CN111413502A

CN111413502A - New application of GP73 and liver tissue inflammation activity degree detection kit based on GP73

Info

Publication number: CN111413502A
Application number: CN202010149333.6A
Authority: CN
Inventors: 鲁凤民; 姚明解
Original assignee: Individual
Current assignee: Peking University
Priority date: 2017-04-27
Filing date: 2017-04-27
Publication date: 2020-07-14
Anticipated expiration: 2037-04-27
Also published as: CN107247148B; CN107247148A; CN111413502B

Abstract

The invention relates to application of serum GP73 concentration in detection of liver tissue inflammation activity, and provides a liver tissue inflammation activity detection kit, which can realize detection and classification of liver tissue inflammation activity of a subject by utilizing the GP73 concentration in the serum, and can be suitable for patients with moderate inflammation necrosis above and normal A L T and viral hepatitis patients adopting antiviral treatment.

Description

New application of GP73 and liver tissue inflammation activity degree detection kit based on GP73

The application is a divisional application, the invention name of a primary application is a new application of GP73 and a liver tissue inflammation activity degree detection kit based on the GP73, the application number is 201710289636.6, and the application date is 2017, 4 and 27.

Technical Field

The invention belongs to the field of biomedicine, and relates to a new application of Golgi protein 73(GP73) and a liver tissue inflammation activity detection kit prepared based on the detection of the concentration of the Golgi protein 73(GP 73).

Background

Kladney et al found Golgi protein 73(GP73) in studies of adult cytomegalohepatitis, as of 17 years so far (Kladney RD, Bulla GA, Guo L, et al. Gene.2000.249(1-2):53-65), GP73 is a transmembrane glycoprotein with 402 amino acids and a relative molecular weight of 73kDa, also known as Golgi protein type II (Golgiphosphoprotein 2, Golph2) and Golgi membrane protein I (Golgi membrane protein 1, Golgi 1). it is usually located in trans-Golgi complex, the gene encoding GP73 protein is located in chromosome 9, total length contains 3080 nucleotides, the coding region 199 is located in laid-open mouse 1404, GP73 is expressed in various tissues but not equally strong, as high as in small intestine, colon and stomach, but as minimal as of liver expression in heart, as of kidney, spleen, testis, uterine, liver cancer, kidney cancer, liver cancer, kidney cancer, liver cancer, kidney cancer, liver cancer, kidney cancer, liver cancer, kidney cancer, liver cancer, kidney cancer, liver cancer, kidney cancer, liver cancer, kidney cancer, liver cancer, kidney cancer, liver cancer, kidney cancer, liver cancer, kidney cancer, liver cancer, kidney cancer, liver cancer, kidney cancer, liver cancer, kidney cancer, liver cancer.

In conclusion, the researches of the documents show that the GP73 has a better distinguishing capability for distinguishing liver cancer from hepatitis and cirrhosis patients than AFP, but the sensitivity for diagnosing cirrhosis is higher than that of serum AFP.

Clinically for the diagnosis of hepatitis, A L T (full name alanine aminotransferase) and AST (full name aspartate aminotransferase) are commonly used, A L T and AST are mainly distributed in liver cells, A L T and AST are increased, which shows that liver cells are damaged, A L T is most sensitive, A L T in serum is increased by 1 time, which shows that 1% of liver cells are necrotic, A L T and AST are increased to the same extent as that of liver cells are damaged, the two enzymes are distributed in liver cells differently, A L T is mainly distributed in liver cell paste, A L T is increased to reflect damage of liver cell membranes, AST is mainly distributed in liver cell paste and liver cell mitochondria, therefore, the A L T, AST increase condition of different hepatitis conditions is different, and the ratio of A L T/AST is also different.

