TWI426269B - Method of gastric juice protein analysis for stomach cancer diagnosis - Google Patents
Method of gastric juice protein analysis for stomach cancer diagnosis Download PDFInfo
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一種偵測胃與十二指腸疾病的生物標誌和方法。A biomarker and method for detecting gastric and duodenal diseases.
在人類基因體計劃完成之後,蛋白質體的研究獲得高度的重視,蛋白質體分析提供一個研究疾病發生的有效工具。利用健康者與病患間全部蛋白質種類和數量的比較,可發現和特定疾病具顯著相關性的蛋白質,利用此類蛋白質可做為診斷和治療該疾病的標的。After the completion of the human genome program, protein body research has received a high degree of attention, and protein body analysis provides an effective tool for studying disease occurrence. By comparing the total protein species and quantity between healthy people and patients, proteins that are significantly associated with specific diseases can be found, and such proteins can be used as a target for diagnosis and treatment of the disease.
質譜儀,理論上其偵測之敏感度和解析度沒有限制,可應用於分析單一測試樣本之全部蛋白質組成,並產生該測試樣本蛋白質體之質量譜圖,藉由比較不同蛋白質疾病標誌的量,可於單一測試樣本(來自於動物或人類)中完成一種或多種疾病的診斷。然而,施行此類綜合性的質譜分析目前並不可行,因為質譜分析的敏感度和解析度經常受限並隨不同蛋白質而異。首先,擁有類似分子量的蛋白質難以區分,即使是由敏感度最高的質譜儀來偵測。第二,在不同的器官和血液中有各種不同的蛋白質,而且大部分的這些蛋白質存在的量都很少,使得利用這些蛋白質進行質譜儀分析,作為疾病篩檢或是診斷都很花時間和金錢,更不用說只有少部分的病人能夠負擔起這筆費用。質譜儀的直接分析通常不夠充份,需要結合生物資訊分析以減少時間和金錢的花費,及省略詳細蛋白質鑑定和/或胺基酸序列決定的步驟。Mass spectrometry, theoretically there is no limit to the sensitivity and resolution of the detection, can be applied to analyze the entire protein composition of a single test sample, and produce a mass spectrum of the protein body of the test sample, by comparing the amount of different protein disease markers The diagnosis of one or more diseases can be done in a single test sample (from an animal or human). However, performing such comprehensive mass spectrometry is currently not feasible because the sensitivity and resolution of mass spectrometry are often limited and vary from protein to protein. First, proteins with similar molecular weights are difficult to distinguish, even by the most sensitive mass spectrometer. Second, there are different kinds of proteins in different organs and blood, and most of these proteins are present in small amounts, making it possible to use these proteins for mass spectrometry analysis, as a disease screening or diagnosis. Money, not to mention only a small percentage of patients can afford the cost. Direct analysis of mass spectrometers is often inadequate, combined with bioinformatics analysis to reduce time and money costs, and omitting detailed protein identification and/or amino acid sequence determination steps.
現階段利用血清樣本進行質譜分析其成功度有限,部份原因是血清中大量的蛋白質於質譜儀中會產生複雜的訊號,而存在於血清中並確實來自癌症細胞的特異性蛋白,相對地變得罕見且不易精確測量。藉由比較正常人和癌症病患的血清樣本,質譜分析目前可以應用於卵巢癌和前列腺癌的診斷,雖然如此,其精確地分析依然困難重重。利用血清樣本分析的變因包含:血清中特異性蛋白含量的天然變動性,其變化可能取決於不同的營養、環境和心理的狀態。即使同一人每日規律的生理與心理的活動所產生的變化,某特定蛋白質於血清樣本中變動的量可能比疾病和正常時的相異量來得更大,而在每日中變化量有時也會較正常人與癌症患者間的差異更為顯著。At this stage, the use of serum samples for mass spectrometry has limited success, in part because a large amount of protein in the serum produces complex signals in the mass spectrometer, and the specific proteins present in the serum and indeed from cancer cells are relatively variable. It is rare and difficult to measure accurately. By comparing serum samples from normal and cancer patients, mass spectrometry can now be applied to the diagnosis of ovarian and prostate cancers. However, accurate analysis is still difficult. The causes of analysis using serum samples include: the natural variability of specific protein content in serum, which may depend on different nutritional, environmental, and psychological states. Even if the same person's daily physiological and psychological activities change, the amount of a particular protein in the serum sample may vary more than the difference between the disease and normal, and the amount of change in the daily is sometimes It is also more significant than normal and cancer patients.
癌症早期在血清中相對地僅能發現少量癌症相關或腫瘤相關的蛋白質,因此利用蛋白質體技術分析血清樣本進行癌症診斷,一般均受限於晚期的癌症病患,早期癌症診斷的期望並不容易達成。另一方面,由患處而非血液取得試驗樣本是高度侵入性的,雖然可能獲得含量較高的腫瘤相關蛋白質,大部份病人非在萬不得已的情況並不願意給予此類的採樣。相反地,從胃液中取得供質譜儀分析的蛋白質/多肽相對簡單許多。In the early stage of cancer, relatively few cancer-related or tumor-associated proteins can be found in serum. Therefore, the use of proteomic techniques to analyze serum samples for cancer diagnosis is generally limited to advanced cancer patients. The expectation of early cancer diagnosis is not easy. Achieved. On the other hand, obtaining test samples from the affected area rather than the blood is highly invasive. Although it is possible to obtain higher levels of tumor-associated proteins, most patients are not willing to give such samples as a last resort. Conversely, obtaining proteins/peptides for analysis by mass spectrometry from gastric juice is relatively simple.
胃癌(stomach cancer or gastric cancer;GC),係為世界上第二常見之癌症且於早期相當難診斷。胃癌可以存在於胃部一段很長的時間,在症狀發生前生長成大型的尺寸。在胃癌的早期,該患者可能會有消化不良、胃部不適、飯後撐脹感、輕微噁心、食慾喪失或胃灼熱。在胃癌的進階時期,可能會有血便、嘔吐、體重減輕或更嚴重的疼重。到目前為止,並沒有胃癌良好的生物標誌(biomarker)。近期,活體組織的內視鏡檢查仍是胃癌診斷的選擇測試方式,但其侵入性使其無法作為一篩檢的工具而被廣泛地使用。到目前為止,仍然無可靠的、非侵入性的篩檢或診斷胃癌的方式。Stomach cancer or gastric cancer (GC) is the second most common cancer in the world and is quite difficult to diagnose in the early stage. Gastric cancer can be present in the stomach for a long period of time and grows into large sizes before symptoms occur. In the early stages of gastric cancer, the patient may have indigestion, stomach upset, post-meal sensation, mild nausea, loss of appetite, or heartburn. In the advanced stage of gastric cancer, there may be bloody stools, vomiting, weight loss or more serious pain. So far, there is no good biomarker for gastric cancer. Recently, endoscopic examination of living tissue is still the selective test for the diagnosis of gastric cancer, but its invasiveness makes it incapable of being widely used as a screening tool. To date, there is still no reliable, non-invasive way to screen or diagnose gastric cancer.
一般而言,偵測和監視癌症的一般試驗係測量其腫瘤相關抗原或癌症生物標誌。此類試驗係測量腫瘤細胞顯著釋出或分泌於血清中之蛋白質或醣蛋白(glycoprotein)。例如,僅有少部分的胃癌細胞釋出癌胚胎抗原(carcinoembryonic antigen,CEA),碳水化合物抗原(CA19-9)或其他目前已知的生物標誌於循環系統中。然而,許多癌症特異的或癌症相關的蛋白質在進入血液前己被消化分解。此類可偵測的抗原(detected antigen)的量一般隨該疾病的演進而上升,同時於癌症發生的晚期為最易偵測,但其於癌化疾病的早期鮮少提升,所以,它們不適合早期篩檢或診斷。In general, a general test for detecting and monitoring cancer measures its tumor-associated antigen or cancer biomarker. Such tests measure proteins or glycoproteins that are significantly released or secreted by tumor cells in serum. For example, only a small fraction of gastric cancer cells release carcinoembryonic antigen (CEA), carbohydrate antigen (CA19-9) or other currently known biomarkers in the circulatory system. However, many cancer-specific or cancer-related proteins have been digested and decomposed before entering the bloodstream. The amount of such detectable antigen generally increases with the progression of the disease, and is most easily detected in the late stages of cancer, but it rarely increases in the early stages of cancerous disease, so they are not suitable. Early screening or diagnosis.
因而,一個可鑑定的胃癌的生物標誌是需要的。質譜分析係可做為此一早期和快速癌症診斷之工具。Thus, an identifiable biomarker for gastric cancer is needed. Mass spectrometry can be used as a tool for early and rapid cancer diagnosis.
本發明之一個或多個具體實施例係提供胃癌偵測的方法和生物標誌。該方法係包含質譜儀和/或二維凝膠電泳之應用於分析胃液與偵測胃病相關蛋白質和多肽之增加和/或受抑制程度。生物資訊分析和/或神經網路分析係用於支持質譜分析。該定義於此之生物標誌、多肽和蛋白質係可被應用於胃與十二指腸疾病之試驗和診斷套組(kits)。One or more embodiments of the invention provide methods and biomarkers for gastric cancer detection. The method comprises the use of a mass spectrometer and/or two-dimensional gel electrophoresis for the analysis of the increase and/or suppression of gastric juice and detection of gastric disease-related proteins and polypeptides. Bioinformatics analysis and/or neural network analysis are used to support mass spectrometry. The biomarkers, polypeptides and protein lines defined herein can be applied to trial and diagnostic kits for gastric and duodenal diseases.
在一具體實施例,其係提供一可鑑定胃與十二指腸疾病之方法。該方法包含收集一個或多個人類受測者的胃液樣本,對一個或多個人類受測者的胃液樣本施行質譜分析和/或二維凝膠電泳以產生一個或多個質譜,比較該一個或多個質譜與基礎線質量的質譜,並鑑定一個或多個分子異常質量的量,該一個或多個分子係為該胃或十二指腸疾病之生物標誌。In a specific embodiment, it provides a method for identifying gastric and duodenal diseases. The method comprises collecting a gastric fluid sample of one or more human subjects, performing mass spectrometry and/or two-dimensional gel electrophoresis on a gastric fluid sample of one or more human subjects to generate one or more mass spectra, comparing the one Or a mass spectrum of mass spectrometry and baseline quality, and identifying an amount of one or more molecular abnormalities, the one or more molecules being a biomarker of the stomach or duodenal disease.
在另一具體實施例,其係提供一篩檢胃與十二指腸疾病之方法。該方法包含收集一個或多個人類受測者的胃液樣本,藉由對一個或多個胃液樣本的質譜分析篩檢該胃與十二指腸疾病之一個或多個生物標誌的量以產生一個或多個質譜圖,比較該一個或多個質譜與基礎線質量的質譜圖,並定義該一個或多個生物標誌之異常質量的量為胃或十二指腸疾病陽性。In another embodiment, it provides a method of screening for gastric and duodenal diseases. The method comprises collecting a gastric fluid sample of one or more human subjects, and screening one or more biomarkers of the stomach and duodenal diseases by mass spectrometric analysis of one or more gastric fluid samples to generate one or more A mass spectrum is obtained by comparing the mass spectrum of the one or more mass spectra to the baseline quality and defining an abnormal mass of the one or more biomarkers for positive gastric or duodenal disease.
在本發明之一部份,與基礎線量相較後,一個或多個減少的多肽係被鑑定。該一個或多個多肽之例子係包含一選自序列辨識號1-3之胺基酸序列。在本發明之另一部份,與基礎線量相較後,一個或多個多肽增加的量係被鑑定。該一個或多個多肽之例子係包含一選自序列辨識號4-5之胺基酸序列。在本發明之另一其他部份,與基礎線量相較後,一個或多個蛋白質增加的量係被鑑定。該一個或多個蛋白質之例子係包含α-抗胰蛋白酶前驅物。In one part of the invention, one or more reduced polypeptides are identified as compared to the amount of baseline. An example of such one or more polypeptides comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3. In another part of the invention, an increased amount of one or more polypeptides is identified as compared to the amount of baseline. An example of such one or more polypeptides comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4-5. In still another part of the invention, an increased amount of one or more proteins is identified as compared to the amount of baseline. An example of such one or more proteins is an alpha-antitrypsin precursor.
在另一具體實施例,一個或多個胃或十二指腸疾病之生物標誌係被鑑定。例如,α-抗胰蛋白酶前驅物係被鑑定為一新的胃癌和胃潰瘍的生物標誌。再者,胃癌的一個或多個生物標誌被鑑定為包含一胺基酸序列,其至少90%相似於選自序列辨識號1-5之胺基酸序列。In another embodiment, one or more biomarkers of the stomach or duodenal disease are identified. For example, the alpha-antitrypsin precursor system has been identified as a novel biomarker for gastric cancer and gastric ulcer. Furthermore, one or more biomarkers of gastric cancer are identified as comprising an amino acid sequence at least 90% similar to the amino acid sequence selected from Sequence Numbers 1-5.
本發明之具體實施例係提供胃與十二指腸疾病之早期篩檢和分析。其應用質譜分析為一快速且簡易的方式,比較患者與健康者樣本在多肽和/或蛋白質表現的異常量。此外,應用二維凝膠電泳比較患者和健康者樣本多肽和/或蛋白質表現的異常量。在一方面,利用生物資訊的神經網路分析以幫助分析並解釋該質譜分析的結果。在另一方面,利用胃液為質譜儀分析的試驗樣本,以與其他型式的試驗樣本相較,獲得對胃與十二指腸疾病較高的敏感度和特異性。在一具體實施例,利用質譜分析比較患者和健康者的試驗樣本並鑑定一個或多個多肽和蛋白質表現量增加和抑制的量。Specific embodiments of the invention provide for early screening and analysis of gastric and duodenal diseases. Its application of mass spectrometry is a quick and easy way to compare abnormal amounts of peptide and/or protein expression in patients and healthy subjects. In addition, two-dimensional gel electrophoresis was used to compare abnormal amounts of peptide and/or protein expression in patient and healthy subjects. In one aspect, neural network analysis of biological information is utilized to aid in the analysis and interpretation of the results of the mass spectrometry. On the other hand, test samples analyzed by gas chromatography for mass spectrometry have higher sensitivity and specificity for gastric and duodenal diseases than other types of test samples. In a specific embodiment, mass spectrometric analysis is used to compare test samples of patients and healthy individuals and to identify the amount of increase and inhibition of one or more polypeptides and proteins.
鑑定於此之胃與十二指腸疾病相關的一個或多個多肽和/或蛋白質,可被當作生物標誌,並利用此做為發展胃與十二指腸疾病的偵測/診斷試驗或套組。例如,該胃與十二指腸疾病係可為胃癌、胃潰瘍、十二指腸潰瘍和其他。在一具體實施例,藉由一質譜議偵測或監視鑑定於此之一個或多個生物標誌的量,進行常態化分析後,達成胃與十二指腸疾病之早期偵測。在另一具體實施例,調整該鑑定之一個或多個生物標誌於生物晶片試驗以偵測相對應之胃與十二指腸疾病。在另一具體實施例,該鑑定之相對應胃與十二指腸疾病之一個或多個生物標誌係可被應用為發展治療和/或預防檢測之標的。One or more polypeptides and/or proteins associated with the duodenal disease identified herein can be used as biomarkers and used as a detection/diagnostic test or kit for developing gastric and duodenal diseases. For example, the gastric and duodenal diseases may be gastric cancer, gastric ulcer, duodenal ulcer, and others. In one embodiment, an early detection of gastric and duodenal diseases is achieved after a normalized analysis by detecting or monitoring the amount of one or more biomarkers identified by a mass spectrometer. In another embodiment, the identified one or more biomarkers are adjusted in a biochip assay to detect corresponding gastric and duodenal diseases. In another embodiment, the identified one or more biomarker lines corresponding to gastric and duodenal diseases can be used to develop a therapeutic and/or prophylactic test.
