中华消化外科杂志
中華消化外科雜誌
중화소화외과잡지
Chinese Journal of Digestive Surgery
2015年
11期
948-952
,共5页
李平昂%江宇星%杨世伟%郝迎学%余佩武
李平昂%江宇星%楊世偉%郝迎學%餘珮武
리평앙%강우성%양세위%학영학%여패무
胃肿瘤%白细胞介素17%上皮间质转化%迁移%侵袭
胃腫瘤%白細胞介素17%上皮間質轉化%遷移%侵襲
위종류%백세포개소17%상피간질전화%천이%침습
Gastric neoplasms%Interleukin-17%Epithelial-mesenchymal transition%Migration%Invasion
目的 探讨IL-17对胃癌细胞上皮间质转化(EMT)和侵袭迁移的影响及其可能作用机制.方法 (1)取对数生长期的胃癌细胞株MGC-803,分别运用浓度为0、1 ng/mL、10 ng/mL、100 ng/mL、1 μg/mL IL-17干预48 h,观察细胞形态学变化.取细胞形态变化最为明显的浓度作为后续实验最适浓度.浓度为100 ng/mL的IL-17干预胃癌细胞48h,设为实验组;加入等量PBS干预胃癌细胞48 h,设为对照组.(2) RT-PCR检测两组胃癌细胞中钙粘附蛋白E(E-cadherin)、波形蛋白(Vimentin)的RNA表达水平.(3) Western blot检测两组胃癌细胞中E-cadherin和Vimentin的相对蛋白表达量.(4)划痕实验和Transwell实验检测两组胃癌细胞的侵袭和迁移能力.正态分布的计量资料采用-x±s表示,两组比较采用t检验.结果 (1)胃癌细胞EMT形态变化:不同浓度IL-17处理MGC-803胃癌细胞48 h后,细胞形态发生显著改变.主要是细胞由多角紧密连接状态逐渐向连接松散,梭形形态转变,细胞粘附能力明显下降,且随着IL-17浓度从0增加至100 ng/mL时,细胞形态改变逐渐明显;当浓度达到100 ng/mL时,细胞形态改变最明显;但当IL-17浓度继续增加至1μg/mL时,细胞形态改变不再显著,部分细胞出现死亡,漂浮现象.(2) RT-PCR检测结果:实验组和对照组胃癌细胞中E-cadherin mRNA相对表达量分别为0.45 ±0.13和1.06±0.23;Vimentin mRNA相对表达量分别为2.39 ±0.55和1.23 ±0.41,上述指标两组比较,差异均有统计学意义(t=3.811,2.923,P＜0.05).(3)Western blot检测结果:实验组和对照组胃癌细胞中E-cadherin蛋白相对表达量分别为0.86 ±0.17和1.56±0.29;Vimentin蛋白相对表达量分别为1.01 ±0.12和0.56±0.17,上述指标两组比较,差异均有统计学意义(t=3.551,3.601,P＜0.05).(4)划痕实验结果显示:划痕36 h后,实验组和对照组胃癌细胞均发生了迁移,其划痕宽度分别为(0.76±0.13) mm和(0.40 ±0.15)mm,两组比较,差异有统计学意义(t=3.095,P＜0.05).Transwell法检测结果显示:实验组和对照组穿膜细胞数计数为(159±28)个和(94±18)个,两组比较,差异有统计学意义(t=3.307,P＜0.05).结论 IL-17通过促进胃癌细胞发生EMT转变,进而增强胃癌细胞迁移侵袭能力.
