TW202308668A - 含有粒線體的組合物修復軟骨損傷或改善退化性關節炎的用途 - Google Patents
含有粒線體的組合物修復軟骨損傷或改善退化性關節炎的用途 Download PDFInfo
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Abstract
本發明一實施例提供包含粒線體的組合物，藉由對軟骨提供粒線體，可透過改善軟骨細胞中粒線體的功能以提升軟骨細胞的修復能力並修復軟骨細胞老化導致的軟骨損傷，進而達到改善並治療退化性關節炎的目的。
Description
本發明係關於粒線體的用途及含有粒線體的組合物，尤其係關於粒線體用於製備修復軟骨損傷或改善退化性關節炎之組合物的用途以及含有粒線體及高濃度血小板血漿的組合物。
退化性關節炎是人類老化最常見的關節疾病，主要病變的地方在於骨頭與骨頭之間的關節接觸面，也就是所謂的關節軟骨。在退化性關節炎病患的X光照片中可觀察到軟骨受損、關節表面凹凸不平、關節腔變窄以及骨刺產生。退化性關節炎主要的症狀包含疼痛、僵硬、腫脹、發炎或變形。傳統的退化性關節炎治療方式包含藥物治療、物理治療、玻尿酸注射、置換人工關節等。
然而，除了人工關節置換手術外，其他療法大都只能短暫緩解發炎與疼痛，無法有效改善或延緩病程的進展。因此，如何改善並治療退化性關節炎為目前的研究目標。
根據本發明一實施例，透過對軟骨提供粒線體以修復軟骨損傷，進而改善並治療退化性關節炎。
本發明一實施例提供一種粒線體用於製備修復軟骨損傷之組合物的用途。
本發明一實施例提供一種粒線體用於製備改善退化性關節炎之組合物的用途。
本發明一實施例提供一種組合物，包含粒線體及來自血液的生長因子。
本發明一實施例提供包含粒線體的組合物，藉由對軟骨提供粒線體，可透過改善軟骨細胞中粒線體的功能以提升軟骨細胞的修復能力並修復軟骨細胞老化導致的軟骨損傷，改善軟骨損傷或退化性關節炎所造成之疼痛現象、發炎腫脹反應或僵硬現象，進而達到改善並治療退化性關節炎的目的。
於以下實施方式中詳細敘述本發明之詳細特徵及優點，其內容足以使任何熟習相關技藝者了解本發明之技術內容並據以實施，且根據本說明書所揭露的內容、申請專利範圍及圖式，任何熟習相關技藝者可輕易理解本發明相關之目的及優點。以下實施例係進一步詳細說明本發明之觀點，但非以任何觀點限制本發明之範疇。
粒線體是細胞內進行氧化磷酸化反應及合成三磷酸腺苷（ATP）的場所，除了提供細胞正常代謝所需的能量外，還負責調控細胞內氧化壓力處理、訊號傳遞等功能。與正常的軟骨細胞相比，在損傷或老化的軟骨細胞中粒線體的功能明顯下降，具體表現在ATP產能效率下降、膜電位下降電子傳遞鏈的效率變差等。伴隨著粒線體的功能下降，各項生物反應會開始衰退，亦即是軟骨細胞開始老化，使軟骨細胞逐漸無法維持正常的運作，進而出現發炎或關節炎的症狀。因此，透過將健康的粒線體提供給軟骨細胞，可改善軟骨老化現象並提升軟骨修復能力，進一步治療退化性關節炎等老化引起的疾病。
本發明一實施例提供用於修復軟骨損傷或改善退化性關節炎的一組合物，包含粒線體。在本實施例中，粒線體的濃度可為每毫升1微克至4000微克(μg/mL)，但不限於此。在另一實施例中，粒線體的濃度可為每毫升1微克至200微克。在又一實施例中，粒線體的濃度可為每毫升1微克至100微克。在再一實施例中，粒線體的濃度可為每毫升1微克至40微克。在其他實施例中，粒線體的濃度可為每毫升15微克至40微克。在本實施例中，粒線體的有效劑量可為每1平方公分的關節腔7.9毫克至2.11克，但不限於此。在其他實施例中，粒線體的有效劑量可為每1平方公分的關節腔7.9毫克至21.1毫克。
