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WO2012109977A1 - Complexe protéique et spectre et utilisations de celui-ci dans le diagnostic de maladies, l'évaluation de la progression de celles-ci, et évaluation de l'efficacité - Google Patents

Complexe protéique et spectre et utilisations de celui-ci dans le diagnostic de maladies, l'évaluation de la progression de celles-ci, et évaluation de l'efficacité Download PDF

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WO2012109977A1
WO2012109977A1 PCT/CN2012/071113 CN2012071113W WO2012109977A1 WO 2012109977 A1 WO2012109977 A1 WO 2012109977A1 CN 2012071113 W CN2012071113 W CN 2012071113W WO 2012109977 A1 WO2012109977 A1 WO 2012109977A1
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cancer
gel
protein complex
disease
benign
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Chinese (zh)
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李智立
王彦英
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Institute of Basic Medical Sciences of AMMS
Institute of Basic Medical Sciences of CAMS and PUMC
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Institute of Basic Medical Sciences of AMMS
Institute of Basic Medical Sciences of CAMS and PUMC
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    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • 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
    • G01N2333/4716—Complement proteins, e.g. anaphylatoxin, C3a, C5a
    • 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
    • G01N2333/775—Apolipopeptides

Definitions

  • the present invention relates to protein complexes and their profiles and their use in the diagnosis, progression assessment and efficacy evaluation of chronic diseases.
  • the present invention relates to human serum/plasma protein complexes and their separation profiles, and their use in inflammatory and/or immune-related chronic disease screening and/or prognosis assessment, and in the evaluation of drug efficacy, and in the use of Protein Complex Profile Diagnosis and/or Prognosis
  • cancer markers are mainly substances secreted or exfoliated into body fluids or tissues by diseases (such as cancer), or substances which are produced by the host in response to new substances in the body to enter and enter body fluids or tissues.
  • Clinically commonly used cancer markers such as prostate specific antigen PSA (can assist in the diagnosis of prostate cancer); alpha-fetoprotein AFP (can assist in the diagnosis of acute and chronic hepatitis, primary liver cancer, etc.); carcinoembryonic antigen CEA (in colon cancer, High expression in pancreatic cancer, gastric cancer, lung cancer and breast cancer); glycoprotein antigen CA125 (can assist in the diagnosis of ovarian cancer); CA19-9 (in pancreatic cancer, colon cancer, liver cancer, gastric cancer, gallbladder cancer, lung cancer and breast cancer, etc.) High expression); squamous cell-associated antigen SCC (can assist in the diagnosis of squamous cell carcinoma); cytokeratin 19 (combined with CEA can assist in the diagnosis of lung cancer) is a single protein
  • Serum tumour markers how to order and interpret them. BMJ. 2009, 339: 852-858. Hartwell, L. et Al. Cancer biomarkers: a systems approach. Nat Biotechnol. 2006, 24(8): 905-908.). By measuring the presence or amount of a marker, it can aid in diagnosing cancer, analyzing the course of the disease, guiding treatment, monitoring recurrence or metastasis, and judging prognosis. Cancer markers should have the following characteristics: high sensitivity, specificity, organ specificity, serum concentration related to tumor size and clinical stage, can reflect the dynamic changes of cancer, and the accuracy of the determination method is high. However, to date, no cancer markers have been found that meet these criteria.
  • Protein complexes are formed by the interaction of two or more polypeptide chains with non-covalent bonds. In the process of life activities, proteins mostly function through intermolecular interactions, which exist in the process of material metabolism, transfer, information transmission, etc. Different protein complexes have different biological functions (Babusiak, M ., et al. Native proteomic analysis of protein complexes in murine intestinal brush border membranes. Proteomics. 2007, 7 (1): 121-129. Protein complexes in the blood play important biological functions in regulating the process of blood coagulation and disease. For example, the alpha-fetoprotein-IgM complex is elevated in the serum of patients with primary hepatocellular carcinoma, and is detected by immunological methods.
  • This complex is useful for the diagnosis of primary hepatocellular carcinoma, and its sensitivity and specificity are superior to those of alpha-fetoprotein-IgM immune complexes on the diagnosis of primary (Jingting, J., et al. Hepatocellular carcinoma. J Clin Lab Anal. 2009, 23(4): 213-218.) Test results.
