WO2015103743A1 - Lectin chip for identifying liver series of disease based on salivary glycoprotein sugar chain and application thereof - Google Patents
Lectin chip for identifying liver series of disease based on salivary glycoprotein sugar chain and application thereof Download PDFInfo
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- WO2015103743A1 WO2015103743A1 PCT/CN2014/070300 CN2014070300W WO2015103743A1 WO 2015103743 A1 WO2015103743 A1 WO 2015103743A1 CN 2014070300 W CN2014070300 W CN 2014070300W WO 2015103743 A1 WO2015103743 A1 WO 2015103743A1
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- WIPO (PCT)
- Prior art keywords
- lectin
- saliva
- chip
- sugar chains
- glycoprotein sugar
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- 108091000699 pea lectin Proteins 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 108010030511 potato lectin Proteins 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 235000004252 protein component Nutrition 0.000 description 1
- 238000000751 protein extraction Methods 0.000 description 1
- 230000020978 protein processing Effects 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 108010066476 ribonuclease B Proteins 0.000 description 1
- 208000007442 rickets Diseases 0.000 description 1
- 210000003079 salivary gland Anatomy 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 125000005630 sialyl group Chemical group 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 108010076805 snowdrop lectin Proteins 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 108010002645 sophora japonica agglutinin Proteins 0.000 description 1
- 108010048090 soybean lectin Proteins 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
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- 238000003786 synthesis reaction Methods 0.000 description 1
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4724—Lectins
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/08—Hepato-biliairy disorders other than hepatitis
- G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin
Definitions
- liver series diseases based on salivary glycoprotein sugar chains
- the present invention relates to a lectin chip for identifying a disease, and particularly to a lectin chip for identifying a liver series disease based on a salivary glycoprotein sugar chain. Background technique
- liver cancer is one of the five most common malignant tumors in the world.
- 2008 there were 749,000 patients with liver cancer in the world, 695,000 patients who died of liver cancer, and 4,200,000 patients with liver cancer in China. , 3,200,000 patients died of liver cancer (http: glob globocan. iarc. fr /).
- 2012 China Health Statistics Yearbook of the Ministry of Health of China malignant tumors ranked first in China's major disease mortality rate in 2011, and liver cancer ranks second in malignant tumor mortality (http: ⁇ www. moh. gov. cn ).
- liver cancer is a specific indicator for the diagnosis of liver cancer, but there are problems with false positives and false negatives in AFP. About 20% of patients with advanced liver cancer, until the time of the disease, AFP is still negative. Histopathological examination is still the gold standard for current diagnosis, but in view of the inherent defects of histological examination such as injury examination, inability to detect dynamically, and differences in sampling. Therefore, seeking non-invasive examination, early diagnosis of liver cancer, is prevention and control The key to liver cancer.
- AFP Alpha-fetoprotein
- saliva collection is safe, convenient, non-invasive and risk free of blood-borne diseases.
- saliva has been widely used as a clinical sample for the monitoring of various levels of diseases such as AIDS, autoimmune diseases, alcoholic cirrhosis, cystic fibrosis, diabetes, cardiovascular disease, rickets, etc. Monitoring and efficacy evaluation.
- the literature and our previous research work show that saliva is rich in l-linked glycoproteins and glycosylated proteins, and the alteration of salivary protein glycosylation has a high correlation with the occurrence and development of diseases.
- carbohydrates are important bioinformatics molecules and are a continuation of genetic information. With the development of molecular biology and cell biology, many other biological functions of carbohydrates are constantly being recognized. Sugars can not only directly participate in the life process in the form of polysaccharides or free oligosaccharides, but also as sugar complexes, such as Glycoproteins, proteoglycans and glycolipids are involved in many important life activities. In addition, glycoconjugates are closely related to many diseases such as cancer, bacteria and viral infections.
- HCC hepatocel lular carcinoma
- 75% ⁇ 80% of HCC is associated with chronic viral infections in the liver, worldwide.
- 80% to 90% of patients with HCC are accompanied by liver fibrosis.
- Current research indicates that changes in glycoprotein sugar chain structure and function occur in liver tissue and serum of patients with liver disease during inflammation, fibrosis, and canceration. Due to its complex structure and versatility, sugar chains contain a huge amount of information. Once the sugar chain modification disorder occurs during the post-translational modification process, serious consequences will occur. For example, under pathological conditions, due to changes or defects in the activity of sugar-metabolizing enzymes, the expression level of sugar chains on glycoproteins may be abnormal, leading to cell dysfunction and even malignant development.
- hepatitis B chronic hepatitis
- the level of Galectile double-antenna type _ sugar chain is significantly higher than that of healthy volunteers, and the level of double-antenna and three-antenna type sugar chains in serum is accompanied by the degree of liver fibrosis. The increase is reduced.
- Other studies have shown that with the development of liver fibrosis, in serum The bisecting core ⁇ (1,6 ⁇ 11 (the double antenna type - the sugar chain level is elevated, and the three antenna type sugar chain is decreased, indicating that the change in the specific sugar chain level in the serum can be used to monitor the development of fibrosis. Kam et al.
- peaks 1, 2, 8 and 10 in the glycan chain spectrum can distinguish liver fibers to some extent. Different stages of the study. The study also found a correlation between changes in serum hyaluronic acid (HA) concentration and different stages of liver fibrosis.
- HA hyaluronic acid
- ⁇ Acetylglucosyltransferase III (GnT-111), acetylglucosaminyltransferase V (GnT-V) and ⁇ 1,6-fucosyltransferase ( a 1-6FT) in serum of patients with liver cancer
- high expression in cancerous tissues which are related to liver cancer
- An important aberrant expression of the enzyme which can cause changes in glycoprotein sugar chain structure in liver cancer patients.
- Sia ⁇ -2, 6 sugar chain structure is highly expressed in liver cancer tissues, but there is no significant change in liver cirrhosis tissue, which can be used as one of the basis for distinguishing liver cirrhosis and liver cancer.
- ion gamma mass spectrometry was used to analyze the glycoprotein sugar chain structure in serum of healthy people, liver cirrhosis and liver cancer patients, and it was found that there are different sugar chains in cirrhosis and liver cancer.
- Liu et al. used a lectin chip (containing 16 lectins) to analyze the difference in glycoprotein chain structure in serum of patients with HCC and cirrhosis, and found that the abundance of Fucylated glycoprotein in serum of HCC patients was higher.
- MALDI-TOF-MS was used to quantitatively compare 83 glycoproteins on serum glycoproteins of 77 randomly selected healthy volunteers, 52 patients with chronic liver disease and 73 patients with liver cancer, and found 57 glycans on serum glycoprotein in patients with liver cancer.
- the abundance of the chain has changed significantly.
- Six of the sugar chains can be used for the diagnosis of liver cancer, and the sensitivity is from 36% to 91%.
- the combination of three sugar chains can diagnose liver cancer patients from patients with chronic liver disease with a sensitivity of 90% and a specificity of 89%.
- GPC3 serum phosphatidylinositol-3
- GPC3 not only helps early detection of liver cancer, but also has strong liver cancer specificity, such as AFP. Supplements can help improve the diagnosis rate of HCC.
- Saliva is an ideal diagnostic medium for non-invasive diseases, but the relationship between changes in glycoprotein sugar chains in saliva and the development of liver cancer remains to be studied.
- Human saliva is secreted by the parotid gland, submandibular gland, sublingual gland and other small glands, containing nucleic acids, proteins, lipids, minerals and other small molecules.
- Saliva can maintain the stability of the oral environment, lubricate the oral cavity, facilitate chewing, talking and swallowing food.
- salivary basic constitutive proteins proteins or glycoproteins that are almost indistinguishable in different sexes and age groups
- saliva also contains some proteins that reflect the health and physiological conditions of the human body.
- Proteins are often differentially expressed in populations of different ages, genders, pathologies, and physiological conditions. Analysis of these differentially expressed proteins in saliva for human health Condition assessment and the basis of disease-related biomarker research. Some of the protein components in the blood are also present in the saliva, which reflects changes in the level of some proteins in the blood. Therefore, it is possible to diagnose the disease by detecting saliva.
- Tumors like endocrine organs, secrete hormones, lymphokines, and cytokines that travel to the distal organs through blood transport, affecting the whole body. Once these factors reach the salivary glands, or secrete into the oral cavity with saliva or cause a change in the transcriptional profile of the gene in the saliva, causing abundance or species changes in certain proteins, thereby becoming a tumor biomarker in saliva.
- the current study found that there are 1939 proteins in human saliva, 3 020 proteins in human blood paddles, and 27% of the salivary proteome coincides with the plasma proteome. This suggests that many biomarkers in the blood circulation may be identified in saliva, further indicating that saliva can be used to diagnose disease.
- Lectin chip technology High-throughput detection of changes in glycoprotein sugar chain structure and attachment patterns in samples.
- Lectins are widely used in the study of glycomics because of their ability to specifically recognize different sugar chain structures and can bind to sugar chains with high affinity in a multivalent form.
- Lectin chip The lectin probe immobilized on the chip specifically binds to the glycoprotein sugar chain in the sample, which can detect the change of the glycoprotein sugar chain structure and the connection mode in the sample with high throughput, and is the most effective study on the glycoprotein sugar chain structure change.
- One of the analytical tools that contribute to the development of new methods for diagnosing and monitoring diseases.
- the application number is 201110021447.
- 3 invention patent we used the chip spotter to prepare the lectin on the epoxy-derived slide carrier, and fixed the high-coverage lectin chip.
- the specific binding of glycoprotein sugar chains to lectins in clinical samples was studied by lectin chip technology, and the glycoprotein sugar chain profiles and the identified lectins in various samples were analyzed.
- the gene chip data analysis method was used to analyze the expression profile of glycoprotein sugar chain, and the difference structure of glycoprotein sugar chain in clinical samples and normal control group was determined.
- glycoprotein has polyvalent Sia or GlcNAc, terminal ⁇ -1, 3 man, GalNAc and Gal ⁇ l-4GlcNAc sugar chain structures.
- the high-coverage lectin chip was then used to study the changes in glycoprotein glycophores in transcriptional growth factor-activated hepatic stellate cells (LX-2), which were found to be: AAL, PHA-E (Cloth Bean-E) and ECA-recognized
- LX-2 transcriptional growth factor-activated hepatic stellate cells
- AAL AAL
- PHA-E Cloth Bean-E
- ECA-recognized The expression of Fuc, N_GlcNAc and Gal sugar chain structure was significantly enhanced on the membrane of activated cells.
- the correlation between glycogene expression and hepatocyte surface glycan profile in liver fibrosis model induced by CC1 4 was studied by using chip technology. It was found that there were 10 levels of glycosyltransferase gene transcriptional changes in the fibrotic liver.
- the present invention intends to use the established glycosics techniques and methods in three stages of progression of "hepatitis B-hepatitis B cirrhosis - liver cancer" to find common in saliva of patients with hepatitis B, hepatitis B and cirrhosis and liver cancer. And a unique glycoprotein sugar chain; will provide a new method for non-invasive identification of hepatitis, cirrhosis and liver cancer. Summary of the invention
- the object of the present invention is to provide a lectin chip for identifying liver diseases based on salivary glycochain and its application, which can non-injury, rapidly identify changes in glycoprotein sugar chains in saliva, and identify non-invasive hepatitis and liver. Hardening and liver cancer provide new methods.
- the technical solution of the present invention is: a lectin chip for identifying a liver-series disease based on a salivary glycochain, comprising testing a lectin probe set and a control probe, which are special in: the test lectin probe
- the group includes at least a combination of Jacalin, GSL_II, PTL_I, SJA, GSL_I, LCA, Con_A, PTL_II, DSA, WA, GNA, PHA_E+L, MAL_II, AAL, PSA, WGA, and UEA-I lectin probes.
- the above test lectin probe set also includes a combination of WFA, LTL, LEL, EEL, and PWM lectin probes.
- An application of the above lectin chip characterized in that it is used for detecting changes in glycoproteins and sugar chains in saliva.
