TWI694828B - 利用神經幹細胞與IL12p40以增強神經再生的方法 - Google Patents
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Abstract
本發明係提供利用神經幹細胞或IL12p40之至少一組成分以促進神經再生之組成物及方法。該組成物係包括神經幹細胞及神經滋養因子，其係以IL12p40作為至少一次單元所構成。該促進神經再生之方法係包括提供一種神經再生組成物到一個體，其中該組成物係包括至少含有IL12p40作為一個次單元的神經滋養因子。於該方法中，該組成物可進一步包括神經幹細胞。
Description
本案係關於利用神經幹細胞及IL12p40促進神經再生之組成物及方法。
嚴重的周邊神經傷害會造成運動及感知神經活動減少，以及神經纖維及周圍組織退化。受損之周邊神經再生係一複雜的過程，以瓦勒氏變性(Wallerian degeneration，WD)為最基本反應，而許旺細胞(Schwann cells)則扮演一重要角色(Ren Z.et al.,Reviews in the Neurosciences 2012,23：135-143)。瓦勒氏變性可以創造出微環境以供存活神經元再生，且對功能復原(functional recovery)有利(Navarro X.et al.,Progress in Neurobiology2007,82：163-201.)。調控瓦勒氏變性包含有許旺細胞之存在、神經滋養因子(neurotrophic factor)之分泌、以及作為神經細胞之支架之特定胞外基質之分泌(Gaudet A.D.et al.,Journal of Neuroinflammation 2011,8：110；Kehoe S.et al.,Injury 2012,43：553-572.)。
神經導管係提供機械性支撐及引導該受損神經殘餘處之間的軸突萌芽。導管可用以保留由該受損細胞分泌或補充之神經滋養因子(Kehoe S.et al,Injury 2012,43：553-572.)並避免受傷處之纖維組織向內生長。最近的研究顯示，植入含有神經幹細胞(NSC)之導管可促進受損之
周邊神經之再生(Zhang H.et al.,Journal of Translational Medicine2008,6：67；Shi Y.et al.,Acta Oto-Laryngologica 2009,129：906-914.)。
可依賴下列方式促進神經之再生：植入的神經幹細胞分化成許旺細胞、或本身分泌神經滋養因子、或產生能增加來自本身環境(milieu)的神經滋養因子之微環境、及有助於髓鞘形成作用之能力(Ren Z.et al.,Reviews in the Neurosciences 2012,23：135-143)。然而，尚不清楚涉及此過程之細胞激素或生長因子之特性。該植入之神經幹細胞分化成許旺細胞並參與新軸突再生之分子機轉亦未良好地建構。
於本案中，目標是鑑定出參與神經幹細胞調控之神經再生及功能復原之因子。發明人利用蛋白質抗體陣列，檢索在小鼠坐骨神經傷害修復模式中使用含有或不含有神經幹細胞之導管中蛋白質濃度之差異。IL12p80(為IL12p40具生物活性的同型二聚體形式)(Heinzel F.P.et al.,Journal of Immunology 1997,158：4381-4388)的濃度，在含有神經幹細胞導管為不含神經幹細胞導管之將近兩倍高。於小鼠坐骨神經傷害修復模式中，植入含有神經幹細胞及IL12p80之神經導管可改善運動功能。
提供IL12p80可以進一步促進神經再生，可由該再生神經直徑增加而獲得證實。與僅有導管組相較，該再生神經中段之厚度增加達4.5倍，並可改善神經傳導。又，IL12p80可誘導體外小鼠神經幹細胞之神經膠質細胞分化，其訊號傳導係經由轉錄活化子3(Stat3)之磷酸化途徑。如Kettenmann及Verkhratsky所報告，該神經膠質細胞包括星狀細胞、寡樹突細胞、及許旺細胞(Kettenmann H,Verkhratsky A,Fortschritte der Neurologie-Psychiatrie 2011,79：588-597.)。
本申請案主張美國臨時申請案之優先權，美國臨時申請案案號為62/037,612，申請日為西元2014年8月15日，該案之揭露內容以參考文獻之方式併入此處。
本發明係提供一種神經再生之組成物，包括神經幹細胞及神經滋養因子，其IL12p40為構成神經滋養因子的一個次單元。
本發明係提供一種神經再生之組成物，包括神經幹細胞、神經滋養因子、及神經再生促進成分(nerve regeneration enhancing element)，該神經滋養因子係由包含至少一個IL12p40次單元所構成。
本發明進一步提供一種神經再生之組成物，係包括神經幹細胞、神經滋養因子，其係由包含至少一個IL12p40次單元所構成、及神經導管，其帶有該神經幹細胞及該神經滋養因子之至少一者。
本發明係提供一種再生神經之方法，包括提供一種神經再生組成物予一對象，該神經再生組成物係包括包含IL12p40為至少一個次單元之神經滋養因子。
本發明進一步提供一種再生神經之方法，包括提供一種神經再生組成物予一對象，該神經再生組成物包括神經幹細胞及包含IL12p40為至少一個次單元之神經滋養因子。
第1圖是顯示在小鼠中植入該含有神經幹細胞(NSC)之神經導管可改善小鼠之坐骨神經傷害之功能復原，且較僅有導管組為佳。(A)SFI係用於評估自第一週至第八週每週之功能復原。統計分析顯示，導管+NSC
