關節內注射高分子量玻尿酸治療膝部骨關節炎之實證醫學與分子機轉研究

Therapeutic Effects of Intra-Articular Injection of High-Molecular-Weight Hyaluronic Acid on Knee Osteoarthritis: Evidence-based Medicine and Molecular Mechanisms

王貞棣(Chen-Ti Wang)

指導教授 ： 侯勝茂

共同指導教授 ： 江伯倫

國立臺灣大學/醫學院/臨床醫學研究所/博士(2006年)

https://doi.org/10.6342/NTU.2006.00025

摘要

關節內注射高分子量玻尿酸，已經被用來作為膝部骨關節炎的關節液黏稠度補充療法，然而其療效仍有待以實證醫學的方法來證實。活化的滑膜細胞，在人類骨關節炎的病程進展中扮演極重要的角色，然而高分子量玻尿酸對骨關節炎病人滑膜細胞的影響至今仍不清楚。最近的研究顯示，取自關節液的纖維母細胞樣滑膜細胞 (fibroblast-like synoviocytes)，其表現型及功能，與取自手術滑膜組織檢體的滑膜細胞是相同的。由於早期骨關節炎病人很少會進行手術取得組織檢體，因此，取自關節液的纖維母細胞樣滑膜細胞，是研究早期骨關節炎病人的滑膜細胞一個很有用的細胞模式。本研究之目標，是以實證醫學的系統綜述 (systematic review) 和統合分析 (meta-analysis) 方法分析關節內注射高分子量玻尿酸對膝部骨關節炎的療效及其安全性，以期在臨床上能正確地使用高分子量玻尿酸來治療膝部骨關節炎，並進一步以分子生物學研究方法探討高分子量玻尿酸對早期骨關節炎病人的纖維母細胞樣滑膜細胞之骨關節炎相關細胞激素與酶之基因表現的影響，以助於了解高分子量玻尿酸治療骨關節炎的分子機轉。
本研究分為二個部分：第一部分的研究，是關節內注射高分子量玻尿酸治療膝部骨關節炎之實證醫學研究。我們利用實證醫學的系統綜述方法學收集了比較關節內注射高分子量玻尿酸與注射安慰劑 (placebo) 對膝部骨關節炎療效的20個單盲或雙盲隨機對照臨床試驗，並使用統合分析方法分析關節內注射高分子量玻尿酸對膝部骨關節炎的療效及其安全性，而且進一步利用次群體分析 (subgroup analysis) 和統合迴歸分析 (meta-regression analysis) 的統計分析技巧，探討病人特質及高分子量玻尿酸性質和療效之間的相關性。我們將臨床療效指標分成三類：活動時的疼痛指標 (pain with activities)、不活動時的疼痛指標 (pain without activities)、以及功能性指標 (functioning)，並定義了三種效力分數 (efficacy scores)，以高分子量玻尿酸組與安慰劑組之間效力分數平均值的差異來作為高分子量玻尿酸的療效計算，以發生藥物不良反應事件的相對危險性 (relative risk) 來作為高分子量玻尿酸的安全性計算。第二部分的研究，是關節內注射高分子量玻尿酸治療膝部骨關節炎之分子機轉研究。我們由抽取自15位早期膝部骨關節炎病人的膝關節液中，分離培養出纖維母細胞樣滑膜細胞，在有或沒有interleukin (IL)-1刺激的情況下，分別給予或不給予600-800 kDa高分子量玻尿酸處理，再以real-time定量polymerase chain reaction (PCR) 方法同時定量16種骨關節炎相關細胞激素與酶，包括IL-1β、IL-6、IL-8、leukemia inhibitory factor (LIF)、tumor necrosis factor (TNF)-α、TNF-α converting enzyme (TACE)、matrix metalloproteinase (MMP)-1、MMP-2、MMP-3、MMP-9、MMP-13、tissue inhibitor of metalloproteinase (TIMP)-1、TIMP-2、aggrecanase-1、aggrecanase-2、及inducible nitric oxide synthase (iNOS) 等，在各實驗組中的基因表現。此外，我們也利用抗CD44阻斷抗體 (anti-CD44 blocking antibodies) 以阻斷高分子量玻尿酸和其細胞受器CD44接合，以探討CD44在高分子量玻尿酸對骨關節炎相關細胞激素與酶的影響中所扮演的角色。
在第一部分的研究中，我們發現關節內注射高分子量玻尿酸能使膝部骨關節炎病人的疼痛指標及功能指標達到統計上顯著的改善，且很少發生藥物不良反應。然而，高分子量玻尿酸的療效估計值在不同試驗之間有顯著的異質性 (between-study heterogeneity)。次群體分析與統合迴歸分析顯示如單盲或單一中心的試驗設計等較低的方法學品質 (methodological quality) 可能會造成高分子量玻尿酸療效的高估；在臨床試驗中使用acetaminophen作為解救止痛劑 (escape analgesics) 可能會造成高分子量玻尿酸療效的低估；高分子量玻尿酸對年齡大於65歲的患者或放射線學上最嚴重 (X光上膝關節間隙完全喪失) 的骨關節炎較無療效。在第二部分的研究中，我們發現在未刺激的纖維母細胞樣滑膜細胞中，高分子量玻尿酸可以抑制IL-8及iNOS的基因表現。在以IL-1刺激的纖維母細胞樣滑膜細胞中，高分子量玻尿酸可以抑制aggrecanase-2及TNF-α的基因表現。利用抗CD44阻斷抗體以阻斷高分子量玻尿酸和其細胞受器CD44接合，可以降低高分子量玻尿酸對這些細胞激素與酶之基因表現的抑制效果。這些結果意味著藉由抑制纖維母細胞樣滑膜細胞的aggrecanase-2之基因表現，高分子量玻尿酸可能對骨關節炎有structure-modifying的效果。而藉由抑制纖維母細胞樣滑膜細胞的TNF-α、IL-8、及iNOS之基因表現，高分子量玻尿酸也可能有抗發炎的效果。高分子量玻尿酸的這些效果可能是透過和CD44之間的作用。
總括而言，我們證實了關節內注射高分子量玻尿酸能使膝部骨關節炎病人的疼痛及功能達到統計上顯著的改善，且很少發生藥物不良反應；而且，高分子量玻尿酸可以抑制早期骨關節炎病人的纖維母細胞樣滑膜細胞之骨關節炎相關細胞激素與酶的基因表現。但是仍然需要具有良好實驗設計及高方法學品質的隨機對照臨床試驗，以闡明不同高分子量玻尿酸製品在不同臨床情況和不同病人族群中對膝部骨關節炎的療效；我們也需要更進一步研究來闡明在骨關節炎的滑膜細胞上，高分子量玻尿酸抑制這些骨關節炎相關細胞激素與酶之基因表現的分子機轉。未來，我們希望將這個系統綜述和統合分析研究的結果應用在臨床上，進一步進行關節內注射高分子量玻尿酸治療膝部骨關節炎的經濟效益分析，以期有助於實證醫學臨床決策、臨床醫學治療方針的發展、和醫療政策的制定。我們也期望在活體外和活體內進一步探討在骨關節炎的滑膜細胞上，高分子量玻尿酸和其細胞受器CD44作用的訊息傳遞途徑，以及各種骨關節炎相關細胞激素與酶之間的因果關係和相互影響。