目的 探討IL-17對胃癌細胞上皮間質轉化(EMT)和侵襲遷移的影響及其可能作用機製.方法 (1)取對數生長期的胃癌細胞株MGC-803,分彆運用濃度為0、1 ng/mL、10 ng/mL、100 ng/mL、1 μg/mL IL-17榦預48 h,觀察細胞形態學變化.取細胞形態變化最為明顯的濃度作為後續實驗最適濃度.濃度為100 ng/mL的IL-17榦預胃癌細胞48h,設為實驗組;加入等量PBS榦預胃癌細胞48 h,設為對照組.(2) RT-PCR檢測兩組胃癌細胞中鈣粘附蛋白E(E-cadherin)、波形蛋白(Vimentin)的RNA錶達水平.(3) Western blot檢測兩組胃癌細胞中E-cadherin和Vimentin的相對蛋白錶達量.(4)劃痕實驗和Transwell實驗檢測兩組胃癌細胞的侵襲和遷移能力.正態分佈的計量資料採用-x±s錶示,兩組比較採用t檢驗.結果 (1)胃癌細胞EMT形態變化:不同濃度IL-17處理MGC-803胃癌細胞48 h後,細胞形態髮生顯著改變.主要是細胞由多角緊密連接狀態逐漸嚮連接鬆散,梭形形態轉變,細胞粘附能力明顯下降,且隨著IL-17濃度從0增加至100 ng/mL時,細胞形態改變逐漸明顯;噹濃度達到100 ng/mL時,細胞形態改變最明顯;但噹IL-17濃度繼續增加至1μg/mL時,細胞形態改變不再顯著,部分細胞齣現死亡,漂浮現象.(2) RT-PCR檢測結果:實驗組和對照組胃癌細胞中E-cadherin mRNA相對錶達量分彆為0.45 ±0.13和1.06±0.23;Vimentin mRNA相對錶達量分彆為2.39 ±0.55和1.23 ±0.41,上述指標兩組比較,差異均有統計學意義(t=3.811,2.923,P＜0.05).(3)Western blot檢測結果:實驗組和對照組胃癌細胞中E-cadherin蛋白相對錶達量分彆為0.86 ±0.17和1.56±0.29;Vimentin蛋白相對錶達量分彆為1.01 ±0.12和0.56±0.17,上述指標兩組比較,差異均有統計學意義(t=3.551,3.601,P＜0.05).(4)劃痕實驗結果顯示:劃痕36 h後,實驗組和對照組胃癌細胞均髮生瞭遷移,其劃痕寬度分彆為(0.76±0.13) mm和(0.40 ±0.15)mm,兩組比較,差異有統計學意義(t=3.095,P＜0.05).Transwell法檢測結果顯示:實驗組和對照組穿膜細胞數計數為(159±28)箇和(94±18)箇,兩組比較,差異有統計學意義(t=3.307,P＜0.05).結論 IL-17通過促進胃癌細胞髮生EMT轉變,進而增彊胃癌細胞遷移侵襲能力.
목적 탐토IL-17대위암세포상피간질전화(EMT)화침습천이적영향급기가능작용궤제.방법 (1)취대수생장기적위암세포주MGC-803,분별운용농도위0、1 ng/mL、10 ng/mL、100 ng/mL、1 μg/mL IL-17간예48 h,관찰세포형태학변화.취세포형태변화최위명현적농도작위후속실험최괄농도.농도위100 ng/mL적IL-17간예위암세포48h,설위실험조;가입등량PBS간예위암세포48 h,설위대조조.(2) RT-PCR검측량조위암세포중개점부단백E(E-cadherin)、파형단백(Vimentin)적RNA표체수평.(3) Western blot검측량조위암세포중E-cadherin화Vimentin적상대단백표체량.(4)화흔실험화Transwell실험검측량조위암세포적침습화천이능력.정태분포적계량자료채용-x±s표시,량조비교채용t검험.결과 (1)위암세포EMT형태변화:불동농도IL-17처리MGC-803위암세포48 h후,세포형태발생현저개변.주요시세포유다각긴밀련접상태축점향련접송산,사형형태전변,세포점부능력명현하강,차수착IL-17농도종0증가지100 ng/mL시,세포형태개변축점명현;당농도체도100 ng/mL시,세포형태개변최명현;단당IL-17농도계속증가지1μg/mL시,세포형태개변불재현저,부분세포출현사망,표부현상.(2) RT-PCR검측결과:실험조화대조조위암세포중E-cadherin mRNA상대표체량분별위0.45 ±0.13화1.06±0.23;Vimentin mRNA상대표체량분별위2.39 ±0.55화1.23 ±0.41,상술지표량조비교,차이균유통계학의의(t=3.811,2.923,P＜0.05).(3)Western blot검측결과:실험조화대조조위암세포중E-cadherin단백상대표체량분별위0.86 ±0.17화1.56±0.29;Vimentin단백상대표체량분별위1.01 ±0.12화0.56±0.17,상술지표량조비교,차이균유통계학의의(t=3.551,3.601,P＜0.05).(4)화흔실험결과현시:화흔36 h후,실험조화대조조위암세포균발생료천이,기화흔관도분별위(0.76±0.13) mm화(0.40 ±0.15)mm,량조비교,차이유통계학의의(t=3.095,P＜0.05).Transwell법검측결과현시:실험조화대조조천막세포수계수위(159±28)개화(94±18)개,량조비교,차이유통계학의의(t=3.307,P＜0.05).결론 IL-17통과촉진위암세포발생EMT전변,진이증강위암세포천이침습능력.