組較僅導管組具有明顯較佳之復原。(B)滾輪測試係於手術後第四及第八週進行。與對照組與導管+NSC組相較，僅導管組在滾輪上具有明顯較差之平衡與步態。導管+NSC組具有較好的行動復原且與對照組無差別。所有數據均以平均值±與平均值之標準差(SEM)(假性對照組為n=3、僅導管組為n=8、導管+NSC組為n=8、及對照組於第四週為n=8且第八週為n=8)。統計顯著性顯示為*p<0.05及**p<0.01。
第2圖係顯示來自植入之含有或不含神經幹細胞(NSC)之導管之細胞萃取物之抗體陣列之表及圖。存在於該細胞萃取物之IL12p40之訊號(以紅色虛線框指示)，導管+NSC組係為僅導管組之1.89倍高(僅導管組與導管+NSC組均為n=4)。該等實驗係重複三次，且導管+NSC組之IL12p40濃度係大幅高於僅導管組。
第3圖係顯示，於含有或不含IL12p80(mIL12)之神經幹細胞(NSC)移植中，利用SFI及滾輪測試進行之功能評估之比較性分析。(A)於手術後第八週，與僅導管組相較，導管+NSC組及導管+NSC+mIL12組之小鼠係顯示明顯較高的SFI分數。(B)於滾輪測試中，與僅導管組相較，導管+NSC組及導管+NSC+mIL12組之小鼠係展現較佳的在滾輪上之平衡與步態之能力。該等數據指出，在SFI及滾輪分析兩者之功能評估上，與不含IL-12p80之組相較(*p<0.05)，該導管+NSC+mIL12組顯示了統計學上較顯著之結果(**p<0.01)。數據係以平均值±SEM表示(於SFI中，僅導管組為n=3、導管+NSC組為n=3、及導管+NSC+mIL12組為n=4。於滾輪測試中，僅導管組為n=7、導管+NSC組為n=5、及導管+NSC+mIL12組為n=8)。
第4圖係顯示再生之坐骨神經區段之H&E染色及免疫組織化學。(A-C)再生之新軸突之區段係以蘇木精及伊紅染色，而“P”與“D”意指該殘餘神經兩端(“P”係距導管近側端1.0mm，而“D”為距“P”之遠側3.0mm)。與導管+NSC組(B)及導管+NSC+mIL12組(C)相較，僅導管組(A)顯示較低之新生軸突之接合。又，發明人以抗-神經絲200之抗體(NF200，神經纖維之標記)及抗蛋白質零之抗體(PZO，髓鞘化之許旺細胞之標記)之免疫組織化學染色驗證再生神經之特徵。染色結果顯示，僅導管組(D)之軸突再生失敗。發明人可觀察到，在導管+NSC(E)中，髓鞘化之許旺細胞(PZO陽性細胞)伴隨神經纖維(NF200陽性細胞)存在於導管之中間區域，且於導管+NSC+mIL12組(F)存在更多。D、E及F之相對位置係以虛線方框分別表示於A、B及C中。不同位置之再生神經直徑之定量結果顯示於(G)，其中，近側位置意指離該導管之近側端之1.0mm，遠側位置意指離該近側位置之3.0mm，而中間位置意指該近側位置與該遠側位置之間之中心(僅導管組為n=3、導管+NSC組為n=4、導管+NSC+mIL12組為n=3)。(H)顯示不同處理之組別之再生作用簡圖。(A)、(B)及(C)圖之比例尺為1mm，(D)、(E)及(F)圖之比例尺為100μm。統計顯著性顯示為*p<0.05及**p<0.01。
第5圖係顯示以複合肌肉動作電位(CMAP)測試之坐骨神經傷害復原。係測量傷腿與對側之正常腿之CMAPs。CMAP之復原係以傷腿除以正常腿進行計算。不同組之CMAP之復原係顯示於圖中(僅導管組為n=4、導管+NSC組為n=4、導管+NSC+mIL12組為n=6、及假性對照組為n=3)。
第6圖係顯示免疫細胞化學法之結果，顯示IL12p80可取代CNTF+T3以誘導小鼠神經幹細胞之寡樹突細胞分化作用。(A)以CNTF+T3或IL12p80將小鼠神經幹細胞誘導分化為寡樹突細胞(Y軸所列)。將分化的細胞以下列進行染色，寡樹突細胞標記：抗-Galc抗體及抗-OSP抗體、或髓鞘化之許旺細胞標記：抗-PZO抗體(X軸所列)。標記陽性之細胞之倍數係經定量及摘要於(B)。各圖之比例尺為100μm。統計顯著性顯示為*p<0.05及**p<0.01。
第7圖係顯示西方墨點法之結果，顯示IL12p80可將小鼠神經幹細胞分化為寡樹突細胞及許旺細胞。以CNTF+T3或IL12p80將小鼠神經幹細胞誘導分化為寡樹突細胞。以不同細胞激素處理條件命名分化組別。(A)細胞分化作用係以西方墨點法及種系專一性抗體拮抗星狀細胞標記GFAP、寡樹突細胞標記OSP、及髓鞘化之許旺細胞標記PZO進行鑑定。(B)定量化結果。統計顯著性顯示為*p<0.05及**p<0.01。
第8圖係顯示IL12p80於神經幹細胞中誘導Stat3磷酸化。以西方墨點分析測定於對照培養基(對照)或於包含人類IL12p80(hIL12)或小鼠IL12p70(mIL12)之分化培養基中，於不同時間點(球體、0min、15min、30min、1h、2h、4h、及8h)下，Stat3於Y705及S727位置之磷酸化程度(pStat3)。結果顯示，當與IL12p80培養時，pStat3之濃度於係於15分鐘達到高峰。以Stat3總量及α-微管蛋白作為對照組。
於本發明中，一種神經再生之組成物係包括神經幹細胞(NSC)及神經滋養因子，該神經滋養因子是由至少包含一個IL12p40次單
元所構成。於一些實施例中，IL12p40具有如NCBI編錄號碼(NCBI Accession No.)：NM_008352(小鼠)或1F42_A(人類)所示之蛋白質序列。
