EGCG衍生物Y6联合柔红霉素对人肝癌裸鼠移植瘤生长及血管生成的影响

Effect of EGCG derivative Y6 combined with daunorubicine on the growth and angiogenesis of human hepatocellular carcinoma in nude mice

目的:探讨EGCG乙基化衍生物Y6联合柔红霉素(DNR)对HepG2细胞裸鼠移植瘤生长及血管生成的影响。方法:建立人肝癌HepG2细胞移植瘤模型,随机分为8组,即对照组、EGCG组、Y6中剂量(Y6-M)组、DNR组、DNR+Y6高剂量(Y6-H)组、DNR+Y6-M组、DNR+Y6低剂量(Y6-L)组和DNR+EGCG组。Y6高、中、低剂量分别为110 mg/kg、55 mg/kg和27.5mg/kg,灌胃给药;EGCG单用及联合给药剂量均为40mg/kg,灌胃给药;DNR单用及联合给药剂量均为2mg/kg,腹腔注射;对照组灌胃给予0.5%羧甲基纤维素钠溶液。给药期间每4d测定裸鼠移植瘤体积。观察各组裸鼠肿瘤生长状况,称取瘤重并计算抑瘤率;苏木精—伊红(HE)染色观察肿瘤组织形态结构改变,TUNEL法观察肿瘤组织细胞凋亡,免疫组化法检测CD34标记的微血管密度(MVD),分别采用荧光定量PCR(qPCR)法和western blotting法检测瘤组织中低氧诱导因子-1α(HIF-1α)和血管内皮生长因子(VEGF)mRNA和蛋白的表达。结果:除EGCG组和Y6-M组外,其他各组瘤体体积均较对照组显著减小,抑瘤率升高(P<0.05或P<0.01),与DNR组相比,DNR+Y6-M组和DNR+Y6-H组的抑瘤率显著升高(P<0.05或P<0.01);与EGCG组比较,Y6-M组MVD明显降低,细胞凋亡指数(AI)升高(P<0.05或P<0.01),与DNR组相比,各联合给药组MVD显著降低,AI显著升高(P<0.01);同时,随着Y6剂量的增加,DNR联合Y6组瘤组织HIF-1α和VEGF的表达降低,具有一定的剂量依赖性。Y6与DNR联用对肿瘤生长具有协同抑制作用。结论:Y6可增强DNR抑制裸鼠移植瘤生长及肿瘤血管生成的作用,其机制可能与下调HIF-1α和VEGF的表达有关。

Objective：To investigate the effect of epigallocatechin gallate（EGCG）ethyl derivative Y6 combined with daunorubicin（DNR）on the growth and angiogenesis of HepG2 transplanted tumor tissue in nude mice.Methods：BALB/C nude mice were injected subcutaneously with the suspension of HepG2 cells to establish the human hepatocellular carcinoma transplantation tumor model,and then were randomly divided into 8 groups：the control group,EGCG group,middle-dose of Y6（Y6-M）group,DNR group,DNR plus high-dose of Y6（DNR＋Y6-H）group,DNR＋Y6-M group,DNR plus low-dose of Y6（DNR＋Y6-L）group and DNR＋EGCG group.DNR was administered intraperitoneally to the animals every 4 days,while EGCG or Y6 was given orally to the animals daily.The tumor growth in each group was observed.The tumor tissues were weighed and the tumor inhibition rate was calculated.The morphological changes of tumor were observed by hematoxylin-eosin（HE）staining and the apoptosis of tumor cells was detected byterminal deoxynucletidyl transferase-mediated dUTP neck end labeling（TUNEL）assay.The microvessel of tumor tissue were marked by CD34 and the microvessel density（MVD）were counted.The mRNA and protein expressions of hypoxia inducible factor-1α（HIF-1α）and vascular endothelial growth factor（VEGF）in tumor tissue were detected by qPCR and western blotting,respectively.Results：Except for the EGCG group and Y6-M group,the growth of tumors was significantly inhibited in other groups（P〈0.05 or P〈0.01）.DNR combined with Y6-M or Y6-H had stronger inhibitory effect on the growth of tumors compared with DNR only（P〈0.05 or P〈0.01）.MVD was decreased,whereas the apoptotic index（AI）was increased in Y6-M group,compared with EGCG group（P〈0.05 or P〈0.01）.The combination of DNR and Y6/EGCG resulted in lower MVD and higher AI than DNR only（P〈0.01）.Moreover,Y6 significantly suppressed HIF-1αand VEGF expressions in a dose-dependent manner.The combination of DNR and Y6 exerted a synergistic inhibitory effect on tumor growth.Conclusion：Y6 combined with DNR could enhance the inhibition effect on tumor growth and angiogenesis,and the mechanism might be related to the down-regulation of HIF-1αand VEGF expressions.