KR102453551B1 - Method of Providing Information for Diagnosing Acute Pancreatitis Severity - Google Patents

Abstract

The present invention relates to a biomarker for diagnosing the severity of acute pancreatitis and a use thereof, and the biomarker according to the present invention has excellent diagnostic effects on the severity of acute pancreatitis, especially the mild, and thus accurately determines the level of the condition as well as the presence or absence of acute pancreatitis. By quickly identifying it, it can be usefully used in the treatment of patients with mild acute pancreatitis.

Description

Method of Providing Information for Diagnosing Acute Pancreatitis Severity

The present invention relates to a method for providing information for diagnosing the severity of acute pancreatitis.

There are two major inflammatory diseases of the pancreas, which can be classified into acute pancreatitis (AP) and chronic pancreatitis (CP).

Acute pancreatitis is a life-threatening disease with varying degrees of symptoms. More than 80% of acute pancreatitis cases are mild, self-limiting, and resolve without serious complications, but 20% have severe morbidity or mortality, and temporary organ failure or local/systemic complications. is known to be Persistent organ failure leads to severe acute pancreatitis with a high mortality rate, with an overall mortality rate of about 10-15%, but up to 30%-40% in patients with severe disease. The sepsis complications associated with pancreatic necrosis leading to multiorgan failure are a leading cause of death later in the disease, and the severity of acute pancreatitis can fluctuate rapidly and unpredictably.

Since about 50% of all deaths in acute pancreatitis patients occur within 2 weeks of onset, it is important to evaluate the severity at the time of diagnosis and select patients who are expected to show a mild or severe course and provide intensive treatment from the beginning. In addition, even mild pancreatitis can progress to severe, so initial treatment and continuous evaluation are very important.

However, in most cases of acute pancreatitis, early diagnosis of pancreatitis is difficult because there are often no symptoms or vague symptoms. Nevertheless, even using these various diagnostic methods to date, an improvement in the early diagnosis rate of pancreatitis cannot be expected. Since pancreatitis without specific symptoms has almost no treatment after it has already developed, further research is needed for early diagnosis and treatment of pancreatitis.

In addition, despite advances in current research techniques, the exact etiology of pancreatitis is still unclear, and biomarkers targeting diagnosis and treatment are almost non-existent. Moreover, since most of the known markers only play the role of general risk factors, little is known about the genes related to the severity of pancreatitis.

Therefore, the discovery of diagnostic markers that can establish the diagnosis and prediction of the pathophysiology and severity of acute pancreatitis as a new risk factor and therapeutic factor marker that can target acute pancreatitis, where the effectiveness of treatment is greatly affected by early diagnosis, That is, there is a need to establish an improved technical means capable of detecting serum markers specific for acute pancreatitis and performing diagnosis in acute pancreatitis more accurately compared with tests used in the art. In particular, there is an urgent need for a method to differentiate mild AP, which is an early stage.

Under these circumstances, the present inventors made intensive research efforts to develop a method for diagnosing acute pancreatitis according to the severity. As a result, the present inventors discovered that there was a difference in the expression levels of C5a and ANGPTL-4 depending on the degree of acute pancreatitis during the study on patients with acute pancreatitis. Accordingly, by analyzing C5a and ANGPTL-4 expression as biomarkers targeting acute pancreatitis, in particular, early mild acute pancreatitis, the correlation with mild acute pancreatitis was identified, and not only the onset of acute pancreatitis, but also the severity, that is, mild By confirming that it can be accurately diagnosed, the present invention was completed.

Accordingly, one object of the present invention is to provide a method for providing information for diagnosing the severity of acute pancreatitis.

Another object of the present invention is to provide a kit that provides information on the severity of acute pancreatitis.

The terms used herein are used for the purpose of description only and should not be construed as limiting. The singular expression includes the plural expression unless the context clearly dictates otherwise. In the present specification, terms such as “comprise” or “have” are intended to designate that a feature, number, step, operation, component, part, or combination thereof described in the specification exists, but one or more other features It is to be understood that this does not preclude the possibility of the presence or addition of numbers, steps, operations, components, parts, or combinations thereof.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiment belongs. Terms such as those defined in a commonly used dictionary should be interpreted as having a meaning consistent with the meaning in the context of the related art, and should not be interpreted in an ideal or excessively formal meaning unless explicitly defined in the present application. does not

Hereinafter, the present invention will be described in more detail.

