CN110736840A - Application of MMP7, CTSE or LAMC2 protein in preparation of intrahepatic cholangiocellular carcinoma diagnostic reagent - Google Patents
Application of MMP7, CTSE or LAMC2 protein in preparation of intrahepatic cholangiocellular carcinoma diagnostic reagent Download PDFInfo
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- CN110736840A CN110736840A CN201911002168.5A CN201911002168A CN110736840A CN 110736840 A CN110736840 A CN 110736840A CN 201911002168 A CN201911002168 A CN 201911002168A CN 110736840 A CN110736840 A CN 110736840A
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- 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
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- 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
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- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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Abstract
The invention relates to the technical field of medical bioengineering, and aims to provide application of MMP7, CTSE or LAMC2 protein in preparation of a reagent for diagnosing intrahepatic cholangiocellular carcinoma, wherein at least proteins are used for preparing a reagent capable of identifying and diagnosing intrahepatic cholangiocellular carcinoma and distinguishing primary hepatocellular carcinoma, namely, the identification and the distinguishing of hepatocellular carcinoma and intrahepatic cholangiocellular carcinoma are realized by using a single or combined use mode of the three proteins.
Description
Technical Field
The invention belongs to the technical field of medical bioengineering, and particularly relates to application of MMP7, CTSE or LAMC2 protein in preparation of a diagnosis reagent for intrahepatic cholangiocellular carcinoma.
Background
The intrahepatic cholangiocellular carcinoma accounts for 10% -15% of primary malignant tumors of the liver, is the second most common primary liver cancer, has no obvious clinical symptoms at the early stage, develops to the late stage when most patients are diagnosed, and loses the chance of operation, the intrahepatic cholangiocellular carcinoma is not sensitive to chemotherapy, immunotherapy and radiotherapy, and the effective treatment method is surgical resection.
At present, the marker for early diagnosis of intrahepatic cholangiocellular carcinoma is clinically lacked. Although carbohydrate antigen 125, carbohydrate antigen 19-9, carcinoembryonic antigen and the like are clinically used as reference markers for diagnosing cholangiocellular carcinoma, the carbohydrate antigen, carbohydrate antigen 19-9, carcinoembryonic antigen and the like have high expression values in the ductal carcinoma, and have poor sensitivity and specificity. Therefore, if the reagent for detecting the intrahepatic cholangiocellular carcinoma can be prepared and provided, the reagent can provide great help for clinically and accurately identifying and diagnosing the intrahepatic cholangiocellular carcinoma and distinguishing the intrahepatic cholangiocellular carcinoma from the primary liver cancer.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects in the prior art and provides application of MMP7, CTSE or LAMC2 protein in preparation of a liver cholangiocellular carcinoma diagnostic reagent.
In order to solve the technical problem, the solution of the invention is as follows:
the application of MMP7, CTSE or LAMC2 proteins in preparing a diagnostic reagent for intrahepatic cholangiocellular carcinoma is provided, wherein at least proteins are used for preparing a reagent capable of differentially diagnosing intrahepatic cholangiocellular carcinoma and distinguishing primary hepatocellular carcinoma (namely, the method for distinguishing and distinguishing hepatocellular carcinoma from intrahepatic cholangiocellular carcinoma is realized by using , any two or three proteins singly or in combination).
The invention further provides the use of the reagent for detecting the MMP7, CTSE or LAMC2 protein expression level, which is to use the reagent for detecting at least proteins in the reagent to prepare the reagent kit for distinguishing the intrahepatic cholangiocellular carcinoma to distinguish the primary hepatocellular carcinoma.
In the invention, the reagent for specifically detecting the expression level of the MMP7 protein is an antibody of an anti-MMP 7 protein; the reagent for specifically detecting the expression level of the CTSE protein is an antibody against the CTSE protein; the reagent for specifically detecting the expression level of the LAMC2 protein is an antibody against LAMC2 protein.
In the present invention, the antibody is a monoclonal antibody or a polyclonal antibody.
