中华消化外科杂志
中華消化外科雜誌
중화소화외과잡지
CHINESE JOURNAL OF DIGESTIVE SURGERY
2013年
2期
139-144
,共6页
结直肠肿瘤%胃泌素%丙谷胺%细胞外信号调节蛋白激酶%K-ras
結直腸腫瘤%胃泌素%丙穀胺%細胞外信號調節蛋白激酶%K-ras
결직장종류%위비소%병곡알%세포외신호조절단백격매%K-ras
Colorectal neoplasms%Gastrin%Proglumide%Extracellular-signal protein kinase%K-ras
目的 探讨细胞外信号调节蛋白激酶(ERK)-丝裂素活化蛋白激酶(MAPK)信号转导通路在胃泌素促进人结直肠癌细胞株HT-29增殖与凋亡中的作用及其机制.方法 体外培养结直肠癌HT-29细胞株,将细胞分为对照组、胃泌素组、丙谷胺组和胃泌素+丙谷胺组,胃泌素组设6.25、12.50、25.00、50.00、100.00 mg/L 5个浓度梯度,丙谷胺组设8.00、16.00、32.00、64.00、128.00 mg/L 5个浓度梯度,并设胃泌素(25.00 mg/L)+丙谷胺组(8.00、16.00、32.00、64.00、128.00 mg/L)浓度梯度.对照组不加药,其余各组加不同浓度的药物处理.运用MTT法观察细胞增殖活力改变情况,确立5-肽胃泌素和丙谷胺处理HT-29细胞的最佳浓度；流式细胞仪检测各组细胞增殖指数及凋亡率的变化；RT-PCR检测结直肠癌HT-29细胞株胃泌素/胆囊收缩素B受体(CCK-BR)mRNA表达情况以及各组细胞ERK1/2、K-rasmRNA表达水平的变化；Western blot检测ERK1/2和K-ras蛋白表达及其磷酸化水平.采用方差分析和SNK-q检验进行统计学分析.结果 胃泌素浓度在6.25～ 100.00 mg/L能促进结直肠癌HT-29细胞的增殖,且当浓度达25.00 mg/L时为最佳浓度(F=31.36,P＜0.05)；单独丙谷胺对结直肠癌HT-29细胞增殖无明显影响,但丙谷胺浓度在8.00 ～ 128.00 mg/L能明显拮抗胃泌素促进HT-29细胞的增殖作用,其最佳浓度为32.00 mg/L(F =24.31,P＜0.05).胃泌素(25.00 mg/L)组细胞增殖指数为37.5％±5.2％,显著高于对照组的27.7％±5.0％和胃泌素(25.00 mg/L)+丙谷胺(32.00 mg/L)组的27.3％±5.8％(q=4.56,4.75,P＜0.05)；胃泌素(25.00 mg/L)组细胞凋亡率为1.9％±0.4％,低于对照组的2.5％±0.4％和胃泌素(25.00 mg/L)+丙谷胺(32.00 mg/L)组的2.4％±0.3％(q＝4.23,4.06,P＜0.05).HT-29细胞中存在明显的CCK-BR mRNA表达.胃泌素(25.00 mg/L)组磷酸化ERK1/2、磷酸化K-ras蛋白相对表达量分别为0.43％±0.04％和0.45％±0.06％,高于对照组的0.32％±0.02％和0.31％±0.05％(q =7.78,4.95,P＜0.05),也高于胃泌素(25.00 mg/L)+丙谷胺(32.00 mg/L)组的0.36％±0.01％和0.35％±0.04％(q=5.72,4.08,P＜0.05)；但对照组、胃泌素(25.00 mg/L)组、丙谷胺(32.00 mg/L)组、胃泌素(25.00 mg/L)+丙谷胺(32.00 mg/L)组间ERK1/2 mRNA和蛋白表达水平以及K-ras mRNA和蛋白表达水平比较,差异均无统计学意义(F=0.52,0.72,0.78,0.28,P＞0.05).结论 胃泌素可以促进结直肠癌细胞株HT-29的增殖,抑制细胞凋亡,其作用可能是通过ERK-MAPK信号通路中Ras→ Raf→ MEK1/2→ERK1/2途径上调ERK和K-ras的磷酸化水平来实现的,但能够被其受体拮抗剂丙谷胺抑制.