組合物中包含的粒線體可為外源性粒線體。外源性粒線體係取自不同於施用組合物者的細胞。施用組合物者與提供外源性粒線體者較佳為同屬，更佳為同種。在其他實施例中，粒線體可為取自施用組合物者本身的細胞的自體粒線體。提供粒線體的細胞被取出後，可直接從細胞分離出粒線體或將細胞經體外培養增殖後再分離出粒線體。前述提供外源性粒線體或提供自體粒線體的細胞可為脂肪幹細胞、單核球細胞、胚胎幹細胞、間質幹細胞、造血幹細胞、CD34+幹細胞、骨髓幹細胞等具有粒線體的細胞。
本實施例之組合物可透過口服、注射等方式給予關節。透過注射的方式將組合物給與關節時，係將組合物注入到關節腔中，使組合物在關節腔中與軟骨細胞接觸而進入軟骨細胞中。
在另一實施例中，組合物更包含來自血液的生長因子。生長因子可來自高濃度血小板血漿（platelet-rich plasma，PRP）。生長因子可包含TGF-β1、PDGF-AA、PDGF-AB或PDGF-BB。於此所述之高濃度血小板血漿每毫升含有1×10
9個血小板以及多種生長因子，其中TGF-β1的濃度可為7767皮克/毫升（pg/ml）以上，PDGF-AA的濃度可為9.73皮克/毫升（pg/ml）以上，PDGF-AB的濃度可為10皮克/毫升（pg/ml）以上，PDGF-BB的濃度可為590皮克/毫升（pg/ml）以上。在此組合物中，粒線體與高濃度血小板血漿的比例可為每10微升的高濃度血小板血漿含有1微克至40微克的粒線體，即1微克：10微升至40微克：10微升，但不限於此。在其他實施例中，粒線體與高濃度血小板血漿的比例可為每10微升的高濃度血小板血漿含有15微克至40微克的粒線體，即15微克：10微升至40微克：10微升。在此組合物中，粒線體的濃度可為每毫升1000微克至4000微克（μg/mL），但不限於此。在其他實施例中，粒線體的濃度可為每毫升1500微克至4000微克。在本實施例中，粒線體的有效劑量可為每1平方公分的關節腔7.9毫克至2.11克，但不以此為限。在其他實施例中，粒線體的有效劑量可為每1平方公分的關節腔7.9毫克至21.1毫克。
在其他實施例中，組合物可更包含藥學上可接受的載體，藥學上可接受的載體包含用於任何標準醫療產品或美容產品的載體，依據組合物的形式載體可為半固體或液體。舉例而言，載體包含但不限於玻尿酸、明膠、乳化劑、水、生理食鹽水、緩衝鹽水或乙醇等不影響粒線體活性之物質。
以下說明如何製備本發明實施例之組合物。
［粒線體萃取］
本發明實施例所使用之粒線體取自人類脂肪幹細胞（adipose-derived stem cell，ADSC）。幹細胞培養液包含Keratinocyte SFM 1X溶液（Gibco）、bovine pituitary extract（BPE，Gibco）、重量百分濃度10%之胎牛血清（HyClone）。在培養皿中將人類脂肪幹細胞培養至細胞數為1.5×10
8個細胞，以杜氏磷酸鹽緩衝液（DPBS）沖洗細胞。接著，移除杜氏磷酸鹽緩衝液後，加入細胞剝離用之胰蛋白酶（Trypsin），在37℃下反應3分鐘後，再加入幹細胞培養液以終止反應。接著，將細胞沖洗下來後打散，以600 g離心10分鐘，移除上清液。接著，於細胞中加入80毫升之IBC-1緩衝液（225 mM甘露醇、75mM蔗糖、0.1 mM EDTA、30 mM Tris-HCl pH 7.4），在均質器中於冰上研磨15次。接著，以1000 g離心15分鐘，將上清液收集至另一離心管，再以9000 g離心10分鐘，移除上清液。所獲得之沉澱物即為粒線體。在粒線體沉澱物中加入1.5毫升之IBC-2緩衝液（225 mM甘露醇、75mM蔗糖、30 mM Tris-HCl pH 7.4）及蛋白質分解酶抑制劑，並置於4℃下保存。
