摘要
目的探讨蛋白激酶B（Akt）对Eca109细胞生物学功能的影响及其与血管生成拟态（VM）相关基因表达的关系。方法应用倒置荧光显微镜观察Akt的干扰质粒转染食管癌细胞Eca109后绿色荧光蛋白的表达；平板克隆形成实验检测转染前后细胞增殖能力的变化；Transwell方法检测干扰前后细胞迁移能力的变化；三维培养观察并计数转染前后各组细胞的管状结构数量；将Eca109（未转染组）、Ecal09／Neo（空载体组）、Ecal09／小干扰RNA（siRNA）Akt细胞（稳定转染组）培养后检测细胞凋亡率，了解抑制Akt基因对细胞凋亡的影响；以Western印迹检测各株细胞中Akt蛋白与基质金属蛋白酶2（MMP-2）基因蛋白表达的关系。结果Western印迹结果显示稳定转染组Akt蛋白表达低于空载体组及未转染组（0．03±0．0l比1．49±0．39和1．47±0．41，均P〈0．05）。Transwell实验结果，稳定转染组穿过人工膜的细胞数明显少于空载体组与未转染组[（48-4-9）比（122±11）和（128±10）个，均P〈0．05]；稳定转染组的克隆形成数目显著低于空载体组及未转染组[（63±7）比（148±11）和（163±15）个，均P〈0．05]。流式细胞仪以膜联蛋白V／AAD双标法显示稳定转染组凋亡率高于空载体组与未转染组（12．2％4-1．6％比4．2％±0．8％和4．8％4-0．8％，均P〈0．05）。空载体组及未转染组在基质胶上均能形成典型的血管网状样结构，稳定转染组管状结构数目明显少于未转染组与空载体组[（14．0±1．2）比（30．0±1．2）和（27．74-1．5）个，均P〈0．05]；稳定转染组MMP-2蛋白表达均低于未转染组及空载体组（均P〈0．05）。结论磷脂酰肌醇三磷酸激酶（PI，K）／Akt通路参与调控了食管鳞癌VM的形成，可能是PI，K／Akt通路通过调节MMP-2来影响食管癌细胞VM的形成。阻断Akt通路有可能成为治疗人食管鳞癌的新靶点。
Objective To investigate the inhibitory effect of siRNA targeting Akt on the biological behavior of esophagus squamous cell carcinoma cell line in vitro and to explore the relationship between Akt and vasculogenic mimicry （VM）-related genes in esophageal squamous cell carcinoma.Methods The plasmid-harboring small interfering RNA targeting Akt was introduced into Eca109 cells by liposome- mediated transfection. The proliferation of Eca109 cell was determined by colony formation assay. The cellular migration was evaluated by Transwell migration assay. And three dimensional cell culture was employed to observe and count the number of capillary structure for each cell groiJp. Flow cytometry （FCM） was used to detect the apoptotic rate of Eca109, Eca109/Neo and Eca109/siRNA Akt cells under normoxia exposure. The apoptotic rate was assessed by Annexin V/7-AAD double labeling. And the expressions of Akt and matrix metalloproteinase-2 （MMP-2） protein were detected by Western blotting. Results The results of Western blotting showed that the expression of Akt in stably transfected group were significantlylower than empty carrier and untransfected groups （0. 03 ±0. 01 vs 1.49 ±0. 39 and 1.47 ±0. 41, both P 〈 0. 05）. Transwell migration assay showed that fewer Ecal09/8 cells could move through the artificial basement membrane as compared with untransfected and empty carrier groups （48 ± 9 vs 128 ± 10 and 122 ± 11, both P 〈 0. 05 ）. Clone formation number of stably transfected group was significantly lower than empty carrier and untransfected groups （63 ＋7 vs 148 ± 11 and 163 ± 15, both P 〈0. 05）. Annexin V/7-AAD double standard method demonstrated that the apoptotic rate of stably transfeeted group was much more than those of untransfected and empty carrier groups （ 12. 2% ± 1.6% vs 4. 8% ± 0. 8% and 4. 2% ＋ 0. 8% , both P 〈 0. 05 ）. Ecal09 and Eeal09/Neo cells were capable of forming the in vitro structures of VM. And the number of tube-shaped structure in stably transfected group was markedly less than those of untransfected and empty carrier groups （14.0 ±1.2 vs 30.0 ±1.2 and 27.7 ±1.5, both P〈0.05）. MMP-2 protein expression in stably transfected group was less than those of untransfected and empty carrier groups （both P 〈0. 05 ）. Conclusions The PI3K/Akt pathway is involved in the regulation of VM formation in esophageal squamous cell carcinoma through the action of MMP-2. Blockade of this pathway may provide a new therapeutic approach to human esophagus squamous cell carcinoma.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2012年第36期2556-2560,共5页
National Medical Journal of China
基金
江苏省“六大人才高峰”项目（2009C09）
关键词
食管肿瘤
RNA干扰
基质金属蛋白酶2
血管生成拟态
Esophageal neoplasms
RNA interference
Matrix metalloproteinase 2
Vasculogenic mimicry
