中华消化杂志
中華消化雜誌
중화소화잡지
Chinese Journal of Digestion
2013年
2期
115-118
,共4页
唐娜娜%朱宏%何桂钧%郝波%施瑞华
唐娜娜%硃宏%何桂鈞%郝波%施瑞華
당나나%주굉%하계균%학파%시서화
缺氧诱导因子1,α亚基%糖酵解%RNA,小分子干扰%食管肿瘤
缺氧誘導因子1,α亞基%糖酵解%RNA,小分子榦擾%食管腫瘤
결양유도인자1,α아기%당효해%RNA,소분자간우%식관종류
Hypoxia-inducible factor 1,alpha subunit%Glycolysis%RNA,small interfering%Esophageal neoplasms
目的 观察人食管鳞状细胞癌细胞中缺氧诱导因子-1α(HIF-1α)对糖酵解的影响并探讨其可能的作用机制.方法 TEl3、Ecal09细胞常氧(O2体积分数为0.20)及缺氧(O2体积分数为0.01)培养,缺氧时间为6、12、24、48 h,RNA干扰法稳定静默HIF-1α获得TE13/小干扰(si)HIF、Eca109/siHIF细胞,培养方法和时间同TE13、Eca109.Western印迹法检测HIF-1α的表达变化；分光光度法检测培养上清液中乳酸浓度变化；实时定量PCR检测葡萄糖转运蛋白-1(GLUT-1)、乳酸脱氢酶A(LDHA)的mRNA表达变化；Western印迹法检测GLUT-1、LDHA蛋白表达变化.t或t'检验用于分析不同缺氧时间对HIF-1α蛋白表达的影响,组间差异比较采用单因素方差分析.结果 TE13、Eca109细胞缺氧后HIF-1α表达增强,12h达高峰(t=6.11,8.31,P＜0.05).常氧条件下,TE13/siHIF和Eca109/siHIF细胞乳酸分泌分别为(1.24±0.33)、(1.28±0.37) mmol/L,较TE13和Eca109细胞显著下降[(3.25±1.34)、(4.91±1.69) mmol/L,t=2.53、3.59,P均＜0.05]；TE13、Eca109细胞缺氧后乳酸分泌增多[(6.48±1.73)、(8.02±1.95) mmol/L,t=2.715、2.050,P均＜0.05],TE13/siHIF、Eca109/siHIF细胞缺氧后乳酸分泌无明显增多(P＞0.05).TE13/siHIF和Eca109/siHIF细胞中,GLUT-1、LDHA的mRNA表达均明显受抑(常氧:t=6.98、3.92、7.25、3.67,P均＜0.05),GLUT-1蛋白表达明显减弱(常氧:t=4.57、16.56,缺氧:t=6.19、6.09,P均＜0.05),LDHA蛋白表达稍减弱(P＞0.05).结论 抑制人食管鳞状细胞癌中HIF-1α可降低细胞的糖酵解水平,该途径可能涉及GLUT-1、LDHA的表达抑制；除HIF-1α以外,LDHA的蛋白表达可能同时存在其他调控因素.
目的 觀察人食管鱗狀細胞癌細胞中缺氧誘導因子-1α(HIF-1α)對糖酵解的影響併探討其可能的作用機製.方法 TEl3、Ecal09細胞常氧(O2體積分數為0.20)及缺氧(O2體積分數為0.01)培養,缺氧時間為6、12、24、48 h,RNA榦擾法穩定靜默HIF-1α穫得TE13/小榦擾(si)HIF、Eca109/siHIF細胞,培養方法和時間同TE13、Eca109.Western印跡法檢測HIF-1α的錶達變化；分光光度法檢測培養上清液中乳痠濃度變化；實時定量PCR檢測葡萄糖轉運蛋白-1(GLUT-1)、乳痠脫氫酶A(LDHA)的mRNA錶達變化；Western印跡法檢測GLUT-1、LDHA蛋白錶達變化.t或t'檢驗用于分析不同缺氧時間對HIF-1α蛋白錶達的影響,組間差異比較採用單因素方差分析.結果 TE13、Eca109細胞缺氧後HIF-1α錶達增彊,12h達高峰(t=6.11,8.31,P＜0.05).常氧條件下,TE13/siHIF和Eca109/siHIF細胞乳痠分泌分彆為(1.24±0.33)、(1.28±0.37) mmol/L,較TE13和Eca109細胞顯著下降[(3.25±1.34)、(4.91±1.69) mmol/L,t=2.53、3.59,P均＜0.05]；TE13、Eca109細胞缺氧後乳痠分泌增多[(6.48±1.73)、(8.02±1.95) mmol/L,t=2.715、2.050,P均＜0.05],TE13/siHIF、Eca109/siHIF細胞缺氧後乳痠分泌無明顯增多(P＞0.05).TE13/siHIF和Eca109/siHIF細胞中,GLUT-1、LDHA的mRNA錶達均明顯受抑(常氧:t=6.98、3.92、7.25、3.67,P均＜0.05),GLUT-1蛋白錶達明顯減弱(常氧:t=4.57、16.56,缺氧:t=6.19、6.09,P均＜0.05),LDHA蛋白錶達稍減弱(P＞0.05).結論 抑製人食管鱗狀細胞癌中HIF-1α可降低細胞的糖酵解水平,該途徑可能涉及GLUT-1、LDHA的錶達抑製；除HIF-1α以外,LDHA的蛋白錶達可能同時存在其他調控因素.