- the above test lectin probe set also includes a combination of PHA-E, PNA, MPL, DBA, NPA, BS_I and SNA lectin probes.
- An application of the above lectin chip characterized in that it is used for detecting changes in glycoproteins and sugar chains in saliva.
- a lectin chip for identifying a liver-series disease based on a salivary glycoprotein sugar chain comprising testing a lectin probe set and a control probe, the special one being: the test lectin probe set comprises groups I, II and III
- the groups I, II and III are Jacal in, GSL-II, PTL-I, SJA, GSL-I, IXA, Con_A, PTL-II, DSA, WA, GNA, PHA-E+ MAL-II, AAL , PSA, WGA, and UEA-I lectin probe combinations, W, LTL, LEU EEL, and PWM lectin probe combinations, as well as PHA-E, PNA, MPL, DBA, NPA, BS-I, and SNA agglutinin A combination of needles.
- An application of the above lectin chip characterized in that it is used for detecting changes in glycoproteins and sugar chains in saliva.
- FIG. 1 is a layout diagram of a lectin probe on a lectin chip
- Figure 2 shows the results of fluorescence detection of salivary glycoproteins in healthy people, hepatitis B, cirrhosis and liver cancer.
- Figure 3 shows the results of saliva chip verification.
- A is a saliva chip layout
- N1-N7 is healthy human saliva
- HB1-HB6 is saliva of hepatitis B patients
- HC1-HC7 is saliva of patients with liver cirrhosis
- HCC1-HCC7 is saliva of liver cancer patients.
- B is a graph showing the results of fluorescence detection of lectin LTL combined with a saliva chip.
- C is a comparison of saliva binding strength of LTL corresponding to healthy people, hepatitis B, cirrhosis and liver cancer patients.
- Glycine, disodium hydrogen phosphate, sodium dihydrogen phosphate, Tween-20 and protease inhibitors were purchased from Sigma-Aldrich, USA.
- Bovine serum albumin (BSA) was purchased from Merck, Germany. Cy3 fluorescence was purchased from Amerhsma, USA.
- the T-PER animal tissue protein extraction reagent was purchased from PIERCE, USA. All other chemical reagents were of analytical grade and were not further purified prior to use. All experimental water was ultrapure water treated with a Milli-Q50 pure water system (Millipore, USA). Sephadex G-25 column desalting was purchased from GE Healthcare, USA.
- the chip hybridization kit was purchased from Bio-Rad Bole Company, USA.
- PVDF membranes were purchased from Millipore, USA. Other common glassware are made in China. 37 lectins (see Table 1 for specific names) were purchased from Vector Laboratories, Sigma-Aldrich, USA The company and the company Calbiochem, Germany. Table 1 List of specific names of lectins
- ConA Canavalia ensiformis Agglutinin with concanavalin
- Electric blast drying oven Tianjin Taisite Company; Autoclave: Japan TOMY company; Ultra-speed refrigerated centrifuge 5804R: Germany Eppendorf company; Micronucleic acid protein analyzer: Germany Implen company; Biochip scanner 4000B: American Axon company Chip spotter: Boao crystal core SmartArrayer48 spotting instrument; chip hybrid box HL-2000: US UVP company.
- protease inhibitor add ⁇ per ml of saliva
- ALT alanine aminotransferase
- AST aspartate aminotransferase
- the saliva chip was made from a sample of saliva samples.
- the samples were divided into four groups according to healthy people, hepatitis B, cirrhosis and liver cancer patients, 7 cases, 6 cases, 7 cases and 7 cases respectively.
- Cy3-labeled lectins were used to detect the expression levels of glycoprotein structures specifically recognized by lectins in these saliva samples.
- Salivary proteins were dissolved in spotting buffer (0.5 mg/mL BSA in l x PBS, pH 7.4) to a final concentration of 1 mg/mL and plated on epoxidized modified slides using a chip spotter.
- the layout of the saliva chip is shown in section A of Figure 3. Repeat three times in each sample zone, with three replicate zones per base. The spotted substrate was incubated overnight at 50% humidity, then vacuum-dried at 37 °C for 3 h, and the fixed chip was sealed at 4 °C in the dark.
- the chip was first blocked in the blocking buffer at room temperature for 1 h, l xPBST and l xPBS were washed twice, each time for 5 min, centrifuged and dried. After adding the prepared incubation buffer containing Cy3 labeled lectin, slowly rotate 3 rpm in the chip hybridization box at 4 rpm, l xPBST and l XPBS was washed twice each time, 10 min each time, centrifuged and dried.
- the photomultiplier tube was set to 70% and the laser intensity was 100% by a chip scanner, and the chip image was scanned at a wavelength of 532 nm. The images were analyzed by Genepix 3.0 software to obtain raw data.
- the lectin chip was used to detect the saliva of healthy volunteers, hepatitis B, cirrhosis and liver cancer patients. After obtaining the chip data by professional software and normalizing the treatment, the results of the three groups of liver diseases were compared with the results of the health group.
- the normalized post-fluorescence intensity (NFI) of the lectin obtained Fold-change values in the hepatitis B (HB), cirrhosis (HC), and liver cancer (HCC) groups, respectively, compared to the healthy group (H).
- NFI normalized post-fluorescence intensity
- HB hepatitis B
- HC cirrhosis
- HCC liver cancer
- the polyvalent sialic acid and (GlcNAc) n structure recognized by WGA are lowly expressed in the saliva of three liver disease patients.
- SNA-recognized Sia2-6Gal/GalNAc is highly expressed only in hepatitis B;
- PHA-E recognizes the split-type GlcNAc and dual-antenna N-glycans, and MPL-recognized Gai 1 -3 GalNAc is only expressed in hepatitis B .
- Elan-recognized Gala 1 -3 (Fuca 1 -2)Gal, PWM-recognized branched (LacNAc) n is highly expressed only in liver cancer; WFA recognizes the terminal GalNAca/pi-3/6Gal, LTL recognizes Fucal-3 (Gaipi -4) GlcNAc is only expressed low in liver cancer.
- Table 3 Expression of salivary glycoprotein glycoprotein profiles in patients with hepatitis B, cirrhosis and liver cancer
- PHA-E+L N-glycans tri- and tetra-antennary 100 100 100
- the data in the table indicates the Fold-change value of each lectin in the lectin chip results for this group relative to the NFI of the control group.
- HB hepatitis B
- HC cirrhosis
- HCC liver cancer
- - no significant difference
- 100 the denominator of Fold-change value is zero.
- the lectin probe was verified to recognize the binding of sugar chains in saliva samples from healthy people, hepatitis B, cirrhosis and liver cancer patients.
- lectin LTL Cy3 fluorescently labeled LTL was incubated with a spotted saliva chip, scanned by a chip scanner (see Figure 3B) and analyzed by Genepix 3.0 software, and the raw data was analyzed in depth according to the method described in 1.6.
- the results showed that the binding strength of LTL to the sugar chain of 7 liver cancer saliva samples was significantly lower than that of other samples (see Figure 3C), and the P value was less than 0.05 by Student t-test.
- the remaining lectin probe verification methods were based on this method, and the results were consistent with the results of the lectin chip. It has been demonstrated that these lectins can be used as probes to identify liver diseases by detecting glycoprotein sugar chains in saliva.
Abstract
The present invention provides a lectin chip for identifying a liver series of disease based on a salivary glycoprotein sugar chain, wherein the lectin chip comprises at least a combination of Jacalin, GSL_II, PTL-I, SJA, GSL-I, LCA, Con-A, PTL-II, DSA, VVA, GNA, PHA-E+L, MAL-II, AAL, PSA, WGA and UEA-I lectin probes, a kit prepared using the lectin chip for detecting a change of a glycoprotein sugar chain in saliva, and an application of detecting the glycoprotein sugar chain in the saliva by using the lectin chip.
Description
基于唾液糖蛋白糖链鉴别肝系列病 Identification of liver series diseases based on salivary glycoprotein sugar chains
的凝集素芯片及其应用 技术领域 本发明涉及一种鉴别疾病的凝集素芯片，具体涉及一种基于唾液 糖蛋白糖链鉴别肝系列病的凝集素芯片。 背景技术 BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a lectin chip for identifying a disease, and particularly to a lectin chip for identifying a liver series disease based on a salivary glycoprotein sugar chain. Background technique
国际癌症研究机构的最新资料表明，肝癌是全球最常见的五种恶 性肿瘤之一， 2008年全球肝癌患者 74. 9万， 死于肝癌的患者 69. 5 万， 其中中国肝癌患者 40. 2 万， 死于肝癌的患者 37. 2 万 (http：〃 globocan. iarc. fr/)。 据中国***《2012中国卫生统计 年鉴》 显示， 2011 年恶性肿瘤高居我国主要疾病死亡率的首位， 而 其中肝癌位居恶性肿瘤死亡率的第二位 （http：〃 www. moh. gov. cn)。 肝癌死亡率高的原因之一是当前肝癌的确诊通常是在晚期，因而错过 了最佳的治疗时期，而且临床诊断后患者平均生存时间不到 12个月。 甲胎蛋白（Alpha-fetoprotein, AFP )是目前肝癌诊断的特异性指标， 但 AFP测定存在假阳性和假阴性的问题。 约 20%的晚期肝癌患者， 直 至病故前， AFP测定仍为阴性。 病理组织学检查仍是目前诊断的金标 准， 但鉴于组织学检查固有的缺陷如损伤性检查、 不能动态检测、 存 在取样差异等。 因此寻求非损伤性检查， 进行肝癌早期诊断， 是防控
肝癌的关键。 According to the latest information from the International Agency for Research on Cancer, liver cancer is one of the five most common malignant tumors in the world. In 2008, there were 749,000 patients with liver cancer in the world, 695,000 patients who died of liver cancer, and 4,200,000 patients with liver cancer in China. , 3,200,000 patients died of liver cancer (http: glob globocan. iarc. fr /). According to the 2012 China Health Statistics Yearbook of the Ministry of Health of China, malignant tumors ranked first in China's major disease mortality rate in 2011, and liver cancer ranks second in malignant tumor mortality (http: 〃 www. moh. gov. cn ). One of the reasons for the high mortality rate of liver cancer is that the current diagnosis of liver cancer is usually in the advanced stage, so the optimal treatment period is missed, and the average survival time of patients after clinical diagnosis is less than 12 months. Alpha-fetoprotein (AFP) is a specific indicator for the diagnosis of liver cancer, but there are problems with false positives and false negatives in AFP. About 20% of patients with advanced liver cancer, until the time of the disease, AFP is still negative. Histopathological examination is still the gold standard for current diagnosis, but in view of the inherent defects of histological examination such as injury examination, inability to detect dynamically, and differences in sampling. Therefore, seeking non-invasive examination, early diagnosis of liver cancer, is prevention and control The key to liver cancer.
