背景：
骨髓間質乾細胞（MSCs）是首先在骨髓中發現的非造血幹細胞群。各種外部因素有助於MSCs分化的微妙平衡。近年來，人們發現在人工製造的不同硬度的基質上培養MSCs可以推動MSCs分化為特定的細胞類型。此外，已知微管（或與微管相關的蛋白質）似乎在MSC成骨分化過程中具有調節作用，並且中心體是微管組織中心之一。這促使我們研究中心體在MSCs分化成不同細胞類型中的作用。為此，我們在MSC上進行了兩組14天的實驗：對照（control）組和加入0.5μM centronone以抑制培養基中的中心體生長的(treatment)組。為了更深入地了解我們的實驗，還在我們的進一步計算研究中對RNA序列進行了取樣和分析。新的證據表明系統生物學中的動態建模和網絡構建可以幫助研究複雜生物系統內的複雜相互作用和機制。我們將系統生物學方法應用於我們的MSCs研究，以探索在中心體存在和不存在期間如何誘導蛋白質 - 蛋白質相互作用（PPI）和途徑。我們的主要目標是研究找出MSCs分化中複雜的細胞內機制。具體而言，我們要找出在中心體存在與否的情況下關鍵生物途徑的交互作用。
結果：
在對照實驗組中，我們觀察到MSC主要分化為成骨細胞，而觀察到處理組主要分化為脂肪細胞。根據時間序列RNA序列數據，我們構建了兩個PPI網絡，捕獲在具有或不具有中心體下MSC分化期間被激活的關鍵途徑和蛋白質。在建構出的MSC control PPI網絡由2396種蛋白質和3997種相互作用組成; MSC treatment PPI網絡由2734種蛋白質和4704種相互作用組成。然後，我們通過PANTHER途徑分類數據庫分析我們建構的PPI網絡。我們的結果顯示，眾所周知的MSCs成骨轉錄因子RUNX2周遭連接到的蛋白多與硬骨發育有關。而MSCs脂肪轉錄因子PPARG與脂肪形成和脂肪相關的蛋白質，例如BMP4，SMAD3和NLK有密切的連接。此外，我們推理了TGF-β/ BMP信號傳導和JAK / STAT信號傳導途徑，是在中心體存在期間觸發以促進MSCs成骨分化。我們還發現據IGF信號通路在沒有中心體的情況下被促進MSCs脂肪形成分化。
結論：
基於我們構建的PPI網絡，途徑分析，我們研究了MSCs分化相對於中心體存在/不存在下的潛在細胞內機制。我們相信，我們的研究結果不僅可以提供對MSCs分化的深入了解，還可以為未來的再生醫學提供更好的組織修復和再生治療策略。

Background:
Mesenchymal stem cells (MSCs) are a non-hematopoietic stem cell population first discovered in bone marrow. A variety of external factors contributes to the delicate balance of MSCs differentiation. In recent years, it was discovered that growing MSCs on artificially made substrates of different stiffness can drive MSCs differentiation into specific cell types over time. Moreover, it is known that microtubules (or proteins associated to microtubules) appears to have a regulatory role on the process of MSCs osteogenic differentiation and centrosome is one of the microtubule-organizing centres. This motivates us to study the role of centrosome in MSCs differentiation into different cell types. For this, we conducted two sets of 14 days experiments on MSCs: the control set and the set in which 0.5μM centrinone was added to inhibit centrosome growth in the culture medium (treatment). To have a deeper understanding into our experiments, RNA-sequence was also sampled and analysed in our further computational studies.
There have been emerging evidences indicating that dynamic modeling and network constructions in systems biology can assist the investigation of complex interactions and mechanisms within complicated biological systems. In this thesis, we applied a systems biology approach into our study of MSCs to explore how protein-protein interactions (PPIs) and pathways are induced during the presence and absence of centrosome. Our main goal is to investigate the complex intracellular mechanisms that dictate the differentiation linages of MSCs. Specifically, we seek to study the crucial signalling pathways and possible cross-talks between these pathways that are involved in the presence and absence of centrosome.

Result:
On the control experiment set, the MSCs were observed to differentiate into mainly osteoblasts, while the treatment set was observed to differentiate mainly into adipocytes. Based on time-course RNA sequence data, we have constructed two refined PPI networks, which capture the crucial pathways and proteins activated during MSCs differentiation with/without centrosome. The resulting MSC control PPI network consists of 2396 proteins and 3997 interactions; the MSC treatment PPI network consists of 2734 proteins and 4704 interactions. We then analyzed the constructed PPI networks through Protein ANalysis THrough Evolutionary Relationships (PANTHER) online pathway classification database. Our results showed that the well-known MSCs osteogenesis marker, RUNX2, were identified to interact with proteins highly related to osteogenic differentiation. MSCs adipogenesis marker, PPARG, was also identified to interact with proteins related to adipogenic and fats related proteins, such as BMP4, SMAD3 and NLK. Furthermore, we also identified TGF-β/BMP signaling and JAK/STAT signaling pathways that may be triggered during the presence of centrosome to promote MSCs osteogenic differentiation. We also identified evidences suggesting IGF signaling pathway is triggered to promote MSCs adipogenic differentiation in the absence of centrosome.
Conclusion:
Based on our constructed PPI networks, pathway analysis, we investigated the underlying mechanism of MSCs differentiation in relation to the presence/absence of centrosome. We believe that our findings could not only provide insight into the differentiation of MSCs but also shed lights towards a better tissue repair and regeneration treatment strategies for future regenerative medicine.

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