該神經滋養因子可為IL12p40之單體(monomer)或同型二聚體(homodimer)或異型二聚體(heterodimer)，其中至少一次單元為IL12p40。於實施例中，該神經滋養因子可為IL12p40之異型二聚體、IL12p40之同型二聚體、IL12p40之三聚體、IL12p40之四聚體、或前述任意之組合。於一實施例中，該同型二聚體可為IL12p80。於實施例中，該異型二聚體可為IL12p70。
於本發明中，該用於神經再生之組成物之神經幹細胞之特徵在於，為CD133及GFAP為陽性表現之腦細胞、或為F1B-GFP+細胞。
本發明係提供一組成物，其進一步包括神經再生促進成分。該神經再生促進成分係包括細胞、生長因子、及其組合。於實施例中，該神經再生促進成分可為許旺細胞(Schwann cells)、間葉幹細胞(MSCs)、脂肪源幹細胞(ADSCs)、羊水幹細胞(AFSCs)、經誘導之神經元(iNs)、經誘導之神經幹細胞(iNSCs)、或源自於經誘導之多能幹細胞(iPSCs)之細胞。
於一些實施例中，該神經再生促進成分可為纖維母細胞生長因子1(FGF1)、纖維母細胞生長因子2(FGF2)、血小板衍生生長因子(PDGF)、腦衍生神經滋養因子(BDNF)、神經膠細胞衍生神經滋養因子(GDNF)、神經生長因子(NGF)、及前述任意之組合。
於一實施例中，該組成物可進一步包括神經導管，其帶有該神經幹細胞及該神經滋養因子之至少一者。於一些實施例中，該神經導管
進一步帶有該神經再生促進成分。
本發明亦提供一種再生神經之方法，係包括提供一種神經再生組成物予一對象，該神經再生組成物係包括神經幹細胞及至少包含一個IL12p40次單元的神經滋養因子。於一些實施例中，該神經再生組成物可進一步包括神經幹細胞。於實施例中，該神經再生組成物係被包含於一神經導管中。於實施例中，該神經導管係植入於標的神經或該標的神經周圍之組織中。
該神經滋養因子可為IL12p40之單體、IL12p40之異型二聚體、IL12p40之同型二聚體、IL12p40之三聚體、IL12p40之四聚體、或前述任意之組合。於一些實施例中，該神經滋養因子為IL12p80。
該對象為哺乳類。於一實施例中，該哺乳類可為小鼠或人類。於本發明中，該組成物係以注射、植入、經皮途徑、及/或口服之方式提供。
實施例
材料與方法
IL12p40之序列
IL12p40之蛋白質序列係如NCBI編錄號碼：NM_008352(小鼠)或1F42_A(Human)所示，該等資料可由美國國家生物技術訊息中心之網站(http：//www.ncbi.nlm.nih.gov/)獲得。
細胞培養及神經幹細胞分離
小鼠神經幹細胞分離與細胞培養係依據兩篇參考文獻(Hsu Y.C.et al.,Developmental Dynamics2009,238：302-314；Lee D.C.et al.,
Molecular and Cellular Neurosciences2009,41：348-363；此兩篇以參考文獻之方式將全文併入本案)所述之方式進行。
將KT98/F1B-GFP細胞培養於含有10%胎牛血清(FBS)、1%青黴素/鏈黴素及500μg/ml G418(Merck,USA)之經杜貝卡氏修飾依格氏培養基(DMEM)/F12(1：1)。對於神經幹細胞分離，將GFP-陽性之KT98/F1B-GFP(KT98/F1B-GFP+)細胞以FACSAria細胞分選儀(BD Bioscience)進行區分，並於神經球形成培養基(DMEM/F12，包含1X B27(Gibco)、20ng/ml EGF(PeproTech Inc.)、20ng/ml FGF2(PeproTech Inc.)、2μg/ml肝素(Sigma)及500μg/ml G418)中培養7天，其係誘導KT98/F1B-GFP+神經球形成。接著，將KT98/F1B-GFP+神經球衍生之單一細胞用於後續動物實驗及細胞分化試驗中。所有細胞係培養於37℃及5% CO2下。
動物外科手術：坐骨神經傷害及導管植入
於本發明中，所有動物實驗流程均遵照倫理準則，並經由國家衛生研究院之實驗動物照護及使用委員會(IACUC)(計畫編號NHRI-IACUC-101067A)核准。FVB小鼠(8-10週大)係用於動物實驗，飼養於國家衛生研究院(NHRI)動物中心。手術前，將小鼠以5%異氟烷(isoflurane，鹵碳)以空氣吸入麻醉，於手術期間以2%異氟烷空氣吸入維持麻醉。在坐骨神經傷害手術中，以顯微手術剪切斷3mm之小鼠坐骨神經段。對於該手術植入之神經導管，係使用聚(L-乳酸)(PLA)導管，如先前技術所述(Hsu S.H.et al.,Artif Organs2009,33：26-35，本篇以參考文獻之方式將全文併入本案)。
將該5mm導管(包含或不包含神經幹細胞及/或小鼠IL12p80)植入該坐骨神經傷害處。使用6-0尼龍顯微縫線將該坐骨神經傷害處之近側及末端神經以1mm餘留神經錨接至該導管中。未進行坐骨神經傷害之小鼠則定義為假性對照組(Sham control group)(n=3)。
外科手術植入組包含僅導管組(n=8)、導管+NSC組(n=8)、及導管+NSC+mIL12組(n=8)。於僅導管組中，導管係以5μl基質膠(matrigel)(BD Bioscience)/磷酸鹽緩衝食鹽水(PBS)之混合物(1：1)填充。於導管+NSC組(n=8)中，導管係以5μl基質膠/PBS之混合物(1：1)及1×106神經幹細胞填充。於導管+NSC+mIL12組(n=8)中，導管係以5μl基質膠/PBS之混合物(1：1)及1×106神經幹細胞及100ng之小鼠IL12p80(BioLegend)填充。
