TW202237169A - 巨噬細胞活化劑 - Google Patents
巨噬細胞活化劑 Download PDFInfo
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Abstract
本發明以簡單的製程提供一種由Gc蛋白組成的巨噬細胞活化劑。巨噬細胞活化劑包括未附加N-乙醯半乳胺糖(N-acetylgalactosamine)的Gc蛋白。
Description
本發明係關於一種巨噬細胞活化劑。
巨噬細胞是白血球的一種,存在於皮膚、肺、腸和大腦等身體的所有組織中。已知巨噬細胞在先天性免疫和後天性免疫兩者中都具有重要的功能。在先天性免疫中,將噬中性球與侵入生物體的異物一起吞噬,透過巨噬細胞內的區域蛋白質分解酵素或脂肪酶消化。在後天性免疫中,來自已吞噬異物的分子作為抗原呈遞在細胞表面以活化輔助型T細胞。進一步地,已知巨噬細胞與發炎性疾病、動脈硬化、肥胖、癌症等各種疾病狀況的形成相關。
Gc蛋白是一種存在於血漿中的蛋白質,因為與維生素D結合,所以也被稱為維生素D結合蛋白質。Gc蛋白雖然具有活化巨噬細胞的能力,但一直以來認為在Gc蛋白的轉譯後所添加的糖鏈結構(sugar chain structure)對巨噬細胞的活化是很重要的。Gc蛋白以下述的狀態表現:由唾液酸(sialic acid)和半乳糖(galactose)與GalNAc(N-乙醯半乳胺糖)結合的三糖所構成的O型糖鏈結合在第418位或第420位的蘇胺酸(Thr)上的狀態。在唾液酸或半乳糖與GalNAc結合的狀態下,或是在GalNAc未與第418位或第420位的蘇胺酸結合的狀態下,巨噬細胞不能被活化。因此,專利文獻1~2中揭示了一種使β-半乳糖苷酶(β-galactosidase)或唾液酸酶(sialidase)作用於Gc蛋白來除去唾液酸和半乳糖的方法。
當在特定的細胞中重組表現Gc蛋白時,能夠製造出在第418位或第420位的蘇胺酸(Thr)上只有GalNAc結合的Gc蛋白(專利文獻3)。然而,能夠使用的細胞種類是有限的。此外,尚未充分明瞭Gc蛋白的糖鏈結構與活化巨噬細胞的能力之間的關係。
[先行技術文獻]
[專利文獻]
[專利文獻1] 日本特表平6-510908號公報
[專利文獻2] 日本特表平11-511962號公報
[專利文獻3] 國際公開WO2019-117295號
[發明所欲解決的問題]
本發明的課題在於以簡單的製程提供由Gc蛋白組成的巨噬細胞活化劑。
[用以解決問題的手段]
本發明人對Gc蛋白的糖鏈結構與巨噬細胞活化能的關係進行研究的結果發現,Gc蛋白的第418位或第420位的Thr中的N-乙醯半乳胺糖對於巨噬細胞的活化能不是必需的,從而完成了本發明。
亦即,本發明係關於一種巨噬細胞活化劑,其包括未附加N-乙醯半乳胺糖的Gc蛋白。
所述Gc蛋白以來自人、牛、山羊、或小鼠的血清或乳汁之純化物為佳。
未附加N-乙醯半乳胺糖的胺基酸以序列識別號1中的第418位或第420位的蘇胺酸為佳。
此外,本發明係關於一種醫藥組成物,其包括前述巨噬細胞活化劑,且用於抗癌、抗感染症、抗自體免疫疾病、抗自閉症、抗發炎性疾病、抗腦・神經變性(degenerative)疾病、改善皮膚、或治療心臟病。
又,本發明係關於一種巨噬細胞活化劑的製造方法,包括使Gc蛋白與N-乙醯半乳胺糖酶(N-acetylgalactosaminidase)接觸的步驟。
前述製造方法以在與N-乙醯半乳胺糖酶接觸的步驟之前,包括使Gc蛋白與神經胺糖酸苷酶(neuraminidase)接觸的步驟為佳。
[發明功效]
本發明的巨噬細胞活化劑所包含的Gc蛋白中未附加N-乙醯半乳胺糖,能夠經由簡單的酵素處理來製造。
[用以實施發明的形態]
<<巨噬細胞活化劑>>
Gc蛋白是存在於哺乳動物的血漿、乳汁等體液中的蛋白質,因為與維生素D結合,所以也被稱為維生素D結合蛋白質。Gc蛋白在血漿、乳汁等體液中第418位或第420位的蘇胺酸(Thr)上,以結合有由唾液酸和半乳糖與GalNAc(N-乙醯半乳胺糖)結合的三糖所構成的O型糖鏈的形態存在,這種形態不具有活化巨噬細胞的能力。另一方面,已知在第418位或第420位的蘇胺酸上只結合有GalNAc的形態具有活化巨噬細胞的能力,稱為GcMAF(Gc蛋白衍生的巨噬細胞活化因子;Gc protein-derived macrophage activating factor)。相較於此,本發明的巨噬細胞活化劑的特徵在於包括未附加N-乙醯半乳胺糖的Gc蛋白。
