軟骨細胞的分化、細胞表面之超微結構與高磁場環境對軟骨復原的影響之研究

Differentiation of chondrocytes, characterization of cell surface of chondrocytes and deleterious effects of MRI on cartilage repair

謝昌訓(Chang-Hsun Hsieh)

指導教授 ： 吳忠勳

國立臺灣大學/醫學院/分子醫學研究所/博士(2007年)

https://doi.org/10.6342/NTU.2007.00077

摘要

背景：軟骨組織的自我修復以及再生能力皆十分的不足，成年人比年輕人的軟骨組織自我修復能力更是低弱。隨著年紀的增長：軟骨組織內的自由基會增多、軟骨細胞數目會減少以及軟骨細胞之間的基質變少使得軟骨組織漸漸稀薄，導致退化性關節炎的產生。基於上述原因，軟骨細胞的特性一直被探討研究以期能在組織工程與臨床上有所應用。(一)由於核磁共振儀為非入侵的醫療評估方式，目前在臨床醫學的使用極為普遍，包括腦部斷層掃瞄、腫瘤追蹤及外科復原評估等等。然而，在近幾年的文獻中不斷探討高磁場環境與無線電電波對細胞、生理和心理的影響，包括c-jun、c-fos基因的表現，鈣離子濃度的增加以及幽閉空間恐懼症等等，顯示磁場環境與無線電電波會對細胞有某種程度的影響，而這方面的研究一直未有明確的結果。所以我們在第一部份先探討高磁場環境下對於軟骨細胞的影響。(二)原子力顯微鏡是目前探討奈米世界的最佳工具。主要是利用探針與雷射的折射角度變化來觀察活著細胞的奈米結構，並可監測細胞膜的物理特性，如黏著力和硬度。所以，我們利用原子力顯微鏡來探討正常（成年人）軟骨細胞與退化性關節炎（老年人）軟骨細胞其細胞膜物理特質的差異性。(三)間葉幹細胞有分化成不同種類細胞的能力，如可以分化成脂肪細胞、肌肉細胞、骨細胞與軟骨細胞。而軟骨細胞在長期的單層細胞培養系統中之初代培養下，其軟骨細胞的特性會漸漸消失並轉型成纖維細胞，所以我們在第三部分建立其間葉幹細胞的分化系統並觀察軟骨細胞的立體培養系統下的生長情況。
研究方法：(一)我們利用3T 強度的核磁共振儀製造高磁場環境，觀察高磁場對軟骨細胞的生長、鈣離子濃度變化與蛋白質表現變化的差異性。另外，建立迷你豬（李宋豬）的軟骨修復評估之動物模式，利用組織切片染色觀察高磁場環境對於軟骨修復之影響。(二)我們利用原子力顯微鏡直接觀測軟骨細胞的細胞膜之物理特性，如黏著力和硬度。另外，利用流式細胞儀檢測軟骨細胞上蛋白質的表現量來佐證原子力顯微鏡觀測的結果。（三）我們建立間葉幹細胞的多型性分化能力，並利用免疫染色來檢驗各個分化細胞型態的成果。此外我們利用反轉錄聚合酶連鎖反應來觀察在單層細胞培養之繼代培養系統下軟骨細胞特性的變化。我們也利用掃瞄式電子顯微鏡來觀察軟骨細胞在聚乳酸與聚羥基乙酸材質所構成的立體培養環境之情況。
結果與討論：(一)在核磁共振儀環境中，軟骨細胞會因為高磁場或是高磁場加上無線電能量的刺激下而造成細胞內鈣離子濃度升高和誘導細胞凋亡相關蛋白質的大量表現進而使軟骨細胞進入細胞程式化死亡。另外在豬隻軟骨修復與評估的計畫中，相同的實驗條件下，利用核磁共振評估與沒有利用核磁共振評估的豬隻相較下發現，使用核磁共振之豬隻軟骨修復情況極為不好，修復位置（即原造成損傷位置）有一嚴重的凹陷損傷。推測原因可能在軟骨修復過程中，因為暴露在核磁共振環境下造成細胞死亡或細胞轉性而影響到軟骨之修復。(二)透過原子力顯微鏡的測量，正常（成年人）軟骨細胞的細胞表面起伏變化比退化性關節炎（老年人）之軟骨細胞劇烈。正常（成年人）軟骨細胞其細胞膜的黏著度與硬度均大於退化性關節炎（老年人）之軟骨細胞。另外，我們也發現正常（成年人）軟骨細胞其表面蛋白質表現量（integrin beta1 and type II collagen）也明顯比退化性關節炎（老年人）之軟骨細胞表現量多。這可以建立一觀測細胞膜物理特質之系統，用來觀測軟骨細胞隨著年紀增長之健康度變化。（三）我們成功地建立利用間葉幹細胞分化成其他細胞型態之系統。另外，我們也觀察到軟骨細胞在單層細胞培養下，約第八代便會失去其軟骨細胞之特質。而軟骨細胞在聚乳酸與聚羥基乙酸材質所構成的立體培養環境下生長，會誘導軟骨細胞分泌更多的細胞外間質。

