TWI387750B - Biomarker and detection method for detecting kidney disease - Google Patents
Biomarker and detection method for detecting kidney disease Download PDFInfo
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Description
本發明係有關於一種檢測腎臟疾病之用途及方法，尤指使用一Annexin A1(AnxA1)為生物標記或早期生物標記(Early Biomarker)以檢測腎臟疾病之用途及方法。
按，腎臟是由數百萬顆腎絲球所組成的一對器官，這個數目是從出生就決定的，腎絲球是微血管叢組織，一旦有發炎，造成微血管通透性改變，血中的紅血球或蛋白滲入尿中，即所謂的血尿或蛋白尿。
慢性腎衰竭的病因，包含：一、腎絲球腎炎：這是一種炎症，不是細菌感染，它會造成蛋白尿，一般視腎絲球腎炎的種類之不同，可能過數月甚至十年、二十年後才會變成慢性腎衰竭。二、高血壓：因為高血壓會造成腎臟的血管硬化，故如有長期及嚴重的高血壓就會產生腎臟衰竭。三、糖尿病：在洗腎的患者當中，大概有20~25%是因糖尿病而引起腎臟衰竭，至於其確實導因尚不是很清楚，但是臨床醫師們認為，血糖控制是可以減緩腎臟衰竭發生的。四、藥物：長期使用未經醫師處方的一些止痛藥、抗生素、利尿劑或減肥藥，甚至一些草藥或偏方都可能會傷害到腎臟。五、多囊腎病變：這是一種遺傳病，腎臟本身會有很多囊泡，大部份的患者要到四、五十歲才會引起腎臟衰竭。六、腎結石：長期結石會導致泌尿道阻塞。
慢性腎衰竭(Chronic Renal Failure)或***(Uremia)是腎小球疾病(Glomerular Disorders)極常見的重大合併症，導致病患只能靠洗腎等保守治療維持生命。醫界目前係主要依賴病史、是否出現蛋白尿血尿、腎功能異常、及/或腎組織穿刺等傳統方法來推斷是否已有腎小球疾病之發生，尚無疾病生物標記可供早期診斷和疾病追蹤治療或惡化用，亟待加速開發早期診斷用之疾病生物標記。
又，尿蛋白可區分為兩類：腎小球(glomerular)及腎小管(tubular)。腎小
球蛋白尿(glomerular proteinuria)是由於腎小球微細血管壁之穿透力改變，使大分子物質由腎小球濾出，其中以白蛋白為主要成份，其他如IgG，IgA亦會不正常升高；若超過腎小管對這些分子的再吸收能力，則尿中這些成份濃度升高，例如：α1-microalbumin，β2-microglobulin。利用高靈敏度方法測定尿中微量白蛋白是否升高，可以早期偵測腎小球之病變，尤其是對於胰島素依賴性糖尿病(IDDM)患者。一般而言，糖尿病腎病變可分為五階段：第一階段：腎小球機能亢進及肥大。第二階段：早期腎小管損害，白蛋白排除率大多正常，無其它臨床症狀。第三階段：初期腎病，微量蛋白出現於尿中。第四階段：臨床糖尿性腎病(clinical diabetic nephropathy)。第五階段：末期腎病變。
尿中微量白蛋白排除變化，由正常→微量白蛋白尿→腎病變。若能在第三階段以前偵測出微量白蛋白尿，給予及時治療，則腎病變是可以復原的。胰島素依賴性糖尿病病人早期之微量白蛋白若大於20 μg/min，其後併發腎衰竭的機率為早期無尿中微量白蛋白異常者之24倍強，因此尿中微量白蛋白之定量可以做為糖尿性腎病的指標。另外，尿中微量白蛋白亦可以做為妊娠糖尿病之控制與追蹤指標，早期診斷腎小球蛋白尿症和懷孕血毒病(toxaemia in pregnancy)。
本發明之主要目的，係在於提供一種檢測腎臟疾病之生物標記及檢測方法，使用AnxA1以做為生物標記或早期生物標記(Early Biomarker)，在發生腎纖維化之前，即可檢測腎小球疾病。
本發明之次要目的，係在於提供一種檢測腎臟疾病之生物標記及檢測方法，使用尿液檢測或腎臟組織進行分析，以做為檢定腎臟疾病之方法。