<Gc蛋白>
Gc蛋白沒有特別限制,只要是來自哺乳類即可,可列舉例如來自人、牛、山羊、小鼠的Gc蛋白,但為了在投予至人類時不產生免疫反應,以來自人為佳。
作為具體的Gc蛋白,可列舉與序列表的序列識別號1所示的胺基酸序列具有85%以上的序列同一性的多肽,或是由序列表的序列識別號1所示的胺基酸序列中有一個或複數個胺基酸經缺失、插入、取代及/或添加的胺基酸序列組成的多肽。
與序列識別號1所示的胺基酸序列的序列同一性以90%以上為佳,以95%以上為較佳,以98%以上為更佳,以99%以上為特別佳。
在序列識別號1所示的胺基酸序列中,經缺失、插入、取代及/或添加的胺基酸數量以68個以下為佳,以45個以下為較佳,以22個以下為更佳,以9個以下為進一步較佳,以4個、3個、或2個以下為特別佳。
未附加N-乙醯半乳胺糖的胺基酸的位置沒有特別限制,但以Gc蛋白的胺基酸序列中的蘇胺酸為佳,以序列識別號1中第418位或第420位的蘇胺酸為佳。
<血清、乳汁>
能夠使用前述哺乳類的乳汁或血清等體液中所包含的Gc蛋白作為Gc蛋白。能夠直接使用含有Gc蛋白的乳汁或血清,也能夠從乳汁或血清純化出Gc蛋白並使用。
血清沒有特別限制,只要是由從哺乳類採取的血液製備的即可。能夠透過常用方法來製備血清。此外,為了降低因使用他人的血清而引起免疫反應的風險或感染的風險,能夠使用由被投予本發明巨噬細胞活化劑的患者自身的血液所製備的血清。此外,也能夠使用從健康的他人的血液所製備的血清。
作為從血清純化Gc蛋白的方法,可列舉例如從血清除去白蛋白(albumin)等,經由維生素D結合性管柱吸附並洗提Gc蛋白並進行透析的方法。
乳汁以只在分娩後預定天數內分泌的初乳為佳。從乳汁純化Gc蛋白時,能夠透過例如減少水量、除去酪蛋白(casein)和脂肪來進行純化。
<基因重組>
Gc蛋白可以是經由基因重組表現的蛋白質。作為經由基因重組表現Gc蛋白的方法,可列舉培養導入了編碼Gc蛋白的基因的宿主細胞之方法、使用包括編碼Gc蛋白的基因的無細胞表現系統之方法等。宿主細胞並沒有特別限制,可列舉鏈絲菌屬(Streptomyces)、紅球菌屬(Rhodococcus)、大腸桿菌屬(Escherichia)、芽孢桿菌屬(Bacillus)、假單胞菌屬(Pseudomonas)、短桿菌屬(Brevibacterium)、鏈球菌屬(Streptococcus)、乳酸桿菌屬(Lactobacillus)、酵母菌屬(Saccharomyces)、克魯維酵母屬(Kluyveromyces)等的微生物、高等真核生物的細胞等。作為高等真核生物的細胞,可列舉來自人、倉鼠、雞、昆蟲的細胞,作為具體的細胞株,可列舉CHO細胞、HeLa細胞、HEK293細胞、sf9細胞、DT40細胞等。細胞培養的方法可以是懸浮培養,也可以是附著培養,但以懸浮培養為佳。此外,為了避免雜質的混入,以使用無血清培養基為佳。
<酵素處理>
當Gc蛋白中附加有N-乙醯半乳胺糖時,能夠經由除去N-乙醯半乳胺糖來獲得本發明的Gc蛋白。N-乙醯半乳胺糖的除去方法沒有特別限制,但以經由酵素處理進行為佳。作為使用的酵素,可列舉內-α-N-乙醯半乳胺糖酶(endo-α-N-acetylgalactosaminidase)、外-α-N-乙醯半乳胺糖酶(exo-α-N-acetylgalactosaminidase)。
作為內-α-N-乙醯半乳胺糖酶,可列舉例如來自大腸桿菌(Escherichia coli)、來自雙叉桿菌(Bifidobacterium longum)、來自肺炎鏈球菌(Streptococcus pneumoniae)、來自牛肝(bovine liver)的內-α-N-乙醯半乳胺糖酶等。作為市售物,可列舉例如默克(MERCK)公司的目錄號#324716等。內-α-N-乙醯半乳胺糖酶可以單獨使用,也可以組合使用2種以上。
此外,在唾液酸與N-乙醯半乳胺糖結合的情況下,由於內-α-N-乙醯半乳胺糖酶對N-乙醯半乳胺糖的切斷效果降低,因此以預先切割唾液酸為佳。可以透過唾液酸酶處理Gc蛋白來進行唾液酸的切割。
