穀胱甘肽對CISD2基因缺陷之誘導式多能性幹細胞分化之類軟骨細胞於氧化壓力下的影響

Abstract

骨關節炎（OA）是一種和老化相關的疾病，全世界的病患數約有影響數百萬之多，而在美國，骨關節炎也是造成失能的主因之一。此外，它不是一個單一的疾病，而是多種疾病，導致整個關節及其周圍組織的破壞的最終結果。關節的結構和功能衰竭是伴隨著慢性疼痛導致人們無力進行他們的日常活動。 具體而言，骨關節炎是由特定類型的軟骨的關節（關節軟骨）內的故障引起的。它是已知的關節軟骨僅由滑液包圍;因此，它被認為是無血管組織。這股骨頭缺血性自然有助於其無法承受的毒性和自我修復。因此，為了防止OA devolvement就必須停止關節軟骨的破壞以及細胞外基質（ECM）的降解。 事實上，醫學界中一個共同的信念是，骨性關節炎是衰老的副產品。然而，新的研究指出了有毒的活性氧的干預（ROS）作為該疾病的發展的根本原因。此外，CDGSH鐵硫域2（CISD2）基因的突變似乎是負責在ROS產生的釋放。因此，我們利用小鼠誘導具有CISD2缺陷基因通過細胞分化過程中獲得軟骨細胞樣細胞的OA多能幹細胞（的mIPSC）。 因此，我們的主要目標是成功地分化的mIPSC CISD2 - / - ，+ / +成軟骨細胞樣細胞的OA不影響其基因型和表型。同樣重要的是獲得一種抗氧化劑具有軟骨保護作用，能抑制或過氧化氫曝光（H2O2）降低升高的stress-induced/apoptosis。因此，我們採用了熱敏殼聚醣/明膠/甘油（C / G / GP）水凝膠，並用它作為藥物（穀胱甘肽，穀胱甘肽）攜帶者治療軟骨細胞樣細胞的OA。 本研究的第一部分是誘導的mIPSC CISD2的分化 - / - + / +成軟骨細胞樣細胞的OA。在適當的條件下才會產生子代細胞的上調的三個主要軟骨細胞標記一致：SRY（性別決定區Y） - 盒9（SOX-9），聚集蛋白聚醣和Ⅱ型膠原。我們的結果表明，當的mIPSC CISD2 - / - + / +的誘導生長因子骨形態發生蛋白4（BMP4），它引起了細胞中顯示的軟骨細胞樣細胞的特徵。 本研究的第二部分是把氧化應激下的細胞中設置野生型和突變型的細胞之間的基線條件。用於誘導的氧化應激的化合物是過氧化氫（250μM）。因此，GHS（100μM）是用來降低抑制誘導的ROS的氧化損傷。實時聚合酶鍊式反應（RT-PCR）表明：像白細胞介素-1（IL-1），白細胞介素-6（IL-6），基質金屬蛋白酶-3（MMP-3）和腫瘤壞死因子α（TNF-α）的炎性基因的表達被下調治療GHS（100微米）之後。 本研究的第三部分是要找到一個合適的水凝膠（C / G / GP）與合適的物理特性，允許GHS的加載（100微米）到凝膠基質，並隨後發布到終端脫氧核苷酸轉移酶缺口末端標記（TUNEL）分析表明，實際上，細胞凋亡水平時，細胞與GHS（100微米）+（C / G / GP）水凝膠治療顯著下降。 從本研究中進行的分析所獲得的結果強烈地表明，BMP-4（25μM）生長因子成功誘導的mIPSC CISD2的分化 - / - + / +成軟骨細胞樣細胞的OA。同樣，我們可以自信地說，GHS（100μM）對來自的mIPSC CISD2軟骨細胞樣細胞的OA抗氧化作用 - / - ，+ / +。此外，很明顯的TUNEL結果（C / G / GP）水凝膠似乎是穀胱甘肽有效的控制釋放系統。