  • methods for detecting protein complexes in serum or plasma are primarily immunological methods, which have the disadvantage of being used only to determine known protein complexes.
  • Ideal disease markers should be applied to simple, non-invasive methods, using them or they can predict the risk of malignant diseases, early detection of malignant diseases and monitoring of disease duration; secondly, changes in markers and clinical phenomena Together, it provides new ideas for individualized treatment and drug development.
  • the technical object of the present invention is to investigate the use of human serum or plasma protein complexes and their profiles in the diagnosis and/or prognosis and/or molecular typing and/or evaluation of drug efficacy of chronic diseases.
  • a first aspect of the invention relates to a group of protein complexes having the general formula I:
  • the protein complex has a molecular weight ranging from 140 to 1400 kDa.
  • the protein complex has a formula selected from the group consisting of the following formula ⁇ to formula VII, wherein:
  • the above protein complex is used for the preparation of a reagent for diagnosing and/or prognosing human cancer, preferably the cancer is selected from the group consisting of lung cancer, colorectal cancer, pancreatic cancer, gastric cancer or thyroid cancer.
  • a second aspect of the invention relates to the use of a protein complex as described above for the preparation of a medicament for the diagnosis and/or prognosis assessment of human cancer, preferably selected from the group consisting of lung cancer, colorectal cancer, pancreatic cancer, gastric cancer or thyroid cancer.
  • a third aspect of the invention relates to a group of human serum or plasma protein complexes comprising a complement system protein, an immunoglobulin, an acute phase response protein and an apolipoprotein, the molecular mass of said protein complex
  • the range is 140 to 1400 kDa
  • the expression of the protein complex is composed of the following expression: [(HPT) (CFH) (C3) (IgGl) (IgAl)], [(HPT) (CFH) (C3) (C4A) (IgGl) (IgAl)], [(HPT) (C3) (C4A) (C5) (C7) (IgGl) (IgAl)], [(HPT) (C3) (C4A) (IgGl) (IgAl )], [(HPT) (C3) (C4A) (IgGl) (IgAl )], [(HPT) (C3) (C4A) (IgGl) (ApoA-I)
  • C4A stands for complement 4A
  • C5 stands for complement 5
  • C7 stands for complement 7
  • CFH stands for complement H
  • HPT haptoglobin
  • IgG1 stands for immunoglobulin G1
  • IgAl stands for immunoglobulin A1
  • TF stands for transferrin.
  • a fourth aspect of the invention relates to a non-denaturing polyacrylamide gel separation spectrum of a human serum protein complex as described in Figure 1 of the present specification, wherein a, b, c, d, e, f and g represent The main type of protein complex in different human serum, wherein the main protein complex distribution type is a, b, (!
  • the fifth aspect of the invention relates to a group of human serum protein complexes as shown in Figure 2 of the present specification
  • Non-denaturing polyacrylamide gel electrophoresis spectrum of the substance wherein the relationship between the protein complex strip number and the corresponding protein complex type and protein complex expression is as shown in Table 3 of the present specification, wherein the protein complex strip number
  • the main components in 1 ⁇ 18 are immune and/or inflammatory and/or complement system related proteins, which are called immune inflammation-related protein complexes, abbreviated as IRPPC.
  • Immunologically related proteins are closely related to changes in the immune system; inflammation-related proteins It is closely related to the occurrence of inflammation. Therefore, changes in the amount of these protein complexes can be used with immunity and/or inflammation.
  • the protein complex strip number 19 is composed of transferrin and apolipoprotein AI, and is called a transferrin protein-related protein complex, abbreviated as TRPC.
  • TRPC transferrin protein-related protein complex
  • the human serum or plasma protein complex according to the third aspect of the invention is used for the preparation of individualized diagnosis and/or individualized prognostic evaluation and/or individualized monitoring and/or molecular typing of human immunity and/or inflammation.