功能性評估：步態分析與滾輪測試
利用踏車/踏步掃瞄系統(CleverSys)進行步態分析，並以手術後每週之坐骨功能指數(SFI)表示。SFI公式如下所示：
SFI計算係依據正常足(N)與實驗足(E)，其中PL意指足印長度(最長的腳趾尖端至腳跟之縱向長度)、TS意指總腳趾延展度(第一與第五腳趾之間的橫向距離)、及IT意指中數腳趾延展度(第二與第四腳趾之間的橫向距離)。利用FVB成鼠獲得該正常步態錄影(一隻小鼠之完整步行期間係總共收集1500個畫面)且該等影像數據係用以校准該踏步掃瞄軟體(每踏步10-12個略圖才足以訓練該軟體識別該腳掌位置)。校准後，將
該建構良好之程式用以於坐骨神經傷害之小鼠之完整步行期間排除異常步態及非正規之腳趾延展度。
手術後第四週及第八週，以RT系列滾輪踏車(SINGA)執行該滾輪測試。於對照組中，小鼠係定義為無神經切開及肌肉損傷(第四週時n=8，且第八週時n=8)。於正式收集數據前，每隻小鼠先跑過滾輪三次，分別為10rpm、12rpm、及15，作為測試前訓練。針對數據收集，小鼠以20rpm跑滾輪6次。記錄時間最大值為120秒。
蛋白質分析
以1X RIPA緩衝液[50mM HEPES,pH 7.3,150mM NaCl,2mM EDTA,20mM β-甘油磷酸鹽，0.1mM Na3VO4,1mM NaF,0.5mM DTT,and 0.5% NP-40]包含1X蛋白酶抑制劑混合物(Roche)，由該植入之導管萃取得蛋白質樣本。依據製造商之流程書，將100μg蛋白質樣本用於小鼠血管新生蛋白質抗體陣列(RayBiotech)分析。以化學螢光法檢測蛋白質含量。
神經分化分析
以1X HyQTase(Hyclone)由該神經球分離出單一細胞。將2×103細胞植於聚-D-離胺酸(BD Bioscience)-塗覆之腔室培養玻片上，以神經分化培養基(含有2% FBS之DMEM/F12)包含或不含誘導因子培養。對於CNTF+T3組，神經分化培養基含有50ng/ml CNTF與10ng/ml T3。對於hIL12及mIL12組，神經分化培養基係分別含有100ng/ml之人類IL12p80與小鼠IL12p80。對於CNTF+T3+hIL12組，神經分化培養基含有50ng/ml CNTF、10ng/ml T3及100ng/ml人類IL12p80。對於CNTF+T3+mIL12組，神經分化
培養基含有50ng/ml CNTF、10ng/ml T3及100ng/ml小鼠IL12p80。CNTF、T3及人類IL12p80係購自PeproTech。每三天更換培養基。
免疫螢光染色
將細胞培養於腔室培養玻片(Nunc,Naperville,IL,USA)上，於37℃及5% CO2下。對於免疫螢光染色，以PBS洗滌細胞並於室溫下以含4%(v/v)三聚甲醛(PFA；Electron Microscopy Sciences)之PBS固定15分鐘。接著於室溫下將細胞以0.1%(v/v)曲拉通(Triton)X-100之PBS滲透15分鐘。接著於室溫下將細胞以阻斷溶液(含1% BSA之1X PBS)阻斷1小時，接著於室溫下與專一性初級抗體共同培養1小時。於本發明中，利用免疫螢光染色，以抗半乳糖腦苷脂之小鼠單株抗體(Galc,1：1000,Millipore)、抗寡樹突細胞專一性蛋白質之兔多株抗體(OSP,1：1000,Abcam)、及抗髓磷脂蛋白質零之雞多株抗體(PZO,1：1000,GeneTex)，驗證分化能力。隨後將該細胞於室溫下與玫瑰紅共軛之二級抗體(Millipore,Billerica,MA,USA)共培養1小時。所有區段均以4',6-二脒基-2-苯基吲哚二鹽酸鹽(DAPI)(Molecular Probes)將核染色，並於螢光顯微鏡(Olympus,Tokyo,Japan)下觀察。計算Galc/DAPI、OSP/DAPI及PZO/DAPI雙陽性細胞並以對照組進行標準化。
西方墨點法
抗膠質原纖維酸性蛋白之兔多株抗體(GFAP,1：1000；Abcam)、抗寡樹突細胞專一性蛋白質之兔多株抗體(OSP,1：4000；Abcam)、抗髓磷脂蛋白質零之雞多株抗體(PZO,1：4000；GeneTex)、及抗α-微管蛋白之兔多株抗體(α-tubulin,1：8000稀釋；GeneTex)係用於以免疫印漬法檢
測已分化細胞中之該對應蛋白質濃度，該等已分化細胞係由神經球分離出，並以包含或不包含誘導因子之神經分化培養基(表1)培養7天。取自已分化細胞之蛋白質樣本係以含有1X蛋白酶抑制劑混合物(Roche)之1X RIPA分解緩衝液(Millipore,Billerica,MA,USA)獲得。
抗Stat3之小鼠單株抗體(1：1000；Cell Signaling)、抗pStat3-Y705位置之兔多株抗體(1：1000；Cell Signaling)、抗pStat3-S727位置之兔多株抗體(1：1000；Cell Signaling)、及抗α-微管蛋白之兔多株抗體(α-tubulin,1：8000稀釋；GeneTex)係用於分析經IL12p80處理之小鼠神經幹細胞之Stat3磷酸化狀態。由神經球分離出單一細胞，將細胞離心3分鐘以獲得實驗信號細胞，於不同時間點萃取細胞溶胞物(球體，0min、15min、30min、1h、2h、4h、及8h)。因此，於時間點「0min」，細胞係於離心期間處於IL12p80存在下達3分鐘。以西方墨點法分析細胞溶胞物。