關鍵字

膝部 ； 骨關節炎 ； 玻尿酸 ； 實證醫學 ； 分子生物學

並列摘要

Background and objective: Intra-articular injection of high molecular weight hyaluronic acid (HMW-HA) has been used as viscosupplementation for knee osteoarthritis (OA). However, the actual magnitude of the therapeutic effects of intra-articular injection of HMW-HA on knee OA is still a question. The aim of the first-part study was to elucidate the therapeutic efficacy and safety of intra-articular injection of HMW-HA for knee OA by conducting a systematic review and a meta-analysis. In addition, activated synoviocytes play important roles in the progression of human OA. However, the effect of HMW-HA on synoviocytes remains undisclosed. The aim of the second-part study was to investigate the effects of HMW-HA on the gene expression of 16 OA-associated cytokines and enzymes, including interleukin (IL)-1β, IL-6, IL-8, leukemia inhibitory factor (LIF), tumor necrosis factor (TNF)-α, TNF-α converting enzyme (TACE), matrix metalloproteinase (MMP)-1, MMP-2, MMP-3, MMP-9, MMP-13, tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, aggrecanase-1, aggrecanase-2, and inducible nitric oxide synthase (iNOS), in fibroblast-like synoviocytes (FLS) from patients with early stage OA.
Methods: In the first-part study, we conducted a meta-analysis of 20 blinded randomized controlled trials which compared the therapeutic effect of intra-articular injection of HMW-HA with that of intra-articular injection of placebo on knee OA. The outcome endpoints were classified into three categories: pain with activities, pain without activities, and functioning. The outcome measures of HMW-HA efficacy were the mean differences between HMW-HA and placebo groups for the efficacy scores. The outcome measure of HMW-HA safety was relative risk for adverse events. In the second-part study, synovial fluid-derived FLS were obtained from the knees of 15 patients with early stage OA. IL-1-stimulated or unstimulated FLS were cultured with or without the treatment of 600-800 kDa HMW-HA. Moreover, blocking experiments with anti-CD44 monoclonal antibodies (mAb) were used to examine the involvement of CD44 in HMW-HA effects. We designed and validated the real-time quantitative polymerase chain reaction (Q-PCR) assays with SYBR Green dyes for simultaneous quantification of the gene expression of 16 OA-associated cytokines and enzymes.
Results: In the systematic review of the first-part study, intra-articular injection of HMW-HA can improve symptoms of knee OA. We found statistically significant improvements in pain and functional outcomes with few adverse events. However, there was significant between-study heterogeneity in the estimates of HMW-HA efficacy. Subgroup analysis and meta-regression analysis showed that lower methodological quality such as single-blind or single-center design would result in higher estimates of HMW-HA efficacy, introduction of acetaminophen as escape analgesics in trials would result in lower estimates of HMW-HA efficacy, and the patients over 65 years of age or those with the most advanced radiographic stage of OA, which is defined as complete loss of joint space, were less likely to benefit from intra-articular injection of HMW-HA. In the second-part study, we found that HMW-HA down-regulated IL-8 and iNOS gene expression in unstimulated FLS and down-regulated aggrecanase-2 and TNF-α gene expression in IL-1-stimulated FLS. CD44 blocking inhibited the down-regulatory effects of HMW-HA on the expression of these genes.
Conclusions: The results of the first-part study confirm the therapeutic efficacy and safety of intra-articular injection of HMW-HA for knee OA. Further well-designed randomized controlled trials with high methodological quality are needed to resolve the continued uncertainty about the therapeutic effects of different types of HMW-HA products on knee OA in various clinical situations and patient populations. The results of the second-part study suggest that HMW-HA may have a structure-modifying effect for OA by down-regulation of aggrecanase-2 in FLS. HMW-HA also has an anti-inflammatory effect by down-regulation of TNF-α, IL-8, and iNOS in FLS. These effects may be mediated through the interaction of CD44 and HMW-HA. These results may clarify the molecular mechanism of HMW-HA in the treatment of OA.

並列關鍵字

Knee ； osteoarthritis ； hyaluronic acid ； evidence-based medicine ； molecular biology

參考文獻

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