The kit is a detection kit based on an immune principle, and comprises a monoclonal antibody or a polyclonal antibody of at least proteins in MMP7, CTSE or LAMC2, a solid phase carrier coated with the antibody, a detection antibody (secondary antibody) capable of combining with a detectable signal molecule, a chromogenic substrate or a luminescent substrate, and a reactant for reacting with the detection antibody and the signal molecule so as to realize qualitative and quantitative detection.
In the present invention, the detectable signal molecule is any of common in the art, such as horseradish peroxidase, alkaline phosphatase, glucose oxidase, β -D-galactosidase, urease, ruthenium complex.
In the present invention, the chromogenic substrate or the luminescent substrate is a reaction substrate for a detectable signal molecule; for example, the chromogenic substrate may be Tetramethylbenzidine (TMB), o-phenylenediamine (OPD) or p-nitrophenyl phosphate and the luminescent substrate may be luminol or tripropylamine.
Compared with the prior art, the invention has the beneficial effects that:
1.MMP7 and/or CTSE and/or LAMC2 protein can be used for preparing a reagent or a kit product, and the expression level of MMP7, CTSE and LAMC2 protein in blood is detected by adopting an immunology related method to be used as a basis for diagnosing intrahepatic cholangiocellular carcinoma markers.
2. The expression measurement of the three proteins is combined, so that the possibility of judging intrahepatic cholangiocellular carcinoma of the subject can be accurately and specifically provided for a doctor to carry out diagnosis and treatment schemes.
3. The expression measurement of the three proteins is combined, so that the possibility of judging benign biliary tract diseases for the testee can be accurately and specifically determined, and a basis is provided for a diagnosis and treatment scheme of steps by a doctor.
4. The expression measurement of the three proteins is combined, so that the possibility of judging the liver cell liver cancer of the tested person can be accurately and specifically provided for a doctor to carry out diagnosis and treatment schemes.
5. The expression measurement of the three proteins is combined, so that the possibility of judging the health state of the testee can be accurately and specifically provided for a doctor to make an -step diagnosis and treatment scheme.
Drawings
FIG. 1 shows the results of the detection of marker protein against MMP7 protein in the examples of the present invention.
FIG. 2 shows the results of detection of marker proteins against CTSE protein in the examples of the present invention.
FIG. 3 shows the results of the detection of a marker protein against LAMC2 protein in the examples of the present invention.
FIG. 4 is an operator characteristic curve (in a patient with intrahepatic cholangiocellular carcinoma VS healthy person) in an example of the present invention.
FIG. 5 is an operator's profile (in a patient with intrahepatic cholangiocellular carcinoma, VS hepatocellular carcinoma) in an example of the present invention.
FIG. 6 is an operator's profile (in patients with intrahepatic cholangiocellular carcinoma VS benign biliary disease) in an embodiment of the present invention.
In fig. 1-3, the dots are labeled 39 patients with intrahepatic cholangiocellular carcinoma, the squares are labeled 22 healthy persons, the triangles are labeled 20 patients with benign biliary tract disease, and the diamonds are labeled 17 patients with hepatocellular carcinoma.
Detailed Description
The MMP7 protein is shown in the literature to be matrix metalloproteases, the main role of which is extracellular matrix protein degradation, associated diseases including glioblastoma and spastic eyelid inversion, the CTSE protein is aspartic endopeptidases which are reported to be associated with gastrointestinal adenocarcinoma but not disease diagnosis, and the LAMC2 protein is laminin which can bind to heparin, and the function of which is related to cell migration and motility.
The applicant has further studied that MMP7 and/or CTSE and/or LAMC2 protein has high expression level in blood of a patient with intrahepatic cholangiocellular carcinoma, and the expression level is different from the protein level in blood of healthy people, benign biliary tract diseases and hepatocellular carcinoma patients according to a receiver operation characteristic curve (ROC curve), proteins or protein combinations can obtain high specificity and sensitivity, therefore, or more proteins can be combined into a marker for specific early diagnosis of intrahepatic cholangiocellular carcinoma based on the characteristic.
