蝎毒多肽提取物联合5-氟尿嘧啶对H_(22)肝癌血管生成拟态形成的作用及机制研究

Research on the Effect and Mechanism of Polypeptide Extract from Scorpion Venom Combined with 5-fluorouracil on Vasculogenic Mimicry of H 22 Hepatoma

目的观察蝎毒多肽提取物(polypeptide extract from scorpion venom,PESV)联合5-氟尿嘧啶(5-fluorouracil,5-Fu)对小鼠H22肝癌移植瘤血管生成拟态(vasculogenic mimicry,VM)形成的抑制作用并探讨其可能的作用机制。方法 60只昆明小鼠,皮下接种H22肝癌细胞悬液建立荷瘤模型,随机分为荷瘤对照组、5-氟尿嘧啶组(5-Fu组)、联合组(PESV+5-Fu),每组20只,连续干预14天,隔日测量肿瘤体积,绘制肿瘤体积增殖曲线,并计算抑瘤率;HE染色法观察肿瘤组织的形态学改变;免疫组织化学法和过碘酸雪夫氏染色(PAS)双标法检测各组肿瘤组织VM密度;免疫组织化学法检测缺氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)、基质金属蛋白酶2(matrix metalloproteinase-2,MMP-2)的蛋白表达水平,用LeicaQwin V3图像分析软件进行灰度值半定量分析。结果联合组和5-Fu组H22肝癌移植瘤的生长较荷瘤对照组均受到明显抑制(P<0.05),且联合组和5-Fu组瘤重和肿瘤体积亦存在显著差异(P<0.05);免疫组织化学法和PAS双标法检测显示,联合组VM密度明显低于荷瘤对照组和5-Fu组(P<0.01);与荷瘤对照组比较,联合组HIF-1α和MMP-2的蛋白表达水平均显著降低(P<0.01)。结论 PESV联合5-Fu可抑制小鼠H22肝癌移植瘤VM形成,其机制可能与抑制肿瘤微环境中HIF-1α和MMP-2的表达有关。

Objective To observe the inhibition effects of polypeptide extract from scorpion venom （PESV） combined 5-fluorouracil （5-Fu） on vasculogenic mimicry （VM） of H 22 hepatoma carcinoma cells in mice and its possible mechanisms. Methods The H 22 carcinoma cell suspension was subcutaneously inoculated into 60 Kunming mice. Then tumor-bearing mice were randomly divided into three groups, i.e., the control group, the 5-Fu group, and the combination group （PESV＋5-Fu）, 20 in each group. The tumor volume was measured once every other day after 14 successive days of intervention. Then the tumor volume growth curve was drawn, and the tumor inhibitory rate was calculated. The morphological changes of the tumor tissue were observed by HE staining. The VM density of each tumor tissue were detected by immunohistochemical assay and periodic acid-schiff stain （PAS）. The protein expression levels of hypoxia inducible factor-1α （HIF-1α） and matrix metalloproteinase-2 （MMP-2） were detected using immunohistochemical assay. The gray value was semi-quantitatively analyzed using LeicaQwinV3 Image Analysis Software. Results The growth of H 22 hepatoma transplantation tumor was inhibited more obviously in the combination group and the 5-Fu group than in the control group （P0.05）. There was statistical difference in the tumor weight and the tumor volume between the combination group and the 5-Fu group （P0.05）. Immunohistochemical assay and PAS showed that the VM density was obviously lower in the combination group than in the control group and the 5-Fu group （P0.01）. Compared with the control group, the protein expressions of HIF-1α and MMP-2 significantly decreased in the combination group （P0.01）. Conclusions PESV combined 5-Fu could inhibit the generation of VM of H 22 hepatoma transplantation tumor in mice. Its mechanisms might be associated with inhibiting the expressions of HIF-1α and MMP-2 in the microenvironment of tumors.