随着社会的进步， 科学技术的发展， 人们对医学检查的要求也 越来越高， 要求无创、 简便、 快速的疾病检查诊断方法。 与血清标 本相比， 唾液采集安全方便、 无创伤和无血源性疾病传播的危险。 而且近年来， 唾液作为临床样本已被广泛应用于多种疾病诸如艾滋 病、 自身免疫性疾病、 酒精性肝硬变、 囊性纤维病、 糖尿病、 心血管 病、 龋病等的药物水平监测、 病情监控和疗效评价当中。文献和我们 以往的研究工作表明唾液中含有丰富的 ^连接糖蛋白和 连接糖蛋 白， 而唾液蛋白糖基化的改变与疾病的发生发展具有较高的关联性。 从变化的唾液糖蛋白糖链中可以找到与疾病相关的生物标志物，而基 于唾液检测的新技术和新方法也会逐渐成为今后非侵袭性临床诊断 发展的一个方向。糖类物质同核酸一样是重要的生物信息分子， 而且 是基因信息的延续。随着分子生物学及细胞生物学的发展， 糖类物质 的其它诸多生物功能不断被认识，糖类物质不仅可以以多糖或游离寡 糖的形式直接参与生命过程， 而且可以作为糖复合物， 如糖蛋白、 蛋 白多糖及糖脂等参与许多重要的生命活动。此外， 糖复合物还与许多 疾病， 如癌症、 细菌和病毒感染等疾病有着密切的关系。 With the advancement of society and the development of science and technology, people are increasingly demanding medical examinations, requiring non-invasive, simple and rapid diagnosis and diagnosis methods for diseases. Compared to serum samples, saliva collection is safe, convenient, non-invasive and risk free of blood-borne diseases. In recent years, saliva has been widely used as a clinical sample for the monitoring of various levels of diseases such as AIDS, autoimmune diseases, alcoholic cirrhosis, cystic fibrosis, diabetes, cardiovascular disease, rickets, etc. Monitoring and efficacy evaluation. The literature and our previous research work show that saliva is rich in l-linked glycoproteins and glycosylated proteins, and the alteration of salivary protein glycosylation has a high correlation with the occurrence and development of diseases. Disease-related biomarkers can be found in the changing salivary glycoprotein sugar chains, and new techniques and new methods based on saliva detection will gradually become a direction for the development of non-invasive clinical diagnosis in the future. As with nucleic acids, carbohydrates are important bioinformatics molecules and are a continuation of genetic information. With the development of molecular biology and cell biology, many other biological functions of carbohydrates are constantly being recognized. Sugars can not only directly participate in the life process in the form of polysaccharides or free oligosaccharides, but also as sugar complexes, such as Glycoproteins, proteoglycans and glycolipids are involved in many important life activities. In addition, glycoconjugates are closely related to many diseases such as cancer, bacteria and viral infections.
我国 90%以上的肝癌为肝细胞癌 （Hepatocel lular carcinoma, HCC )， 75%〜80%的 HCC发病与肝脏慢性病毒性感染有关， 世界范围内
80%〜90%的 HCC患者伴随有肝纤维化。 目前研究表明： 在肝病的炎症、 纤维化、癌变的过程中患者肝组织和血清中发生了糖蛋白糖链结构和 功能的改变。 糖链因其结构复杂和功能多样， 包含了巨大的信息量， 一旦在翻译后的修饰过程中发生糖链修饰紊乱， 就会产生严重的后 果。 如在病理状态下， 由于糖代谢酶类活力的改变或缺陷， 可使糖蛋 白上的糖链表达水平发生异常， 导致细胞功能失常， 甚至出现恶性发 展。 More than 90% of liver cancers in China are hepatocel lular carcinoma (HCC), and 75%~80% of HCC is associated with chronic viral infections in the liver, worldwide. 80% to 90% of patients with HCC are accompanied by liver fibrosis. Current research indicates that changes in glycoprotein sugar chain structure and function occur in liver tissue and serum of patients with liver disease during inflammation, fibrosis, and canceration. Due to its complex structure and versatility, sugar chains contain a huge amount of information. Once the sugar chain modification disorder occurs during the post-translational modification process, serious consequences will occur. For example, under pathological conditions, due to changes or defects in the activity of sugar-metabolizing enzymes, the expression level of sugar chains on glycoproteins may be abnormal, leading to cell dysfunction and even malignant development.
国内外研究现状及分析： Research status and analysis at home and abroad:
( 1 ) 肝炎、 肝纤维化 /肝硬化与肝癌发生发展过程中患者血清 / 肝组织中糖蛋白糖链发生变化， 表明血清和组织中糖蛋白糖链变化 与肝癌发生发展具有关联性。 (1) Changes in glycoprotein sugar chains in serum/liver tissue of patients with hepatitis, liver fibrosis/cirrhosis and liver cancer, indicating that changes in glycoprotein sugar chains in serum and tissues are associated with the development of liver cancer.
我国是慢性肝炎特别是乙型肝炎的高流行区，约有 1. 2亿人长期携 带乙肝病毒， 慢性肝炎患者大约有 3000万。约有 30万慢性肝炎患者发 展为肝硬化和肝癌患者。 研究发现， 80%-90%的肝癌患者经历 "慢性 肝炎一肝硬化一肝癌" 的过程。 已有研究表明， 在肝炎到肝纤维化 / 肝硬化过程中患者血清中糖蛋白糖链发生变化 [5]。 如在乙肝病毒 (HBV)感染后肝纤维化患者血清中 Gal化双天线型 _糖链水平明显高 于健康志愿者，同时血清中双天线型和三天线型 ^糖链水平伴随肝纤 维化程度的加重而降低。另有研究表明随着肝纤维化的发展， 血清中
平分型核心 α (1，6^11(化双天线型 -糖链水平升高， 而三天线型 糖链降低，表明血清中特定 ^糖链水平的变化可以用来监测纤维化的 发展。 Kam等利用 MALDI-T0F质谱技术定量分析了慢性 HBV感染和不同 程度的肝纤维化患者血清中的 ^连接糖链谱的变化，发现了 17个糖链 质谱峰可以作为肝纤维化 /肝硬化检测的潜在生物标志物，而 1341. 5、 1829. 7、 1933. 3和 2130. 3 m/z四个糖链质谱峰能鉴别诊断肝纤维化和 肝硬化， 准确率达 85%。 Qu等运用 DNA测序仪的荧光糖链电泳技术 (DSA-FACE) 评估了慢性 HBV患者血清中的 ^糖链谱的变化， 发现糖 链谱中峰 1， 2， 8和 10在一定程度上可以区分肝纤维化的不同阶段。 研究也发现血清中透明质酸（HA)浓度变化与肝纤维化不同阶段的之 间具有相关性。 China is a highly endemic area of chronic hepatitis, especially hepatitis B. About 120 million people carry hepatitis B virus for a long time, and there are about 30 million patients with chronic hepatitis. About 300,000 patients with chronic hepatitis develop cirrhosis and liver cancer patients. The study found that 80%-90% of patients with liver cancer experience the process of "chronic hepatitis-cirrhosis-hepatic cancer". Studies have shown that glycoprotein sugar chains in patients' serum change during hepatitis to liver fibrosis/cirrhosis [5] . For example, in the serum of patients with liver fibrosis after hepatitis B virus (HBV) infection, the level of Galectile double-antenna type _ sugar chain is significantly higher than that of healthy volunteers, and the level of double-antenna and three-antenna type sugar chains in serum is accompanied by the degree of liver fibrosis. The increase is reduced. Other studies have shown that with the development of liver fibrosis, in serum The bisecting core α (1,6^11 (the double antenna type - the sugar chain level is elevated, and the three antenna type sugar chain is decreased, indicating that the change in the specific sugar chain level in the serum can be used to monitor the development of fibrosis. Kam et al. used MALDI-T0F mass spectrometry to quantitatively analyze the changes in the ligated glycoproteins in the serum of patients with chronic HBV infection and different degrees of hepatic fibrosis. It was found that 17 sugar chain mass spectrometry peaks can be used as liver fibrosis/cirrhosis detection. The potential biomarkers, and 1341. 5, 1829. 7, 1933. 3 and 2130. 3 m / z four sugar chain mass spectrometry peak can be used to differentially diagnose liver fibrosis and cirrhosis, the accuracy rate is 85%. The DNA sequencer's fluorescent sugar chain electrophoresis technique (DSA-FACE) evaluated the changes in the glycan profile of serum in patients with chronic HBV. It was found that peaks 1, 2, 8 and 10 in the glycan chain spectrum can distinguish liver fibers to some extent. Different stages of the study. The study also found a correlation between changes in serum hyaluronic acid (HA) concentration and different stages of liver fibrosis.
在肝纤维化 /肝硬化与肝癌发生发展过程中患者血清和肝组织中 糖蛋白糖链发生变化， 如 Liu等运用 DSA-FACE检测肝纤维化、 肝硬化 和肝癌患者血清中的 9种糖链结构，发现 α -1， 3Fuc化三天线糖链结构 在肝癌患者血清中的丰度最高， 而平分型 α -1， 6核心 Fuc化双天线糖 链结构在肝硬化患者血清中的丰度较高，证明这两种糖链在血清中的 丰度变化与肿瘤发展阶段密切相关。 ^乙酰葡糖胺基转移酶 III (GnT- 111)、 乙酰葡糖胺基转移酶 V (GnT- V ) 和 α 1， 6-岩藻糖基转移酶 ( a 1-6FT) 在肝癌患者血清及癌变组织中高表达， 是肝癌相关的三
种重要异常表达酶， 能引起肝癌患者体内糖蛋白糖链结构的改变。研 究也发现 Sia α -2， 6糖链结构在肝癌组织中高表达， 但在肝硬化组织 中并无明显变化， 可作为区分肝硬化与肝癌的依据之一。 Changes in glycoprotein sugar chains in serum and liver tissues during liver fibrosis/cirrhosis and liver cancer development, such as Liu, using DSA-FACE to detect 9 kinds of sugar chains in serum of liver fibrosis, liver cirrhosis and liver cancer patients Structure, it was found that the α-1, 3Fuc three-antennary sugar chain structure has the highest abundance in the serum of liver cancer patients, and the abundance of the split-type α-1, 6-core Fucized double-antennary sugar chain structure in the serum of patients with liver cirrhosis Higher, it is proved that the abundance changes of these two sugar chains in serum are closely related to the stage of tumor development. ^Acetylglucosyltransferase III (GnT-111), acetylglucosaminyltransferase V (GnT-V) and α 1,6-fucosyltransferase ( a 1-6FT) in serum of patients with liver cancer And high expression in cancerous tissues, which are related to liver cancer An important aberrant expression of the enzyme, which can cause changes in glycoprotein sugar chain structure in liver cancer patients. The study also found that Sia α -2, 6 sugar chain structure is highly expressed in liver cancer tissues, but there is no significant change in liver cirrhosis tissue, which can be used as one of the basis for distinguishing liver cirrhosis and liver cancer.
目前， 在肝癌诊断标志物的研究中， 一些新技术用于了肝癌患者 血清中糖蛋白糖链结构的研究，发现了一些在肝癌发生发展过程中患 者血清中特有的糖蛋白糖链。 如， 运用离子淌度质谱 （ion mobil ity mass spectrometry, IM- MS) 技术分析了健康人、 肝硬化和肝癌患者 血清中糖蛋白糖链结构， 发现肝硬化和肝癌中存在差异的糖链。 Liu 等运用凝集素芯片 （含有 16种凝集素） 技术分析 HCC和肝硬化患者 血清中糖蛋白糖链结构的差异，发现 HCC患者血清中 Fuc化的糖蛋白 丰度较高。 应用 MALDI-TOF- MS定量比较了 77名随机选取的健康志 愿者、 52名慢性肝病患者和 73名肝癌患者血清糖蛋白上的 83个 ^ 糖链， 发现肝癌患者血清糖蛋白上 57个 ^糖链的丰度发生了显著变 化， 其中六种糖链可分别用于肝癌诊断， 检测敏感性从 36%到 91%。 三种 ^糖链的联用能从慢性肝病患者中诊断出肝癌患者， 敏感性为 90%, 特异性为 89%。 在肝癌 （肿瘤直径小于 3厘米） 早期诊断标志 物的研究中， 我国科学家发现血清磷脂酰肌醇蛋白聚糖 -3 (Glypican-3, GPC3)可作为检出早期肝癌的分子标志物。 GPC3 不 仅有助于早期发现肝癌， 而且具有很强的肝癌特异性， 如作为 AFP的
补充， 有助于提高 HCC的确诊率。 At present, in the research of liver cancer diagnostic markers, some new techniques have been applied to the study of glycoprotein sugar chain structure in the serum of patients with liver cancer, and some glycoprotein sugar chains unique to the serum in the process of liver cancer development have been discovered. For example, ion gamma mass spectrometry (IM-MS) was used to analyze the glycoprotein sugar chain structure in serum of healthy people, liver cirrhosis and liver cancer patients, and it was found that there are different sugar chains in cirrhosis and liver cancer. Liu et al. used a lectin chip (containing 16 lectins) to analyze the difference in glycoprotein chain structure in serum of patients with HCC and cirrhosis, and found that the abundance of Fucylated glycoprotein in serum of HCC patients was higher. MALDI-TOF-MS was used to quantitatively compare 83 glycoproteins on serum glycoproteins of 77 randomly selected healthy volunteers, 52 patients with chronic liver disease and 73 patients with liver cancer, and found 57 glycans on serum glycoprotein in patients with liver cancer. The abundance of the chain has changed significantly. Six of the sugar chains can be used for the diagnosis of liver cancer, and the sensitivity is from 36% to 91%. The combination of three sugar chains can diagnose liver cancer patients from patients with chronic liver disease with a sensitivity of 90% and a specificity of 89%. In the study of early diagnosis markers of liver cancer (tumor diameter less than 3 cm), Chinese scientists found that serum phosphatidylinositol-3 (GPC3) can be used as a molecular marker for detecting early liver cancer. GPC3 not only helps early detection of liver cancer, but also has strong liver cancer specificity, such as AFP. Supplements can help improve the diagnosis rate of HCC.
