CAJ | 학술논문

目的构建高尔基磷酸化蛋白-3(GOLPH-3)基因过表达的胰腺癌稳定细胞株BxPC-3,观察GOLPH-3基因过表达对胰腺癌细胞迁移能力的影响.方法制备逆转录病毒pMSCV-GOLPH-3和pMSCV-vector,并分别感染胰腺癌细胞株BxPC-3,用0.5 mg/L嘌呤霉素筛选得到GOLPH-3过表达及空载对照稳定细胞株.Western blot技术在蛋白水平上检测BxPC-3稳定细胞株中GOLPH-3及β-连环蛋白(β-catenin)的蛋白表达水平,细胞迁移实验(Transwell)检测GOLPH-3对胰腺癌细胞迁移能力的影响.结果胰腺癌细胞BxPC-3表达GOLPH-3蛋白；空白对照组、空载对照组及过表达组的GOLPH-3蛋白相对含量分别为0.32 ±0.13、0.46 ±0.12、1.02±0.30,过表达组较空载对照组上调50.9％,其差异有统计学意义(P＜0.05).β-catenin蛋白相对含量分别为0.42 ±0.06、0.63±0.34、1.77 ±0.19,与空载对照组比较,上调GOLPH-3能明显促进过表达组β-catenin蛋白的表达,且上调64.4％ (P ＜0.01).每200倍随机计数5个视野下空白对照组、空载对照组、过表达组穿膜胰腺癌细胞数分别为(14.33 ±6.30)个、(16.25±10.45)个、(83.08±62.57)个,过表达组的迁移细胞数明显多于空载对照组(P＜0.001),而两对照组间差异无统计学意义(P＞0.05).结论上调GOLPH-3能影响胰腺癌细胞的迁移能力,GOLPH-3过表达增强胰腺癌细胞的迁移机制可能是上调Wnt/β-catenin信号通路β-catenin表达的结果.
목적 구건고이기린산화단백-3(GOLPH-3)기인과표체적이선암은정세포주BxPC-3,관찰GOLPH-3기인과표체대이선암세포천이능력적영향.방법 제비역전록병독pMSCV-GOLPH-3화pMSCV-vector,병분별감염이선암세포주BxPC-3,용0.5 mg/L표령매소사선득도GOLPH-3과표체급공재대조은정세포주.Western blot기술재단백수평상검측BxPC-3은정세포주중GOLPH-3급β-련배단백(β-catenin)적단백표체수평,세포천이실험(Transwell)검측GOLPH-3대이선암세포천이능력적영향.결과 이선암세포BxPC-3표체GOLPH-3단백；공백대조조、공재대조조급과표체조적GOLPH-3단백상대함량분별위0.32 ±0.13、0.46 ±0.12、1.02±0.30,과표체조교공재대조조상조50.9％,기차이유통계학의의(P＜0.05).β-catenin단백상대함량분별위0.42 ±0.06、0.63±0.34、1.77 ±0.19,여공재대조조비교,상조GOLPH-3능명현촉진과표체조β-catenin단백적표체,차상조64.4％ (P ＜0.01).매200배수궤계수5개시야하공백대조조、공재대조조、과표체조천막이선암세포수분별위(14.33 ±6.30)개、(16.25±10.45)개、(83.08±62.57)개,과표체조적천이세포수명현다우공재대조조(P＜0.001),이량대조조간차이무통계학의의(P＞0.05).결론 상조GOLPH-3능영향이선암세포적천이능력,GOLPH-3과표체증강이선암세포적천이궤제가능시상조Wnt/β-catenin신호통로β-catenin표체적결과.
Objective To establish stable overexpression of golgi phosphorylation protein 3 (GOLPH-3) gene in the pancreatic cancer cell line BxPC-3 and observe the effect of GOLPH-3 gene overexpression on migration of pancreatic cancer cells.Methods The BxPC-3 cells were transfected by retroviruses of pMSCV-GOLPH and pMSCV-vector respectively.The stable cell lines were obtained after screening by 0.5 mg/L puromycin.The protein expression levels of GOLPH-3 and β-catenin were detected by using Western blotting.The migration of BxPC-3 stable cells was tested by transwell assay.Results BxPC-3 cells could express GOLPH-3 protein.The relative protein expression of GOLPH-3 was 0.32 ± 0.13,0.46 ±0.12 and 1.02 ± 0.30 in the blank,vector control and overexpression groups,respectively.As compared with the vector control group,the protein levels of the GOLPH-3 in overexpression group was significantly up-regulated by 50.90％ (P ＜ 0.05).The gray value of β-catenin protein expression in the blank,vector control and overexpression groups was 0.42 ± 0.06,0.63 ± 0.34 and 1.77 ± 0.19 respectively.As compared with the vector control group,up-regulated GOLPH-3 could promote the protein expression of β-catenin in the overexpression group which was up-regulated by 64.4％ (P ＜ 0.01).The mean migrating cell number was 14.33 ±6.30 in the blank control group,16.25 ± 10.45 in the vector control group and 83.08 ± 62.57 in the overexpression group.The number of cells positive for GOLPH-3 in the overexpression group was significantly greater than that in the vector control group (P ＜ 0.001).Conclusion Overexpresssion of GOLPH-3 could increase the migration of pancreatic cancer cells,which might be related to the up-regulated expression of β-catenin in Wnt-/β-catenin signal pathway.