重组人血管内皮抑制素对小鼠移植瘤及正常组织中血管相关因子影响的比较

Effect of rh-endostatin on Factors Associated with Vascular Xanocraft Tumor and Normal Tissue

目的:观察重组人血管内皮抑制素(rh-endostatin,商品名:恩度,endostar)的抗肿瘤及抗血管生成效应,比较其对移植瘤、心肌及肾脏组织中神经型一氧化氮合成酶(neuron NOS,nNOS)、内皮型一氧化氮合成酶(endothelial NOS,eNOS)mRNA、血管生成因子表达和微血管密度及结构的影响,探讨其对心血管系统不良反应的原因。方法:将36只小鼠随机分为空白对照组(不接种瘤块,注射生理盐水)、药物对照组(不接种瘤块,注射恩度)、模型组(接种瘤块,注射生理盐水)和实验组(接种瘤块,注射恩度),每日1次,处理28天。实验前、后称量小鼠体重,测量移植瘤体积;Real-time PCR法检测各组小鼠移植瘤、心脏及肾脏组织中nNOS、eNOSmRNA的表达量,免疫组化法检测小鼠移植瘤及肾脏组织中基质金属蛋白酶MMP-2、MMP-9、缺氧诱导因子HIF-1α、血管内皮生长因子(VEGF)的表达情况,CD34标记血管内皮细胞计数微血管密度(MVD);以CD34与Masson双色染色法观察微血管结构的变化结果:1)实验组移植瘤体积增长(75.60 mm^3)小于模型组(131.89mm^3),差异有统计学意义(P=0.030);各组小鼠体重变化无显著性差异(P=0.609)。2)药物对照组及实验组小鼠肾脏组织中nNOS及eNOSmRNA表达量均下调(P<0.05),心肌组织中nNOSmRNA表达下调(P<0.05)、eNOSmRNA却未见明显变化;各组小鼠移植瘤组织中nNOS及eNOSmRNA表达的差异均无统计学意义。3)移植瘤组织中MMP-9和VEGF蛋白的表达显著降低,MMP-2、HIF-1α及各组小鼠肾脏组织MMP-2、MMP-9、HIF-1α、VEGF表达无明显变化;移植瘤组织的MVD显著降低,胶原覆盖血管的比例升高,而肾脏组织中MVD和结构几乎无变化。结论:重组人血管内皮抑制素可下调移植瘤组织中MMP-9和VEGF蛋白的表达、降低MVD,抑制移植瘤的生长,并可使移植瘤血管趋向成熟,但不能降低肾脏组织中血管生成相关因子的表达、亦不能改变肾脏组织的MVD及微血管结构;其可下调心脏及肾脏组织NOSmRNA的表达,可能为应用中导致血压变化的原因之一。

Objective： To observe and analyze the antitumor and anti-angiogenesis effects ofrh-endostatin, to compare the effects of rh-endostatin on the microvasculature in tumor, myocardium, and renal tissue, and to investigate the underlying mechanism of endostar, the adverse effect of the cardiovascular system, for the purpose of directing its clinical application. Methods： Nude mice were randomized into four groups： blank control group （ without tumor, NS 100 μL.d^-1 ）, drug control group （ mice without tumor, rh-endostatin 400μg.d^-1 ）, model group （ mice with tumor, NS 100μg.d^-1 ）, and treatment group （ mice with tumor, rh-endostatin 400 μg.d^-1 ）. The drug was administered on days 1 to 28. The volume of the tumor and the weight of mice were measured before and after administration. QRT-PCR assay was conducted to detect the level of eNOS and nNOS mRNA in the tumor, myocardium, and kidney. The expressions of CD34, MMP-2, MMP-9, HIF-lct, VEGF, and iNOS in the tumor and kidney were detected through immunohistochemistry. The structure of vasculature was observed through immunoenzymatic double staining with CD34 and Masson. Results： 1） Results of the animal experiment： the tumor volume in the treatment group （ 75.60 mm3 ） was less than that in the model group （ 131.89 mm3）. The discrepancy in weight was insignificant among the four groups. 2） Results of the Real-time PCR： after treatment with endostar, the eNOS and nNOS mRNA levels in the mouse kidney significantly decreased. In the myocardium, the nNOS level significantly decreased. However, the eNOS level in the myocardium and the nNOS and eNOS levels in the tumor did not change significantly. 3） After treatment with endostar, the expressions of MMP-9 and VEGF in the tumor were obviously downregulated. However, the same results were not found in the MMP-2 and HIF-1 α of the tumor. MVD in the tumor of the treatment group significantly decreased compared with the model group. The proportion of tumor vessels covered by collagen in the treatment group increased compared with the model group. However, MVD and the microvasculature in the kidney did not change significantly. Conclusion： Rh-endostatin can reduce the expressions of MMP-9, VEGF, and MVD to inhibit the growth of tumor and to normalize the micrangium in the tumor. However, it cannot weaken the MMP and MVD of mature micrangium in the kidney. The eNOS and nNOS mRNA levels in the mouse kidney significantly decreased after treatment with endostar, which may be responsible for endostar causing hypertension.