Kit for detecting metabolic activity of related enzyme and application thereof
阅读说明：本技术 一种相关酶代谢活性检测试剂盒及其应用 (Kit for detecting metabolic activity of related enzyme and application thereof ) 是由 乔海灵 方艳 于 2019-04-12 设计创作，主要内容包括：本申请公开了一种相关酶代谢活性检测试剂盒,包括所述相关酶对应的特异性探针,所述试剂盒用于检测相关酶代谢活性、检测至少两种相关酶代谢活性的比值中的至少一种。该试剂盒为肝癌高危人群的筛查及预测肝癌患者的预后提供了新的更准确、更灵敏的标准,对肝癌高危人群的筛查及肝癌防治具有重大的意义。本申请还提供了基于上述试剂盒的相关酶代谢活性及活性比值检测方法及该方法在筛查肝癌易感性和/或高危人群、肝癌患者预后评估中的应用,该应用准确性高、灵敏度优异,且易于操作,便于推广应用。(The application discloses a kit for detecting metabolic activity of related enzymes, which comprises specific probes corresponding to the related enzymes, and is used for detecting the metabolic activity of the related enzymes and at least one of the ratios of at least two related enzymes. The kit provides a new more accurate and sensitive standard for screening of high risk group of liver cancer and prediction of prognosis of liver cancer patient, and has great significance for screening of high risk group of liver cancer and prevention and treatment of liver cancer. The application also provides a detection method of the metabolic activity and the activity ratio of related enzymes based on the kit and application of the method in screening prognosis evaluation of liver cancer susceptibility and/or high risk groups and liver cancer patients, and the application has the advantages of high accuracy, excellent sensitivity, easy operation and convenient popularization and application.)
1. A kit for detecting metabolic activity of a related enzyme is characterized by comprising a specific probe corresponding to the related enzyme;
the kit is used for detecting the metabolic activity of related enzymes and detecting at least one of the ratios of the metabolic activities of at least two related enzymes.
2. The kit of claim 1, wherein the related enzymes comprise at least one of CYP2E1, CYP2C 8;
the CYP2E1 is cytochrome P450 enzyme 2E1, and the CYP2C8 is cytochrome P450 enzyme 2C 8;
preferably, the ratio of the metabolic activities of the at least two related enzymes comprises: at least one of the ratio of the metabolic activities of liver microsome CYP2E1 and CYP2C8, and the ratio of the metabolic activities of human CYP2E1 and CYP2C 8.
3. The kit according to claim 1 or 2, wherein the specific probe comprises at least one of a substance involved in CYP2E1 metabolism/transformation, a substance involved in CYP2C8 metabolism/transformation;
preferably, the specific probe comprises at least one of chlorzoxazone, diethylnitrosamine, 4-nitrophenol, aniline, paclitaxel, amodiaquine, repaglinide, montelukast sodium, rosiglitazone and pioglitazone.
4. The kit of claim 2, comprising:
an incubation system;
wherein the incubation system comprises specific probes corresponding to the related enzymes; or
The kit comprises at least one of 125-1200 mg of chlorzoxazone, 0.1-16 mg of repaglinide, 2-10 mg of montelukast sodium, 2-8 mg of rosiglitazone and 7.5-45 mg of pioglitazone;
preferably, the kit comprises 600mg of chlorzoxazone tablet and 4mg of repaglinide.