According to one aspect of the present invention, there is provided a method for providing information for diagnosing the severity of acute pancreatitis, comprising the following steps:

(a) the expression level of angiopoietin like 4 (ANGPTL-4) protein in a biological sample; and measuring the expression level of the C5a (complement component 5a) protein;

(b) predicting or diagnosing the severity of acute pancreatitis with a value calculated by comparing the expression level of each protein with the expression level of each protein in the normal group sample.

The expression levels of the angiopoietin like 4 (ANGPTL-4) protein and the complement component 5a (C5a) protein are biomarkers for diagnosing the severity of acute pancreatitis, and the measurement thereof is performed using a method known in the art.

In the present invention, the term "diagnosis" refers to determining the susceptibility of an individual to a specific disease or disorder, determining whether an individual currently has a specific disease or disorder, or an individual suffering from a specific disease or disorder determining the prognosis of, or therametrics (eg, monitoring a subject's condition to provide information about treatment efficacy).

For the purpose of the present invention, diagnosis is to determine whether acute pancreatitis occurs, whether or not there is a possibility of the onset, and the severity, such as mild or mild, of acute pancreatitis distinguished by severity, such as mild or severe.

In the present invention, acute pancreatitis can be largely divided into two types, mild type acute pancreatitis in which interstitial edema and peripancreatic fat necrosis around the pancreas are found; There are extensive fatty necrosis of the peripancreatic and intrapancreatic regions, pancreatic parenchymal necrosis, and severe type of pancreatitis with bleeding (Bank PA, Am J gastroenterol, 89, pp151). -152, 1994; Bradley EL, Arch Surg, 128, pp586-590, 1993; Changdeok Kim, Journal of the Korean Society of Gastroenterology, 46, pp321-332, 2005).

In the present invention, "marker" is a substance capable of diagnosing mild to moderate severity of acute pancreatitis, and polypeptides, nucleic acids (eg, mRNA, etc.) related to acute pancreatitis, lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides) organic biomolecules and the like). For the purposes of the present invention, the markers are ANGPTL-4 (angiopoietin like 4) and C5a (complement component 5a), which can be diagnosed in patients with mild acute pancreatitis, characterized in that they show a significant expression level.

In the present invention, "biological sample" includes whole blood, serum, plasma, saliva, urine, sputum, lymph fluid, cells, etc. isolated from a subject, preferably pancreatic tissue, but is not limited thereto.

In the present invention, "measurement of protein expression level" refers to a process of confirming the presence and expression level of proteins expressed from the ANGPTL-4 and C5a genes related to acute pancreatitis in a biological sample in order to diagnose the severity of acute pancreatitis, particularly a mild condition. , by measuring the amount of protein.

Methods for measuring the expression level of the protein in the present invention include Western blot, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion method, Rocket immunoelectrophoresis, tissue immunostaining, Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Cell Sorter (FACS), protein chip, etc. It is not limited, and in the present invention, ELISA (enzyme linked immunosorbent assay) was used.

Since the expression of the marker of the present invention is significantly increased in acute pancreatitis patients compared to the control group, it is possible to accurately and quickly diagnose the severity of acute pancreatitis as well as the onset of acute pancreatitis through comparison with the control sample.

Therefore, the value calculated by measuring the expression level of ANGPTL-4 and C5a can be usefully used to provide information for the diagnosis of mild acute pancreatitis.

In a specific embodiment of the present invention, the present inventors confirmed that the levels of ANGPTL-4 and C5a protein in the serum of patients with mild acute pancreatitis were significantly increased compared to the control group.

That is, it was confirmed that the ratio of ANGPTL-4 and C5a content in severe acute pancreatitis was significantly increased compared to mild acute pancreatitis.

Therefore, by measuring the expression level of ANGPTL-4 and C5a, it can be usefully used for diagnosing a mild condition based on the onset of acute pancreatitis.

According to an embodiment of the present invention, the value calculated in step (b) of the present invention is the logistic regression analysis result of the ELISA cut-off value, and in the case of ANGPTL-4, 40.98 ng/ ml and 5.4 ng/ml for C5a, determine that the subject has mild acute pancreatitis.