上述的研究表明： 在肝炎、 肝纤维化 /肝硬化与肝癌发生发展过 程中患者血清 /肝组织中糖蛋白糖链发生变化， 表明血清和组织中糖 蛋白糖链变化与肝癌发生发展具有关联性， 通过血清区别检测肝 炎、 肝硬化与肝癌是可行的。但是鉴于血检固有的缺陷， 如损伤和存 在血源性疾病传播的危险， 因此有必要进一步研究和寻求非损伤性 鉴别诊断肝炎、 肝硬化与肝癌的新技术和新方法。 The above studies have shown that changes in glycoprotein sugar chains in serum/liver tissue during the development of hepatitis, liver fibrosis/cirrhosis and liver cancer indicate that glycoprotein sugar chain changes in serum and tissues are associated with the development of liver cancer. It is feasible to detect hepatitis, cirrhosis and liver cancer by serum. However, in view of the inherent defects of blood tests, such as injury and the risk of transmission of blood-borne diseases, it is necessary to further study and seek new techniques and methods for non-invasive differential diagnosis of hepatitis, cirrhosis and liver cancer.
(2) 唾液是一种理想的非损伤性疾病诊断介质， 但唾液中糖蛋白 糖链的变化与肝癌发生发展的关联性有待研究。 (2) Saliva is an ideal diagnostic medium for non-invasive diseases, but the relationship between changes in glycoprotein sugar chains in saliva and the development of liver cancer remains to be studied.
人体唾液由腮腺、 下颌下腺、 舌下腺和其他一些小腺体分泌， 包 含核酸、 蛋白质、 脂类、 矿物质和其他小分子物质。 唾液能起到维持 口腔内环境稳定、 润滑口腔壁、 方便咀嚼、 说话和吞咽食物的作用。 另外， 唾液中还存在抑制和消灭细菌和病毒的物质。唾液含量和组成 上的变化， 不管是由口腔局部病变还是机体整体状况引起的， 都可能 会造成唾液功能缺陷。研究发现， 唾液中除了含有一些唾液基本组成 性蛋白质（在不同性别和不同年龄段都几乎无差别表达的蛋白质或糖 蛋白）外， 还含有一些可以反应人体身体健康和生理状况的一些蛋白 质， 这些蛋白质往往在不同年龄、 性别、 病理和生理状况的人群中出 现差异性表达。分析唾液中这些差异表达的蛋白质是进行人体健康状
况评估和疾病相关生物标记物研究的基础。血液中的部分蛋白质成分 同样存在于唾液中， 唾液能反映出血液中部分蛋白质水平的变化。 因 此， 就有可能通过唾液的检测来进行疾病的诊断。 Human saliva is secreted by the parotid gland, submandibular gland, sublingual gland and other small glands, containing nucleic acids, proteins, lipids, minerals and other small molecules. Saliva can maintain the stability of the oral environment, lubricate the oral cavity, facilitate chewing, talking and swallowing food. In addition, there are substances in the saliva that inhibit and destroy bacteria and viruses. Changes in saliva content and composition, whether caused by local lesions in the mouth or the overall condition of the body, may cause defects in saliva function. The study found that in addition to some salivary basic constitutive proteins (proteins or glycoproteins that are almost indistinguishable in different sexes and age groups), saliva also contains some proteins that reflect the health and physiological conditions of the human body. Proteins are often differentially expressed in populations of different ages, genders, pathologies, and physiological conditions. Analysis of these differentially expressed proteins in saliva for human health Condition assessment and the basis of disease-related biomarker research. Some of the protein components in the blood are also present in the saliva, which reflects changes in the level of some proteins in the blood. Therefore, it is possible to diagnose the disease by detecting saliva.
肿瘤和内分泌器官一样， 可以分泌激素、 淋巴因子和细胞因子， 通过血液运输到达远端器官， 对全身产生影响。 一旦这些因子到达 唾液腺， 或随唾液分泌进入口腔或导致唾液中的基因转录谱发生改 变， 引起某些蛋白质的丰度或种类改变， 从而成为唾液中的肿瘤生 物标志物。 目前研究发现， 人唾液中有 1939种蛋白质， 人血桨中有 3 020种蛋白质， 27%的唾液蛋白质组与血桨蛋白质组重合。 这表明许 多血液循环中的生物标志物可能在唾液中得到鉴定， 也进一步说明 唾液可被用于诊断疾病。 血桨蛋白质组学发现的 177个与心血管疾病 相关的潜在生物标志物的蛋白质中， 40%的蛋白质同样出现在唾液蛋 白质组中； 在已列出的 1058个潜在癌症相关生物标志物的蛋白质中 ， 34%的蛋白质同样出现在唾液蛋白质组中。 唾液和血桨的蛋白质组 比较结果显示， 有些蛋白质只在唾液蛋白质组中出现， 而没有出现 在血桨蛋白质组中， 表明两者蛋白质种类并不完全一样。 通过对唾 液、 血桨和人类全蛋白质组的 GO ( geneontology ) 注释比较， 发现 唾液和血桨蛋白质组中细胞外基质组分较多， 而细胞内液组分较少 ， 提示唾液和血桨蛋白质组具有分泌蛋白质组的特征； 在生物学过
程和分子功能方面的分析结果提示， 唾液蛋白质组和血桨蛋白质组 具有相似的生物学功能。 因此， 从唾液中筛选肿瘤标志物具有科学 性和可行性， 并具有非损伤性检测的优势。 Tumors, like endocrine organs, secrete hormones, lymphokines, and cytokines that travel to the distal organs through blood transport, affecting the whole body. Once these factors reach the salivary glands, or secrete into the oral cavity with saliva or cause a change in the transcriptional profile of the gene in the saliva, causing abundance or species changes in certain proteins, thereby becoming a tumor biomarker in saliva. The current study found that there are 1939 proteins in human saliva, 3 020 proteins in human blood paddles, and 27% of the salivary proteome coincides with the plasma proteome. This suggests that many biomarkers in the blood circulation may be identified in saliva, further indicating that saliva can be used to diagnose disease. Of the 177 proteins associated with cardiovascular disease-associated biomarkers found in blood proteomics, 40% of proteins are also present in the salivary proteome; proteins listed in 1058 potential cancer-associated biomarkers Of these, 34% of the protein also appears in the salivary proteome. The proteome comparison of saliva and blood paddles showed that some proteins appeared only in the salivary proteome and did not appear in the plasma proteome, indicating that the protein types were not exactly the same. By comparing the GO (geneontology) annotations of saliva, blood paddles, and human whole proteomes, it was found that there are more extracellular matrix components in the saliva and blood proteome, and fewer intracellular fluid components, suggesting saliva and blood protein. The group has the characteristics of a secreted proteome; Analysis of the process and molecular function suggests that the salivary proteome and the plasma proteome have similar biological functions. Therefore, screening tumor markers from saliva is scientific and feasible, and has the advantage of non-invasive detection.
随着凝集素亲和纯化、 化学修饰方法捕获糖蛋白及串联质谱分析 技术的发展， 近 5年唾液糖蛋白质组的研究得到了较快的发展， 推动 了肿瘤相关唾液糖蛋白的筛选和鉴定。 2006年 Ramachandran等运用 酰肼化学方法从唾液中鉴定到 45个 _糖蛋白， 2年后该研究组又通 过分别鉴定唾液、 腮腺、 下颌下腺和舌下腺的 糖蛋白， 将鉴定的 -糖蛋白数目提高到 77个。 最新研究显示， 运用基于 6肽库的动态 范围压缩技术、 酰肼化学和串联质谱的方法， 从唾液中共鉴定到 193 个 -糖蛋白。 目前， 在唾液中寻找年龄和性别相关的标志性糖蛋白 也成为一个新的研究热点。如免疫球蛋白 A, 糖蛋白 340和粘蛋白 5B 等在唾液中的丰度随着年龄的增加而增加，而粘蛋白 7的丰度却随着 年龄的增加而减少。 其中粘蛋白 5B上的糖链谱随着血型的不同而有 所不同。 With the development of lectin affinity purification, chemical modification method to capture glycoprotein and tandem mass spectrometry, the research on salivary glycoprotein group has been developed rapidly in the past 5 years, which promoted the screening and identification of tumor-related salivary glycoprotein. In 2006, Ramachandran et al. identified 45 glycoproteins from saliva using hydrazide chemistry. Two years later, the group identified glycoproteins by salivary, parotid, submandibular gland and sublingual gland, respectively. The number has increased to 77. Recent studies have shown that a total of 193-glycoproteins have been identified from saliva using a dynamic range compression technique based on the 6-peptide library, hydrazide chemistry and tandem mass spectrometry. At present, finding age- and sex-related marker glycoproteins in saliva has also become a new research hotspot. The abundances of immunoglobulin A, glycoprotein 340 and mucin 5B in saliva increase with age, while the abundance of mucin 7 decreases with age. The glycan profile on mucin 5B varies with blood type.
凝集素芯片技术：可以高通量地检测出样品中糖蛋白糖链结构和 连接方式的变化。 Lectin chip technology: High-throughput detection of changes in glycoprotein sugar chain structure and attachment patterns in samples.
凝集素由于其可特异性识别不同糖链结构并可以多价形式与糖 链高亲和性结合的特性被广泛用于糖组学的研究。凝集素芯片通过固
定于芯片上的凝集素探针与样品中糖蛋白糖链特异性结合，可以高通 量地检测出样品中糖蛋白糖链结构和连接方式的变化，是研究糖蛋白 糖链结构变化最有效的分析工具之一，其有助于发展新的诊断和监测 疾病的方法。 Lectins are widely used in the study of glycomics because of their ability to specifically recognize different sugar chain structures and can bind to sugar chains with high affinity in a multivalent form. Lectin chip The lectin probe immobilized on the chip specifically binds to the glycoprotein sugar chain in the sample, which can detect the change of the glycoprotein sugar chain structure and the connection mode in the sample with high throughput, and is the most effective study on the glycoprotein sugar chain structure change. One of the analytical tools that contribute to the development of new methods for diagnosing and monitoring diseases.