5. The kit according to claim 4, wherein the incubation system in step (1) comprises specific probes and buffers corresponding to the relevant enzymes;
the buffer solution is selected from one of 50-100 mM phosphate buffer solution and 50-100 mM Tris-HCL buffer solution;
preferably, the concentration of the buffer is 100 mM;
preferably, the pH of the buffer is 7.4;
preferably, the incubation system comprises 100mM phosphate buffer solution with pH7.4, and at least one of 2.5-80 mu M paclitaxel, 0.78-100 mu M diethylnitrosamine, 7.8-1000 mu M chlorzoxazone, 0.022-20 mu M repaglinide, 5-2000 mu M4-nitrophenol, 5-6000 mu M aniline, 0.1-200 mu M amodiaquine, 0.025-50 mu M montelukast, 0.25-1000 mu M rosiglitazone and 2.06-5272 mu M pioglitazone;
preferably, the incubation system further comprises one of 1mM NADPH or NADPH regeneration system;
wherein the NADPH regenerating system comprises 1.3mM NADP +,3.3mM 6-phosphoglucose, 0.4U/ml glucose dehydrogenase, 3.3mM magnesium chloride;
preferably, the kit further comprises:
terminating the system;
the termination system comprises at least one of ethyl acetate, dichloromethane, acetonitrile, methanol, perchloric acid;
further preferably, the termination system comprises at least one of 1mL ethyl acetate, 1mL dichloromethane, 100 μ L acetonitrile, 100 μ L methanol, 10 μ L perchloric acid;
still more preferably, the stop system comprises at least one of 1mL ethyl acetate, 1mL methylene chloride, 100 μ L acetonitrile.
6. The kit of claim 4, comprising:
(a) 2.5-80 mu M of paclitaxel, 0.78-100 mu M of diethylnitrosamine, 7.8-1000 mu M of chlorzoxazone, 0.022-20 mu M of repaglinide, 5-2000 mu M of 4-nitrophenol, 5-6000 mu M of aniline, 0.1-200 mu M of amodiaquine, 0.025-50 mu M of montelukast, 0.25-1000 mu M of rosiglitazone and 2.06-5272 mu M of pioglitazone;
(b) at least one of 50mM phosphate buffer, 100mM phosphate buffer, 50mM Tris-HCl buffer, and 100mM Tris-HCl buffer;
(c)1mM NADPH, NADPH regeneration system;
(d)1mL of ethyl acetate, 1mL of dichloromethane, 100. mu.L of acetonitrile, 100. mu.L of methanol, and 10. mu.L of perchloric acid.
7. A method for detecting metabolic activity of a related enzyme, comprising: measuring the metabolic activity of the related enzyme on a specific probe corresponding to the related enzyme;
the method employs at least one of the kits of any one of claims 1 to 6.
8. The method of claim 7, wherein the metabolic activity comprises at least one of a Michaelis constant, a maximum turnover rate, a clearance rate, a drug peak concentration, a time to peak, a terminal elimination rate, a half-life, an area under a drug time curve, a clearance rate, an apparent distribution volume, and an average residence time.
9. The method of claim 7, wherein the method comprises:
b) mixing at least one of a system containing related enzyme and a specific probe corresponding to the related enzyme with a buffer solution, pre-incubating, adding reduced coenzyme, incubating, terminating the reaction, and analyzing to obtain a substance to be detected to obtain the metabolic activity of the related enzyme;
the related enzyme comprises at least one of CYP2E1 and CYP2C 8;
preferably, the system comprising the relevant enzyme comprises liver microsomes and the human body;
preferably, the method of analysis comprises: measuring the amount of metabolites produced by chromatography, and calculating the metabolic activity of the relevant enzyme by a nonlinear regression analysis method;
preferably, the metabolic activities of at least two related enzymes are respectively obtained according to the method, and the metabolic activity ratio of different related enzymes is obtained through calculation;
preferably, the ratio of CYP2E1 and CYP2C8 metabolic activities is obtained by the method.
10. Use of the kit according to any one of claims 1 to 6 and/or the method according to any one of claims 7 to 9 for screening liver cancer susceptibility and/or high risk population, prognosis evaluation of liver cancer patients.
Technical Field
The application relates to a detection kit for metabolic activity of a related enzyme, a detection method for metabolic activity of the related enzyme based on the detection kit, and application of the detection kit in screening liver cancer susceptibility and/or high risk group and prognosis evaluation of liver cancer patients, belonging to the field of detection and medical health.
