TW202320836A - 缺血性疾病之治療劑 - Google Patents
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Abstract
本發明提供一種缺血性疾病之治療劑,其包含巨噬細胞凋亡抑制劑(Apoptosis inhibitor of macrophage,AIM)、具有AIM之生物活性之AIM片段、或編碼該AIM或AIM片段之核酸。
Description
本發明係關於一種缺血性疾病之治療劑等,詳細而言,係關於含有巨噬細胞凋亡抑制劑(AIM)之缺血性疾病之治療劑等。
若死細胞或其壞死組織碎片之去除不充分,則會釋放細胞內炎症物質,即損傷相關分子模式(damage-associated molecular patterns,DAMPs)。DAMPs係通常之內源性細胞內蛋白質,因此無法被免疫系統識別,若一旦釋放至細胞外環境,則該等就會與模式識別受體(pattern recognition receptors)結合,該模式識別受體主要包含以巨噬細胞為首之免疫細胞上之Toll樣受體(TLRs)或最終糖化產物之受體,並在不存在下微生物之情況下誘導炎症性細胞激素之產生,激活自然免疫。該過程被稱為無菌性炎症,經常發生於缺血再灌注損傷(ischemia-reperfusion injury,IRI)、外傷、或化學誘發之損傷(chemically induced injury)中(非專利文獻1)。
缺血性腦梗塞係世界範圍導致重度病變及死亡之最常見原因之一,係與無菌性炎症相關之典型例子,現在亦被列為COVID-19之重要併發症(非專利文獻2~5)。缺血性腦梗塞之預後受梗塞發病後患處之腦中無菌性炎症狀態較大影響,這已經成為廣泛接受之共識。由於血栓溶解或血栓切除等當前可利用之治療法未必會較大程度地改善梗塞患者之預後,故近年來,對作為治療目標之梗塞後無菌性炎症(post-stroke sterile inflammation)之關注不斷提高(非專利文獻5~7)。近年來之研究表明,各種類型之DAMPs中,過氧化物酶(PRDX;尤其是PRDX1)、高遷移率族蛋白1(HMGB1)、及S100鈣結合蛋白(S100)與梗塞後無菌性炎症有較大關聯(非專利文獻8)。最近有報告稱,於修復過程中,DAMPs經由巨噬細胞清道夫受體1(該表現受MAF bZIP轉錄因子B(MafB)控制),被微神經膠質細胞或浸潤於梗塞部之巨噬細胞吸收、去除(非專利文獻9)。然而,目前尚未確立基於去除DAMP之腦梗塞治療。
巨噬細胞凋亡抑制劑(AIM,亦稱為CD5抗原樣(CD5L))係由組織巨噬細胞產生之血液蛋白,該蛋白質係本發明者首次鑑定為支持巨噬細胞存活之物質。AIM現在被認為係在許多疾病中誘導修復過程之分子(非專利文獻10~12)。AIM包含3個半胱胺酸富含區(稱為SRCR區),於羧基末端之第3個SRCR區內,存在獨特之帶正電荷之胺基酸簇。該簇與死細胞形成了基於電荷之相互作用(非專利文獻10、11),該死細胞由於其表面暴露於高水準之磷脂絲胺酸而帶較強之負電。AIM經由複數個清道夫受體而被巨噬細胞有效率地吸收,因此該結合較強地亢進了巨噬細胞對死細胞之吞噬(非專利文獻12)。本發明人研究了缺血性腦梗塞之修復過程中AIM之作用與其治療作用,該缺血性腦梗塞之主要病情生理為因短暫性之缺血引起之腦神經細胞之部分壞死、及因從壞死細胞所釋放之DAMPs引起之無菌性炎症。
[先前技術文獻]
[非專利文獻]
[非專利文獻1]Zindel, J. and Kubes, P. (2020). DAMPs, PAMPs, and LAMPs in Immunity and Sterile Inflammation. Annu. Rev. Pathol. 15, 493-518.
[非專利文獻2]Trejo-Gabriel-Galan, J.M. (2020). Stroke as a complication and prognostic factor of COVID-19. Neurologia 35, 318-322.
[非專利文獻3]Oxley, T.J., Mocco, J., Majidi, S., Kellner, C.P., Shoirah, H., Singh, I.P., De Leacy, R.A., Shigematsu, T., Ladner, T.R., Yaeger, K.A. et al. (2020). Large-Vessel Stroke as a Presenting Feature of Covid-19 in the Young. N. Engl. J. Med. 382, e60 (2020).
[非專利文獻4]Tan, Y.K., Goh, C., Leow, A.S.T., Tambyah, P.A., Ang, A., Yap, E.S., Tu, T.M., Sharma, V.K., Yeo, L.L.L., Chan, B.P.L. et al. (2020). COVID-19 and ischemic stroke: a systematic review and meta-summary of the literature. J. Thromb. Thrombolysis 50, 587-595.
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[發明所欲解決之問題]
本發明之目的在於提供一種以腦梗塞為首之缺血性疾病之新穎治療劑及新穎治療方法。
[解決問題之技術手段]
本發明人對上述課題進行了努力研究,結果發現:(1)AIM以2種結合模式與各種DAMPs(例如PRDX1~6、HMGB1、S100A8、S100A8/9、S100A9、及HSP70等)結合;(2)AIM對DAMPs之結合能中和DAMPs之生物活性;(3)AIM對DAMPs之結合能使DAMPs被微神經膠質細胞與浸潤於梗塞病灶之巨噬細胞有效率地吞噬或去除,(4)作為(2)及(3)之結果,AIM抑制了無菌性炎症之擴散,及(5)AIM亢進了腦梗塞區域中死細胞碎片之吞噬去除,且抑制了無菌性炎症之擴散。基於該見解進行進一步深入研究而完成本發明。
即,本發明如下所示。
[1]一種缺血性疾病之治療劑,其包含巨噬細胞凋亡抑制劑(AIM)、具有AIM之生物活性之AIM片段、或編碼該AIM或AIM片段之核酸。
[2]如[1]之治療劑,其中缺血性疾病係選自由腦梗塞、心肌梗塞、肢體缺血、肺梗塞、脾梗塞、腸梗塞、及柏格氏病所組成之群。
[3]一種損傷相關分子模式(DAMPs)介導之無菌性炎症之抑制劑,其包含巨噬細胞凋亡抑制劑(AIM)、具有AIM之生物活性之AIM片段、或編碼該AIM或AIM片段之核酸。
[4]如[3]之抑制劑,其中DAMPs係選自由PRDX1、PRDX2、PRDX3、PRDX4、PRDX5、PRDX6、HMGB1、S100A8、S100A8/9、S100A9、及HSP70所組成之群中之至少一種。
[5]一種缺血性疾病之治療方法,其包含將巨噬細胞凋亡抑制劑(AIM)、具有AIM之生物活性之AIM片段、或編碼該AIM或AIM片段之核酸投予至罹患缺血性疾病之對象。
[6]如[5]之治療方法,其中缺血性疾病係選自由腦梗塞、心肌梗塞、肢體缺血、肺梗塞、脾梗塞、腸梗塞、及柏格氏病所組成之群。
[7]一種DAMPs介導之無菌性炎症之抑制方法,其包含向患有損傷相關分子模式(DAMPs)介導之無菌性炎症之對象投予巨噬細胞凋亡抑制劑(AIM)、具有AIM之生物活性之AIM片段、或編碼該AIM或AIM片段之核酸。
[8]如[7]之抑制方法,其中DAMPs係選自由PRDX1、PRDX2、PRDX3、PRDX4、PRDX5、PRDX6、HMGB1、S100A8、S100A8/9、S100A9、及HSP70所組成之群中之至少一種。
[發明之效果]
根據本發明,可將對象之梗塞區域之死細胞碎片有效率地去除,同時抑制無菌性炎症。因此,本發明可治療腦梗塞、心肌梗塞、肢體缺血、肺梗塞、脾梗塞、腸梗塞、或柏格氏病等缺血性疾病。
以下對本發明進行詳細說明。
1. 缺血性疾病之治療劑本發明提供一種缺血性疾病之治療劑(以下有時稱為「本發明之治療劑」),其包含巨噬細胞凋亡抑制劑(AIM)、具有AIM之生物活性之AIM片段、或編碼該AIM或AIM片段之核酸。
缺血性疾病係由於對器官提供營養之動脈阻塞或狹窄而導致器官之缺血,組織由於氧或營養之不足而壞死或功能不全之疾病。作為利用本發明之治療劑治療之缺血性疾病,可例舉:腦梗塞、心肌梗塞、肢體缺血、肺梗塞、脾梗塞、腸梗塞、或柏格氏病等,但並不限定於該等。於較佳之一態樣中,缺血性疾病可為腦梗塞。
腦梗塞係基於發病機制而分類的。將於頭或腦中比較粗之血管進行動脈硬化,結果由血流受阻而引起之腦梗塞稱為「動脈粥樣硬化性腦梗塞」。將由於腦之深處中細血管堵塞而引起之腦梗塞稱為「腔隙性梗塞」。將於心臟中形成之血栓被輸送至腦血管,結果由腦血管堵塞而引起之腦梗塞稱為「心源性腦栓塞」。本發明之治療劑可用於任何類型之腦梗塞之治療。
本發明中所使用之AIM係包含與序列編號1(源自人類之AIM蛋白之胺基酸序列)所表示之胺基酸序列同一或與其實質上同一之胺基酸序列之蛋白質。AIM例如可以是從恆溫動物(例如:人類、小鼠、大鼠、兔、羊、豬、牛、馬、貓、狗、猴、黑猩猩、鳥等)之作為免疫細胞之巨噬細胞所單離、純化之蛋白質。又,亦可為藉由化學合成或無細胞轉譯系統而生化合成之蛋白質,或亦可為由轉形體所產生之重組蛋白質,該轉形體導入有包含編碼上述胺基酸序列之鹼基序列的核酸。於某些情況下,較佳為成為本發明中所使用之AIM之來源的生物種與罹患神經退化性疾病之對象生物種一致。例如,於本發明之治療劑之適用對象為人類之情形時,較佳為使用人類AIM。