목적 관찰인식관린상세포암세포중결양유도인자-1α(HIF-1α)대당효해적영향병탐토기가능적작용궤제.방법 TEl3、Ecal09세포상양(O2체적분수위0.20)급결양(O2체적분수위0.01)배양,결양시간위6、12、24、48 h,RNA간우법은정정묵HIF-1α획득TE13/소간우(si)HIF、Eca109/siHIF세포,배양방법화시간동TE13、Eca109.Western인적법검측HIF-1α적표체변화；분광광도법검측배양상청액중유산농도변화；실시정량PCR검측포도당전운단백-1(GLUT-1)、유산탈경매A(LDHA)적mRNA표체변화；Western인적법검측GLUT-1、LDHA단백표체변화.t혹t'검험용우분석불동결양시간대HIF-1α단백표체적영향,조간차이비교채용단인소방차분석.결과 TE13、Eca109세포결양후HIF-1α표체증강,12h체고봉(t=6.11,8.31,P＜0.05).상양조건하,TE13/siHIF화Eca109/siHIF세포유산분비분별위(1.24±0.33)、(1.28±0.37) mmol/L,교TE13화Eca109세포현저하강[(3.25±1.34)、(4.91±1.69) mmol/L,t=2.53、3.59,P균＜0.05]；TE13、Eca109세포결양후유산분비증다[(6.48±1.73)、(8.02±1.95) mmol/L,t=2.715、2.050,P균＜0.05],TE13/siHIF、Eca109/siHIF세포결양후유산분비무명현증다(P＞0.05).TE13/siHIF화Eca109/siHIF세포중,GLUT-1、LDHA적mRNA표체균명현수억(상양:t=6.98、3.92、7.25、3.67,P균＜0.05),GLUT-1단백표체명현감약(상양:t=4.57、16.56,결양:t=6.19、6.09,P균＜0.05),LDHA단백표체초감약(P＞0.05).결론 억제인식관린상세포암중HIF-1α가강저세포적당효해수평,해도경가능섭급GLUT-1、LDHA적표체억제；제HIF-1α이외,LDHA적단백표체가능동시존재기타조공인소.
Objective To investigate the effects of hypoxia-inducible factor (HIF)-1α on glycolysis of human esophageal squamous carcinoma cells and the possible mechanism.Methods TE13 and Eca109 cells were cultured under normal oxygen (20％O2) and hypoxia (1％O2) conditions.The hypoxia was duration 6 hours,12 hours,24 hours and 48 hours.HIF-1α gene was stable silented by RNA interference method and TE13/small interfering HIF cells and Eca109/siHIF cells were obtained.The cell culture condition and time was same as TE13 and Eca109 cells.The changes of HIF-1α expression were detected by Western-blot.The changes of lactic acid concentration in cell culture supernatant were determined by Spectrophotometry.The changes of glucose transporter-1 (GLUT-1) and lactic dehydrogenase A (LDHA) expression at mRNA level were examined by realtime polymerase chain reaction.The changes of GLUT-1 and LDHA expression at protein level were tested by Western blot.Using t or t' test to analyze the effects of hypoxia duration on HIF-1αexpression at protein level.One-way ANOVA was applied for the difference analysis between the groups.Results In TE13 and Eca109 cells,the HIF-1α expression significantly increased under hypoxia condition and reached the peak at 12 hour (t=6.11,8.31; both P＜0.05).The lactic acid secretion of TE13/siHIF cells and Eca109/siHIF cells was (1.24±0.33) and (1.28±0.37) mmol/L,which significantly decreased when compared with TE13 and Eca109 cells [(3.25±1.34) and (4.91±1.69) mmol/L,t=2.53,3.59,both P＜0.05].The lactic acid secretion of TE13 and Eca109 cells significantly increased after hypoxia [(6.48±1.73) and (8.02± 1.95) mmol/L,t=2.715,2.050,both P＜0.05].There was no significant lactic acid secretion in TE13/siHIF cells and Eca109/siHIF cells after hypoxia (P ＞ 0.05).The expressions of GLUT-1 and LDHA at mRNA level were significantly suppressed in TE13/siHIF cells and Eca109/siHIF cells (normal oxygen:t=6.98,3.92,7.25,3.67,all P＜0.05).The expression of GLUT-1 at protein level remarkably weaked (normal oxygen:t=4.57、16.56,hypoxia:t=6.19、6.09,all P＜0.05),while the expression of LDHA at protein level slightly decreased (P＞0.05).Conclusions The level of glycolysis can be lowered by suppression HIF-1α expression in human esophageal squamous carcinoma cells.The pathway may be involved in the suppression of GLUT-1 and LDHA expression.Except for HIF-1α,there may be other regulating factors in LDHA protein expression at same time.