Background
Liver cancer is one of common malignant tumors in China, is a disease seriously harming the life safety of human beings, and has the characteristics of high malignancy, fast progress, strong invasiveness, poor prognosis, high mortality and the like. Recent data show that liver cancer is third-ranked in death due to malignancy worldwide. 13.5 thousands of patients with liver cancer newly increase in China each year, about 11 thousands of patients die, and account for 45 percent of the dead people with liver cancer all over the world.
The existing methods for treating liver cancer mainly comprise surgical resection, chemotherapy, radiotherapy and the like. Because liver cancer is found in middle and late stages clinically, the treatment method has been greatly developed, but the best treatment opportunity is missed when a liver cancer patient is in a clinic, and the prognosis of the current treatment method is poor. Therefore, early screening, early diagnosis and treatment are the key points for reducing the incidence rate of liver cancer and improving the survival rate of liver cancer patients. In addition, most liver cancer patients experience a pathological evolution process from chronic hepatitis to cirrhosis, eventually progressing to liver cancer. Therefore, screening and finding early liver cancer in high risk population has important significance for reducing social cost and medical cost.
At present, the clinically common indexes related to the early diagnosis and prognosis of liver cancer mainly include: alpha-fetoprotein (AFP), aspartate Aminotransferase (AST), Prothrombin Time (PT), glutamyl transpeptidase (GGT), Total Bilirubin (TBIL), and the like. However, the above indexes may be changed in patients with hepatitis, liver cirrhosis, and liver cancer, and chronic liver disease and liver cancer cannot be specifically distinguished. Such as AFP, which is widely used clinically, its specificity is about 80%. However, a large number of clinical studies find that the AFP level in serum is not remarkably increased until 3-40% of liver cancer patients develop liver cancer in the late stage; some patients with liver cirrhosis also have serum AFP levels as high as 1000U or more, but do not suffer from liver cancer after multiple periodic examinations. Therefore, the current method for diagnosing liver cancer lacks high sensitivity and specificity, and becomes a bottleneck for early screening, diagnosis and treatment of liver cancer.
The inventor finds out in long-term research that in order to screen high risk group of liver cancer at early stage and to make preventive diagnosis and treatment of liver cancer, the conventional consistent research thought of comparing cancer tissues and tissues beside cancer needs to be broken through, and the difference between liver fibrosis tissues of normal human liver tissues and liver cancer patients needs to be screened in turn to search and develop specific protein and function in cancer occurrence soil (environment).
At present, the application of screening liver cancer high risk population by measuring the activity of related enzymes in human bodies is not available; the inventor aims to test the application of human liver cytochrome P450 enzymes CYP2E1 and CYP2C8 to screen liver cancer susceptibility and/or high risk group and liver cancer patient prognosis evaluation so as to achieve early discovery, early diagnosis and early treatment of liver cancer.
Disclosure of Invention
According to one aspect of the application, the kit for detecting the metabolic activity of the related enzyme is provided, has higher sensitivity and specificity in the aspect of detecting the metabolic activity of the related enzyme, is easy to operate and is convenient to popularize and apply.
The kit for detecting the metabolic activity of the related enzyme is characterized by comprising a specific probe corresponding to the related enzyme;
the kit is used for detecting the metabolic activity of related enzymes and detecting at least one of the ratios of the metabolic activities of at least two related enzymes.
Optionally, the kit comprises reagents for detecting at least one of metabolic activity of the related enzymes and detecting a ratio of metabolic activity of at least two related enzymes.
Optionally, the kit comprises reagents for detecting the ratio of CYP2E1 and CYP2C8 metabolic activity.
Optionally, the relevant enzyme is a human liver cytochrome P450 enzyme.
Optionally, the related enzymes include at least one of CYP2E1, CYP2C 8;
the CYP2E1 is cytochrome P450 enzyme 2E1, and the CYP2C8 is cytochrome P450 enzyme 2C 8.
Optionally, the CYP2E1 includes at least one of liver microsome CYP2E1, human CYP2E 1.
Optionally, the CYP2C8 includes at least one of liver microsome CYP2C8, human CYP2C 8.