  • An agent for assessing the efficacy of a chronic disease and/or a drug, preferably, the immune and/or inflammation-related chronic disease From lung cancer, benign lung disease, colorectal cancer, benign colorectal disease, pancreatic cancer, benign pancreatic disease, gastric cancer, benign gastric disease, thyroid cancer, benign thyroid disease, type 2 diabetes or hypertension.
  • a sixth aspect of the invention relates to the preparation of individualized diagnosis and/or individualized prognostic and/or individualized monitoring and/or molecular typing human immunity and/or human serum or plasma protein complexes according to the above third aspect.
  • the immune and/or inflammation-related chronic disease is selected from the group consisting of lung cancer, benign lung disease, colorectal cancer, benign colorectal disease, pancreas Cancer, benign pancreatic disease, stomach cancer, benign gastric disease, thyroid cancer, benign thyroid disease, type 2 diabetes or high blood pressure.
  • a seventh aspect of the invention relates to a method of diagnosing and/or prognosing a human cancer, comprising the steps of:
  • the gel is stained by a gel staining method known in the art, and then the gel is scanned to obtain a gel electrophoresis pattern.
  • the gel staining method is silver staining or Coomassie blue staining;
  • step D) according to the above step C) gel strip in the electropherogram, the sample serum is classified according to the strip type in Fig. 1 of the specification of the present invention, wherein the molecular weight of the strip is marked from the largest to the smallest when labeling the gel strip The order starts from 1;
  • An eighth aspect of the invention relates to a method for detecting the diagnosis and/or prognosis of serum and plasma IIRPC and TRPC in a patient with a human immune and/or inflammation-related chronic disease (e.g., cancer), comprising the steps of:
  • step C) Coomassie brilliant blue or silver staining, scanning the gel to obtain a gel electropherogram; D) According to the gel strip in the electrophoresis pattern according to step C), the samples are classified according to the strip type in Fig. 1 of the specification of the present invention, and the gel strips are labeled in the order of the molecular weight from the largest to the smallest Start tag
  • the subject's health is judged according to the fold change in the amount of IIRPC in Tables 7 to 12 of the present specification.
  • the present invention has discovered a novel protein complex (IIRPC) and its spectrum in human serum or plasma, which include complement system proteins, immunoglobulins, acute phase response proteins and apolipoproteins.
  • the molecular weight of the IIRPC ranges from 140 to 1400 kDa, and the spectrum thereof is shown in Table 3 and Figure 1 of the specification of the present invention.
  • the main component of the protein complex strips 1 to 18 is an immune and/or inflammation-related protein, so it is called an immunoinflammation-related protein complex (IIRPC) o immune-related protein and immunity.
  • IIRPC immunoinflammation-related protein complex
  • Systemic changes are closely related; inflammation-related proteins are closely related to the development of inflammation. Therefore, changes in the amount of these protein complexes can be used for the diagnosis, prognosis assessment, and disease progression of chronic diseases associated with immunity and/or inflammation.
  • the IIRPC spectrum can classify the population into a large group, and the steroid IRPPS spectrum accounts for about 99% of the total population, wherein the type II population does not have the IRPPC, which accounts for about 13% of the total population, as shown in Figure 1 of the present specification. And Table 1 (continued).
  • the population can be subjected to individualized diagnosis of molecular typing, individualized diagnosis of immune-related chronic diseases, individualized treatment, and individualized disease progression.
  • the protein complex band number 19 is composed of transferrin and apolipoprotein AI, and is called a transferrin-related protein complex (TRPC), its content, its existence form and the sex of the individual, Age, health status is independent of the type of IRPPC, and it can be used as an internal reference for the quantitative analysis of IIRPC as described in the present invention.
  • TRPC transferrin-related protein complex
  • the IIRPC spectrum has the chronic disease selected from the group consisting of lung cancer and benign diseases thereof, colorectal cancer and benign diseases thereof, pancreatic cancer and benign diseases thereof, gastric cancer and benign diseases thereof, thyroid cancer and benign diseases thereof, type 2 diabetes and high Blood pressure is verified.
  • the presence of the amount of the IIRPC and its content change can distinguish five cancers (lung cancer, colorectal cancer, pancreatic cancer, gastric cancer, thyroid cancer), type 2 diabetes, hypertension, etc. and healthy controls; can distinguish cancer and corresponding thereto Benign disease. Therefore, the IIRPC and its spectrum in the present invention can be used for screening, early detection and early diagnosis of chronic diseases associated with inflammation and/or immunity.