申请号为 201110021447. 3发明专利， 是我们在前期用芯片点样 仪将凝集素点制于环氧衍生化的玻片载体上， 经固定化处理， 制备出 高覆盖率凝集素芯片。用凝集素芯片技术对临床样本中糖蛋白糖链与 凝集素进行特异性结合的研究，分析各种样本中的糖蛋白糖链谱及其 识别的凝集素。并借鉴基因芯片数据分析方法对糖蛋白糖链的表达谱 进行数据统计分析，确定临床样本和正常对照组中糖蛋白糖链的差异 结构。 首先我们用标准糖蛋白 RNase B 和 Fetuin, 验证了凝集素芯 片在检测糖蛋白糖链方面的可行性和可靠性， 并应用凝集素芯片对 Chang' s liver 正常肝细胞总蛋白中的糖蛋白进行了初步分析， 发 现糖蛋白上有多价 Sia 或 GlcNAc、 末端 α -1， 3 man, GalNAc和 Gal β l-4GlcNAc 糖链结构。然后应用高覆盖率凝集素芯片研究转录生长 因子活化的肝星状细胞（LX-2 ) 中糖蛋白糖链谱的变化， 发现： AAL, PHA-E (菜豆凝集素 -E) 和 ECA识别的 Fuc、 N_GlcNAc和 Gal糖链结 构在活化态细胞的胞膜上表达明显增强。应用芯片技术研究 CC14诱导 小鼠肝纤维化模型中糖基因表达与肝细胞表面糖链谱之间的关联性。
发现纤维化肝脏中有 10个糖基转移酶基因转录水平发生改变。 首次 揭示在肝纤维化发生过程中 ^Glycan的合成通路" Tn antigen → T antigen (core-1) → sialyl-T antigen"被激活。 从糖组学角度揭 示了肝纤维化形成过程中蛋白糖基化发生变化的分子机理。 综上所述， 本发明拟从 "乙肝一乙肝后肝硬化一肝癌"进展的三 个阶段， 利用已建立的糖组学技术和方法， 寻找乙肝、 乙肝后肝硬化 和肝癌患者唾液中共有的和特有的糖蛋白糖链；将为非损伤性鉴别肝 炎、 肝硬化与肝癌提供新方法。 发明内容 The application number is 201110021447. 3 invention patent, we used the chip spotter to prepare the lectin on the epoxy-derived slide carrier, and fixed the high-coverage lectin chip. The specific binding of glycoprotein sugar chains to lectins in clinical samples was studied by lectin chip technology, and the glycoprotein sugar chain profiles and the identified lectins in various samples were analyzed. The gene chip data analysis method was used to analyze the expression profile of glycoprotein sugar chain, and the difference structure of glycoprotein sugar chain in clinical samples and normal control group was determined. First, we validated the feasibility and reliability of the lectin chip in detecting glycoprotein sugar chains using the standard glycoproteins RNase B and Fetuin, and applied glycoproteins in the total protein of Chang's liver normal hepatocytes using a lectin chip. A preliminary analysis revealed that the glycoprotein has polyvalent Sia or GlcNAc, terminal α-1, 3 man, GalNAc and Gal β l-4GlcNAc sugar chain structures. The high-coverage lectin chip was then used to study the changes in glycoprotein glycophores in transcriptional growth factor-activated hepatic stellate cells (LX-2), which were found to be: AAL, PHA-E (Cloth Bean-E) and ECA-recognized The expression of Fuc, N_GlcNAc and Gal sugar chain structure was significantly enhanced on the membrane of activated cells. The correlation between glycogene expression and hepatocyte surface glycan profile in liver fibrosis model induced by CC1 4 was studied by using chip technology. It was found that there were 10 levels of glycosyltransferase gene transcriptional changes in the fibrotic liver. It was first revealed that the synthesis pathway of Tly antigen → T antigen (core-1) → sialyl-T antigen was activated during the process of liver fibrosis. From the perspective of glycomics, the molecular mechanism of protein glycosylation changes during the formation of liver fibrosis was revealed. In summary, the present invention intends to use the established glycosics techniques and methods in three stages of progression of "hepatitis B-hepatitis B cirrhosis - liver cancer" to find common in saliva of patients with hepatitis B, hepatitis B and cirrhosis and liver cancer. And a unique glycoprotein sugar chain; will provide a new method for non-invasive identification of hepatitis, cirrhosis and liver cancer. Summary of the invention
本发明的目的在于提供一种基于唾液糖蛋白糖链鉴别肝系列病 的凝集素芯片及其应用， 其能非损伤， 快速鉴别唾液中糖蛋白糖链的 变化， 为非损伤性鉴别肝炎、 肝硬化与肝癌提供新方法。 本发明的技术解决方案是： 一种基于唾液糖蛋白糖链鉴别肝系列病的凝集素芯片，包括测试 凝集素探针组和对照探针， 其特殊之处在于： 所述测试凝集素探针组 至少包括 Jacalin、 GSL_II、 PTL_I、 SJA、 GSL_I、 LCA、 Con_A、 PTL_II、 DSA、 WA、 GNA、 PHA_E+L、 MAL_II、 AAL、 PSA、 WGA以及 UEA-I凝集 素探针的组合。 The object of the present invention is to provide a lectin chip for identifying liver diseases based on salivary glycochain and its application, which can non-injury, rapidly identify changes in glycoprotein sugar chains in saliva, and identify non-invasive hepatitis and liver. Hardening and liver cancer provide new methods. The technical solution of the present invention is: a lectin chip for identifying a liver-series disease based on a salivary glycochain, comprising testing a lectin probe set and a control probe, which are special in: the test lectin probe The group includes at least a combination of Jacalin, GSL_II, PTL_I, SJA, GSL_I, LCA, Con_A, PTL_II, DSA, WA, GNA, PHA_E+L, MAL_II, AAL, PSA, WGA, and UEA-I lectin probes.
一种上述凝集素芯片的应用， 其特征在于： 用于检测唾液中糖蛋 白及糖链的变化。
上述测试凝集素探针组还包括 WFA、 LTL、 LEL、 EEL以及 PWM凝 集素探针的组合。一种上述凝集素芯片的应用， 其特征在于： 用于检 测唾液中糖蛋白及糖链的变化。 An application of the above lectin chip, characterized in that it is used for detecting changes in glycoproteins and sugar chains in saliva. The above test lectin probe set also includes a combination of WFA, LTL, LEL, EEL, and PWM lectin probes. An application of the above lectin chip, characterized in that it is used for detecting changes in glycoproteins and sugar chains in saliva.
上述测试凝集素探针组还包括 PHA-E、 PNA、 MPL、 DBA, NPA、 BS_I 以及 SNA凝集素探针的组合。 一种利用上述凝集素芯片制备检测唾液中糖蛋白及糖链的变化 的试剂盒。 The above test lectin probe set also includes a combination of PHA-E, PNA, MPL, DBA, NPA, BS_I and SNA lectin probes. A kit for detecting changes in glycoproteins and sugar chains in saliva using the above lectin chip.
一种上述凝集素芯片的应用， 其特征在于： 用于检测唾液中糖蛋 白及糖链的变化。 An application of the above lectin chip, characterized in that it is used for detecting changes in glycoproteins and sugar chains in saliva.
一种基于唾液糖蛋白糖链鉴别肝系列病的凝集素芯片，包括测试 凝集素探针组和对照探针， 其特殊之处在于： 所述测试凝集素探针组 包括 I、 II及 III组，所述 I、 II及 III组分别是 Jacal in、GSL-II、PTL-I、 SJA、 GSL-I , IXA、 Con_A、 PTL-II , DSA、 WA、 GNA、 PHA - E+ MAL-II , AAL、 PSA、 WGA以及 UEA-I凝集素探针的组合， W 、 LTL、 LEU EEL 以及 PWM凝集素探针的组合以及 PHA-E、 PNA、 MPL、 DBA, NPA、 BS-I 以及 SNA凝集素探针的组合。 一种利用上述凝集素芯片制备检测唾液中糖蛋白及糖链的变化 的试剂盒。 A lectin chip for identifying a liver-series disease based on a salivary glycoprotein sugar chain, comprising testing a lectin probe set and a control probe, the special one being: the test lectin probe set comprises groups I, II and III The groups I, II and III are Jacal in, GSL-II, PTL-I, SJA, GSL-I, IXA, Con_A, PTL-II, DSA, WA, GNA, PHA-E+ MAL-II, AAL , PSA, WGA, and UEA-I lectin probe combinations, W, LTL, LEU EEL, and PWM lectin probe combinations, as well as PHA-E, PNA, MPL, DBA, NPA, BS-I, and SNA agglutinin A combination of needles. A kit for detecting changes in glycoproteins and sugar chains in saliva using the above lectin chip.
一种上述凝集素芯片的应用， 其特征在于： 用于检测唾液中糖蛋 白及糖链的变化。 An application of the above lectin chip, characterized in that it is used for detecting changes in glycoproteins and sugar chains in saliva.
本发明的有益效果是： 能非损伤， 高通量， 快速鉴别唾液中糖蛋 白及糖链的变化， 为非损伤性鉴别肝炎、 肝硬化与肝癌提供新方法。
附图说明 图 1为凝集素芯片上凝集素探针布局图； The beneficial effects of the invention are: non-injury, high-throughput, rapid identification of changes in glycoproteins and sugar chains in saliva, providing a new method for non-invasive identification of hepatitis, cirrhosis and liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a layout diagram of a lectin probe on a lectin chip;
图 2为健康人， 乙肝， 肝硬化和肝癌患者唾液糖蛋白糖链荧光检 测结果。 Figure 2 shows the results of fluorescence detection of salivary glycoproteins in healthy people, hepatitis B, cirrhosis and liver cancer.
图 3为唾液芯片验证结果。 A为唾液芯片布局图， N1-N7为健康 人唾液， HB1-HB6 为乙肝患者唾液， HC1-HC7 为肝硬化患者唾液， HCC1-HCC7为肝癌患者唾液。 B为凝集素 LTL与唾液芯片结合的荧光 检测结果图。 C为 LTL对应健康人、 乙肝、 肝硬化和肝癌患者唾液结 合强度比较。 Figure 3 shows the results of saliva chip verification. A is a saliva chip layout, N1-N7 is healthy human saliva, HB1-HB6 is saliva of hepatitis B patients, HC1-HC7 is saliva of patients with liver cirrhosis, and HCC1-HCC7 is saliva of liver cancer patients. B is a graph showing the results of fluorescence detection of lectin LTL combined with a saliva chip. C is a comparison of saliva binding strength of LTL corresponding to healthy people, hepatitis B, cirrhosis and liver cancer patients.