The hepatitis B or HBsAg has a history or acute hepatitis course of more than half a year, and patients with symptoms, signs and abnormal liver function can be diagnosed as chronic hepatitis, and can possibly turn into cirrhosis or liver cancer, if the disease date is unknown or no history of hepatitis exists, the imaging, laparoscope or liver biopsy pathology accords with the change of chronic hepatitis, or the corresponding diagnosis can be made according to comprehensive analysis of symptoms, signs and tests, the patients are clinically divided into mild, moderate and severe according to the liver damage degree, the division standard is as follows, 1. mild disease, the symptoms are not obvious or symptoms and signs exist, biochemical indexes are only 1-2 mild abnormal patients, 2. severe patients have obvious or continuous hepatitis symptoms, such as weakness, poor appetite, abdominal distension, stool and the like, can be accompanied by liver disease face, liver disease nevus, spider nevus or liver splenomegaly, other reasons are eliminated, and no portal hypertension is avoided, 2. the laboratory examination A L T repeatedly or continuously increases, albumin or the A/G proportion is abnormal, the hepatitis C-globulin ratio is increased, the liver cancer cell count is obviously increased, the liver cancer cell count is increased, the normal liver cancer cell count is increased, the liver cancer.

The requirements mainly include 1) moderate and severe liver inflammation is a main factor of chronic liver disease liver injury, and once positive treatment is determined, sensitive, specific and quantifiable serological indexes are needed for diagnosis and degree determination of the moderate and severe liver inflammation, 2) A L T is an important index for clinically reflecting liver cell injury, obvious liver inflammation exists in a certain proportion of chronic liver diseases, but A L T is normal, and the condition of judging the liver tissue inflammation activity degree through an A L T method is not needed, and 3) after antiviral treatment is adopted, sensitive and specific judgment indexes for judging whether the liver tissue inflammation activity degree is controlled/improved are lacked.

At present, the clinically used index is liver tissue inflammation activity, and the levels thereof are classified according to G0, G1, G2, G3 and G4, which is specifically shown in table 1 below. As liver tissue destruction progresses, the grading of liver tissue inflammatory activity levels will tend to rise; however, with the remission of the condition of the liver tissue, the grading of the activity level of the inflammation of the liver tissue tends to decrease.

Many documents research and discussion about the clinical diagnosis of liver tissue inflammation activity, such as "analysis of correlation between serum A L T level of chronic hepatitis B patient and liver tissue inflammation activity", Dorsen, Qiao, Zhonghua Miao Chi & Miao, 2007, and "analysis of correlation between serum hepatic fibrosis marker level and liver tissue inflammation activity fibrosis degree", admission to people, etc. ", journal of clinical liver and gall disease, 2005,21(3): 175. 176", clinical diagnosis and treatment of liver tissue inflammation activity of chronic hepatitis B patient ", hong Cao, Wan, Suting, Zhonghua Hospital, J of infectious diseases in Chinesis 2014, 17 th, clinical value of chronic hepatitis B patient alanine aminotransferase in diagnosing liver tissue inflammation activity, Wang Bing, Chinese prescription, 2014, 10 th, etc., but no suitable marker is found to indicate the liver tissue inflammation activity level and grading.

At present, there is no sensitive and efficient method for detecting the inflammatory activity of liver tissue by serum rather than tissue. Therefore, development of such a detection kit is urgently required.

Disclosure of Invention

For the three conditions, 1) moderate and severe liver inflammation is a main factor of chronic liver disease liver injury, once positive treatment is determined, sensitive, specific and quantifiable serological indexes are needed for diagnosing and judging the degree of the moderate and severe liver inflammation, 2) A L T is an important index for clinically reflecting liver cell injury, obvious liver inflammation exists in a certain proportion of chronic liver diseases, but A L T is normal, and the condition of liver tissue inflammation activity is not judged by an A L T method, and 3) for viral hepatitis, after antiviral treatment, a sensitive and specific judgment index for judging whether the liver tissue inflammation activity is controlled or improved is lacked, and at present, a sensitive and efficient method for detecting the liver tissue inflammation activity by serum instead of a tissue is not available, and liver tissue biopsy detection always brings trauma to a subject, so that in order to reduce the liver tissue injury, the development of a serum detection kit for detecting the liver tissue inflammation activity and grading treatment guidance and post-treatment effects is urgently needed.