作為與序列編號1所表示之胺基酸序列實質上同一之胺基酸序列,可例舉具有與序列編號1所表示之胺基酸序列約60%以上、較佳為約70%以上、進而較佳為約80%以上、特佳為約90%以上、最佳為約95%以上之同一性或類似性的胺基酸序列等。此處所謂「同一性」係指使用該技術領域中公知之數學演算法對2個胺基酸序列進行比對之情形時,最佳之序列比對(較佳為該演算法可以考慮向序列之一方或雙方導入間隙以實現最佳序列比對)中之同一胺基酸及類似胺基酸殘基相對於重疊之所有胺基酸殘基之比率(%)。又,「類似性」係指對2個胺基酸序列進行序列比對時,於該兩個序列中存在同一或類似胺基酸殘基之位置數相對於全長胺基酸殘基數之比率(%)。「類似胺基酸」係指於物理化學性質上類似之胺基酸,例如可例舉:芳香族胺基酸(Phe、Trp、Tyr)、脂肪族胺基酸(Ala、Leu、Ile、Val)、極性胺基酸(Gln、Asn)、鹼性胺基酸(Lys、Arg、His)、酸性胺基酸(Glu、Asp)、具有羥基之胺基酸(Ser、Thr)、側鏈較小之胺基酸(Gly、Ala、Ser、Thr、Met)等分類於同一組之胺基酸。預測此種類似胺基酸之置換不會改變蛋白質之表型(即,保守性胺基酸置換)。保守性胺基酸置換之具體例於本技術領域係眾所周知者,且於各種文獻中有所記載(例如參照Bowie等人,Science, 247: 1306-1310(1990))。
本說明書中之胺基酸序列之同一性或類似性,可使用同一性或類似性計算演算法NCBI BLAST(National Center for Biotechnology Information Basic Local Alignment Search Tool,美國國家生物技術信息中心基本局部比對搜索工具),於以下之條件下(預期值=10;允許空隙;矩陣=BLOSUM62;過濾=OFF)計算。作為用以確定胺基酸序列之同一性或類似性之其他演算法,例如可例舉:於Karlin等,Proc. Natl. Acad. Sci. USA, 90:5873-5877(1993)記載之演算法[該演算法被納入至NBLAST及XBLAST程式(2.0版)(Altschul等,Nucleic Acids Res., 25:3389-3402(1997))]、於Needleman等,J .Mol. Biol., 48:444-453(1970)記載之演算法[該演算法被納入至GCG軟體封裝中之GAP程式],於Myers及Miller, CABIOS, 4: 11-17(1988)記載之演算法[該演算法被納入至CGC序列比對軟體封裝之一部分即ALIGN程式(2.0版)]、於Pearson等,Proc. Natl. Acad. Sci. USA, 85:2444-2448(1988)記載之演算法[該演算法被納入至GCG軟體封裝中之FASTA程式]等,該等亦可同樣地較佳使用。更佳為與序列編號1所表示之胺基酸序列實質上同一之胺基酸序列係與序列編號1所表示之胺基酸序列具有約60%以上、較佳為約70%以上、進而較佳為約80%以上、特佳為約90%以上、最佳為約95%以上之同一性之胺基酸序列。
作為包含與序列編號1所表示之胺基酸序列實質上同一之胺基酸序列之蛋白質,例如可例舉包含與上述之序列編號1所表示之胺基酸序列實質上同一之胺基酸序列,且具有與野生型AIM所具有之生物活性實質上同質之活性的蛋白質。作為野生型AIM所具有之生物活性,例如可例舉:對巨噬細胞(包括微神經膠質細胞)之內噬活性、巨噬細胞之細胞凋亡抑制活性、動脈硬化之維持/促進活性、脂肪細胞分化抑制活性、脂肪細胞之脂肪滴融解活性、脂肪細胞縮小活性、CD36結合活性、對脂肪細胞之內噬活性、FAS結合活性、FAS功能抑制活性、抗肥胖活性、肝臟疾病(脂肪肝、NASH、肝硬變、肝癌)之預防或治療活性、腎臟疾病(急性腎功能衰竭、慢性腎炎、慢性腎功能衰竭、腎病症候群、糖尿病性腎病、腎硬化症、IgA腎病、高血壓性腎病、伴隨膠原病之腎病或IgM腎病)之預防或治療活性等,於本發明中,特別是對巨噬細胞之內噬活性可成為較佳之指標。該活性可使用於本案之實施例中所詳述之體外之巨噬細胞(或微神經膠質細胞)之吞噬試驗而確認,但並不限定於此。又,本說明書中「實質上同質」係表示該等活性在定性上(例如生理學或藥理學)相同。因此,較佳為上述活性同等,但該等活性之程度(例如約0.1~約10倍,較佳為約0.5~約2倍)或蛋白質之分子量等之定量因素亦可不同。上述活性之測定可依照本身已知之方法進行。
又,本發明中所使用之AIM亦包含例如:(1)序列編號1所表示之胺基酸序列中缺失1個或2個以上(較佳為1~100個左右,較佳為1~50個左右,進而較佳為1~10個左右,特佳為1~數(2、3、4或5)個)胺基酸之胺基酸序列、(2)於序列編號1所表示之胺基酸序列上附加1個或2個以上(較佳為1~100個左右,較佳為1~50個左右,進而較佳為1~10個左右,特佳為1~數(2、3、4或5)個)胺基酸之胺基酸序列、(3)於序列編號1所表示之胺基酸序列***1個或2個以上(較佳為1~50個左右,較佳為1~10個左右,進而較佳為1~數(2、3、4或5)個)胺基酸之胺基酸序列、(4)序列編號1所表示之胺基酸序列中之1個或2個以上(較佳為1~50個左右,較佳為1~10個左右,進而較佳為1~數(2、3、4或5)個)胺基酸被其他胺基酸置換之胺基酸序列、或(5)包含該等組合而成之胺基酸序列的蛋白質等。如上所述,於對胺基酸序列進行***、缺失或置換之情形時,該***、缺失或置換之位置只要保持該蛋白質之所需之生物活性(例如對巨噬細胞(包括微神經膠質細胞)之內噬活性),就沒有特別限定。
本發明之AIM較佳為具有序列編號1所表示之胺基酸序列之人類AIM蛋白(GenBank登錄號:AAD01446),或其在其他哺乳動物中之同源物[例如於GenBank中以登錄號:AAD01445所登錄之小鼠同源物等],更佳為由序列編號1所表示之胺基酸序列構成之人類AIM蛋白。
於本說明書中,蛋白質及肽係依照肽標記物之慣例,以左端為N末端(胺基末端),右端為C末端(羧基末端)之方式記載。作為以包含序列編號1所表示之胺基酸序列的蛋白質為首的本發明中所使用之AIM,C末端可為羧基(-COOH)、羧酸酯(-COO-)、醯胺(-CONH
2)或酯(-COOR)之任一者。
此處,作為酯中之R,可使用:例如甲基、乙基、正丙基、異丙基、正丁基等C
1-6烷基;例如環戊基、環己基等C
3-8環烷基;例如苯基、α-萘基等C
6-12芳基;例如苄基、苯乙基等苯基-C
1-2烷基,α-萘基甲基等α-萘基-C
1-2烷基等C
7-14芳烷基;特戊醯氧基甲基等。
於本發明中所使用之AIM除了C末端以外還具有羧基(或羧酸酯)之情形時,羧基被醯胺化或酯化者亦包含於本發明之蛋白質中。作為該情形之酯,例如可使用上述之C末端之酯等。
進而,本發明中所使用之AIM中亦包含:N末端之胺基酸殘基之胺基經保護基(例如甲醯基、乙醯基等C
1-6烷醯基等C
1-6醯基等)保護而成者、可於生物體內切斷而生成之N末端之麩醯胺殘基經焦麩胺酸化而成者、分子中之胺基酸之側鏈上之取代基(例如-OH、-SH、胺基、咪唑基、吲哚基、胍基等)經適當之保護基(例如甲醯基、乙醯基等C
1-6烷醯基等C
1-6醯基等)保護而成者、或與糖鏈結合之所謂糖蛋白等複合蛋白質等。
本說明書中所謂「AIM」之概念不僅包含野生型AIM,亦包含具有與野生型AIM之生物活性實質上同質或有所提昇之活性的該等改型體等。此處,「實質上同質之活性」係表示與上述相同之含義。又,「實質上同質之活性」之測定可與AIM之情形同樣地進行。
作為AIM之改型體之一例,可例舉以下者,但不限定於該等。
本發明之變異型人類AIM較佳為包含以下之(1b)至(5b)之任1個胺基酸序列。
(1b)序列編號1所表示之胺基酸序列之胺基酸編號191之半胱胺酸被絲胺酸置換之胺基酸序列。
(2b)序列編號1所表示之胺基酸序列之胺基酸編號300之半胱胺酸被絲胺酸置換之胺基酸序列。
(3b)序列編號1所表示之胺基酸序列之胺基酸編號191之半胱胺酸被絲胺酸置換,且序列編號1所表示之胺基酸序列之胺基酸編號300之半胱胺酸被絲胺酸置換之胺基酸序列。
(4b)與(1b)至(3b)之任1個胺基酸序列實質上同一,且存在於(1b)至(3b)之任1個胺基酸序列中之半胱胺酸及該被置換之絲胺酸殘留之胺基酸序列。
(5b)存在於(1b)至(3b)之任1個胺基酸序列中之半胱胺酸及該被置換之絲胺酸以外之部位中,進而包含1個至數個胺基酸之缺失、附加、***或置換或其組合之胺基酸序列。
再者,關於具有與野生型重組AIM同等或有所提昇之功能的AIM改型體,可使用於日本專利特願2017-220733等所揭示者。
又,作為本發明之治療劑之成分,不僅可使用AIM,亦可使用具有AIM之生物活性之AIM片段。AIM片段是否具有野生型AIM之生物活性,藉由上述之方法確定即可。
作為AIM片段之一例,例如完整之AIM蛋白包含3個富含半胱胺酸之SRCR(Scavenger-Receptor Cysteine-Rich,清道夫受體富含半胱胺酸)區,因此可將各個SRCR區作為具有與野生型AIM所具有之生物活性實質上同質之活性的AIM片段之例而例舉。更具體而言,例如可將具有如下部分胺基酸序列之片段用作AIM片段:分別包含序列編號1所表示之胺基酸序列中之SRCR1區(序列編號1所表示之胺基酸序列中之胺基酸編號24~125)、SRCR2區(序列編號1所表示之胺基酸序列中之胺基酸編號138~239)、SRCR3區(序列編號1所表示之胺基酸序列中之胺基酸編號244~346)之部分胺基酸序列,或包含SRCR區之任意組合之部分胺基酸序列等。具有與野生型AIM之生物活性實質上同質之活性之AIM片段只要包含上述之功能區即可,其大小並無特別限制,可例舉較佳為包含50個以上之部分胺基酸序列者,更佳為包含100個以上之部分胺基酸序列者,進而較佳為包含200個以上之部分胺基酸序列者。該部分胺基酸序列可為一個連續之部分胺基酸序列,或亦可為不連續之複數個部分胺基酸序列連結而成者。
