2017-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/658147摘要：神經母細胞瘤是源自於胚胎時期交感神經系統神經脊細胞的腫瘤，其腫瘤形成的分子機制有可能是胚胎神經母細胞無法分化或無法凋亡而造成。神經生長因子 (Nerve growth factor,NGF)則是在胚胎發育過程調控交感神經細胞分化或凋亡最重要的因子。過去的研究發現NGF的調控異常會造成神經母細胞瘤的產生，因此，NGF 在神經母細胞瘤的分化上扮演了必要的角色。血管內皮生長因子(VEGF)亦被證實在神經母細胞瘤的形成具有重要的角色。而在我們最近發表的研究利用神經母細胞瘤細胞株進行實驗發現鈣網蛋白(Calreticulin, CRT)會正向調控VEGF 並促進神經母細胞瘤的細胞分化。CRT 為一種常儲存於內質網上的伴隨蛋白，也在神經母細胞瘤的分化上扮演了重要的角色。此外，在我過去一年多的研究，我們發現無論在神經母細胞瘤的細胞株、神經母細胞瘤的異體移植腫瘤與病人的腫瘤組織檢體中，VEGF 的表現量與CRT 的表現量及腫瘤的分化程度皆呈顯著正相關，但與血管的生成無相關性。此外，我們發現若VEGF 在病人腫瘤組織的表現量較高，病人的預後較佳。我們過去一年多的研究首次支持VEGF 在神經母細胞瘤的分化應該扮演著重要的角色，此研究結果目前已投稿中。根據以上研究，我們推論過去建議在神經母細胞瘤所使用的抗VEGF 藥劑治療應該是不恰當。此外，因為NGF 是神經母細胞瘤分化最重要的調控因子，我們亦推論VEGF 應在NGF調控神經母細胞瘤分化的過程扮演重要角色。因此本計畫希望能夠在細胞實驗與動物實驗探討VEGF 對於神經母細胞瘤的神經分化調控機轉及其與NGF 的關聯性。本兩年期的計劃將提出四個主要的工作目標：一、在細胞研究部分，我們的前驅實驗顯示若用NGF 刺激神經母細胞瘤細胞，的確會造成VEGF mRNA 的表現量顯著上升。我們將進一步探討NGF 對於VEGF 蛋白表現量、分泌量與神經分化表現的影響，我們亦將利用VEGF 接受器抗體去抑制VEGF 的作用，以了解VEGF 是否為參與NGF刺激神經分化的重要因子。二、在動物實驗部分，我們藉由異體移植的方式將此神經母細胞瘤細胞株打入老鼠體內，再給予NGF 的刺激，研究將觀察其腫瘤體積與控制組的差異，並進行腫瘤切片染色來分析VEGF 與血管生成、腫瘤分化之間的關聯。接著我們將老鼠注射抗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。三、我們將在利用shRNA 抑制CRT 表現的神經母細胞瘤細胞株中，給予NGF 刺激，探討CRT是否參與在此調控分化的過程中。四、我們的前驅實驗顯示若以VEGF 刺激神經母細胞瘤細胞，的確會造成神經分化指標的表現量上升，我們將以RNA 定序與次世代基因檢測在此過程中的相關基因變化，進行GO、KEGG 與PPI 的分析，以找出VEGF 在神經母細胞瘤成因中所可能參與的機轉。本計劃的完成將有助於釐清VEGF 在神經母細胞腫瘤分化所扮演的角色，同時也有助於對神經母細胞瘤新治療的發展。<br> Abstract: Neuroblastoma (NB) is derived from the sympatho-adrenal lineage of embryonic neural crest cells. Thepathogenesis of NB may be due to failure of differentiation or apoptosis of precursor cells. The nerve growthfactor (NGF) and its receptor, TrkA, constitute the most crucial signaling to control the differentiation andapoptosis of sympathetic neurons during development. Previous studies revealed that aberrant NGF/TrkAsignaling contributed to the tumorigenesis of NB and NGF/Trk A signaling is essential for neuronaldifferentiation in NB. However, how NGF affects NB differentiation needs further clarification.Recent evidence suggested that vascular endothelial growth factor (VEGF), a key regulator ofangiogenesis, also plays a crucial role in the behavior of NB. Our recently published studies havedemonstrated that VEGF is involved in calreticulin (CRT), a moelcular chaperon found to be essential for NBbiology, -related neuronal differentiation in NB cells. Furthermore, my study in the past year found thatVEGF expression positively correlated with CRT expression and differentiated status in human NB tumors,NB mice xenografts and inducible-CRT stNB-V1 cells. Moreover, Kaplan-Meier analysis revealed thatVEGF expression in tumor tissues predicted a better survival in NB patients. Our work, for the first time,demonstrated that VEGF may affect NB behavior by playing a role in neuronal differentiation rather thanangiogenesis. According to our studies, we postulate that previously suggested anti-VEGF agents may beinappropriate approaches in NB and need further investigation. Because NGF is most crucial for NBdifferentiation, VEGF may also participate in NGF-related neuronal differentiation in NB. Further studies arealso warranted to clarify their relationship.Our proposed studies hope to investigate the role of VEGF in governing neuronal differentiation of NBfocusing on associated molecular mechanism and its relationship with NGF signaling in vitro and in vivo.Specifically, we propose the following four aims in two years：Aim I. Determine the role of VEGF in NGF-mediated differentiation of NB in NB cells. Ourpreliminary results reveal that mRNA expression of VEGF is upregulated in NGF-stimulated differentiationof NB cells. The expression of VEGF and neuronal markers in NB cells treated with NGF will be examined.Blockage of VEGF signaling will also be performed.Aim II. Determine the the role of VEGF and anti-VEGF treatment in NGF-mediateddifferentiation in mice xenograft models. We will establish mice xenograft models of NB for comparisonof the effects of NGF on VEGF expression, angiogenesis and neuronal differentiation. Anti-VEGF treatmentwill also be performed in mice xenograft models.Aim III. Examine that VEGF mediates NGF-related differentiation of NB through CRT-dependentor CRT-independent pathway in NB cells. The expression of VEGF and neuronal markers inCRT-knockdown NB cells treated with NGF will be examined in mRNA and protein levels, as well as thesecretion levels of VEGF in conditioned media.Aim IV. Determine the molecular mechanism that mediates the VEGF-related neuronaldifferentiation of NB. Our preliminary study reveal that VEGF could induce neuronal markers expression inNB cells. Associated gene changes during these processes will be evaluated by NGS RNA-sequencing assay.Gene expression differences will be compared in these sample using GO and KEGG enrichment analysis.Protein-protein interactions analysis will also been performed in order to pick-up 3-5 candidate genes forfurther pathway studies.Altogether, our present studies could not only shed light to the molecular mechanism underlying NBdifferentiation, but also provide important information for developing a new therapeutic strategy to improvethe outcome of NB patients.