具体实施方式 detailed description
1、 实验部分 1. 1 试剂与材料 1. Experimental part 1. 1 Reagents and materials
甘氨酸， 磷酸氢二钠， 磷酸二氢钠， 吐温 -20和蛋白酶抑制剂均购 自美国 Sigma-Aldrich公司。 牛血清白蛋白 (BSA)购自德国 Merck公 司。 Cy3荧光均购自美国 Amerhsma公司。 T-PER动物组织蛋白提取 试剂购自美国 PIERCE公司。其他化学试剂均为分析纯级别， 在使用 前没有经过进一步纯化。所有实验用水都为经 Milli-Q50纯水***（美 国 Millipore 公司） 处理的超纯水。 Sephadex G-25 柱脱盐购自美国 GE Healthcare公司。 芯片杂交盒购自美国 Bio-Rad伯乐公司。 PVDF 膜购自美国 Millipore公司。 其它常见玻璃器皿均为国产。 37种凝集 素 （具体名称见表 1 ) 分别购自 Vector实验室， 美国 Sigma-Aldrich
公司和德国 Calbiochem公司。 表 1 凝集素具体名称表 Glycine, disodium hydrogen phosphate, sodium dihydrogen phosphate, Tween-20 and protease inhibitors were purchased from Sigma-Aldrich, USA. Bovine serum albumin (BSA) was purchased from Merck, Germany. Cy3 fluorescence was purchased from Amerhsma, USA. The T-PER animal tissue protein extraction reagent was purchased from PIERCE, USA. All other chemical reagents were of analytical grade and were not further purified prior to use. All experimental water was ultrapure water treated with a Milli-Q50 pure water system (Millipore, USA). Sephadex G-25 column desalting was purchased from GE Healthcare, USA. The chip hybridization kit was purchased from Bio-Rad Bole Company, USA. PVDF membranes were purchased from Millipore, USA. Other common glassware are made in China. 37 lectins (see Table 1 for specific names) were purchased from Vector Laboratories, Sigma-Aldrich, USA The company and the company Calbiochem, Germany. Table 1 List of specific names of lectins
縮略语 英文全称 中文全称 Abbreviation Full name in English Chinese full name
Jacalin Artocapus integrifolia 木菠萝凝集素Jacalin Artocapus integrifolia
ECA Erythrina cristagalli Letin 鸡冠刺桐凝集素ECA Erythrina cristagalli Letin cockscomb lectin
HHL Hippeastrum Hybrid Lectin 孤挺花凝集素HHL Hippeastrum Hybrid Lectin Amaryllis Lectin
WFA Wisteria Floribunda Agglutinin 紫藤花凝集素WFA Wisteria Floribunda Agglutinin Wisteria Lectin
GSL- II Griffonia Simplicifolia Lectin II 加纳子凝集素—ΠGSL- II Griffonia Simplicifolia Lectin II Ghana sub-agglutinin - Π
MAL- II Maackia Amurensis Lectin II 马鞍树凝集素- IIMAL- II Maackia Amurensis Lectin II Saddle Tree Lectin - II
PHA-E Phaseolus vulgaris Agglutinin(E) 菜豆凝集素 -EPHA-E Phaseolus vulgaris Agglutinin(E) Bean Lectin-E
PTL- I Psophocarpus Tetragonolobus Lectin I 四棱豆凝集素- IPTL- I Psophocarpus Tetragonolobus Lectin I Winged Bean Lectin - I
SJA Sophora Japonica Agglutinin 槐凝集素SJA Sophora Japonica Agglutinin 槐 lectin
PNA Peanut Agglutinin 花生凝集素PNA Peanut Agglutinin Peanut Agglutinin
EEL Euonymus Europaeus Lectin 欧洲卫矛凝集素EEL Euonymus Europaeus Lectin European Euonymus Lectin
AAL Aleuria Aurantia Lectin 橙黄网胞盘菌凝集素AAL Aleuria Aurantia Lectin Phytophthora aureus lectin
LTL Lotus Tetragonolobus Lectin 四棱莲凝集素LTL Lotus Tetragonolobus Lectin
MPL Madura Pomifera Leetin 橙桑凝集素MPL Madura Pomifera Leetin Orange Mulberry Lectin
LEL Lycopersicon 番 凝集素 LEL Lycopersicon
Eseulentum(Tomato)Lectin Eseulentum (Tomato) Lectin
GSL- I Griffonia SimPlicifolia Lectin I 加纳子凝集素 I GSL- I Griffonia SimPlicifolia Lectin I Ghana sub-agglutinin I
DBA Dolchos Biflorus Agglutinin 双花扁豆凝集素DBA Dolchos Biflorus Agglutinin Double Flower Lentil Agglutinin
LCA Lens Culinaris Agglutinin 扁豆凝集素LCA Lens Culinaris Agglutinin Lentil agglutinin
RCA 120 Ricinus Communis Agglutinin I 蓖麻凝集素 RCA 120 Ricinus Communis Agglutinin I Ricin agglutinin
STL Solanum Tuberosum(Potato)Lectin 马铃薯凝集素 STL Solanum Tuberosum (Potato) Lectin Potato Lectin
BS- I Bandeiraea simplicifolia Lectin I 西非单叶豆凝集素 - IBS-I Bandeiraea simplicifolia Lectin I West African Single Leaf Lectin - I
ConA Canavalia ensiformis Agglutinin 伴刀豆凝集素ConA Canavalia ensiformis Agglutinin with concanavalin
PTL- II Psophocarpus Tetragonolobus Lectin II 四棱豆凝集素 - IIPTL- II Psophocarpus Tetragonolobus Lectin II Winged Bean Lectin - II
DSA Datura stramonium Agglutinin 曼陀罗凝集素
SBA Soybean Agglutinin 大豆凝集素DSA Datura stramonium Agglutinin Mandala Lectin SBA Soybean Agglutinin Soy Lectin
VVA Vicia Villosa Agglutinin 蚕豆凝集素VVA Vicia Villosa Agglutinin Broad Bean Lectin
NPL Narcissus Pseudonarcissus Lectin 水仙花凝集素NPL Narcissus Pseudonarcissus Lectin Narcissus Lectin
PSA Pisum Sativum Agglutinin 豌豆凝集素PSA Pisum Sativum Agglutinin Pea Agglutinin
ACA Amaranthus caudatus Agglutinin 尾穗觅凝集素ACA Amaranthus caudatus Agglutinin
WGA Triticum vulgaris Agglutinin 麦胚凝集素WGA Triticum vulgaris Agglutinin Wheat Germ Agglutinin
UEA- I Ulex Europaeus Agglutinin I 荆豆凝集素- 1UEA- I Ulex Europaeus Agglutinin I Jingdou Lectin - 1
PWM Phytolacca americana Agglutinin 美洲商陆凝集素PWM Phytolacca americana Agglutinin American Pokeweed
MAL- I Maackia Amurensis Lectin I 马鞍树凝集素- 1MAL- I Maackia Amurensis Lectin I Saddle Tree Lectin - 1
GNA Galanthus nivalis Agglutinin 雪莲花凝集素GNA Galanthus nivalis Agglutinin Snowdrop Lectin
BPL Bauhinia Purpurea Lectin 羊蹄甲凝集素BPL Bauhinia Purpurea Lectin
PHA-E+L Phaseolus vulgaris Agglutinin(E+L) 菜豆凝集素 -E+LPHA-E+L Phaseolus vulgaris Agglutinin (E+L) Bean agglutinin-E+L
SNA Sambucus Nigra Agglutinin 西洋接骨木凝集素 SNA Sambucus Nigra Agglutinin Western elder agglutinin
1.2 实验仪器 1.2 Experimental instruments
电热鼓风干燥箱： 天津泰斯特公司； 高压灭菌锅： 日本 TOMY公 司； 超速冷冻离心机 5804R: 德国 Eppendorf公司； 微量核酸蛋白测 定仪： 德国 Implen公司； 生物芯片扫描仪 4000B: 美国 Axon公司； 芯片点样仪：博奥晶芯 SmartArrayer48点样仪；芯片杂交箱 HL-2000: 美国 UVP公司。 Electric blast drying oven: Tianjin Taisite Company; Autoclave: Japan TOMY company; Ultra-speed refrigerated centrifuge 5804R: Germany Eppendorf company; Micronucleic acid protein analyzer: Germany Implen company; Biochip scanner 4000B: American Axon company Chip spotter: Boao crystal core SmartArrayer48 spotting instrument; chip hybrid box HL-2000: US UVP company.
1.3 研究人群和全唾液采集 1.3 Study population and total saliva collection
志愿者无其它疾病， 一周之内没有服用任何药物。 已确诊的乙肝 患者 36例， 乙肝后肝硬化患者 27例和乙肝致肝癌患者 27例。 由于 肝病患者平均年龄为 57-65岁， 20例健康志愿者平均年龄为 62岁。 每组中 A, B, AB和 0型血型的个体比例一致， 以保证唾液糖蛋白糖
链不受血型的影响。 饭后两小时， 约 9点到 10点之间， 生理盐水漱 口三次后迅速采集自然分泌的全唾液。 唾液采集至少 1 ml并立即置 于冰上， 加入蛋白酶抑制剂 （每毫升唾液加入 ΙμΙ^) 防止蛋白降解。 Volunteers had no other illnesses and did not take any medications within a week. 36 patients with confirmed hepatitis B, 27 patients with post-hepatitis B cirrhosis and 27 patients with hepatitis B-induced liver cancer. Since the average age of patients with liver disease is 57-65 years, the average age of 20 healthy volunteers is 62 years. The proportion of individual A, B, AB and 0 blood types in each group is the same to ensure salivary glycoprotein sugar The chain is not affected by blood type. Two hours after the meal, between 9:00 and 10:00, the natural saliva was quickly collected after three times of normal saline gargle. Collect at least 1 ml of saliva and immediately place on ice, add protease inhibitor (add ΙμΙ^ per ml of saliva) to prevent protein degradation.
表 2 乙肝患者， 乙肝后肝硬化患者和肝癌患者情况表 Table 2 Hepatitis B patients, post-hepatitis B cirrhosis patients and liver cancer patients
附注表 1 : ALT, 丙氨酸转氨酶； AST, 天冬氨酸转氨酶。 Note Table 1: ALT, alanine aminotransferase; AST, aspartate aminotransferase.
1.4 唾液蛋白处理和荧光标记
收集到的全唾液经 12 000 rpm 4 °C离心 10 min后吸取上清弃去 不溶沉淀物。上清再经 0.22 μπι孔径的滤膜过滤掉细菌和其他微生物。 为了减小个体差异并归一化个体样本， 按照不同分组每例样本各取 100 μΐ混合， BCA法蛋白定量。 混合样本经 Cy3荧光染料标记后用 Sephadex G-25除盐柱去掉游离荧光。 标记好的蛋白准备用于凝集素 芯片孵育。 个例样本用于唾液芯片的点制。 1.4 Salivary protein processing and fluorescent labeling The collected total saliva was centrifuged at 12 000 rpm for 4 min at 4 ° C, and the supernatant was aspirated to discard the insoluble precipitate. The supernatant was then filtered through a 0.22 μm pore size filter to remove bacteria and other microorganisms. In order to reduce individual differences and normalize individual samples, 100 μM of each sample was mixed according to different groups, and BCA protein was quantified. The mixed sample was labeled with Cy3 fluorescent dye and the free fluorescence was removed using a Sephadex G-25 desalting column. The labeled protein is ready for lectin chip incubation. A sample of samples was used for the spotting of saliva chips.
1.5 凝集素芯片和数据分析 1.5 Lectin chip and data analysis
凝集素芯片的制备， Cy3 荧光标记的样本蛋白与凝集素芯片的孵 育步骤及凝集素芯片数据获取与归一化分析与之前专利申请号 201110021447.3发明专利描述一致。 The preparation of the lectin chip, the incubation procedure of the Cy3 fluorescently labeled sample protein and the lectin chip, and the data acquisition and normalization analysis of the lectin chip are consistent with the previous patent application No. 201110021447.3.
1.6唾液芯片的制备和数据分析 1.6 Preparation and data analysis of saliva chips
参见图 3， 唾液芯片由个例唾液样本点制而成， 样本根据健康人、 乙肝、 肝硬化和肝癌患者分成四组， 分别 7例， 6例， 7例和 7例。 Referring to Figure 3, the saliva chip was made from a sample of saliva samples. The samples were divided into four groups according to healthy people, hepatitis B, cirrhosis and liver cancer patients, 7 cases, 6 cases, 7 cases and 7 cases respectively.
Cy3 标记的凝集素用以检测这些唾液样本中凝集素特异识别的糖链 结构表达水平。 唾液蛋白溶于点样缓冲液 （0.5 mg/mL BSA溶于 l xPBS, pH 7.4 )中至终浓度 l mg/mL， 并用芯片点样仪点制于环氧化 修饰的玻片上。 唾液芯片的布局图显示在图 3中 A部分。 每个样本 区内重复三次，每张片基三个重复区。点好的片基在 50%湿度中孵育 过夜， 然后 37 °C真空干燥 3 h固定， 固定好的芯片可 4 °C避光密封 保存备用。芯片首先在封闭缓冲液中常温封闭 lh， l xPBST和 l xPBS 各清洗两次， 每次 5min， 离心甩干。 后加入配好的含 Cy3标记凝集 素的孵育缓冲液， 在芯片杂交箱内 4 rpm缓慢旋转 3h， l xPBST和
l xPBS各清洗两次， 每次 10min， 离心甩干。 通过芯片扫描仪设置光 电倍增管 70%和激光强度 100%, 在 532 nm波长处扫描芯片图像。 图像经 Genepix 3.0软件分析获取原始数据。 原始数据中小于两倍背 景标准偏差的值去掉，每张芯片上每个样本九个重复点的有效值再取 平均值 （As)， 每组平均值表示为组内每个样本平均值 （As) 的均值 (AG) ±标准偏差 （SDCJ)。 任意两组或多组间借助 SPSS statistics 19 软件进行 t检验或单方面方差分析（one-way ANOVA)寻找显著差异， 参见图 3中 B和 C部分。 Cy3-labeled lectins were used to detect the expression levels of glycoprotein structures specifically recognized by lectins in these saliva samples. Salivary proteins were dissolved in spotting buffer (0.5 mg/mL BSA in l x PBS, pH 7.4) to a final concentration of 1 mg/mL and plated on epoxidized modified slides using a chip spotter. The layout of the saliva chip is shown in section A of Figure 3. Repeat three times in each sample zone, with three replicate zones per base. The spotted substrate was incubated overnight at 50% humidity, then vacuum-dried at 37 °C for 3 h, and the fixed chip was sealed at 4 °C in the dark. The chip was first blocked in the blocking buffer at room temperature for 1 h, l xPBST and l xPBS were washed twice, each time for 5 min, centrifuged and dried. After adding the prepared incubation buffer containing Cy3 labeled lectin, slowly rotate 3 rpm in the chip hybridization box at 4 rpm, l xPBST and l XPBS was washed twice each time, 10 min each time, centrifuged and dried. The photomultiplier tube was set to 70% and the laser intensity was 100% by a chip scanner, and the chip image was scanned at a wavelength of 532 nm. The images were analyzed by Genepix 3.0 software to obtain raw data. The value of less than twice the background standard deviation in the original data is removed, and the effective value of nine replicate points per sample on each chip is averaged (A s ), and the average value of each group is expressed as the average value of each sample in the group ( Mean (A G ) ± standard deviation (SDCJ) of A s ). A significant difference was found between any two or more groups using the SPSS statistics 19 software for t-test or one-way ANOVA, see sections B and C of Figure 3.