〔實驗一，修復軟骨細胞損傷〕
在本實驗中，使用75 μM之tBHP誘導人類軟骨肉瘤細胞（SW-1353）出現損傷。透過阿爾瑪藍檢測試劑來評估包含粒線體的組合物對人類軟骨肉瘤細胞之損傷的修復效果，並以細胞存活率來表示。
人類軟骨肉瘤細胞常被用於探討軟骨損傷的機轉，係常見的軟骨細胞評估模式。本實驗使用人類軟骨肉瘤細胞培養液在0%二氧化碳的環境下進行人類軟骨肉瘤細胞的培養。人類軟骨肉瘤細胞培養液包含90% Leibovitz’s L-15培養基、2 mM L-麩醯胺酸及重量百分濃度10%之胎牛血清。
過氧化三級丁醇（tert-butyl hydroperoxide，tBHP）為一種有機過氧化物，常用作為誘導細胞氧化壓力損傷、老化與細胞凋亡的物質。本實驗使用tBHP作為誘導人類軟骨肉瘤細胞老化或損傷之物質。
阿爾瑪藍（Alamar blue）係用於檢測細胞活力的檢測試劑。檢測套組內的刃天青（resazurin）是一種氧化還原指示劑，其為無毒、可穿透細胞膜且低螢光性之深藍色染料。當刃天青進入健康的細胞中，會因活細胞體內的還原環境而被還原成粉紅色且具高螢光性的試鹵靈（resorufin）。可藉由量測試鹵靈的光吸收值或螢光值來評估細胞的活力。試鹵靈的光吸收值或螢光值愈高，表示細胞活力愈高。細胞活力愈高表示細胞愈健康、增生能力愈強。細胞增生能力愈強，表示細胞量愈多。因此，本實驗使用阿爾瑪藍作為評估細胞存活率及細胞增生率的指標。
以下說明實驗流程。首先，取培養代數為4至10代間的人類軟骨肉瘤細胞進行實驗。將人類軟骨肉瘤細胞培養至體積為培養皿的八分滿時，移除培養皿中的培養液並使用磷酸鹽緩衝液（phosphate buffered saline，PBS）潤洗細胞。接著，在培養皿中加入0.25%之胰蛋白酶並使胰蛋白酶在37℃下反應5分鐘，再加入人類軟骨肉瘤細胞培養液以終止胰蛋白酶的反應。接者，將培養皿中的人類軟骨肉瘤細胞及人類軟骨肉瘤細胞培養液移至離心管中，以每分鐘轉速1000（Revolutions Per Minute，rpm）離心5分鐘後，移除上清液。接著，再加入新的人類軟骨肉瘤細胞培養液至離心管中，進行細胞計數。接著，將人類軟骨肉瘤細胞以每孔2×10
4個細胞的密度於盛有人類軟骨肉瘤細胞培養液的24孔盤中培養16小時。接著，於24孔盤中加入tBHP，使人類軟骨肉瘤細胞在含有濃度為75 μM的tBHP的培養液中培養4小時。接著，將含有tBHP的人類軟骨肉瘤細胞培養液移除，加入含有不同濃度粒線體的組合物的培養液，使人類軟骨肉瘤細胞在含有不同粒線體濃度的組合物的培養液中培養20小時。孔盤中粒線體的濃度為1 μg/mL、15 μg/mL或40 μg/mL。培養完成後，使用磷酸鹽緩衝液清洗人類軟骨肉瘤細胞，並將培養液更換為含有阿爾瑪藍的培養液，培養3小時。培養完成後，以OD530/595的波長量測螢光，計算人類軟骨肉瘤細胞的細胞存活率。