Therefore, the invention relates to the application of GP73 concentration in liver tissue inflammation activity detection, and provides a liver tissue inflammation activity detection kit, which can realize detection grading of liver tissue inflammation activity of a subject by using GP73 concentration in serum, and can be suitable for patients with moderate inflammation necrosis above and normal A L T and patients with viral hepatitis adopting antiviral treatment.

Specifically, the technical scheme of the invention relates to application of GP73 concentration in detection of liver tissue inflammation activity, wherein the GP73 concentration is serum GP73 concentration, and can realize detection of liver tissue inflammation activity of a subject and classify the liver tissue inflammation activity into mild degree, moderate degree and severe degree, wherein the mild degree is more than or equal to 1 and less than 2 in liver tissue inflammation activity classification, the moderate degree is more than or equal to 2 and less than 3 in liver tissue inflammation activity classification, and the severe degree is more than or equal to 3 in liver tissue inflammation activity classification.

Specifically, the subjects are judged to have mild hepatic tissue inflammatory necrosis in the range of 40-57ng/m L of the GP73 concentration, moderate hepatic tissue inflammatory necrosis in the range of 57ng-94ng and severe hepatic tissue inflammatory necrosis in the range of 94ng/m L.

Specifically, the subject is a patient with viral hepatitis, autoimmune liver disease, and other etiological liver disease, including non-alcoholic fatty liver disease, drug-induced liver disease, and liver disease of unknown etiological factor.

In particular, the patient has a value of a L T within the normal range and inflammatory necrosis of the liver tissue above moderate.

Specifically, the use includes determining an improvement in inflammatory activity of liver tissue in the subject after treatment, and determining that the disease is controlled or inhibited by a decrease in the GP73 concentration by greater than 20% after treatment relative to before treatment.

The invention also provides a technical scheme of the liver tissue inflammation activity degree detection kit, wherein the liver tissue inflammation activity degree of a subject is detected based on the GP73 concentration in serum and is classified into mild degree, moderate degree and severe degree, wherein the mild degree is classified into the liver tissue inflammation activity degree of more than or equal to 1 and less than 2, the mild degree is G0-1, the moderate degree is classified into the liver tissue inflammation activity degree of more than or equal to 2 and less than 3, the moderate degree is G2, the severe degree is classified into the liver tissue inflammation activity degree of more than or equal to 3, and the severe degree is G3/4.

Specifically, the liver tissue inflammation activity degree detection kit is suitable for subjects suffering from viral hepatitis, autoimmune liver diseases and liver diseases of other causes, wherein the liver diseases of other causes comprise non-alcoholic fatty liver disease, drug-induced liver disease and liver diseases of unknown causes.

Specifically, the liver tissue inflammation activity degree detection kit is suitable for the subjects with more than moderate liver tissue inflammation necrosis with A L T values in a normal range.

Specifically, the liver tissue inflammation activity degree detection kit is suitable for the subjects of hepatitis patients after antiviral treatment.

Specifically, the liver tissue inflammation activity degree detection kit can be used for judging improvement of liver tissue inflammation activity degree after treatment, and disease control or inhibition is judged by that the GP73 concentration after treatment is reduced by more than 20% compared with that before treatment.

The present invention achieves the following effects, including but not limited to:

1) the method for detecting GP73 by serum instead of GP73 by tissue is a sensitive and efficient method for detecting the inflammation activity of liver tissue, is more rapid, convenient and reliable, has clinical guidance significance, and is worthy of popularization. The method is rapid and convenient, the tissue GP73 obtained by biopsy can bring a wound far exceeding the skin of a subject, the tissue injury after biopsy repair is frosted on the surface of the liver with inflammation, the blood serum extraction technology is mature, and the wound surface is recovered rapidly. The reliability of the method is shown in that serum GP73 provides information consistent with tissue GP73, because tissue GP73 is a precursor and serum GP73 is a product after Furin enzyme digestion processing, the two are related to the activity of liver tissue inflammation.