根據本發明之一個或多個具體實施例,係提供一鑑定胃與十二指腸疾病相關之生物標誌的方法。在一具體實施例,係利用胃液樣本。在另一具體實施例,係利用質譜分析和/或二維凝膠電泳分析以鑑定胃與十二指腸疾病之生物標誌。在另一具體實施例,決定該鑑定之生物標誌之序列,例如,藉由質譜儀和其他胺基酸序列分析方法。此外,提供一利用鑑定之生物標誌以篩選人類病患之胃與十二指腸疾病。In accordance with one or more specific embodiments of the present invention, a method of identifying a biomarker associated with a stomach and duodenal disease is provided. In a specific embodiment, a gastric fluid sample is utilized. In another embodiment, mass spectrometry and/or two-dimensional gel electrophoresis analysis is utilized to identify biomarkers of gastric and duodenal diseases. In another embodiment, the sequence of the identified biomarker is determined, for example, by mass spectrometry and other amino acid sequence analysis methods. In addition, an identification biomarker is provided for screening gastric and duodenal diseases in human patients.
藉由各式樣本收集的方法收集胃液樣本。一方法係利用伸入患者胃部之內視鏡吸取胃液,亦可利用其他非內視鏡的方法。對胃癌和其他胃與十二指腸疾病而言,獲得胃液樣本相對地簡單和容易,因為數以百萬的患者和一般受測者係進行年度的內視鏡檢查。亦可利用不需內視鏡的非侵入性方法以收集胃液樣本。例如,收集胃液樣本時利用一非侵入性的線繩試驗(string test)。該非侵入性的線繩試驗係應用於胃幽門螺旋桿菌(H.pylori )感染的診斷以幫助流行病學的研究以評估無症狀受測者的胃幽門螺旋桿菌情況。另一獲得胃液樣本的例子包括利用一小型的膠囊,其包含一沿著吸收紙片的穿孔塑膠覆蓋和一40至45公分長連結至內部膠囊的尼龍線,吞嚥該膠囊並使其停留於胃腔60分鐘以使吸收紙片飽合吸收胃液,接著,拉動尼龍線由胃腔移出該膠囊並由該吸收紙片獲得胃液。Gastric fluid samples were collected by various sample collection methods. One method utilizes an endoscope that extends into the patient's stomach to extract gastric fluid, and other methods that are not endoscopic. For gastric cancer and other gastric and duodenal diseases, obtaining a sample of gastric juice is relatively simple and easy, as millions of patients and the general subject undergo annual endoscopy. Non-invasive methods that do not require an endoscope can also be used to collect gastric fluid samples. For example, a non-invasive string test is used when collecting gastric fluid samples. This non-invasive cord test was applied to the diagnosis of H. pylori infection to help epidemiological studies to assess the condition of H. pylori in asymptomatic subjects. Another example of obtaining a gastric fluid sample includes the use of a small capsule comprising a perforated plastic cover along the absorbent sheet and a nylon thread attached to the inner capsule 40 to 45 cm long, swallowing the capsule and allowing it to remain in the stomach cavity. The gastric paper was absorbed for 60 minutes to saturate the absorbent sheet, and then the nylon thread was pulled out of the stomach cavity to remove the capsule and the gastric juice was obtained from the absorbent sheet.
胃液係包含數個化合物,包含鹽酸、胃蛋白酶、脂肪酶、黏蛋白、內部因子、多肽、核苷酸和電解質等等。此外,其可能由於吞嚥包含唾液成份、胃及十二指腸的逆流包含膽汁、和來自於受損胃壁的免疫調控因子或血液及來自於胃癌的致癌蛋白。該胃液樣本係計劃地利用於蛋白質/多肽的質譜分析,因為胃液樣本中蛋白質或多肽的數量比其他種類的樣本,例如血清樣本,相對的少量。藉由胃部的酵素,大部份的蛋白質和多肽能在非常酸性的環境下被消化分解。即使大部份完整的蛋白質不能存在,我們假設某些多肽和蛋白質仍可能存在於胃液中。The gastric fluid system contains several compounds including hydrochloric acid, pepsin, lipase, mucin, internal factors, polypeptides, nucleotides, electrolytes and the like. In addition, it may contain bile by swallowing a component containing saliva, a countercurrent to the stomach and duodenum, and an immunoregulatory factor or blood from the damaged stomach wall and an oncogenic protein derived from gastric cancer. This gastric fluid sample is intended to be utilized for mass spectrometric analysis of proteins/polypeptides because the amount of protein or polypeptide in the gastric fluid sample is relatively small compared to other types of samples, such as serum samples. Most of the proteins and peptides are digested and decomposed in a very acidic environment by the enzymes in the stomach. Even though most intact proteins cannot be present, we hypothesized that certain peptides and proteins may still be present in the gastric juice.
大部份的蛋白質具高分子量並因此而具高質量,於高質量時典型質譜儀的解析度非常有限,所以在高質量的範圍內分辨蛋白質是相當困難的。然而,一但蛋白質被消化分解,如胃液樣本的例子,較短的蛋白質或多肽的鑑定係被認為是較容易的,相對地可以獲得高解析度的多肽質譜。某些多肽的序列可推估其原始蛋白質前驅物的存在,少數特異性蛋白質選擇性片段的存在也可能反應某些酵素性消化和/或不同生物環境所產生。本發明藉由提供多肽體(peptidomic)和蛋白質體的分析於搜索生物標誌以做為疾病之診斷。Most proteins have high molecular weight and therefore high quality. The resolution of typical mass spectrometers is very limited at high quality, so it is quite difficult to distinguish proteins in a high quality range. However, once the protein is digested and decomposed, as in the case of gastric fluid samples, the identification of shorter proteins or polypeptides is considered to be relatively easy, and relatively high resolution polypeptide mass spectra can be obtained. The sequence of certain polypeptides can be used to estimate the presence of their original protein precursors, and the presence of a few specific protein-selective fragments may also be reflected in certain enzymatic digestions and/or in different biological environments. The present invention provides for the diagnosis of a disease by providing an analysis of a polypeptide (peptidomic) and a proteosome to search for a biomarker.
本發明之具體實施例係提供分析多肽和蛋白質分佈態樣以做為疾病診斷。值得注意的是,藉由本發明方法鑑定之多肽係可被利用為某些疾病的生物標誌,即使當該原始蛋白質含量與此類疾病具有或不具有任何可分辨之差異。因為多肽常係藉由某些蛋白質的消化而製造,某些多肽的含量係與特定酵素含量相關,該酵素為此類特異多肽製造之決定因子。大部份多肽的功能仍未詳知。然而,多種多肽之比較測定對疾病診斷是具有價值的。Specific embodiments of the invention provide for the analysis of polypeptide and protein profiles for use as a disease diagnosis. Notably, the polypeptides identified by the methods of the invention can be utilized as biomarkers for certain diseases, even when the original protein content has or does not have any discernible difference from such diseases. Because polypeptides are often produced by digestion of certain proteins, the content of certain peptides is related to the specific enzyme content, which is the determining factor for the manufacture of such specific polypeptides. The function of most peptides is still not known. However, comparative assays for multiple polypeptides are of value for disease diagnosis.
根據本發明之一或數方面,胃液中蛋白質和多肽的含量和型式構成一蛋白質和多肽的組合態樣,其係藉由本發明之質譜分析作圖並與不同的胃與十二指腸疾病相關。例如,藉由比較健康者和胃癌病患的胃液樣本,可鑑定一關於胃癌的特殊質譜和特異的蛋白質及多肽。其他胃與二指腸疾病,例如胃潰瘍、十二指腸潰瘍、和其他結腸相關或小腸相關之疾病亦可利此述方法分析。In accordance with one or more aspects of the invention, the amount and form of protein and polypeptide in gastric fluid constitute a combination of a protein and a polypeptide which are mapped by mass spectrometry of the present invention and associated with different gastric and duodenal diseases. For example, by comparing gastric samples from healthy and gastric cancer patients, a specific mass spectrometer and specific proteins and polypeptides for gastric cancer can be identified. Other gastric and digestive diseases, such as gastric ulcers, duodenal ulcers, and other colon-related or small intestine-related diseases can also be analyzed by the methods described herein.
可發現胃癌病患的胃液平均而言較一般人具較高之pH值(較不酸)和較高程度之蛋白質和多肽的含量。根據本發明之一個或多個具體實施例,多肽的質譜態樣和蛋白質質譜態樣係被利用於分析胃液樣本,以做為胃與二指腸疾病之偵測與診斷。於此,提供胃癌病患之實驗結果,並成功地鑑定胃癌相關之多肽片段和蛋白質。該結果亦暗指胃液分析優於血清分析根據其敏感性和可靠性。It can be found that the gastric juice of patients with gastric cancer has a higher pH value (less acid) and a higher degree of protein and polypeptide content than the average person. According to one or more embodiments of the invention, the mass spectrometric and protein mass spectrometric profiles of the polypeptide are utilized in the analysis of gastric fluid samples for detection and diagnosis of gastric and duodenal diseases. Herein, the experimental results of gastric cancer patients are provided, and the polypeptide fragments and proteins related to gastric cancer are successfully identified. The results also imply that gastric juice analysis is superior to serum analysis based on its sensitivity and reliability.
根據本發明之一個或多個具體實施例,當收集胃液樣本,可實行蛋白質純化以萃取胃液樣本之蛋白質和/或多肽並移除污染物。例如,離心可被利用於移除污染物。此外,磁性的奈米粒子係為一簡單、快速且容易的方式由胃液樣本中萃取蛋白質。其他蛋白質萃取的方式亦被利用,包括鑽石奈米粒子純化、HPLC、離子交換管柱色層分析、二維凝膠、西方墨點法、毛細管電泳等等,蛋白質萃取方法並不限於上述。In accordance with one or more embodiments of the invention, when a gastric fluid sample is collected, protein purification can be performed to extract proteins and/or polypeptides from the gastric fluid sample and remove contaminants. For example, centrifugation can be utilized to remove contaminants. In addition, magnetic nanoparticles are a simple, fast and easy way to extract proteins from gastric fluid samples. Other methods of protein extraction are also utilized, including diamond nanoparticle purification, HPLC, ion exchange column chromatography, two-dimensional gelation, Western blotting, capillary electrophoresis, etc., and protein extraction methods are not limited to the above.
一實施例係利用基質-輔助雷射脫附離子化(matrix-assisted laser desorption/ionization;MALDI)飛行時間質譜儀(time-of-flight mass spectrometer;TOF-MS)以獲得多肽/蛋白質之質譜。藉由MALDI-TOF-MS,一個質譜分析可於數秒中內獲得。一般而言,經由MALDI,僅單一或雙電荷可被獲得,該結果圖譜較其他種類之質譜明顯地簡易。然而,其他種類之質譜儀亦被利用包括,但不受限於上述質譜,電噴霧質譜儀(electrospray mass spectrometry)、脫附電噴霧質譜儀(desorption electrospray mass spectrometry)、雷射誘發原聲脫附質譜儀(laser induced acoustic desorption mass spectrometry)和其他。各式的質譜儀亦被利用。One embodiment utilizes a matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometer (TOF-MS) to obtain a mass spectrum of a polypeptide/protein. With MALDI-TOF-MS, a mass spectrometric analysis can be obtained in a matter of seconds. In general, only single or double charges can be obtained via MALDI, which is significantly easier than other types of mass spectra. However, other types of mass spectrometers are also utilized, but are not limited to the above-mentioned mass spectrometry, electrospray mass spectrometry, desorption electrospray mass spectrometry, and laser-induced acoustic desorption mass spectrometry. Laser induced acoustic desorption mass spectrometry and others. Various mass spectrometers are also utilized.
電噴霧屬於溫和的離子化方式,並容易在大蛋白質產生複電荷,使其適於分析具高分子量之蛋白質和多肽。雖然電噴霧質譜儀通常很複雜,然而多肽質譜態樣並不如蛋白質態樣複雜,因此電噴霧質譜儀可利用此優點進行分析,因為一般而言,多肽具有少於3之電荷。根據質量選擇、離子阱(ion trap)、磁力部份和傅葉爾轉換(Fourier transform),質譜儀係被可替換。因為傅葉爾轉換質譜儀具有非常高之質量解析度(M/△M>200,000),其係為一測量質譜間差異非常可靠之方法。Electrospray is a mild ionization mode and tends to generate complex charges in large proteins, making it suitable for the analysis of proteins and peptides with high molecular weight. While electrospray mass spectrometers are often complex, peptide mass spectrometry is not as complex as proteinaceous, electrospray mass spectrometers can take advantage of this advantage because, in general, polypeptides have a charge of less than three. The mass spectrometer is replaceable based on mass selection, ion trap, magnetic portion, and Fourier transform. Because the Fourier transform mass spectrometer has a very high mass resolution (M/ΔM > 200,000), it is a very reliable method for measuring the difference between mass spectra.
多肽和蛋白質的質譜態樣係可被用於分辨胃癌樣本和一般測試樣本。質譜態樣亦可被用於分辨胃潰瘍患者的樣本和一般無潰瘍人類受測者的樣本。當胃與十二指腸之特異標誌,藉由質譜儀分析、二維電泳或其他傳統之方法被鑑定出來,胃液樣本之比較質譜亦可被用於篩檢或診斷該胃與十二指腸疾病。例如,針對癌症,應直接比較收集自癌細胞的樣本和一般細胞的樣本。然而,自癌細胞獲得樣本的過程是侵入性的,且鑑定腫瘤的位置以於癌症早期獲得癌細胞通常是困難的。然而,收集自疾病位置、血清樣本、血漿樣本、體液樣本和其他樣本之細胞的試驗樣本亦被利用於本發明中之質譜分析。Mass spectrometric images of peptides and proteins can be used to resolve gastric cancer samples and general test samples. Mass spectrometry can also be used to distinguish samples from patients with gastric ulcers and samples from generally unacquired human subjects. When the specific markers of the stomach and duodenum are identified by mass spectrometry, two-dimensional electrophoresis or other conventional methods, comparative mass spectrometry of gastric juice samples can also be used to screen or diagnose the stomach and duodenal diseases. For example, for cancer, samples collected from cancer cells and samples from normal cells should be directly compared. However, the process of obtaining a sample from a cancer cell is invasive, and identifying the location of the tumor to obtain cancer cells early in the cancer is often difficult. However, test samples collected from disease sites, serum samples, plasma samples, body fluid samples, and other samples are also utilized for mass spectrometry in the present invention.
