| Abstract: | 由先前的研究已經證實由骨膜衍生而來之未分化的細胞仍具有成骨功能，不論是體內自體或異體骨膜移植，或是體外細胞培養，或以細胞移植於體內，均可觀察到骨質沉積的現象。可達成骨膜移植的路徑及材料雖多，但宿主排斥免疫現象仍或多或少地造成移植的干擾或失敗。環孢靈是目前使用最廣泛的一種免疫抑制劑，他可以經由抑制白血球間質-2的產生，來抑制宿主產生免疫排斥反應。但是他也有副作用，如肝、腎、心臟毒性，及牙齦增生等現象，近來亦有因器官移植使用環孢靈素而導致患者顏面畸形的病例報告。因此如何既可以避免環孢靈的毒性，又可同時確保或提升骨膜移植的成功率，長久以來便是醫學界極為重視的課題。本論文之目的，即在於建立一個最適切的體外培養的環境，將由大白屬脛骨骨膜取得之骨膜細胞作成繼帶培養，使這些骨膜細胞在經過長期體外培養之後，既能仍然保有骨膜細胞的成股分化能力，又能在環孢靈的作用下保持及促進骨膜細胞的分化造骨能力。本研究的結果顯示，骨膜細胞在初代與繼代的培養下，細胞成多角形似纖維母細胞型態。在細胞長滿時，上皮狀細胞巨集，而纖維狀細胞則散落於周圍，骨膜細胞的培養液在添加維他命C 即β-GP後，不論是低密度細胞培養，或高密度細胞培養均有細胞團塊的出現。這種細胞團塊即是具有造骨細胞潛能的細胞團稱為 Colony forming unit osteoprogenitor (CFU-O)，而且在密度呈 1x105/cc 的情況下，仍可見到細胞團的出現。
鹼性磷酸梅( Alkaline phosphatase ALP ) 是在成骨過程中最早出現的標誌，本研究室以免亦螢光對ALP做了定位及定性分析，以可見光質譜儀做了定量的研究。並研究免疫抑制劑環孢靈(cyclosporin A CsA)對骨膜細胞在生長、分化及對ALP活性的影響。結果顯示，CsA 並不會影響骨膜細胞型態，也對骨膜細胞生長沒有明顯作用。但是他可增加CFU-O的數目，再免疫螢光法中，CsA 可增加 ALP的螢光呈色，在可見光質譜儀的定量分析下，CsA亦可增加ALP活性的表現。在亞致死劑量之環孢靈存在作用下，骨膜細胞表現出鹼性磷酸活性大增，胞內分布擴展，孢外磷及鈣的沉積也提升，這些反應在長達四星期的觀察中，並表現出與時遽增的特性，環孢靈素也如預期的並不會造成明顯的細胞增生。本研究初步結果提供了我們將來進一步作動物實驗的設計基礎，也增加了我們對其可行性的信心。
Previous studies have demonstrated that undifferentiated periosteum-derived osteoprogenitor cells (POC) retain their osteogenic capacity of POC cells can be maintained in various situations, be it as autograft or heterotopic allografts in vivo; or cells cultured in vitro; or as transplanted cell mass into recipients. Although diversified ways and materials for transplants are available, each and every one of them poses few inheritant disadvantages, i.e. the host-vs-graft immune rejections. Cyclosporin A (CsA), one of the most commonly used immunosupressants nowadays, inhibits host rejections. CsA, however, has many undesirable side effects, to name a few, renotoxicity, hepatotoxicity, and the overgrowth of gingiva. It has, therefore, been a challenging subject of medicine advancements to develop a regimen by which not only side-effect of CsA can be reduced or eliminated, but also the success rate of transplantation can be raised. The purpose of the present study was to set up and find out the optimal conditions for an in vitro model system of explant culture of POC cells isolated from rate tibiae. Small modifications like modifying the medium composition, the density of cells inoculated on plastics, and other extrinsic factors as well, were attempted such that only the differentiated markers of POC cells cab be faithfully retained for a long period of time in vitro, but also some intrinsic factors can be promoted or enhanced by CsA, e. g. the osteogenic capacity. Our data show that in the presence of sublethal maximal dosage of CsA, the expression of alkaline phosphatase, the major marker of osteogenic capacity, by POC cells was enhanced as a function of time. Also, extracellular deposits of calcium and phosphate in the form of mineralizing nodules were increased. Furthermore, as expected, the proliferation rate was not significantly stimulated. Results from the above-mentioned studies demonstrate that the plasticity of differentiated phenotypes ca be modified by the synergistic reactions of bioactive factors and matrix milieu. This conceptual findings will pave the way to use cultured cells in cell therapy in the future |