Optionally, the ratio of the metabolic activities of the at least two related enzymes comprises: at least one of the ratio of the metabolic activities of liver microsome CYP2E1 and CYP2C8, and the ratio of the metabolic activities of human CYP2E1 and CYP2C 8.
Optionally, the specific probe comprises at least one of a substance involved in metabolism/transformation by CYP2E1, a substance involved in metabolism/transformation by CYP2C 8.
Optionally, the specific probe comprises at least one of chlorzoxazone, 4-nitrophenol, aniline, diethylnitrosamine, paclitaxel, repaglinide, amodiaquine, montelukast, rosiglitazone, pioglitazone.
CYP2E1 can metabolize or convert many drugs, chemicals, and endogenous substances, such as: chlorzoxazone, acetaminophen, fluoxetine, theophylline, sulfadiazine, ethanol, 4-nitrophenol, 1, 3-butadiene, benzene, toluene, chloroform, vinyl chloride, dimethylnitrosamine, diethylnitrosamine, acetone, arachidonic acid.
CYP2C8 can be involved in the metabolic transformation of many drugs and endogenous substances, such as: paclitaxel, amodiaquine, repaglinide, montelukast, pioglitazone, rosiglitazone, cerivastatin, ibuprofen, epoxyeicosatrienoic acid, docosahexaenoic acid, eicosapentaenoic acid.
As a specific embodiment, the specific probe of CYP2E1 comprises at least one of chlorzoxazone, diethylnitrosamine, 4-nitrophenol and aniline, and the specific probe of CYP2C8 comprises at least one of paclitaxel, repaglinide, amodiaquine, montelukast, rosiglitazone and pioglitazone.
As a specific embodiment, the specific probe of CYP2E1 is chlorzoxazone,
the specific probe of the CYP2C8 is repaglinide.
Optionally, the kit comprises:
an incubation system;
wherein the incubation system comprises specific probes corresponding to the related enzymes; or
The kit comprises 125-1200 mg of chlorzoxazone, 0.1-16 mg of repaglinide, 2-10 mg of montelukast sodium, 2-8 mg of rosiglitazone and 7.5-45 mg of pioglitazone.
Optionally, the kit comprises 600mg of chlorzoxazone tablet and 4mg of repaglinide.
Optionally, the incubation system comprises a specific probe corresponding to the relevant enzyme and a buffer solution;
the buffer solution is selected from one of 50-100 mM phosphate buffer solution and 50-100 mM Tris-HCL buffer solution;
the pH value of the buffer solution is 4.4-10.4.
Optionally, the concentration of the buffer solution is 50-100 mM.
Optionally, the buffer has a concentration of 100 mM.
Optionally, the buffer is selected from one of phosphate buffer, Tris-HCL buffer.
Optionally, the buffer has a pH of 7.4.
Optionally, the incubation system comprises 100mM phosphate buffer solution with pH of 7.4, 2.5-80 mu M of paclitaxel, 0.78-100 mu M of diethylnitrosamine, 7.8-1000 mu M of chlorzoxazone, 0.022-20 mu M of repaglinide, 5-2000 mu M of 4-nitrophenol, 5-6000 mu M of aniline, 0.1-200 mu M of amodiaquine, 0.025-50 mu M of montelukast, 0.25-1000 mu M of rosiglitazone and 2.06-5272 mu M of glipizide.
Optionally, the incubation system further comprises at least one reduced coenzyme.
Optionally, the incubation system comprises one of NADPH or NADPH regenerating system at a concentration of 1 mM;
wherein the NADPH regenerating system comprises 1.3mM NADP +,3.3mM glucose-6-phosphate, 0.4U/ml glucose dehydrogenase, 3.3mM magnesium chloride
Optionally, the kit further comprises:
the system was terminated.
The termination system includes at least one of ethyl acetate, dichloromethane, acetonitrile, methanol, perchloric acid.
Optionally, the stop system comprises at least one of 1mL ethyl acetate, 1mL dichloromethane, 100 μ L acetonitrile, 100 μ L methanol, 10 μ L perchloric acid.