比較正常和疾病樣本間的各式多肽/蛋白質的量。經由質譜鑑定其異常量,其係包含表現量增加或抑制的量(藉由蛋白質表現表現量增加和抑制)、特異酵素性的消化和/或其他機轉。Compare the amount of each polypeptide/protein between normal and disease samples. The abnormal amount is identified by mass spectrometry, which includes an amount of increase or inhibition of expression (by an increase in expression and inhibition of protein expression), specific enzyme-induced digestion, and/or other mechanisms.
在質譜態樣的比較中,生物資訊分析,例如神經網路程式和其他生物資訊軟體,係被利用於快速地和有效地分析大量獲得的數據,以於質譜中比較一特異質量平均程度之相異。該同樣分析可被施行於正常者和病患的樣本。In the comparison of mass spectrometry, bioinformatics analysis, such as neural network programs and other bioinformatics software, is used to quickly and efficiently analyze large amounts of acquired data to compare the average degree of a specific mass in the mass spectrum. different. This same analysis can be performed on normal and patient samples.
在另一替代之具體實施例,施行二維電泳以分辨病患和正常者胃液樣本間的蛋白質差異。除了分子量,二維電泳係藉由pI值提供胃液樣本中蛋白質程度之精確具像化,然而質譜分析僅藉由分子量提供分離。In another alternative embodiment, two-dimensional electrophoresis is performed to resolve protein differences between patient and normal gastric fluid samples. In addition to molecular weight, two-dimensional electrophoresis provides precise visualization of the extent of protein in gastric fluid samples by pI values, whereas mass spectrometry provides separation only by molecular weight.
施行於健康者和不同胃與十二指腸疾病患者胃液樣本的二維電泳結果呈現3種不同的條帶態様,包含基礎條帶態様、特殊條帶態様和非特殊條帶態様。一統計分析之結果如第14圖所示。Two-dimensional electrophoresis results of gastric juice samples from healthy people and patients with different stomach and duodenal diseases showed three different strip states, including basic strip states, special strip states, and non-special strip states. The results of a statistical analysis are shown in Figure 14.
比較病患樣本之條帶態様之特殊條帶的結果或強度與健康者樣本基礎線之二維電泳的條帶態様,並鑑定異常蛋白質量,其係藉由二維凝膠上之條帶強度所呈現。例如,二維凝膠蛋白的態樣係被利用於分辮胃癌樣本和一般健康受測者的試驗樣本。二維凝膠蛋白的態樣亦可被用於分辮胃潰瘍患者的樣本和正常非胃潰瘍健康受測者的樣本。此外,亦比較胃十二指腸病患的樣本和健康者的樣本。Comparing the results or intensity of the strips of the patient's sample strips with the two-dimensional electrophoresis of the healthy person's sample base line, and identifying the abnormal protein content by the strip intensity on the two-dimensional gel Presented. For example, a two-dimensional gel protein pattern is used in a test sample of a bisected gastric cancer sample and a general healthy subject. The two-dimensional gel protein can also be used for samples of patients with gastric ulcer and healthy non-gastric ulcer healthy subjects. In addition, samples of patients with gastroduodenal diseases and samples of healthy people were also compared.
當鑑定胃與十二指腸疾病病患樣本的二維凝膠條帶態様,並與一正常樣本之二維凝膠條帶態様相較,該出現或消失之某一或多個蛋白質條帶或該改變蛋白質條帶之強度係暗指其一個或多個蛋白質條帶係為該胃與十二指腸疾病之良好生物標誌。藉由自二維凝膠上切除該一個或多個蛋白質條帶,其一個或多個蛋白質條帶的特性可經由胺基酸定序分析。When identifying a two-dimensional gel strip state of a patient with a stomach and duodenal disease and comparing it to a two-dimensional gel strip state of a normal sample, the one or more protein bands that appear or disappear or the change The strength of the protein band implies that one or more of its protein bands are good biomarkers for the stomach and duodenal diseases. By excising the one or more protein bands from the two-dimensional gel, the properties of one or more of the protein bands can be analyzed via amino acid sequencing.
可以發現,來自健康者和十二指腸潰瘍樣本之特殊條帶態様的頻率相對較低,兩者皆約為6%,然而,來自胃潰瘍和胃癌病患樣本之特殊條帶態様的頻率則為相對較高,約為42%和93%。因此,來自胃潰瘍和胃癌樣本之蛋白質條帶係可被分析以做為胃潰瘍和胃癌之特殊蛋白質生物標誌。It can be found that the frequency of special bandings from healthy people and duodenal ulcer samples is relatively low, both of which are about 6%. However, the frequency of special bandings from gastric ulcer and gastric cancer patients is relatively high. , about 42% and 93%. Therefore, protein bands from gastric ulcer and gastric cancer samples can be analyzed as specific protein biomarkers for gastric ulcers and gastric cancer.
我們也發現,胃潰瘍和胃癌病患胃液的蛋白質濃度較健康者明顯地增高(約1.06 mg/ml和約2.61 mg/ml vs約0.48 mg/ml,個別地),且十二指腸潰瘍患者具有較低的胃液的蛋白質濃度與健康者相較(約0.26 mg/ml vs約0.48 mg/ml)。此外,胃部低酸度和老化係為影響胃液蛋白質濃度之獨立因子,其奇異率(odds ratios)分別為32.9(95% CI:11.8-90.9)以及3.2(95% CI:1.3-8.3)。We also found that the gastric protein concentration of gastric ulcer and gastric cancer patients was significantly higher than that of healthy people (about 1.06 mg/ml and about 2.61 mg/ml vs about 0.48 mg/ml, individually), and patients with duodenal ulcer had lower The protein concentration of gastric juice was comparable to that of healthy people (about 0.26 mg/ml vs about 0.48 mg/ml). In addition, the low acidity and aging of the stomach were independent factors affecting the protein concentration of gastric juice, and the odds ratios were 32.9 (95% CI: 11.8-90.9) and 3.2 (95% CI: 1.3-8.3), respectively.
鑑定自本發明方法之生物標誌係可適用於胃與十二指腸疾病之診斷試驗或篩檢套組。此外,該鑑定自本發明方法之生物標誌係可應用於設計胃與十二指腸疾病之治療方式或胃與十二指腸疾病治療方式之標的。一組特殊的多肽可做為可靠之疾病診斷有價值之生物標誌。例如,本發明係提供胃液中特殊的多肽和蛋白質做為胃癌診斷之生物標誌,包含多肽2187 m/z、多肽2387 m/z、多肽3573 m/z、多肽2573 m/z、多肽4132 m/z、α-抗胰蛋白酶前驅物和其他。Biomarkers identified from the methods of the invention are applicable to diagnostic tests or screening kits for gastric and duodenal diseases. Furthermore, the biomarker identified from the method of the invention can be used to design a treatment for gastric and duodenal diseases or a treatment for gastric and duodenal diseases. A special set of peptides can be used as a valuable biomarker for reliable disease diagnosis. For example, the present invention provides specific polypeptides and proteins in gastric juice as biomarkers for diagnosis of gastric cancer, comprising polypeptide 2187 m/z, polypeptide 2387 m/z, polypeptide 3573 m/z, polypeptide 2573 m/z, polypeptide 4132 m/ z, alpha-antitrypsin precursors and others.
這些多肽的胺基酸序列係列舉為序列辨識號NOS:1-5。多肽2187 m/z、多肽2387 m/z和多肽3573 m/z(序列辨識號NOS:1-3)的量被發現與一般健康者相較其於胃癌病患中較低。然而,多肽2573 m/z和多肽4132 m/z(序列辨識號NOS:4-5)的量被發現與一般健康者相較其於胃癌病患中增高。The amino acid sequence series of these polypeptides is given by the sequence number NOS: 1-5. The amount of polypeptide 2187 m/z, polypeptide 2387 m/z, and polypeptide 3573 m/z (SEQ ID NO: 1-3) was found to be lower in patients with gastric cancer than in healthy subjects. However, the amount of polypeptide 2573 m/z and polypeptide 4132 m/z (SEQ ID NO: 4-5) was found to be increased in patients with gastric cancer compared with those in general health.
多肽2187 m/z(序列辨識號:1,FLKKHNLNPARKYFPQW)和多肽2387 m/z(序列辨識號:2,FLKKHNLNPARKYFPQWEA)係為胰蛋白酶原之一部份。多肽3573 m/z(序列辨識號:3,ETKKTEDRFVPSSSKSEGKKSREQPSVLSRY)係為一亮氨酸拉鍊結構蛋白(leucine zipper protein)之多肽片段。此類小型多肽片段受抑制量的重要性仍在研究中。The polypeptide 2187 m/z (SEQ ID NO: 1, FLKKHNLNPARKYFPQW) and the polypeptide 2387 m/z (SEQ ID NO: 2, FLKKHNLNPARKYFPQWEA) are part of the trypsinogen. The polypeptide 3573 m/z (SEQ ID NO: 3, ETKKTEDRFVPSSSKSEGKKSREQPSVLSRY) is a polypeptide fragment of a leucine zipper protein. The importance of the amount of inhibition of such small polypeptide fragments is still under investigation.
多肽2573 m/z(序列辨識號:4,DAHKSEVAHRFKDLGEENFKALVL)係為一白蛋白之多肽片段。普遍認為,癌症病患具較高出血的可能性使白蛋白由血管中進入胃部。亦可能是,白蛋白在非常酸性之胃十二指腸環境下被消化分解成小的多肽且此類多肽和癌細胞交互作用以維持高含量而受本發明之方法偵測並於胃液中觀察到。該多肽4132 m/z(序列辨識號NOS:5 SIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK)表現量增加的量的重要性仍在研究中。The polypeptide 2573 m/z (SEQ ID NO: 4, DAHKSEVAHRFKDLGEENFKALVL) is a polypeptide fragment of an albumin. It is generally believed that cancer patients have a higher likelihood of bleeding, allowing albumin to enter the stomach from the blood vessels. It is also possible that albumin is digested into small polypeptides in a very acidic gastric duodenal environment and such polypeptides interact with cancer cells to maintain high levels and are detected by the methods of the invention and observed in gastric juice. The importance of the amount of increased expression of the polypeptide 4132 m/z (SEQ ID NO: 5 SIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK) is still under investigation.
至少一個來自胃癌樣本二維電泳特殊條帶態様條帶之蛋白質體分析和胺基酸序列分析顯示,α-抗胰蛋白酶前驅物係為一表現於胃癌樣本特殊條帶之主要蛋白質,如第18圖之B1條帶所呈現。該α-抗胰蛋白酶前驅物的量於胃癌病患明顯地較健康者為高。有趣的是,該特殊α-抗胰蛋白酶前驅物之條帶可於早期的胃癌(100%)和進階的胃癌(90%)被鑑定,如第15圖所示,其暗指該α-抗胰蛋白酶前驅物係可作為早期和進階的胃癌的生物標誌。At least one protein body analysis and amino acid sequence analysis of a special banded band of two-dimensional electrophoresis from gastric cancer samples showed that the α-antitrypsin precursor is a major protein expressed in a special band of gastric cancer samples, such as the 18th. The B1 strip of the figure is presented. The amount of the alpha-antitrypsin precursor is significantly higher in patients with gastric cancer than in healthy subjects. Interestingly, the band of this particular alpha-antitrypsin precursor was identified in early gastric cancer (100%) and advanced gastric cancer (90%), as shown in Figure 15, which implies the alpha- The antitrypsin precursor system serves as a biomarker for early and advanced gastric cancer.
第2圖係介紹本發明以篩選患有胃與十二指腸疾病之人類受測者,其係藉由本發明鑑定之生物標誌。收集胃液試驗樣本,來自於收集之試驗樣本的多肽和蛋白質係可被部份地純化。Figure 2 is a diagram showing the present invention for screening human subjects suffering from gastric and duodenal diseases, which are biomarkers identified by the present invention. The gastric juice test sample is collected and the polypeptide and protein lines from the collected test sample can be partially purified.
藉由各式的偵測方法,與胃與十二指腸疾病相關之生物標誌的量係可被篩選。例如,一特殊的質譜胃液態樣係可作為一特徵態樣利用於篩選胃十二指腸疾病,例如,胃癌、胃潰瘍、十二指腸潰瘍和其他。在另一實施例,抗該鑑定之生物標誌的單株抗體和多株抗體或抗血清係可被製作以為疾病偵測和診斷之用。具有至少一胺基酸延展,對於序列辨識號NOS:1-5和/或α-抗胰蛋白酶前驅物具有高於90%序列特徵之多肽係可利用於篩檢胃與十二指腸疾病和/或胃癌與其他的應用。藉由本發明之方法,胃液中生物標誌之發現係提供一簡易胃癌診斷和胃與十二指腸疾病診斷之方式。The amount of biomarkers associated with gastric and duodenal diseases can be screened by various detection methods. For example, a special mass spectrometry gastric fluid sample can be used as a feature to screen for gastroduodenal diseases such as gastric cancer, gastric ulcer, duodenal ulcer and others. In another embodiment, monoclonal antibodies and polyclonal antibodies or antisera against the identified biomarkers can be made for disease detection and diagnosis. A polypeptide having at least one amino acid extension and having a sequence identity of NOS: 1-5 and/or an α-antitrypsin precursor having greater than 90% sequence characteristics can be used for screening gastric and duodenal diseases and/or gastric cancer With other applications. By the method of the present invention, the discovery of biomarkers in gastric juice provides a means for the diagnosis of simple gastric cancer and the diagnosis of gastric and duodenal diseases.
該鑑定之生物標誌亦可應用於篩選調節此類生物標誌表現量增加或抑制的酵素或蛋白質。本發明之生物標誌、鑑定於此之酵素和蛋白質,對於其相關胃與十二指腸疾病之發展係為重要,並可做為該胃與十二指腸疾病之治療或偵測之可能標的。此外,許多生物標誌、蛋白質、酵素和多肽的量化偵測係可同時作為一疾病診斷之可靠方式。The identified biomarker can also be used to screen for enzymes or proteins that modulate the increase or inhibition of the expression of such biomarkers. The biomarkers of the present invention, the enzymes and proteins identified therein, are important for the development of their associated gastric and duodenal diseases and may serve as a possible target for the treatment or detection of such gastric and duodenal diseases. In addition, many biomarkers, proteins, enzymes, and peptides can be used as a reliable means of disease diagnosis.
生物標誌的量與之前收集正常受測者樣本的基礎線量相較。當胃液生物標誌之異常量被鑑定,該受測者試驗樣本係可能為胃與十二指腸疾病陽性。如果該試驗樣本的生物標誌的量和基礎線的量相同,則該受測者係為陰性。The amount of biomarker is compared to the amount of baseline used to collect samples from normal subjects. When the abnormal amount of gastric biomarker is identified, the test sample of the subject may be positive for gastric and duodenal diseases. If the amount of the biomarker of the test sample is the same as the amount of the baseline, the subject is negative.
本發明以下列實施例做進一步闡明,但發明的範圍不受於此限The invention is further illustrated by the following examples, but the scope of the invention is not limited thereto.