作為唾液酸酶,可列舉例如來自產氣莢膜梭菌(Clostridium perfringenes)、來自鏈球菌(Streptococcus 6646K)、來自霍亂弧菌(Vibrio cholerae)、來自產脲節桿菌(Arthrobacter ureafaciens)的唾液酸酶等。作為市售物,可列舉例如SIGMA-ALDRICH公司的產品編號(SIGMA Prod. Nos.)N2876、N2133、N2904、N3001、N5631、生化學工業股份有限公司(Biochemical Biobusiness)的代碼編號(Code Number)120052、BioLabs公司目錄編號(Catalog #)P0720L、P0720S等。唾液酸酶可以單獨使用,也可以組合使用2種以上。
Gc蛋白與內-α-N-乙醯半乳胺糖酶或唾液酸酶的接觸,以透過使用充足量的酵素並接觸充足的時間,直到酵素反應實質上不再進行的程度為佳。內-α-N-乙醯半乳胺糖酶的使用量,相對於1 μg的Gc蛋白,以1~500 mU為佳,以100~200 mU為較佳。唾液酸酶的使用量,相對於1 μg的Gc蛋白,以1~500 mU為佳,以10~200 mU為較佳。無論使用哪一種酵素,酵素處理的溫度以35~39°C為佳。酵素處理時間以60~200分鐘為佳。
酵素處理可以任意地在緩衝液的存在下進行。作為這種緩衝液,可列舉生理食鹽水、磷酸鹽緩衝生理食鹽水(PBS)等。
酵素處理後,可以透過熱處理使酵素失活。熱處理條件沒有特別限制,只要能夠使酵素失活即可,例如,在60°C左右的溫度下加熱約10分鐘。此外,酵素處理後,可以經由組合維生素D結合性管柱、凝膠過濾管柱、超濾(ultrafiltration)等從Gc蛋白除去酵素。
也能夠使用固定在固相上的酵素(固定化酵素)來進行酵素處理。將酵素固定在固相上的方法是本技術領域具有通常知識者已知的,例如,能夠透過溴化氰等的偶合劑將內-α-N-乙醯半乳胺糖酶或唾液酸酶固定在瓊脂糖珠(agarose beads)上。可以透過將Gc蛋白應用至固定有酵素的固相上來進行酵素反應。透過使用固定化酵素,在酵素處理後,能夠在使酵素不因熱處理而失活的情況下將其回收,並且能夠除去雜質(因熱處理失活的酵素等的蛋白質等)。
透過上述方法獲得的Gc蛋白可以進一步冷凍乾燥成固體或粉末。此外,能夠經由與凝集素結合的有無來確認是否從Gc蛋白除去N-乙醯半乳胺糖。作為凝集素,可列舉來自多花紫藤(Wisteria floribunda)的凝集素、來自羅馬蝸牛(Helix pomatia)的凝集素。在Gc蛋白不與凝集素結合的情況下,就能夠判斷為N-乙醯半乳胺糖已被除去。
<巨噬細胞活化能力>
能夠透過測定巨噬細胞的吞噬能力來評價巨噬細胞活化能力。將Gc蛋白添加至巨噬細胞的培養液中,使巨噬細胞吞噬螢光乳膠珠粒、酵母聚糖(zymosan)等的測試物質,透過計算已吞噬的巨噬細胞的比例、被吞噬的測試物質的粒子數等來進行巨噬細胞吞噬能力的測定。當已吞噬的巨噬細胞的比例或被吞噬的測試物質的粒子數高於比較對照時,能夠判斷具有巨噬細胞活化能力。作為巨噬細胞株,能夠使用例如RAW264.7、NR8383、J774.1等。
本發明的巨噬細胞活化劑以不增加巨噬細胞的NO生產為佳。能夠透過將巨噬細胞活化劑添加至巨噬細胞中,並在靜置24小時左右後測定NO生產量來評價NO生產能力。例如,能夠透過實施例中記載的Griess法來測定NO生產量。
本發明的巨噬細胞活化劑以抑制巨噬細胞的NO生產為佳。能夠透過將巨噬細胞活化劑和脂多醣(Lipopolysaccharide; LPS)等的NO產生刺激劑添加至巨噬細胞中,並在靜置24小時左右後測定NO生產量來評價NO生產的抑制能力。抑制巨噬細胞的NO生產意味著具有抗發炎作用。
本發明的巨噬細胞活化劑以不增加巨噬細胞的TNF-α的產生為佳。能夠透過將巨噬細胞活化劑、脂多醣(LPS)等的NO產生刺激劑、和IFN-γ添加至巨噬細胞中,並在靜置24小時左右後測定TNF-α生產量來評價TNF-α的生產能力。例如,能夠透過實施例中記載的ELISA法來測定TNF-α的生產量。不增加巨噬細胞的TNF-α生產意味著具有抗發炎作用。
