摘要
目的血管生成对肿瘤的生长和转移是至关重要的,而血管抑素(AS)是已知的最强的血管生成抑制因子之一,血管内皮生长因子(VEGF)是最重要的血管生成因子,而两者在人舌鳞癌中的关系,尚无报导。本课题的目的则是探讨AS对人舌鳞癌细胞VEGF的表达的影响。
方法将Tca8113人舌鳞癌细胞进行常规培养传代,收集正处于指数增生期的第6代Tca8113细胞系, 18只BALB/C裸鼠,年龄均为6周大,将悬浮于100μl的PBS液中的2×106个肿瘤细胞于每只裸鼠近中线的背部进行皮下注射。然后随机分成三组(每组6只):PBS组注射PBS、AS(250ng)组注射250ng纯化AS,AS(30μg)组注射30μg纯化AS,一天两次腹腔注射,同时一周两次测量肿瘤的大小,21天后处死小鼠,将取出的每个肿瘤一分为二,一半用免疫组织化学检测VEGF及其两个受体(VEGFR1、VEGFR2)和CD34,用TUNEL法检测肿瘤细胞的凋亡,另一半用半定量反转录聚合酶链反应检测VEGF、VEGFR1和VEGFR2的mRNA的表达。
结果AS(250ng)和AS(30μg)组中:凋亡指数(AI)明显增加(P<0.05);而肿瘤体积,微血管密度(MVD,用CD34抗体标记并用来评价肿瘤血管生成), VEGF、VEGFR1和VEGFR2在蛋白质及mRNA水平表达,都有明显降低(P<0.05);且AS(30μg)组更为显著(P<0.05)。
结论AS下调人舌鳞癌细胞VEGF的表达且呈剂量依赖性。(另外,AS抑制人舌鳞癌的生长、抑制人舌鳞癌的血管生成、促进人舌鳞癌细胞的凋亡、下调人舌鳞癌细胞VEGFR1和VEGFR2的表达,且呈剂量依赖性。)
Objective angiogenesis plays a critical role in tumor growth and tumor metastais, Angiostatin(AS) is one of the known the strongest angiogenesis inhibitors while vascular endothelial growth factor(VEGF) is the most important angiogenesis activator,the link between AS and VEGF in human tongue squamous cell carcinoma has never been reported.The purpose of these experiments is to evaluate the effect of angiostatin on va-scular endothelial growth factor expression in human tongue squamous cell carcinoma cells.
Methods Human tongue squamous cell carcinoma (Tca8113) cell line was cultured and subcultured according to routine.cells of the sixth generation during the cell growth period were collected and BALB/C nude mice,6 weeks of age,received s.c. injections of 2×106 tumor cells suspended in 100μl of PBS in the proximal dorsum. then eighteen mice were randomly divided into three groups (6 mice each): the PBS group in- jected with PBS, the AS(250ng) group injected with 250ng purified angiostatin,and the AS(30μg) group in - jected with 30μg purified angiostatin . reagents were injected intraperitoneally twice daily and tumor vo- lume was calculated twice weekly for 21 days ,then animals were sacrificed and After surgical resection each tumor specimen was divided into two parts. The first part was submitted to both Immunocytochemistry for VEGF、VEGFR1、VEGFR2 and CD34,and TUNEL apoptosis assay. The second part was prepared for Semi-Quantitative RT-PCR Analysis of VEGF、VEGFR1 and VEGFR2 mRNA Abundance.
Results in the AS(250ng) group and the AS(30μg) group,there was an increase in apoptotic index (AI) (P<0.05) and there was a decrease in Protein and mRNA expression levels of VEGF、VEGFR1 and VEGFR2、in tumor volume and in MVD(tumor angiogenesis was estimated by determining microvessel density with the use of CD34 antibody) (P<0.05). These changes were more obvious in the the AS(30μg) group than in the AS(250ng) group(P<0.05).
Conclusion AS down-regulates VEGF expression in human tongue squamous cell carcinoma cells in a dose-dependent fasion。(in addition,AS leads to a growth and angiogenesis suppression and promotes apoptosis and down-regulates VEGFR1 and VEGFR2 in human tongue squamous cell carcinoma in a dose-dependent fasion。)
引文
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