又,作為本發明中所使用之AIM片段,C末端可為羧基(-COOH)、羧酸酯(-COO
-)、醯胺(-CONH
2)或酯(-COOR)之任一者。此處,作為酯中之R,可例舉與AIM中所述之R相同者。本發明之部分肽除了C末端以外還具有羧基(或羧酸酯)之情形時,羧基被醯胺化或酯化者亦包含於本發明之部分肽中。作為該情形之酯,例如可使用與C末端之酯相同者。
進而,於本發明中所使用之AIM片段中,與上述之AIM同樣地亦包含:N末端之胺基酸殘基之胺基經保護基保護而成者、N末端之麩醯胺殘基經焦麩胺酸化而成者、分子中之胺基酸之側鏈上之取代基經適當之保護基保護而成者、或與糖鏈結合之所謂糖肽等複合肽等。
本發明中所使用之AIM(包括AIM片段)亦可為鹽之形態。例如可使用與生理學上所容許之酸(例如:無機酸、有機酸)或鹼(例如:鹼金屬鹽)等之鹽,特別較佳為生理學上所容許之酸加成鹽。作為此種鹽,例如可使用與無機酸(例如:鹽酸、磷酸、氫溴酸、硫酸)之鹽、或與有機酸(例如:乙酸、甲酸、丙酸、反丁烯二酸、順丁烯二酸、琥珀酸、酒石酸、檸檬酸、蘋果酸、草酸、苯甲酸、甲磺酸、苯磺酸)之鹽等。
AIM可藉由本身已知之蛋白質之純化方法,由上述哺乳動物之巨噬細胞製造。具體而言,使哺乳動物之巨噬細胞均質化,藉由低速離心而去除細胞碎片後,對上清液進行高速離心而使含有細胞膜之組分沈澱,對該上清液實施逆相層析法、離子交換層析法、親和層析法等層析法等,藉此製備AIM或其鹽。
AIM(包括AIM片段)亦可依照公知之肽合成法而製造。肽合成法例如可為固相合成法、液相合成法之任一者。於將可構成AIM之部分肽或胺基酸與殘餘部分縮合,產物具有保護基之情形時,可藉由將保護基脫離而製造目標蛋白質。
此處,縮合或保護基之脫離係依照本身已知之方法,例如以下之(1)及(2)所記載之方法進行。
(1)M. Bodanszky及M. A. Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966年)
(2)Schroeder及Luebke, The Peptide, Academic Press, New York(1965年)
如此所得到之AIM可藉由公知之純化法進行純化單離。此處,作為純化法,例如可例舉:溶劑萃取、蒸餾、管柱層析法、液相層析法、再結晶、該等之組合等。
於藉由上述方法而得到之AIM為游離體之情形時,可藉由公知之方法或基於其之方法而將該游離體轉化為適當之鹽,反之,於以鹽之形式而得到AIM之情形時,可藉由公知之方法或基於其之方法而將該鹽轉化為游離體或其他鹽。
進而,AIM亦可藉由如下方式而製造:培養含有編碼AIM之核酸的轉形體,並從所得到之培養物分離純化AIM。編碼AIM或AIM片段之核酸可為DNA,亦可為RNA,或亦可為DNA/RNA嵌合體。較佳為DNA。又,該核酸可為雙股,亦可為單股。於雙股之情形,可為雙股DNA、雙股RNA或DNA:RNA之雜合體。於單股之情形,可為正義股(即編碼鏈),亦可為反義股(即非編碼鏈)。
作為編碼AIM(包括AIM片段)之DNA,可例舉:基因組DNA、源自恆溫動物(例如:人類、牛、猴、馬、豬、羊、山羊、狗、貓、豚鼠、大鼠、小鼠、兔、倉鼠、鳥等)之巨噬細胞之cDNA、合成DNA等。若為編碼AIM或AIM片段之基因組DNA,則可使用由上述動物之所有細胞[例如:肝細胞、脾細胞、神經細胞、神經膠細胞、胰腺β細胞、骨髄細胞、腎小球膜細胞、蘭格漢氏細胞、表皮細胞、上皮細胞、杯狀細胞、內皮細胞、平滑肌細胞、纖維母細胞、纖維細胞、肌細胞、脂肪細胞、免疫細胞(例如:巨噬細胞、T細胞、B細胞、自然殺手細胞、肥胖細胞、嗜中性球、嗜鹼性球、嗜酸性球、單核球)、巨核細胞、滑膜細胞、軟骨細胞、骨細胞、成骨細胞、破骨細胞、乳腺細胞、肝細胞或間質細胞、或該等細胞之前驅細胞、幹細胞或癌細胞等]或該等細胞所在之所有組織[例如:腦、腦之各部位(例如:嗅球、扁桃核、大腦基底球、海馬、丘腦、下丘腦、大腦皮質、髓質、小腦)、脊髄、下垂體、胃、胰腺、腎臟、肝臟、生殖腺、甲狀腺、膽嚢、骨髄、腎上腺、皮膚、肺、消化道(例如:大腸、小腸)、血管、心臟、胸腺、脾臟、顎下腺、末梢血、***、睾丸、卵巢、胎盤、子宮、骨骼、關節、脂肪組織(例如:褐色脂肪組織、白色脂肪組織)、骨骼肌等]製備之基因組DNA組分作為模板,藉由聚合酶鏈反應(以下簡稱為「PCR法」)而直接擴增,若為編碼AIM或AIM片段之cDNA,則亦可分別將由巨噬細胞製備之全RNA或mRNA組分用作模板,藉由PCR法及逆轉錄酶-PCR(以下簡稱為「RT-PCR法」)而直接擴增。或者編碼AIM或其肽片段之基因組DNA及cDNA亦可藉由群落或噬菌斑雜交法或PCR法等,由將上述基因組DNA及全RNA或mRNA之片段***至適當之載體中而製備之基因組DNA基因庫及cDNA基因庫分別選殖。基因庫所使用之載體可為噬菌體、質體、黏接質體、噬菌粒等之任一者。
作為編碼AIM之核酸,例如可例舉包含與序列編號2所表示之鹼基序列同一或實質上同一之鹼基序列之核酸等。作為包含與序列編號2所表示之鹼基序列實質上同一之鹼基序列之核酸,例如可使用包含具有與序列編號2所表示之鹼基序列約60%以上、較佳為約70%以上、進而較佳為約80%以上、特佳為約90%以上之同一性或類似性之鹼基序列,且編碼具有與上述之AIM實質上同質之活性之蛋白質的核酸等。於一態樣中,作為包含與序列編號2所表示之鹼基序列實質上同一之鹼基序列的核酸,係包含具有與序列編號2所表示之鹼基序列約60%以上、較佳為約70%以上、進而較佳為約80%以上、特佳為約90%以上之同一性之鹼基序列,且編碼具有與上述之AIM實質上同質之活性之蛋白質的核酸。編碼AIM之核酸亦包含為了提高在成為適用對象之生物中的表現效率而進行了密碼子最佳化之核酸序列等。
作為本說明書中之鹼基序列之同一性或類似性,可使用同一性或類似性計算演算法NCBI BLAST(National Center for Biotechnology Information Basic Local Alignment Search Tool,美國國家生物技術信息中心基本局部比對搜索工具),於以下之條件下(預期值=10;允許空隙;過濾=ON;匹配得分=1;失配得分=-3)進行計算。作為用以確定鹼基序列之同一性或類似性之其他演算法,同樣較佳地例示上述之胺基酸序列之同源性計算演算法。
作為編碼AIM之核酸,較佳為包含由序列編號2所示之鹼基序列表示的編碼人類AIM蛋白之鹼基序列的核酸(GenBank登錄號:AF011429)或其在其他哺乳動物中之同源物[例如於GenBank中以登錄號:AF011428所登錄之小鼠同源物等]。
進而,作為本發明之治療劑之成分,亦可使用編碼AIM、或具有AIM之生物活性之AIM片段之核酸。
本發明中所使用之編碼AIM或AIM片段之核酸若包含編碼肽之鹼基序列則可為任意者,該肽包含與序列編號1所表示之胺基酸序列之一部分同一或實質上同一之胺基酸序列。具體而言,作為編碼AIM片段之核酸,例如可使用:(1)包含序列編號2所表示之鹼基序列之部分鹼基序列之核酸、或(2)包含與含有序列編號2所表示之鹼基序列之部分鹼基序列的核酸具有約60%以上、較佳為約70%以上、進而較佳為約80%以上、特佳為約90%以上之同一性或類似性之鹼基序列,且編碼具有與上述之AIM實質上同質活性之蛋白質之核酸等。
編碼AIM或AIM片段之核酸可使用具有編碼該AIM或AIM片段之鹼基序列的一部分的合成DNA引子,藉由PCR法進行擴增,或藉由將被納入至適當之表現載體之DNA、與對編碼AIM之一部分或全區域之DNA片段或合成DNA進行標記而成者雜交而進行選殖。雜交可依照本身已知之方法或基於其之方法,例如於分子選殖(Molecular Cloning)第2版(J. Sambrook et al., Cold Spring HarborLab. Press, 1989)中記載之方法等進行。又,於使用市售之基因庫之情形時,雜交可依照隨附之使用說明書中記載之方法進行。雜交較佳為能依照嚴格之條件進行。
作為高度嚴格之條件,例如可例舉於6×SSC(氯化鈉/檸檬酸鈉)中以45℃進行雜交反應後,於0.2×SSC/0.1%SDS中以65℃進行一次以上之洗浄等。業者可將雜交溶液之鹽濃度、雜交反應之溫度、探針濃度、探針之長度、失配數、雜交反應之時間、洗浄液之鹽濃度、洗浄之溫度等適當變更,藉此容易地調節至所需之嚴格度。又,於使用市售之基因庫之情形時,雜交可依照該基因庫所隨附之使用說明書中記載之方法進行。
編碼AIM或AIM片段之核酸亦可與具有腦特異性表現之啟動子之表現載體等功能性連結。藉由將包含編碼AIM或AIM片段之核酸的表現載體傳遞至腦內,可腦特異性地表現AIM或AIM片段。腦特異性啟動子包括SCG10、GFAP啟動子、突觸蛋白1啟動子、微管蛋白α1啟動子、鈣離子/鈣調素依賴性蛋白激酶II啟動子、神經元特異性烯醇化酶啟動子、PDGF(血小板衍生生長因子β)-β鏈啟動子等,但並不限定於該等。
又,於較佳之一態樣中,可將編碼AIM或AIM片段之核酸搭載於病毒載體。作為合適之病毒載體,可例舉:腺相關病毒、腺病毒、慢病毒、及仙台病毒,但並不限定於該等。若考慮於基因治療中之利用,則就使導入基因長期表現之方面或源自非病原性病毒而安全性較高等理由而言,較佳為腺相關病毒。又,作為腺相關病毒之血清型,只要能夠獲得本發明之所需之效果,就沒有特別限定,可使用血清型1、2、3、4、5、6、7、8、9、及10之任一者。於本發明之一態樣中,於以腦梗塞為治療對象之情形時,特別是考慮到於神經組織中表現效率較高之方面,較佳為血清型1、2、5、9、或10(關於AAV之各種血清型,請參照WO2005/033321),進而,就高表現效率之觀點而言,更佳為AAV5;就具備有效率地透過血腦障壁之特性之觀點而言,更佳為血清型9(AAV9)(Iwata N et al, Sci Rep. 2013;3:1472)。又,在本發明中所使用之病毒載體中,亦包括其衍生物。作為病毒載體之衍生物,公知有衣殼經修飾者等,特別是作為AAV之衍生物,例如可例示WO2012/057363中所揭示者等,但並不限定於該等。