以蛋白質分析染料試劑(Bio-Rad Laboratories)檢測各樣本之蛋白質濃度。等量之蛋白質樣本係以於十二烷基硫酸鈉聚丙烯醯胺凝膠(SDS-PAGE)進行尺寸分餾，接著轉移至聚偏二氟乙烯(PVDF)薄膜(Amersham)上，接著，於室溫下，以含5%牛血清白蛋白(Bio Basic,Markham Ontario,Canada)之阻斷緩衝液阻斷該薄膜1小時。接著將該薄膜與於含5%牛血清白蛋白之阻斷緩衝液中的專一性初級抗體共培養，接著與對應之HRP共軛之二級抗體(1：10000)(Millipore)共培養。利用ECL試劑(Millipore)測定蛋白質濃度，以α-微管蛋白之X-射線膜作為內部對照組。以ImageJ軟體定量三組獨立實驗之免疫活性鍵，並以對照組標準化。
複合肌肉動作電位測量
複合肌肉動作電位係以BIOPAC MP36及BIOPAC BSL 4.0軟體(BIOPAC system Inc.)記錄及分析。該不銹鋼電極之直徑為0.22mm。將刺激電極置於坐骨切痕，記錄電極置於腓腸肌(距離該坐骨切痕約2cm)。刺激電壓為6伏特，刺激期間為0.1毫秒，且獲取長度為200毫秒。以游標卡尺測量距離，在室溫維持於恆定溫度25℃下將皮膚溫度維持於36℃。
蘇木精與伊紅(H&E)染色與免疫組織化學染色
於手術後第八週收集植入之導管。簡言之，該植入之導管係以4% PFA於4℃下固定隔夜。經固定之樣本以含30%蔗糖之1X PBS於4℃下脫水隔夜。將樣本包埋入Tissue-Tek O.C.T.(Sakura,Netherlands)，並以液態氮冷凍後儲存於-80℃。
對於蘇木精與伊紅(H&E)與免疫組織化學染色，以冷凍切片機(MICROM HM550)將冷凍包埋之神經導管切成10μm薄片。免疫組織化學染色係利用專一性抗體進行：抗神經絲200之兔多株抗體(NF-200,1：500，神經纖維之標記)及抗髓磷脂蛋白質零之雞多株抗體(PZO,1：500，髓鞘化之許旺細胞之標記)。於PBS洗滌後，將樣本與個別之二級抗體於室溫下共培養1小時。將所有樣本以DAPI(1：5000)在室溫下進行核染色3分鐘，並以FluorSave試劑(Merck)固定。以螢光顯微鏡(Olympus)與共軛顯微鏡(Leica)觀察並收集所有再生組織之影像數據。
統計
數據係以平均值±標準差(SEM)表示。以學生t檢驗(Student’s t-test)比較兩個組別，並以單向ANOVA比較複數組別。當p<0.05時，統計顯著性為可接受。
結果
與僅有導管相較，含有神經幹細胞之神經導管之植入更能改善小鼠之坐骨神經傷害之功能性復原
左側坐骨神經(3mm)切除後，所有小鼠喪失左後肢之運動功能，表現為腳趾蜷縮及拖行。於本實驗中，發明人植入含神經幹細胞之導管以修復該受傷之坐骨神經。利用非侵入式方法，即步態分析與滾輪測試，檢測再生時期之期間之功能性復原。坐骨功能指數(SFI)為來自步態分析之數據經計算之分數，步態分析係結合步伐分析及在步行期間，一足印與其他足印之間在時間與空間上的關連性。SFI為自0至-100之一分數，其中0表示正常步行功能而-100表示完全喪失功能。於追蹤分析中，假性對照組之小鼠係定義為接受相同手術程序但並未實質傷害坐骨神經。與手術前SFI分數相較，此種小鼠顯示在手術後第一週至第八週之SFI分數並無明顯差異(第1圖，假性對照組)。對於植入含神經幹細胞之導管(第1A圖，導
管+NSC組)或僅有導管(第1A圖，僅導管組)，於手術後第一週，該兩組之間之SFI分數並無顯著差別(-73.2±2.5 vs.-77.0±3.9)。自手術後第二週起，該導管+NSC組顯示較僅導管組高的SFI分數(p<0.01)。與僅導管組相較，該導管+NSC組持續至第八週均呈現更佳的功能性(第1A圖)。
滾輪測試亦用於評估坐骨神經受傷之小鼠之運動功能與平衡能力之復原。於滾輪測試中，對照組小鼠係定義為未接受神經切開及肌肉傷害者。對照組、導管+NSC組及僅導管組於第四週之滾輪測試之結果分別為55±8、40±7、及16±1秒，而第八週則分別為62±14、53±9、及25±4秒(第1B圖)。與對照組、導管+NSC組相較，僅導管組在手術後第四與第八週顯示了於滾輪上明顯較差之平衡與步行能力。有趣的是，對照組與導管+NSC組之間，在手術後第四與第八週之滾輪測試並未觀察到差異性。該等結果暗示了，植入含有神經幹細胞之導管可幫助坐骨神經傷害之小鼠之功能復原。
IL12p80係涉及神經幹細胞-調控之坐骨神經修復
神經幹細胞具有分化為全部三種神經外胚層細胞系(包含許旺細胞)之潛力，且亦可分泌或吸收神經再生之營養因子(Ren Z.et al.,Reviews in the Neurosciences 2012,23：135-143)。因此，為鑑定涉及神經幹細胞-調控之神經再生之因子，以小鼠抗體陣列檢視僅導管組與導管+NSC組之間的蛋白質表現量。以由該植入之含有或不含神經幹細胞之導管之神經(於手術後第四週)萃取所得之蛋白質裂解液用於蛋白質抗體陣列。依據該抗體陣列之定量數據，於導管+NSC組所得之細胞萃取液中之IL12p40之表現為僅導管組之1.89倍高，導管+NSC組之IL12p35表現則與僅導管組類似