目的 探討細胞外信號調節蛋白激酶(ERK)-絲裂素活化蛋白激酶(MAPK)信號轉導通路在胃泌素促進人結直腸癌細胞株HT-29增殖與凋亡中的作用及其機製.方法 體外培養結直腸癌HT-29細胞株,將細胞分為對照組、胃泌素組、丙穀胺組和胃泌素+丙穀胺組,胃泌素組設6.25、12.50、25.00、50.00、100.00 mg/L 5箇濃度梯度,丙穀胺組設8.00、16.00、32.00、64.00、128.00 mg/L 5箇濃度梯度,併設胃泌素(25.00 mg/L)+丙穀胺組(8.00、16.00、32.00、64.00、128.00 mg/L)濃度梯度.對照組不加藥,其餘各組加不同濃度的藥物處理.運用MTT法觀察細胞增殖活力改變情況,確立5-肽胃泌素和丙穀胺處理HT-29細胞的最佳濃度；流式細胞儀檢測各組細胞增殖指數及凋亡率的變化；RT-PCR檢測結直腸癌HT-29細胞株胃泌素/膽囊收縮素B受體(CCK-BR)mRNA錶達情況以及各組細胞ERK1/2、K-rasmRNA錶達水平的變化；Western blot檢測ERK1/2和K-ras蛋白錶達及其燐痠化水平.採用方差分析和SNK-q檢驗進行統計學分析.結果 胃泌素濃度在6.25～ 100.00 mg/L能促進結直腸癌HT-29細胞的增殖,且噹濃度達25.00 mg/L時為最佳濃度(F=31.36,P＜0.05)；單獨丙穀胺對結直腸癌HT-29細胞增殖無明顯影響,但丙穀胺濃度在8.00 ～ 128.00 mg/L能明顯拮抗胃泌素促進HT-29細胞的增殖作用,其最佳濃度為32.00 mg/L(F =24.31,P＜0.05).胃泌素(25.00 mg/L)組細胞增殖指數為37.5％±5.2％,顯著高于對照組的27.7％±5.0％和胃泌素(25.00 mg/L)+丙穀胺(32.00 mg/L)組的27.3％±5.8％(q=4.56,4.75,P＜0.05)；胃泌素(25.00 mg/L)組細胞凋亡率為1.9％±0.4％,低于對照組的2.5％±0.4％和胃泌素(25.00 mg/L)+丙穀胺(32.00 mg/L)組的2.4％±0.3％(q＝4.23,4.06,P＜0.05).HT-29細胞中存在明顯的CCK-BR mRNA錶達.胃泌素(25.00 mg/L)組燐痠化ERK1/2、燐痠化K-ras蛋白相對錶達量分彆為0.43％±0.04％和0.45％±0.06％,高于對照組的0.32％±0.02％和0.31％±0.05％(q =7.78,4.95,P＜0.05),也高于胃泌素(25.00 mg/L)+丙穀胺(32.00 mg/L)組的0.36％±0.01％和0.35％±0.04％(q=5.72,4.08,P＜0.05)；但對照組、胃泌素(25.00 mg/L)組、丙穀胺(32.00 mg/L)組、胃泌素(25.00 mg/L)+丙穀胺(32.00 mg/L)組間ERK1/2 mRNA和蛋白錶達水平以及K-ras mRNA和蛋白錶達水平比較,差異均無統計學意義(F=0.52,0.72,0.78,0.28,P＞0.05).結論 胃泌素可以促進結直腸癌細胞株HT-29的增殖,抑製細胞凋亡,其作用可能是通過ERK-MAPK信號通路中Ras→ Raf→ MEK1/2→ERK1/2途徑上調ERK和K-ras的燐痠化水平來實現的,但能夠被其受體拮抗劑丙穀胺抑製.
목적 탐토세포외신호조절단백격매(ERK)-사렬소활화단백격매(MAPK)신호전도통로재위비소촉진인결직장암세포주HT-29증식여조망중적작용급기궤제.방법 체외배양결직장암HT-29세포주,장세포분위대조조、위비소조、병곡알조화위비소+병곡알조,위비소조설6.25、12.50、25.00、50.00、100.00 mg/L 5개농도제도,병곡알조설8.00、16.00、32.00、64.00、128.00 mg/L 5개농도제도,병설위비소(25.00 mg/L)+병곡알조(8.00、16.00、32.00、64.00、128.00 mg/L)농도제도.대조조불가약,기여각조가불동농도적약물처리.운용MTT법관찰세포증식활력개변정황,학립5-태위비소화병곡알처리HT-29세포적최가농도；류식세포의검측각조세포증식지수급조망솔적변화；RT-PCR검측결직장암HT-29세포주위비소/담낭수축소B수체(CCK-BR)mRNA표체정황이급각조세포ERK1/2、K-rasmRNA표체수평적변화；Western blot검측ERK1/2화K-ras단백표체급기린산화수평.채용방차분석화SNK-q검험진행통계학분석.결과 위비소농도재6.25～ 100.00 mg/L능촉진결직장암HT-29세포적증식,차당농도체25.00 mg/L시위최가농도(F=31.36,P＜0.05)；단독병곡알대결직장암HT-29세포증식무명현영향,단병곡알농도재8.00 ～ 128.00 mg/L능명현길항위비소촉진HT-29세포적증식작용,기최가농도위32.00 mg/L(F =24.31,P＜0.05).위비소(25.00 mg/L)조세포증식지수위37.5％±5.2％,현저고우대조조적27.7％±5.0％화위비소(25.00 mg/L)+병곡알(32.00 mg/L)조적27.3％±5.8％(q=4.56,4.75,P＜0.05)；위비소(25.00 mg/L)조세포조망솔위1.9％±0.4％,저우대조조적2.5％±0.4％화위비소(25.00 mg/L)+병곡알(32.00 mg/L)조적2.4％±0.3％(q＝4.23,4.06,P＜0.05).HT-29세포중존재명현적CCK-BR mRNA표체.위비소(25.00 mg/L)조린산화ERK1/2、린산화K-ras단백상대표체량분별위0.43％±0.04％화0.45％±0.06％,고우대조조적0.32％±0.02％화0.31％±0.05％(q =7.78,4.95,P＜0.05),야고우위비소(25.00 mg/L)+병곡알(32.00 mg/L)조적0.36％±0.01％화0.35％±0.04％(q=5.72,4.08,P＜0.05)；단대조조、위비소(25.00 mg/L)조、병곡알(32.00 mg/L)조、위비소(25.00 mg/L)+병곡알(32.00 mg/L)조간ERK1/2 mRNA화단백표체수평이급K-ras mRNA화단백표체수평비교,차이균무통계학의의(F=0.52,0.72,0.78,0.28,P＞0.05).결론 위비소가이촉진결직장암세포주HT-29적증식,억제세포조망,기작용가능시통과ERK-MAPK신호통로중Ras→ Raf→ MEK1/2→ERK1/2도경상조ERK화K-ras적린산화수평래실현적,단능구피기수체길항제병곡알억제.