為了達到上述之目的，本發明係揭示生物標記以做為檢測腎臟疾病之用途及檢測方法，使用AnxA1之蛋白質或訊息核糖核酸(mRNA)做為腎臟疾病之生物標記，其檢測方法為使用尿液或腎臟組織進行檢測AnxA1之蛋白質或訊息核糖核酸(mRNA)，以在發生腎纖維化之前用者，檢測腎小球疾病。
再者，本發明係揭示建立腎臟疾病之早期生物標記(Early Biomarker)，亦即在發生前述之嚴重腎病變之前，即可測出，本發明同時亦供可預測發生纖維化等嚴重之腎臟病變，提供醫師進行早期防治之依據。
茲為使 貴審查委員對本發明之結構特徵及所達成之功效有更進一步之瞭解與認識，謹佐以較佳之實施例及配合詳細之說明，說明如後：
實施例：
分別收集55名各種腎臟疾病病人和10個健康自願者之尿液樣品、腎臟組織切片等進行分析。
首先，以組織病理學分析(H&E)各病人之腎臟組織之變化。
本發明之一實施例係以免疫組織化學染色法(IHC)分析腎組織中AnxA1蛋白質於何種腎臟細胞表現。腎臟組織切片於福爾馬林固定-石蠟包埋後，進行IHC，步驟為：去除石蠟，並以微波加熱法加熱10 min作抗原回覆，冷卻20分鐘後，再將腎臟組織切片浸置於5%之牛血清白蛋白以阻隔背景，並將初級抗人AnxA1(BD biosciences)抗體加入，靜置於隔夜4℃中，再分別加入生物素化次抗體(biotinylated secondary antibody)，及鏈黴卵白素-過氧化物酶反應物，最後加入AEC試劑(DakoCytomation)進行呈色反應。並於顯微鏡底下觀察AnxA1蛋白質之程度。
本發明之另一實施例係以原位雜交染色法(ISH)分析腎組織中AnxA1核醣核酸於何種腎臟細胞表現。腎臟組織切片於福爾馬林固定-石蠟包埋後，進行ISH，步驟為：以AnxA1核醣核酸序列設計出人類AnxA1之引子序列(primer sequence，如表一)，並以聚合酵素鏈鎖反應(RT-PCR)獲得AnxA1互補去氧核醣核酸產物，並將此產物置入pGEM-T EASY載體(Promega,WI,USA)，並進行活體外之轉錄反應，將AnxA1互補去氧核醣核酸轉成核醣核酸，製備成AnxA1 dig-labeled mRNA探針。
表一
去除石蠟，腎臟切片加入20 μg/ml蛋白酶K(Sigma,MO,USA)進行反應，並以25-50 μl之雜交混合物(2 mmol/L之乙二胺四乙酸，20 mmol/L Tris,pH7.5,0.6 mol/L NaCl,2x Denhardt’s solution,20%dextran sulfate,0.1 mg/mL tRNA,and 0.2 mol/LDTT)進行去乙醯，再加入AnxA1 dig-labeled mRNA探針，於42℃下反應16小時，清洗後，腎臟切片置於1X封閉液(Roche,IN,USA)，並以anti-daioxigenin鹼性磷酸酶作用1小時，NBT/BCIP溶液呈色，及複染甲基綠做核染色，最後玻片於顯微鏡下觀察。
本發明之另一實施例係以西方墨點分析法進行分析AnxA1於尿液之表現。首先將各種腎臟疾病病人之尿液收集後，離心1000xg，10分鐘後，分別加入1%之抗蛋白質酶(Roche,IN,USA)，冰存於-70冰箱待分析，再取出尿液檢體分別加入1X檢體溶劑(Sample Buffer，10%SDS，2ml glycerol，1MDTT，145mgTris，0.05%bromophenol blue)，混合均勻並置於冰上反應15-20分鐘，不可煮沸檢體，將此檢體，以10% SDS-PAGE(SDS-Polyacrylamide Gel Eletrophoresis)進行分析，將此凝膠轉漬於硝化纖維素膜後，於阻隔緩衝液(5%脫酯乳)作用2小時，再加入初級抗人AnxA1(BD biosciences)抗體，於隔夜4℃中進行反應。硝化纖維素膜被洗滌三次後，加入次級抗體辣根過氧化物酶標記(horseradish peroxidase-conjugated)於室溫下作用1小時。硝化纖維素膜洗了三次，以化學冷光試劑(chemiluminescent reagent)(PerkinElmer Life Sciences,Boston,MA)進行反應，以X光片進行訊息檢測。數據以目標蛋白質密度的比率對尿的肌氨酸酐含量做表示。