Objective To investigate the effect of interleukin-17 (IL-17) in the gastric cancer cell migration and invasion via regulating epithelial-mesenchymal transition (EMT) and its potential function.Methods (1) Human gastric cancer cell MGC-803 lines in the logarithmic growth phase were stimulated by 0, 1 ng/mL, 10 ng/mL,100 ng/mL and 1μg/mL of IL-17 for 48 hours, and the phenotypic changes were observed.The concentration of IL-17 was selected for follow-on experiments based on the most obvious phenotypic changes.Gastric cancer cell MGC-803 which were stimulated by 100 ng/mL of IL-17 and PBS for 48 hours were allocated into the experimental group and control group, respectively.(2) The expressions of E-cadherin and Vimentin mRNA in gastric cancer cells were assayed through real-time PCR (RT-PCR).(3) The relative expressions of E-cadherin and Vimentin proteins in gastric cancer cells were assayed by the Western blot.(4) The scratch test and Transwell detection were also utilized to study the migration and invasion of gastric cancer cell MGC-803 in vitro.Measurement data with normal distribution were presented as-x ± s and comparison between groups was analyzed using the t test.Results (1) There were significant phenotypic changes in the gastric cancer cell after the different concentration of IL-17 stimulated gastric cancer cell MGC-803 for 48 hours.Cells were changed from polygonal and tight junction to spindle and loosely junction with a deterioration of cell adhesion.Cell phenotypes were gradually changed as the concentration of IL-17 was changed from 0 to 100 ng/mL.Phenotypic changes were the most obvious when 100 ng/mL of IL-17 was used, but these were non-significant as the concentration of IL-17 increased to 1 μg/mL with the death and floating of some cells.(2) The relative expressions of E-cadherin mRNA and Vimentin mRNA in RT-PCR were 0.45 ±0.13 and 1.06 ±0.23 in the experimental group and 2.39 ±0.55 and 1.23±0.41 in the control group, respectively, with significant differences (t =3.811, 2.923, P ＜0.05).(3) The results of Western blot showed the relative expressions of E-cadherin and Vimentin proteins were 0.86 ± 0.17 and 1.56 ± 0.29 in the experimental group and 1.01 ± 0.12 and 0.56 ± 0.17 in the control group, respectively, with significant differences (t =3.551, 3.601, P ＜ 0.05).(4) Cell migration in the 2 groups were detected by the scratch test at 36 hours after scratch test, and the width of scratch in the experimental and control groups were (0.76 ± 0.13) mm and (0.40 ± 0.15) mm, showing a significant difference (t =3.095, P ＜ 0.05).Transwell detection showed number of transmembrane cell in the experimental and control groups were 159 ±28 and 94 ± 18, respectively, with a significant difference (t =3.307, P ＜ 0.05).Conclusion IL-17 can promote the migration and invasion of gastric cancer cells via stimulating alteration of EMT.