2、 结果部分 2, the results section
2.1 乙肝、 肝硬化和肝癌患者唾液糖蛋白糖链谱的变化 2.1 Changes in salivary glycoprotein glycoprotein profiles in patients with hepatitis B, cirrhosis and liver cancer
利用凝集素芯片分别对健康志愿者、 乙肝、肝硬化和肝癌患者唾 液进行检测， 通过专业软件获取芯片数据并归一化处理后， 首先将三 组肝病结果与健康组结果进行比较，即每个凝集素对应的归一化后荧 光强度 （NFI) 在乙肝 （HB)、 肝硬化 （HC) 和肝癌组 （HCC) 分别 比健康组 （H) 得到 Fold-change值。 我们认为 Fold-change > 2 和 Fold-change < 0.5为肝病患者相较于健康人在唾液中上调和下调表达 的糖链。 The lectin chip was used to detect the saliva of healthy volunteers, hepatitis B, cirrhosis and liver cancer patients. After obtaining the chip data by professional software and normalizing the treatment, the results of the three groups of liver diseases were compared with the results of the health group. The normalized post-fluorescence intensity (NFI) of the lectin obtained Fold-change values in the hepatitis B (HB), cirrhosis (HC), and liver cancer (HCC) groups, respectively, compared to the healthy group (H). We believe that Fold-change > 2 and Fold-change < 0.5 are up-regulated and down-regulated in saliva by patients with liver disease compared to healthy people.
结果发现， 分别有 23种， 20种和 24种凝集素识别糖链在 HB、 HC和 HCC患者唾液中差异表达（表 3和图 2)。其中 Jacalin和 VVA 识别的 T/Tn抗原，唾液酸化 Τ/Τη抗原; PTL-L PTL-IL SJA和 GSL-I 等识别的 aGalNAc和 aGal (特别是末端） 糖链结构； LCA、 Con-A
和 GNA识别的高甘露糖， α-D-Man, Manal-3Man结构； 以及 GSL-Π 和 DSA识别的 β-D-GlcNA, (GlcNAcpi-4)n等在三种肝病患者唾液中 高表达。 而 AAL、 PSA 和 UEA-1 识别的核心岩藻糖， Fuca-N-acetylchitobiose-Man 和 Fucal-2Gaipi-4Glc(NAc)等结构； MAL-II识别的 Siaa2-3Gaipi-4Glc(NAc)/Glc; WGA识别的多价唾液 酸和 (GlcNAc)n结构等则在三种肝病患者唾液中低表达。 As a result, 23, 20, and 24 lectin-recognizing sugar chains were differentially expressed in saliva of HB, HC, and HCC patients, respectively (Table 3 and Figure 2). Among them, the T/Tn antigen recognized by Jacalin and VVA, the sialyl/Τη antigen; the aGalNAc and aGal (especially the terminal) sugar chain structures recognized by PTL-L PTL-IL SJA and GSL-I; LCA, Con-A High mannose, α-D-Man, Manal-3Man structure recognized by GNA; and β-D-GlcNA recognized by GSL-Π and DSA, (GlcNAcpi-4) n are highly expressed in saliva of three liver disease patients. The core fucose recognized by AAL, PSA and UEA-1, Fuca-N-acetylchitobiose-Man and Fucal-2Gaipi-4Glc (NAc); MAL-II recognized Siaa2-3Gaipi-4Glc(NAc)/Glc; The polyvalent sialic acid and (GlcNAc) n structure recognized by WGA are lowly expressed in the saliva of three liver disease patients.
除此之外， SNA识别的 Sia2-6Gal/GalNAc只在乙肝中高表达； PHA-E 识别的平分型 GlcNAc 和双天线 N-糖链， MPL 识别的 Gai 1 -3 GalNAc只在乙肝中低表达。 EEL识别的 Gala 1 -3 (Fuca 1 -2)Gal， PWM识别的分支型 （LacNAc)n只在肝癌中高表达； WFA识别的末 端 GalNAca/pi-3/6Gal， LTL识别的 Fucal-3(Gaipi-4)GlcNAc只在肝 癌中低表达。 In addition, SNA-recognized Sia2-6Gal/GalNAc is highly expressed only in hepatitis B; PHA-E recognizes the split-type GlcNAc and dual-antenna N-glycans, and MPL-recognized Gai 1 -3 GalNAc is only expressed in hepatitis B . Elan-recognized Gala 1 -3 (Fuca 1 -2)Gal, PWM-recognized branched (LacNAc) n is highly expressed only in liver cancer; WFA recognizes the terminal GalNAca/pi-3/6Gal, LTL recognizes Fucal-3 (Gaipi -4) GlcNAc is only expressed low in liver cancer.
2.2 乙肝、 肝硬化和肝癌患者唾液糖蛋白糖链谱之间的比较 2.2 Comparison of salivary glycoprotein glycoprotein profiles in patients with hepatitis B, cirrhosis and liver cancer
通过对乙肝、 肝硬化和肝癌唾液的凝集素芯片结果比较， 发现 PNA识别的 T抗原， MPL识别的 Gaipi-3GalNAc， NPA识别的高甘 露糖和 Manal-6Man在肝硬化患者唾液中的表达水平显著高于乙肝 患者，而 DSA和 WGA识别的多价唾液酸，（GlcNAc ^PfGlcNAcpi-^n 在肝硬化患者唾液中的表达水平显著低于乙肝患者。 PHA-E 识别的 平分型 GlcNAc和双天线 N-糖链， LEL识别的 (GlcNAc)n， WGA识 别的多价唾液酸， （GlcNAc ^PfGlcNAcpi-^在肝癌患者唾液中的表 达水平显著高于肝硬化患者 ， 而 WFA 识别 的末端 GalNAca/ i-3/6Gal, PNA识别的 T抗原， NPA识别的高甘露糖和
Manal-6Man, PSA识别的 Fuca-N-acetylchitobiose-Man在肝癌患者 唾液中的表达水平显著低于肝硬化患者。 By comparing the results of lectin microarray on hepatitis B, cirrhosis and liver cancer saliva, it was found that the T antigen recognized by PNA, the Gaipi-3GalNAc recognized by MPL, the high mannose and Manal-6Man recognized by NPA were significantly expressed in the saliva of patients with liver cirrhosis. Higher than hepatitis B patients, and DSA and WGA recognized polyvalent sialic acid, (GlcNAc ^ PfGlcNAcpi-^n expression level in saliva of patients with liver cirrhosis is significantly lower than that of hepatitis B patients. PHA-E recognition of bisecting GlcNAc and dual antenna N-glycans, LEL-recognized (GlcNAc) n , multivalent sialic acid recognized by WGA, (GlcNAc ^PfGlcNAcpi-^ is significantly higher in saliva of liver cancer patients than in patients with cirrhosis, and WFA recognizes the terminal GalNAca/i -3/6Gal, T-antigen recognized by PNA, high mannose and NPA-recognition Manal-6Man, PSA-recognized Fuca-N-acetylchitobiose-Man was significantly lower in saliva than liver cirrhosis patients.
表 3 : 乙肝、 肝硬化和肝癌患者唾液糖蛋白糖链谱的表达 Table 3: Expression of salivary glycoprotein glycoprotein profiles in patients with hepatitis B, cirrhosis and liver cancer
HB/N HC/N HCC/N HC/HB HCC/HC HB/N HC/N HCC/N HC/HB HCC/HC
T/Tn antigen, sialyl-T(ST), T/Tn antigen, sialyl-T(ST),
Jacalin 4.6 3.9 2.9 ― ― sialyl-Tn (STn) Jacalin 4.6 3.9 2.9 ― ― sialyl-Tn (STn)
WFA terminating in GalNAca/pi-3/6Gal ― ― 0.4 ― 0.3 WFA terminating in GalNAca/pi-3/6Gal ― ― 0.4 ― 0.3
GlcNAc and agalactosylated GlcNAc and agalactosylated
GSL-II 2.0 2.3 2.7 ― ― tri/tetra antennary glycans GSL-II 2.0 2.3 2.7 ― ― tri/tetra antennary glycans
Siaa2-3Gaipi-4Glc(NAc)/Glc, Siaa2-3Gaipi-4Glc(NAc)/Glc,
MAL-II Siaa2-3Gal, Siaa2-3, 0.4 0.4 0.5 ― ― MAL-II Siaa2-3Gal, Siaa2-3, 0.4 0.4 0.5 ― ―
Siaa2-3GalNAc Siaa2-3GalNAc
Bisecting GlcNAc, biantennary Bisecting GlcNAc, biantennary
PHA-E complex-type N-glycan with outer 0.5 ― ― ― 2.1 PHA-E complex-type N-glycan with outer 0.5 ― ― ― 2.1
Gal Gal
GalNAc, GalNAca-l,3Gal, GalNAc, GalNAca-l, 3Gal,
PTL-I 4.5 4.2 4.2 ― ― PTL-I 4.5 4.2 4.2 ― ―
GalNAca- 1 ,3ϋα1β- 1 ,3/4Glc GalNAca-1, 3ϋα1β-1, 3/4Glc
Terminal in GalNAc and Gal, Terminal in GalNAc and Gal,
SJA anti-A and anti-B human blood 2.2 2.5 2.6 ― ― group SJA anti-A and anti-B human blood 2.2 2.5 2.6 ― ― group
PNA Gaip 1 -3GalNAca-Ser/Thr(T) 0.1 ― 0.3 23.9 0.2 PNA Gaip 1 -3GalNAca-Ser/Thr(T) 0.1 ― 0.3 23.9 0.2
Gala 1 -3 (Fuca 1 -2)Gal (blood group Gala 1 -3 (Fuca 1 -2)Gal (blood group
EEL ― ― 2.4 ― ― EEL ― ― 2.4 ― ―
B antigen) B antigen)
Fuc l -6GlcNAc(core fucose), Fuc l -6GlcNAc (core fucose),
AAL 0.4 0.2 0.3 0.5 ― AAL 0.4 0.2 0.3 0.5 ―
Fucal-3(Gaipi-4)GlcNAc Fucal-3 (Gaipi-4) GlcNAc
LTL Fucal-3(Gaipi-4)GlcNAc ― ― 0.5 ― ― LTL Fucal-3(Gaipi-4)GlcNAc ― ― 0.5 ― ―
MPL Gaipi-3GalNAc 0.2 ― ― 4.8 MPL Gaipi-3GalNAc 0.2 ― ― 4.8
LEL (GlcNAc)n 0.2 0.5 ― ― 2.6 LEL (GlcNAc)n 0.2 0.5 ― ― 2.6
GSL-I aGalNAc, aGal, anti-A and B 2.3 2.2 2.6 ― ―GSL-I aGalNAc, aGal, anti-A and B 2.3 2.2 2.6 ― ―
DBA aGalNAc, Tn antigen ― 2.6 2.7 ― ― α-D-Man, Fuca-l,6GlcNAc, DBA aGalNAc, Tn antigen ― 2.6 2.7 ― ― α-D-Man, Fuca-l, 6GlcNAc,
LCA 3.1 5.1 5.0 ― ― a-D-Glc LCA 3.1 5.1 5.0 ― ― a-D-Glc
a-Gal, α-GalNAc, Gala-l,3Gal, a-Gal, α-GalNAc, Gala-l, 3Gal,
BS-I 100 ― ― 0.2 ― BS-I 100 ― ― 0.2 ―
Gala-l,6Glc Gala-l, 6Glc
High-Mannose, High-Mannose,
Con-A Mana 1 -6(Mana 1 -3)Man, terminal 2.4 3.1 3.6 ― ― Con-A Mana 1 -6 (Mana 1 -3) Man, terminal 2.4 3.1 3.6 ― ―
GlcNAc GlcNAc
PTL-II Gal, blood group H， T-antigen 2.0 2.5 2.1 ― ― β-D-GlcNA, (GlcNAcpi-4)n, PTL-II Gal, blood group H, T-antigen 2.0 2.5 2.1 ― ― β-D-GlcNA, (GlcNAcpi-4)n,
DSA 5.5 2.7 3.4 0.5 ― DSA 5.5 2.7 3.4 0.5 ―
Gaipi-4GlcNAc
terminal GalNAc, Gaipi-4GlcNAc Terminal GalNAc,
VVA GalNAca-Ser/Thr(Tn), 8.5 7.5 4.8 VVA GalNAca-Ser/Thr(Tn), 8.5 7.5 4.8
GalNAcal-3Gal GalNAcal-3Gal
NPA High-Mannose, Manal-6Man ― 8.14 2.83 7.2 0.4 NPA High-Mannose, Manal-6Man ― 8.14 2.83 7.2 0.4
PSA Fuca-N-acetylchitobiose-Man 0 0.2 0.1 100 0.2PSA Fuca-N-acetylchitobiose-Man 0 0.2 0.1 100 0.2
WGA Multivalent Sia and (GlcNAc)n 0.1 0.02 0.3 0.2 15.8WGA Multivalent Sia and (GlcNAc) n 0.1 0.02 0.3 0.2 15.8
UEA-I Fuca 1 -2Gaip 1 -4Glc(NAc) 0.4 0.2 0.4 UEA-I Fuca 1 -2Gaip 1 -4Glc(NAc) 0.4 0.2 0.4
PWM Branched (LacNAc)n ― ― 2.0 PWM Branched (LacNAc) n ― ― 2.0
GNA High-Mannose, Manal-3Man 3.9 3.9 3.3 GNA High-Mannose, Manal-3Man 3.9 3.9 3.3
Bisecting GlcNAc, bi-antennary Bisecting GlcNAc, bi-antennary
PHA-E+L N-glycans, tri- and tetra-antennary 100 100 100 PHA-E+L N-glycans, tri- and tetra-antennary 100 100 100
complex-type N-glycan Complex-type N-glycan
SNA Sia2-6Gal/GalNAc 2.3 ― ― SNA Sia2-6Gal/GalNAc 2.3 ― ―
附注： 表中数据表示该组的凝集素芯片结果中每个凝集素对应 NFI 相对于对照组 NFI的 Fold-change值。 HB, 乙肝； HC,肝硬化； HCC, 肝癌； -， 无显著差异； 100， Fold-change值的分母为零。 Note: The data in the table indicates the Fold-change value of each lectin in the lectin chip results for this group relative to the NFI of the control group. HB, hepatitis B; HC, cirrhosis; HCC, liver cancer; -, no significant difference; 100, the denominator of Fold-change value is zero.
