PTEN-PI3K/AKT信号传导途径对胃癌细胞株MKN28凋亡的影响
PTEN-PI3K/AKT신호전도도경대위암세포주MKN28조망적영향
Effects of PTEN-PI3K/AKT pathway on the apoptosis of gastric cancer MKN28 cells
  • 万方数据
    中华消化外科杂志 중화소화외과잡지
    CHINESE JOURNAL OF DIGESTIVE SURGERY
    2012年 4期 377-381 ,共5页

    许小涛%陶泽璋%宋启斌%姚颐%阮鹏

    허소도%도택장%송계빈%요이%원붕

    胃肿瘤%PTEN%PI3K%AKT%凋亡 胃腫瘤%PTEN%PI3K%AKT%凋亡
    위종류%PTEN%PI3K%AKT%조망
    Gastric neoplasms%PTEN%PI3K%AKT%Apoptosis
    目的 探讨PTEN-PI3 K/AKT信号传导途径对胃癌细胞株MKN28凋亡的调控作用及其机制.方法 构建PTEN真核表达载体,转染至胃癌细胞株MKN28中(转染组);同样方法转染空载体和等量的PBS分别作为阴性对照组和空白对照组,检测对胃癌细胞株MKN28凋亡的影响及对PI3K、AKT、Caspase-3、Caspase-9的影响.MTT检测细胞生长曲线,TUNEL染色法检测细胞凋亡,Western blot检测蛋白的表达.PI3K抑制剂LY294002处理未转染PTEN的胃癌细胞株MKN28(处理组);对照组加入等量的PBS,抑制PI3K活性后检测细胞凋亡蛋白及凋亡相关蛋白的表达.组间比较采用t检验,时间依赖性检验采用相关分析方法.结果 成功构建PTEN真核表达质粒并转染至胃癌细胞株MKN28中,获得稳定过表达PTEN的细胞模型.MTT检测发现转染组胃癌细胞株MKN28生长明显受到抑制,且呈时间依赖性(r =0.938,P＜0.05).转染组平均凋亡率达到27.86％ ±4.78％,明显高于阴性对照组的0.01％±0.01％(t=9.527,P＜0.05).转染PTEN后,转染组PI3K蛋白表达量为0.25±0.03,明显低于空白对照组的0.93 ±0.16及阴性对照组的0.96±0.15(t=7.235,8.883,P＜0.05).转染组活化的AKT (P-AKT)蛋白表达量为0.21±0.03,明显低于空白对照组的0.93 ±0.13及阴性对照组的0.91±0.12(t =9.347,9.802,P＜0.05).而转染组凋亡相关蛋白Caspase-3及Caspase-9表达量分别为0.86±0.11和0.87±0.12,明显高于阴性对照组的0.16±0.03和0.18 ±0.04及空白对照组的0.15±0.02和0.16 ±0.03(t=10.634,10.999,9.448,9.942,P＜0.05).抑制PI3K活性后,处理组细胞凋亡率为28.60％ ±4.50％,明显高于对照组的0.12％±0.06％(t=10.961,P＜0.05).Western blot检测发现处理组PI3K和P-AKT的蛋白表达量分别为0.18 ±0.02和0.11 ±0.01,明显低于对照组的0.93 ±0.14和0.90 ±0.12(t =9.186,11.363,P＜0.05);同时处理组PTEN的蛋白相对表达量为1.15 ±0.15,明显高于对照组的0.21±0.08(t =9.577,P＜0.05).处理组的凋亡相关蛋白Caspase-3及Caspase-9相对表达量分别为0.86 ±0.12和0.88 ±0.11,明显高于对照组的0.25±0.02和0.21±0.03(t =8.685,10.178,P＜0.05).结论 高表达PTEN可以抑制PI3K/AKT途径,促进胃癌细胞凋亡;抑制PI3K途径可以促进PTEN的表达,两者之间相互作用,共同调控着胃癌细胞的凋亡.
    목적 탐토PTEN-PI3 K/AKT신호전도도경대위암세포주MKN28조망적조공작용급기궤제.방법 구건PTEN진핵표체재체,전염지위암세포주MKN28중(전염조);동양방법전염공재체화등량적PBS분별작위음성대조조화공백대조조,검측대위암세포주MKN28조망적영향급대PI3K、AKT、Caspase-3、Caspase-9적영향.MTT검측세포생장곡선,TUNEL염색법검측세포조망,Western blot검측단백적표체.PI3K억제제LY294002처리미전염PTEN적위암세포주MKN28(처리조);대조조가입등량적PBS,억제PI3K활성후검측세포조망단백급조망상관단백적표체.조간비교채용t검험,시간의뢰성검험채용상관분석방법.결과 성공구건PTEN진핵표체질립병전염지위암세포주MKN28중,획득은정과표체PTEN적세포모형.MTT검측발현전염조위암세포주MKN28생장명현수도억제,차정시간의뢰성(r =0.938,P＜0.05).전염조평균조망솔체도27.86％ ±4.78％,명현고우음성대조조적0.01％±0.01％(t=9.527,P＜0.05).전염PTEN후,전염조PI3K단백표체량위0.25±0.03,명현저우공백대조조적0.93 ±0.16급음성대조조적0.96±0.15(t=7.235,8.883,P＜0.05).전염조활화적AKT (P-AKT)단백표체량위0.21±0.03,명현저우공백대조조적0.93 ±0.13급음성대조조적0.91±0.12(t =9.347,9.802,P＜0.05).이전염조조망상관단백Caspase-3급Caspase-9표체량분별위0.86±0.11화0.87±0.12,명현고우음성대조조적0.16±0.03화0.18 ±0.04급공백대조조적0.15±0.02화0.16 ±0.03(t=10.634,10.999,9.448,9.942,P＜0.05).억제PI3K활성후,처리조세포조망솔위28.60％ ±4.50％,명현고우대조조적0.12％±0.06％(t=10.961,P＜0.05).Western blot검측발현처리조PI3K화P-AKT적단백표체량분별위0.18 ±0.02화0.11 ±0.01,명현저우대조조적0.93 ±0.14화0.90 ±0.12(t =9.186,11.363,P＜0.05);동시처리조PTEN적단백상대표체량위1.15 ±0.15,명현고우대조조적0.21±0.08(t =9.577,P＜0.05).처리조적조망상관단백Caspase-3급Caspase-9상대표체량분별위0.86 ±0.12화0.88 ±0.11,명현고우대조조적0.25±0.02화0.21±0.03(t =8.685,10.178,P＜0.05).결론 고표체PTEN가이억제PI3K/AKT도경,촉진위암세포조망;억제PI3K도경가이촉진PTEN적표체,량자지간상호작용,공동조공착위암세포적조망.
