2015-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/658146摘要：神經母細胞瘤是幼兒期最常見的惡性腫瘤，其腫瘤形成的分子機制依然不清楚，有可能是胚胎神經母細胞無法分化或無法凋亡而造成。鈣網蛋白（Calreticulin，CRT)為一種常儲存於内質網上的伴隨蛋白，過去的研究發現CRT是神經母細胞瘤一項重要的預後指標，CRT的大量表現與神經母細胞瘤的分化程度呈正相關性，病人並有較佳的預後表現。因此，CRT也在神經母細胞瘤的分化上扮演了重要的角色。血管内皮生長因子（VEGF-A)亦被證實在神經母細胞瘤的形成具有重要的角色。研究顯示VEGF-A引導的血管新生現象不僅與神經母細胞瘤的惡化相關，也在神經母細胞瘤的分化與成熟扮演重要角色。而在我們最新的研究利用神經母細胞瘤細胞株進行實驗發現鈣網蛋白會正向調控VEGF-A並促進神經母細胞瘤的細胞分化。在這篇研究當中，我們希望能夠在動物實驗與臨床病人探討CRT與VEGF-A對於神經母細胞瘤的分化調控。因此本計劃將提出四個主要的工作目標：一、在臨床應用研究部分，希望由本院神經母細胞瘤病患接受手術切除治療後剩下的腫瘤組織檢體中，檢查CRT與VEGF-A的表現量與其分化程度、臨床表徵及預後的關聯性。二、在動物實驗部分，我們利用慢病毒的感染以及嘌呤霉素篩選出能經由四環黴素誘導CRT表現之stNB-V1神經母細胞瘤細胞株，並藉由異體移植的方式將此細胞株打入老鼠體内，再以餵食的方式讓小鼠攝入多西霉素來誘導CRT之表現。研究將觀察CRT被誘導表現之組別，其腫瘤體積與控制組的差異，並進行腫瘤切片染色來分析CRT、VEGF-A與血管生成、腫瘤分化之間的關聯。三、我們進一步將誘導CRT表現之老鼠注射抗VEGF接受器抗體，觀察VEGF訊號被抑制後其腫瘤大小、血管生成與腫瘤分化的情形。四、我們將以為陣列基因晶片檢測神經母細胞瘤細胞與檢體在CRT或VEGF-A表現量改變之後的相關基因變化，以找出CRT和VEGF-A在神經母細胞瘤成因中所可能參與的分子角色。本計劃的完成將有助於釐清CRT與VEGF-A在神經母細胞腫瘤形成原因與預後指標所扮演的角色，同時也有助於對神經母細胞瘤新治療的發展。<br> Abstract: Neuroblastoma (NB) is the most common malignant tumor of infancy. The molecular mechanism underlying the tumorigenesis of NB remains largely elusive. It has been suggested that the pathogenesis of NB is due to failure of differentiation or apoptosis of precursor cells.Previous studies have identified calreticulin (CRT), a molecular chaperone primarily localized to the endoplasmic reticulum, as an independent prognostic marker in NB. Studies suggest that increased CRT expression in NB positively correlates with tumor differentiation and therefore predicts favorable outcome. CRT is also found to be essential for NGF-elicited neuronal differentiation. Thus, CRT could play an important role in neuronal differentiation in NB.Recent evidence suggested that vascular endothelial growth factor (VEGF)-A, a key regulator of physiological and pathologic angiogenesis, plays a crucial role in the behavior of NB. Studies have revealed that VEGF-A-associated angiogenesis is not only associated with tumor aggressiveness but also plays an essential role for NB differentiation and maturation. Our recent studies have further demonstrated that VEGF-A is involved in CRT-related neuronal differentiation in NB cells through angiogenesis-independent mechanism. Taken together, VEGF-A may participate in the CRT-mediated NB differentiation via both angiogenesis-dependent and angiogenesis-independent pathways. Further studies are warranted to clarify the role of CRT and VEGF-A in NB behavior and biology in v/vo. Our proposed studies are translational researches in which we want to investigate the role of CRT and VEGF in regulating NB tumorigenesis focusing on angiogenesis and neuronal differentiation in animal models and clinical patients. Specifically, we propose the following four aims in two years ：Aim I. Examine the CRT&VEGF-A-related tumorigenesis of NB in human NB tumors focusing on VEGF-A expression, angiogenesis, neuronal differentiation and their relationship with clinical parameters and prognostic relevance. CRT, VEGF-A and CD31 expression will be examined in mRNA and protein levels and by immunohistochemistry (IHC).Aim II. Examine the CRT-related tumorigenesis of NB in mice xenograft models focusing on VEGF-A expression, angiogenesis and neuronal differentiation. Inducible CRT stNB-V1 cells will be used to establish mice xenograft models of NB for comparison of the effects of up-regulated CRT on VEGF-A expression, angiogenesis, neuronal differentiation.Aim III. Examine the role of VEGF-A in CRT-related tumorigenesis of NB on angiogenesis and neuronal differentiation in mice transplanted with inducible CRT NB cells. We further examined the role of VEGF-A in the angiogenesis and neuronal differentiation of NB via VEGF receptor blockade in mice xenograft models.Aim IV. Determine the molecular mechanism that mediates the interaction between CRT, VEGF-A and neuronal differentiation of NB. Associated gene changes will be evaluated by cDNA microarray and tissue oligonucleotide microarrays. Gene expression differences will be compared in these samples to evaluate the possible mechanism involved in CRT and VEGF-A-regulated neuronal differentiation of NB.Altogether, our present studies could not only shed light to the molecular mechanism underlying NB differentiation, but also provide important information for developing a new therapeutic strategy to improve the outcome of NB patients.