Optionally, the stop system comprises at least one of 1mL ethyl acetate, 1mL dichloromethane, 100 μ L acetonitrile.
Optionally, the kit comprises:
(a) 2.5-80 mu M of paclitaxel, 0.78-100 mu M of diethylnitrosamine, 7.8-1000 mu M of chlorzoxazone, 0.022-20 mu M of repaglinide, 5-2000 mu M of 4-nitrophenol, 5-6000 mu M of aniline, 0.1-200 mu M of amodiaquine, 0.025-50 mu M of montelukast, 0.25-1000 mu M of rosiglitazone and 2.06-5272 mu M of pioglitazone;
(b) at least one of 50mM phosphate buffer, 100mM phosphate buffer, 50mM Tris-HCl buffer, and 100mM Tris-HCl buffer;
(c)1mM NADPH or NADPH regeneration system;
(d) at least one of 1mL ethyl acetate, 1mL methylene chloride, or 100. mu.L acetonitrile.
Optionally, the kit is used for detecting at least one of metabolic activities of related enzymes and detecting a ratio of metabolic activities of at least two related enzymes.
According to another aspect of the application, a method for detecting metabolic activity of related enzymes is provided, and the method has high sensitivity and specificity, is easy to operate and is convenient to popularize and apply. The method provides a new more accurate and sensitive standard for screening the high risk group of liver cancer and predicting the prognosis of the liver cancer patient, and has great significance for screening the high risk group of liver cancer and preventing and treating liver cancer.
The method for detecting the metabolic activity of the related enzyme comprises the following steps: measuring the metabolic activity of the related enzyme on a specific probe corresponding to the related enzyme;
the method employs at least one of the kits described above.
Optionally, the metabolic activity comprises at least one of a michaelis constant, a maximum rate of conversion, clearance, a concentration of drug peak, time to peak, rate of terminal elimination, half-life, area under the drug time curve, clearance, apparent volume of distribution, mean residence time.
As a specific embodiment, the metabolic activities of liver microsomes CYP2E1 and CYP2C8 include the Michaelis constant (K)m) Maximum conversion rate (V)max) Clearance rate (CL)int)。
As a specific embodiment, the metabolic activity of human CYP2E1 and CYP2C8, including the drug peak concentration (C)max) Time to peak (T)max) End elimination rate (K)e) Half life (T)1/2) Area under the curve (AUC), Clearance (CL), apparent volume of distribution (V)d) Average residence time (MRT).
Optionally, the method comprises:
mixing a system containing related enzyme, at least one of specific probes corresponding to the related enzyme and a buffer solution, pre-incubating, adding reduced coenzyme, incubating, terminating the reaction after the reaction is finished, and analyzing the obtained organic phase to obtain the metabolic activity of the related enzyme;
the related enzyme comprises at least one of CYP2E1 and CYP2C 8.
Optionally, the system comprising the relevant enzyme comprises liver microsomes or the human body.
Optionally, the method of analyzing comprises: the amount of the metabolite produced was measured by chromatography, and the metabolic activity of the relevant enzyme was calculated by a non-linear regression analysis method.
Optionally, the metabolic activities of at least two related enzymes are obtained according to the method, and the metabolic activity ratio of different related enzymes is obtained through calculation.
Optionally, the ratio of CYP2E1 and CYP2C8 metabolic activities is obtained by the method.
As a specific embodiment, the method for detecting the metabolic activity of the human liver cytochrome P450 enzyme is implemented by measuring the metabolic activity of the human liver cytochrome P450 enzyme on a specific probe thereof and determining a ratio.
As a specific embodiment, the human hepatocyte cytochrome P450 enzyme ratio is the ratio of the metabolic activities of CYP2E1 and CYP2C 8.
According to another aspect of the application, an application of a reagent for detecting metabolic activities of related enzymes and/or detecting a ratio of metabolic activities of at least two related enzymes in preparation of a reagent for screening liver cancer susceptibility and/or high risk group and liver cancer patient prognosis evaluation is provided.
