- 學位論文
經由cAMP-PKA路徑調控人類間葉幹細胞脂肪和骨頭的行成
Reulation of adipogensis and osteogensis in human mesenchymal stem cells via cAMP-PKA pathway
摘要
人類間葉幹細胞是多功能的幹細胞，可以分化成一些不同的個體。我們實驗室 過去報導過不死化的間葉幹細胞：KP-hMSCs.此細胞能夠在添加IBMX之脂肪誘導的 培養基中形成脂肪。而IBMX是一種phosphodiesterases 的抑制劑， 能夠刺激 cAMP-dependent protein kinase 的活性 (cAMP-PKA)。由此研究，我們進一步推測幹細胞是經由cAMP-PKA路徑上的作用進行脂肪分化。我們檢視在脂肪形成時，一些 牽涉到此路徑上的調控物質，包含刺激物 (forskolin, Sp-cAMP) ；抑制物 (PKI) 和 刺激物及抑制物的組合物。 當KP人類間葉幹細胞培養在誘導脂肪形成的培養基時，添加cAMP-PKA刺激物 能夠增強脂肪的分化。而且也能增加脂肪標記基因(PPARγ2 and LPL)的表現和減少 骨頭標記基因(Runx2, Alk-p and Op)的表現。相對的，KP人類間葉幹細胞培養在誘導 脂肪形成的培養基時，添加含有cAMP-PKA的抑制劑時，會降低脂肪標記基因的表 現，和增加骨頭標記基因的表現。 我們更發現於培養基中，KP間葉幹細胞再進行脂肪分化時，會合成瘦素mRNA 同時也會釋放瘦素，且瘦素mRNA表現及瘦素的釋放會經由添加PKA的刺激物而抑 制基因上的表現,相對地也會因添加PKA的抑制物而促進基因上的表現。因為瘦素， 它的角色被我們推測是藉由cAMP-PKA路徑來調控KP間葉幹細胞脂肪形成和骨頭 形成。我們在脂肪誘導的培養基中添加forskolin及瘦素，會發現在添加的劑量上，在 脂肪標記基因及骨頭標記基因上，會有相對的表現的差異. KP人類間葉幹細胞經轉殖DN (dominant negative) CREB被發現會增強CREB蛋 白程度的表現。然而，在轉殖DN CREB至人類間葉幹細胞經過脂肪誘導的培養基添 加forskolin處理三天，不僅幹細胞脂肪的分化PPARγ2基因會被抑制，相對也會增強 Runx2及leptin的基因表現。此外, DN CREB會增加RANKL和OPG在基因表現上的比 值。由這些結果，KP人類間葉幹細胞可能是使用此分子訊號來調控成人脂肪的形成 和骨頭的形成。
並列摘要
Mesenchymal stem cells (MSCs) are pluripotent stem cells that can differentiate into several distinct lineages. We have reported an immortalized line of human MSCs (hMSCs), KP-hMSCs, which undergo adipogenesis in adipocyte induction medium (AIM) with IBMX, a phosphodiesterases inhibitor, which was reported to stimulate cAMP-dependent protein kinase activity (cAMP-PKA). In current study, we further hypothesized the involvement of cAMP-PKA pathway in adipogenesis of hMSC. We investigated the involvement of several modulators of cAMP-PKA pathway, including stimulators (forskolin, Sp-cAMP), inhibitors PKI, and combination of both in the adipogenesis of hMSC. We demonstrated that KP-hMSCs when cultured in AIM with the stimulators of PKA enhance the differentiation into adipocytes, and also increased the expression of markers for adipocyte (PPARγ2 and LPL) and decreased the expression of makers for osteoblast (Runx2, Alk-p and Op). On the other hand, KP-hMSCs when cultured in AIM with the inhibitors of PKA decreased the expression of markers for adipocytes and increased the expression of makers for osteoblast. We further found that the secretion of leptin and the mRNA expression of leptin by KP-hMSCs decreased as the addition of PKA stimulators and increased as the addition of PKA inhibitors. Because we speculated that leptin played a role in cAMP-PKA mediated regulation of adipogenesis and osteogenesis in KP-hMSCs, we added leptin into AIM with forskolin and found leptin reversed the effects of forskolin on adipogenesis and osteogenesis in a dose-dependent manner. KP-hMSCs transfected with DN (dominant negative) CREB was found to have an increase in CREB protein level, however, not only underwent adipogenesis of hMSC but also amplified mRNA expressions of Runx2 or leptin in AIM with forskolin at three day. On the other hand, mRNA expression of PPARγ2 was downregulated by DN CREB. Beside, DN CREB also increased RNAKL/OPG ratio in mRNA level. From the current results, KP-hMSCs may be used to elucidate molecular signaling on the regulation of adult adipogenesis and osteogenesis.