rmhTNF对Δ133p53表达阳性胃癌细胞系MKN45的影响

Effect of rmh TNF on Human Gastric Cancer Cell Lines MKN45 with Δ133p53 Status

[目的]利用rmh TNF-MKN45细胞模型,观察p53异构体Δ133p53及p53下游基因的表达。[方法]不同浓度(50、100、200、400、500 IU/ml)重组改构人肿瘤坏死因子(rmh TNF)作用应用于MKN45,采用CCK-8法,观察其细胞增殖抑制率;通过巢式逆转录多聚酶链反应(n RT-PCR法)检测Δ133p53、p53、Gadd45α、PTEN、Mdm2、Bax和Cyclin B1 m RNA的表达变化。[结果]随rmh TNF作用浓度增加和作用时间(24、48、72h)延长,MKN45细胞抑制率明显增加(F=35.683,P<0.001;F=60.328,P<0.001)。随rmh TNF浓度增加,MKN45细胞中,p53、Gadd45α、PTEN、Bax基因表达上调,Δ133p53、Mdm2、Cyclin B1基因表达下调。Δ133p53与p53、Gadd45α、PTEN、Bax表达呈负相关(r=-0.916,-0.894,-0.872,-0.971,P均<0.01),与Mdm2、Cyclin B1表达呈正相关(r=0.924,0.943,P均<0.01)。[结论]在肿瘤生长抑制效应中,Δ133p53异构体表现出与野生型p53拮抗作用,其机制可能与调控Gadd45α、PTEN、Mdm2、Bax、Cyclin B1等下游基因表达有关。

[Purpose] To observe the expression and significant of the p53 isoforms A133p53 and p53 downstream molecules by rmhTNF-MKN45 gastric cancer cell line model. [Methods] The dif- ferent concentrations （50,100,200,400,500 IU/ml） of recombined and modified human Tumor Necrosis Factor （rmhTNF） were used in gastric carcinoma cell lines MKN45. The growth inhibitory effect of rmhTNF was observed by CCK8 assay. The expression of p53 isoforms A133p53 and p53 downstream molecule Gadd45ot,PTEN, Mdm2, Bax and CyclinB 1 on the mRNA level was detected by nested reverse transcription PCR （nRT-PCR）. [Results] With the increased concentration of rmhTNF and the role of the extension of time（24,48,72h）,the cell inhibition rate increase gradually （F=35.683,P〈0.001;F=60.328,P〈0.001）.In mRNA measurement,down-regulation of A133p53, Mdm2 and CycliuB1, up-regulation of p53, Gadd45α,PTEN and Bax was significant in MKN45 cells treated by rmhTNF. The expression of A133p53 was negatively related to p53,Gadd45α, PTEN and Bax （r=-0.916,-0.894,-0.872,-0.971 ;all P〈0.01）,but positively related to Mdm2 and CyclinB1 （r=0.924, =0,943;all P〈0.01）. [Conclusion] A133p53 is shown as an anti-p53 factor, which might be related to regulate Gadd45a,PTEN,Mdm2,Bax and CyclinB1 in the inhibitory ef- fects of rmhTNF to gastric carcinoma.