  • the IIRPC profile is characterized in that the cancer is selected from the group consisting of lung cancer in prognostic assessment or disease progression monitoring and drug efficacy assessment.
  • prognostic assessment, progression monitoring, and assessment of drug efficacy can be performed using changes in the amount of IIRPC for immune and/or inflammation-related diseases.
  • the present invention also relates to a method for detecting and/or prognosing the detection of IIRPC and its spectrum and TRPC in serum or plasma of a patient with immune and/or inflammation-related chronic diseases such as human cancer, comprising the following steps:
  • step C According to the gel strip in the electrophoresis pattern according to step C), the samples are classified according to the strip type in Fig. 1 of the specification of the present invention, and the gel strips are labeled in the order of the molecular weight from the largest to the smallest Start tag
  • Figure 1 shows the results of separation of protein complexes in serum by a non-denaturing polyacrylamide gel, and gives the main 7 types of spectrum in which protein complexes are predominant.
  • Figure 2 shows the band of protein complexes to be separated and identified on a non-denaturing polyacrylamide gel.
  • Figure 3 shows the results of denaturing polyacrylamide gel separation of protein complexes of type a distribution in serum.
  • Figure 4 shows the results of denaturing polyacrylamide gel separation of protein complexes with b and e type distribution in serum.
  • Figure 5 shows the results of the separation of the protein complex of the d-type distribution in serum and the denatured polyacrylamide gel of TRPC.
  • Figure 6 Identification band showing the composition of the protein complex.
  • Figure 7 shows the amount of marker expressed in the sample analyzed and the ability to distinguish cancer patients.
  • Figure 8 shows the trends in the partial complexes during treatment of patients with non-small cell lung cancer. detailed description
  • the invention utilizes non-denaturing polyacrylamide gel electrophoresis (Native-PAGE) to separate protein complexes in serum or plasma, and is combined with denatured sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS).
  • -PAGE non-denaturing polyacrylamide gel electrophoresis
  • SDS denatured sodium dodecyl sulfate polyacrylamide gel electrophoresis
  • TRPC iron-protein-related protein complex
  • IIRPC immunoinflammatory-related protein complex
  • TRPC transferrin complex
  • Fig. 2 The present invention finds that the expression of the transferrin complex (TRPC) in Fig. 2 is relatively stable, and has nothing to do with age, sex, health status, and band type, and can be used as an internal reference for quantitative analysis of IIRPC in Native-PAGE. ; (3) normalize the optical density value of the 19th band (TRPC, Figure 2) in each person's Native-PAGE gel, and divide by the optical density value of each IIRPC gel strip, which is the protein. The relative expression level of the complex;
  • the present invention screens corresponding complexes according to different protein complex expression profile types and relative expression levels of IIRPC, and provides lung cancer, colorectal cancer, pancreatic cancer, gastric cancer, and thyroid gland. Range of changes in marker values for screening, early detection, assisted diagnosis, and/or prognostic assessment of chronic diseases such as cancer, type 2 diabetes, and hypertension.
  • the sample used in the present invention has a small serum volume (2 ⁇ , and the operation method is simple, easy, and inexpensive.
  • the present invention screens protein complex markers associated with chronic diseases such as tumors, and targets different protein complexes in tumor patients' serum.
  • the type of distribution, the selection of the corresponding IIRPC can effectively improve the sensitivity, specificity and accuracy of diagnosis of chronic diseases such as tumors, and can be used for disease progression monitoring, prognosis evaluation and guiding clinical rational drug use.
  • Protein complex separation method Discovery of protein complex spectrum, separation and identification of protein complex components
  • the corresponding benign diseases of gastric cancer and thyroid cancer were 60, 31, 52, 54 and 53 cases, respectively; 100 cases of diabetes and hypertension. See Table 1.
  • the Native-PAGE composition and electrophoresis conditions are as follows:
  • Composition 20 mM Tris-HCl (pH 7.5), 5 mM Magnesium Chloride , 0.1% by volume of tetramethylethylenediamine and 0.001 g/mL of ammonium persulfate.