將編碼AIM或AIM片段之核酸搭載於病毒載體時,較佳為使用腦特異性啟動子作為控制編碼AIM或AIM片段之核酸之表現的啟動子,以達到使AIM或AIM片段於作為目標之腦內特異性表現之目的。腦特異性啟動子包括SCG10、GFAP啟動子、突觸蛋白1啟動子、微管蛋白α1啟動子、鈣離子/鈣調素依賴性蛋白激酶II啟動子、神經元特異性烯醇化酶啟動子、PDGF(血小板衍生生長因子β)-β鏈啟動子等,但不限定於該等。又,除啟動子外,亦可將Poly A附加訊號、Kozak一致序列、標籤序列、連接序列、及NLS等公知之序列視需要適當與編碼AIM或AIM片段之核酸一同搭載於病毒載體。
包含編碼AIM或AIM片段之核酸的病毒載體可藉由公知之方法製備。簡而言之,製備編碼AIM或AIM片段之核酸,視需要製備***有具有所需功能之核酸(例如腦特異性啟動子等)之病毒表現用質體載體,將其轉染至合適之宿主細胞,短暫性產生包含本發明之聚核苷酸之病毒載體,將其回收即可。
例如於製備AAV載體之情形時,首先,製成如下之載體質體:留下野生型AAV之基因組序列中兩端之ITR,***編碼AIM或AIM片段之核酸,以代替除此以外之編碼Rep蛋白質及衣殼蛋白質之DNA。另一方面,將編碼形成病毒粒子所需之Rep蛋白質及衣殼蛋白質之DNA***至其他質體。進而,製成包含負責AAV之增殖所需之腺病毒輔助作用的基因(E1A、E1B、E2A、VA、及E4orf6)之質體作為腺病毒輔助質體。藉由將該等3種質體共轉染至宿主細胞,導致於該細胞內產生重組AAV(即AAV載體)。作為宿主細胞,較佳為使用能夠供給負責上述輔助作用之基因的基因產物(蛋白質)中之一部分的細胞(例如293細胞等),於使用此種細胞之情形時,無需將編碼可由該宿主細胞供給之蛋白質之基因搭載於上述腺病毒輔助質體。由於所產生之AAV載體存在於細胞核中,故將宿主細胞冷凍融解並回收,藉由使用氯化銫之密度梯度超速離心法或管柱法等進行分離及純化,由此製備所需之AAV載體。
於使用本發明之治療劑而治療或預防對象之缺血性疾病之情形時,其投予路徑除實現作為有效成分之AIM蛋白向患處之傳遞外,並無特別限定。作為較佳之投予路徑,可例舉:靜脈內投予、動脈內投予、皮下投予、腹腔內投予等,但並不限定於該等。
於一態樣中,於本發明之治療劑之有效成分為AIM蛋白或其功能性片段(以下有時稱為「AIM蛋白等」),且缺血性疾病為腦梗塞之情形時,已知AIM蛋白等通常不透過血腦障壁(Blood-Brain Barrier、BBB)。然而,如以下之實施例所證實,於腦梗塞發病時,BBB暫時部分地被破壞,其結果,可藉由靜脈投予而將AIM蛋白等傳遞至腦梗塞部位。於BBB發揮功能之情形時,例如可於對象之顱骨開設針孔,藉由顯微注射等本身已知之方法將本發明之治療劑直接導入至腦組織。或者,亦可將AIM蛋白封入至經修飾以可透過血腦障壁之脂質體中,並藉由靜脈投予等將其投予至對象,藉此將AIM蛋白傳遞至腦內。又,於本發明之治療劑所含有之成分為編碼AIM蛋白之核酸之情形時,亦可藉由從設於對象之顱骨之針孔直接導入本發明之治療劑之方法或使用上述之脂質體之方法等,而將編碼AIM蛋白之核酸傳遞至腦內。
於本發明之治療劑之成分為搭載有編碼AIM之核酸的病毒載體之情形時,可於對象之顱骨設置針孔,藉由顯微注射等本身已知之方法而將本發明之治療劑經由該針孔直接導入至腦組織。又,如上所述,若使用AAV9,則無需於對象之顱骨設置針孔,僅將本發明之治療劑注入至循環血液中便可使AIM於對象之腦內特異性表現,因此尤佳。即,於使用脂質體或AAV9等能夠透過血腦障壁之方法的態樣中,本發明之治療劑可非經口地投予,例如:靜脈內投予、動脈內投予、皮下投予、腹腔內投予。
於本發明之治療劑被製劑化為非經口投予用之情形時,例如可製劑化為注射劑、栓劑等。注射劑可包括靜脈注射劑、皮下注射劑、皮內注射劑、肌肉注射劑、點滴注射劑等劑型。該等注射劑可依照公知之方法製備。作為注射劑之製備方法,例如可藉由將AIM、編碼AIM之核酸、及/或搭載有編碼AIM之核酸的病毒等成分溶解、懸浮或乳化於通常用於注射劑之無菌之水性液、或油性液中來製備。作為注射用之水性液,例如可使用生理鹽水、含有葡萄糖或其他輔助劑之等張液等,亦可與適當之增溶劑,例如醇(例如乙醇)、多元醇(例如丙二醇、聚乙二醇)、非離子界面活性劑[例如聚山梨醇酯80、HCO-50(氫化蓖麻油之聚氧乙烯(50 mol)加合物)]等併用。作為油性液,例如使用芝麻油、大豆油等,亦可併用苯甲酸苄酯、苄醇等作為增溶劑。所製備之注射液較佳為填充於適當之安瓿中。
本發明之治療劑向對象之投予量只要是治療有效量,就沒有特別限定,可根據有效成分之種類及形態、對象之年齡及體重、投予排程、以及投予方法等適當地最佳化。
作為將本發明之治療劑投予至對象之時機,只要能夠治療缺血性疾病,就沒有特別限定。作為本發明之治療劑之投予時機,例如可例舉:缺血性疾病剛發病後、發病後1小時以內、2小時以內、3小時以內、4小時以內、5小時以內、6小時以內、7小時以內、8小時以內、9小時以內、10小時以內、11小時以內、12小時以內、24小時以內、36小時以內、48小時以內、72小時以內、96小時以內、120小時以內,但並不限定於該等。
本發明之治療劑亦可與用以治療缺血性疾病之其他治療劑併用。例如,亦可將用於治療梗塞之血栓溶解劑(t-PA、尿激酶等)、抗凝固劑(肝素、阿加曲班等)、抗血小板劑(奧紮格雷鈉、阿斯匹靈等)、腦保護劑(依達拉奉等)、抗腦水腫劑(甘油、甘露醇等)與本發明之治療劑組合使用。
藉由將本發明之治療劑應用於罹患缺血性疾病之對象,AIM或AIM片段被傳遞至對象之梗塞區域,或於對象之梗塞區域中表現。梗塞內之AIM或AIM片段與存在於梗塞內之死細胞碎片及/或DAMPs結合,促進該死細胞碎片之去除,且抑制無菌性炎症之擴散。其結果可治療對象之缺血性疾病。
再者,本說明書中疾病之「治療」,除疾病之治癒外,亦包括疾病之緩解及疾病程度之改善。
2. 無菌性炎症之抑制劑本發明亦提供一種損傷相關分子模式(DAMPs)介導之無菌性炎症之抑制劑(以下有時稱為「本發明之抑制劑」),其包含:巨噬細胞凋亡抑制劑(AIM)、具有AIM之生物活性之AIM片段、或編碼該AIM或AIM片段之核酸。
關於本發明之抑制劑中之作為有效成分AIM等,與「1.缺血性疾病之治療劑」相同。若本發明之抑制劑中所含之AIM等之量為可抑制對象中之無菌性炎症之量即可,並無特別限定。
至於本發明之抑制劑,於其有效成分為AIM蛋白或其功能性片段之情形時,該等與DAMPs結合而中和DAMPs之生物活性,及/或DAMPs被微神經膠質細胞/巨噬細胞有效率地吞噬,藉此抑制炎症之擴散。此處,作為AIM或其功能性片段所結合之DAMPs,可例舉:PRDX1、PRDX2、PRDX3、PRDX4、PRDX5、PRDX6、HMGB1、S100A8、S100A8/9、S100A9、及HSP70,但並不限於該等。又,於本發明之抑制劑之有效成分為編碼AIM或其功能性片段之核酸之情形時,該核酸所編碼之AIM或其功能性片段於細胞內轉譯,產生AIM蛋白或其功能性片段,由此獲得所需之效果。
若投予本發明之抑制劑之對象係可產生DAMPs介導之無菌性炎症之對象,則並無特別限定。作為投予本發明之抑制劑之對象,例如可例舉:恆溫動物(例如:人類、小鼠、大鼠、兔、羊、豬、牛、馬、貓、狗、猴、黑猩猩、鳥等),但並不限定於該等。於一態樣中,對象為人類。
作為將本發明之抑制劑投予至對象之時機,只要獲得所需之效果,就沒有特別限定。作為將本發明之抑制劑投予至對象之時機,例如可例舉:炎症剛發病後、發病後1小時以內、2小時以內、3小時以內、4小時以內、5小時以內、6小時以內、7小時以內、8小時以內、9小時以內、10小時以內、11小時以內、12小時以內、24小時以內、36小時以內、48小時以內、72小時以內、96小時以內、120小時以內,但並不限定於該等。
本發明之治療劑亦可與其他無菌性炎症之抗炎劑併用。
3. 缺血性疾病之治療方法本發明亦提供一種缺血性疾病之治療方法(以下有時稱為「本發明之治療方法」),其包含將巨噬細胞凋亡抑制劑(AIM)、具有AIM之生物活性之AIM片段、或編碼該AIM或AIM片段之核酸投予至罹患缺血性疾病之對象。
本發明之治療方法係藉由將本發明之治療劑投予至罹患缺血性疾病之對象而達成。關於本發明之治療方法中之AIM等或投予對象、投予時機等,與「1.缺血性疾病之治療劑」相同。
4. 無菌性炎症之抑制方法本發明提供一種DAMPs介導之無菌性炎症之抑制方法(以下有時稱為「本發明之抑制方法」),其包含向患有損傷相關分子模式(DAMPs)介導之無菌性炎症之對象投予巨噬細胞凋亡抑制劑(AIM)、具有AIM之生物活性之AIM片段、或編碼該AIM或AIM片段之核酸。
本發明之抑制方法係藉由將本發明之抑制劑投予至發病由DAMPs介導之無菌性炎症之對象而達成。關於本發明之抑制方法中之AIM等或投予對象、投予時機等,與「2.無菌性炎症之抑制劑」相同。
於以下之實施例中對本發明進行更具體之說明,但本發明並不受該等例子任何限制。
[實施例]
[實驗順序]
小鼠為了於純C57BL/6背景下製作AIM
-/-小鼠,將具有5'-caccgaacaatggagccatggccc-3'(序列編號3)或5'-caccggtgagtgtccctgcttctg-3'(序列編號4)之2種類型之pX335載體(Addgene、MA、USA)顯微注射至C57BL/6小鼠之受精卵中之前核,其後將2細胞胚胎移植至假孕之雌性小鼠之子宮。藉由PCR及序列確定,對從後代之尾部所單離之基因組DNA進行關於包含cd51(AIM)基因座之缺失之調查。使於最初之ATG區域具有適當缺失之AIM
