中文摘要：

目的探讨葡萄糖调节蛋白78（GRP78）对人结直肠癌RKO细胞增殖和迁移能力的影响。方法构建编码GRP78的shRNA慢病毒表达载体pLV。shRNAGRP78及阴性对照慢病毒表达载体pLV—control，分别转染人结直肠癌细胞系RKO。四甲基偶氮唑盐法实验和克隆形成实验检测细胞的增殖能力，流式细胞术检测细胞周期的分布情况，划痕实验检测细胞迁移能力，裸鼠皮下成瘤实验检测细胞体内成瘤能力。Affymetrix人全基因组表达芯片检测GRP78表达抑制后RKO细胞中基因表达谱的变化。Westernblot技术验证部分差异基因以确定结果的可靠性。结果转染pLV—shRNAGRP78后，RKO细胞增殖能力及克隆形成数目明显受到抑制（P〈0．05），但迁移能力没有明显变化（P〉0．05）；流式细胞术检测结果显示，沉默GRP78导致G，期细胞比例显著增加，而s期细胞比例显著降低（P〈0．05）；裸鼠成瘤实验结果显示，沉默GRP78导致肿瘤细胞在裸鼠皮下成瘤能力显著减弱（P〈0．05）。基因芯片检测结果显示，GRP78表达抑制后，共有397个基因的表达发生改变（与对照组相比，变化表达倍数≥1．2），其中上调的基因258个，下调139个，这些基因主要涉及信号转导、细胞代谢、细胞凋亡等功能。通过生物信息学分析，筛选3个（CDKN2B、MTOR及BIRC3）特异相关的差异基因进行Westernblot验证，验证结果与芯片结果相符。结论沉默GRP78可通过抑制人结直肠癌RKO细胞周期G，／S期转换，从而抑制细胞的体内外增殖生长能力，但对细胞的迁移能力无显著影响。通过基因芯片技术能够筛选和鉴定与GRP78表达改变相关的基因。GRP78可能通过调控这些基因的表达从而影响结直肠癌细胞的增殖能力。

英文摘要：

Objective To investigate the roles of glucose regulated protein 78 （GRP78） in proliferation and migration of human colorectal carcinoma cell RKO. Methods The colorectal carcinoma cell line RKO was transfected by lentiviral vector pLV-shRNA GRP78 and lentivirus vector pLV-control. MTT test and colony formation assay were used to evaluate the cell proliferation ability. Distribution of cell cycle was analyzed by flow cytometry. Cell migration ability was detected by scratches migration experiment. In vivo tumorigenicity ability was measured using subcutaneous tumor assay. Differentially expressed genes were detected by Affymetrix human genome-wide expression profile chip and confirmed by Western blot analysis. Results Compared with the negative vector transfection group, cell proliferation was inhibited in vitro and in vivo （P 〈 0. 05 ）, while there was no significant difference in migration （ P 〉 0. 05 ）. Flow cytometry results showed that silencing GRP78 resulted in a significant increase in the proportion of cells in G1 phase, while the proportion of cells in S phase was significantly lower （P 〈 0. 05）. The gene chip results showed that 397 genes were differentially expressed by at least 1.2 folds in GRP78 knocked-out RKO cells, including 258 up-regulated and 139 down-regulated ones. Bioinformatics analysis identified 3 genes （CDKN2B, MTOR and BIRC3） with specific expression in GRP78 down-regulated RKO cells, and the result was verified by Western blot. Conclusions GRP78 knock-out inhibits the proliferation and growth of colorectal cancer cell,but has no obvious effect on migration invasion. Down regulation of GRP78 results in expression changes of lots of genes in RKO cells. GRPTg may exert its role in proliferation of RKO cell through regulating these genes.