本發明的巨噬細胞活化劑可以具有M2型巨噬細胞的分化能力。能夠透過將巨噬細胞活化劑添加至巨噬細胞中,並在靜置24小時左右後,經由測定巨噬細胞中標記基因的表現量來評價M2型巨噬細胞的分化能力。作為標記基因,能夠使用例如ARG-1。例如,能夠透過實施例中記載的螢光顯微鏡觀察固定化細胞來評價標記基因的表現量。一般來說,已知發炎性M1型和抗發炎性M2型為巨噬細胞的分化類型。透過投予本發明的巨噬細胞活化劑被認為能夠促進抗發炎反應。
<<醫藥組成物>>
本發明的醫藥組成物,其特徵在於包括前述的巨噬細胞活化劑,用於抗癌、抗感染症、抗自體免疫疾病、抗自閉症、抗發炎性疾病、抗腦・神經變性疾病、改善皮膚、或治療心臟病。
<組成>
除了巨噬細胞活化劑之外,醫藥組成物可以適當地添加藥學上可接受的載體。作為藥學上可接受的載體,可列舉例如稀釋劑、穩定劑、防腐劑、緩衝劑等。
醫藥組成物的形態沒有特別限制,可列舉注射用組成物、口服組成物、點眼劑組成物、輸液組成物、點鼻劑組成物、點耳劑組成物、栓劑(suppository)、腸內營養組成物。其中,以注射用組成物為佳。作為注射的形態,可列舉靜脈內注射、皮下注射、皮內注射、肌肉內注射、腹腔內注射等,以肌肉內注射為佳。作為口服組成物的形態,可列舉散劑、顆粒劑、錠劑(包括舌下錠等)、膠囊劑、丸劑、腸溶劑、內用液劑(包括懸浮劑、乳劑、糖漿劑等)、吸入劑等。
醫藥組成物的投予量依患者的年齡、性別、體重和症狀、投予方法等而不同,但作為典型例子,例如,作為醫藥組成物中所包含的蛋白質總量,每1次的投予,每1kg體重以0.1 mg~4.0 mg為佳,以0.2 mg~2.0 mg為較佳,以0.3 mg~1.3 mg為更佳。
在醫藥組成物以上述的單次投予量投予的情況下,投予間隔和投予次數可列舉一週1~2次的投予間隔、共投予12~24次。此外,也可以在投予初期(例如1~2個月)一週投予2次,之後一週投予1次。
<適應症狀>
醫藥組成物能夠適當地使用於抗癌、抗感染症、抗自體免疫疾病、抗自閉症、抗發炎性疾病、抗腦・神經變性疾病、改善皮膚、治療心臟病。
癌症包括癌(carcinoma)、肉瘤(sarcoma)、其他任一種惡性腫瘤(malignant tumor),可列舉例如皮膚癌、支氣管癌、肺癌、非小細胞肺癌、乳癌、卵巢癌、舌癌、咽癌、食道癌、胃癌、小腸癌、大腸癌、直腸癌、結腸癌、肝癌、胰臟癌、腎臟癌、腎細胞癌、膀胱癌、前列腺癌、子宮癌、子宮頸癌、Wilms氏瘤(Wilms tumor)、惡性黑色素瘤、腦脊髓膜瘤(meningioma)、神經母細胞瘤(neuroblastoma)、骨肉瘤、卡波西肉瘤(Kaposi’s sarcoma)、淋巴瘤、白血病等。除了這些惡性腫瘤之外,癌症也包括其轉移。
作為感染症,可列舉例如病毒感染症、細菌感染症等,具體來說,除了新型冠狀病毒感染症(COVID-19)、人類免疫不全病毒(HIV)感染症、愛滋病之外,可列舉B型肝炎、C型肝炎、皰疹、流行性感冒、肺炎、肺結核、EB病毒感染症等。
自閉症是一種行為障礙,其特徵在於難以與他人形成社會關係、語言發展遲緩等。
發炎性疾病是經由物理刺激、化學刺激、微生物的感染所引起的生物體防禦反應中因發炎而產生的疾病。此外,本發明的醫藥組成物對於因先天性免疫的異常而自然地產生發炎反應並導致臟器損傷的自體發炎性疾病也有效。
作為腦・神經變性疾病,可列舉阿茲海默氏症(Alzheimer's disease)、帕金森氏病(Parkinson disease)、脊髓小腦變性症、肌肉萎縮性脊髓側索硬化症、路易體失智症(Dementia with Lewy bodies)等。
作為皮膚改善,可列舉皮膚的美白、色素沉殿的抑制或改善、除去角質或促進角質更新(turnover)、防止老化、皺紋的抑制或改善、保濕、再生、脫髮(alopecia)的治療或預防等。
作為心臟病,可列舉鬱血性心衰竭(congestive heart failure)、心律不整(arrhythmia)、心絞痛(angina pectoris)、心肌梗塞(myocardial infarction)等。
<<醫藥部外品(quasi drug)用組成物>>
能夠根據需要藉由添加助劑(auxiliary agent)等將巨噬細胞活化劑製成醫藥部外品用組成物。該醫藥部外品用組成物可採取溶液狀、懸浮液狀、糖漿狀、顆粒狀、乳膏狀、糊狀、凝膠狀等各種形態,也能夠根據需要成形為所期望的形狀。能夠經由任一種常用方法來製造醫藥部外品用組成物。