-/-小鼠系統繁殖,並用於本實驗。小鼠於SPF條件下,於東京大學飼養。藉由Lysm-Cre基因轉殖小鼠與MafB
flox/flox小鼠之雜交而獲得之巨噬細胞特異性MafB缺失小鼠於準SPF(semi-SPF condition)條件下,於築波大學飼養。所有動物實驗嚴格按照NIH之「實驗動物護理及使用指南(the Guide for the Care and Use of Laboratory Animals)」之建議來進行。本協定經東京大學之動物實驗倫理委員會核准(許可證號:P10-143)。所有外科手術均於戊巴比妥鈉麻醉下進行,並盡最大努力來使痛苦最小化。
缺血性腦梗塞之誘發 (MCAO)將8~14週齡具有20~25 g體重之雄性小鼠用於MCAO。依照Shichita等人(2017,非專利文獻9)之順序,於麻醉(異氟醚;Pfizer)下將塗佈有矽橡膠之尼龍單絲(Doccol)橫向***至中大腦動脈45分鐘。於MCAO治療期間,使用雷射都卜勒血流儀(OMEGA)監測腦血流量,且將於顳葉之腦血流中顯示70%以上之減少之小鼠用於本實驗。
抗體及試劑組織學實驗中所使用之抗體及試劑如下所示:
一次抗體:AIM(用於小鼠及人類腦標本之IHC的rab2 兔多株抗體);由本研究實驗室製作之#35(用於小鼠AIM ELISA)、#6(用於人類AIM ELISA)、部分可從Transgenic Inc.購得)、HMGB1(選殖GT383、Gene-Tex、LA、USA)、PRDX1(兔多株抗體、abcam、Cambridge、UK)、S100A9(AF2065、R&D systems、NE、USA)、Doublecortin(Dcx;雞多株抗體、ab153668、abcam、Cambridge、UK)、Iba1(山羊多株抗體、abcam、Cambridge、UK)、生物素化6xHis標籤(D291-6、MBL、日本)、NF-κB p65(D14E12、Cell signaling technology)、磷酸化NF-κB p65(93H1、Cell signaling technology)、及CD11b微珠(選殖:M1/70.15.11.5、Miltenyi Biotec.Bergisch gladbach、德國)。二次抗體及相關試劑:Alexa fluor 488或594複合抗兔或大鼠 IgG(Molecular Probes)、抗人IgG-Fc-HRP(A80-104P、Bethyl laboratories, Inc.)、Streptavidin-Alexa fluor 488(Molecular Probes)、Streptavidin-HRP(554066、BD Pharmingen)、DAPI或Hoechst33342(Molecular Probes)、G-Block(Genostaff、日本東京)及HISTOFINE簡易染色小鼠MAX-PO(R)(用於細胞核;NICHIREI、日本)。標本使用共聚焦顯微鏡進行解析:FV10i-DOC及載玻片掃描器:SLIDEVIEW VS200(Olympus、東京)。
DAMPs 及相關蛋白質結合檢定中所使用之具有HA標籤之小鼠PRDXs由本研究實驗室製作。該等於HEK293T細胞中產生,其後使用抗HA抗體柱自細胞溶解物進行純化。其他DAMPs及相關蛋白質購自:
小鼠PRDX1-His(RPF757Mu01、USCN)、人類PRDX1-His(NBC118543、Novus Biologics)、小鼠S100A8(9877-S8-050、R&D Systems)、小鼠S100A9(2065-S9-050、R&D Systems)、小鼠S100A8/A9(8916-S8-050、R&D Systems)、人類S100A8(9876-S8-050、R&D Systems)、人類S100A9(9254-S9-050、R&D Systems)、人類S100A8/A9(8226-S8-050、R&D Systems)、小鼠HSP70-A1(低內毒素;ADI-ESP-502-D、Enzo)、人類HSP70(無內毒素;SPR-117A、StressMarq)、人類HMGB1(1690-HMB-050、R&D Systems)、小鼠TLR2-Fc(1530-TR、R&D Systems)、人類RAGE-Fc(1145-RG、R&D Systems)。
自腦中單離之 CD11b
+ 細胞 腦組織之單細胞分離:利用異氟醚對小鼠實施深度麻醉,經心臟注射1x 磷酸鹽緩衝鹽水(PBS)並放血。切除腦組織,將梗塞側或非梗塞側之腦切成小片,其後藉由在1xHBSS中含有2.5 U/mL膠原酶D、8.5 U/mL分離酶、25 mg/mL DNase I及Complete Mini(Roche; Basel, Switzerland)的酵素溶液,將該小片於37℃下處理1小時。使消化之組織多次通過a18 G-needle,以70 μm過濾器(Miltenyi Biotec、Bergisch gladbach、德國)進行過濾,其後以400 g、15分鐘、4℃進行離心分離。使細胞顆粒重新懸浮於35%Percoll之PBS溶液,以800 g、45分鐘、4℃進行離心分離。使顆粒重新懸浮於0.5%胎牛血清(FBS)之PBS溶液。最後,依照製造商之協定,使用CD11b微珠(Miltenyi Biotec、Bergisch gladbach、德國)單離出所有細胞中之CD11
+細胞。
AIM 之 ELISA所有ELIASA檢定均進行2次。作為腦組織溶胞物中之小鼠AIM,使用2種不同之大鼠抗小鼠AIM單株抗體(大鼠IgG、選殖#35;由本研究實驗室製作)藉由ELISA進行測定。使用C57BL/6小鼠血清所評價之檢定間變異係數相對於小鼠AIM為4.8%,且檢定間變異係數始終未達4.1%。使用以重組AIM蛋白作為標準而評價的定量下限相對於小鼠AIM為0.0625 ng/ml。
AIM 變異體蛋白質2CS變異體係藉由將小鼠AIM中之194位之半胱胺酸置換為絲胺酸(於核酸中將TGC置換為TCC)而製作。ΔSRCR3變異體係藉由使SRCR3區之胺基酸(從242位之天冬胺酸至352位之纈胺酸)缺失而製作。
rAIM 之純化將CHO-S細胞用pcDNA3.1-mAIM質體轉染,在CD Forti CHO培養基(Invitrogen、CA)中培養3天。rAIM使用複合有蛋白質G瓊脂糖(GE Healthcare Life Sciences、PA)之大鼠抗小鼠AIM單株抗體(自製),藉由培養上清液而進行純化。所結合之蛋白質於0.1 M Glycin-HCl、pH值3.0下溶出,於1 M Tris-HCl、pH值8.5下進行中和。蛋白質視需要使用Amicon超濾濃縮器(Millipore、MA)進行濃縮,於PBS中以-80℃進行保管。內毒素水準係依照製造商之協定,使用顯色LAL內毒素檢測系統(Genscript、NJ)進行測定。蛋白質濃度係依照製造商之協定,藉由BCA(二辛可寧酸)檢定(Pierce,Rockford、IL)而確定。重組AIM/IgM-Fc及IgM-Fc蛋白質如以前所記載之方式製作(Hiramoto et al.,(2018), The IgM pentamer is an asymmetric pentagon with an open groove that binds the AIM protein. Sci. Adv. 4, eaau1199.)。
向 AIM 之 DAMPs 之結合效率之評價除非另有明確記載,否則將96孔ELISA板在4℃下,整夜包覆以2 μg/mL溶解於碳酸氫鹽緩衝液(0.1 M NaHCO
3/Na
2CO
3,pH值9.6)之DAMPs及對照蛋白質。用含有tween之tris鹽緩衝液(TBS-T)將板清洗4次後,於室溫下用緩衝液(1%酪蛋白/TBS)包覆該等2小時。用TBS-T清洗後,將以各種濃度溶解於稀釋緩衝液(0.2%酪蛋白/TBS/2 mM CaCl
2)中之AIM添加於孔中,將板於室溫下培育1小時。用TBS-T/2 mM CaCl
2清洗5次後,將生物素化之抗小鼠或人類AIM抗體(小鼠用選殖#35、人類用選殖7)添加於孔中,於室溫下培育1小時。用TBS-T/2 mM CaCl
2清洗4次後,將於0.2%酪蛋白/TBS/2 mM CaCl
2中稀釋之Streptavidin-HRP添加於孔中,於室溫下培育1小時。用TBS-T/2 mM CaCl
2清洗4次後,添加TMB,於室溫下培育10~25分鐘。藉由添加1 N H
2SO
4來停止反應,使用多功能讀板儀以OD450nm解析吸光度。
藉由巨噬細胞捕捉 DAMPs 之活體外評價將自AIM
-/-小鼠單離出之腹腔巨噬細胞鋪在Lab-Tek II腔室載玻片(NalgeNunk)上,培養2小時以使巨噬細胞可攻擊載玻片。將該細胞在伴隨或不伴隨小鼠或人類rAIM(100 μg/mL)之情況下,與10 μg/mL之DAMPs(小鼠PRDX1-His、人類PRDX1-His、人類HMGB1、或小鼠S100A9)於37℃下培育10分鐘。對於小鼠PRDX1,亦使用2CS變異體或ΔSRCR3變異體。各配體及AIM均用對於His(對於小鼠及人類PRDX1)、HMGB1、或S100A9之抗體,其後用螢光複合之2級抗體進行免疫染色。細胞核以4',6-二脒基-2-苯基吲哚(DAPI)進行染色。於螢光共聚焦顯微鏡下解析細胞。