In the present invention, either monoclonal antibodies or polyclonal antibodies can be achieved by conventional techniques. Since the preparation of antibodies is within the skill of those skilled in the art, further description of the invention is omitted.
After obtaining reagents for specifically detecting the expression level of MMP7 protein or CTSE protein or LAMC2 protein, it is convenient to prepare an immunoassay kit for diagnosing intrahepatic bile duct cell carcinoma, which comprises, in addition to the corresponding antibodies, a detectable signal molecule and a detection antibody capable of binding to the detectable signal molecule, a monoclonal or polyclonal antibody for identifying specifically detecting MMP7 protein or CTSE protein or LAMC2 protein, and a reactant reactive with the detection antibody and the signal molecule.
Based on the present invention, various immunologically relevant methods can be used to detect the expression level of MMP7 protein or CTSE protein or LAMC2 protein in a sample, and these methods can all utilize the prior art. The present invention will not be described in detail as it is within the skills of a person skilled in the art.
The kit prepared by the invention can realize the diagnosis of intrahepatic cholangiocellular carcinoma, and the use method comprises the following steps:
1. the kit is used for detecting the expression level of MMP7, CTSE and LAMC2 proteins in a blood sample of a subject.
2. Analyzing the expression level of MMP7, CTSE and LAMC2 proteins to determine possible disease states:
if the expression of MMP7 protein is increased, the subject can be prompted to have intrahepatic cholangiocellular carcinoma.
If expression of the CTSE protein is increased, the pathological state of the intrahepatic bile duct can be suggested to the subject.
If the expression of LAMC2 protein is increased, the subject can be prompted to have tumor at intrahepatic bile duct.
The following describes the implementation of the present invention in detail with reference to the data of the specific embodiment. This example is intended to illustrate the invention only and not to limit the scope of the invention.
Example 1, 98 volunteers were selected in a hospital, including 39 patients with intrahepatic cholangiocellular carcinoma, 22 healthy people, 20 patients with benign biliary tract disease, and 17 patients with hepatocellular carcinoma. Biological samples were extracted from volunteers, characteristic parameters were recorded, and specific detection was performed using the kit. The characteristic parameters of the test groups are shown in table 1.
Table 1 shows the clinical characteristics of 39 patients with intrahepatic cholangiocellular carcinoma, 17 patients with hepatocellular carcinoma, and 20 patients with benign biliary tract disease.
(in the table: a Fisher's exact test; b chi-square test; c variance test)
The three proteins were detected using a commercially available antibody sandwich method, the sample was added to the antibody coated wells, incubated at 37 ℃ for 1 hour, biotinylated antibody (diluted 100 fold) was added, incubated at 37 ℃ for 1 hour again, washed, added to the detection solution, incubated at 37 ℃ for half an hour again, washed, added to the chromogenic substrate, developed at 37 ℃ away from light for 10 minutes, the reaction was stopped and the optical density was measured. Specific detection results are shown in fig. 1-3, which show the detection results of MMP7, CTSE, LAMC2 proteins in blood of 39 intrahepatic cholangiocellular carcinoma patients, 22 healthy persons, 20 benign biliary tract disease patients, and 17 hepatocellular carcinoma patients, and it can be seen from the receiver operating characteristic curve (ROC curve) of fig. 4-6 that MMP7, CTSE, LAMC2 proteins can effectively distinguish intrahepatic cholangiocellular carcinoma, hepatocellular carcinoma, benign biliary tract disease, and health status.
As can be seen from the above examples, the reagent according to the present invention can accurately and specifically predict the possibility of the patient suffering from intrahepatic cholangiocellular carcinoma, thereby confirming the need for further examinations and treatments.
Claims (7)
- The application of MMP7, CTSE or LAMC2 protein in the preparation of a reagent for diagnosing intrahepatic cholangiocellular carcinoma is characterized in that at least proteins are used for preparing a reagent capable of differentially diagnosing intrahepatic cholangiocellular carcinoma and distinguishing primary hepatocellular carcinoma.