關鍵字

軟骨細胞 ； 核磁共振儀 ； 原子力顯微鏡 ； 黏著度 ； 硬度 ； 間葉幹細胞 ； 聚乳酸與聚羥基乙酸

並列摘要

Background: The proliferation and self-repair of articular cartilage are both poor. In fact, a number of age-related changes occur in the articular cartilage, including the free radical increase, a decrease in the number of chondrocytes and an increase in the degradation of matrix components. Such an age-related degeneration of articular cartilage could be a major risk factor for the development of osteoarthritis. Therefore, a lot of medical researches focused on the characters of chondrocytes in tissue engineering and clinic research. For instance, (1) As the MRI is a non-invasive physical assessment method, it is prevailing in clinic, including brain CT, tumor tracing, and assessment of surgery recovery…etc. In recent years, many papers discuss about the influence of high magnetic field or radiation on the cell, physiology and psychology, including expression of c-jun and c-fos genes, Ca2+ flux change, particularly Ca2+ entry from the extracellular environment through the plasma membrane, and induction of cloustrophobia syndrome…etc. Studies with cellular systems using different exposure setups, exposure durations, amplitudes, frequencies and wave forms indicate that biological effects of magnetic fields on cellular systems are at hand, but there is no definite conclusion in this field. In the first aim, we want to monitor the effect of the high magnetic field on human chondrocytes. (2) Atomic force microscope (AFM) permits the characterization of biologic samples ranging from a single molecules to whole cells (nanometers to micrometers), it is a powerful tool for exploring the shape of a single cell, the properties of a cellular membrane, or the interaction of intermolecular forces, such as adhesion force and stiffness. The second aim of this study is to use AFM technique to differentiate the mechanical behavior of the chondrocytes between the young modulus (normal) and the old modulus (OA). (3) Mesenchymal stem cells have multi-potential differentiation capacities, such as fat, muscle, bone and cartilage. In addition, primary culture of chondrocytes tend to lose the chondro-property and de-differentiation in monolayer culture condition during subculture. The third aim is to evaluate the characterization of MSCs differential capacities and establishing the 3D culture system for chondrocytes in vitro.
Method: (1) The 3T magnetic field was provided by the machine of Magnetic Resonance Imaging. The human chondrocytes were directly exposed to a 3-tesla (T) magnetic field (MF group) or a 3-T static magnetic field plus 125.3 MHz radio frequency (MF+RF group), and cell proliferation, apoptosis, cytosolic Ca2+ ([Ca2+]i) fluxes and expression of the apoptosis-related proteins of the treated cells were examined to assess the effects of the treatments. In the pig study, we examined the effects of the treatments on the recovery of surgically damaged pig knees. (2) We used AFM to observe a single chondrocyte cell directly and measured the dimensions of the cells. In addition, the receptors of cellular membrane were monitored by FACS. (3) The MSCs were isolated from human bone marrow. The MSCs were induced to adipogenesis, myogenesis, osteogenesis and chondrogenesis separately by respective inducers. The differentiated cell lineages were confirmed by specific markers expression by immunochemical staining. In addition, the RT-PCR system was applied for monitoring the property of chondrocytes in monolayer culture condition during subculture. Besides, chondrocytes which were seeded in the PGA/PLA scaffold was evaluated by using SEM observation.
Results and discussions: (1) A 3-T static magnetic field and radio frequency suppressed cell growth and induced apoptosis through p53, p21, p27 and Bax protein expression. In the pig model, we found that MRI surveillance had a deleterious effect on the recovery of the damaged knee cartilage. Magnetic strength, with or without concurrent radio frequency, suppressed chondrocyte growth in vitro and affected recovery of damaged knee cartilage in vivo in the pig model. The possible reason is chondrocytes exposing to the high magnetic field inducing cell apoptosis or cell transformation in the cartilage repair process. (2) The AFM revealed differences in the sizes and structures between the young modulus (normal) and the old modulus (OA). These findings suggested that the mechanical properties of normal chondrocytes substantially differed from those of OA chondrocytes. This new approach could be a useful technique for investigating age-related changes in the properties of human chondrocytes. (3) The MSCs from human bone marrow could differentiate into adipogenesis, myogenesis, osteogenesis and chondrogenesis processes. The morphology of human chondrocytes can be maintained after six passages both in petri dish and on scaffold but the cells were easily to be transformed to fibroblasts after eight passages. The scaffold laminated by PLA-coated PGA fiber was suitable for chondrocytes growth and showed better cell attachment under SEM investigation.

並列關鍵字

chondrocytes, MRI ； AFM ； adhesion force ； stiffness ； MSCs and PLA/PGA

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