2017-08-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/707456摘要:脊椎椎間盤退化及相關脊椎病變是造成頸背疼痛及醫療支出增加的重要因素,目前的研究證據顯示椎間盤退化應是源自於本核部位(Nucleus Pulposus)。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。自體椎間盤本核細胞移植目前已顯示其作為椎間盤退化治療的潛力,而臨床上最方便取得的細胞來源是自椎間盤手術中取得之椎間盤組織,但在不同發育與退化階段的細胞俱有相當不同的特性。為增加細胞移植治療的成功機會,本研究團隊長期專注如何於培養過程中維持甚或增進本核細胞的組織再生能力,近來尤其聚焦於自組織中獲取具最佳組織再生能力之細胞族群。目前我們執行中的科技部計劃(MOST 104-2314-B-002-049)中發現,自人類本核組織分離的細胞有高達百分之九十以上會表現間質幹細胞的細胞標記(CD90+、CD73+、CD105+、CD166+、CD45-、CD34-、HLA-DR-)。但在脊索細胞或未成熟本核細胞標記部分,僅有brachyury T、CD155、 CD90呈陽性,CD221、cytokeratin-19 並無表現。因CD90 被認為是脊索細胞的反向指標,故在成熟人類本核組織中發現大量類脊索細胞的機會渺茫,但確有間質前驅細胞之存在。但在CD24 表現上我們同時發現一個特殊現象,相異於其他細胞標記的一致化表現,同時有相當數量的人類本核細胞呈現CD24陽性或陰性(CD24+或CD24-)。CD24 會表現在神經元、血液細胞和腫瘤細胞,也在人類本核細胞和脊索瘤細胞中表現,因此被認為可作為本核細胞和脊索細胞的特殊標記。文獻中發現具有CD24 陽性表現的小鼠本核細胞被發現增生能力較差,人類細胞株研究顯示呈CD24 陰性者具較為不成熟的細胞特性且對軟骨性分化之刺激反映較佳,流式細胞儀分析則顯示自本核分離間質前驅細胞可能持續維持CD24 陰性或是隨增殖代數增加或是成長激素處理而增加CD24 陽性之表現。然而CD24 的表現與否對人類本核細胞之組織再生特性的重要性至今仍不明確。此兩年期計劃目的在研究CD24 表現與否在人類本核細胞及間質前驅細胞特性與功能上之意涵,尤其是針對組織再生能力之部分。預計將不同CD24 表現之細胞個別純化後增殖,進行細胞增生速率、細胞標記表現、多向分化潛力、細胞激素表現、三維培養下醣蛋白及膠原蛋白合成等特性進行分析。並將研究可增殖特定細胞族群(CD24+或CD24-)的培養方法及條件,期望進一步應用於椎間盤退化之治療。<br> Abstract: Regeneration of the nucleus pulposus (NP) tissues in the early stages of degeneration can theoreticallyretard or even reverse the process and possibly restore healthy NP. Transplantation of autologous NP cellspresents potential for cell-based therapies for IVD degeneration. The most clinically applicable source ofcells is human NP tissue from surgeries to treat disc herniation and degenerative discs. The cells in the NPat different stage of development, growth and degeneration are heterogeneous. To enhance the chances oftherapeutic success, efforts should focus on preserving the phenotype of cells before transplantation. Ourgroup has done a series of studies for improving regenerative potential of human NP cells. Further effortshave been focused on retrieving desirable cells from the NP in order to achieve better regenerative results.Mesenchymal stem/progenitor cells (MSCs/MPCs) have been identified in the IVDs of humans and otheranimals. In our research project granted by the Ministry of Science and Technology (MOST104-2314-B-002-049), more than 90% of the analyzed primary cells from human degenerated NP tissuesexpressed a panel of markers for multipotent MSCs (CD90+, CD73+, CD105+, CD166+, CD45-, CD34-,HLA-DR-). Regarding cell surface markers for cells in the notochord or immature NP, cells from maturehuman NP tissues expressed positively to brachyury T, CD155, CD90, but not CD221 and cytokeratin(KRT)-19. Since CD90 has been proposed as a negative notochordal cell marker, high CD90 positivityindicates that notochordal cells are not likely to be populated in mature human NP tissues. A uniquemanifestation of CD24 expression was found in the cells from human degenerative NP tissues. Unlike othersurface markers were rather uniform in expressions/positivity, substantial numbers of cells from human NPtissues of a same subject are either positive or negative to CD24.CD24 is expressed in neuron, hemopoietic cells, and cancer cells. CD24 is also highly expressed in rat andhuman NP cells and human chordoma, therefore it has been considered as a specific marker for NP andnotochordal cells. CD24 expression was associated with inferior proliferation in mouse NP cells.CD24-negative clones of immortal human NP cell lines showed responses to chondrogenic differentiation,suggesting a more immature phenotype compared to CD24+ clones. In flow cytometric analysis,MSCs/MPCs from human degenerated NP tissue were found negative for CD24 even at the third passage inone study but were negative for CD24 just at the primary passage but gradually increased with everypassage and after TGF-β1 stimulation in the other study. It is still unclear about the significance of CD24expression in human NP cells especially on the regenerative potential.This 2-year research project is conducted for further understanding the implications of CD24 expression inNP cells or MSCs/MPCs from human degenerative NP tissues especially on the aspect of regenerativepotentials. After separation of CD24+ and CD24- cells from human NP tissues, serial assays will beperformed including proliferation, endogenous expressions and anabolic cytokines, expressions of specificsurface markers, multilinage differentiation, and matrix synthesis in 3-dimensional culture. Specific cultureconditions for isolation and propagation of desirable cell population (CD24- or CD24+) will also beinvestigated. Upon completion of this 2-year project, the appropriate way to propagate and to collect aspecific subpopulation within human NP tissues will hopefully be discovered. The information onCD24+/CD24- cells from human NP tissues can provide further insights on cell-based therapy for IVDdegeneration.dentification, Characteristics, Purification and Optimization of Culture Condition for Cell Subpopulations in Human Nucleus Pulpous (Ii)