本发明凝集素探针筛选的结果， 参见表 4， For the results of the screening of the lectin probe of the present invention, see Table 4.
表 4: Table 4:
Ml (确定患有肝病） HB HC HCC Ml (determined to have liver disease) HB HC HCC
Jacalin † † † Jacalin † † †
GSL-II † † † GSL-II † † †
PTL-I † † † PTL-I † † †
SJA † † † SJA † † †
GSL-I † † † GSL-I † † †
LCA † † † LCA † † †
Con-A † † † Con-A † † †
PTL-II † † †
DSA † † †PTL-II † † † DSA † † †
VVA † † †VVA † † †
GNA † † †GNA † † †
PHA-E+L † † †PHA-E+L † † †
MAL-II Ϊ Ϊ ΪMAL-II Ϊ Ϊ Ϊ
AAL Ϊ Ϊ ΪAAL Ϊ Ϊ Ϊ
PSA Ϊ Ϊ ΪPSA Ϊ Ϊ Ϊ
WGA Ϊ Ϊ ΪWGA Ϊ Ϊ Ϊ
UEA-I Ϊ Ϊ Ϊ UEA-I Ϊ Ϊ Ϊ
M3(M1后确定 HB和 HC) HB HC M3 (determined HB and HC after M1) HB HC
PHA-E Ϊ ― PHA-E Ϊ ―
PNA Ϊ ―PNA Ϊ ―
MPL Ϊ ―MPL Ϊ ―
DBA ― †
NPA ― † DBA ― † NPA ― †
BS-I † ― BS-I † ―
SNA † ― SNA † ―
2.3 凝集素芯片结果验证 2.3 Lectin chip results verification
为进一步验证凝集素芯片的结果， 验证凝集素探针与健康人、 乙 肝、 肝硬化和肝癌患者唾液样本中识别糖链的结合。 以凝集素 LTL 为例， 将 Cy3荧光标记的 LTL与点制好的唾液芯片孵育， 经芯片扫 描仪扫描 （见图 3B) 和 Genepix 3.0软件分析， 根据 1.6所述方法对 原始数据深入分析。结果显示， LTL与 7例肝癌唾液样本中识别糖链 的结合强度明显低于其与其它样本的结合（见图 3C),经 student t-test 发现 P值小于 0.05。其余凝集素探针验证方法参照此方法， 结果均显 示与凝集素芯片结果一致。证明这些凝集素可以作为探针通过对唾液 中的糖蛋白糖链的检测鉴别肝系列病。
To further validate the results of the lectin chip, the lectin probe was verified to recognize the binding of sugar chains in saliva samples from healthy people, hepatitis B, cirrhosis and liver cancer patients. Using lectin LTL as an example, Cy3 fluorescently labeled LTL was incubated with a spotted saliva chip, scanned by a chip scanner (see Figure 3B) and analyzed by Genepix 3.0 software, and the raw data was analyzed in depth according to the method described in 1.6. The results showed that the binding strength of LTL to the sugar chain of 7 liver cancer saliva samples was significantly lower than that of other samples (see Figure 3C), and the P value was less than 0.05 by Student t-test. The remaining lectin probe verification methods were based on this method, and the results were consistent with the results of the lectin chip. It has been demonstrated that these lectins can be used as probes to identify liver diseases by detecting glycoprotein sugar chains in saliva.
Claims
1、 一种基于唾液糖蛋白糖链鉴别肝系列病的凝集素芯片， 包括测试 凝集素探针组， 其特征在于： 所述测试凝集素探针组至少包括 1. A lectin chip for identifying a series of liver diseases based on salivary glycoprotein sugar chains, including a test lectin probe set, characterized in that: the test lectin probe set at least includes
Jacal in, GSL- II、 PTL- I、 SJA、 GSL- I、 LCA、 Con- A、 PTL- II、 DSA、 WA、 GNA、 PHA-E+ MAL_II、 AA PSA、 WGA以及 UEA-I凝集素探 针的组合。 Jacalin, GSL-II, PTL-I, SJA, GSL-I, LCA, Con-A, PTL-II, DSA, WA, GNA, PHA-E+ MAL_II, AA PSA, WGA and UEA-I lectin probes The combination.
2、 根据权利要求 1所述基于唾液糖蛋白糖链鉴别肝系列病的凝集素 芯片， 其特征在于： 所述测试凝集素探针组还包括 WFA、 LTL、 LEL、 EEL以及 PWM凝集素探针的组合。 2. The lectin chip for identifying liver diseases based on salivary glycoprotein sugar chains according to claim 1, characterized in that: the test lectin probe set also includes WFA, LTL, LEL, EEL and PWM lectin probes The combination.
3、 根据权利要求 1所述基于唾液糖蛋白糖链鉴别肝系列病的凝集素 芯片， 其特征在于： 所述测试凝集素探针组还包括 PHA-E、 PNA、 MPL、 DBA, NPA、 BS-I以及 SNA凝集素探针的组合。 3. The lectin chip for identifying liver diseases based on salivary glycoprotein sugar chains according to claim 1, characterized in that: the test lectin probe set also includes PHA-E, PNA, MPL, DBA, NPA, BS -I as well as a combination of SNA lectin probes.
4、 一种基于唾液糖蛋白糖链鉴别肝系列病的凝集素芯片， 包括测试 凝集素探针组和对照探针， 其特征在于： 所述测试凝集素探针组包括 4. A lectin chip for identifying a series of liver diseases based on salivary glycoprotein sugar chains, including a test lectin probe set and a control probe, characterized in that: the test lectin probe set includes
I、 II及 III组， 所述 I、 II及 III组分别是 Jacal in、 GSL- II、 PTL- 1、 SJA、 GSL-I , IXA、 Con_A、 ΡΊ1 - II、 DSA、 WA、 GNA、 PHA - E+ MAL-II , AAL、 PSA、 WGA以及 UEA-I凝集素探针的组合， W 、 LTL、 LEU EEL 以及 PWM凝集素探针的组合以及 PHA-E、 PNA、 MPL、 DBA, NPA、 BS-I 以及 SNA凝集素探针的组合。 Groups I, II and III, the I, II and III groups are Jacalin, GSL-II, PTL-1, SJA, GSL-I, IXA, Con-A, PΊ1-II, DSA, WA, GNA, PHA- E+ MAL-II, combination of AAL, PSA, WGA and UEA-I lectin probes, combination of W, LTL, LEU EEL and PWM lectin probes and PHA-E, PNA, MPL, DBA, NPA, BS- I and a combination of SNA lectin probes.
5、 一种权利要求 1所述凝集素芯片的应用， 其特征在于： 用于检测 唾液中糖蛋白糖链的变化。 5. An application of the lectin chip according to claim 1, characterized in that: used to detect changes in glycoprotein sugar chains in saliva.
6、 一种权利要求 2所述凝集素芯片的应用， 其特征在于： 用于检测
唾液中糖蛋白糖链的变化。 6. An application of the lectin chip according to claim 2, characterized in that: used for detection Changes in glycoprotein sugar chains in saliva.
7、 一种权利要求 3所述凝集素芯片的应用， 其特征在于： 用于检测 唾液中糖蛋白糖链的变化。 7. An application of the lectin chip according to claim 3, characterized in that: used to detect changes in glycoprotein sugar chains in saliva.
8、 一种权利要求 4所述凝集素芯片的应用， 其特征在于： 用于检测 唾液中糖蛋白糖链的变化。 8. An application of the lectin chip according to claim 4, characterized in that: used to detect changes in glycoprotein sugar chains in saliva.
9、 一种利用权利要求 2所述凝集素芯片制备检测唾液中糖蛋白糖链 的变化的试剂盒。 9. A kit using the lectin chip of claim 2 to prepare a kit for detecting changes in glycoprotein sugar chains in saliva.
10、一种利用权利要求 4所述凝集素芯片制备检测唾液中糖蛋白糖链 的变化的试剂盒。
10. A kit using the lectin chip of claim 4 to prepare a kit for detecting changes in glycoprotein sugar chains in saliva.
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| PCT/CN2014/070300 WO2015103743A1 (en) | 2014-01-08 | 2014-01-08 | Lectin chip for identifying liver series of disease based on salivary glycoprotein sugar chain and application thereof |
| CN201480058936.8A CN105849278A (en) | 2014-01-08 | 2014-01-08 | Lectin chip for identifying liver series of disease based on salivary glycoprotein sugar chain and application thereof |
| JP2016545829A JP2017502307A (en) | 2014-01-08 | 2014-01-08 | Lectin chip for identifying liver diseases based on glycoprotein sugar chains of saliva and use thereof |
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| PCT/CN2014/070300 WO2015103743A1 (en) | 2014-01-08 | 2014-01-08 | Lectin chip for identifying liver series of disease based on salivary glycoprotein sugar chain and application thereof |
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