Optionally, the reagent is used to measure the ratio of CYP2E1 and CYP2C8 metabolic activities.
Optionally, the reagent is used for detecting at least one of the ratio of the metabolic activities of liver microsomes CYP2E1 and CYP2C8 and the ratio of the metabolic activities of human CYP2E1 and CYP2C 8.
According to still another aspect of the present application, there is provided an application of any one of the above-mentioned kits and/or any one of the above-mentioned detection methods in screening for liver cancer susceptibility and/or high risk group, and prognosis evaluation of liver cancer patients.
As an embodiment of the application, the metabolic activity of CYP2E1 is measured to screen susceptibility and high risk groups of liver cancer and predict prognosis of liver cancer patients.
As an embodiment of the application, the method is used for screening susceptibility and high risk groups of liver cancer and predicting prognosis of liver cancer patients by measuring the value of the metabolic activity of CYP2C 8.
As an implementation mode of the application, the ratio of the metabolic activities of human liver cytochrome P450 enzymes CYP2E1 and CYP2C8 is measured to screen the susceptible and high risk groups of liver cancer and predict the prognosis of liver cancer patients.
As an embodiment of the application, the ratio of the metabolic activities of human liver cytochrome P450 enzymes CYP2E1 and CYP2C8 is used for screening susceptibility and high risk groups of liver cancer and predicting prognosis of patients with liver cancer. The specific findings are as follows:
the ratio of the liver microsome CYP2E1 to CYP2C8 metabolic activity of a liver cancer patient is obviously increased;
shown by a receiver working characteristic curve (ROC) of the ratio of the CYP2E1 to the CYP2C8 metabolic activity, the ratio of the CYP2E1 to the CYP2C8 metabolic activity can be used for very accurately screening high risk groups of liver cancer, and the sensitivity and specificity are high;
and the ratio of the metabolic activity of CYP2E1 to CYP2C8 is closely related to the survival time of the liver cancer patient, the survival time of the patient with high ratio is obviously shortened compared with that of the patient with low ratio, and the death risk of the patient with high ratio is 2.49 times that of the patient with low ratio.
As an embodiment of the present application, the present inventors found that the ratio of CYP2E1 and CYP2C8 metabolic activities was significantly increased by about 4.8 times in liver fibrosis tissues of liver cancer patients,
as an embodiment of the present application, the relationship between the ratio of CYP2E1 and CYP2C8 metabolic activities and liver cancer susceptibility and prognosis was studied. The ratio of the CYP2E1 to CYP2C8 metabolic activity is found to be closely related to the susceptibility of the liver cancer, and the ratio of the CYP2E1 to CYP2C8 metabolic activity is obviously related to the life cycle of the liver cancer patient, so that a new more accurate and sensitive standard is provided for screening and predicting the prognosis of the liver cancer patient for the high risk group of the liver cancer, and the method has great significance for screening and preventing and treating the liver cancer for the high risk group of the liver cancer. In addition, the detection method of the ratio of the metabolic activities of CYP2E1 and CYP2C8 is easy to operate and convenient to popularize and apply in hospitals.
In the present application, "CYP 2E 1" and "CYP 2C 8" are human hepatocyte cytochrome P450-related enzymes.
In the present application, CYP2E1 (cytochrome P450 enzyme 2E1) is a protein mainly present in the endoplasmic reticulum of the liver, and accounts for about 24.8% of the total amount of CYP450 in the liver. CYP2E1 has the main function of metabolism, and participates in the biotransformation of drugs, precancerogen and environmental toxicants, for example, more than 85 exogenous substances can be metabolically activated to generate hepatotoxic or carcinogenic substances, including carcinogens such as nitrosamine, benzene, 1, 3-butadiene and the like, and CYP2E1 is related to the biotransformation and induced tumors of nitrosamine, toluene, chloroform, acetone, tobacco-specific carcinogen NNK and the like. Therefore, the metabolic activation of CYP2E1 is involved in the development and progression of liver cancer and other diseases.