人類軟骨肉瘤細胞的細胞存活率的實驗結果如表1及圖1所示。圖1為使用75 μM之tBHP處理再以實施例之組合物修復的人類軟骨肉瘤細胞的細胞存活率之圖表。控制組為未經tBHP誘導損傷的人類軟骨肉瘤細胞。對照組為經tBHP誘導損傷但未施以實施例之組合物的人類軟骨肉瘤細胞。試驗組為經tBHP誘導損傷且分別施以包含1 μg/mL、15 μg/mL或40 μg/mL之粒線體的組合物處理的人類軟骨肉瘤細胞。縱軸為相對於控制組的細胞數，以細胞存活率（%）表示。實驗結果顯示試驗組的細胞存活率皆大於對照組的細胞存活率，且細胞存活率隨著粒線體濃度增加而提升，表示實施例之含有粒線體的組合物確實可改善tBHP對軟骨細胞造成的損傷及死亡。
〔表1〕
| 組別 | 粒線體 （μg/mL） | tBHP （μM） | 細胞存活率（%） | |||
| 控制組 | - | - | 100 | |||
| 對照組 | - | 75 | 69.9±5.5 | |||
| 試驗組 | 實施例一 | 1 | 71.2±4.2 | |||
| 實施例二 | 15 | 73.1±3.1 | ||||
| 實施例三 | 40 | 76.3±6.1 | ||||
〔實驗二，修復軟骨細胞老化〕
在本實驗中，使用50 μM之tBHP誘導人類軟骨肉瘤細胞老化。透過SA-β-gal套組來評估包含粒線體的組合物對tBHP所造成的老化的修復效果，並以老化程度來表示。
在老化的細胞中，老化相關-β-半乳糖苷酶（Senescence-associated beta-galactosidase，SA-β-gal）會被過度表達，因此SA-β-gal可作為細胞衰老的標記之一。本實驗使用SA-β-gal套組（Senescence β-Galactosidase Staining Kit #9860，Cell Signaling technology）來評估人類軟骨肉瘤細胞的老化狀態。
本實驗的實驗流程與上述實驗一的實驗流程大致相同。差異在於將人類軟骨肉瘤細胞以每孔4×10
4個細胞的密度於盛有人類軟骨肉瘤細胞培養液的12孔盤中培養16小時。接著，於12孔盤中加入tBHP，使人類軟骨肉瘤細胞在含有濃度為50 μM的tBHP的培養液中培養4小時。接著，將含有tBHP的人類軟骨肉瘤細胞培養液移除，加入含有不同濃度粒線體的組合物的培養液，使人類軟骨肉瘤細胞在含有不同粒線體濃度的組合物的培養液中培養20小時。孔盤中粒線體的濃度為1 μg/mL、15 μg/mL或40 μg/mL。培養完成後，使用磷酸鹽緩衝液清洗人類軟骨肉瘤細胞，並使用SA-β-gal套組進行細胞老化的評估。
人類軟骨肉瘤細胞的老化程度的實驗結果如表2、圖2及圖3所示。圖2為使用50 μM之tBHP處理再以實施例之組合物修復的人類軟骨肉瘤細胞的老化程度之圖表。圖3為使用50 μM之tBHP處理再以實施例之組合物修復的人類軟骨肉瘤細胞的染色照片。控制組為未經tBHP誘導損傷的人類軟骨肉瘤細胞。對照組為經tBHP誘導損傷但未施以實施例之組合物的人類軟骨肉瘤細胞。試驗組為經tBHP誘導損傷且分別施以包含1 μg/mL、15 μg/mL或40 μg/mL之粒線體的組合物處理的人類軟骨肉瘤細胞。縱軸為老化程度（%），老化程度為老化的細胞數占所有的細胞數的百分比。###表示相較於對照組具有顯著差異（P＜0.01）。實驗結果顯示試驗組的老化程度皆小於對照組的老化程度，且細胞老化程度隨著粒線體濃度增加而降低，表示實施例之含有粒線體的組合物確實可改善tBHP對軟骨細胞造成的老化。
〔表2〕
| 組別 | 粒線體 （μg/mL） | tBHP （μM） | 老化程度（%） | |||
| 控制組 | - | - | 16.2±2.3 | |||
| 對照組 | - | 50 | 38.2±3.8 | |||
