磷酸肌酸对小鼠移植性S180肉瘤血管生成的作用及机制研究

Effects of creatine phosphate on angiogenesis in mice with transplanted S180 sarcoma and its possible mechanism

目的探讨磷酸肌酸(creatine phosphate,Cp)对小鼠移植性S180肉瘤血管生成的作用及其相关机制。方法建立昆明小鼠S180肉瘤模型,60只小鼠随机分为4组:0.9%氯化钠溶液对照组(对照组)、Cp低剂量组(200 mg/kg)、Cp中剂量组(400 mg/kg)、Cp高剂量组(800 mg/kg),观察4组小鼠移植瘤重量;流式细胞术测定基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)及金属蛋白酶组织抑制因子-2(tissue inhibitors of metalloproteinases,TIMP-2)蛋白的表达;免疫组化检测肿瘤组织微血管密度(microvasvular density,MVD);RT-PCR检测移植瘤组织中血管内皮生长因子(vascular endothlial growth factor,VEGF)及碱性成纤维细胞生长因子(base fibroblast growth,b FGF)mRNA的表达水平。结果对照组、Cp低剂量组和中剂量组移植瘤重量、VEGF、b FGFmRNA、MMP-2、TIMP-2水平比较,差异均无统计学意义(P>0.05);Cp高剂量组与对照组、Cp低、中剂量组比较,小鼠移植性肉瘤质量明显减小,MVD数显著减少,VEGF、bFGFmRNA和MMP-2蛋白表达明显减少,TIMP-2蛋白表达明显增加,差异均有统计学意义(P<0.01)。结论 Cp低、中剂量对肿瘤血管生成无明显影响;Cp高剂量对肿瘤血管生成具有明显的抑制作用,其机制可能与通过下调MMP-2、VEGF和bFGF、上调TIMP-2的表达水平有关。

Objective To investigate the effects of creatine phosphate（ Cp） on angiogenesis in mice with transplanted S180 sarcoma,and to explore its related action mechanism. Methods The animal models with S180 sarcoma were established in Kunming mice. The 60 mice were randomly divided into four groups： control group,low-dose group（ Cp200mg/kg）,middle-dose group（ Cp 400mg/kg）,high-dose group（ Cp 800mg/kg）. The changes of transplanted tumors weight were observed,moreover,the expression levels of（ matrix metalloproteinase-2（ MMP-2） and tissue inhibitors of metalloproteinases（ TIMP-2） protein in transplanted sarcoma tissues were detected by flow cytometry,and the microvessel density（ MVD） of tumors was detected by immunohistochemistry（ IHC）,besides the expression levels of VEGF and b FGF mRNA in transplanted tumor were detected by RT-PCR. Results There were no significant differences in transplantation tumor weight,the expression levels of VEGF,b FGFmRNA,MMP-2,TIMP-2 among control group,low-dose group and middle-dose group（ P 〉0. 05）. However the transplantation tumor weight in high-does group was significantly decreased,as compared with those in control group,low-dose group and middle-dose group,moreover MVD was obviously decreased,and the expression levels of VEGF,b FGFmRNA and MMP-2 protein were significantly decreased,however,the expression levels of TIMP-2 protein were significantly increased（ P 〈0. 01）. Conclusion The low-dose and middle-dose Cp has no obvious inhibitory effects on angiogenesis in mice with transplanted S180 sarcoma,however high-dose Cp has obvious inhibitory effects on angiogenesis in mice with transplanted S180 sarcoma,and its action mechanism might be correlated to down-regulation of expression levels of MMP-2,VEGF,b FGF and up-regulation of TIMP-2 expression.