论文部分内容阅读
目的探讨去甲斑蝥素(NCTD)对胆囊癌肿瘤血管生成的抑制作用。方法通过建立人脐静脉内皮细胞(HUVEC)、鸡胚绒毛尿囊膜(CAM)新生血管生成模型和裸鼠胆囊癌荷瘤模型,分别对HUVEC、CAM新生血管和裸鼠荷瘤血管生成进行随机干预实验,实验分对照组、5-氟尿嘧啶(5-Fu)组、不同浓度NCTD组、内皮抑素(ES)组、NCTD十5-Fu组和NCTD＋ES组,应用病理学检查、FCM、免疫细胞化学SABC法、CD34染色和HoechSt 33258荧光染色、荧光显微镜等,观测、分析HUVEC的凋亡形态改变、凋亡率,CAM血管被抑制、破坏作用,荷瘤裸鼠胆囊癌微血管密度(MVD)、瘤周血管破坏形态。结果(1)NCTD对HUVEC的ED_(50)为0．352 g/L。NCTD组HU—VEC凋亡率明显高于对照组[(76．86±5．67)%vs(4．60±1．45)%,P＜0．05],荧光显微镜下见血管内皮细胞不同程度凋亡；NCTD和ES一样,随时间延长其诱导血管内皮细胞凋亡作用加强,呈时间效应关系。(2)在CAM实验中,NCTD浓度与其抑制CAM血管生成作用呈正相关(r=0．89,t=3．45,P＜0．05),即随浓度增高NCTD鸡胚存活率下降(55%～60%比80%,P＜0．05)、CAM毛细树状血管减少、血管生成抑制率增高(90．9%～100．0%比0～53%,P＜0．05),与ES差异无统计学意义,NCTD对CAM血管生成抑制作用强。(3)NCTD应用后,荷瘤MVD与5-Fu组(4．12±1．40比15．80±5．90,P＜0．01)、对照组(4．12±1．40比17．60±3．20,P＜0．01)比较明显减少,与ES差异无统计学意义(P＞0．05),显微镜下见凋亡、片状坏死肿瘤细胞周围有血管破坏现象；且荷瘤MVD与荷瘤体积(r=0．985,t=8．069,P＜0．05)、细胞增殖/凋亡比(r=0．956,t=4．61,P＜0．05)相关。结论NCTD可有效抑制、破坏胆囊癌肿瘤血管生成,进而有效抑制胆囊癌增殖与生长。
Objective To investigate the inhibitory effect of norcantharidin (NCTD) on tumor angiogenesis in gallbladder carcinomas. Methods HUVEC, CAM neovascularization and nude mice bearing tumor angiogenesis were randomly divided into three groups: HUVEC, CAM, and nude mice with gallbladder carcinoma The experimental groups were divided into control group, 5-Fu group, NCTD group, endostatin group, NCTD group and NCTD + ES group. Pathological examination, FCM, immune cells (SABC) method, CD34 staining, Hoechst 33258 fluorescence staining and fluorescence microscopy were used to observe and analyze the morphological changes of apoptotic HUVECs, the apoptosis rate, the inhibition and destruction of CAM vessels, the microvessel density (MVD) of gallbladder carcinoma in nude mice, Peripheral blood vessel destruction morphology. Results (1) The ED_ (50) of NCTD to HUVEC was 0.352 g / L. The apoptosis rate of HU-VEC in NCTD group was significantly higher than that in control group [(76.86 ± 5.67)% vs (4.60 ± 1.45)%, P <0.05] With different degrees of apoptosis; NCTD and ES, with the prolonged role in inducing apoptosis of vascular endothelial cells to strengthen, showing a time-dependent relationship. (2) In the CAM experiment, the NCTD concentration had a positive correlation with the inhibition of CAM angiogenesis (r = 0.89, t = 3.45, P <0.05) % ~ 60% vs 80%, P <0.05). The number of capillary vessel dendrites and the inhibition rate of angiogenesis in CAM increased (90.9% -100.0% vs 0-53%, P <0.05) No significant difference with ES, NCTD on CAM angiogenesis inhibition. (3) After the application of NCTD, the tumor-bearing MVD and 5-Fu group (4.12 ± 1.40 vs 15.80 ± 5.90, P <0.01), the control group (4.12 ± 1.40 ratio 17.60 ± 3.20, P <0.01), but there was no significant difference with ES (P> 0.05) .Apoptosis was found under the microscope, The tumor-bearing MVD and tumor volume (r = 0.985, t = 8.069, P <0.05) .05). Conclusion NCTD can effectively inhibit and destroy the tumor angiogenesis of gallbladder carcinoma, and then effectively inhibit the proliferation and growth of gallbladder carcinoma.