| 試驗組 | 實施例一 | 1 | 35.5±2.6 | |||
| 實施例二 | 15 | 26.3±2.5 | ||||
| 實施例三 | 40 | 25.6±2.4 | ||||
〔實驗三，改善軟骨細胞的粒線體功能〕
在本實驗中，使用50 μM之tBHP誘導人類軟骨肉瘤細胞老化，並透過粒線體的膜電位來評估人類軟骨肉瘤細胞中粒線體的功能。
粒線體功能分析係透過測量粒線體的膜電位改變來判斷粒線體功能的好壞。TMRE（tetramethylrhodamine ethyl ester）是一種帶正電的螢光染劑，會聚集在具有活性的粒線體上，故可使用TMRE來標記健康的粒線體。當粒線體活性較低或呈現去極化現象時粒線體的膜電位會降低，造成TMRE無法保留於粒線體上。FCCP（Carbonylcyanide 4-(trifluoromethoxy) phenylhydrazone）為可跨越粒線體內膜的離子載體，會與質子結合來破壞ATP的合成造成膜電位改變。FCCP可用來消除粒線體的膜電位，因此常被用於粒線體去活性或去極化的對照組。藉由TMRE與FCCP染色處理，分析螢光強度以得知粒線體的膜電位變化，以此作為判斷粒線體功能的依據。透過流式細胞儀偵測以TMRE處理後的上清液，可根據TMRE標記分析有功能之粒線體佔上清液中所有粒子的比例。
本實驗的實驗流程與上述實驗一的實驗流程大致相同。差異在於將人類軟骨肉瘤細胞以每孔1×10
5個細胞的密度於盛有人類軟骨肉瘤細胞培養液的6孔盤中培養16小時。接著，於6孔盤中加入tBHP，使人類軟骨肉瘤細胞在含有濃度為50 μM的tBHP的培養液中培養4小時。接著，將含有tBHP的人類軟骨肉瘤細胞培養液移除，加入含有不同濃度粒線體的組合物的培養液，使人類軟骨肉瘤細胞在含有不同粒線體濃度的組合物的培養液中培養20小時。孔盤中粒線體的濃度為1 μg/mL、15 μg/mL或40 μg/mL。培養完成後，使用磷酸鹽緩衝液清洗人類軟骨肉瘤細胞，並使用TMRE與FCCP進行染色處理，以流式細胞儀進行偵測。
人類軟骨肉瘤細胞的膜電位分析的實驗結果如表3及圖4所示。圖4為使用50 μM之tBHP處理再以實施例之組合物修復的人類軟骨肉瘤細胞的粒線體膜電位分析圖。控制組為未經tBHP誘導損傷的人類軟骨肉瘤細胞。對照組為經tBHP誘導損傷但未施以實施例之組合物的人類軟骨肉瘤細胞。試驗組為經tBHP誘導損傷且分別施以包含1 μg/mL、15 μg/mL或40 μg/mL之粒線體的組合物處理的人類軟骨肉瘤細胞。實驗結果顯示，相較於對照組，試驗組之具有功能的粒線體的數量較多，且恢復程度隨著組合物中粒線體濃度增加而更接近控制組，表示實施例之含有粒線體的組合物確實可改善軟骨細胞的粒線體功能，粒線體功能提升進一步表示細胞具有較佳的抗老化及修復能力。
〔表3〕
| 組別 | 粒線體 （μg/mL） | tBHP （μM） | 膜電位分析（%） | |||
| 控制組 | - | - | 62.27 | |||
| 對照組 | - | 50 | 56.66 | |||
| 試驗組 | 實施例一 | 1 | 60.01 | |||
| 實施例二 | 15 | 60.23 | ||||
| 實施例三 | 40 | 61.65 | ||||
〔實驗四，改善退化性關節炎〕
在本實驗中，使用碘乙酸單鈉（monosodium iodoacetate，MIA）誘導小鼠產生退化性關節炎，並透過旋轉輪測試（rotarod analysis）評估包含粒線體的組合物以及包含粒線體及高濃度血小板血漿的組合物對退化性關節炎小鼠的運動能力的修復效果。本實驗使用八周齡、體重約18至22公克的C57BL/6雄性小鼠作為實驗對象。