  • the composition of the running buffer was: 25 mM Tris and 192 mM glycine solution.
  • Electrophoresis equipment DYCZ-24DN vertical electrophoresis instrument of Beijing Liuyi Instrument Factory. In this example, the glass plate parameters of Native-PAGE are 130mmx l00mmx l.5mm.
  • Sample processing method Whole blood at 3000 °g at 4 °C, centrifuge for 10 minutes, pipette the supernatant into a new centrifuge tube; centrifuge at 102000 xg for 10 minutes, dispense the small volume of the supernatant into a new centrifuge tube. Store in a -80 ° ⁇ refrigerator. Native knot s good - PAGE before each sample taken 2 and 8 1.25x loading buffer [25 mM
  • Tris (hydroxymethyl) aminomethane-salt enteric acid citrate (pH 6.8), 12.5% glycerol (volume ratio), xylene blue trace amount] was mixed and added to each well.
  • the same mixed healthy human serum was added to each gel as an external reference.
  • Electrophoresis conditions Set the constant current mode, continue to separate for 2 hours according to the current of each piece of glue, and then continue to separate for 3.5 hours according to the current of each piece of glue, and the whole separation process is carried out at 4 °C.
  • Mass spectrometry was performed using a mass spectrometer (Apex ultra MALDI-FTICR MS) manufactured by Bruker, with a laser wavelength of 355 nm. Mass spectrometry parameter setting: positive ion mode, acquisition range 800 ⁇ 4000 m/z (mass-to-charge ratio);
  • bands 9 and 12 are the beta and oc2 chains of haptoglobin, respectively; and band 13 is the alpha chain of complement C5.
  • Table 2 Identification results of protein complex components No. Protein name theory standard theoretical sequence score * Molecular weight isoelectric point coverage *
  • Serotransferrin 79280 6.81 57.00% 150 The serial number corresponds to the protein identification result of the band labeled in Figure 6.
  • the table compares the identification of protein bands with the corresponding polypeptide chains.
  • the composition of each protein complex in Fig. 2 is determined. According to the results of the identification of Table 2 and the SDS-PAGE separation of each protein complex, see Figures 3 to 5.
  • the composition of the protein complex is shown in Table 3.
  • the main components of the protein complexes 1 to 18 are immunological and/or inflammatory related proteins, so they are called immunoinflammation-related protein complex (IIRPC).
  • IIRPC immunoinflammation-related protein complex
  • 0 Immunologically related proteins and complement system-associated proteins are closely related to changes in the immune system; inflammation-associated proteins are closely related to the development of inflammation. Therefore, these changes in the amount of IIRPC can be used for the diagnosis, prognosis assessment, and disease progression of chronic diseases associated with immunity and/or inflammation.
  • Table 3 Strip number, type, composition and expression of protein complex
  • the protein complex strip number and type correspond to the protein complex number and type in Figure 2, respectively.
  • the composition of the protein complex in other IIRPC distribution types is the same as that of the a, b, d, and e positions.
  • Embodiment 2 Application Example 1
  • Variable lung cancer colorectal cancer pancreatic cancer gastric cancer thyroid cancer control health status ⁇ 0.394 0.401 0.836 0.105 0.129
  • Embodiment 3 (Application Example 2)
  • the normalized content after TRPC normalization (set to 100) is the internal reference, and the relative content of IRPPC (1-18, Fig. 2) is obtained.
  • Statistical analysis is performed on cancer, cancer-related benign diseases, and other chronic diseases (type 2 diabetes). And hypertension) differences in levels between patients and healthy controls.
  • the samples used in this step are all from the samples used in the above Application Example 1.
  • the number of specific samples used is 1620, including 378 healthy controls and 200 cases.
  • the c type Since the number of samples of d, e, f, g and other types is small, the c type has almost no target IIRCC band, so the main statistical analysis in the present invention is the content of IRPC in the types a and b, see Table 6. Table 6 Statistical characteristics of gender and age in different types of study samples*
  • the Mann-Whitney U test showed that there was no significant difference in the target IIRPC content between the case group and the healthy control group in terms of gender and age.