- 2. The application of the reagent for specifically detecting the expression level of MMP7, CTSE or LAMC2 protein is characterized in that the reagent for specifically detecting at least proteins is used for preparing a kit capable of identifying intrahepatic cholangiocellular carcinoma to distinguish primary hepatocellular carcinoma.
- 3. The use of claim 2, wherein the agent for specifically detecting the expression level of MMP7 protein is an antibody against MMP 7; the reagent for specifically detecting the expression level of the CTSE protein is an anti-CTSE antibody; the reagent for specifically detecting the expression level of the LAMC2 protein is an antibody against LAMC2 protein.
- 4. The use of claim 3, wherein the antibody is a monoclonal antibody or a polyclonal antibody.
- 5. The use according to claim 2, wherein the kit is an immuno-principle based detection kit; the kit comprises:a monoclonal or polyclonal antibody to at least proteins of MMP7, CTSE, or LAMC 2;a solid support coated with an antibody;a detection antibody capable of binding a detectable signal molecule;the chromogenic substrate or the luminescent substrate is used for reacting with the detection antibody and the signal molecule to realize qualitative and quantitative detection.
- 6. The use according to claim 5, wherein the detectable signal molecule is any of horseradish peroxidase, alkaline phosphatase, glucose oxidase, β -D-galactosidase, urease, ruthenium complex.
- 7. Use according to claim 5, wherein the chromogenic substrate is tetramethylbenzidine, o-phenylenediamine or p-nitrophenylphosphate and the luminescent substrate is luminol or tripropylamine.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911002168.5A CN110736840A (en) | 2019-10-21 | 2019-10-21 | Application of MMP7, CTSE or LAMC2 protein in preparation of intrahepatic cholangiocellular carcinoma diagnostic reagent |
| CN202011130894.8A CN112129954B (en) | 2019-10-21 | 2020-10-21 | Application of MMP7, CTSE or LAMC2 protein in preparation of intrahepatic cholangiocellular carcinoma diagnostic reagent |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911002168.5A CN110736840A (en) | 2019-10-21 | 2019-10-21 | Application of MMP7, CTSE or LAMC2 protein in preparation of intrahepatic cholangiocellular carcinoma diagnostic reagent |
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| CN202011130894.8A Expired - Fee Related CN112129954B (en) | 2019-10-21 | 2020-10-21 | Application of MMP7, CTSE or LAMC2 protein in preparation of intrahepatic cholangiocellular carcinoma diagnostic reagent |
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| SG11201810670VA (en) * | 2016-06-14 | 2018-12-28 | Agency Science Tech & Res | Use of mi r-198 in treating and diagnosing cutaneous squamous cell carcinoma |
| WO2018020530A1 (en) * | 2016-07-25 | 2018-02-01 | 静岡県 | Diagnostic biomarker for extrahepatic bile duct cancer, intrahepatic bile duct cancer or gallbladder cancer |
| CN108267585A (en) * | 2017-12-28 | 2018-07-10 | 华中科技大学同济医学院附属协和医院 | Diagnosis of biliary atresia kit based on blood serum designated object MMP-7 |
| CN110736840A (en) * | 2019-10-21 | 2020-01-31 | 浙江大学 | Application of MMP7, CTSE or LAMC2 protein in preparation of intrahepatic cholangiocellular carcinoma diagnostic reagent |
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| CN112129954A (en) * | 2019-10-21 | 2020-12-25 | 杭州道合天孚生物科技有限公司 | Application of MMP7, CTSE or LAMC2 protein in preparation of intrahepatic cholangiocellular carcinoma diagnostic reagent |
| CN112129954B (en) * | 2019-10-21 | 2021-08-03 | 杭州道合天孚生物科技有限公司 | Application of MMP7, CTSE or LAMC2 protein in preparation of intrahepatic cholangiocellular carcinoma diagnostic reagent |
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