2011-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/644357摘要：組織工程是一個嶄新且深具潛力的脊椎椎間盤退化治療的研究方向，若在退化的初期進行椎間盤組織再生，理論上可以延遲甚至逆轉椎間盤的退化進程。自體椎間盤軟骨細胞移植(Autologous disc chondrocyte transplantation)是目前唯一臨床上應用作為脊椎椎間盤退化治療的方法，其細胞是分離自椎間盤突出症手術中取得之椎間盤組織，然後在於體外以單層培養方式增加其細胞量，期望大量增生的細胞能製造足夠的細胞外基質，得以填補退化的椎間盤組織。由於椎間盤是極為缺乏血管支配的組織，位於中心的本核（Nucleus pulposus）細胞尤其必須進行調適以存活於此低氧且相對缺乏養分的環境，而本核組織中的氧氣濃度約介於百分之二至五間。為發展對椎間盤退化有效之生物性治療方式，這些研究應該於可以反應實際內狀況的環境中進行。然而儘管目前以有為數不少的椎間盤組織再生研究被發表，但這些體外培養的研究是在傳統的培養環境（百分之21氧濃度）下所進行，並不能反應實際體內的環境。因此，我們設計此三年期研究計畫以探討低氧培養環境對人類椎間盤本核細胞的組織再生能力的影響。計畫第一年將研究人類椎間盤本核細胞進行單層培養以增加細胞數時，低氧培養環境會對其增生率與細胞分化喪失（Dedifferentiation）現象會造成什麼影響，以選取較佳的細胞增生模式。計畫第二年將運用三組實驗模式，評估人類椎間盤本核細胞經過單層培養增殖後再以三維方式培養的過程，低氧環境對細胞外基質生成、重要基質蛋白及促進合成細胞激素（anabolic cytokines）的基因表現所產生的影響。計畫第三年將再進一步評估人類椎間盤本核細胞在低氧與高氧培養環境中對不同促進合成因子（包括TGF-β1, BMP-2, lovastatin）反應的差異。計畫的結果將有助於瞭解並建立針對人類椎間盤本核細胞最適當的培養與再生研究的實驗評估環境。<br> Abstract: Regeneration of nucleus pulposus (NP) tissues in the early stages of degeneration theoretically can retard or even reverse the degenerative process with possible restoration of healthy intervertebral disc (IVD). Autologous disc chondrocyte transplantation (ADCT) is the only tissue engineering method practiced clinically for treatment of IVD degeneration. The most clinically applicable source of cells is IVD tissue from surgeries to treat lumbar disc herniation and degenerative disc disease. Retrieved disc cells need to be expanded in monolayer culture through several passages, with a goal of establishing a population of disc cells capable of producing matrix and sustaining an expanded volume within the damaged IVD.As the IVD is a highly avascular compartment, NP cells have necessarily adapted to survive within a hypoxic and relatively nutrient-poor environment. The concentration of O2 within NP compartment is reported to be between 2 and 5%. To develop effective future biological therapeutics, it is vital that such interventions be designed and studied in a biologically relevant system. Although there are a number of potential biologically based therapies currently under development and reported in the literature, the studies that reflect in vitro experimentation have been largely preformed under classical tissue-culture O2 saturation (21%) - a condition that does not mirror the in vivo condition. Therefore, we conduct this 3-year research project intended to explore the effects of hypoxic culture environment to human NP cells especially on their regenerative potentials.The first-year project intends to disclose the effect of hypoxia on human NP cells regarding the proliferation rate and the tendency of dedifferentiation in monolayer. The second-year project is going to reveal the effect of hypoxia on human NP cells in 3-dimensional culture environment. The regenerative potential of human NP cells will be examined by determining synthesis of extracellular matrix, expression of important matrix components and anabolic cytokines. Three different settings of monolayer expansion and 3-dimensional culture will be utilized to disclose the effect of hypoxia and normoxia on human NP cells. The third-year project will further evaluate the effect of hypoxia on human NP cells regarding the responsiveness to different anabolic factors including TGF-β1, BMP-2, and lovastatin. Three experimental settings will also be employed to evaluate how human NP cells react to anabolic factors under hypoxic and normoxic culture conditions. This project will help to establish a better assessment system of regenerative researches for nucleus pulpous cells.椎間盤本核細胞培養低氧細胞增殖Nucleus PulposusCell CultureHypoxiaIsolationExpansion