實施例：
為了判斷AnxA1是否與形成腎臟纖維化具關聯性，將本發明所採用之腎臟疾病病人之年齡、性別、臨床診斷(是否具腎臟纖維化病徵)、數據，包括蛋白尿、腎功能指標(血肌酸酐、血尿素氮、肌酸酐過濾率)統計分析，請參閱表二。
表二、臨床的數據，請參閱下表：
腎臟病理學分析腎臟病變(Nephropathy)之腎臟疾病病人：新月型腎小球腎炎(crescentic glomerulonephritis；Crescentic GN)5例、腎硬化8例、局部性腎絲球硬化症4例、糖尿病腎纖維化病變5例、及第4c級腎臟狼瘡腎病變6例。並同時分析5位腎微小變化症(此病症為無顯微病理變化之類型，Minimal change disease，MCD)。請參閱圖1A~1D係病理組織型態學分析法之腎疾病圖；圖1A~1B係為新月型腎絲球腎炎，而圖3C係為腎微小變化症圖，1D係為腎小管腎病變。
再以ISH分析AnxA1 mRNA於各病人中之mRNA表現量；請參閱圖1E~1H係ISH分析法之腎疾病圖；圖1A~1B係為新月型腎絲球腎炎，而圖1C係為腎微小變化症圖，1D係為腎小管腎病變。請參閱圖1C，其係為腎微小變化症之病人腎組織，AnxA1 mRNA僅有微量於腎小管中或腎小球量中表現；但其餘腎病變類型病人之腎組織，AnxA1 mRNA大量表現於腎小球之纖維化區域(如第3E及3F圖所示之)、新月結構、腎小球臟層上皮細胞或腎小球腎膈細胞；同時，AnxA1mRNA於腎小管間質、再生腎小管或浸潤之發炎細胞之表現亦具高表現(如第1H圖所示)。
IHC係用來確認AnxA1蛋白質之含量與其於組織中之表現分布狀況，如第1I至1L圖所示，AnxA1蛋白質之分布及強度與相似於ISH染色之分佈與強度。
尿液樣品係從55名患者和10個健康自願者中為分析以進行收集。AnxA1蛋白質於尿液樣品由西部墨點分析法檢驗。如圖2A所顯示，除腎微小變化症(無腎組織之顯微病理變化，亦不會進一步惡化成腎衰竭或***)和正常人之尿液外，AnxA1(37 kDa)在所有腎病變病人之尿液樣品被檢測出。
由於高死亡率和發病率，腎臟纖維化成為代表一個主要醫療保健問題。多數腎病變是由免疫和非免疫機制所造成的一個多因子的疾病過程，會導致腎絲球硬化、腎間質纖維化、腎臟發炎細胞浸潤(inflammatory infiltration)、腎實質受損和腎血管病變等。許多因素在腎臟纖維化都有相關聯性，其中腎臟發炎細胞浸潤是一重要形成因子。本發明自人類之腎臟切片中，發現AnxA1 mRNA和蛋白質主要表現於多種病變類型，及腎絲球周
邊淋巴球中，與腎微小變化症或正常人相比，具統計上顯著差異。
同時，藉由西方墨點法偵測人類之尿液檢體，本發明首次驗證AnxA1蛋白質可於尿液中被偵測到，更於比較容易發生腎臟纖維化病變之尿液檢體中具高表現，與腎微小變化症及正常人相比，具統計上顯著之差異。
綜合以上發現，AnxA1可作為檢測多種重要之腎臟病變、腎纖維化以及腎硬化之生物標記，作為日後臨床病人之診斷用之疾病標記。
以往研究顯示，AnxA1在表現於腎包氏囊(Bowman’s capsule)的上皮細胞、緻密斑(macula densa)或是延髓(medullary)乳突(papillary)集尿管(collecting ducts)。但於本發明，以IHC和ISH驗證，首次驗證AnxA1在腎臟病變之病灶中具明顯表現，並於腎臟新月結構(crescent，腎臟發生纖維化之一種初期病理特徵)、Bowman’s capsule、足細胞及腎絲球之腎膈細胞(mesangial cell)、再生之腎小管及腎絲球周邊發炎浸潤細胞亦有AnxA1mRNA和蛋白質之表現。