NFκB 活化檢定將RAW264.1細胞於96孔細胞培養板中,在小鼠AIM(10 μg/mL)之存在下或不存在下,與溶解於DMEM+0.1%FBS中之小鼠S100A9或RPDX(5 μg/mL)一同於37℃下培育30分鐘。用PBS清洗後,將細胞溶解於含有SDS之擔載緩衝液中,煮沸並擔載於SDS-PAGE凝膠。其後,使用抗磷酸化NFκB p65抗體(3033S、Cell Signaling Technology, Inc.)藉由免疫墨點法解析NFκB之磷酸化。合計之NFκB亦使用抗NFκB抗體(8242S、Cell Signaling Technology, Inc.)解析。
組織學檢查 梗塞腦之 AIM 檢測及其他 IHC:切除腦組織,於4%PFA之PBS溶液中固定24小時,其後包埋於石蠟中。將6 μm厚之切片以兔抗AIM多株抗體(Rab2;可用於人類及小鼠AIM),隨後以HITOFINE簡單染色小鼠MAX-PO(R)(NICHIREI、日本)培育30分鐘,並免疫染色。以二胺基聯苯胺四鹽酸鹽(DAB)染色後,將切片以蘇木精進行對比染色。為了阻擋自發螢光,於免疫染色前,將載玻片於用70%乙醇稀釋之0.5%蘇丹黑B(199664-25G、SIGMA-ALDRICH)中培育25分鐘。
DAMPs 染色:藉由將切片與兔抗PRDX1多株抗體(ab41906)、小鼠抗HMGB1單株抗體(GTX628834)、或山羊抗S100A9多株抗體(AF2065)與其後之HISTOFINE簡單染色小鼠MAX-PO(R、M或G)進行30分鐘培育而免疫染色。
MAP2 及 Iba1 檢測:藉由將切片與小鼠抗MAP2單株抗體(M9942)或山羊抗Iba1多株抗體(ab5076),與其後之HISTOFINE簡單染色小鼠MAX-PO(M或G)進行30分鐘培育而免疫染色。於與一級抗體反應之前,以各條件處置切片以進行抗原恢復。S100A9、HMGB1、及MAP2:於煮沸之含有0.05%Tween-20之Tris/EDTA緩衝液(pH值9.0)中20分鐘。PRDX1:於20 μg ProK之PBS溶液中在37℃下20分鐘。Iba1:於煮沸之含有0.05%Tween-20之檸檬酸緩衝液(10 mM、pH值6.0)中20分鐘。用DAB染色後,切片以蘇木精進行對比染色。
Oil red-O 染色:將腦組織於4%多聚甲醛(PFA)之PBS溶液中固定24小時,於30%蔗糖溶液中固定24~48小時,並與OCT化合物一同冷凍。10 μm切片以Oil red-O溶液(MUTO OURE CHEMICALS CO., LTD;日本東京)進行染色。人腦AIM之組織學資料購自Genostaff Co. Ltd(日本東京)。Genostaff Co. Ltd之資料係從FUNAKOSHI Co. Ltd(日本)購買市售之來自人類腦梗塞患者之腦組織塊之石蠟塊,並針對人類AIM及MAP2之連續切片進行染色而獲得者。
腦中之 DAMPs 之定量使用具備20×物鏡之載玻片掃描儀VS200(OLYMPUS,德國)獲得DAMPs-DAB染色影像,並進行數字記錄。數字影像解析係藉由市售之軟體HALO(IndicaLabs、Corrales、NM、USA)進行。使用基於對象共定位之演算法(object colocalization-based algorithms)檢測DAB及蘇木精訊號,細胞外之DAMPs之量係藉由自合計之DAB訊號減去與蘇木精共定位之DAB訊號而推定。
神經病變之評價 ( 神經學評分 )行動評價係於MCAO後每24小時進行一次。神經病變以先前所說明之方式(Jiang et al., 2005. Chlortetracycline and demeclocycline inhibit calpains and protect mouse neurons against glutamate toxicity and cerebral ischemia. J. Biol. Chem. 280, 33811-33818.)進行評分。標準概述如下:0分:正常;1分:將尾巴拉起時,伴隨/不伴隨不一致之捲曲的較弱之盤旋運動,<50%嘗試捲曲到對側部位;2點:較弱之有一致性之捲曲,>50%嘗試捲曲到對側部位;3分:較強、即刻之有一致性之捲曲,小鼠保持捲曲之姿勢1~2秒以上,小鼠之鼻子幾乎到達尾巴;4分:從極度捲曲發展為桶狀、缺乏歩行或翻正反射;5分:昏迷或瀕死。
統計學分析資料使用BellCurve for Excel(Social Survey Research Information Co.,Ltd.)進行分析,除非有特別說明,否則以平均值±s.d.示出。所有資料均藉由兩端檢定進行分析。成對之結果藉由Welch's t檢定進行評價。複數個組之間的比較使用伴隨Bonferroni事後比較檢定之單因子或多因子變異數分析進行分析。對於Kaplan-Meier曲線,使用廣義Wilcoxon檢驗確定P值。除非有特別說明,否則顯著性代碼如圖式簡單說明中說明所示。
定量 PCR 檢定使用ΔΔC
T法進行mRNA之定量評價,該ΔΔC
T法使用QuantStudio 3 實時PCR系統(Thermo Fisher Scientific)。以下例舉所使用之寡核苷酸序列。
名稱 序列 (5' → 3')f-GAPDH AGAACATCATCCCTGCATTC(序列編號5)
r-GAPDH CACATTGGGGGTAGGAACAC(序列編號6)
f-mAIMGAGGACACATGGATGGAATGT(序列編號7)
r-mAIMACCCTTGTGTAGCACCTCCA(序列編號8)
f-IL1B CAGGCAGGCAGTATCACTCA(序列編號9)
r-IL1B AGGCCACAGGTATTTTGTCG(序列編號10)
f-IL6 ATGGATGCTACCAAACTGGAT(序列編號11)
r-IL6 TGAAGGACTCTGGCTTTGTCT(序列編號12)
f-TNFA CTTCTGTCTACTGAACTTCGGG(序列編號13)
r-TNFA TGATCTGAGTGTGAGGGTCTG(序列編號14)
f-IL23 CAACTTCACACCTCCCTAC(序列編號15)
r-IL23 CCACTGCTGACTAGAACT(序列編號16)
f-MCP1 CATCCACGTGTTGGCTCA(序列編號17)
r-MCP1 GATCATCTTGCTGGTGAATGAGT(序列編號18)
f-CD11b CAATAGCCAGCCTCAGTGCC(序列編號19)
r-CD11b GAGCCCAGGGGAGAAGTGG(序列編號20)
f-β-肌動蛋白 CTAAGGCCAACCGTGAAAAG(序列編號21)
r-β-肌動蛋白 ACCAGAGGCATACAGGGACA(序列編號22)
f-18SrRNA CGCCGCTAGAGGTGAAATTCT(序列編號23)
r-18SrRNA CATTCTTGGCAAATGCTTTCG(序列編號24)
[ 實施例 1] 缺血性腦梗塞之發病後的腦 AIM 之增加AIM(=cd51)mRNA之表現於健康之小鼠之腦中未檢測出,但於藉由短暫性中大腦動脈阻塞術(MCAO)而於單側產生梗塞之小鼠之腦中,藉由定量PCR(QPCR)於梗塞側觀察到AIM mRNA表現顯著增加(圖1A)。進而,藉由對從產生梗塞之側之腦中所單離之巨噬細胞系細胞之細胞表面標記物CD11b陽性及陰性之細胞進行解析,可知於梗塞側之腦中增加之AIM mRNA由浸潤於CD11b陽性之微神經膠質細胞及梗塞病灶之巨噬細胞而表現(圖1A)。使用將MCAO後第7天之腦組織溶解為液狀者,藉由酵素結合免疫吸附檢查法(ELISA)進行評價,於未引起梗塞之側之腦中未觀察到AIM蛋白之增加,但於引起梗塞之側之腦中,每1 mg腦組織中存在有最大250 ng之AIM蛋白(圖1B)。以免疫組織化學染色(IHC)評價於產生此種梗塞之腦內之AIM表現之增加,於MCAO後第7天,於因微管相關蛋白2(MAP2)陰性之梗塞而死亡之神經細胞之區域,AIM被較強地染色(圖1C)。於腦梗塞之人類患者之腦中亦觀察到此種梗塞區中之AIM之累積(圖1D)。若以高倍率進行觀察,則於小鼠及人類之腦中之Iba1陽性巨噬細胞樣細胞(藉由箭頭指示)中,AIM被較強地染色(圖1C、1D),證實了QPCR結果顯示圖1A之CD11b陽性之巨噬細胞系細胞中AIM mRNA之表現增加。浸潤於梗塞區(MAP2陰性)之Iba1陽性巨噬細胞被oil-red-O較強地染色,表示其積極地吞噬死細胞碎片(圖1E)。相比之下,於實施MCAO之AIM缺失(AIM
-/-)小鼠中,梗塞區域中之Iba1陽性巨噬細胞大部分為oil-red-O陰性,表示其不能吞噬死細胞碎片(圖1E)。即,提示AIM有助於吞噬去除梗塞區域中死細胞碎片。
[ 實施例 2]AIM 降低由 DAMPs 引起之梗塞後之腦之炎症,改善腦梗塞之預後進行因MCAO而發病梗塞之腦之IHC解析,AIM
-/-小鼠之梗塞區之細胞外PRDX1(與細胞體及細胞核共定位者)之量與野生型小鼠之腦相比而顯著更多。進而,從MCAO後即刻起,一天一次經靜脈投予0.5 mg/小鼠之rAIM,結果野生型及AIM
-/-小鼠之雙方之PRDX1均降低(圖2A)。已知於缺血性腦梗塞之急性期,血腦障壁(blood-brain-barrier、BBB)會被暫時、部分地破壞(55. Zou, R. et al., (2015). Electroacupuncture pretreatment attenuates blood-brain barrier disruption following cerebral ischemia/reperfusion. Mol. Med. Rep. 12, 2027-2034.; Choi, K.H. et al., (2016). Overrexpressionof caveolin-1 attenuates brain edema by inhibiting tight junction degradation. Oncotarget. 7, 67857-67867.)。實際上,若將0.5 mg之rAIM經靜脈投予至MCAO後第3天之AIM-/-小鼠,則發生梗塞之腦組織被抗AIM抗體廣泛染色,故表明所投予之rAIM通過BBB(圖5)。於MCAO後第3天及第7天,PRDX1之減少與腦內之AIM量之關聯很明顯(圖2A,圖表)。較多之AIM