(1.04倍)(參照第2圖，IL12p40與IL12p35)。IL12p40為IL12p70之次單元之一，而IL12p40次單元整體之20%至40%會以同型二聚體形式(IL12p80)分泌，其在生物功能之誘導上的活性為IL12p40單體的25-50倍高(Heinzel F.P.et al.,Journal of Immunology 1997,158：4381-4388；Gillessen S.et al.,European Journal of Immunology1995,25：200-206；Jacobson N.G.et al.,Journal of Experimental Medicine1995,181：1755-1762.)。在該抗體陣列數據中，導管+NSC組增加了IL12p40之表現量而非IL12p35，暗示了IL12p80(IL12p40生物活性形式)可能為涉及神經再生之因子。IL12p40係鍵結至IL12受體β1，並造成Stat3磷酸化及活化Stat3下游訊息(Heinzel F.P.et al.,Journal of Immunology 1997,158：4381-4388；Gillessen S.et al.,European Journal of Immunology1995,25：200-206；Jacobson N.G.et al.,Journal of Experimental Medicine1995,181：1755-1762.)。先前的研究暗示，活化Stat3係誘導神經前驅細胞分化成星狀細胞系(Wang B.et al.,PLoS One2008,3：e1856.)。此等結果暗示，IL12p80在活化Stat3下游訊息及參與神經再生中的角色。
含神經幹細胞之導管之移植及IL12p80促進功能復原及神經再生
為測試IL12p80對神經再生之功能，比較以下列條件處理之經坐骨神經傷害之小鼠：僅導管組、含神經幹細胞之導管組(導管+NSC組)、及含神經幹細胞及小鼠IL12p80之導管組(導管+NSC+mIL12組)。於移植後第八週，以步態分析及滾輪測試進行功能評估。導管+NSC及導管+NSC+mIL12組之小鼠顯示了明顯高於僅導管組之SFI分數(第3A圖)。於滾輪測試中，導管+NSC及導管+NSC+mIL12組之小鼠顯示了優於僅導管組
之步態及平衡能力(第3B圖)。又，該等數據證實，在步態分析及滾輪測試兩者之功能評估上，與不含IL12p80之組別相較(p<0.05)，導管+NSC+mIL12組展現了更為顯著的結果(p<0.01)(第3A及3B圖)。然而，額外給予IL12p80(導管+NSC+mIL12組)之效果太微弱，以致於在功能評估上無法與未提供IL12p80(導管+NSC組)組區分該神經修復之輔助能力。因此，IL12p80之神經修復效果係藉由組織切片評估(第4圖)及神經傳導研究(第5圖)進行判定。
於神經再生研究中，植入5mm之神經導管以連接該3mm之坐骨神經傷害間隙。將該導管縫合至該受傷之坐骨神經之近端與遠端，並於兩端各保留1mm之殘留神經。因此，存在於該導管中央區域之軸突可被視為新生之再生神經。藉由蘇木精與伊紅(H&E)染色及利用辨識特定神經細胞專一性標記之抗體之免疫組織化學可觀察到軸突再生。於第4A、4B、及4C圖中，“P”與“D”係指示殘餘神經端點(“P”為距離該植入導管之近端之1.0mm，“D”為距離“P”之遠端之3.0mm)。在H&E染色中，與僅導管組(第4A圖)相較，導管+NSC組(第4B圖)與導管+NSC+mIL12組(第4C圖)顯示新生軸突之整合之明顯促進效果。藉由免疫組織化學染色，利用抗-神經絲200抗體(NF200，神經纖維之標記)及抗蛋白質零抗體(PZO，髓鞘化之許旺細胞之標記)進一步證實神經再生之狀況。免疫組織化學染色結果證實，於導管+NSC組(第4E圖)與導管+NSC+mIL12組(第4F圖)發生導管中之再生神經產生髓鞘化之許旺細胞(PZO陽性細胞)伴隨神經纖維(NF200陽性細胞)但僅導管組(第4D圖)則未發生。更進一步而言，藉由重建以H&E染色於植入之神經導管之近側、中間、及遠側位置染色之系
列樣本切片測量再生神經之直徑：近側位置(距導管之近端之1.0mm)、遠側位置(距該近側處3.0mm)、及中間位置(該近側位置與該遠側位置之間之中心)。於近側位置及遠側位置，三組的再生神經直徑並無顯著區別(近側位置分別為262±44μm、284±42μm及302±97μm，而遠側位置分別為279±56μm、298±75μm及322±123μm)。反之，於中間位置之直徑則為：與僅導管組(65±21μm)相較，導管+NSC組(189±18μm)與導管+NSC+mIL12組(295±47μm)分別為2.9倍及4.5倍厚。很明顯地，於再生坐骨神經之中間位置，與未添加IL12p80之組別相較，添加IL12p80者之神經直徑係增加1.6倍(第4G圖)。於該三組之再生之示意圖係顯示於第4H圖。藉由電刺激坐骨神經並記錄該複合肌肉動作電位(CMAP)以測量神經傳導，CMAP為來自個別肌肉纖維動作電位之總和電壓反應(Mallik A.et al.,J Neurol Neurosurg Psychiatry 2005,76 Suppl 2：ii23-31.)。於本發明中，測量傷腿與對側正常腿之CMAP。以傷腿之CMAP除以對側正常腿之CMAP，以計算復原百分比(第5圖)。導管+NSC+mIL12組顯示較佳的復原狀態，無論是與導管+NSC組或僅導管組相較(分別為17.7±4.3%、8.5±2.1%及6.6±0.2%)。又，導管+NSC+mIL12組顯示較僅導管組少的腓腸肌肌肉萎縮(傷腿/正常腿分別為0.39±0.02及0.26±0.07，p<0.01)。該等數據證實，植入含有神經幹細胞之導管可幫助功能復原及神經再生。重要的是，額外給予IL12p80，在SFI及滾輪分析之功能評估、神經傳導改善、新生再生神經之直徑增加，均具有統計顯著性之結果。