Osteoarthritis (OA) is a disease that affects millions of people around the world and it is catalogued as one of the major disabilities in the US. In addition, it’s not a single disease but rather the end result of a variety of disorders that lead to the destruction of the entire joint and its nearby tissues. As the disease progresses, the joint’s structure and functionality became limited and chronic pain ensued, which results on people’s inability to perform their daily activities. Specifically, osteoarthritis is caused by the breakdown of a particular type of cartilage inside the joint (articular cartilage). It is known that articular cartilage is only surrounded by synovial fluid; therefore, it is considered an avascular tissue. This avascular nature contributes to its inability to withstand toxicity and self-repair. Thus, to prevent OA devolvement it is imperative to stop the destruction of articular cartilage along with extracellular matrix (ECM) degradation. Indeed, a common belief among the medical community was that osteoarthritis was a byproduct of aging. Nevertheless, new studies pointed to the intervention of toxic reactive oxygen species (ROS) as the underlying cause for the development of this disease. Furthermore, the mutation of the CDGSH Iron Sulfur Domain 2 (Cisd2) gene appears to be responsible in the release of ROS production. Therefore, we utilized mouse induce pluripotent cells (miPSCs) that have a Cisd2 deficiency gene to obtain a disease model that can provided chondrocyte-like OA cells through a cell differentiation process. Hence our main goal was to successfully differentiate miPSCs Cisd2 -/-, +/+ into chondrocyte-like OA cells without compromising its genotype and phenotype. Equally important was attaining an antioxidant with chondroprotective effects that can inhibit or reduce elevated stress-induced/apoptosis by hydrogen peroxide exposure (H2O2 250μM). Consequently, we used a thermosensitive chitosan/gelatin/glycerol (C/G/GP) hydrogel and used it as a drug (glutathione, GSH 100 μM) carriers to treat chondrocyte-like OA cells. The first part of this study was to induce the differentiation of miPSCs Cisd2 -/-, +/+ into chondrocyte-like OA cells. Under the appropriate conditions it will generate progeny cells consistent with the up-regulation of the main three chondrocyte markers: SRY (sex determining region Y)-box 9 (SOX-9), Aggrecan and Collagen II. Our results indicated that when miPSCs Cisd2 -/-, +/+ are induced with growth factor bone morphogenetic protein 4 (BMP-4 25 ng/ml) it gave rise to cells that display characteristics of a chondrocyte-like cells. The second part of this study was to put the cells under oxidative stress to set a baseline condition between wild type and mutant type cells (miPSCs Cisd2 -/-, +/+). The compound used to induce oxidative stress was hydrogen peroxide H2O2 (250μM). Consequently, glutathione (GHS) 100μM is used to reduce or inhibit the oxidative damage cause by ROS. Real-time polymerase chain reaction (RT-PCR) indicated that inflammatory gene expression like Interleukin 1s (IL-1s), Interleukin-6 (IL-6), Metalloproteinase 3 (MMP-3) and Tumor Necrosis Factor alpha (TNF-α) were down regulated after treated with GHS (100 μM). The third part of this study was to find a suitable hydrogel (C/G/GP) with the right physical properties that permit the loading of GHS (100 μM) into the gel matrix and subsequent release of GHS (100 μM) into chondrocyte-like cells. Analysis with Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) revealed that in effect, apoptosis level decreased significantly when cells were treated with GHS (100 μM) + (C/G/GP) hydrogel. The results obtained from the analysis performed in this study strongly indicated that BMP-4 (25 μg/ml) growth factor successfully induce the differentiation of miPSCs Cisd2 -/-, +/+ into chondrocyte-like OA cells. Likewise, we confidently can state that GHS (100μM) have antioxidant effects on chondrocyte-like OA cells derived from miPSCs Cisd2 -/-, +/+. Furthermore, TUNEL results clearly indicated that (C/G/GP) hydrogel appears to be an effective control-released system for GSH loading.