【作者】 秦龙；

【导师】 张才全；

【作者基本信息】 重庆医科大学 ， 外科学， 2010， 硕士

【摘要】 目的:通过Akt信号通路抑制剂渥曼青霉素(wortmannin)对人胃癌细胞SGC7901的干预,以研究经干预后SGC7901细胞增殖活性的改变及对核转录因子NF-κB表达的影响,从而探讨wortmannin对人胃癌细胞的作用及机制,以期为胃癌的临床治疗提供新的思路。方法:(1)分别采用10,30,60,90nmol/L的Akt通路抑制剂wortmannin,以3,6,12,24h不同时间对胃癌细胞SGC7901进行干预,并设置对照组运用甲基偶氮唑蓝(MTT)法检测细胞的增殖抑制情况。(2)将60nmol/L的wortmannin作用胃癌细胞SGC7901后,分别于3,6,12,24h提取NF-κB细胞核蛋白,运用western blot法检测NF-κB核蛋白表达水平的变化。(3)将60nmol/L的wortmannin作用胃癌细胞SGC7901后,分别于3,6,12,24h提取RNA,运用RT-PCR法检测NF-κB基因的转录水平的变化。结果:(1)四种不同浓度的wortmannin均对胃癌细胞SGC7901的生长具有抑制作用;随着作用时间的延长及作用浓度的增加,与对照组相比,胃癌细胞的存活率明显下降(P<0.05);且60nmol/L的作用浓度对胃癌细胞的抑制效应与90nmol/L作用浓度的抑制效应无明显差异(P>0.05)。(2)随着wortmannin作用时间的延长,NF-κB核蛋白的表达水平均显著下降;与对照组相比,6h后其表达已下降至67%,24h后下降至21%(p<0.05)。(3)随着wortmannin作用时间的延长,NF-κBmRNA的转录表达水平亦均显著下降;与对照组相比,作用3h以后,NF-κBmRNA表达水平已下降至66%,24h后下降至5%(P<0.05)。结论:(1)wortmannin能够抑制胃癌细胞SGC7901的生长,当其作用浓度在一定范围内时,其抑制效应呈明显剂量和时间依赖性。(2)wortmannin对胃癌细胞SGC7901干预后能够下调NF-κB的核蛋白及mRNA转录的活性,且随着作用时间的延长其抑制效应更加显著。(3)wortmannin抑制胃癌细胞SGC7901的生长其机制可能是通过胃癌中存在的PI3K/Akt/NF-κB信号通路调节NF-κB的表达而发挥作用。更多还原

【Abstract】 Objective:Through the effect of Akt signal pathway inhibitor wortmannin on the human gastric carcinoma cell SGC7901,which is to study and research the change of the cell viability and the expression of nuclear factor NF-κB. Accordingly , we can make an approach to the effection and mechanism of wortmannin on the human gastric carcinoma cell,which is hope to provid a new thought of the clinical therapy of gastric carcinoma.Methods:(1)SGC7901 cells were treated with 10nmol/L, 30nmol/L, 60nmol/L, 90nmol/L of specific inhibitor of wortmannin for 3h,6h,12h,24h.And set up a control group,using MTT assay to estimated the cell viability.(2)SGC7901 cells were treated with 60nmol/L of wortmannin for 3h, 6h, 12h,24h,then extracting the NF-κB nucleoprotein. Furthermore, weste- rn blot was used to detect the expression level of NF-κB nucleoprotein.(3)SGC7901 cells were treated with 60nmol/L of wortmannin for 3h, 6h,12h,24h, then extracting the RNA.Reverse transcriptasepolymerase ch- ain reaction(RT-PCR) was used to detect transcription of NF-κB mRNA. Results:(1)Four different concentration of wortmannin could both inhibit the growth of SGC7901 cells.Comparing to the control group, the survival rat- e of gastric carcinoma cells was significant descended along with prolong- ing action time and increasing the drug dose(P<0.05).However,the depres- sion effect of 60nmol/L group was almost similar with 90nmol/L group (P >0.05).(2)By prolonging the action time,the nucleoprotein expression of NF-κB were significant decreased.Comparing to the control group,it was decr- eased to 67% after six hours.And after twenty four hours,it was decreased to 21%(P<0.05).(3)By prolonging the action time,the mRNA expression of NF-κB we- re also obviously decreased.Comparing to the control group,it was decrea- sed to 66% after three hours.And after twenty four hours,it was decreased to 5%(P<0.05).Conclusions:(1)Wortmannin could inhibit the growth of gastric carcinoma cells S- GC7901,and the depression effect is obviously depend on the action time and drug dose when the action time in some scope.(2)The nucleoprotein and mRNA transription activity of NF-κB were significant turned down compared with the control group after the wortm- annin intervention,and the depression effect became more and more stro- nger by prolonging the action time.(3)The mechanism of depression effect may be depend on an PI3K/ Akt/NF-κB pathway in gastric carcinoma cells to regulate the exprssion of NF-κB.更多还原