碘乙酸單鈉（monosodium iodoacetate，MIA）為3-磷酸甘油醛脫氫酶（Glyceraldehyde 3-phosphate dehydrogenase）的活性抑制劑，可誘導出與人類骨關節炎（Osteoarthritis，OA）症狀極為相似的軟骨損傷，使軟骨失去蛋白聚醣基質並誘導軟骨細胞死亡。本實驗使用MIA作為誘導退化性關節炎之物質。
旋轉輪測試（rotarod analysis）為評估小鼠的平衡與協調能力的分析方式。在測試前一周，將小鼠放至旋轉輪上訓練，使小鼠能在旋轉輪上的時間超過三分鐘，進行實驗處理後，再藉由小鼠在旋轉輪上的時間評估小鼠的運動能力。
高濃度血小板血漿（platelet-rich plasma，PRP）為全血經離心後移除紅血球成為富含血小板的血漿蛋白濃縮液。高濃度血小板血漿含有豐富的生長因子，因此被認為對軟骨、肌腱、韌帶等的修復有所幫助。本實驗之高濃度血小板血漿來自Sprague-Dawley（SD）大鼠。首先以3%戊巴比妥鈉（pentobarbital sodium）麻醉大鼠，劑量為每公斤30毫克。接著，以含有檸檬酸右旋糖（acid citrate dextrose）抗凝劑的針筒對大鼠抽取全血約7至8毫升，其中血小板的濃度約為每毫升0.5×10
9至1.5x10
9個。將血液轉移至15毫升離心管在室溫下以150 g進行第一次離心10分鐘，將血小板及血漿收集至另一15毫升離心管在室溫下以1500 g進行第二次離心10分鐘，第二次離心後移除四分之三的上清液，剩下的四分之一即為本實驗所使用之高濃度血小板血漿。於此所述之高濃度血小板血漿每毫升含有1×10
9個血小板以及多種生長因子，所包含的生長因子的種類及濃度如下：TGF-β1 (≧7767 pg/ml)、PDGF-AA (≧9.73 pg/ml)、PDGF-AB (≧10pg/ml)及PDGF-BB (≧590 pg/ml)。
以下說明實驗流程。首先，將小鼠分籠並給予數天適應期，避免小鼠因環境不適造成緊張焦慮而影響實驗結果。注射MIA前先對小鼠進行旋轉輪測試，作為第0天的數據並作為後續比較的基準。進行手術前10分鐘給予小鼠0.25毫升之4%水合氯醛（cholra hydrate）。在手術途中，給予異氟醚（isoflurane）以維持麻醉避免小鼠甦醒。將0.1毫克MIA溶解於10微升之生理食鹽水中，使用30 G（gauge）之針頭注入小鼠的關節腔。在注射MIA後的第7天，以同樣的注射方式將實施例之組合物注入小鼠的關節腔，並在第7、14天進行旋轉輪測試，每次測試最長為1200秒鐘，轉速為20 rpm。
小鼠的運動能力的實驗結果如表4所示。對照組為經MIA誘導退化性關節炎但未施以實施例之組合物的小鼠。PRP組為經MIA誘導退化性關節炎且施以高濃度血小板血漿的小鼠。試驗組為經MIA誘導退化性關節炎且分別施以包含不同濃度之粒線體及/或10微升之高濃度血小板血漿的組合物處理的小鼠。實驗結果顯示，相較於第0天尚未受退化性關節炎影響，在第7天時各組別的小鼠的運動能力下降至30%至40%。在第14天時，對照組僅恢復至40%，而PRP組及試驗組的小鼠的運動能力皆恢復至50%以上。此外，相較於PRP組（10微升之高濃度血小板血漿）及實施例四（15 μg之粒線體），施以實施例六之組合物（15 μg之粒線體及10 μL之高濃度血小板血漿）的小鼠的運動能力明顯提升。小鼠運動能力的恢復表示軟骨損傷或退化性關節炎所造成之疼痛現象、發炎腫脹反應或僵硬現象受到舒緩及改善。由上述結果可知含有粒線體的組合物可改善患有退化性關節炎的動物的運動能力，且隨著粒線體濃度提高可具有較佳的效果。此外，含有粒線體及高濃度血小板血漿的組合物相較於僅含有粒線體或僅含有高濃度血小板血漿的組合物可具有更優異的改善效果。