2) By detecting and grading the quantified index liver tissue inflammation activity, the sensitive and efficient diagnosis is realized for moderate and severe hepatitis, and the disease development stage of a subject is determined for corresponding treatment;

3) for chronic hepatitis patients with normal A L T index and failure in judging the liver tissue inflammation activity, the detection of the quantifiable index liver tissue inflammation activity is adopted, so that sensitive and efficient diagnosis is realized;

4) after antiviral treatment is adopted for viral hepatitis, the treatment effect is judged sensitively and efficiently by detecting the quantifiable index of the liver tissue inflammation activity.

Drawings

FIG. 1 shows the expression level and correlation of serum GP73 in different degrees of liver tissue inflammation activity, FIGS. 1(A) and (B) show the included population, FIGS. 1(C) and (D) show the viral hepatitis patients, FIGS. 1(E) and (F) show the autoimmune liver disease patients, and FIGS. 1(G) and (H) show the liver disease patients with other etiologies (nonalcoholic steatohepatitis, drug-induced liver disease and liver disease with unknown etiology). The horizontal axis represents the degree of inflammation activity, and the vertical axis represents the concentration of GP73 in serum.

FIG. 2 is a ROC curve of GP73 on liver tissue inflammatory activity.

FIG. 3 shows serum GP73 and A L T at levels above moderate inflammatory necrosis (G.gtoreq.2) and in patients with normal A L T.

FIG. 4 shows a comparison of GP73 concentration changes before and after antiviral treatment of patients, based on whether the inflammatory activity of liver tissue was improved or not.

Detailed Description

The present invention is described in detail below by embodiments in conjunction with data diagrams, the scope of protection of which includes but is not limited to what is claimed in the claims, and the data ranges can be modified and adapted accordingly as will be appreciated by those skilled in the art.

The present invention will be further described below by way of specific embodiments and experimental data. Although specific terms are used below for the sake of clarity, these terms are not meant to define or limit the scope of the invention.

As used herein, the terms "liver tissue inflammatory activity" and "liver tissue inflammatory activity degree" are used interchangeably, with the liver tissue inflammatory activity degree being graded abbreviated as G, and the following table being the corresponding pathological diagnostic criteria with a specific grade of 0-4.

TABLE 1 pathological diagnosis criteria for liver tissue inflammation activity

As used herein, the term "mild" is used interchangeably with "liver tissue inflammatory activity rating ≧ 1 and < 2", abbreviated as G0-1 or G1; "moderate" is used interchangeably with "liver tissue inflammatory activity rating ≧ 2 and < 3", abbreviated as G2; "Severe" is used interchangeably with "liver tissue inflammation activity rating ≧ 3", abbreviated as G3/4, i.e., G3 and G4. Thus, the terms "mild", "moderate" and "severe" refer specifically to the above pathological diagnosis criteria.

As used herein, the term "sensitivity", also called "true positive rate", refers to the percentage of actual diseased that is correctly judged to be diseased according to the test criteria, with greater sensitivity being better, and ideal sensitivity being 100%,

as used herein, the term "specificity," also called "true negative rate," refers to the percentage of actual disease-free that is correctly judged to be disease-free according to the test standard, with greater specificity being better and ideal specificity being 100%.

The receiver (relative) operating characteristics (ROC) reflects the balance between sensitivity and specificity, and the area under the ROC curve is an important test accuracy index. The ROC of each test is calculated and compared by the area under the line (AUCROC), and the large area has large diagnostic value of the test.

The experimental procedures in the following examples are conventional unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 design of the experiment

1.1 study object

642 chronic liver disease patients who were diagnosed with liver puncture under row B ultrasound guidance in Beijing 302 hospital from 12 months to 2016 months in 2013 were examined.