IL12p80刺激小鼠神經幹細胞分化為寡樹突細胞系及許旺細胞系
本發明接著研究於小鼠神經幹細胞中，IL12p80對於分化為寡樹突細胞及許旺細胞之誘導潛力。將自神經球分離之細胞分別培養於作為分化對照培養基之含有2% FBS之DMEM/F12(對照組)及寡樹突細胞分化培養基(CNTF+T3組)中(Hsu Y.C.et al.,Developmental Dynamics 2009,238：302-314；Lee D.C.et al,Molecular and Cellular Neurosciences 2009,41：348-363.)。為測試IL12p80於啟動寡樹突細胞及許旺細胞分化之潛力，將100ng/ml之人類IL12p80(hIL12p80)或小鼠IL12p80(mIL12p80)添加至對照組或CNTF+T3組中，並命名為hIL12、mIL12、CNTF+T3+hIL12、及CNTF+T3+mIL12組。以初級抗體(辨識特定細胞標記：半乳糖腦苷脂(Galc；寡樹突細胞)、寡樹突細胞專一性蛋白質(OSP；寡樹突細胞)、及抗髓磷脂蛋白質零(PZO；髓鞘化之許旺細胞))及螢光染劑共軛之二級抗體進行免疫螢光染色以證實該經分化之細胞(第6A圖)。標記陽性細胞之百分比係定量並摘要於第6B圖。細胞表型與免疫螢光染色結果顯示，於小鼠神經幹細胞中，IL12p80可取代CNTF+T3以誘導分化為寡樹突細胞或髓鞘化之許旺細胞。以西方墨點法，分別利用拮抗膠質原纖維酸性蛋白(GFAP，星狀細胞標記)、OSP、及PZO之種系專一性抗體，驗證細胞分化與成熟狀態(第7A圖)。定量結果顯示，於包含IL12p80之分化條件中，GFAP、OSP及PZO之蛋白質含量係經上游調控(第7B圖)。該等結果證實，IL12p80可誘導神經幹細胞分化為寡樹突細胞及髓鞘化之許旺細胞。
IL12p80係於神經幹細胞中誘導Stat3之磷酸化
於神經幹細胞中，已顯示Stat3磷酸化作用會參與寡樹突細胞分化過程(Wang B.et al.,PLoS One2008,3：e1856.)。於T細胞中，IL12p40
次單元可鍵結至IL12受體β1，接著誘導Stat3磷酸化作用及下游訊息途徑(Jacobson N.G.et al.,Journal of Experimental Medicine 1995,181：1755-1762.)。因此，IL12p80可於神經幹細胞中經由Stat3活化而啟動寡樹突細胞分化作用。以西方墨點法分析經hIL12p80或mIL12p80處理之神經幹細胞之Stat3磷酸化狀態。於本研究中，Y705及S727(Stat3磷酸化位置)兩者之磷酸化狀態，於神經球中為輕微表現(第8圖，神經球列)。人類及小鼠IL12p80所誘導之Y705位置之Stat3磷酸化作用，其強度尖峰出現在15分鐘，且於30分鐘開始減弱。類似地，S727位置之Stat3磷酸化作用會藉由IL12p80而增加，並維持達8小時(第8圖，hIL12組、mIL12組)。該等結果顯示，Stat3磷酸化作用於神經幹細胞之IL12p80-誘導之寡樹突細胞分化上至為重要。
結論
於本發明中，證實植入含有IL12p80及神經幹細胞之神經導管能增進運動功能復原、促進神經再生、增進神經傳導、及增加新生再生神經之直徑。實驗證實導管+NSC+IL12可以使該再生神經之中央區段的直徑大小較僅導管的實驗組厚4.5倍。於體外試驗，IL12p80可誘導神經幹細胞分化為寡樹突細胞或髓鞘化之許旺細胞。又，本發明進一步顯示，該分化作用之誘導係經由Stat3磷酸化而達成。單獨給予IL12p80可達成一定程度之神經修復。
討論
IL12p80為IL12p40之同型二聚體，而IL12p70為IL12p40及IL12p35之異型二聚體。IL12p40係鍵結至IL12受體β1，而IL12p35係鍵結至
IL12受體β2。亦報導IL23為IL12p40及IL23p19之異型二聚體，IL23p19係鍵結至IL23受體。本發明已顯示含有IL12p80及神經幹細胞之神經導管能增進受損神經之修復。單獨給予IL12p80可達成一定程度之神經修復。
已有報導，於體外試驗，IL12p70可增進小鼠交感上頸神經節神經元之軸突向外生長(Lin H.et al.,Neurosci.Lett.2000,278：129-132.)。因此，含有IL12p40次單元之蛋白質，包括IL12p80、IL12p70、及IL23均可作用於神經修復。神經修復可涵蓋之範疇，不僅是神經受損(如周邊神經損傷、脊髓損傷、及中風)之神經發生，更包括神經退化性疾病(如阿茲海默症、帕金森氏症、及多發性硬化症)之神經發生(Beckervordersandforth R.et al.,Stem Cell Reports2014,2：153-162.)。
上述特定實施例之內容係為了詳細說明本發明之利用神經幹細胞及IL12p40促進神經再生之組成物及方法，然而，該等實施例係僅用於說明，並非意欲限制本發明。熟習本領域之技藝者可理解，在不悖離後附申請專利範圍所界定之範疇下針對本發明所進行之各種變化或修改係落入本發明之一部分。
Claims (8)
- 一種神經再生之組成物，係包括：神經幹細胞，及神經滋養因子，係IL12p40單體及/或其同型二聚體IL12p80。
- 如申請專利範圍第1項之組成物，其中IL12p40具有如NCBI編錄號碼NM_008352(小鼠)或1F42_A(人類)所示之蛋白質序列。
- 如申請專利範圍第1項之組成物，其中該神經幹細胞之特徵在於，為腦細胞，其CD133及GFAP為陽性表現。