文獻記載小鼠關節腔中軟骨組織的面積約為1896.91平方微米。本實驗證實對小鼠的關節腔給予15微克至40微克的粒線體，可有效改善軟骨損傷或退化性關節炎所造成之疼痛現象、發炎腫脹反應或僵硬現象，因此，經換算，粒線體的有效劑量可為每1平方公分的軟骨組織7.9毫克至2.11克，較佳為每1平方公分的軟骨組織7.9毫克至21.1毫克。
〔表4〕
| 組別 | 粒線體 （μg） | PRP （μL） | 第0天 | 第7天 | 第14天 | |
| 對照組 | - | - | 100% | 36% | 40% | |
| PRP組 | - | 10 | 100% | 33% | 58% | |
| 試驗組 | 實施例四 | 15 | - | 100% | 30% | 55% |
| 實施例五 | 40 | - | 100% | 32% | 80% | |
| 實施例六 | 15 | 10 | 100% | 32% | 84% |
本發明一實施例提供包含粒線體的組合物，藉由對軟骨提供粒線體，可透過改善軟骨細胞中粒線體的功能以提升軟骨細胞的修復能力並修復軟骨細胞老化導致的軟骨損傷，改善軟骨損傷或退化性關節炎所造成之疼痛現象、發炎腫脹反應或僵硬現象，進而達到改善並治療退化性關節炎的目的。
雖然本發明以前述之實施例揭露如上，然其並非用以限定本發明。在不脫離本發明之精神和範圍內，所為之更動與潤飾，均屬本發明之專利保護範圍。關於本發明所界定之保護範圍請參考所附之申請專利範圍。
無
圖1為使用75 μM之tBHP處理再以實施例之組合物修復的人類軟骨肉瘤細胞的細胞存活率之圖表。
圖2為使用50 μM之tBHP處理再以實施例之組合物修復的人類軟骨肉瘤細胞的老化程度之圖表。
圖3為使用50 μM之tBHP處理再以實施例之組合物修復的人類軟骨肉瘤細胞的染色照片。
圖4為使用50 μM之tBHP處理再以實施例之組合物修復的人類軟骨肉瘤細胞的粒線體膜電位分析圖。
Claims (10)
- 一種粒線體用於製備修復軟骨損傷之組合物的用途。
- 如請求項1所述之用途，其中修復軟骨損傷為修復軟骨細胞老化導致的軟骨損傷。
- 如請求項1所述之用途，其中修復軟骨損傷為透過改善軟骨細胞中粒線體的功能以提升軟骨細胞的修復能力。
- 如請求項1所述之用途，其中該組合物改善軟骨損傷所造成之疼痛現象或發炎腫脹反應。
- 一種粒線體用於製備改善退化性關節炎之組合物的用途。
- 如請求項5所述之用途，其中改善退化性關節炎為修復因關節處的軟骨細胞老化導致的軟骨損傷。
- 如請求項5所述之用途，其中改善退化性關節炎為透過改善關節處的軟骨細胞中粒線體的功能以提升關節處的軟骨細胞的修復能力。
- 如請求項5所述之用途，其中該組合物改善退化性關節炎所造成之疼痛現象或發炎腫脹反應。
- 如請求項1或5所述之用途，其中該組合物中的粒線體的濃度為每毫升1微克至4000微克（μg/mL）。
- 如請求項1或5所述之用途，其中該組合物更包含高濃度血小板血漿。
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