中华医学杂志
中華醫學雜誌
중화의학잡지
National Medical Journal of China
2012年
36期
2556-2560
,共5页
丁宗励%王璐%朱宏%凌亭生%郝波%施瑞华
丁宗勵%王璐%硃宏%凌亭生%郝波%施瑞華
정종려%왕로%주굉%릉정생%학파%시서화
食管肿瘤%RNA干扰%基质金属蛋白酶2%血管生成拟态
食管腫瘤%RNA榦擾%基質金屬蛋白酶2%血管生成擬態
식관종류%RNA간우%기질금속단백매2%혈관생성의태
Esophageal neoplasms%RNA interference%Matrix metalloproteinase 2%Vasculogenic mimicry
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)、Eca109/Neo(空载体组)、Eca109/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 ±0.01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±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％±1.6％比4.2％±0.8％和4.8％±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(27.7±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( PI3 K)/Akt通路参与调控了食管鳞癌VM的形成,可能是PI3K/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.
目的 探討蛋白激酶B(Akt)對Eca109細胞生物學功能的影響及其與血管生成擬態(VM)相關基因錶達的關繫.方法 應用倒置熒光顯微鏡觀察Akt的榦擾質粒轉染食管癌細胞Eca109後綠色熒光蛋白的錶達；平闆剋隆形成實驗檢測轉染前後細胞增殖能力的變化；Transwell方法檢測榦擾前後細胞遷移能力的變化；三維培養觀察併計數轉染前後各組細胞的管狀結構數量；將Eca109(未轉染組)、Eca109/Neo(空載體組)、Eca109/小榦擾RNA(siRNA) Akt細胞(穩定轉染組)培養後檢測細胞凋亡率,瞭解抑製Akt基因對細胞凋亡的影響；以Western印跡檢測各株細胞中Akt蛋白與基質金屬蛋白酶2(MMP-2)基因蛋白錶達的關繫.結果 Western印跡結果顯示穩定轉染組Akt蛋白錶達低于空載體組及未轉染組(0.03 ±0.01比1.49 ±0.39和1.47 ±0.41,均P＜0.05).Transwell實驗結果,穩定轉染組穿過人工膜的細胞數明顯少于空載體組與未轉染組[(48±9)比(122±11)和(128 ±10)箇,均P＜0.05]；穩定轉染組的剋隆形成數目顯著低于空載體組及未轉染組[(63±7)比(148±11)和(163±15)箇,均P＜0.05].流式細胞儀以膜聯蛋白V/AAD雙標法顯示穩定轉染組凋亡率高于空載體組與未轉染組(12.2％±1.6％比4.2％±0.8％和4.8％±0.8％,均P＜0.05).空載體組及未轉染組在基質膠上均能形成典型的血管網狀樣結構,穩定轉染組管狀結構數目明顯少于未轉染組與空載體組[(14.0±1.2)比(30.0±1.2)和(27.7±1.5)箇,均P＜0.05]；穩定轉染組MMP-2蛋白錶達均低于未轉染組及空載體組(均P＜0.05).結論 燐脂酰肌醇三燐痠激酶( PI3 K)/Akt通路參與調控瞭食管鱗癌VM的形成,可能是PI3K/Akt通路通過調節MMP-2來影響食管癌細胞VM的形成.阻斷Akt通路有可能成為治療人食管鱗癌的新靶點.
목적 탐토단백격매B(Akt)대Eca109세포생물학공능적영향급기여혈관생성의태(VM)상관기인표체적관계.방법 응용도치형광현미경관찰Akt적간우질립전염식관암세포Eca109후록색형광단백적표체；평판극륭형성실험검측전염전후세포증식능력적변화；Transwell방법검측간우전후세포천이능력적변화；삼유배양관찰병계수전염전후각조세포적관상결구수량；장Eca109(미전염조)、Eca109/Neo(공재체조)、Eca109/소간우RNA(siRNA) Akt세포(은정전염조)배양후검측세포조망솔,료해억제Akt기인대세포조망적영향；이Western인적검측각주세포중Akt단백여기질금속단백매2(MMP-2)기인단백표체적관계.결과 Western인적결과현시은정전염조Akt단백표체저우공재체조급미전염조(0.03 ±0.01비1.49 ±0.39화1.47 ±0.41,균P＜0.05).Transwell실험결과,은정전염조천과인공막적세포수명현소우공재체조여미전염조[(48±9)비(122±11)화(128 ±10)개,균P＜0.05]；은정전염조적극륭형성수목현저저우공재체조급미전염조[(63±7)비(148±11)화(163±15)개,균P＜0.05].류식세포의이막련단백V/AAD쌍표법현시은정전염조조망솔고우공재체조여미전염조(12.2％±1.6％비4.2％±0.8％화4.8％±0.8％,균P＜0.05).공재체조급미전염조재기질효상균능형성전형적혈관망상양결구,은정전염조관상결구수목명현소우미전염조여공재체조[(14.0±1.2)비(30.0±1.2)화(27.7±1.5)개,균P＜0.05]；은정전염조MMP-2단백표체균저우미전염조급공재체조(균P＜0.05).결론 린지선기순삼린산격매( PI3 K)/Akt통로삼여조공료식관린암VM적형성,가능시PI3K/Akt통로통과조절MMP-2래영향식관암세포VM적형성.조단Akt통로유가능성위치료인식관린암적신파점.
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 Ecal09 cells by liposomemediated 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 group.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 significantly lower 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-AADdouble standard method demonstrated that the apoptotic rate of stably transfected 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 ).Eca109 and Eca109/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.