- 如申請專利範圍第1項之組成物，其中該神經幹細胞之特徵在於，為F1BGFP+細胞。
- 如申請專利範圍第1項之組成物，進一步包括一神經再生促進成分，該神經再生促進成分係包括纖維母細胞生長因子1(FGF1)、許旺細胞、及/或經誘導之神經幹細胞(iNSCs)。
- 如申請專利範圍第1項之組成物，進一步包括一神經導管，其帶有該神經幹細胞及該神經滋養因子之至少一者。
- 如申請專利範圍第5項之組成物，進一步包括一神經導管，其帶有該神經幹細胞、該神經滋養因子、及該神經再生促進成分之至 少一者。
- 如申請專利範圍第6或7項之組成物，其中該神經導管係植入於標的神經或該標的神經周圍之組織中。
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- 2019-07-17 US US16/514,701 patent/US20190351022A1/en not_active Abandoned
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1374898B1 (en) * | 2001-03-12 | 2007-06-27 | Institute of Gene and Brain Science | Use of interleukin-12 for treating nerve damage |
Non-Patent Citations (4)
| Title |
|---|
| Ehtesham, Moneeb, et al. "The use of interleukin 12-secreting neural stem cells for the treatment of intracranial glioma." Cancer research 62.20 (2002): 5657-5663. |
| Ehtesham, Moneeb, et al. "The use of interleukin 12-secreting neural stem cells for the treatment of intracranial glioma." Cancer research 62.20 (2002): 5657-5663. Lin, Haiyan, et al. "Interleukin-12 promotes neurite outgrowth in mouse sympathetic superior cervical ganglion neurons." Neuroscience letters 278.3 (2000): 129-132. Hsu, Yi-Chao, et al. "Stem cell-based therapy in neural repair." Biomedical journal 36.3 (2013): 98-105. * |
| Hsu, Yi-Chao, et al. "Stem cell-based therapy in neural repair." Biomedical journal 36.3 (2013): 98-105. |
| Lin, Haiyan, et al. "Interleukin-12 promotes neurite outgrowth in mouse sympathetic superior cervical ganglion neurons." Neuroscience letters 278.3 (2000): 129-132. |
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| CA2958398C (en) | 2019-05-14 |
| TW201613623A (en) | 2016-04-16 |
| US10391149B2 (en) | 2019-08-27 |
| EP3180019A1 (en) | 2017-06-21 |
| CN107148278A (zh) | 2017-09-08 |
| CA2958398A1 (en) | 2016-02-18 |
| CN107148278B (zh) | 2021-06-29 |
| KR20170065504A (ko) | 2017-06-13 |
| HK1243648A1 (zh) | 2018-07-20 |
| WO2016023130A1 (en) | 2016-02-18 |
| JP6339737B2 (ja) | 2018-06-06 |
| JP2017529329A (ja) | 2017-10-05 |
| KR101897422B1 (ko) | 2018-09-10 |
| AU2015303798B2 (en) | 2020-03-12 |
| EP3180019A4 (en) | 2018-05-09 |
| SG11201700971XA (en) | 2017-03-30 |
| US20170224776A1 (en) | 2017-08-10 |
| EP3180019B1 (en) | 2019-12-25 |
| US20190351022A1 (en) | 2019-11-21 |
| AU2015303798A1 (en) | 2017-03-02 |
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