評估不同分子量玻尿酸對創傷性膝關節炎與原發退化性膝關節炎的軟骨保護效果與抗發炎效果

Abstract

背景 關節面骨折的病人，在受傷後產生創傷性關節炎，是很常見的併發症，特別是在需要負重的關節更是如此。一旦發生創傷性關節炎變化之後，接下來的補救重建手術都無法達到預期中的復原。唯有在病變的早期作預防，來改變創傷性關節炎的自然病程，才是為合理且最有效的解決方式。 玻尿酸注射在退化性關節內，已經被證實能有效的緩解疼痛，以及改善關節功能。目前世界上有很多國家都已核准並廣泛使用。但是，玻尿酸是否能用在關節面骨折癒合後的病人，來預防創傷性關節炎，仍是未知數。除此之外，先前有關這方面的研究都是以動物實驗的方式進行，在人體創傷性關節炎的預防效果，需要更多研究與實驗印證。 許多學者發現，玻尿酸濃度以及分子量會隨著退化性關節炎的進展而下降。並意指高分子量玻尿酸比低分子量玻尿酸對關節的保護效果較好。但是，正常人的身體中就有三種玻尿酸合成酶基因。有兩種基因分泌高分子量玻尿酸，一種分泌低分子量玻尿酸。如果低分子量玻尿酸，對關節沒有保護作用，在人的演化過程中，分泌低分子量玻尿酸的基因就應該被淘汰。此外，最近的研究顯示玻尿酸不僅僅只有緩解症狀的效果而已，它可能也具有軟骨保護作用，進而改變退化性關節炎的病程，但是玻尿酸分子量在其中扮演的角色為何，仍不清楚。 本篇論文提出一個假說，闡述高分子量玻尿酸和低分子量玻尿酸在人體的關節內扮演著不同角色的作用。研究實驗分成三個部分，首先，我們檢測玻尿酸對於從關節面骨折的病人身上所取出的類纖維母細胞之滑液細胞在六種與關節炎相關的蛋白質分泌之影響。第二部分，我們檢測不同分子量玻尿酸對於從關節面骨折的病人身上所取出的類纖維母細胞之滑液細胞在六種與關節炎相關的蛋白質分泌之差異。最後，我們檢測三種不同分子量玻尿酸對於從退化性關節炎的病人身上所取出的類纖維母細胞之滑液細胞在七種與關節炎相關的蛋白質分泌之差異。 方法 從不同病人，不管是關節面骨折或是退化性關節炎的病人身上所取出的類纖維母細胞之滑液細胞，同時和不同分子量的玻尿酸一起培養。將培養液取出，並進行與關節炎有關的蛋白質分析。 結果 1.在有關節面骨折的病人，玻尿酸可以降低一些對關節不好的降解性蛋白質的分泌，例如：IL-1，MMP3，TNFα。此外，玻尿酸可以促進對關節有益的蛋白質分泌，例如：IL-10，TIMP-1，TIMP-2。 2.在有關節面骨折的病人，高分子量玻尿酸可以有效降低一些引起發炎的蛋白質之分泌如：IL-1，TNFα。而低分子量玻尿酸可以有效降低間質蛋白酶MMP-3之分泌，並促進抗間質蛋白酶TIMP-1與TIMP-2分泌。 3.在退化性關節炎的病人身上，在未被IL-1誘導的實驗中，低分子量玻尿酸，無論做在抗發炎作用以及在軟骨保護作用上都比高分子量玻尿酸好。然而，被IL-1誘導之後，高分子量玻尿酸在抗發炎作用的表現比低分子量玻尿酸好。整體而言，低分子量玻尿酸的軟骨保護效果好，高分子量玻尿酸的抗發炎作用好。 結論 1.在有關節面骨折的病人，玻尿酸可以有效調整滑液細胞所分泌的蛋白質，並對預防創傷性關節炎有幫助。 2.在有關節面骨折的病人，高分子量玻尿酸可明顯調控並改善發炎蛋白質的表現，而低分子量玻尿酸可以明顯改善保護軟骨蛋白質的表現。 3.在退化性關節炎病人身上，高分子量玻尿酸比較偏向抗發炎效果，低分子量玻尿酸則比較偏向軟骨保護效果。

Background: Post-traumatic osteoarthritis (OA) is a common sequela in patients following intra-articular fracture, especially in weight-bearing joints. Subsequent reconstructive surgeries may not correct all the problems that relate to patient’s disability. Prevention at the very early stage and changing the nature history of the disease will be the more reasonable and feasible modality of resolution. Intra-articular injections of hyaluronic acid (HA) into OA joints have been reported to relieve joint pain and improve function, which are now licensed worldwide for the treatment of OA. However, few studies evaluate the feasibility of using HA to prevent OA progression in early stage traumatic arthritis. In addition, previous studies concerning this issue were all conducted in animal models. There is still limited understanding regarding this efficacy in human. Many investigators have reported that the molecular weight (MW) of HA in diseased joint is much lower than that in normal joint, implying joints with higher MW HA get better function. However, there are three human HA synthase (HAS) genes synthesizing different MW HAs in human synovial joint. HA with higher MW (2 x 106 Da) is synthesized by HAS 1 and HAS 2, whereas HA with lower MW (2-3 x 105 Da) is synthesized by HAS 3. If low MW HA is nothing but a depolymerized product, HAS 3 that synthesizes LMW HA should be deleted during the human evolution process. Increasing evidences suggest that HA may not only provide an anti-inflammatory effect but also a chondro-protective effect, which modify the progression of OA. Controversy exists concerning whether these effects are molecular weight dependent. In this thesis, whether HA would induce expression of OA-related factors associated with improved outcomes in patients with tibia plateau fracture was examined in the first part experiments. Besides, the effects of two different MW HAs on six OA-related proteins expressed in fibroblast-like synoviocytes (FLS) from patients with tibia plateau fracture were compared and analyzed in the second part experiments. Furthermore, in the third part experiments, it was hypothesized that HA with different MW may have different contributions in the maintenance of normal joint physiology. The effects of three different MW HAs on seven OA-related proteins expression in FLS from patients with early stage OA were evaluated in this part. Methods: The interleukin (IL)-1-β induced or un-induced FLS from patients with tibia plateau fracture and early stage OA and were cultivated with or without the treatment of HA. Quantifications of these factors are performed with sandwich Enzyme-Linked Immuno-Sorbent Assay (ELISA) by using commercially available kits. Results: (1). HA downregulated the expression of catabolic factors, including IL-1, MMP-3, and TNF-α, and upregulated the production of anticatabolic factors such as TIMP-1 and TIMP-2. HA promoted the creation of interleukin-10, an anti-inflammatory cytokine, in FLS. (2). HA with a high MW (HMW) is more effective in down-regulating proinflammatory cytokines such as IL-1β and TNF–