    Objective To investigate the regulatory effects of PTEN-PI3K/AKT pathway on the apoptosis of gastric cancer MKN28 cells and the possible mechanisms.Methods The specific sequence of PTEN was transfected to MKN28 cells by eukaryotic expression vector (transfection group),and then vacant vector (negative control group) and PBS (blank control group) were transfected to the MKN28 cells,respectively.The effects of PTEN-PI3K/AKT pathway on the apoptosis of MKN28 cells and the expressions of PI3K,AKT,Caspase-3 and Caspase-9 were investigated.The growth curve and apoptosis of the MKN28 cells were detected by MTT assay and TUNEL staining,respectively,and the protein expression was detected by the Western blot.MKN28 cells which did not transfected by the PTEN were processed by inhibitor of PI3K (LY294002) (treated group),and MKN28 cells in the control group were processed by PBS.The expressions of apoptosis protein and apoptosis-related protein were detected after inhibition of PI3 K.All data were analyzed using the t test.Results The model of over-expression of PTEN was obtained and transfected into MKN28 cells.The survival of MKN28 cells in the transfection group was significantly inhibited in a time-dependant manner ( r =0.938,P ＜ 0.05 ).The mean apoptotic rate of the transfection rate was 27.86％ ± 4.78％,which was significantly higher than 0.01％ ± 0.01％ of the negative control group ( t =9.527,P ＜ 0.05 ).The protein expression of PI3K in the transfection group was 0.25 ± 0.03,which was significantly lower than 0.93 ± 0.16 of the blank control group and 0.96 ± 0.15 of the negative control group (t =7.235,8.883,P ＜ 0.05 ).The protein expression of P-AKT in the transfection group was 0.21 ± 0.03,which was significantly lower than 0.93 ± 0.13 of the blank control group and 0.91 ± 0.12 of the negative control group (t =9.347,9.802,P ＜ 0.05 ).The expressions of Caspase-3 and Caspase-9 of the transfection group were 0.86 ± 0.11 and 0.87 ± 0.12,which were significantly higher than 0.16 ± 0.03 and 0.18 ± 0.04 of the negative control group,and 0.15 ± 0.02 and 0.16 ± 0.03 of the blank control group ( t =10.634,10.999,9.448,9.942,P ＜0.05).The apoptotic rate of the MKN28 cells of the treated group was 28.60％ ± 4.50％,which was significantly higher than 0.12％ ± 0.06％ of the control group (t =10.961,P ＜ 0.05 ).The protein expression of PI3K and P-AKT of the treated group were 0.18 ± 0.02 and 0.11 ± 0.01,which were significantly lower than 0.93 ± 0.14 and 0.90 ± 0.12 of the control group (t =9.186,11.363,P ＜ 0.05 ).The protein expression of PTEN of the treated group was 1.15 ± 0.15,which was significantly higher than 0.21 ± 0.08 of the control group (t =9.577,P ＜ 0.05 ).The relative expressions of Caspase-3 and Caspase-9 of the treated group were 0.86 ± 0.12 and 0.88 ± 0.11,which were significantly higher than 0.25 ± 0.02 and 0.21 ± 0.03 of the control group (t =8.685,10.178,P ＜ 0.05).Conclusions Over-expression of PTEN may enhance the apoptosis of gastric cancer cells through inhibition of PI3K/AKT pathway.Inhibition of PI3K can enhance the expression of PTEN.PI3K and PTEN regulate the apoptosis of gastric cancer cells.
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