Objective To investigate the effects and mechanisms of extracellular-signal regulated protein kinase-motogenactived protein kinase(ERK-MAPK)signaling pathway in gastrin-induced cell proliferation and apoptosis of colorectal cancer cells.Methods HT-29 cells were incubated in different media,and then were divided into the control group,gastrin group,proglumide group and gastrin + proglumide group.No reagent was added in the control group,and other groups were dealed with reagent in different concentrations.The changes of proliferation of the HT-29 cells were detected by MTT assay,and the optimal concentration of gastrin and proglumide were determined.The changes of proliferation index and apoptotic rates of HT-29 cells were detected by cell cytometry.The mRNA expressions of gastrin receptor/cholecystokinin-B receptor(CCK-BR),ERK1/2 and K-ras were detected by RT-PCR.The protein of ERK1/2,K-ras protein and phosphorylation levels were detected by Western blot.All data were analyzed by analysis of variance and SNK-q test.Results The proliferation of HT-29 was stimulated by gastrin when the concentration of the gastrin was 6.25-100.00 mg/L,and the optimal concentration of gastrin was 25.00 mg/L(F =31.36,P ＜ 0.05).Proglumide had no obvious effects on the proliferation of HT-29 cells,while it significantly inhibited the proliferation of HT-29 cells stimulated by gastrin when the concentration of proglumide was 8.00-128.00 mg/L,and the optimal concentration was 32.00 mg/L(F =24.31,P ＜ 0.05).The proliferation index of the gastrin(25.00 mg/L)group was 37.5 ％ ± 5.2％,which was significantly higher than 27.7％ ± 5.0％ of the control group and 27.3％ ± 5.8％ of the gastrin(25.00 mg/L)+ proglumide(32.00 mg/L)group(q =4.56,4.75,P ＜ 0.05).The apoptotic index of the gastrin(25.00 mg/L)group was 1.9％ ± 0.4％,which was significantly lower than 2.5％ ± 0.4％ of the control group and 2.4％ ± 0.3％ of the gastrin(25.00 mg/L)+ proglumide(32.00 mg/L)group(q =4.23,4.06,P＜0.05).The mRNA expression of CCK-BR was detected in the HT-29 cells.The levels of phosphorylated ERK1/2 protein and phosphorylated K-ras protein were 0.43％ ± 0.04％ and 0.45％ ± 0.06％,which were significantly higher than 0.32％ ± 0.02％ and 0.31％ ± 0.05 ％ of the control group(q =7.78,4.95,P ＜ 0.05),and they were also higher than 0.36％ ± 0.01％ and 0.35 ％ ± 0.04％ of the gastrin(25.00 mg/L)+ proglumide(32.00 mg/L)group(q =5.72,4.08,P ＜0.05).There were no significant differences in the mRNA and protein expressions of ERK1/2 and K-ras among the control group,gastrin(25.00 mg/L)group,proglumide(32.00 mg/L)group and gastrin (25.00 mg/L)+ proglumide(32.00 mg/L)group(F =0.52,0.72,0.78,0.28,P ＞0.05).Conclusion Gastrin could stimulate the proliferation of HT-29 cells and inhibit their apoptosis by upregulate the phosphorylation levels of ERK and K-ras through the Ras→Raf→ MEK1/2→ ERK1/2 pathway,while the effect can be restrained by gastrin receptor antagonist proglumide.
