鈣網蛋白(Calreticulin, CRT)是一個在內質網中的多功能蛋白質，可調控細胞中的多種功能，諸如鈣離子平衡、細胞黏附和基因表達等。許多的研究已指出鈣網蛋白被大量地表達於癌症組織中，這顯示鈣網蛋白在癌症組織可能扮演重要角色，目前尚未有鈣網蛋白在胃癌中的研究報導，從臺大醫院陳炯年醫師的臨床結果顯示，和正常組織相比，胃癌組織中可偵測到大量表達的鈣網蛋白，且鈣網蛋白的表現量和腫瘤轉移、侵襲、血管密度、胎盤生長因子(placenta growth factor, PlGF)的表達量皆有高度正相關，此外，若病人的胃癌組織中有較高的鈣網蛋白表達量，其存活率較低，這顯示了胃癌的惡性度的確和鈣網蛋白相關，本研究的目的為鈣網蛋白對於胃癌細胞所造成的生理現象影響的探討。為了研究鈣網蛋白的作用，我們首先建立了鈣網蛋白大量表達及少量表達的穩定胃癌細胞株，由這些細胞株中顯示，大量表達鈣網蛋白可使胃癌細胞的形狀改變，變得較為伸展，同時也可加快細胞生長及爬行的速度；至於少量表達鈣網蛋白的細胞則可觀察到相反的情形。我們更進一歩利用cDNA微陣列法中發現結締組織生長因子(connective growth factor, CTGF)，此一和癌症轉移相關的蛋白質，是可能被鈣網蛋白所調控的分子，我們的實驗發現鈣網蛋白在轉錄及轉譯層級皆有著負向調控結締組織生長因子的作用。再者，在臨床上所觀察到和鈣網蛋白有著正相關的促血管新生因子-胎盤生長因子也能夠被鈣網蛋白所調控，亦即在大量表達鈣網蛋白的胃癌細胞株中，其胎盤生長因子的信使核糖核酸(mRNA)及蛋白質會增加。綜合上述所論，我們的實驗結果清楚地解釋為何在較惡性的胃癌組織中著有較大量的鈣網蛋白表現量，因為在胃癌細胞株中，大量表達鈣網蛋白可促使胃癌細胞伸展，生長及爬行速率較快，並且可調節使得癌症惡化的因子如結締組織生長因子和胎盤生長因子等。我們的研究顯示鈣網蛋白可當作預測病人預後的一個分子，也可作為控制癌症病情的治療標靶。

Calreticulin (CRT) is a multiple functional chaperone protein mainly expressed in the endoplasmic reticulum (ER), and it regulates various cellular functions including Ca2+ homeostasis, adhesion, and gene expression. Although it is reported that CRT is overexpressed in several cancer tissues, the relationship between CRT expression and gastric cancer has not been revealed. Dr. Chung-Nien Chen’s clinical results indicated that CRT expression is correlated with tumor metastasis, invasion, microvessel density and expression of placenta growth factor (PlGF). Furthermore, survival rate of gastric cancer patients with high CRT expression is lower than low CRT expression counterpart. Therefore, it is suggested that CRT might play crucial roles in regulating gastric cancer behavior. In this study, we select CRT stably overexpressed- and knockdowned-AGS gastric cancer cell lines to investigate the physiological effects of CRT in these cells. We demonstrated that overexpression of CRT increases cell spreading, proliferation and migration. In contrast, knockdown of CRT suppresses these effects. In addition, by using cDNA microaraay analysis and compared with clinical results, we found that connective tissue growth factor (CTGF) and PlGF might be regulated by the expression of CRT. CTGF, a molecule related to cancer progression, is negatively regulated by CRT expression at both transcriptional and translational levels. Meanwhile, PlGF mRNA and protein expressions are significantly up-regulated in CRT-overexpressed cells. In conclusion, CRT could positively regulate cell spreading, proliferation, migration, and PlGF expression in gastric cancer cells, while CTGF is negatively modulated by CRT. Taken together, our data explained the correlation between CRT overexprssion and worse clinicopathological factors in clinical observations. In the future, CRT might used as a potential marker for prognosis prediction and a therapeutic target in gastric cancer.

中文摘要…………………………………………………………………I

Abstract...…………………………...……………………………………II

Introduction………………………………………………………………1
Calreticulin (CRT) structure and functions………………………………………1
CRT in cancer tissues…………………………………………………………….2
Gastric cancer…………………………………………………………………….3
Connective tissue growth factor (CTGF)………………………………………...4
Placenta growth factor (PlGF)……………………………………………………5
Tumor angiogenesis, metastasis, and invasion…………………………………...6

Material and Methods…………………………………………………….8
Reagents………………………………………………………………………….8
Cell culture……………………………………………………………………….9
Plasmid constructions……………………………………………………………9
Transfection and stable clones selection…………………………………………9
Cell cycle analysis………………………………………………………………10
Chemotaxis assay……………………………………………………………….10
RNA isolation and RT-Q-PCR…………………………………………………..10
Western blot……………………………………………………………………..11
Microarray………………………………………………………………………12
ELISA…………………………………………………………………………...12
Statistics analysis………………………………………………………………..12

Results………..…………………………………………………………14
Plasmid constructions and stable cell lines selection…………………………...14
CRT mRNA and protein expression in CRT differentially expressed stable cell lines……………………………………………………………………………...14
Cell morphology of CRT differentially expressed stable cell lines…………….15
EGFP protein and CRT-EGFP fusion protein distribution in stable cell lines….15
Cell cycle analysis of CRT differentially expressed stable cell lines…………...15
Cell migration analysis of CRT differentially expressed stable cell lines……...16
Microarray analysis of genes regulated by CRT expression……………………17
CTGF mRNA and protein is down-regulated in CRT-overexpressed cells and up-regulated in CRT-knockdowned cells………………………………………17
PlGF mRNA and protein secretion is up-regulated in CRT-overexpressed cells18
Physiological Effects of CRT expression in gastric cancer cells are associated to clinical observations…………………………………………………………….18
Discussion……………………………………………………………….20

References………………………………………………………………25

Tables……………………………………………………………………34

Figures…………………………………………………………………..38

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