中华耳鼻咽喉头颈外科杂志
中華耳鼻嚥喉頭頸外科雜誌
중화이비인후두경외과잡지
CHINESE JOURNAL OF OTORHINOLARYNGOLOGY HEAD AND NECK SURGERY
2012年
1期
53-57
,共5页
刘勇%张欣%余长云%邱元正%黄东海%周小娟%谭平清%肖健云%田勇泉
劉勇%張訢%餘長雲%邱元正%黃東海%週小娟%譚平清%肖健雲%田勇泉
류용%장흔%여장운%구원정%황동해%주소연%담평청%초건운%전용천
头颈部肿瘤%癌,鳞状细胞%受体,EphA2%肿瘤转移%新生血管化,病理性
頭頸部腫瘤%癌,鱗狀細胞%受體,EphA2%腫瘤轉移%新生血管化,病理性
두경부종류%암,린상세포%수체,EphA2%종류전이%신생혈관화,병이성
Head and neck neoplasms%Carcinoma,squamous cell%Receptor,EphA2%Neoplasms metastasis%Neovascularization,pathologic
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.
目的 研究EphA2對頭頸部鱗狀細胞癌(簡稱鱗癌)血管新生及頸淋巴轉移的影響.方法 利用慢病毒介導的短髮夾RNA( short hairpin RNA,shRNA)沉默EphA2在高轉移性頭頸鱗癌細胞株M2中的錶達,嘌呤黴素篩選穩定剋隆,反轉錄PCR及Western blot技術檢測EphA2沉默效果；構建頭頸鱗癌高轉移動物模型,通過HE染色驗證其成瘤及頸淋巴轉移情況；免疫組織化學技術檢測移植瘤微血管密度,Western blot技術檢測移植瘤標本中EphA2及血管內皮生長因子(VEGF)的錶達情況.結果 篩選齣EphA2基因穩定沉默的M2細胞(定義為M2 EphA2 RNA+即實驗組),併成功構建頭頸鱗癌高轉移動物模型,成瘤率達100％.25 d後,實驗組移植瘤體積為(430.7±190.0) mm3(x±s,以下同)較對照組的(1179.0 ±289.4) mm3明顯減少(t =5.597,P＜0.01),質量顯著減輕(t=-4.560,P＜0.05),且雙側頸淋巴轉移率明顯降低(Mann-Whitney U=10.0,P＜0.05).Western blot 顯示實驗組移植瘤組織中EphA2及VEGF蛋白錶達顯著下調,免疫組織化學技術髮現實驗組瘤體微血管密度為(4.74±0.67)箇/視野,顯著少于對照組的(14.17±0.59)箇/視野(t=26.751,P＜0.01).結論 EphA2沉默能抑製頭頸部鱗癌細胞的生長及轉移,併能明顯減少頭頸部鱗癌組織內的腫瘤新生血管,且該腫瘤新生血管的抑製可能與促血管生成因子VEGF的減少有關.
목적 연구EphA2대두경부린상세포암(간칭린암)혈관신생급경림파전이적영향.방법 이용만병독개도적단발협RNA( short hairpin RNA,shRNA)침묵EphA2재고전이성두경린암세포주M2중적표체,표령매소사선은정극륭,반전록PCR급Western blot기술검측EphA2침묵효과；구건두경린암고전이동물모형,통과HE염색험증기성류급경림파전이정황；면역조직화학기술검측이식류미혈관밀도,Western blot기술검측이식류표본중EphA2급혈관내피생장인자(VEGF)적표체정황.결과 사선출EphA2기인은정침묵적M2세포(정의위M2 EphA2 RNA+즉실험조),병성공구건두경린암고전이동물모형,성류솔체100％.25 d후,실험조이식류체적위(430.7±190.0) mm3(x±s,이하동)교대조조적(1179.0 ±289.4) mm3명현감소(t =5.597,P＜0.01),질량현저감경(t=-4.560,P＜0.05),차쌍측경림파전이솔명현강저(Mann-Whitney U=10.0,P＜0.05).Western blot 현시실험조이식류조직중EphA2급VEGF단백표체현저하조,면역조직화학기술발현실험조류체미혈관밀도위(4.74±0.67)개/시야,현저소우대조조적(14.17±0.59)개/시야(t=26.751,P＜0.01).결론 EphA2침묵능억제두경부린암세포적생장급전이,병능명현감소두경부린암조직내적종류신생혈관,차해종류신생혈관적억제가능여촉혈관생성인자VEGF적감소유관.
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.