在病患和健康受測者禁食隔夜之後,以Olympus GIF XVIO和GIF XQ200(Olympus Corp.,Tokyo,Japan)施行內視鏡檢查。在插入內視鏡於胃部之後,藉由內視鏡的抽吸管吸出胃液,例如,5ml或其他適合的量/體積,並收集於吸取線之無菌阱(sterile trap)。一般而言,受測者,例如人類受測者,禁食隔夜以確保精確性和避免在樣本分析時的複雜性。其他非內視鏡的方法亦被利用。例如,一高度吸收力的線繩纏繞於一具重量的膠囊內部。隨後吞嚥該膠囊並於15分鐘後由胃部取出。該線段之遠端係被剪裁以做為胃液樣本。各式收集胃液的方法亦可被使用。Endoscopic examinations were performed with Olympus GIF XVIO and GIF XQ200 (Olympus Corp., Tokyo, Japan) after fasting overnight for patients and healthy subjects. After the endoscope is inserted into the stomach, the gastric fluid is aspirated through the suction tube of the endoscope, for example, 5 ml or other suitable amount/volume, and collected in a sterile trap of the suction line. In general, subjects, such as human subjects, are fasted overnight to ensure accuracy and avoid complexity in sample analysis. Other methods of non-endoscopes are also utilized. For example, a highly absorbent cord is wound inside a weighted capsule. The capsule was then swallowed and removed from the stomach after 15 minutes. The distal end of the line segment is clipped for use as a sample of gastric fluid. Various methods of collecting gastric juice can also be used.
接著施行定期上胃十二指腸管道的檢視,並由較大彎曲之竇室(antrum)獲得活體組織切片行快速尿素酵素分析以決定H.pylori 感染情形。對同意進行地形組織病理研究(topographical histopathological study)的受測者採取額外活體組織以行胃部的組織檢視。於中竇室(midantrum)(幽門腺區)和中體(middle body)(胃底腺區)較小和較大彎曲的部份採得4個樣本。該活體組織樣本以10%的緩衝福馬林固定、以石臘包埋並切片。該切片以H&E染色。該切片進行盲目病患臨床診斷檢視。藉由升級的Sydney系統評估胃炎的活動(親神經浸潤(neutrophil infiltration)、單核細胞細胞浸潤(mononuclear cell infiltration)、腺體萎縮(glandular atrophy)和小腸變性生長(intestinal metaplasia))。為了調整臨床的特徵,記錄每個受測者下列的資料:年齡、性別、胃癌家族歷史、抽煙歷史和飲酒情形。A regular examination of the upper gastroduodenal duct is then performed, and a large amount of biochemical tissue is obtained from the larger curved antrum to perform a rapid urease assay to determine the H. pylori infection. Subjects who consented to a topographical histopathological study were given additional living tissue to perform a tissue examination of the stomach. Four samples were taken from the smaller and larger curved portions of the midantrum (pyloric gland) and middle body (stomach gland). The living tissue sample was fixed with 10% buffered formalin, embedded in paraffin and sectioned. The sections were stained with H&E. The section was used for clinical diagnosis of blind patients. Gastritis activity (neutrophil infiltration, mononuclear cell infiltration, glandular atrophy, and intestinal metaplasia) was assessed by an upgraded Sydney system. To adjust the clinical characteristics, the following data were recorded for each subject: age, gender, family history of gastric cancer, smoking history, and drinking status.
在以玻璃電極pH測量儀(glass-electrode pH meter)收集之後,測量胃液的pH值。該收集之胃液樣本接著於10,000x g,4℃,離心約10分鐘以移除細胞破片和汙染物。平均分配該上清液並儲存於-70℃直至進一步的試驗分析。以2μl 150 mM的氫氧化銨中和約10μl離心後的胃液樣本上清液以調整至一所需的pH值。The pH of the gastric juice was measured after collection by a glass-electrode pH meter. The collected gastric juice samples were then centrifuged at 10,000 xg, 4 °C for about 10 minutes to remove cell fragments and contaminants. The supernatant was distributed equally and stored at -70 °C until further experimental analysis. The gastric juice sample supernatant after centrifugation of about 10 μl was neutralized with 2 μl of 150 mM ammonium hydroxide to adjust to a desired pH.
根據本發明之一個或多個具體實施例,當收集該胃液樣本,可施行部份蛋白質純化以萃取該胃液樣本的蛋白質或多肽並移除污染物。例如,藉由奈米粒子珠球(nanoparticle beads)施行進一步的蛋白質萃取。In accordance with one or more embodiments of the invention, when the gastric fluid sample is collected, partial protein purification can be performed to extract the protein or polypeptide of the gastric juice sample and remove contaminants. For example, further protein extraction is performed by nanoparticle beads.
第3圖係為一收集胃液樣本以行質譜分析的範例圖表介紹,其係根據本發明之一具體實施例。該胃液樣本係結合銅磁性珠球以形成混合物。當該混合物暴露於一磁力時,洗去不需要的或未結合的部份。結合的部份可藉由一緩衝液或一溶液,例如10μl的析出緩衝液(elution buffer),在磁力消失之後被析出。一使用之析出緩衝液係為50%之乙腈(acetonitrile)。Figure 3 is an illustration of a sample chart for collecting a sample of gastric juice for mass spectrometry, in accordance with an embodiment of the present invention. The gastric fluid sample is combined with copper magnetic beads to form a mixture. When the mixture is exposed to a magnetic force, the unwanted or unbonded portions are washed away. The bound portion can be precipitated after a magnetic force is lost by a buffer or a solution, for example, 10 μl of an elution buffer. One of the precipitation buffers used was 50% acetonitrile.
各式表面修飾的磁性珠球,例如C8、C18、磁性奈米粒子、鑽石奈米粒子或其他金屬奈米粒子係可被利用。第4圖介紹胃液樣本收集於50%乙腈析出緩衝液之中並準備好進行質譜分析。部份蛋白質純化、HPLC、離子交換、二維凝膠、西方墨點和毛細管電泳係使癌症和非癌症病患間的質譜比較更簡易。Various surface-modified magnetic beads, such as C8, C18, magnetic nanoparticles, diamond nanoparticles or other metal nanoparticles, can be utilized. Figure 4 shows that the gastric juice sample was collected in 50% acetonitrile precipitation buffer and prepared for mass spectrometry. Partial protein purification, HPLC, ion exchange, two-dimensional gels, Western blots, and capillary electrophoresis systems make mass spectrometry between cancer and non-cancer patients easier.
120個連續健康受測者(HSs)、39個胃潰瘍病患(GU)、38個十二指腸潰瘍病患(DU)和31個胃癌(GC)病患係參與本2D凝膠電泳研究。該健康受測者招募於健康檢查門診且不具胃十二指腸疾病病史。健康受測者的內視鏡檢查結果為正常或僅呈現輕微胃炎。病患排除原則包含於本研究前4週內質子幫浦抑制劑、H2 受體頡頏劑(H2 receptor antagonists)或非類脂醇抗發炎藥物的使用、兩種胃十二指腸病灶的共同存在、上胃十二指腸出血的表現和嚴重系統性疾病的共同存在。120 consecutive healthy subjects (HSs), 39 gastric ulcer patients (GU), 38 duodenal ulcer patients (DU), and 31 gastric cancer (GC) patients were involved in this 2D gel electrophoresis study. The healthy subject was recruited at a health check-up clinic and did not have a history of gastroduodenal disease. Endoscopic examinations of healthy subjects were normal or showed only mild gastritis. Patients excluded principles contained in the four weeks prior to the study of proton pump inhibitors, using H 2 receptor antagonistic agent (H 2 receptor antagonists) anti-inflammatory drugs or lipid alcohols of the two co-exist gastroduodenal lesions, The presence of upper gastroduodenal hemorrhage and the common systemic disease coexist.
藉由內視鏡檢查確認胃潰瘍和十二指腸潰瘍的診斷,一般指具有於直徑約5公釐或大於5公釐之環形黏膜損傷且具有明顯定義之潰瘍。藉由在潰瘍前張開一己知寛度之活體組織切片鉗子測量該潰瘍的尺寸。The diagnosis of gastric ulcer and duodenal ulcer confirmed by endoscopy is generally referred to as having an annular ulcer with a diameter of about 5 mm or more and having a well defined ulcer. The size of the ulcer was measured by opening a well-known biopsy forceps before the ulcer.
藉由組織學確認胃癌病患並根據Lauren的分類方法分類為腸型胃癌(n=19)、擴散性胃癌(n=9)和混合性胃癌(n=3)。手術後胃癌病患(n=27)腫瘤侵害的程度根據日本胃癌研究學會(the Japan Research Society for Gastric Cancer)提出的標準進一步區分為早期或進階的胃癌樣本。本研究經高雄榮民總醫院醫學研究委員會核准。全部病患和控制組係知悉同意。The gastric cancer patients were confirmed by histology and classified into intestinal type gastric cancer (n=19), diffuse type gastric cancer (n=9), and mixed gastric cancer (n=3) according to Lauren's classification method. The degree of tumor invasion in patients with gastric cancer after surgery (n=27) was further classified into early or advanced gastric cancer samples according to the criteria proposed by the Japan Research Society for Gastric Cancer. The study was approved by the Medical Research Committee of Kaohsiung Veterans General Hospital. All patients and control groups were informed of their consent.
近期研究指出,腸型胃癌的發生為一多重步驟事件,其係包含由淺表性胃炎(superficial gastritis)、萎縮性胃炎(atrophic gastritis)、小腸變性生長(intestinal metaplasia)、發育異常(dysplasia)演變成惡性腫瘤,透過累積的多重遺傳改變和蛋白質富含量、結構或功能的改變。關於擴散性胃癌,其黏附分子(adhesion molecules),例如,上皮性鈣黏素(E-cadherin)、連接素(catenin)和膜聯蛋白-7(annexin-7)的喪失亦參與其癌化過程。Recent studies have pointed out that the occurrence of intestinal type gastric cancer is a multi-step event, which includes superficial gastritis, atrophic gastritis, intestinal metaplasia, and dysplasia. It evolves into a malignant tumor through cumulative multiple genetic alterations and changes in protein richness, structure or function. With regard to diffuse gastric cancer, the loss of adhesion molecules such as E-cadherin, catenin and annexin-7 is also involved in the canceration process. .
因為胃癌發生過程中,胃部蜂窩狀上皮細胞的接續性改變將導致胃液成份改變,於此所描述之胃液蛋白質體研究係提供發現新型腫瘤特殊標誌以篩檢胃惡性腫瘤之一巨大潛力。此外,各式胃與十二指腸病患胃液之蛋白質濃度和成份係可被用於發現新型疾病的生物標誌。Because gastric cancer epithelial cell changes in the process of gastric cancer will lead to changes in gastric juice composition, the gastric juice proteosome research system described here provides great potential for the discovery of new tumor specific markers to screen for gastric malignancies. In addition, the protein concentration and composition of gastric juices in various stomach and duodenal diseases can be used to discover biomarkers of novel diseases.
藉由Bradford方法(21)決定胃液之全部蛋白質濃度。一固定之蛋白含量(約50μg每片膠)係被使用於二維電泳,有興趣之蛋白質墨點藉由質譜加以分析鑑定。一含有約50μg蛋白質的樣本以丙酮(1至2 v/v)於約-20℃沉澱約2個小時,且該混合液於10,000x g,4℃,離心10分鐘。該沈澱物以再水合溶液(9M尿素,35mM Tris,42 mM DTT,2% CHAPS,0.66% SDS,2% IPG緩衝液,微量溴酚藍)混合,並加入於固定的pH梯度IPG膠條,11公分,非線性,pH 4-7(Amersham Phamacia Biotech,Immobiline Dry-Strip,Uppsala,Sweden)在30V條件下進行隔夜覆水反應。The total protein concentration of the gastric juice is determined by the Bradford method (21). A fixed protein content (about 50 μg per gel) was used for two-dimensional electrophoresis, and the protein dots of interest were identified by mass spectrometry. A sample containing about 50 μg of protein was precipitated with acetone (1 to 2 v/v) at about -20 ° C for about 2 hours, and the mixture was centrifuged at 10,000 x g, 4 ° C for 10 minutes. The precipitate was mixed with a rehydration solution (9 M urea, 35 mM Tris, 42 mM DTT, 2% CHAPS, 0.66% SDS, 2% IPG buffer, trace bromophenol blue) and added to a fixed pH gradient IPG strip. 11 cm, non-linear, pH 4-7 (Amersham Phamacia Biotech, Immobiline Dry-Strip, Uppsala, Sweden) was subjected to an overnight water-repellent reaction at 30V.
透過第一維和第二維凝膠電泳施行二維電泳。利用商業上可得的、專門的設備,例如,IPGphor(Amersham Phamacia Biotech),施行第一維凝膠分離。蛋白質被連續地集中於約200V,約1個小時、約500V,約1個小時、約1000V,約1個小時。接著施行一約1000V至8000V之梯度約30分鐘。接著施行8000V之繼續集中約8個半小時以於IPGphor獲得一總量70kVh。Two-dimensional electrophoresis was performed by first-dimensional and second-dimensional gel electrophoresis. The first dimensional gel separation is performed using commercially available, specialized equipment, such as IPGphor (Amersham Phamacia Biotech). The protein is continuously concentrated at about 200 V for about 1 hour, about 500 V, about 1 hour, about 1000 V, about 1 hour. A gradient of about 1000V to 8000V is then applied for about 30 minutes. Then continue to concentrate on 8000V for about 8 and a half hours to get a total of 70kVh for IPGphor.
於第二維,該IPG係平衡於50 mM tris-CL,pH 8.8,6M尿素,30%甘油,1%DDT,和溴酚藍約12分鐘,並接著於50 mM tris-CL,pH 8.8,6M尿素,30%甘油,1.2%SDS,2%iodoacetamide,和溴酚藍約12分鐘。將該膠條置於15%梯度聚丙烯胺凝膠平板之上(190 x 140 x 1mm)且施行該凝膠電泳至隔天。該凝膠係以硝酸銀染色。In the second dimension, the IPG is equilibrated at 50 mM tris-CL, pH 8.8, 6 M urea, 30% glycerol, 1% DDT, and bromophenol blue for about 12 minutes, followed by 50 mM tris-CL, pH 8.8, 6 M urea, 30% glycerol, 1.2% SDS, 2% iodoacetamide, and bromophenol blue for about 12 minutes. The strip was placed on a 15% gradient polyamine gel gel plate (190 x 140 x 1 mm) and the gel was applied to the next day. The gel was dyed with silver nitrate.
藉由一高解析度的掃描器(GS-710 Calibrated Imaging Densitometer,Bio-Rad)紀錄凝膠中蛋白質之態樣為數位影像。以PDQuest軟體(Bio-Rad)做凝膠影像比對。The protein in the gel was recorded as a digital image by a high resolution scanner (GS-710 Calibrated Imaging Densitometer, Bio-Rad). Gel image alignment was performed using PDQuest software (Bio-Rad).
此外,在自二維凝膠萃取該蛋白質之前,以豬的胰蛋白酶(Promega,Madison,USA)消化分解該凝膠片段可理想地施行膠內胰蛋白酶之消化分解。以三氟乙酸(trifluoroacetic acid)和乙腈(acetonitrile;ACN)萃取該結果的消化分解的多肽片段並將其行質譜分析以決定其質量和胺基酸序列。在質譜分析之前,該多肽係以Zip TipC18(Millipore,Bedford,USA)純化,根據該製造者的說明書。In addition, prior to the extraction of the protein from the two-dimensional gel, digestion of the gel fragment by porcine trypsin (Promega, Madison, USA) is ideally performed by digestion and digestion of the trypsin in the gel. The resulting digested and decomposed polypeptide fragment was extracted with trifluoroacetic acid and acetonitrile (ACN) and subjected to mass spectrometry to determine its mass and amino acid sequence. Prior to mass spectrometry, the polypeptide was purified using Zip Tip C18 (Millipore, Bedford, USA) according to the manufacturer's instructions.
