探討Notch2與Notch4在脂肪前驅細胞增生與分化所扮演的相反角色

The Opposite Role of Notch2 and Notch4 on The Proliferation And Differentiation of 3T3-L1 Preadipocyte Cells

賴芃燁(Peng-Yeh Lai)

指導教授 ： 曾銘仁

國立中正大學/理學院/生命科學系分子生物研究所/博士(2014年)

摘要

肥胖(obesity)是現在全球所面臨的一個能量代謝的疾病，而此疾病最終會導致體內堆積過多的白色脂肪組織，而造成各式各樣的併發症。因此如何避免產生過多的脂肪組織成了解決肥胖問題的方法之一。脂肪組織是由脂肪細胞(adipocyte)所組成，而脂肪細胞是由脂肪前驅細胞經由脂肪細胞形成(adipogenesis)的過程而來。許多的細胞傳導訊號參與了脂肪細胞形成的調控，其中也包含了Notch這個訊息路徑。之前對於參與脂肪細胞形成的Notch路徑都是著重於Notch1與其下游基因，HES1的研究，但對於其他的Notch受體參與於脂肪細胞發展所扮演的角色則仍待釐清。首先我們利用即時定量聚合脢鏈所反應(Q-RT-PCR)來分析脂肪前驅細胞3T3-L1分化成脂肪細胞的過程中Notch1、Notch2、Notch3、Notch4基因的表現量，結果發現Notch4在細胞分化的過程中，其表現量的增加程度是與Notch1的表現量增加相似。另外Notch2在分化過程中，在四種Notch基因裡，表現量最早有顯著增加的現象。為了探討Notch2與Notch4在脂肪細胞形成所扮演的角色，我們先將人類活化態的Notch2與Notch4表現質體短暫的轉染到3T3-L1細胞後，分析基因的表現，結果發現Notch下游基因：HES1和Hey1在轉染活化態Notch4的細胞有表現量增加；且促進脂肪細胞形成的調控基因：C/EBPδ和PPARγ的表現量也增加；另外，對脂肪細胞形成具負調控的基因：Pref-1的表現量則下降。為了進一步釐清Notch2與Notch4在脂肪細胞形成所扮演的角色，我們建立了穩定表現人類活化態Notch2與Notch4的3T3-L1細胞株。經由細胞週期及基因表現(Q-RT-PCR或microarray)的分析，從穩定表現Notch2的細胞株，我們發現Notch2會經由調控細胞週期來減緩細胞的增生速度；且經由降低電子傳遞鏈基因的表現使3T3-L1細胞脂肪分化能力下降。相反的，由Notch4的穩定表現細胞株發現Notch4會經由活化ERK路徑與調控細胞分化的細胞系擴張期(clonal expansion)之細胞週期來促進3T3-L1細胞的增生；且Notch4也會透過增加膽固醇生合成(cholesterol biosynthesis)的基因及脂肪細胞形成的正向調控基因：C/EBPα、PPARγ、aP2、LPL、HSL的表現來加速3T3-L1細胞的分化。

關鍵字

脂肪前驅細胞 ； 脂肪分化

並列摘要

Obesity is an energy balance disorder in the modern world which resulting in excessive white adipose tissue accumulation. Adipose tissue is composed of adipocytes, which differentiates from precursor cells in a process called adipogenesis. Many signal molecules are involved in transcriptional control of adipogenesis, including the Notch pathway. Previous adipogenic studies of Notch have focused on Notch1 and HES1; however, the role of other Notch receptors in adipogenesis remains unclear. Q-RT-PCR analyses showed that the augmentation of Notch4 expression during the differentiation of 3T3-L1 preadipocytes was comparable to that of Notch1. Furthermore, Notch2 expression level was increased significantly in the early differentiation day. To elucidate the role of Notch2 and Notch4 in adipogenesis, the human active form Notch2 (N2IC) and Notch4 (N4IC) were transiently transfected into 3T3-L1 cells. The expression of Notch downstream genes, HES1 and Hey1 and proadipogenic genes, C/EBPδ and PPARγ, were upregulated in N4IC-transfected cells and the expression of Pref-1, an adipogenic inhibitor, was downregulated. To further characterize the effects of N2IC and N4IC in adipogenesis, stable 3T3-L1 cell lines expressing human N2IC or N4IC were established. The expression of N2IC suppressed proliferation and differentiation of 3T3-L1 cells. On the other hand, expression of N4IC promoted proliferation and enhanced differentiation of 3T3-L1 cells compared with those of control cells. Cell cycle and gene expression analyses showed that N2IC suppressed proliferation through cell cycle regulation and suppressed differentiation through downregulating electron transport genes. However, N4IC promoted proliferation through modulating the ERK pathway and the cell cycle during the early stage of 3T3-L1 adipogenesis and facilitated differentiation through upregulating cholesterol biosynthesis genes and adipogenic genes such as C/EBPα, PPARγ, aP2, LPL and HSL during the middle and late stage of 3T3-L1 adipogenesis.

並列關鍵字

3T3-L1 ； Adipogenesis ； Notch

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