  • Statistical Analysis Differences in the levels of IIRPC in the a and b types were in the healthy controls, cancer groups, and corresponding benign disease groups.
  • the receiver operating characteristic curve was used to evaluate the diagnostic efficacy of the protein complex, and the area under the curve (AUC) within the 95% confidence interval (95% CI) was calculated.
  • the content of each IRPPC was significantly higher in the benign lung diseases and lung cancer patients than in the healthy control group ( ⁇ 0.01, Table 7).
  • the content of IRPPC in patients with benign disease is healthy control 1.54 ⁇ 3.42 times; the content of IRPPC in lung cancer patients is 4.33 ⁇ 18.60 times that of healthy controls.
  • the b2 ⁇ b6 complex in the b type has a higher diagnostic performance, which can better distinguish lung cancer from healthy controls and benign lung diseases (the area under the ROC curve is Between 0.95 and 0.99, Table 7).
  • each IIRPC was higher in patients with colorectal benign disease and colorectal cancer than in the healthy control group (Table 8).
  • the content of IIRPC in patients with benign disease was 1.14 ⁇ 2.35 times in healthy controls; the content of IIRPC in patients with colorectal cancer was 3.79 ⁇ 18.14 times in healthy controls.
  • the a ⁇ a9 complex, the b2 ⁇ b6 complex in the b type has a higher diagnostic performance, and can better distinguish colorectal cancer from healthy controls and benign bowel disease (under the ROC curve)
  • the area is between 0.93 and 1.00, Table 8).
  • the content of each IIRPC was significantly higher in the pancreatic benign disease and pancreatic cancer patients than in the healthy control group ( ⁇ 0.01, Table 9).
  • the content of IIRPC in patients with benign disease was 1.72 ⁇ 3.67 times in healthy controls; the content of IIRPC in patients with pancreatic cancer was 1.58 ⁇ 17.23 times in healthy controls.
  • the b2 ⁇ b6 complex in the b type has a higher diagnostic performance, which can better distinguish pancreatic cancer from healthy controls and benign pancreatic diseases (the area under the ROC curve is Between 0.92 and 0.99, Table 9).
  • the content of each IIRPC was higher in the benign gastric diseases and gastric cancer patients than in the healthy control group (Table 10).
  • the content of IIRPC in patients with benign disease was 1.11 ⁇ 1.93 times in healthy controls; the content of IIRPC in patients with gastric cancer was 2.26 ⁇ 28.29 times in healthy controls.
  • the b2 ⁇ b6 complex in the b type has a higher diagnostic performance, which can better distinguish gastric cancer from healthy controls and benign diseases of the stomach (the area under the ROC curve is Between 0.92 and 0.99, Table 10).
  • the content of each IIRPC was higher in the thyroid benign disease and thyroid cancer patients than in the healthy control group (Table 11).
  • the content of IIRPC in patients with benign disease was 1.02 ⁇ 1.89 times that of healthy controls; the content of IIRPC in patients with thyroid cancer was 2.01 ⁇ 3.99 times that of healthy controls.
  • the b2 ⁇ b6 complex in the b type has a higher diagnostic performance, which can better distinguish thyroid cancer from healthy controls and benign thyroid diseases (the area under the ROC curve is Between 0.74 and 1.00, Table 11).
  • the content of each IRPPC was significantly higher in the type 2 diabetic patients than in the healthy control group, which was 1.44 to 3.00 times the healthy control content (Table 12).
  • the content of IRPPC in hypertensive patients was higher than that in healthy controls, which was 1.23 ⁇ 3.87 times that of healthy controls.
  • a type of a1 ⁇ a8 complex, b type b2 ⁇ b6 complex has a certain diagnostic accuracy, can distinguish patients and healthy controls; in hypertensive patients, a type in a ⁇ a9
  • the complex, b2, b5 and b6 complexes of type b have a certain diagnostic accuracy and can distinguish between patients and healthy controls (Table 12).
  • the ⁇ P value is calculated by the Mann-Whitney U test.
  • the fold change is the median of each IRPPC content in patients with benign lung disease and lung cancer divided by the median of healthy controls.