於基質-輔助雷射脫附離子化飛行時間質譜儀(MALDI TOF-MS)分析,一般而言,以一含有2,5-二羥基苯甲酸(2,5-dihydroxybenzoic acid)(50nmol/μl於50%乙腈)和磷酸(phosphoric acid)(終濃度約為0.4%)之約1 μl混合液混合約1 μl之析出的胃液樣本。其他已知適合製備蛋白質離子之基質亦以相同之結果試驗。接著,約1 μl該結果混合物被製成4墨點於MALDI無染色之鋼製樣本盤上並將其於室溫中風乾。藉由一修飾自ABI Voyager DE-STR MALDI-TOF質譜儀(Applied Biosystems,Foster City,CA,USA)之MALDI-TOF質譜儀產生該析出樣本之多肽和蛋白質的質譜態樣。該光譜儀係運作於正離子模式並具一25 kV之加速電壓。該低質量閥設定於500 m/z。蛋白質資料庫搜索工具“Profound”(http://www.129.85.J92/profoundbinIWebProFound.exe)係被利用於比較該胰蛋白酶片段的單一同位素m/z值與其在NCBI資料庫中己知蛋白質的值。Analysis by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF-MS), generally, with 2,5-dihydroxybenzoic acid (50 nmol/μl) Approximately 1 μl of the precipitated gastric juice sample was mixed with about 1 μl of a mixture of 50% acetonitrile and phosphoric acid (a final concentration of about 0.4%). Other substrates known to be suitable for the preparation of protein ions were also tested with the same results. Next, about 1 μl of the resulting mixture was made into 4 dots on a MALDI unstained steel sample pan and allowed to air dry at room temperature. A mass spectrometric profile of the polypeptide and protein of the precipitated sample was generated by a MALDI-TOF mass spectrometer modified from an ABI Voyager DE-STR MALDI-TOF mass spectrometer (Applied Biosystems, Foster City, CA, USA). The spectrometer operates in positive ion mode and has an accelerating voltage of 25 kV. The low mass valve is set at 500 m/z. The protein library search tool "Profound" (http://www.129.85.J92/profoundbinIWebProFound.exe) was used to compare the single isotope m/z value of the trypsin fragment with the value of the known protein in the NCBI database. .
該特殊特徵係為其變更微管道盤(microchannel plate)獲得和雷射能量測量的能力。一外部的雷射係被用於獲得MALDI圖譜(spectra)。以具有質量和電荷比例(M/Z)範圍1000至10,000之陽離子模式獲得大部份的圖譜。以約20 mj/cm2 至約300 mj/cm2 之雷射注量和以100次雷射發射之累積獲得一典型之質量圖譜。正常和癌症病患的樣本係於同樣的雷射注量下施行。This particular feature is the ability to change microchannel plate acquisition and laser energy measurements. An external laser system is used to obtain the MALDI spectrum. Most of the spectra were obtained in a cation mode with a mass to charge ratio (M/Z) ranging from 1000 to 10,000. A typical mass spectrum is obtained with a laser fluence of about 20 mj/cm 2 to about 300 mj/cm 2 and a cumulative of 100 laser shots. Samples of normal and cancer patients were performed under the same laser fluence.
MALDI-TOF無法提供明確蛋白質特性之樣本係藉由電噴霧MS/MS做進一步研究。該胰蛋白酶消化分解之上清液係以C18 HPLC純化和濃縮。該濃縮後的蛋白質/多肽樣本接著利用奈米流電噴霧(nanoflow electrospray)自金包覆玻璃毛細(gold-coated glass capillaries)噴灑。該MS/MS圖譜係藉由一Thermo FT-ICR質譜儀(Thermo Electron,Bremen,Germany)獲得並利用於資料庫搜尋,藉由MASCOT胺基酸序列搜尋引擎(http://www.matrixscience.com)於胺基酸鑑定以和Swissport資料庫做確認。Samples in which MALDI-TOF could not provide clear protein properties were further studied by electrospray MS/MS. The trypsin digested supernatant was purified and concentrated by C18 HPLC. The concentrated protein/polypeptide sample was then sprayed from gold-coated glass capillaries using a nanoflow electrospray. The MS/MS map was obtained by a Thermo FT-ICR mass spectrometer (Thermo Electron, Bremen, Germany) and used in a database search by the MASCOT Amino Acid Sequence Search Engine (http://www.matrixscience.com) Identification of the amino acid was confirmed with the Swissport database.
正常者和癌症病患的典型質譜係個別於第5A圖和第5B圖呈現。藉由比較正常、無疾病受測者胃液樣本的質譜和病患樣本的質譜,特殊分子的異常值(以質量的方式表現)係被鑑定。在癌症和非癌症樣本中,小部份質譜區域的明顯不同係可藉由肉眼觀察之,無需其他額外複雜的分析。因此,關於此質量峰部(peak)之多肽係被鑑定為一可用於胃癌診斷之良好生物標誌。其係共有5個和胃癌相關的質量峰部,其具質量和電荷比例(m/z)於2187、2387、3572、2573和4132。於其中,多肽2187 m/z、多肽2387 m/z和多肽3572 m/z,其峰部的量在癌症樣本中受到抑制,然而,多肽2573 m/z和多肽4132 m/z則表現量增加(參見,第6圖)。Typical mass spectra of normal and cancer patients are presented separately in Figures 5A and 5B. The abnormal value of the specific molecule (expressed in mass) was identified by comparing the mass spectrum of the gastric juice sample of the normal, disease-free tester and the mass spectrum of the patient sample. In cancer and non-cancer samples, a significant difference in the mass spectral region can be observed by the naked eye without additional additional complex analysis. Therefore, the polypeptide of this mass peak was identified as a good biomarker for the diagnosis of gastric cancer. There are 5 mass peaks associated with gastric cancer, which have mass and charge ratios (m/z) of 2187, 2387, 3572, 2573 and 4132. Among them, the polypeptide 2187 m / z, the polypeptide 2387 m / z and the polypeptide 3572 m / z, the amount of the peak is inhibited in the cancer sample, however, the polypeptide 2573 m / z and the polypeptide 4132 m / z increase the performance (See, Figure 6).
部份質譜的詳細內容於第6A-6B圖呈現並比較。明顯地,關於多肽2187 m/z、多肽2387 m/z和多肽3572 m/z 3個質量峰部,其在正常樣本中表現但在癌症樣本中消失或是具較低的量。2個主要的峰部,多肽2573 m/z和多肽4132 m/z在正常樣本中消失或非常微弱,卻表現於癌症樣本之中。The details of the partial mass spectra are presented and compared in Figures 6A-6B. Obviously, there are 3 mass peaks for the polypeptide 2187 m/z, the polypeptide 2387 m/z and the polypeptide 3572 m/z, which are expressed in normal samples but disappear in cancer samples or have lower amounts. The two major peaks, peptide 2573 m/z and peptide 4132 m/z disappeared or were very weak in normal samples, but were present in cancer samples.
收集自大量正常受測者和癌症病患之樣本亦進行統計分析。到目前為止,我們已經分析106個來自於健康正常受測者的樣本、38個來自胃潰瘍病患的樣本、37個來自於十二指腸潰瘍病患的樣本和34個來自胃癌病患的樣本。在癌症和非癌症病患的樣本間,有多於15個多肽質量峰部具重要的差異性。其比較質譜的結果呈現於第1-2表。Statistical analysis was also performed on samples collected from a large number of normal subjects and cancer patients. So far, we have analyzed 106 samples from healthy normal subjects, 38 samples from patients with gastric ulcer, 37 samples from patients with duodenal ulcer, and 34 samples from patients with gastric cancer. There are more than 15 peptide mass peaks between cancer and non-cancer patients with important differences. The results of comparing the mass spectra are presented in Tables 1-2.
如果我們計算胃癌對每個多肽的敏感度(sensitivity)和特異性(specificity),多肽2187 m/z、多肽2387 m/z、多肽3572 m/z和多肽4132 m/z,其質譜敏感度個別係為85%、88%、76%和53%。多肽2187 m/z、多肽2387 m/z、多肽3572 m/z和多肽4132 m/z,其質譜特異度個別係為80%、80%、79%和92%。If we calculate the sensitivity and specificity of gastric cancer for each peptide, peptide 2187 m/z, polypeptide 2387 m/z, peptide 3572 m/z, and peptide 4132 m/z, the mass spectrometry sensitivity is individual. The system is 85%, 88%, 76% and 53%. The polypeptide was 2187 m/z, the polypeptide was 2387 m/z, the polypeptide was 3572 m/z, and the polypeptide was 4132 m/z. The mass spectrometry specificity was 80%, 80%, 79%, and 92%.
比較獲得於此的結果與目前能夠作為胃癌偵測的其他血清生物標誌,該鑑定比其他之多肽生物標誌的敏感度明顯較高。例如,在臨床上,癌胚胎抗原抗原(CEA)和碳水化合抗原(CA19-9)係為胃癌中最常使用之血清生物標記。該血清CEA和CA19-9於胃癌偵測的敏感度僅有18%和29%。一近期之研究顯示,於胃癌偵測時胃液中增加之CEA和CA19-9的敏感度係為27%和17%,個別地。此外,其他近期鑑定之生物標誌,例如,血清p53抗體和基質金屬蛋白酶-1(matrix metalloproteinase-1,TIMP-1)的組織抑制劑,應用於偵測胃癌時,其敏感度非常地低(15%和17%,個別地)。因此,胃液CEA和CA19-9於胃癌診斷的實用性是非常有限的,然而,鑑定於此的多肽生物標誌具有相當高的敏感度且於胃癌偵測非常有用。Comparing the results obtained here with other serum biomarkers currently available for detection of gastric cancer, the identification is significantly more sensitive than other polypeptide biomarkers. For example, clinically, cancer embryo antigen antigen (CEA) and carbohydrate antigen (CA19-9) are the most commonly used serum biomarkers in gastric cancer. The sensitivity of serum CEA and CA19-9 in gastric cancer detection was only 18% and 29%. A recent study showed that the sensitivity of CEA and CA19-9 in gastric juice increased during gastric cancer detection was 27% and 17%, individually. In addition, other recently identified biomarkers, such as serum p53 antibodies and tissue inhibitors of matrix metalloproteinase-1 (TIMP-1), have very low sensitivity when used to detect gastric cancer (15 % and 17%, individually). Therefore, the utility of gastric juice CEA and CA19-9 in the diagnosis of gastric cancer is very limited. However, the biomarkers identified herein have a relatively high sensitivity and are very useful for gastric cancer detection.
鑑定於此的多肽和蛋白質生物標誌的優勢為它們的高敏感度和特異性。例如,於胃癌偵測中,胃液α-抗胰蛋白酶前驅物的敏感度和特異性個別為93%和83%。就我們所知,α-抗胰蛋白酶前驅物係為一胃液中新型的生物標誌,在其它目前可獲得的生物標誌之中,其對於胃癌偵測具有最高的敏感度。The advantages of the polypeptides and protein biomarkers identified herein are their high sensitivity and specificity. For example, in gastric cancer detection, the sensitivity and specificity of gastric juice α-antitrypsin precursors were 93% and 83%, respectively. To the best of our knowledge, the alpha-antitrypsin precursor is a novel biomarker in gastric juice, and among other currently available biomarkers, it has the highest sensitivity for gastric cancer detection.
利用SPSS程式(第10.1版,Chicago,Illinois,USA)施行統計分析。第10圖呈現健康受測者、胃潰瘍(GU)、十二指腸潰瘍(DU)和胃癌(GC)病患的人口特徵。胃癌病患明顯較健康受測者年長(67.5年vs 51.4年,P <0.001)。此外,該胃癌病患族群與健康受測者相較具一較高的男比女之比例(P =0.054)。在鑑定的族群間,胃癌家族歷史和飲酒歷史無明顯的不同。Statistical analysis was performed using the SPSS program (version 10.1, Chicago, Illinois, USA). Figure 10 shows the demographic characteristics of healthy subjects, gastric ulcer (GU), duodenal ulcer (DU), and gastric cancer (GC) patients. Gastric cancer patients were significantly older than healthy subjects (67.5 vs 51.4 years, P < 0.001). In addition, the gastric cancer patient population had a higher ratio of male to female than the healthy subjects ( P = 0.054). There was no significant difference in the family history of gastric cancer and the history of drinking among the identified ethnic groups.
然而,胃潰瘍和十二指腸潰瘍病患的吸煙比例明顯較健康受測者為高(P =0.002和0.001,個別地)。再者,十二指腸潰瘍病患的H.pylori 感染率亦明顯較健康受測者為高((P =0.034)。However, the proportion of smokers with gastric ulcers and duodenal ulcers was significantly higher than that of healthy subjects ( P = 0.002 and 0.001, individually). Furthermore, the H. pylori infection rate in patients with duodenal ulcer was also significantly higher than in healthy subjects ( P = 0.034).
第11圖列舉健康受測者和胃十二指腸疾病病患胃液樣本的特徵。在胃潰瘍(GU)、十二指腸潰瘍(DU)和胃癌(GC)族群間胃液胃酸pH和全部蛋白質濃度的相異係藉由單因子變數分析(one-way ANOVA test)而決定。Figure 11 shows the characteristics of gastric samples from healthy subjects and patients with gastroduodenal diseases. The difference in gastric acid pH and total protein concentration between gastric ulcer (GU), duodenal ulcer (DU) and gastric cancer (GC) groups was determined by one-way ANOVA test.
胃液中多肽含量也是一項重要指標,在胃潰瘍和十二指腸潰瘍病患明顯較健康受測者高(44%和55% vs 27%;P <0.01和<0.001)。然而,在十二指腸潰瘍病患和健康受測者之間胃液量並無明顯的不同。胃液的pH值於健康受測者、胃潰瘍、十二指腸潰瘍和胃癌病患的平均值個別為3.0、4.3、1.9和6.4。其胃液的pH值於胃潰瘍和胃癌病患明顯較健康受測者高(P =0.001和<0.001)。與健康受測者相較十二指腸潰瘍病患具有較低的胃液的pH值(P =0.001)。The amount of peptide in gastric juice is also an important indicator, which is significantly higher in patients with gastric ulcer and duodenal ulcer than in healthy subjects (44% and 55% vs 27%; P <0.01 and <0.001). However, there was no significant difference in gastric fluid volume between patients with duodenal ulcer and healthy subjects. The mean values of gastric juice pH values in healthy subjects, gastric ulcers, duodenal ulcers, and gastric cancer patients were 3.0, 4.3, 1.9, and 6.4, respectively. The pH of the gastric juice was significantly higher in patients with gastric ulcer and gastric cancer than in healthy subjects ( P = 0.001 and < 0.001). Patients with duodenal ulcer had lower pH of gastric juice than healthy subjects ( P = 0.001).