醫藥部外品用組成物中Gc蛋白的使用量沒有特別限制,能夠採用與上述醫藥組成物的情況相同的投予量,或是參照上述而適當設定的量。
<<食品組成物>>
巨噬細胞活化劑能夠根據需要經由適當地添加助劑、或是甜味料、辛香料、調味料、防腐劑、保鮮料、殺菌劑、抗氧化劑等食品和飲料中常用的各種添加劑而作為食品組成物。該食品組成物可以採取溶液狀、懸浮液狀、糖漿狀、顆粒狀、乳膏狀、糊狀、凝膠狀等各種形態。
食品組成物中Gc蛋白的使用量沒有特別限制,能夠採用與上述醫藥組成物的情況相同的投予量,或是參照上述而適當設定的量。
食品組成物能夠作為所謂的健康食品、健康飲料、功能性食品、營養功能食品、健康補助食品、營養補助食品(補充物)、特殊用途食品、特定保健用食品等。
<<巨噬細胞活化劑的製造方法>>
本發明巨噬細胞活化劑的製造方法之特徵在於,包括使Gc蛋白與N-乙醯半乳胺糖酶接觸的步驟。關於N-乙醯半乳胺糖酶和接觸條件,如同以上關於巨噬細胞活化劑的描述。
本發明巨噬細胞活化劑的製造方法,可以在與N-乙醯半乳胺糖酶接觸的步驟之前,包括使Gc蛋白與神經胺糖酸苷酶接觸的步驟。關於神經胺糖酸苷酶和接觸條件,如同以上關於巨噬細胞活化劑的描述。
[實施例]
(1)Gc蛋白的純化(製造例1)
透過將回收在SSTII(含有新血清分離劑)採血管(13 mm×100 mm)(#376528,BD)中的人血離心(4°C,3000 rpm,10分鐘)來獲得血清成分。
然後,使用BioLogic LP Core系統(#731-8300,BIO RAD) 藉由通過以下3個管柱從血清分離純化Gc蛋白。亦即,使用Blue Sepharose 6 Fast Flow(#17094801,GE Healthcare)從血清中除去白蛋白(albumin)等。隨後,藉由將回收的除去完白蛋白的血清通過維生素D結合性管柱,使Gc蛋白吸附在管柱上。將其以胍鹽酸鹽(guanidine hydrochloride)[6M](#077-02435,Wako)洗提並回收。
回收的溶液是藉由使用Snakesin Dialysis Tubing,3.5K MWCO,35 mm I.D(#88244,Thermo Fisher SCIENTIFIC)在SPB [2 mM,5 L] 中經由透析(4°C,15小時)來進行脫鹽。之後,藉由通過Bio-Scale Mini CHT Type II Cartridge(#7324332,BIO RAD)來進行純化。
最後使用Vivaspin Turbo 4,10 kDa,PES,25 pc(#VS04T01,sartorius)將回收的溶液濃縮(4°C,7500 rpm,10分鐘)成為最終回收產物Gc蛋白。藉由與多株兔抗人Gc球蛋白(#A002102-2,Dako)的抗原抗體反應和電泳來確認Gc蛋白的回收。
(2)未附加N-乙醯半乳胺糖的Gc蛋白(GcMMF)的製備(製造例2)
為了切斷與Gc蛋白的第418位或第420位的蘇胺酸殘基結合的糖鏈,使用神經胺糖酸苷酶(#N2876-6U,MeRCK)和內-α-N-乙醯半乳胺糖酶(#324716,MERCK)2種酵素。
透過以神經胺糖酸苷酶[200 mU]與Gc蛋白[10 μg]進行保溫(37°C,180分鐘)來除去與GalNAc結合的唾液酸。接著,透過將唾液酸切斷的Gc蛋白與內-α-N-乙醯半乳胺糖酶[1600 mU]進行保溫(37°C,120分鐘)來切斷蘇胺酸殘基和GalNAc之間的結合。使用多花紫藤(Wisteria floribunda)凝集素、生物素(Biotin)(#B-1355,Funakoshi)、來自羅馬蝸牛(Helix pomatia)的凝集素(#L6512-1MG,SAJ)的抗原抗體反應來確認酵素切斷是否完成。將得到的未附加N-乙醯半乳胺糖的Gc蛋白稱為GcMMF。
接著,從GcMMF除去用於切割的酵素。使用與回收Gc蛋白時一樣的低壓層析法,藉由通過維生素D結合性管柱和Bio-Scale Mini CHT Type II Cartridge(#7324332,BIO RAD)來進行酵素的除去。將得到的溶液藉由使用Vivaspin Turbo 4,10 kDa,PES,25 pc(#VS04T01,sartorius)進行超濾(4°C,7500 rpm,5~10分鐘)成為最終回收產物。