  • the ⁇ P value is calculated by the Mann-Whitney U test.
  • the fold change is the median of each IRPPC content in patients with colorectal benign disease and colorectal cancer divided by the median of healthy controls.
  • the ⁇ P value is calculated by the Mann-Whitney U test.
  • the fold change is the median of each IRPPC content in patients with benign pancreatic disease and pancreatic cancer divided by the median of healthy controls.
  • the ⁇ P value is calculated by the Mann-Whitney U test.
  • the fold change is the median of the IRPPC-containing t in the benign gastric disease and gastric cancer patients divided by the median of the healthy control.
  • the ⁇ P value is calculated by the Mann-Whitney U test.
  • the fold change is the median of each IRPPC content in patients with thyroid benign disease and thyroid cancer divided by the median of healthy controls.
  • the ⁇ P value is calculated by the Mann-Whitney U test.
  • the fold change is the median of the IRPPC levels in patients with type 2 diabetes and hypertension divided by the median of healthy controls.
  • IIRPC can be used to distinguish between chronic diseases such as cancer and healthy controls, cancer and benign diseases, benign diseases and health controls, cancer and other chronic diseases such as type 2 diabetes and hypertension.
  • the present invention selects a2 and a7 in the complex type a, and the b3 and b4 markers in the type b as an example.
  • the content of complex a2 was 2.14, 2.15, 1.84, 5.74 times higher than that of healthy controls in patients with benign disease, type 2 diabetes, hypertension, and cancer, respectively; the content of a7 in the above case group was healthy control. 2.18, 2.22, 2.27 and B 8.22 times; a2 and a7 can better distinguish cancer patients from healthy controls, benign diseases, type 2 diabetes and hypertension patients (area under the ROC curve are 0.91 and 0.93, respectively)
  • IIRPC markers can better distinguish between disease and health controls, cancer and benign diseases and other chronic diseases.
  • Example 4 Using multivariate regression to determine the diagnostic ability of IIRPC using healthy controls and cancer samples in Example 3)
  • the ratio of the optical density value of the remaining 18 gel strips to the gel strip of the external reference is taken as the target IRPC in the gel.
  • the relative content ( ra tiol ⁇ ra ti 0 18), the case number, gender, age, band type, health status (control group and disease type) and ratiol ⁇ ratiol8 were entered into the excel table to establish the database.
  • SPSS 17.0 software statistical analysis data to screen risk factors associated with different disease types, and establish a risk assessment model. Analyze and compare the similarities and differences between different cancers and health care groups. Firstly, the normality test is performed on the 18 ratios of different samples. According to the distribution characteristics of the data, the appropriate statistical methods are used. The statistical methods used mainly include Mann- Whitney U or Kruskal-Wallis H test, Chi-Square test, Spearman rank-order correlation analysis and Binary logistic regression.
  • IIRPC distribution type a IIRPC 1-5, 8 in serum of patients with lung cancer (LC), colorectal cancer (CC), pancreatic cancer (PC), gastric cancer (SC) and thyroid cancer (TC)
  • LC lung cancer
  • CC colorectal cancer
  • PC pancreatic cancer
  • SC gastric cancer
  • TC thyroid cancer
  • IIRPC distribution type b the relative expression levels of IIRPC 5, 7, 9, 12, 14, 16, 18 in serum of patients with LC, CC, PC, SC and TC were significantly higher than those of the control group ( ⁇ 0.01).
  • Univariate logistic regression analysis was performed for different tumors compared with the control group, and multivariate logistic regression analysis was performed to screen the risk factors corresponding to different tumor patients.
  • Multi-factor logistic regression analysis According to the type of IRPC distribution of different tumors, it will pass Regression analysis was performed on the relative content of the differential gel strips after the initial screening in steps 2 and 3, and the risk factors were further screened to establish a regression model comparing different tumors with the control group. The effects of gender and age have been considered in the following equations.
  • IIRPC in the evaluation of prognosis or disease progression in cancer patients
  • the study subjects were 41 patients with non-small cell lung cancer treated with EGFR-targeted drug therapy (Table 14). Plasma samples from different treatment time points were collected and collected every two weeks, from the initial treatment of targeted therapy to the organization of patients. Learn the time of diagnosis of progressive disease (PD).
