神经母细胞瘤细胞在鸡胚尿膜囊模型上生物学性状的研究

Biological characteristics of neuroblastoma transfected with hypoxia inducing factor-laon chicken chorioallantoic membrane model.

目的 探讨神经母细胞瘤（neuroblastoma,NB）鸡胚尿膜囊（chicken chorioallantoic membrane,CAM）血管模型的建立,以及缺氧诱导因子1α（hypoxia inducing factor-1α,HIF-1α）对神经母细胞瘤细胞株SH-SY5Y肿瘤生长和血管生成的影响.方法 含有目的 基因HIF1 α和对照组增强型绿色荧光蛋白（Enhanced green fluorescent protein,EGFP）的非增值性重组5型腺病毒分别转染人神经母细胞瘤细胞株SH-SY5Y,检测目的 基因和目的 蛋白表达.SH-SY5Y细胞株经不同处理后分为两组：A组为空白对照组;SH-SY5Y细胞组;B组为HIF1 α-SH-SY5Y组;EGFP-SH-SY5Y组.不同组别细胞悬液分别种植于CAM,1周后A组提取组织RNA,检测人源性促血管相关基因的表达.B组于1周后测量肿瘤体积,分别测定肿瘤周嗣固定范围内放射状血管所占面积以及血管分支数.结果 RT-PCR和细胞免疫组化检测HIF1α转基因组较对照EGFP组目的 基因和蛋白表达水平显著增高,差异有显著统计学意义（P〈0.05）.A组SH-SY5Y细胞悬液种植于CAM膜后,肿瘤形成、周围放射状血管网络形成、肿瘤组织中检测到人源性促血管生长因子的表达.B组HIF1α转基因组血管计数、血管面积较对照EGFP组显著增多,差异有统计学意义（P〈0.05）,而肿瘤大小无显著统计学差异（P〉0.05）.结论 CAM模型作为NB血管模型有可行性.HIF-1 α与肿瘤血管生成具有显著相关性,可作为神经母细胞瘤患者预后评估的指标.

Objective To construct CAM model of neuroblastoma and test the effect of HIF-1 α on tumor growth and angiogenesis. Methods SH-SYSY were transfeeted with adenovirus either encoding EGFP or HIF-1 α. The transfection efficiency was measured by RT-PCR and Immunohistochemistry of target gene and its related protein. The CAM models were categoried into two groups according to different treatment： Group A： control group; group SH-SYSY;Group B： group HIFI α-SH-SYSY;group EGFP-SH-SYSY. Cell suspensions treated diferently corresponding to different groups above were transplanted onto the CAM. One week later,Total RNA was extracted from tumor nodules of group A and the gene associated angiogenesis were tested. Tumor size, vessels branches and the area of vessels around tumor nodules of group B were measured, All data were analyzed with SPSS software. Results RT-PCR and immunohistochemistry shown that the expression of both target gene and HIF-1α protein were increased. Tumor nodules, surrounded by newly formed vessels, were formed on the CAM. And the existence of human genes related angiogenesis was proved by RT-PCR. The tumor size of HIF-1 α and EGFP group was not significantly different, while the vessels branches and area of vessels around tumor nodules were different significantly （P 〈 0.05）. Conclusions Adenovirus encoding HIF-1 α could be transfected steadily into SH-SY5Y. The tumor formation, vessels branches around tumor nodules, added with the existence of human gene related angiogenesis can demonstrate the feasibility of CAM model. HIF-1 α, although do not associated with the increase of tumor size, contributes to tumor vessel formation, so it might be used as index of prognosis of neuroblatoma.