此時,也透過多花紫藤凝集素、生物素、和來自羅馬蝸牛的凝集素、多株兔抗人Gc-球蛋白(Polyclonal Rabbit Anti-Human Gc-Globulin)的抗原抗體反應進行確認。此外,將電泳後的凝膠以CBB Stain One Super(Ready To Use)(#11642-31,nacalai tesque)進行染色,確認酵素是否除去。
(3)巨噬細胞吞噬活性測試(實施例1、比較例1~4)
本測試中的稀釋全部都使用1% PS中的D-MEM(-)。
[第1天]
經由1% PS的D-MEM(-)回收以10% FBS、1% PS的D-MEM繼代培養至第二代的RAW264.7細胞(#EC91062702-F0,KAC),並稀釋成1×10
6個細胞/ml。將其在96孔黑色透明底部平底TC處理盤(#353219,FALCON)中各接種100 μl並保溫(37°C,5% CO
2,15小時)。
[第2天]
添加10 μl的各種活化劑(GcMMF [10 ng,100 ng](實施例1)、Gc 蛋白[10 ng](比較例2)、GcMAF [10 ng](比較例3)、LPS [100 ng](比較例4))並透過移液(pipetting)使其溫和地懸浮。再者,使用製造例1中純化的蛋白質作為Gc蛋白。使用以國際公開WO2013/038997號中記載的方法,將從人血清純化的Gc蛋白經由β-半乳糖苷酶(β-galactosidase)和唾液酸酶(sialidase)處理而獲得的蛋白質作為GcMAF。此外,未添加活化劑的作為比較例1。之後,透過保溫(37°C、5% CO
2、180分鐘)使巨噬細胞活化。保溫後,添加10 μl 乳膠珠粒(Latex-Beads)IgG-FITC溶液,並透過移液使其溫和地懸浮。操作結束後,以鋁箔避光並保溫(37°C,5% CO
2,24小時),使巨噬細胞吞噬乳膠珠粒。
[第3天]
第三天的全部實驗操作都在避光條件下進行。保溫結束後,除去培養基,加入100 μl的1級甲醇(#136-01837,Wako),靜置10分鐘。之後,除去甲醇並風乾5分鐘。風乾結束後,以100 μl Dulbecco磷酸鹽緩衝生理食鹽水(不含Ca、Mg,液體)(#14249-95,nacalai tesque)進行2次洗滌。之後,將96-孔黑色盤置於螢光顯微鏡(#BZ-X710,KEYENCE)上進行攝影。
在第3天的螢光顯微鏡觀察・拍攝中,使用20倍鏡頭拍攝了相位差圖像、螢光圖像(濾光立方體:BZ-X filter GFP)、Merge圖像共計3張圖像。拍攝地點設置為事先設定好的3~4個位置。但是,由於RAW264.7細胞是半附著性的細胞,因此可能會藉由實驗操作(甲醇固定等)將其剝離或聚集。此時,透過從預定的拍攝地點去除異常點來進行拍攝。
在所拍攝的相位差觀察圖像、螢光觀察圖像、和Merge圖像中,使用螢光觀察圖像並透過BZ-X分析儀進行數據處理。首先,使用拍攝到的對照組的螢光圖像進行混合細胞計數(Hybrid Cell Count),並測定螢光計數和螢光亮度(積分)。此時,設定螢光標記用的閾值,保存數據處理條件。使用這個保存的處理條件,對所有拍攝到的螢光圖像進行宏細胞計數(Macro Cell Count)。
螢光強度顯示於圖1。實施例1的GcMMF顯示出與比較例3的GcMAF相同的巨噬細胞活化能力。從這個結果清楚得知,在GcMAF對於巨噬細胞的活化中,GalNAc糖鏈不是必須的。
(4)NO生產/抑制測試(實施例2~3、比較例5~13)
本實驗中使用Griess法進行NO產生量的測定。
[第1天]
回收以10% FBS、1% PS的D-MEM繼代培養至第二代的RAW264.7細胞(#EC91062702-F0,KAC),並稀釋成0.5×10
6個細胞/ml。將其在24-孔細胞培養盤(#VTC-P24,AS ONE)上以10% FBS、1% PS的D-MEM各接種1 ml並保溫(37°C,5% CO
2,15小時)。
[第2天]
(在NO生產測試的情況下)