  • PD progressive disease
  • Example 2 Perform Native-PAGE electrophoresis according to the method of the above Example 1 (Preparation Example). Take 2 ⁇ L of each sample, and add the same mixed healthy human plasma to each gel as an external reference.
  • the distribution of IIRPC was mainly a, b and c, including 25 cases of a type, 11 cases of b type and 5 cases of c type.
  • the TRPC optical density values of different treatment time points in the same patient were analyzed by SPSS 12.0 software for one-way ANOVA. The results showed that there was no significant difference in TRPC content during the treatment (P ⁇ 0.05).
  • the relative amount of the protein complex in serum or plasma provided in the Native-PAGE gel is an indicator for early detection, auxiliary diagnosis and/or prognosis evaluation of inflammation and/or immune correlation such as cancer. Chronic disease and efficacy assessment, and can distinguish between health conditions.
  • the conventional gel electrophoresis used in the present invention can be replaced by capillary gel electrophoresis, and a picture reflecting the profile of the protein complex in human serum or plasma can be prepared according to the conditions of capillary electrophoresis, and the patient is The capillary gel electrophoresis pattern of the protein complex of the sample is compared with the picture to obtain the health status of the subject and the like.
  • the bands of protein complexes in serum or plasma according to the present invention exhibit different numbers or on gels due to differences in electrophoresis conditions or storage time of serum or plasma.
  • the migration distance but at least the technical information required to obtain the desired information, such as a2 or b3, as indicated in the above description of the present invention, further achieves the technical object of the present invention, and such a case falls within the scope of the claimed invention.

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Abstract

La présente invention concerne un complexe protéique présent dans le sérum ou le plasma humain, ainsi que le spectre de séparation de celui-ci, et ses utilisations dans le criblage et/ou l'évaluation du pronostic d'une maladie chronique d'origine immunitaire et/ou associée à une inflammation. L'invention concerne également l'évaluation de l'efficacité médicinale, ainsi qu'un procédé de détection destiné au diagnostic et/ou à l'évaluation du pronostic de maladies chroniques immunitaires et/ou associées à une inflammation chez l'homme en utilisant le spectre du complexe protéique.
PCT/CN2012/071113 2011-02-14 2012-02-14 Complexe protéique et spectre et utilisations de celui-ci dans le diagnostic de maladies, l'évaluation de la progression de celles-ci, et évaluation de l'efficacité Ceased WO2012109977A1 (fr)

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WO2006121952A2 (fr) * 2005-05-05 2006-11-16 The Regents Of The University Of California Biomarqueurs de diagnostic de troubles neurologiques du developpement
WO2009108073A1 (fr) * 2008-02-28 2009-09-03 Auckland Uniservices Limited Biomarqueurs pour la prédiction d’un éclampsisme et/ou d’une maladie cardiovasculaire
US20100129846A1 (en) * 2006-12-07 2010-05-27 Power3 Medical Products, Inc. Isoform specificities of blood serum proteins and their use as differentially expressed protein biomarkers for diagnosis of breast cancer
US20100167937A1 (en) * 2008-07-08 2010-07-01 Power3 Medical Products, Inc. Multiple forms of Alzheimer's disease based on differences in concentrations of protein biomarkers in blood serum

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006121952A2 (fr) * 2005-05-05 2006-11-16 The Regents Of The University Of California Biomarqueurs de diagnostic de troubles neurologiques du developpement
US20100129846A1 (en) * 2006-12-07 2010-05-27 Power3 Medical Products, Inc. Isoform specificities of blood serum proteins and their use as differentially expressed protein biomarkers for diagnosis of breast cancer
WO2009108073A1 (fr) * 2008-02-28 2009-09-03 Auckland Uniservices Limited Biomarqueurs pour la prédiction d’un éclampsisme et/ou d’une maladie cardiovasculaire
US20100167937A1 (en) * 2008-07-08 2010-07-01 Power3 Medical Products, Inc. Multiple forms of Alzheimer's disease based on differences in concentrations of protein biomarkers in blood serum

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