-/-小鼠於MCAO後第3天前迅速地死於健康狀況不佳。該等之即將死亡之前(從第2天至第3天)之梗塞區域顯著地具有大量S100A9(圖2B)。即,梗塞發病後,一定數量之AIM
-/-小鼠中,大量DAMPs於腦內蓄積而造成死亡。又,野生型小鼠於第3天之前未死亡。在AIM
-/-小鼠、野生型小鼠雙方中,藉由投予rAIM,浸潤於梗塞腦之CD11b陽性之巨噬細胞中炎症性細胞激素之mRNA之表現量均減少(圖2C)。即,提示AIM可減少梗塞區域中之炎症。於未投予rAIM之野生型小鼠與AIM
-/-小鼠中,梗塞病灶之巨噬細胞中炎症性細胞激素mRNA之表現量未發現顯著差異(圖2C)。認為其原因在於:於野生型小鼠中,在第7天之前,腦內之AIM尚未增加(圖1A)。腦中DAMPs之量與炎症狀態相關,在AIM
-/-小鼠中,於MCAO後第7天之死亡率顯著高於野生型小鼠,但若連續幾天經靜脈投予0.5 mg/小鼠之rAIM,則死亡率大大減少(圖2D)。投予相對較少量之rAIM(0.1 mg/小鼠)之野生型小鼠在MCAO後一隻都沒有死亡(圖2D)。與存活率之改善同樣,梗塞後之神經症狀(偏癱)於AIM
-/-小鼠中比野生型小鼠更嚴重,但藉由rAIM投予再次得到改善(圖2E)。綜上所述,於梗塞之急性期靜脈投予rAIM可顯著地改善預後。即,認為於腦內表現、或經靜脈投予到達梗塞病灶之AIM可促進梗塞病灶中死細胞碎片或DAMPs之去除,減少梗塞後之腦內之炎症,從而改善預後。
[ 實施例 3]AIM 亢進了 DAMPs 之吞噬去除,且經由直接結合而中和了 DAMPs 。為了驗證關於該AIM之作用之假設,本發明人首先使用ELISA系統來確認AIM是否以其與死細胞碎片結合相同之方式直接結合於DAMPs。如圖3A及圖4所示,AIM與PRDX1、S100A9、及HMGB1、以及許多其他DAMPs劑量依賴性地結合。由於大部分DAMPs帶負電(Uchida et al., 2014. Natural antibodies as a sensor of electronegative damage-associated molecular patterns (DAMPs). Free Radic. Biol. Med. 72, 156-161.; Knoopset al., 2018. Specific Interactions Measured by AFM on Living Cells between Peroxiredoxin-5 and TLR4: Relevance for Mechanisms of Innate Immunity. Cell Chem. Biol. 25, 550-559.e3.),故認為與AIM與死細胞碎片之結合(Arai et al., 2016; Tomita et al., 2017)同樣,AIM可經由電荷相互作用而與DAMPs結合。為了確認該可能性,本發明人驗證了變異小鼠AIM(ΔSRCR3)與DAMPs之結合,該變異小鼠AIM(ΔSRCR3)缺失了包含帶正電之表面胺基酸簇的AIM之羧基末端。正如所預想那樣,ΔSRCR3並未顯示出與任意DAMP之實質性結合(圖3B)。除此以外,若於高劑量NaCl之存在下抑制電荷之相互作用,則AIM與DAMPs之間的結合顯著地減少(圖3C)。進而,於還原劑三(2-羧乙基)膦(TCEP)之存在下,AIM與DAMPs之結合被抑制(圖3D),提示雙硫鍵之進一步參與。由於存在於AIM之SRCR2區的孤立之半胱胺酸殘基是唯一可用於與其他分子形成雙硫鍵之胺基酸殘基(Miyazaki et al., (1999), Increased susceptibility of thymocytes to apoptosis in mice lacking AIM, a novel murine macrophage-derived soluble factor belonging to the scavenger receptor cysteine-rich domain superfamily. J. Exp. Med. 189, 413-422.; Hiramoto et al., (2018)(上述)),因此該半胱胺酸殘基與DAMPs形成雙硫鍵之可能性較高。為了驗證該可能性,本發明人製作了該半胱胺酸被置換為 絲胺酸之其他小鼠AIM變異體(2CS),評價其與DAMPs之結合。如圖3E所示,2CS變異體失去了已確認之所有與DAMPs結合之能力。因此,認為AIM藉由電荷相互作用及形成雙硫鍵這2種獨立之結合模式而與DAMPs穩定地結合。正如本發明人先前所報告那樣(Hiramoto et al., (2018)(上述)),該等2個結合部位亦用於AIM與IgM五聚物之結合。該事實提示當AIM與IgM五聚物結合時,失去與DAMPs之結合能力。實際上,AIM與IgM五聚物之複合體幾乎未顯示與DAMPs結合(圖3F)。進而,沒有結合AIM之IgM五聚物劑量依賴性地抑制AIM與DAMPs之結合(圖3G)。即,與IgM五聚物之結合在功能上使AIM與DAMPs之結合失活。進而,本發明人於螢光顯微鏡下評價於rAIM之存在下,活體外由巨噬細胞對PRDX1、HMGB1、及S100A9之捕捉與吸收是否受到抑制。如圖3H所示,添加之rAIM與全部種類之DAMPs較強地共定位,證實了ELISA所獲得之結合資料(圖3A)。進而,存在於細胞表面或細胞內之PRDX1、HMGB1、S100A9之量在rAIM之存在下顯著地增加,該結果提示rAIM促進了巨噬細胞對DAMPs之捕捉與吸收(圖3I)。有趣的是,AIM結合較強地誘導了PRDX1之凝集(圖3J),這讓人想起AIM誘導性細菌凝集(Sarrias et al., (2005). A role for human Sp alpha as a pattern recognition receptor. J. Biol. Chem. 280, 35391-35398.)。已知相對較大之分子更容易被巨噬細胞吸收(Aderem & Underhill, (1999). Mechanisms of Phagocytosis in Macrophages. Annu. Rev. Immunol. 17, 593-623.; Sarrias et al., (2005) (上述); Canton et al., Scavenger receptors in homeostasis and immunity. Nat. Rev. Immunol. 13, 621-634.),因此AIM對PRDX1之凝集誘導更有利於PRDX1之吞噬。未與DAMPs結合之ΔSRCR3 AIM變異體與2CSAIM變異體(圖3B、3E)雙方均不誘導PRDX1凝集(圖3J)。進而使用基於ELISA之檢定進行評價,結果與AIM之結合本身抑制PRDX1或HMGB1與TLR2或RAGE之結合(圖3K)。該結果提示AIM與DAMPs之結合具有如下作用:不僅亢進了巨噬細胞對DAMPs之吸收,而且競爭性地妨礙DAMPs與TLR或RAGE之電荷相互作用。此種AIM之作用導致整體DAMPs之炎症活性減少。作為實際之實驗例,如圖3L所示,巨噬細胞中之PRDX及S100NFκB活化能由於rAIM之添加而顯著地降低。
如上述之實驗結果所示,AIM可藉由吞噬而去除與中和因缺血而產生之梗塞區域中之各種DAMPs,從而抑制炎症反應,且促進死細胞碎片之高效率去除,從而治療腦梗塞。該AIM之作用機制不僅可應用於腦梗塞,亦可應用於以心肌梗塞為首之缺血性疾病之治療。因此,本發明可用作缺血性疾病之治療劑。
[產業上之可利用性]
根據本發明,可治療包括腦梗塞之缺血性疾病。因此,本發明於醫療領域極其有用。
本申請基於在日本申請之特願2021-125789(申請日:2021年7月30日),其全部內容包含於本說明書中。
圖1係表示梗塞腦中AIM表現之圖。(A)於大腦中動脈閉塞(Middle Cerebral Artery Occlusion,MCAO)後第1、3、及7天,使用從源自野生型小鼠之全組織或藉由CD11b(巨噬細胞/骨髄性細胞標記物)表現而分離之細胞單離之RNA進行腦中AIM(cd51)mRNA水準之聚合酶鏈反應定量聚合酶鏈反應(quantitative 聚合酶鏈反應,QPCR)解析。L:左葉、非梗塞;R:右葉、梗塞。示出對於MCAO前之全組織之mRNA水準(Pre)之相對值。比例尺:平均值之標準偏差。統計:伴隨Bonferroni事後比較檢定之多因子變異數分析(multi-way ANOVA)。p:*<0.05、**<0.01、***<0.001(左葉或右葉之不同天子的值之間);#<0.05、###<0.001(同一天之左葉及右葉的值之間)。n=3。(B)使用來自梗塞腦(右葉;R)及非梗塞腦(左葉;L)之溶胞物藉由ELISA解析腦中AIM蛋白之量。值以μg AIM/mg腦來表示。n=4。(C)MCAO後第7天之來自野生型小鼠之腦標本中的AIM之免疫組織化學(IHC)分析。將對於微管相關蛋白2(Microtubule-associated protein 2,MAP2)為陰性之梗塞區進行AIM染色。對於紋狀體之一部分(紅色方塊),將AIM及MAP2進一步放大顯示(從左起第3行,藉由紅線包圍四邊之部分)。最右之版面以高放大率示出對Iba1及AIM染色之連續切片。Iba1陽性之巨噬細胞對AIM顯示強陽性。縮寫:ctx:大腦皮層、st:紋狀體、hi:丘腦下部、th:丘腦、mb:中腦、cb:大腦。比例尺:1 mm,其中,冠狀切面之高放大版面中之比例尺表示100 mm,最右之IHC:Iba1及IHC:AIM之版面中之比例尺表示5 mm。(D)對來自梗塞區域之人類腦標本進行AIM染色。一併示出僅使用二次抗體之對照染色(下方之版面)。所有區域均為MAP2陰性(未示出資料)。AIM之強染色表示於巨噬細胞中產生AIM(根據該等之形態判斷;藉由箭頭指示),該等與(A)中示出之QPCR資料一致。比例尺:50 mm。(E)對MCAO後第7天之來自野生型及AIM