在健康受測者、胃潰瘍、十二指腸潰瘍和胃癌病患的全部蛋白質濃度個別為0.48、1.06、0.26和2.61 mg/ml。明顯地,在4個族群中胃癌病患的全部蛋白質濃度係為最高且胃潰瘍病患的全部蛋白質濃度明顯較健康受測者高(P =0.001)。健康受測者相較和十二指腸潰瘍病患具有一較低的全部蛋白質濃度(P =0.046)。如果約為0.5 mg/ml的蛋白質濃度,其係指於健康受測者全部蛋白質濃度之標準差加2,被設定為胃液正常蛋白質濃度之上限,增高之蛋白質濃度係個別標記為健康受測者、胃潰瘍病患、十二指腸潰瘍病患和胃癌病患的22%、36%、8%和71%。The total protein concentrations in healthy subjects, gastric ulcers, duodenal ulcers, and gastric cancer patients were 0.48, 1.06, 0.26, and 2.61 mg/ml, respectively. Obviously, the total protein concentration of gastric cancer patients was highest in the four ethnic groups and the total protein concentration of gastric ulcer patients was significantly higher than that of healthy subjects ( P = 0.001). Healthy subjects had a lower overall protein concentration than patients with duodenal ulcer ( P = 0.046). If the protein concentration is about 0.5 mg/ml, it refers to the standard deviation of the total protein concentration of the healthy subjects plus 2, which is set as the upper limit of the normal protein concentration of gastric juice. The increased protein concentration is individually labeled as a healthy subject. 22%, 36%, 8%, and 71% of patients with gastric ulcer, duodenal ulcer, and gastric cancer.
在適當時候,具有或不具有耶茲校正(Yate's correction for continuity)和費雪精確檢定(Fisher's exact test)之卡方檢定(chi-square test)係被應用於分析人類胃部中影響全部蛋白質濃度的因子。施行具有羅吉斯迴歸(logistic regression)的變異分析以評估影響全部蛋白質濃度的獨立因子為高(高於0.5 mg/ml之全部蛋白質濃度)或為低(低於0.5 mg/ml之全部蛋白質濃度)。這些因子係包括至少11個臨床因子、細菌因子、胃液因子和其他。例如,研究於此的因子係包含年齡(低於或高於60歲)、性別(男或女)、胃癌的家族歷史(有或無)、抽煙歷史(少於或多於1包/週)、飲酒歷史(少於(-)或多於(+)80 g/天)、細菌感染(H.pylori 感染情形的表現或不表現)、胃液特徵(少於或高於5 ml胃液;胃液的酸度低於或高於約pH 3.5)、胃潰瘍(有或沒有)、十二指腸潰瘍(有或沒有)、胃癌(有或沒有)的表現或不表現。When appropriate, chi-square test with or without Yate's correction for continuity and Fisher's exact test is used to analyze the total protein concentration in the human stomach. Factor. Perform a variance analysis with logistic regression to assess whether the independent factor affecting the overall protein concentration is high (all protein concentrations above 0.5 mg/ml) or low (all protein concentrations below 0.5 mg/ml) ). These factors include at least 11 clinical factors, bacterial factors, gastric fluid factors, and others. For example, the factors studied here include age (below or older than 60 years), gender (male or female), family history of gastric cancer (with or without), smoking history (less or more than 1 pack / week) History of drinking (less than (-) or more than (+) 80 g/day), bacterial infection (performance or non-performance of H. pylori infection), gastric fluid characteristics (less or higher than 5 ml gastric juice; gastric juice Acidity below or above about pH 3.5), gastric ulcer (with or without), duodenal ulcer (with or without), gastric cancer (with or without) or not.
在一方面,胃液中影響蛋白質濃度之多個因子被同時地評估。例如,11個臨床因子、細菌因子和胃液因子係被比較並發現獨立參數。In one aspect, multiple factors affecting protein concentration in gastric juice are evaluated simultaneously. For example, 11 clinical factors, bacterial factors, and gastric fluid factor lines were compared and independent parameters were found.
第12圖展現影響人類胃部全部蛋白質濃度因子的單變數分析結果。在P <0.05時相異度係被考慮為有意義。該結果顯示,至少5個不同的因子明顯地與高蛋白質濃度相關(例如,高於約0.5 mg/ml)於胃酸,包括老化(P =0.001)、胃癌家族歷史(P =0.054)、十二指腸潰瘍之不表現(P =0.026)、胃癌的表現(P <0.001)、胃液的低含量(P <0.001)和胃部的低酸度(P <0.001)。Figure 12 shows the results of a single variable analysis of all protein concentration factors affecting the human stomach. Dissimilarity was considered meaningful at P < 0.05. The results showed that at least 5 different factors were significantly associated with high protein concentrations (eg, above about 0.5 mg/ml) in gastric acid, including aging ( P = 0.001), family history of gastric cancer ( P = 0.054), duodenal ulcer. The performance was not ( P = 0.026), the performance of gastric cancer ( P < 0.001), the low content of gastric juice ( P < 0.001), and the low acidity of the stomach ( P < 0.001).
進一步地用羅吉斯線性迴歸施行一多變數分析,以尋找全部蛋白質濃度和獨立因子間的任何關連。第13圖呈現人類胃部影響全部蛋白質濃度獨立因子的多變數分析結果。該結果指出,老化和胃部低酸度為影響人類胃液全部蛋白質濃度的獨立因子。二個參數的奇異率(odds rates)個別係為約3.23(95%信賴區間(CI)):1.26-8.25)和約32.86(95% CI:11.8-90.85)。A multivariate analysis was further performed using Logistic linear regression to find any association between total protein concentration and independent factors. Figure 13 shows the results of a multivariate analysis of the human stomach affecting the independent factors of total protein concentration. The results indicate that aging and low acidity in the stomach are independent factors affecting the total protein concentration of human gastric juice. The odds rates for the two parameters were approximately 3.23 (95% confidence interval (CI)): 1.26-8.25) and approximately 32.86 (95% CI: 11.8-90.85).
人們廣泛地知悉,胰蛋白酶為一胃液中活躍的蛋白質分解酵素,自pH 1.6起具理想的功效,於pH 4-6不活化但是穏定,於pH>6時不可逆。胃液中蛋白質的組成易受於酸性胃酸胰蛋白酶消化分解的影響。因此,低酸度胃液之高蛋白質濃度係可理解。It is widely known that trypsin is an active proteolytic enzyme in gastric juice and has an ideal effect from pH 1.6. It is not activated at pH 4-6 but is determined to be irreversible at pH>6. The composition of the protein in the gastric juice is susceptible to the digestion and decomposition of acid gastric acid trypsin. Therefore, the high protein concentration of low acidity gastric juice is understandable.
雖然老化導致各式胃部生理的改變(例如,黏膜血液流量的減少,前列腺素的合成),然而並無解釋老化過程和胃液高蛋白質濃度之關連性。老化是胃潰瘍和胃癌的發生的主要危險因子,其可能反射於H.pylori 感染、吸煙、其他疾病出現、和使用藥物治療(例如,非類脂醇抗發炎藥物)的高發生率。但是有關於在胃液中高蛋白質含量與年齡之間之關係,仍須進一步的研究來證實。Although aging causes various physiological changes in the stomach (eg, reduction of mucosal blood flow, synthesis of prostaglandins), there is no explanation for the association between the aging process and the high protein concentration of gastric juice. Aging is a major risk factor for the development of gastric ulcers and gastric cancer, which may reflect a high incidence of H. pylori infection, smoking, the appearance of other diseases, and the use of medications (eg, non-lipid alcohol anti-inflammatory drugs). However, there is still a need for further research to confirm the relationship between high protein content in gastric juice and age.
藉由類神經網路(Plausible Neural Network(PNN)態樣辨識軟體系統快速編譯和分析來自數個樣本的結果以做為複雜資料分析。PNN係為一智慧自我組織(self-organized)神經網路系統。根據特徵選擇模式(feature selection module)圖譜間的相異係可被鑑定。來自正常和癌症病患圖譜間的相異係可指出可能的生物標誌,其接著根據PNN的分類模式接續地分析(PNN為一新式一般神經網路模型係根據其神經網路、資訊、不連續組合和統計分干擾原理的介面。PNN於單一網路結構中施行聯合記憶(associative memory),群集(clustering)、分類、功能近似值(function approximation)和簡易評鑑,Reference:1 Chen,Y.Y.(2005).Neural networks,fuzzy logic and statistical inference.Neural Networks((2006) to appear);2.Chen,Y.Y.and Chen,J.J.(2004).Neural networks and belieflogic,Proceedings of the Fourth International Conference on Hybrid Intelligent Systems(HIS’04),IEEE. 460-461;3.Chen,Y.Y.(2002).Plausible neural networks.Advance in Neural Networks World.Ed. Grmela,A.and Mastorakis,N.E.WSEAS Press,180-185.)。我們的結果指出在正常和潰瘍病患間的區別非常清晰且結合胃液樣本的質譜的PNN程式可分辨出癌症樣本和正常樣本。Rapidly compiling and analyzing the results from several samples for complex data analysis by the PLAS Neural Network (PNN) pattern recognition software system. PNN is a self-organized neural network. System. Depending on the feature selection module, the dissimilarity between the maps can be identified. The differences between the normal and cancer patient maps can indicate possible biomarkers, which are then analyzed based on the classification model of the PNN. (PNN is a new general neural network model based on its neural network, information, discontinuous combination and statistical interference interference interface. PNN performs associative memory, clustering, in a single network structure. Classification, function approximation, and easy evaluation, Reference: 1 Chen, YY (2005). Neural networks, fuzzy logic and statistical inference. Neural Networks ((2006) to appear); 2. Chen, YY and Chen, JJ (2004). Neural networks and belieflogic, Proceedings of the Fourth International Conference on Hybrid Intelligent Systems (HIS'04), IEEE. 460-4 61;3.Chen, YY(2002).Plausible neural networks. Advance in Neural Networks World.Ed. Grmela, A. and Mastorakis, NEWSEAS Press, 180-185.) Our results indicate between normal and ulcer patients The difference is very clear and the PNN program combined with the mass spectrometry of the gastric fluid sample can distinguish between cancer samples and normal samples.
因為在人體中有許多蛋白質被製造,MALDI-TOF質譜儀相對低質量解析度的質譜並不精確到足以鑑定這些質量峰部對應的蛋白質或多肽。許多蛋白質具有可合併為一個質量峰部的分子量。一個二步驟的方法係被應用於鑑定這些生物標誌。Because many proteins are produced in the human body, the mass spectra of MALDI-TOF mass spectrometers with relatively low mass resolution are not accurate enough to identify proteins or polypeptides corresponding to these mass peaks. Many proteins have molecular weights that can be combined into one mass peak. A two-step method was applied to identify these biomarkers.
首先,HPLC被利用於先前分離這些析出的樣本。一典型的結果於第7圖呈現,其係介紹一鑑定之多肽生物標誌的HPLC圖譜。自HPLC的每個峰部,我們進一步施行MALDI-TOF質量圖譜分析,以鑑定哪個HPLC區間包含感興趣的質量峰部的物質。該結果於第8A-8C圖呈現,其係介紹在HPLC層柱色層純化之後,胃液樣本的三個區份的質譜於質量範圍500 m/z至5000 m/z。First, HPLC was utilized to previously separate these precipitated samples. A typical result is presented in Figure 7, which is an HPLC chromatogram of an identified polypeptide biomarker. From each peak of the HPLC, we further performed a MALDI-TOF mass spectrogram to identify which HPLC interval contains the mass peaks of interest. The results are presented in Figures 8A-8C, which show that after purification of the HPLC layer column chromatography, the mass spectra of the three fractions of the gastric fluid sample ranged from 500 m/z to 5000 m/z.
標的分子(target molecules)係藉由蛋白質色層分析,例如,C18 HPLC,純化和濃縮以準備進行進一步的胺基酸序列鑑定。該MS/MS圖譜接著以一ABI 4700質譜儀獲得。一典型的圖譜如第9圖所呈現,其顯示MS/MS分析於一實驗的多肽(2187m/z)胺基酸序列決定的結果。以MASCOT胺基酸定序搜尋引擎之搜索,多肽和/或蛋白質的序列可被鑑定。除了多肽2187m/z,多肽2387m/z、多肽3572m/z、多肽2573m/z以及多肽4132m/z的序列亦被鑑定。The target molecules are purified and concentrated by protein chromatography, for example, C18 HPLC, to prepare for further amino acid sequence identification. The MS/MS map was then obtained on an ABI 4700 mass spectrometer. A typical map is presented as Figure 9, which shows the results of MS/MS analysis of the amino acid sequence of an experimental polypeptide (2187 m/z). The search for the MASCOT amino acid sequencing search engine, the sequence of the polypeptide and/or protein can be identified. In addition to the polypeptide 2187 m/z, the polypeptide 2387 m/z, the polypeptide 3572 m/z, the polypeptide 2573 m/z, and the polypeptide 4132 m/z were also identified.
根據本發明之一個或多個具體實施例,以非侵入性的收集方式獲得胃液並續行蛋白質體分析係可做為一實用的工具,其能於大部份胃癌潛在病患和已存在病患,可靠地偵測胃癌。和其他目前可行之試驗方法相較,例如,那些偵測CEA和CA19-9值的血清試驗,描述於此的診斷/篩選方法和胃癌生物標誌於胃癌偵測提供較高之敏感性和特異性。According to one or more embodiments of the present invention, obtaining gastric juice in a non-invasive collection manner and continuing the proteomic analysis system can be used as a practical tool for most potential gastric cancer patients and existing diseases. Suffering, reliable detection of gastric cancer. Compared with other currently available test methods, for example, those that detect CEA and CA19-9 values, the diagnostic/screening methods described and gastric cancer biomarkers provide higher sensitivity and specificity for gastric cancer detection. .
胃液的二維凝膠電泳施行自47位健康受測者、33位胃潰瘍病患、33位十二指腸潰瘍病患和29位胃癌病患的胃液樣本。二維蛋白質電泳圖像的結果分類成為3種態樣的其中1樣,其係包含基礎條帶態樣、特殊條帶態樣和非特殊條帶態樣。Two-dimensional gel electrophoresis of gastric juice was performed on gastric juice samples from 47 healthy subjects, 33 gastric ulcer patients, 33 duodenal ulcer patients, and 29 gastric cancer patients. The results of two-dimensional protein electrophoresis images are classified into one of three kinds of patterns, which include basic strip patterns, special strip patterns and non-special strip patterns.
第14圖介紹健康受測者和各式胃與十二指腸潰瘍病患胃液樣本二維電泳的結果,包含基礎條帶態樣、特殊條帶態樣和非特殊條帶態樣。胃潰瘍和胃癌病患的電泳圖像態樣明顯地和健康受測者不同。特殊條帶態樣的頻率,特別是特殊蛋白質α-抗胰蛋白酶前驅物,高度與胃潰瘍和胃癌樣本相關,且健康受測者、胃潰瘍病患、十二指腸潰瘍病患和胃癌病患樣本的經計算後頻率個別為6%、42%、6%和93%。該資料指出胃液中α-抗胰蛋白酶前驅物係高度與胃潰瘍和胃癌相關(兩者皆P <0.001)。Figure 14 shows the results of two-dimensional electrophoresis of gastric samples from healthy subjects and various gastric and duodenal ulcer patients, including basic strip patterns, special strip patterns, and non-special strip patterns. The electrophoretic images of gastric ulcer and gastric cancer patients are clearly different from those of healthy subjects. The frequency of special strips, especially the special protein α-antitrypsin precursor, is highly correlated with gastric ulcer and gastric cancer samples, and is calculated for healthy subjects, gastric ulcer patients, duodenal ulcer patients, and gastric cancer patients. The post frequencies are 6%, 42%, 6%, and 93%, respectively. This data indicates that the alpha-antitrypsin precursor system in gastric juice is highly associated with gastric ulcer and gastric cancer (both P < 0.001).
