GRP78蛋白在胰腺癌中的表达及对胰腺癌细胞增殖和凋亡的影响研究

目的：检测葡萄糖调节蛋白78（GRP78）在胰腺癌组织中的表达,探讨GRP78蛋白与胰腺癌发生、发展的相关关系.观察GRP78基因表达下调对胰腺癌细胞凋亡和增殖的影响,为以GRP78基因为靶基因治疗胰腺癌提供理论依据.方法：利用免疫组织化学技术检测93例胰腺癌组织及58例胰腺良性病变组织中GRP78蛋白的表达情况;Sh GRP78和Sh Neg克隆人真核表达载体,经脂质体转染胰腺癌细胞系BXPC-3,G418筛选阳性克隆,半定量RT-PCR检测GRP78 mRNA表达,Western blot检测GRP78蛋白表达.采用流式细胞仪、磺酰罗丹明B（SRB）染色法检测各组细胞增殖和凋亡情况.结果：GRP78蛋白在正常胰腺组织表达率为10.8％,在胰腺良性病变组织的表达率为41.4％,在胰腺癌组织中阳性率为88.2％,3组之间差异显著.建立了2组稳定转染细胞系：BXPC-3 sh GRP78和BXPC-3 sh Neg,BXPC-3 sh GRP78细胞中GRP78 mRNA和蛋白表达显著下调.GRP78基因表达抑制后BXPC-3细胞的增殖明显受到抑制,凋亡显著增加.结论：GRP78蛋白在正常胰腺组织,胰腺良性病变组织及胰腺癌组织中的表达水平依次升高;GRP78基因高表达是细胞组织恶性转化的重要标志物.

Study on the expression of GRP78 protein in pancreatic cancer and the effect of GRP78 on the proliferation and apoptosis of pancreatic cancer cells

Objective：To detect the expression of GRP78 in Pancreatic Cancer,Normal pancreatic tissue and Pancreatic benign lesion tissues.Methods：The expression of GRP78 Protein in 93 cases of Pancreatic Cancer,58 cases of Pancreatic benign lesion tissues and 37 cases of Normal pancreatic tissue were detected by using immunohistochemistry.Sh GRP78 and sh Neg based on GRP78 gene sequence were transfected respectively into human Pancreatic Cancer cell BXPC3 using LipofectAMINE2000,then selected the positive clones by G418,detected GRP78 mRNA expression by semiquantitative RT-PCR,detected its protein steady expression by Western blot.The proliferation and apoptosis of three groups cells were detected by SRB and flow cytometer.Results：In Normal pancreatic tissue,Pancreatic benign lesion tissues and Pancreatic Cancer tissues,the positivity of GRP78 is10.8％,41.4％,88.2％ respectively.The difference among three groups is significantly.Established stable transected cell line：BXPC-3 sh GRP78,BXPC-3 sh Neg.The expression inhibitive rates of GRP78 gene mRNA and protein in BXPC-3 Sh GRP78 were inhibited significantly.The proliferation of BXPC-3 cells were inhibited and The apoptosis of BXPC-3 cells were enhanced when the expression of GPR78 were inhibited.Conclusion：GRP78 is gradually over expressed along the normal tissue-adenoma-carcinoma sequence and that its expression may be an indicator for malignant transformation.