用1 mL Dulbecco磷酸鹽緩衝生理食鹽水(不含Ca、Mg,液體)(#14249-95,nacalai tesque)洗滌各個孔,並施加1 mL 1% PS的D-MEM(-)。之後,添加100 μl的各種活化劑(GcMMF [10 ng,100 ng](實施例2)、Gc蛋白[10 ng](比較例6)、GcMAF [10 ng](比較例7)、LPS [100 ng](比較例8))並保溫(37°C,5% CO
2,15小時)。另外,未添加活化劑的作為比較例5。
(在NO抑制測試的情況下)
以1 mL Dulbecco磷酸鹽緩衝生理食鹽水(不含Ca、Mg,液體)(#14249-95,nacalai tesque)洗滌各個孔,並施加1 mL 1% PS的D-MEM(-)。之後,添加100 μl的各種活化劑(GcMMF [10 ng,100 ng](實施例3)、LPS [100 ng](比較例10)、Gc蛋白[10 ng](比較例11)、GcMAF [10 ng](比較例12)、葡萄糖胺(glucosamine)[10 mM](比較例13))並保溫(37°C,5% CO
2,15小時)。此外,未添加活化劑的作為比較例9。在此,除了對照組(比較例9)之外的所有組別均同時添加LPS [100 ng],作為NO抑制的陽性對照組,加入葡萄糖胺至最終濃度為10 mM。
[第3天]
將各個孔的培養上清液分別全量回收至微量離心管(eppendorf)中,並用旋渦混合器(vortex mixer)充分攪拌。之後,各施加100 μl從各個微量離心管回收的培養上清液至96孔細胞培養盤(#TR5003,True Line)中。各添加各100 μl的Griess試劑,在避光條件下進行保溫(室溫,5~10分鐘),然後用微量盤檢測儀(microplate reader)(#infinite M200,TECAN)測定吸光度(550 nm)。使用亞硝酸鈉(sodium nitrite)作為校準曲線。
NO生產測試的結果如圖2A所示,NO抑制測試結果如圖2B所示。實施例2~3的GcMMF和比較例7、比較例12的GcMAF同樣地沒有顯示出NO生產能力,只顯示出NO抑制能力。
(5)TNF-α抑制測試(實施例4、比較例14~18)
使用附有盤的小鼠腫瘤壞死因子α未被覆的ELISA試劑盒(TNF α Mouse Uncoated ELISA Kit with plates)(#88-7324-22,eBioscience)進行測試。也根據試劑盒的內容製作了以下的流程(protocol)。
[第1天]
回收以10% FBS、1% PS的D-MEM繼代培養至第二代的RAW264.7細胞(#EC91062702-F0,KAC)並稀釋成0.4×10
6個細胞/ml。將其在24-孔細胞培養盤(#VTC-P24,AS ONE)上以10% FBS、1% PS中的D-MEM各接種1 ml並保溫(37°C,5% CO
2,15小時)。
[第2天]
用1 mL Dulbecco磷酸鹽緩衝生理食鹽水(不含Ca、Mg,液體)(#14249-95,nacalai tesque)洗滌各個孔,並施加1 mL 1% PS的D-MEM(-)。之後,添加100 μl的各種活化劑(GcMMF [10 ng,100 ng](實施例4)、DMSO中的薑黃素(curcumin)[20 mM](比較例15)、Gc蛋白[10 ng](比較例16)、GcMAF [10 ng,100 ng ](比較例17)、LPS [1 μg]+IFN-γ [10 ng](比較例18))並保溫(37°C,5% CO
2,24小時)。此外,未添加活化劑的作為比較例14。在此,在所有樣品組別中添加LPS [1 μg] + IFN-γ [10 ng],作為TNF-α抑制的陽性對照組,在各個孔中添加2 μl DMSO中的薑黃素[10 mM]。
除了上述操作之外,製作塗覆緩衝液(coating buffer)中的捕獲抗體,並各添加100 μl到附屬的96孔盤中。之後,將盤密封並保溫(4°C,搖動,15小時)。
[第3天]
捨棄96孔盤的溶液,用洗滌緩衝液(wash buffer)[250 μl]進行洗滌共計3次。洗滌後,將製作好的1×ELISA/ELISPOT各添加200 μl在各個孔中,將盤密封並保溫(室溫,60分鐘)。(保溫時製作標準品(1000 pg/ml)並回收刺激24小時後的培養上清液。)