1.2 statistical methods

Statistical analysis is carried out by using SPSS21.0(SPSS, Chicago, Illinois, USA) statistical software, a description method is selected according to the distribution characteristics of continuous variables, and quantitative data approximately complying with normal distribution is adopted

The quantitative data in the skewed distribution adopts a median (M) and a quartile interval to describe the distribution characteristics, the qualitative data group comparison adopts a chi 2 test, the two independent samples adopt a Mann-WhitnyU test, the multiple groups of samples adopt a Kruskal Wallis test, a receiver operating characteristic curve (ROC curve) is established, and evaluation values of different indexes on the liver tissue inflammation activity degree are compared according to the area under the curve so as to obtain the test standard of α -0.05.

1.3 Baseline data for samples

The sample is included in 642 patients, including 266 patients with viral hepatitis, 158 patients with autoimmune liver diseases and 218 patients with other diseases (alcoholic fatty liver, drug-induced liver disease and liver disease with unknown causes). The mean ages of viral hepatitis, autoimmune liver disease and chronic liver disease of other etiology were 41.27 + -11.47 years, 48.46 + -10.33 years and 44.20 + -11.94 years, respectively, as shown in Table 2.

TABLE 2 general conditions of the study subjects

Note that GP73 is Golgi protein 73, A L T is glutamic-pyruvic transaminase and AST is glutamic-oxalacetic transaminase.

Example 2 analysis of test results

2.1 serum GP73, A L T, AST expression levels at varying degrees of liver tissue inflammatory activity

Serum GP73, a L T and AST are described in quartile intervals in terms of the trend of change in the degree of liver tissue inflammatory activity at different degrees serum GP73, at different degrees of liver tissue inflammatory activity levels G0-1 (n-241): 41.43(24.34-58.08) ng/m L, G2 (n-239): 69.26(45.66-115.38) ng/m L, G3/4 (n-46): 122.54(72.19-186.51) ng/m L, Spearman correlation analysis shows that serum GP73 and liver tissue inflammatory activity are significantly correlated (Spearman correlation coefficient r is 0.609, P < 0.001). the results of genetic analysis show that serum GP73 increases with the degree of liver tissue inflammatory activity and is significantly correlated with liver tissue inflammatory activity in chronic patients with viral hepatitis, autoimmune liver diseases and other etiologies, see table 1 and 3.

TABLE 3 interquartile spaces between GP73 and A L T, AST in patients with different degrees of liver tissue inflammation activity

2.2 differential value of serum GP73 on liver tissue inflammation Activity

The pathological diagnosis result is used as a gold standard, an ROC curve is used for calculating the sensitivity, specificity, positive prediction value, negative prediction value, false positive rate and Youden index of serum GP73 to moderate liver inflammatory necrosis (G is more than or equal to 2 and less than 3) and severe inflammatory necrosis (G is more than or equal to 3), the optimal diagnosis value is calculated, the results show that the areas under the ROC curve of serum Golgi protein 73(GP73) to moderate liver inflammatory necrosis (G is more than or equal to 2 and less than 3) and severe inflammatory necrosis (G is more than or equal to 3) are respectively 0.822 (95% CI: 0.793-0.849, P is less than 0.001), 0.832 (95% CI: 0.804-0.858, P is less than 0.001), the areas under the ROC curve of serum GP73 to moderate liver inflammatory necrosis (G is more than or equal to 2 and less than 3) and severe inflammatory necrosis (G is more than or equal to 2 and less than 3) to A32T normal patients are respectively 0.763 (95% CI: 0.708-0.001), the areas under the ROC curve are respectively different from viral hepatitis caused by viral hepatitis and other viral hepatitis caused by the viral hepatitis (0.7.

TABLE 4 comparison of diagnostic value of serum GP73 on liver tissue inflammation activity

2.3 serum GP73 increased in patients with more than moderate inflammatory necrosis (G.gtoreq.2), whether A L T is normal or elevated, at a higher rate than A L T

Statistically, of 642 patients with chronic liver disease, 401 (62.5%) had moderate and above inflammatory necrosis (G.gtoreq.2), 347 (86.5%) of the 401 patients with moderate and above inflammatory necrosis had elevated serum GP73(GP73 > 40), 263 (65.6%) of the patients with A L T (A L T > 40), and 112 (81.2%) of the 138 patients with moderate and above inflammatory necrosis and A L T normal had elevated serum GP 73.

