EphA2调控头颈部鳞状细胞癌血管新生及转移的体内实验

目的 研究EphA2对头颈部鳞状细胞癌(简称鳞癌)血管新生及颈淋巴转移的影响.方法 利用慢病毒介导的短发夹RNA( short hairpin RNA,shRNA)沉默EphA2在高转移性头颈鳞癌细胞株M2中的表达,嘌呤霉素筛选稳定克隆,反转录PCR及Western blot技术检测EphA2沉默效果;构建头颈鳞癌高转移动物模型,通过HE染色验证其成瘤及颈淋巴转移情况;免疫组织化学技术检测移植瘤微血管密度,Western blot技术检测移植瘤标本中EphA2及血管内皮生长因子(VEGF)的表达情况.结果 筛选出EphA2基因稳定沉默的M2细胞(定义为M2 EphA2 RNA+即实验组),并成功构建头颈鳞癌高转移动物模型,成瘤率达100％.25 d后,实验组移植瘤体积为(430.7±190.0) mm3(x±s,以下同)较对照组的(1179.0 ±289.4) mm3明显减少(t =5.597,P＜0.01),质量显著减轻(t=-4.560,P＜0.05),且双侧颈淋巴转移率明显降低(Mann-Whitney U=10.0,P＜0.05).Western blot 显示实验组移植瘤组织中EphA2及VEGF蛋白表达显著下调,免疫组织化学技术发现实验组瘤体微血管密度为(4.74±0.67)个/视野,显著少于对照组的(14.17±0.59)个/视野(t=26.751,P＜0.01).结论 EphA2沉默能抑制头颈部鳞癌细胞的生长及转移,并能明显减少头颈部鳞癌组织内的肿瘤新生血管,且该肿瘤新生血管的抑制可能与促血管生成因子VEGF的减少有关.

EphA2 promotes angiogenesis and metastasis of head and neck squamous cell carcinoma in vivo

Objective To investigate the effects of EphA2 on the angiogenesis and cervical lymph node metastasis of squamous cell carcinoma of the head and neck (SCCHN) in vivo.Methods EphA2 short hairpin (shRNA) lentiviral particles were used to knockdown the expression of EphA2 in SCCHN cell line M2 with high lymph nodes metastasis rate. Stable clones,obtained by puromycin screening,were assayed by RT-PCR and Western blot to validate the gene silencing efficiency and were used to establish SCCHN metastatic xenograft mouse model.Hematoxylin-eosin staining was applied to identify cervical lymph node metastasis of SCCHN in xenografted tumors.Immunohistochemistry was used to observe microvessel density.Western blot was used to investigate the protein expressions of EphA2 and vascular endothelial,growth factor (VEGF). Results EphA2 shRNA lentiviral particles efficiently decreased the mRNA and protein expressions of EphA2 in SCCHN cell line M2,which were further successfully utilized to establish SCCHN metastatic xenograft mouse model.Compared with xenografted tumors in control group,xenografted tumors in M2EphA2RNAi+ group decreased significantly tumor volume (430.7 ± 190.0) mm3 ((x) ± s) vs(1179.0±289.4) mm3] and weight (0.26-±0.10) gvs (0.54±0.12) g] (bothP＜0.05).More importantly,bilateral cervical lymph node metastasis rate in M2EphA2RNAi+ was also greatly declined (Mann-Whitney U =10.0,P ＜ 0.05).Decreased protein expressions of EphA2 and VEGF and microvessel density were observed in M2EphA2RNAi+ group (t =26.751,P ＜0.01).Conclusions Knockdown of EphA2 expression led to the inhibition of tumor growth and metastasis in SCCHN nude mouse model.More importantly,SCCHN angiogenesis was also impeded,which might be associated with the decreased expression of VEGF.