第15圖介紹二維電泳特殊條帶態樣與癌症演進相關的結果。該結果顯示α-抗胰蛋白酶前驅物的特殊條帶態樣高度地被發現於進階胃腫瘤(90%)和早期胃癌(100%)中。此α-抗胰蛋白酶前驅物和早期或晚期胃癌的高度相關性係支持利用α-抗胰蛋白酶前驅物作為一胃癌的生物標誌。Figure 15 shows the results of the two-dimensional electrophoresis special banding pattern associated with cancer evolution. This result shows that the specific banding pattern of the α-antitrypsin precursor is highly found in advanced gastric tumors (90%) and early gastric cancer (100%). The high correlation between this alpha-antitrypsin precursor and early or advanced gastric cancer supports the use of alpha-antitrypsin precursor as a biomarker for gastric cancer.
第16圖係於胃液樣本中的二維電泳態樣,呈現萎縮性胃炎和胃部低酸度的相關性。小體的腺體萎縮和胃部低酸度與特殊α-抗胰蛋白酶前驅物條帶態樣高度相關(兩者皆P <0.001)。基礎條帶、特殊條帶和非特殊條帶的頻率於低酸度胃液二維電泳圖中個別為0%、93%和7%。Figure 16 is a two-dimensional electrophoretic pattern in a gastric fluid sample showing the correlation between atrophic gastritis and low acidity in the stomach. Glandular atrophy and low acidity in the stomach were highly correlated with the specific alpha-antitrypsin precursor strip pattern (both P < 0.001). The frequency of the base strip, the special strip, and the non-special strip are 0%, 93%, and 7% in the two-dimensional electrophoresis pattern of the low acidity gastric juice.
在9位受過胃黏膜組織檢查的健康受測者之中,8位健康受測者不具胃部萎縮,造成胃液二維凝膠電泳的基礎條帶態樣。來自剩餘健康受測者(其具有竇室和小體的腺體萎縮)之樣本於二維凝膠電泳研究中呈現一非特殊條帶態樣。Among the 9 healthy subjects who had undergone gastric mucosal examination, 8 healthy subjects did not have atrophy of the stomach, resulting in a basic strip pattern of gastric dioxide two-dimensional gel electrophoresis. Samples from the remaining healthy subjects (which have sinus and corpus callosum atrophy) present a non-special banding pattern in a two-dimensional gel electrophoresis study.
第17圖為一例示二維凝膠電泳圖像,其係展示一基礎條帶態樣。二個主要條帶在酸性pI(4.0-5.0)可被觀察到。該始於頂端之第一條帶具42 kDa之分子量係被定名為A1,其接著藉由MALDI-TOF和MS/MS質譜儀分析鑑定為胰蛋白A酶前驅物。該始於頂端之第二條帶具35 kDa之分子量係被定名為A2並接著於MALDI-TOF分析中被鑑定為2個明顯的多肽峰部於m/z 527和m/z 538。來自質譜的兩個多肽峰部皆被鑑定為具有相同胺基酸序列(QYFTVFDR)的胰蛋白酶A前驅物。Figure 17 is an illustration of a two-dimensional gel electrophoresis image showing a basic strip pattern. Two major bands are observed in the acidic pI (4.0-5.0). The first band starting at the top with a molecular weight of 42 kDa was designated A1, which was then identified as a trypsin A enzyme precursor by MALDI-TOF and MS/MS mass spectrometry analysis. The second band starting at the top with a molecular weight of 35 kDa was designated A2 and was subsequently identified in the MALDI-TOF analysis as two distinct polypeptide peaks at m/z 527 and m/z 538. Both polypeptide peaks from the mass spectrum were identified as trypsin A precursors with the same amino acid sequence (QYFTVFDR).
第18圖為一例示二維凝膠電泳圖像,其係展示一特殊的條帶態樣。鑑定於基礎條帶態樣的胰蛋白A酶前驅物幾乎偵測不到。相反地,一具有酸性pI(4.5-5.5)和分子量47 kD的明顯黑色條帶定名為B1且可顯著地偵測的到。該來自二維凝膠電泳特殊條帶態樣的B1條帶接著藉由MALDI-TOF和MS/MS質譜儀分析鑑定為α-抗胰蛋白酶前驅物。Figure 18 is an illustration of a two-dimensional gel electrophoresis image showing a particular strip pattern. The trypsin A enzyme precursor identified in the basic banding pattern was barely detectable. Conversely, a distinct black band with an acidic pI (4.5-5.5) and a molecular weight of 47 kD was designated B1 and was significantly detectable. The B1 band from the special strip morphology of the two-dimensional gel electrophoresis was then identified as an alpha-antitrypsin precursor by MALDI-TOF and MS/MS mass spectrometry analysis.
第19圖為一例示二維凝膠電泳圖像,其係展示一非特殊的條帶態樣。該非特殊的條帶態樣並無呈現任何觀察自基礎條帶態樣和特殊條帶態樣的條帶,因此對應於胰蛋白酶A前驅物和α-抗胰蛋白酶前驅物的條帶無法觀察的到。在非特殊性二維凝膠電泳條帶態樣中蛋白質墨點的分布是相當多樣的。部份淋巴球彈性蛋白酶抑制劑(leukocyte elastase inhibitor)(Mr, 43kDa;pI,5.90)、橋粒芯蛋白-1前驅物(desmoglein-1 precursor)(Mr, 114kDa;pI,4.90)、橋粒斑蛋白(desmoplakin)(Mr, 332kDa;pI,6.44)和Ig鍊C部位(Mr, 12kDa;pI,5.58)的片段係由分散於二維凝膠呈現非特殊條帶態樣之墨點所鑑定。Figure 19 is an illustration of a two-dimensional gel electrophoresis image showing a non-special strip pattern. The non-special banding pattern does not exhibit any bands observed from the basic banding pattern and the special banding pattern, so the bands corresponding to the trypsin A precursor and the α-antitrypsin precursor are unobservable. To. The distribution of protein dots in a non-specific two-dimensional gel electrophoresis strip pattern is quite diverse. Part of the leukocyte elastase inhibitor ( Mr, 43kDa; pI, 5.90) , desmoglein-1 precursor ( Mr, 114kDa; pI, 4.90), desmosome Protein (desmoplakin) ( Mr, 332kDa; pI, 6.44) and Ig Fragments of the chain C site ( Mr, 12 kDa; pI, 5.58) were identified by ink dots dispersed in a two-dimensional gel exhibiting a non-special banding pattern.
電泳圖像的基礎條帶態樣係包含二個由胰蛋白酶原A前驅物所組成之主要條帶。此基礎條帶態樣的圖像主要在健康受測者(74%)和十二指腸潰瘍病患(85%)之中。相反地,在胃癌病患中該電泳圖像主要係為特殊條帶態樣(93%)。因為來自胃癌病患的電泳圖像的特殊條帶態樣和非特殊條帶態樣高度地變動,約93%和約3%。該資料係指出,胃液分析中至少93%的胃癌病患具異常蛋白質體態樣,且於胃液中評估α-抗胰蛋白酶前驅物係為一在病患中偵測胃癌出現非常敏銳之方法。The basic strip pattern of the electrophoretic image contains two major bands consisting of the trypsinogen A precursor. The images of this basic strip pattern were mainly among healthy subjects (74%) and duodenal ulcer patients (85%). Conversely, in electrocancerous patients, this electrophoretic image was predominantly a special banding pattern (93%). Because the special banding patterns and non-special banding patterns of electrophoretic images from patients with gastric cancer vary widely, about 93% and about 3%. According to the data, at least 93% of gastric cancer patients have abnormal protein body morphology, and the α-antitrypsin precursor system is evaluated in gastric juice as a very sensitive method for detecting gastric cancer in patients.
在此呈現,胃液中α-抗胰蛋白酶前驅物出現與小體的線體萎縮和胃部的低酸度高度相關。其可能是,一多餘的α-抗胰蛋白酶前驅物,其係來自於各式的免疫細胞並透過癌症的或潰瘍的傷口流入胃部,可在低酸度胃液中避免被快速蛋白質水解。Here, the appearance of the α-antitrypsin precursor in the gastric juice is highly correlated with the atrophy of the body and the low acidity of the stomach. It may be that an excess of alpha-antitrypsin precursor, which is derived from a variety of immune cells and flows into the stomach through a cancerous or ulcerated wound, avoids rapid proteolysis in low acidity gastric fluids.
亦值得注意的是,8位在二維凝膠圖像具有基礎條帶態樣的健康受測者胃部全部不具腺體萎縮。相反地,該具有非特殊條帶態樣的唯一健康受測者具有明顯的腺體萎縮(竇室衰退和小體萎縮)。這些發現顯示在胃液中存在胰蛋白酶原A前驅物,表示不具有胃部萎縮的症狀。在胃液蛋白質分析中具有非特殊條帶態樣的健康受試者,可能在胃中也具有胃癌的潛在條件,例如萎縮和小腸變性生長。It is also worth noting that all of the healthy subjects in the two-dimensional gel image with the basic strip pattern had no glandular atrophy. Conversely, the only healthy subject with a non-specific banding pattern had significant glandular atrophy (sinus chamber degeneration and small body atrophy). These findings indicate the presence of trypsinogen A precursor in gastric fluid, indicating no symptoms of gastric atrophy. Healthy subjects with non-specific banding patterns in gastric juice protein analysis may also have potential conditions for gastric cancer in the stomach, such as atrophy and small intestine degeneration.
利用前述之過程,鑑定胃癌受抑制之多肽,包含一多肽生物標誌,多肽2187 m/z,其具有相對應於胰蛋白酶原多肽片段之序列FLKKHNLNPARKYFPQW,和多肽2387 m/z,亦具有相對應於胰蛋白酶原多肽片段且多2個胺基酸之序列FLKKNHLNPARKYFPQWEA。胰蛋白酶原係由胃部的主細胞(chief cell)所分泌。胃部癌化歷經胃部萎縮的過程,其導致主細胞數目減少和胰蛋白酶原含量減少。因此,胃癌病患胃液中2個胰蛋白酶原片段受抑制量之鑑定與胃部癌化的過程一致。Using the foregoing process to identify a polypeptide inhibited by gastric cancer, comprising a polypeptide biomarker, the polypeptide 2187 m/z, which has a sequence corresponding to the trypsinogen polypeptide fragment FLKKHNLNPARKYFPQW, and the polypeptide 2387 m/z, also has a corresponding The sequence of the trypsinogen polypeptide fragment and two more amino acids FLKKNHLNPARKYFPQWEA. Trypsin is secreted by the chief cell of the stomach. Gastric cancer undergoes atrophic process of the stomach, which leads to a decrease in the number of primary cells and a decrease in trypsinogen content. Therefore, the inhibition of the two trypsinogen fragments in the gastric juice of gastric cancer patients is consistent with the process of gastric canceration.
另一個鑑定而得的胃癌受抑制多肽為多肽3572 m/z,其包含具有序列ETKKTEDRFVPSSSKSEGKKSREQPSVLSRY之亮氨酸拉鏈蛋白質區域(leucine zipper protein domain)。亮氨酸拉鏈蛋白質區域經常表現於腫瘤抑制蛋白質(tumor suppressor protein)中。某些腫瘤抑制蛋白質的表現在人類胃癌細胞株被發現受到抑制。亮氨酸拉鏈蛋白質的抑制亦曾在胰臟癌、口腔鱗狀上皮細胞癌(oral squamous cell carcinoma)和膀胱癌(bladder cancer)被報告。某些亮氨酸拉鏈蛋白質的表現誘發細胞凋亡(apoptosis)並減少在某些人類細胞株的細胞存活度。該亮氨酸拉鏈蛋白質多肽片段,多肽3572 m/z對應於一潛能腫瘤抑制蛋白質,其表現在胃和胰臟癌細胞株中減少。因此,該亮氨酸拉鏈多肽片段(3572 m/z)在胃癌病患胃液中受抑制量的鑑定係暗指在胃癌細胞或萎縮胃部黏膜中相對應亮氨酸拉鏈蛋白質的抑制。Another identified gastric cancer-inhibiting polypeptide is the polypeptide 3572 m/z, which comprises a leucine zipper protein domain having the sequence ETKKTEDRFVPSSSKSEGKKSREQPSVLSRY. Leucine zipper protein regions are often expressed in tumor suppressor proteins. The expression of certain tumor suppressor proteins was found to be inhibited in human gastric cancer cell lines. Inhibition of leucine zipper proteins has also been reported in pancreatic cancer, oral squamous cell carcinoma, and bladder cancer. The performance of certain leucine zipper proteins induces apoptosis and reduces cell viability in certain human cell lines. The leucine zipper protein polypeptide fragment, polypeptide 3572 m/z, corresponds to a latent tumor suppressor protein, which is shown to be reduced in gastric and pancreatic cancer cell lines. Therefore, the identification of the leucine zipper polypeptide fragment (3572 m/z) in the gastric juice of gastric cancer patients implies that the corresponding leucine zipper protein is inhibited in gastric cancer cells or atrophic gastric mucosa.
自質譜分析的胺基酸鑑定結果亦鑑定胃癌向上調控(up-regulated)的多肽。一多肽,多肽2573 m/z,鑑定為序列DAHKSEVAHRFKDLGEENFKALVL之白蛋白多肽片段。因為白蛋白在血液中含量很高且在胃癌裡自形成潰瘍的腫瘤中隱藏出血(occult bleeding)為一常見現象,所以可能是在胃癌發生和腫瘤出血的過程中,來自血液的白蛋白流入胃液。因此,該白蛋白多肽片段(多肽2573 m/z)在胃癌病患胃液中向上調控量之鑑定與胃癌發生過程腫瘤出血一致。再者,胃液中白蛋白的存在可做為一警示,必須更仔細確認胃癌存在之可能性。The amino acid identification results from mass spectrometry also identified up-regulated polypeptides of gastric cancer. A polypeptide, polypeptide 2573 m/z, identified as an albumin polypeptide fragment of the sequence DAHKSEVAHRFKDLGEENFKALVL. Because albumin is high in the blood and occlusion bleeding is a common phenomenon in the formation of ulcers in gastric cancer, it may be that during the process of gastric cancer and tumor hemorrhage, albumin from the blood flows into the gastric juice. . Therefore, the identification of the upward regulation of the albumin polypeptide fragment (polypeptide 2573 m/z) in gastric juice patients is consistent with tumor hemorrhage during gastric cancer. Furthermore, the presence of albumin in gastric juice can be used as a warning, and the possibility of gastric cancer must be more carefully confirmed.