This data shows that serum GP73 has a higher sensitivity for detection than serum a L T for moderate inflammatory necrosis.

2.4 serum GP73 can be used as index for controlling or inhibiting the inflammatory activity of liver tissue after treatment

Patients in the antiviral treatment cohort of chronic hepatitis b patients were classified into a progressive group (n ═ 11), an invariant group (n ═ 31), and an improved group (n ═ 40) according to whether the liver tissue inflammatory activity improved before and after the treatment, and the results are shown in fig. 4: the difference of GP73 concentration between the patients in the inflammation progression group before and after treatment is not statistically significant (P is 0.062), and the GP73 concentration of the patients in the inflammation improvement group and the patients in the unchanged group is reduced (P is 0.017, and P is less than 0.001). See table 5 below for data.

TABLE 5 comparison of serum GP73 before and after antiviral treatment

The improvement is that inflammation is inhibited, and not changed, that inflammation is controlled.

While the present invention has been described with reference to the embodiments, it is to be understood that the present invention is not limited thereto, and those skilled in the art will appreciate that the present invention is capable of modification and variation within the spirit and scope of the present invention, and that such modification and variation are within the scope of the present invention.

Claims

Use of GP73 as a marker in the development and/or design of a kit for liver tissue inflammation activity detection, characterized in that the kit enables detection of liver tissue inflammation activity of a subject based on GP73 concentration in serum and is graded as mild, moderate and severe, wherein the mild liver tissue inflammation activity is graded at least equal to 1 and <2, the moderate liver tissue inflammation activity is graded at least equal to 2 and <3, and the severe liver tissue inflammation activity is graded at least equal to 3.
2. The use of claim 1, wherein the kit judges the subjects to have mild hepatic tissue inflammatory necrosis in the range of 40-57ng/m L of the GP73 concentration, judges the subjects to have moderate hepatic tissue inflammatory necrosis in the range of 57ng-94ng, and judges the subjects to have severe hepatic tissue inflammatory necrosis in the range of 94ng/m L or above.
3. The use according to claim 2, wherein said kit is suitable for use in subjects with viral hepatitis, autoimmune liver disease, other etiologic liver disease including nonalcoholic fatty liver disease, drug-induced liver disease and liver disease of unknown etiology.
4. The use according to claim 3, wherein the kit is suitable for use in subjects with inflammatory necrosis of liver tissue of greater than moderate degree having A L T values within the normal range.
The application of the GP73 monoclonal antibody in preparing the kit for detecting the liver tissue inflammation activity degree is characterized in that the kit can detect the liver tissue inflammation activity degree of a subject based on the GP73 concentration in serum and classify the liver tissue inflammation activity degree into mild degree, moderate degree and severe degree, wherein the mild degree is more than or equal to 1 and less than 2, the moderate degree is more than or equal to 2 and less than 3, and the severe degree is more than or equal to 3.
6. The use of claim 5, wherein the kit judges the subjects have mild liver tissue inflammatory necrosis in the range of 40-57ng/m L of the GP73 concentration, judges the subjects have moderate liver tissue inflammatory necrosis in the range of 57ng-94ng, and judges the subjects have severe liver tissue inflammatory necrosis in the range of 94ng/m L or above.
7. The use of claim 6, wherein said kit is suitable for use in subjects with viral hepatitis, autoimmune liver disease, other etiologic liver disease including nonalcoholic fatty liver disease, drug-induced liver disease, and liver disease of unknown etiology.
8. The use according to claim 7, wherein the kit is suitable for use in subjects with inflammatory necrosis of liver tissue of greater than moderate severity having a value of A L T within the normal range.