保溫結束後,除去1×ELISA/ELISPOT,用洗滌緩衝液[250 μl]洗滌1次。之後,在盤上稀釋標準品,各加入100 μl刺激24小時後的培養上清液並保溫(4°C,15小時)。(在空白組(blank)中添加100 μl的1×ELISA/ELISPOT。)
[第4天]
除去各個孔的溶液,用洗滌緩衝液[250 μl]洗滌5次後,各添加100 μl的TNF-α 1× DetAb,將盤密封並保溫(室溫,60分鐘)。(在保溫時準備Avidin-HRP。 )
保溫結束後,用洗滌緩衝液[250 μl]洗滌3次後,各添加100 μl的Avidin-HRP,將盤密封並保溫(室溫,30分鐘)。保溫結束後,捨棄溶液,用洗滌緩衝液[250 μl]洗滌7次。之後,各添加100 μl的1×TMB後進行保溫(室溫,15分鐘)。(在此期間製作STOP溶液。)
保溫結束後,各添加100 μl的STOP溶液,用微量盤檢測儀(#infinite M200,TECAN)測定吸光度(450 nm(參考570 nm))。
TNF-α抑制測試的結果顯示於圖3。實施例4的GcMMF未顯示出與比較例17的GcMAF同樣的TNF-α抑制能力。
(6)M2分化能力評價測試(實施例5、比較例19~22)
[第1天]
經由1% PS的D-MEM(#044-29765,Wako)回收以10% FBS、1% PS的D-MEM繼代培養至第二代的RAW264.7細胞(#EC91062702-F0,KAC),並稀釋成1×10
6個細胞/ml。將其在96孔黑色透明底部平底TC處理盤(#353219,FALCON)上各接種100 μl並保溫(37°C,5% CO
2,15小時)。
[第2天]
添加10 μl的各種活化劑(GcMMF [10 ng,100 ng](實施例5)、Gc蛋白[10 ng](比較例20)、GcMAF [10 ng](比較例21)、IL-4 [50 ng] + IL-13 [50 ng](比較例22))並透過移液使其溫和地懸浮。此外,未添加活化劑的作為比較例19。之後,保溫(37°C,5% CO
2,24小時)。(陽性對照組是IL-4 [50 ng] + IL-13 [50 ng](比較例22))
[第3天]
保溫結束後,除去培養基,加入100 μl的1級甲醇(#136-01837,Wako),靜置10分鐘。之後,除去甲醇並風乾5分鐘。風乾結束後,以100 μl Dulbecco磷酸鹽緩衝生理食鹽水(不含Ca、Mg,液體)(#14249-95,nacalai tesque)進行2次洗滌。之後,各添加150 μl PBS中的0.1% BSA並進行保溫(室溫,15小時)。
[第4天]
以PBS [100 μl]洗滌3次,各添加30 μl的抗人/鼠精胺酸酶1抗體(anti-h/m Arginase1 Antibody)後進行保溫(室溫,30分鐘)。之後,以PBS [100 μl]洗滌3次,並以微量盤檢測儀測定螢光強度(ex: 488 nm、em: 530 nm)。
螢光強度顯示於圖4。實施例5的GcMMF顯示出與比較例21的GcMAF同樣弱的M2型文化能力。
無
圖1顯示巨噬細胞吞噬活性測試的結果。
圖2A顯示NO生產測試的結果。
圖2B顯示NO抑制測試的結果
圖3顯示TNF-α抑制測試的結果。
圖4顯示M2分化能力評價測試的結果。
Claims (6)
- 一種巨噬細胞活化劑,包括未附加N-乙醯半乳胺糖的Gc蛋白。
- 如請求項1所述之巨噬細胞活化劑,其中該Gc蛋白是來自人、牛、山羊、或小鼠的血清或乳汁之純化物。
- 如請求項1或2所述之巨噬細胞活化劑,其中未附加N-乙醯半乳胺糖的胺基酸為序列識別號1中的第418位或第420位的蘇胺酸。
- 一種醫藥組成物,包括如請求項1到3中任一項所述之巨噬細胞活化劑,且用於抗癌、抗感染症、抗自體免疫疾病、抗自閉症、抗發炎性疾病、抗腦・神經變性疾病、改善皮膚、或治療心臟病。
- 一種巨噬細胞活化劑的製造方法,包括使Gc蛋白與N-乙醯半乳胺糖酶接觸的步驟。
- 如請求項5所述之巨噬細胞活化劑的製造方法,在與N-乙醯半乳胺糖酶接觸的步驟之前,包括使Gc蛋白與神經胺糖酸苷酶接觸的步驟。
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