In the application, CYP2E1 participates in the occurrence and development of diseases such as liver cancer and the like through metabolic activation, and the inflammation causing effect of CYP2E1 also participates in the occurrence and development of liver cancer. CYP2E1 enhances the release of TNF-alpha in Kupffer cells, resulting in inflammatory, necrotic changes in hepatocytes. CYP2E1 knockdown mice were resistant to the inflammatory response induced by chronic alcohol exposure. In an animal model of nonalcoholic steatohepatitis (NASH), inhibition of CYP2E1 activity can be prevented and treated by reducing TNF-alpha expression, restoring endothelial nitric oxide synthase activity, and the like. The CYP2E1 knock-out mouse or CYP2E1 inhibitor allyl thioether can inhibit the release of IL-1 beta and IL-12, thereby preventing and treating NASH.
In the present application, CYP2C8 (cytochrome P450 enzyme 2C8) is a protein mainly present in the endoplasmic reticulum of the liver, and accounts for about 6.8% of the total amount of CYP450 in the liver. CYP2C8 has a main function of metabolism, and is involved in the metabolism of many long-chain unsaturated fatty acids, such as Arachidonic Acid (AA) to generate different eicosatrienoic acids (EETs), docosahexaenoic acid (DHA) to generate different epoxydocosapentaenoic acids (EDPs), and eicosapentaenoic acid (EPA) to generate different epoxyeicosatetraenoic acids (EEQs), in addition to the metabolism of drugs and other exogenous substances. The EETs, EDPs and EEQs not only have the function of regulating the vascular tension, but also have the strong functions of relieving inflammation and eliminating active oxygen. Therefore, the metabolic activation of CYP2C8 is involved in the development and progression of liver cancer and other diseases.
Benefits that can be produced by the present application include, but are not limited to:
1) the kit for detecting the metabolic activity of the related enzyme has high sensitivity and specificity in the aspect of detecting the metabolic activity of the related enzyme, is easy to operate and is convenient to popularize and apply.
2) The enzyme-dependent metabolic activity detection method provided by the application provides a new more accurate and sensitive standard for screening of high risk groups of liver cancer and prediction of prognosis of liver cancer patients, and has great significance for screening of high risk groups of liver cancer and prevention and treatment of liver cancer.
3) The detection method provided by the application is adopted to determine the metabolic activity and the ratio of the related enzymes, particularly the metabolic activity and the ratio of the related enzymes of human liver cytochrome P450, has important guiding significance for screening of high risk groups of liver cancer and prognosis evaluation of liver cancer patients, particularly the ratio of the metabolic activity of CYP2E1 and CYP2C8 is closely related to the survival time of the liver cancer patients, the survival time of the patients with high ratio is obviously shortened compared with that of the patients with low ratio, and the death risk of the patients with high ratio is 2.49 times that of the patients with low ratio.
Drawings
Fig. 1 is a comparison of the ratios of the metabolic activities of CYP2E1 and CYP2C8 in liver fibrosis (HCC group, n 101) and normal human liver (Control group, n 95) in a liver cancer patient according to example 3 of the present application.
FIG. 2 is a ROC curve showing the ratio of CYP2E1 and CYP2C8 metabolic activity in example 3 of the present application and other clinically common indicators associated with early diagnosis and prognosis of liver cancer;
in the figure, V2E1/V2C8: the ratio of CYP2E1 and CYP2C8 metabolic activities; ALT: glutamic-pyruvic transaminase; AST: glutamic-oxalacetic transaminase; AFP: alpha-fetoprotein; PT: prothrombin time.
Fig. 3 is a survival curve (n-87) of liver cancer patients with different ratios of CYP2E1 and CYP2C8 metabolic activities in example 4 of the present application.
Detailed Description
The present application will be described in detail with reference to examples, but the present application is not limited to these examples.
Unless otherwise specified, the raw materials and reagents in the examples of the present application were all purchased commercially.