-/-腦之連續冷凍切片進行oil-red O及Iba1染色。於野生型小鼠中,梗塞之紋狀體區域之較多Iba1陽性巨噬細胞對於oil-red-O為陽性(紅色訊號),但於AIM
-/-小鼠中,該等之大部分對於oil-red-O為陰性。左列之4個版面中之比例尺表示1 mm,中央及右列之8個版面中之比例尺表示100 mm。
圖2係表示AIM降低DAMP水準,抑制梗塞區之炎症,藉此改善梗塞後預後之圖。(A)對MCAO後第3天及第7天之伴隨或不伴隨rAIM之投予(從MCAO後第1天起每天0.5 mg)之野生型(AIM
+/+)及AIM
-/-小鼠之腦標本進行PRDX1、HMGB1、S100A9染色,且為了鑑定細胞核,亦藉由蘇木精(hematoxylin)進行染色。示出於MCAO後第7天染色之PRDX1(上方之版面)及MAP2(下方之版面)之代表性照片。比例尺:500 mm。對在MAP2陰性區域中未與蘇木精訊號共定位之PRDX1的陽性訊號數(棕色之訊號),藉由MAP2陰性區域之面積(mm
2)進行標準化。n=3-8。統計:伴隨Bonferroni事後比較檢定之多因子變異數分析。p:*<0.05;**<0.01(有無AIM投予之間之比較)。##<0.01(AIM
+/+v. s. AIM
- / -)。縮寫:st:紋狀體、hi:丘腦下部、th:丘腦。(B)瀕死之2隻及比較健康之1隻AIM
-/-小鼠及1隻野生型小鼠於第3天之100A9染色。於第3天,與比較健康之AIM
-/-小鼠或野生型小鼠相比,於瀕死之AIM
-/-小鼠之腦中更明顯地觀察到非常強之訊號。縮寫:st:紋狀體、hi:丘腦下部、th:丘腦。由MCAO引起之中大動脈中血流之減少於所試驗之小鼠間沒有差異。比例尺:500 mm。(C)於MCAO後第4天,使用從伴隨或不伴隨rAIM投予(從MCAO後第1天起每天0.5 mg)之野生型及AIM
-/-小鼠之梗塞腦單離之CD11
+巨噬細胞/骨髄性細胞,進行炎症性細胞激素之mRNA之QPCR解析。示出了與CD11
+細胞之相對值。分別是n=4-6。顯示平均值±s.e.m.。統計:伴隨Bonferroni事後比較檢定之多因子變異數分析。*p<0.05。(D)MCAO後之野生型、AIM
-/-、rAIM(0.5 mg)投予AIM
-/-、及rAIM(0.1 mg)投予野生型小鼠之存活率。n=38(野生型)、21(AIM
-/-)、9(野生型+rAIM投予)、12(AIM
-/-+rAIM投予)。從第1天起藉由每天靜脈注射而投予rAIM。使用廣義Wilcoxon檢驗評價MCAO後7天間之存活之統計學有意義差。p=0.0000347(野生型小鼠 vs. AIM
-/-小鼠;0.0780(伴隨rAIM投予之AIM
-/-小鼠vs. 不伴隨rAIM投予之AIM
-/-小鼠)。(E) (D)中4組之小鼠之神經學評分。n=17(野生型)、11(AIM
-/-)、9(野生型+rAIM投予)、6(AIM
-/-+rAIM投予)。顯示平均值±s.e.m.。統計:伴隨Bonferroni事後比較檢定之多因子變異數分析。p:*<0.05;***<0.001(野生型vs.AIM
-/-)、##<0.01;###<0.001(野生型vs.野生型+rAIM投予)、§§§<0.001(AIM
-/-vs. AIM
-/-+rAIM投予)。
圖3係表示藉由使AIM與DAMPs結合來抑制DAMPs之吞噬去除之亢進、及DAMPs對模式識別受體之結合之圖。(A)AIM對各種DAMPs之活體外結合檢定。示出各種濃度之人類或小鼠AIM對於PRDX1、HMGB1、S100A9、及作為對照之牛血清白蛋白(BSA)(以2 μg/mL塗佈於板上)之結合能力。檢定連續進行3次,示出平均值±s.d.。將AIM對DAMPs之結合結果示於圖4。於150 mM(生理)或500 mM(過量)NaCl之存在下,(B)小鼠ΔSRCR3變異體或(C)野生型小鼠AIM(各20 μg/mL)對PRDX1、HMGB1、及S100A9之結合活性。於過量NaCl之存在下,結合大大減少。(D)於50 μM TCEP之存在下之小鼠野生型AIM(20 μg/mL)或(E)小鼠2CS變異體對PRDX1、HMGB1、及S100A9之結合活性。人類AIM亦得到相同之結果(未示出資料)。(F)藉由與(A)相同之手法確認重組小鼠AIM/IgM-Fc五聚物複合體對DAMPs之結合活性。結合減少。(G)小鼠AIM對DAMPs之結合由於小鼠IgM-Fc五聚物而劑量依賴性地減弱。於(B)~(G)中,檢定連續進行3次,示出平均值。統計:伴隨Welch's t檢定(B)-(F)、Bonferroni事後比較檢定之單因子變異數分析、p:*<0.05,**<0.01,***<0.001(vs.對照)。(H)將自AIM
-/-小鼠單離之腹腔巨噬細胞於腔室載玻片上,於rAIM之存在或不存在下,以PRDX1、HMGB1、及S100A9進行攻擊。培育10分鐘後,固定細胞,用各DAMP(綠色)、AIM(紅色)、及細胞核(藍色)進行染色,於螢光共聚焦顯微鏡下進行分析。白色區域表示DAMPs與AIM之共定位。亦觀察到rAIM之附著於一些死細胞上(黃色之箭頭)。於HMGB1染色中觀察到源自二次抗體之非特異性訊號(背景)(橙色之箭頭)。(I)使用軟體Halo確定存在於細胞表面及細胞內之各PRDX1、HMGB1、或S100A9之量,並以圖表顯示。值係3次實驗(n=4-13)之結果之平均值。統計:Welch's t檢定。p:*<0.05,**<0.01。(J)AIM(野生型)、ΔSRCR3變異體、及2CS變異體對PRDX1凝集之誘導。(K)於各種濃度之rAIM之存在或不存在下,PRDX1與S100A9對TLR2之結合及HMGB1對RAGE之結合。rAIM之存在使PRDX1及S100A9對TLR2之結合能力劑量依賴性地降低,且使HMGB1對RAGE之結合能力降低。檢定連續進行3次,示出平均值。統計:伴隨Bonferroni事後比較檢定之單因子變異數分析。p:*<0.05,**<0.01。(L)將RAW264.1小鼠巨噬細胞於rAIM之存在或不存在下,與小鼠PRDX或S100A9在37℃下培育30分鐘。其後,回收細胞,藉由免疫墨點法針對磷酸化(指示NFκB活化)分析細胞溶解物。對訊號強度進行定量,並以圖表顯示。值表示磷酸化之NFκB p65之強度相對於合計之NFκB p65之強度。rAIM之存在使NFκB之磷酸化減少。統計:伴隨Bonferroni事後比較檢定之雙因子變異數分析。p:*<0.05,***<0.001。
圖4係表示AIM與各種DAMPs結合之圖。示出各種濃度之人類或小鼠AIM對於PRDXs、S100A8、S100A8/9、HSP70及作為對照之牛血清白蛋白(BSA)(以2 μg/mL塗佈於板上)之結合能力。檢定連續進行3次,示出平均值±s.d.。
圖5係表示rAIM通過血腦障壁之圖。將實施MCAO之AIM
-/-小鼠於MCAO後第3天藉由靜脈注射而投予0.5 mg之rAIM,於rAIM投予之1小時後宰殺。將腦之梗塞側之矢狀切面以AIM(上部)及MAP2(下部)染色。MAP2陰性之梗塞區進行AIM染色。比例尺:1 mm。
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Claims (8)
- 一種缺血性疾病之治療劑,其包含巨噬細胞凋亡抑制劑(AIM)、具有AIM之生物活性之AIM片段、或編碼該AIM或AIM片段之核酸。
- 如請求項1之治療劑,其中缺血性疾病係選自由腦梗塞、心肌梗塞、肢體缺血、肺梗塞、脾梗塞、腸梗塞、及柏格氏病所組成之群。
- 一種損傷相關分子模式(DAMPs)介導之無菌性炎症之抑制劑,其包含巨噬細胞凋亡抑制劑(AIM)、具有AIM之生物活性之AIM片段、或編碼該AIM或AIM片段之核酸。
- 如請求項3之抑制劑,其中DAMPs係選自由PRDX1、PRDX2、PRDX3、PRDX4、PRDX5、PRDX6、HMGB1、S100A8、S100A8/9、S100A9、及HSP70所組成之群中之至少一種。
- 一種缺血性疾病之治療方法,其包含將巨噬細胞凋亡抑制劑(AIM)、具有AIM之生物活性之AIM片段、或編碼該AIM或AIM片段之核酸投予至罹患缺血性疾病之對象。
- 如請求項5之治療方法,其中缺血性疾病係選自由腦梗塞、心肌梗塞、肢體缺血、肺梗塞、脾梗塞、腸梗塞、及柏格氏病所組成之群。
- 一種DAMPs介導之無菌性炎症之抑制方法,其包含向患有損傷相關分子模式(DAMPs)介導之無菌性炎症之對象投予巨噬細胞凋亡抑制劑(AIM)、具有AIM之生物活性之AIM片段、或編碼該AIM或AIM片段之核酸。
- 如請求項7之抑制方法,其中DAMPs係選自由PRDX1、PRDX2、PRDX3、PRDX4、PRDX5、PRDX6、HMGB1、S100A8、S100A8/9、S100A9、及HSP70所組成之群中之至少一種。
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