而Fan et al.(Fan X,Krahling S,Smith D,et al.：Macrophage surface expression of annexins I and II in the phagocytosis of apoptotic lymphocytes.Mol Biol Cell 15：2863-2872,2004)研究發現，AnxA1具有促進吞噬作用。
故，AnxA1之表現量增加與腎臟纖維化、腎絲球周邊巨噬細胞之浸潤應具密切的相關性。再者，本發明驗證尿中AnxA1蛋白質含量與腎臟纖維化亦具明顯的相關性，可作為腎臟疾病之生物標記，有利於未來發展臨床非侵入性(Non-invasive)之診斷用生物標記。
綜上所述，本發明實為一具有新穎性、進步性及可供產業利用者，應符合我國專利法所規定之專利申請要件無疑，爰依法提出發明專利申請，祈 鈞局早日賜准利，至感為禱。
惟以上所述者，僅為本發明之一較佳實施例而已，並非用來限定本發明實施之範圍，舉凡依本發明申請專利範圍所述之形狀、構造、特徵及精神所為之均等變化與修飾，均應包括於本發明之申請專利範圍內。
圖1A~1D：其係為本發明之另一較佳實施例之以組織病理學分析人類腎病
之腎小球或腎小管間質病變之圖；圖1E~1H：其係為本發明之另一較佳實施例之以ISH分析AnxA1mRNA於人類腎病之腎絲球或腎小管間質病變之圖；圖1I~1L：其係為本發明之另一較佳實施例之以IHC分析AnxA1蛋白質於人類腎病之腎小球或腎小管間質變病之圖；圖2A：其係為本發明之另一較佳實施例之以西方墨點分析法進行AnxA1蛋白質於尿液分析之示意圖；圖2B：其係為本發明之另一較佳實施例之以西方墨點分析法進行AnxA1蛋白質於尿液之分部圖；
Claims (12)
- 一種腎臟疾病之檢測方法，其步驟係包含有：提供一檢體；及進行一檢測方法，以檢驗該檢體是否含有AnxA1之蛋白質或AnxA1之訊息核糖核酸(mRNA)；其中，該腎臟疾病係為腎纖維化。
- 如申請專利範圍第1項所述之腎臟疾病之檢測方法，其中該檢體係為一腎臟切片。
- 如申請專利範圍第2項所述之腎臟疾病之檢測方法，其中該腎臟切片包含腎皮質、腎小囊病變、腎小球臟層上皮細胞、新月結構、腎小管間質、再生腎小管、浸潤之發炎細胞或腎小球膈細胞。
- 如申請專利範圍第1項所述之腎臟疾病之檢測方法，其中該檢體係為尿液。
- 如申請專利範圍第1項所述之腎臟疾病之檢測方法，其中該檢體係為血清。
- 如申請專利範圍第1項所述之腎臟疾病之檢測方法，其中該進行一檢測方法中，該檢測方法係為原位雜交染色法(ISH)以檢測腎臟切片。
- 如申請專利範圍第1項所述之腎臟疾病之檢測方法，其中該進行一檢測方法中，該檢測方法係為免疫組織化學染色法(IHC)以檢測腎臟切片。
- 如申請專利範圍第1項所述之腎臟疾病之檢測方法，其中該進行一檢測方法中，該檢測方法係為即時聚合酵素鏈鎖反應(RT-PCR)以檢測腎臟切片。
- 如申請專利範圍第2項所述之腎臟疾病之檢測方法，其中該腎臟切片係包含腎皮質。
- 如申請專利範圍第1項所述之腎臟疾病之檢測方法，其中該進行一檢測方法中，該檢測方法係為西方墨點染色法以檢驗尿液。
- 申請專利範圍第10項所述之腎臟疾病之檢測方法，其中該西方墨點染色法包含下列步驟： 離心後，加入抗蛋白質酶，加入檢體溶劑，混合均勻並置於冰上反應，不可煮沸檢體，將此檢體，以SDS-PAGE(SDS-Polyacrylamide Gel Eletrophoresis)進行分析，將此凝膠轉漬於硝化纖維素膜後，於阻隔緩衝液作用，再加入初級抗人AnxA1(BD biosciences)抗體，硝化纖維素膜被洗滌後，加入次級抗體辣根過氧化物酶標記(horseradish peroxidase-conjugated)於室溫下作用，將硝化纖維素膜進行清洗，以化學冷光試劑進行反應，以X光片進行訊息檢測。
- 如申請專利範圍第1項所述之腎臟疾病之檢測方法，其中該進行一檢測方法中，該檢測方法係為病理組織型態學分析法。
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