中,RPM抑制了MHCC97H细胞的增殖,并使细胞停滞于G0/G1期,但未促进细胞凋亡。RPM下调了H IF-1α和VEGF的基因表达及MHCC97H培养液上清VEGF蛋白的表达(890.3 pg/m l±25.1 pg/m lvs 1583.7 pg/m l±17.3 pg/m l,P=0.000)。CsA对细胞周期及增殖无影响。体内实验中,RPM及CsA+RPM抑制了LC I-D20模型移植瘤的生长(0.76 g±0.38 g vs 2.09 g±0.75 g,P=0.001;0.40 g±0.22 g vs 2.09 g±0.75 g,P=0.000)及肺部转移的发生(均为2/7 vs 7/7,P=0.021)。CsA组肺部自发转移灶的数目多于对照组(6±2 vs 4±1,P=0.046)。结论RPM能明显抑制肝癌细胞的生长及转移,以RPM为基础的免疫抑制方案在肝癌肝移植中可能有临床应用前景。

Objective To investigate the effects of rapamycin （RPM） in inhibiting the growth and metastasis of hepatocellular carcinoma （HCC）. Methods Human HCC cells of the line MHCC97H with a high potential of metastasis were divided into 3 groups to be cultured with cyclosporine A （CsA） 100 ng/ml, RPM 10 ng/ml, or CsA ＋ RPM for 48 hours. Flow cytometry was used to examine the apoptosis and cell cycle MTT method was used to examine the effect of RMP on the proliferation of the MHCC97H cells. RTPCR was used to detect the mRNA expression of vascular endothelial growth factor （ VEGF）, basic fibroblast growth factor （bFGF）, hypoxia-inducible factor-1α （HIF-1α）, and transforming growth factor b （TGFb）. Another MHCC97H cells were cultured in complete medium without RPM for 48 hours, then the protein expression of VEGF in the supernatant was detected by ELISA. Twenty-eight nude LCI-D20 mice were inoculated with human HCC cells and then divided into 4 groups to be fed with CsA （25 mg/kg）, RPM （2 mg/kg） , CsA ＋ RPM, and normal saline （0. 2 ml, as control group） for35 days. Then the mice were killed to take the weight of inoculated tumor, measure the blood drug concentration, calculate the lung metastasis rate and number of metastatic foci, and observe pathology of the lung. Results CsA showed no effect on the cycle of the MHCC97H cells. The MHCC97H cells of the RPM and CsA ＋ RPM groups arrested at the stage G0/G1 （ both P = 0. 000）. MMT method also showed that the proliferation of the MHCC97 H cells in the RPM and CsA ＋ RPM groups were blocked （P =0. 003 and P =0. 002）. however, CsA did not influence the proliferation of the MHCC97H cells. Flow cytometry showed that RPM did not promote the apoptosis of the MHCC97H cells. RT-PCR showed that RPM down-regulated the mRNA expression of VEGF and HIF-1α （ both P 〈 0.05 ）, however, did not influence the mRNA expression of bFGF, TGFb, and TGFb. The VEGF protein level in the supematant of the culture fluid of MHCC97H cells of the RPM group was （ 890. 3 ± 25.1 ） pg/ml, significantly lower than that of the control group, 1583.7 ± 17.3 ） pg/ml （ P = 0. 000）. The tumor inhibiting rate of the RPM group was 63.7%, not significantly different from that of the RPM ＋ CsA group （80.9%, P = 1. 000）. The metastatic rate of the CsA and control groups were both 100% with a higher number of metastatic tumors in the CsA group （P = 0. 046）. Conclusion RPM significantly inhibits the growth and metastasis of HCC. RPM-based immunosuppressive regimen may be of value in HCC patients receiving liver transplantation.