摘要
目的研究腺病毒介导的反义血管内皮细胞生长因子165(Adenovirus-mediatedaversevascularendothelialgrowthfactor165,Ad-aVEGF165)基因转染对恶性黑色素瘤体内外生长的影响。方法选用感染复数为100的腺病毒介导的绿色荧光蛋白(Adenovirus-mediatedgreenfluorescentprotein,Ad-GFP)和Ad-aVEGF165转染人血管内皮细胞株304(endotheliumcellofvessel304,ECV304)和人恶性黑色素细胞(A375)。ECV304细胞分为3组:A375母系组、Ad-GFP组和Ad-aVEGF165组。A375细胞分为3组:1640组、Ad-GFP组和Ad-aVEGF165组。测定其生长曲线、细胞周期和VEGF基因的表达。于裸鼠皮下接种A375细胞,待成瘤后进行转种和治疗,根据瘤体内注射物的不同随机分为PBS组、Ad-GFP组和Ad-aVEGF165组,每组5只;治疗结束后行大体观察、HE染色观察和微血管密度(micro-vasculardensity,MVD)检测。结果A375母系组和Ad-GFP组上清液均能刺激ECV304细胞的生长,Ad-aVEGF165组上清液能抑制ECV304细胞的生长。A375细胞中3组细胞均呈增殖趋势,各时间点的增殖速度比较无统计学意义(P>0.05)。ECV304细胞Ad-aVEGF165组增殖指数降低(P<0.05),A375细胞3组细胞增殖指数比较无统计学意义(P>0.05)。A375细胞1640组、Ad-GFP组和Ad-aVEGF165组平均灰度值分别为234.41±13.80、222.73±3.67和180.84±6.34。转种后2周Ad-aVEGF165组裸鼠体内肿瘤体积比Ad-GFP组和PBS组明显减小,并随时间延长越来越明显。HE染色Ad-aVEGF165组有凝固性坏死。PBS组、Ad-GFP组、Ad-aVEGF165组的MVD值分别为65±10、52±11和30±6个/视野。结论在体外,Ad-VEGF165通过抑制A375细胞VEGF基因的表达,进而抑制ECV304细胞的生长。在体内,Ad-aVEGF165阻断肿瘤血管的生成,进而抑制人恶性黑色素瘤的生长。
Objective To investigate the effect of Adenovirus-mediated averse vascular endothelial growth factor165 (Ad-aVEGF165)on the growth of human melanoma cells(A375) in vivo and in vitro. Methods In vitro, the 100 multiplicity of infection of Aadenovirus-mediated green fluorescent protein(Ad-GFP)and Ad-aVEGF165 were transfected into human endothelium ceil of vessel304 (ECV304) and A375. ECV304 ceils were divided into 3 groups : A375 group, Ad-GFP group and Ad-aVEGF165 group. A375 cells were also divided into 3 groups : 1640 group, Ad-GFP group and Ad-aVEGF165 group. Their effects were analyzed by proliferation assay, cell cycle, and VEGF expression. In vivo, A375 cells were injected into the axilla of the nude mouse. When the tumor formed, they were transplanted into another 15 mice. After treatment, the tumor was excised for naked eye observation, HE observation and micro-vascular density (MVD) counting. Results The cell supernatant fluid of A375 group and Ad-GFP group could stimulate ECV304 cell growth,but that of Ad-aVEGF165 group could inhibit the growth of ECV304 cell. All the A375 cells in 3 groups had the proliferation trend, showing no statistically significant difference(P〉0.05). ECV304 cell proliferation index(PI) in Ad-aVEGF165 group reduced(P〈0. 05). There was no statistically significant difference(P〉0. 05) in the PI of A375 cell. The A375 cell integral optical densities were 234. 41±13. 8 in 1640 group, 222.73±3. 67 in Ad-GFP group and 180. 84±6.34 in Ad-aVEGF165 group. The tumor volume in Ad-aVEGF165 group was smaller than that in Ad-GFP group and PBS group at 2 weeks after operation, the trend became much obvious with the time delay. Ad-aVEGF165 brought to much tissue necrosis under HE stain. The MVD of PBS group, Ad-GFP group and Ad-aVEGF165 group were 65±10/view,52±11/view and 30±6/view, respectively. Conclusion In Vitro, Ad-VEGF165 gene could inhibited ECV304 cells' growth by weakening VEGF expression of A375 cells. In vivo, Ad-aVEGF165 could inhibit the growth of human melanoma from blocking micro-vascular.
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2006年第5期499-503,共5页
Chinese Journal of Reparative and Reconstructive Surgery