CAJ | 학술논문

目的探讨p38α丝裂原活化蛋白激酶(MAPK)在食管鳞癌细胞Eca109中的可能作用.方法构建p38α特异性的shRNA干扰载体,瞬时转染Eca109细胞,运用qRT-PCR、免疫印迹分别检测p38α干扰后在mRNA、蛋白水平上的沉默效果；运用MTT和流式细胞仪检测p38α干扰后Eca109细胞增殖,细胞周期和凋亡的变化；运用细胞划痕实验检测p38α干扰后细胞迁移能力的改变.作为平行反向验证,转染含有p38α全长cDNA的真核过表达质粒,运用免疫印迹检测p38α过表达后在蛋白水平上的表达效果；运用MTT和流式细胞仪检测p38α过表达后细胞增殖,细胞周期和凋亡的变化；运用细胞划痕实验检测p38α的变化后细胞迁移能力的改变.结果 p38α基因在受干扰后,测定其蛋白水平表达水平(22.970±2.857)较空质粒组(35.658 ±2.286)降低,差异具有统计学意义(t=-4.475,P＜0.01).观察Eca109细胞的增殖变化,发现干扰48 h后吸光度值(0.951±0.086)大于对照组细胞(0.811 ±0.012)(t=3.20,P＜0.05),表明Eca109细胞增殖加快.观察细胞周期和凋亡变化发现,干扰48 h后凋亡率(17.400±5.495)较空质粒组(1.000 ±0.721)增加(t=40.06,P＜0.01)；干扰72 h后细胞迁移速度(0.034±0.031)较空质粒组(0.278±0.021)加快(t=-5.79,P＜0.01),表明浸润能力增加.p38α在过表达后,检测到其内源性p38α蛋白表达水平(41.170±2.357)高于对照组(35.658±2.286)(t=4.41,P=0.005)；48 h过表达组吸光值(0.472±0.089)与对照组细胞吸光值(0.811±0.012)相比,细胞增殖受到抑制(t=-7.50,P＜0.01),细胞生长活动在G1期受阻(t=4.80,P＜0.01),并且其细胞凋亡(32.233±1.457)高于空质粒组(1.000±0.721)(t=17.20,P＜0.01),细胞迁移[(0.770±0.054) mm]较空质粒组[(0.278±0.021) mm]减慢并且浸润受到抑制(t=11.00,P＜0.01).结论 p38dMARK抑制食管鳞癌细胞Eca109的发生发展过程.
목적 탐토p38α사렬원활화단백격매(MAPK)재식관린암세포Eca109중적가능작용.방법 구건p38α특이성적shRNA간우재체,순시전염Eca109세포,운용qRT-PCR、면역인적분별검측p38α간우후재mRNA、단백수평상적침묵효과；운용MTT화류식세포의검측p38α간우후Eca109세포증식,세포주기화조망적변화；운용세포화흔실험검측p38α간우후세포천이능력적개변.작위평행반향험증,전염함유p38α전장cDNA적진핵과표체질립,운용면역인적검측p38α과표체후재단백수평상적표체효과；운용MTT화류식세포의검측p38α과표체후세포증식,세포주기화조망적변화；운용세포화흔실험검측p38α적변화후세포천이능력적개변.결과 p38α기인재수간우후,측정기단백수평표체수평(22.970±2.857)교공질립조(35.658 ±2.286)강저,차이구유통계학의의(t=-4.475,P＜0.01).관찰Eca109세포적증식변화,발현간우48 h후흡광도치(0.951±0.086)대우대조조세포(0.811 ±0.012)(t=3.20,P＜0.05),표명Eca109세포증식가쾌.관찰세포주기화조망변화발현,간우48 h후조망솔(17.400±5.495)교공질립조(1.000 ±0.721)증가(t=40.06,P＜0.01)；간우72 h후세포천이속도(0.034±0.031)교공질립조(0.278±0.021)가쾌(t=-5.79,P＜0.01),표명침윤능력증가.p38α재과표체후,검측도기내원성p38α단백표체수평(41.170±2.357)고우대조조(35.658±2.286)(t=4.41,P=0.005)；48 h과표체조흡광치(0.472±0.089)여대조조세포흡광치(0.811±0.012)상비,세포증식수도억제(t=-7.50,P＜0.01),세포생장활동재G1기수조(t=4.80,P＜0.01),병차기세포조망(32.233±1.457)고우공질립조(1.000±0.721)(t=17.20,P＜0.01),세포천이[(0.770±0.054) mm]교공질립조[(0.278±0.021) mm]감만병차침윤수도억제(t=11.00,P＜0.01).결론 p38dMARK억제식관린암세포Eca109적발생발전과정.
Objective To investigate the role of p38αmitogen-activated potein kinases (MAPK) in human esophageal squamous cell carcinoma cell line Eca109.Methods Specific short hairpin (shRNA)vector as well as eukaryotic expression vector harbouring full length cDNA of human p38α MAPK were transfected into Eca109 cells.Cell proliferation after transfection was detected by MTT,cell cycle and apoptosis were assayed by flow cytometry.The variation of migration and invasion after tranfection was determined using wound healing assay and Transwell assay,respectively.Results The proliferation of Eca109 cells after knock-down for 48 h (0.951 ± 0.086) was significantly increased (t =3.20,P ＜ 0.05)compared with control (0.811 ± 0.012),Sphase was increased but not significantly.Cell apoptosis rate after knock down for 48 h (17.400 ± 5.495) was significantly increased (t =40.06,P ＜ 0.01) compared with control (1.000 ± 0.721).Migration after knock down for 72 h (0.034 ± 0.031) were enhanced pronouncedly (t =-5.79,P ＜ 0.01) compared with control (0.278 ± 0.021) and invasive ability also increased;whereas the proliferation of Eca109 cells after over-expression for 48 h (0.472 ± 0.089) was inhibited significantly (t =-7.50,P ＜0.01) compared with control(0.811 ±0.012),cells arrested at G1 phase (t =4.80,P ＜ 0.01).Cell apoptosis rate (32.233 ± 1.457) were decreased significantly (t =17.20,P ＜0.01) compared with control (1.000 ± 0.721) mm,migration after overexpression for 72 h ((0.770 ±0.054) mm) was suppressed pronouncedly compared with control groups of (0.278 ± 0.021) mm (t =11.00,P ＜ 0.01).Invasion after overexpression was inhibited.Conclusions p38α MAPK plays an antioncogenic role in the pathogenesis of esophageal squamous cell carcinoma cell line Eca109.