在二維聚丙烯胺凝膠電泳後,質譜分析鑑定之胺基酸序列結果鑑定胃癌向上調控的蛋白質。一蛋白質,多肽4132 m/z,α1-抗胰蛋白酶前驅物,序列為SIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK,係被鑑定。因為α1-抗胰蛋白酶在血液中含量很高,普遍認為,α1-抗胰蛋白酶可在胃癌發生過程腫瘤出血時,由血液進入胃液。After two-dimensional polyacrylamide gel electrophoresis, the amino acid sequence identified by mass spectrometry identified the protein regulated by gastric cancer. A protein, polypeptide 4132 m/z, α1-antitrypsin precursor, sequence SIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK, was identified. Because α1-antitrypsin is high in the blood, it is generally believed that α1-antitrypsin can enter the gastric juice from the blood when the tumor is bleeding during the gastric cancer.
因為鑑定於此的多肽和蛋白質,在胃液中偵測胃癌的敏感度和特異性非常的高,高於任何目前可獲得的胃癌生物標誌,在胃液多肽和蛋白質中,此類蛋白質和多肽片段的出現在胃癌病患中是非常常見的,且本發明之具體實施例係提供應用該蛋白質和多肽片段為新型生物標誌以偵測胃癌。例如,此蛋白質和多肽片段的單株抗體係可發展於胃癌篩檢或診斷。Because of the identification of peptides and proteins, the sensitivity and specificity of detecting gastric cancer in gastric juice is very high, higher than any currently available biomarkers of gastric cancer, in gastric juice polypeptides and proteins, such proteins and polypeptide fragments It is very common to appear in patients with gastric cancer, and specific embodiments of the invention provide for the use of the protein and polypeptide fragments as novel biomarkers to detect gastric cancer. For example, a monoclonal antibody system of this protein and polypeptide fragment can be developed for screening or diagnosis of gastric cancer.
此外,僅一個或二個多肽,並非許多個,被鑑定自相同蛋白質,其係暗指一特殊的機轉以消化分解該蛋白質和維持此類特殊的多肽片段,而非一非特殊機轉的消化分解方式,因為胃液係為高度酸性。再者,因為收集該胃液樣本後,並無酵素性的消化分解被施行,該鑑定於此之多肽片段應以多肽型式存在於胃部。雖然胰蛋白酶原和白蛋白為一般蛋白質,且其蛋白質本身並不能做為一特殊癌症之良好生物標誌。當此類多肽的顯著差異實驗結果在癌症病患與正常者間被觀察到時,此特殊多肽片段(而非該全長蛋白質)仍可為良好的生物標誌。In addition, only one or two polypeptides, not many, are identified from the same protein, which implies a special machine to digest and decompose the protein and maintain such a particular polypeptide fragment, rather than a non-special machine. Digestion and decomposition, because the gastric juice system is highly acidic. Furthermore, since no enzymatic digestion and decomposition is carried out after collecting the gastric juice sample, the polypeptide fragment identified therein should be present in the stomach in the form of a polypeptide. Although trypsinogen and albumin are general proteins, the protein itself is not a good biomarker for a particular cancer. This particular polypeptide fragment (rather than the full length protein) can still be a good biomarker when significant differences in the results of such polypeptides are observed between cancer patients and normal subjects.
一可能的考慮是,此類多肽之差異指出不同來源的癌症但非胃癌。因此,癌症和正常者血清樣本的多肽質譜分析係被施行。胃液樣本中鑑定於此的多肽和蛋白質則未於血清樣本觀察到。該結果係指出在癌症病患和正常者間,此特殊蛋白質和多肽片段之相異來自於胃部。其亦指出癌症來自於其他器官的可能性較小。One possible consideration is that differences in such polypeptides indicate cancer from different sources but not gastric cancer. Therefore, peptide mass spectrometry of cancer and normal serum samples is performed. The polypeptides and proteins identified in the gastric fluid samples were not observed in serum samples. The results indicate that this particular protein and polypeptide fragment differs from the stomach between the cancer patient and the normal. It also points out that cancer is less likely to be from other organs.
其他可能的考慮為,胰蛋白酶原的抑制和白蛋白的向上調控亦能存在於胃炎和胃部的萎縮。值得重視的是,萎縮性胃炎和腸變性生長兩者皆為癌化前的情形,且具此傷口之受測者需要被鑑定和緊密地追踪。然而,不確定的是,是否具有胃炎者亦具有相同的多肽片段。如果不具有,此胰蛋白酶原、白蛋白和其特殊多肽同時發生之偵測則提供胃炎和胃癌間之區別。如果胃炎病患具有此類特殊多肽之相同表現,其仍為一確定胃癌或癌前傷口之良好篩檢試驗。Other possible considerations are that inhibition of trypsinogen and upregulation of albumin can also occur in gastritis and atrophy of the stomach. It is worth noting that both atrophic gastritis and intestinal degenerative growth are pre-cancerous conditions, and subjects with this wound need to be identified and closely tracked. However, it is uncertain whether those with gastritis also have the same polypeptide fragment. If not, the simultaneous detection of this trypsinogen, albumin and its specific polypeptide provides a difference between gastritis and gastric cancer. If a gastritis patient has the same performance as such a particular polypeptide, it is still a good screening test to determine gastric cancer or precancerous wounds.
根據本發明之一個或多個觀點,可以發現,胃癌和胃潰瘍病患胃液的蛋白質濃度明顯地較健康受測者為高,然而十二指腸潰瘍病患的胃液蛋白質濃度則較健康受測者為低。此外,胃部低酸度和老化為兩個在胃液影響蛋白質濃度的獨立因子。According to one or more aspects of the present invention, it has been found that gastric cancer and gastric ulcer patients have significantly higher protein concentrations in gastric juice than those in healthy subjects, whereas duodenal ulcer patients have lower gastric juice protein concentrations than healthy subjects. In addition, low acidity and aging in the stomach are two independent factors that affect protein concentration in gastric juice.
在一方面,一個非侵入性獲得胃液並續行蛋白質體分析的方法係可做為一新式工具於篩檢胃部惡性腫瘤。在另一方面,各式胃與十二指腸疾病病患之胃液樣本的蛋白質組成係顯著地不同。利用二維電泳進一步分析顯示,胃液中該主要蛋白質成份明顯在各式胃與十二指腸疾病中有所不同。In one aspect, a non-invasive method of obtaining gastric juice and continuing proteomic analysis can be used as a new tool for screening gastric malignancies. On the other hand, the protein composition of gastric juice samples of various stomach and duodenal diseases is significantly different. Further analysis by two-dimensional electrophoresis showed that the main protein component in gastric juice was significantly different in various gastric and duodenal diseases.
本發明之具體實施例係提供第一次證據,比較各式胃十二指腸疾病中胃液蛋白質濃度的差異。在胃液中,至少一胃癌和胃潰瘍之新式生物標誌被鑑定為α-抗胰蛋白酶前驅物,其係來自於二維凝膠電泳之特殊條帶態樣。在胃液中至少5個新式生物標誌被鑑定為胃癌相關,其係來自於質譜分析。該發現高度指出,測量胃液中蛋白質濃度並結合蛋白質體分析以分析胃液中蛋白質組成係為一鑑定或篩檢各式胃與十二指腸疾病出現的新式診斷工具。Specific embodiments of the present invention provide first evidence for comparing differences in gastric juice protein concentrations in various gastroduodenal diseases. In gastric juice, at least one new biomarker of gastric cancer and gastric ulcer was identified as an alpha-antitrypsin precursor from a special banding pattern of two-dimensional gel electrophoresis. At least 5 new biomarkers in gastric juice were identified as gastric cancer related, which were derived from mass spectrometry. The findings highly suggest that measuring protein concentrations in gastric juice and combining proteomic analysis to analyze protein composition in gastric juice is a new diagnostic tool for identifying or screening for various gastric and duodenal diseases.
以上進展係根據本發明之具體實施例,本發明之其他或進一步具體實施例可在不偏離本發明範圍下被修飾,且其範圍由以下申請專利範圍所界定。The above-described developments are based on the specific embodiments of the present invention, and other or further embodiments of the present invention may be modified without departing from the scope of the invention, and the scope thereof is defined by the following claims.
第1圖為一例示方法之圖示說明,其係根據本發明之一個或多個具體實施例。1 is a pictorial illustration of an exemplary method in accordance with one or more embodiments of the present invention.
第2圖為另一例示方法之圖示說明,其係根據本發明之一個或多個具體實施例。Figure 2 is a pictorial illustration of another exemplary method in accordance with one or more embodiments of the present invention.
第3圖為為利用不同磁性珠球,例如:C8或C18,收集胃液樣本以行質譜分析之例示圖示說明,其係根據本發明之一個具體實施例。Figure 3 is a pictorial illustration of the collection of gastric fluid samples for mass spectrometry using different magnetic bead balls, such as C8 or C18, in accordance with a particular embodiment of the present invention.
第4圖介紹以微量離心管收集之胃液樣本並準備進行質譜分析,其係根據本發明之一個具體實施例。Figure 4 illustrates a sample of gastric juice collected in a microcentrifuge tube and prepared for mass spectrometry, in accordance with a particular embodiment of the present invention.
第5A圖為一收集自正常、未受疾病感染受測者之胃液樣本的例示質譜,其係根據本發明之一個具體實施例。Figure 5A is an exemplary mass spectrum of a gastric fluid sample collected from a normal, unaffected subject, according to a particular embodiment of the invention.
第5B圖為一收集自染病病患之胃液樣本的例示質譜,其係根據本發明之一個具體實施例。Figure 5B is an exemplary mass spectrum of a gastric fluid sample collected from an infected patient, in accordance with a particular embodiment of the present invention.
第6A圖顯示收集自正常、未受疾病感染的受測者胃液樣本的質譜範示區間。Figure 6A shows the mass spectrometric range of gastric fluid samples collected from normal, unaffected subjects.
第6B圖顯示收集自癌症受測者胃液樣本之質譜之例示區間。Figure 6B shows an exemplary interval of mass spectra collected from gastric fluid samples from cancer subjects.
第7圖為一鑑定之多肽生物標誌的HPLC圖表的例示圖示說明,其係根據本發明之一個具體實施例。Figure 7 is an illustration of an HPLC chart of an identified polypeptide biomarker in accordance with a particular embodiment of the present invention.
第8A圖為一胃液樣本經由HPLC層柱色層分析純化後,於質量範圍500 m/z至5000 m/z一區份之質譜,胃液樣本一區份之質譜,其係根據本發明之一個具體實施例。Figure 8A is a mass spectrum of a gastric juice sample in a mass range of 500 m/z to 5000 m/z after purification of a gastric juice sample by HPLC layer chromatography, and is a mass spectrum according to the present invention. Specific embodiment.
第8B圖為一胃液樣本經由HPLC層柱色層分析純化後,於質量範圍500 m/z至5000 m/z一區份之質譜,胃液樣本另一區份之質譜,其係根據本發明之一個具體實施例。Figure 8B is a mass spectrum of a gastric juice sample in a mass range of 500 m/z to 5000 m/z after purification of a gastric juice sample by HPLC layer chromatography, and is based on the present invention. A specific embodiment.
第8C圖為一胃液樣本經由HPLC層柱色層分析純化後,於質量範圍500 m/z至5000 m/z一區份之質譜,胃液樣本之再另一區份之質譜,其係根據本發明之一個具體實施例。Figure 8C is a mass spectrum of a gastric juice sample after purification by HPLC layer chromatography, in a mass range of 500 m / z to 5000 m / z, and another mass fraction of the gastric juice sample, according to the present A specific embodiment of the invention.
第9圖為胺基酸序列決定之例示圖示介紹,其呈現例示多肽(2187 m/z)MS/MS分析之結果,根據本發明之一個具體實施例。Figure 9 is a schematic illustration of the amino acid sequence determination showing the results of an exemplary MS/MS analysis of a polypeptide (2187 m/z), in accordance with a particular embodiment of the invention.
第10圖呈現健康受測者與患有胃潰瘍(GU)、十二指腸潰瘍(DU)和胃癌(GC)之人口統特徵。Figure 10 shows the demographic characteristics of healthy subjects with gastric ulcer (GU), duodenal ulcer (DU), and gastric cancer (GC).
第11圖表列健康受測者和各式胃十二指腸疾病,包括胃潰瘍(GU)、十二指腸潰瘍(DU)和胃癌(GC)的胃液特徵。Figure 11 shows the gastric fluid characteristics of healthy subjects and various gastroduodenal diseases, including gastric ulcer (GU), duodenal ulcer (DU), and gastric cancer (GC).
第12圖呈現與胃液中高蛋白質濃度相關之11個臨床、細菌和胃液因子的單變數分析。Figure 12 presents a single variable analysis of 11 clinical, bacterial, and gastric fluid factors associated with high protein concentrations in gastric juice.
第13圖為在單變數分析後,更進一步羅吉斯迴歸分析的結果,其顯示影響胃部蛋白質濃度之獨立因子。Figure 13 shows the results of a further logistic regression analysis after single variable analysis showing independent factors affecting gastric protein concentration.
第14圖為各式胃十二指腸疾病,包括胃潰瘍(GU)、十二指腸潰瘍(DU)和胃癌(GC),胃液二維電泳態樣的統計分析。Figure 14 shows the various types of gastroduodenal diseases, including gastric ulcer (GU), duodenal ulcer (DU) and gastric cancer (GC), and statistical analysis of two-dimensional electrophoretic patterns of gastric juice.
第15圖為一癌症演進對於α-抗胰蛋白酶前驅物分析的結果,其確認α-抗胰蛋白酶前驅物特殊地和進階癌症(90%)和早期癌症相關(100%)。Figure 15 is a graph showing the results of a cancer evolution assay for alpha-antitrypsin precursors confirming that alpha-antitrypsin precursors are specifically associated with advanced cancer (90%) and early cancer (100%).
第16圖為在胃液的二維凝膠電泳態樣中呈現萎縮性胃炎和胃部低酸度之關連性。Figure 16 is a graph showing the association between atrophic gastritis and low acidity in the stomach in a two-dimensional gel electrophoresis pattern of gastric juice.
第17圖介紹二維凝膠電泳圖像的例示基礎條帶態樣,其係根據本發明之一個具體實施例。Figure 17 illustrates an exemplary basic strip pattern of a two-dimensional gel electrophoresis image in accordance with a particular embodiment of the present invention.
第18圖介紹二維凝膠電泳圖像的例示特殊條帶態樣,其係根據本發明之一個具體實施例。Figure 18 illustrates an exemplary special strip pattern of a two-dimensional gel electrophoresis image in accordance with a particular embodiment of the present invention.
第19圖介紹二維凝膠電泳圖像的例示非特殊條帶態樣,其係根據本發明之一個具體實施例。Figure 19 illustrates an exemplary non-specific banding pattern of a two-dimensional gel electrophoresis image in accordance with a particular embodiment of the present invention.
<110> 中央研究院<110> Academia Sinica
<120> 胃液蛋白分析於胃癌鑑定之方法<120> Method for gastric juice protein analysis in gastric cancer identification
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2004年09月20日,Proteomic analysis revealed a strong association of a high level of alpha1-antitrypsin in gastric juice with gastric cancer,Lee K,Proteomics. 2004 Nov;4(11):3343-52. 2005年02月01日,Histologic and serum risk markers for noncardia early gastric can * |
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