【作者】 彭利； 王顺祥； 张萌； 唐瑞峰； 张凤瑞； 左连富；

【Author】 Peng Li Wang Shun-Xiang Zhang Men Tang Rui-Feng Zhang Feng-Rui Zuo Lian-Fu The Fourth Hospital of Hebei medical university & Hebei provincial tumor hospital, Shijiazhuang, 050011, China

【机构】 河北医科大学第四医院暨河北省肿瘤医院肝胆外科；

【摘要】 目的:流行病学和实验研究表明非甾体抗炎药 (NSAIDs)可通过抑制环氧合酶-2(COX-2)发挥抗肿瘤作用。新近研究表明,COX-2不但可以抑制肿瘤血管生成,还可以改变肿瘤细胞的侵袭性和粘附性来抑制肿瘤的生长。本研究拟通过体外实验和肝细胞癌SMMC-7721细胞株荷瘤裸鼠体内试验,观察NS-398在体内外实验中对SMMC-7721细胞血管内皮生长因子(VEGF)、基质金属蛋白酶-9(MMP-9)基因、蛋白表达的影响,并进一步观察NS-398对裸鼠SMMC- 7721细胞移植瘤生长情况和肿瘤微血管密度(MVD)的的影响。探讨COX-2在肝细胞癌肿瘤生成和血管生成过程的机制及其特异性抑制剂治疗肝细胞癌的可能性。方法:在体外试验中, 将对数生长期的SMMC-7721细胞按5×103个／孔接种于96 孔培养板,孵育24小时待细胞贴壁后NS-398加入培养液中。 NS-398取0、25、50、75、100μmol／L 5个浓度组,每个浓度组选取24h、48h、72h三个作用时间点进行检测,采用 RT-PER和流式细胞术检测肝癌SMMC-7721细胞VEGF、 MMP-9表达。在体内试验中,取SMMC-7721细胞接种于 BALB／c nu／nu裸小鼠的右腋部皮下。16只裸鼠随机分为实验组和对照组(每组8只)。实验组给予3 mg／kg NS-398 (DMSO溶解)腹腔注射,每周3次,对照组以等量DMSO腹腔注射。给药9周后采用拉颈法处死裸鼠,取瘤体称重、测量体积。肿瘤体积(V)=长径×短径2／2,重量抑瘤率=(对照组瘤重-实验组瘤重)／对照组瘤×100％,体积抑瘤率=(对照组体积-实验组体积)／对照组体积×100％。采用免疫组化法检测裸鼠移植瘤VEGF、MMP-9蛋白表达和MVD,RT-PCR 检测裸鼠移植瘤组织中VEGF、MMP-9 mRNA的表达, ELISA法测定血清中PGE2水平。结果:在体外NS-398可以呈剂量和时间依赖性显著抑制SMMC-7721细胞的VEGF、 MMP-9 mRNA和蛋白表达。NS-398在体内可显著降低裸鼠移植瘤MVD和VEGF、MMP-9基因和蛋白表达(P<0． 001),对照组血管为排列规则及分化良好的血管网络,而实验组表现为分散分布的血管内皮细胞及分化较差的血管网络。并能降低裸鼠血清中PGE2水平(P<0．001)。实验组肿瘤体积为(0．21±0．14)cm3,对照组为(1．92±0．94)cm3,体积抑瘤率为89．1％。实验组肿瘤的重量为(0．40±0．34)g,对照组为 (1．45±0．42)g,重量抑瘤率为72．4％。实验组肿瘤重量和体积较对照组明显减小(P<0．001)。结论:COX-2与肝细胞癌血管生成密切相关,NS-398可通过下调VEGF和MMP- 9的表达而抑制肝细胞癌血管生成。更多还原

【Abstract】 Objective: To explore the role of cyclooxygenase-2(COX-2) on angiogenesis of hepatocullar carcinoma. Methods: VEGF and MMP-9 expressions were detected by RT-PCR and flow cytometry. Sixteen female athymic mice inoculated with SMMC-7721 cells were randomly divided into two groups. The treatment group received NS-398 solution at a dose of 3 mg/kg intraperito-neally three times weekly for 9 weeks. Microvascular density of xenograft tissue were detected by immunohis-tochemical staining and the serum PGE2 was detected by ELISA. Results: The expressions of VEGF and MMP-9 were significantly downregulated in SMMC-7721 cells exposed to NS-398 in a dose-dependence and time-dependent manner (P<0.001). Expression of VEGF, MMP-9 mRNA and protein in xenografts treated with NS-398 were lower than those of control group (P<0.001). The density of microvessel was notably lower in xenografts treated with NS-398 than in those without treatment and serum PGs level was also significantly lower than in those without treatment (P<0.001). Conclusion: Expression of COX-2 contributes to angiogenesis in hepatocullar carcinoma. NS-398 may suppress angiogenesis of hepatocellular carcinoma cell via reducing expressions of VEGF and MMP-9.更多还原