In addition, when the cut-off values appearing in the ROC curves of the subject and the normal control calculated in step (b) are 40.98 ng/ml for ANGPTL-4 and 5.4 ng/ml for C5a, the subject is treated with sensitivity (sensitivity) ) is 97 to 100% and specificity is 96 to 98%, indicating mild acute pancreatitis.

If the value calculated in step (b) is less than 40.98 ng/ml for ANGPTL-4 and less than 5.4 ng/ml for C5a as a result of logistic regression analysis of the result from ELISA, The subject is determined to be normal or not having mild acute pancreatitis.

Further, according to another aspect, the present invention provides a kit for diagnosing the severity of acute pancreatitis.

The kit of the present invention can diagnose the severity of acute pancreatitis by checking the expression level of ANGPTL-4 and C5a protein. The kit of the present invention may further include, for example, ANGPTL-4 and C5a antibodies for judging the severity of acute pancreatitis, as well as one or more other component compositions, solutions, or devices suitable for the assay method, preferably protein It may be in the form of a chip kit, but is not limited thereto.

The kit distinguishes whether the subject is acute pancreatitis or not, so that a medical practitioner such as a doctor can diagnose acute pancreatitis, as well as determine or diagnose the degree of severity of acute pancreatitis, that is, a mild condition, and treatment monitor the subject's response to , allowing the treatment to be altered according to the outcome.

In addition, it can be used to identify substances regulating the expression of ANGPTL-4 and C5a proteins in and out of acute pancreatitis animal models including mice and rats.

Since the kit of the present invention uses the above-described markers, descriptions of common contents between the two are omitted in order to avoid excessive complexity of the present specification.

Since the biomarker according to the present invention has an excellent diagnostic effect on the severity of acute pancreatitis, in particular, it can be usefully used in the treatment of patients with mild acute pancreatitis by accurately and rapidly identifying the level of the disease as well as the onset of acute pancreatitis.

1 shows a result of a Receiver Operating Characteristic (ROC) curve for ANGPTL-4 in a serum sample isolated from a target patient.
FIG. 2 shows the results of a Receiver Operating Characteristic (ROC) curve for C5a in a serum sample isolated from a target patient.

Hereinafter, the examples are only for explaining the present invention in more detail, and those of ordinary skill in the art to which the present invention pertains that the scope of the present invention is not limited by these examples according to the gist of the present invention It will be self-evident for

Example 1. Confirmation of ANGPTL-4 and C5a expression levels in samples

In order to develop a biomarker for diagnosing acute pancreatitis according to the severity in humans, the present inventors first confirmed the levels of ANGPTL-4 and C5a in the blood serum of patients with pancreatitis.

Blood samples from 98 normal patients and 90 patients with acute pancreatitis were provided from Chonbuk National University Hospital, Kyungpook National University Hospital, and Inha University Hospital.

All blood samples were collected according to procedures approved by the Institutional Review Board (IRB) of each medical school with written consent. Samples of patients with acute pancreatitis were obtained from 90 patients diagnosed as mild patients through imaging and cytopathological methods.

The blood sample was centrifuged at 12000 rpm at 4° C. for 10 minutes to obtain serum, and ANGPTL-4 and C5a levels in the serum sample were measured using an ELISA method.

Briefly, the ELISA method was as follows: Human ANGPTL4 and C5a were purchased from R&D (ELISA kit) and used. For ANGPTL4 and C5a, the serum separated from blood in a 96-well plate coated with a primary antibody (1 ug) was diluted 1/2 to 50 μl, and 100 μl was put together with the reference protein, and reacted at 4° C. overnight. The sample and the reference solution were discarded, 100 μl of the detection antibody (100 ng) was added, and the reaction was performed at room temperature for 1 hour. The reaction solution was discarded, washed 3 times with washing buffer, 100 μl HRP solution was added, and the reaction was performed at room temperature for 30 minutes. The reaction solution was discarded, washed 3 times with washing buffer, 100 μl TMB substrate was added, and the reaction was performed for 10-30 min after blocking the light. When it started to turn yellow, 50ul of a stop solution was added to stop the reaction, and then the absorbance was measured at 450 nm using a spectrometer.

Example 2. Analysis of ANGPTL-4 and C5a expression levels in patients with acute pancreatitis

The present inventors analyzed the diagnostic power of each marker by performing logistic regression analysis to verify the feasibility and validity of ANGPTL-4 and C5a as diagnostic markers for mild acute pancreatitis based on the measurement results of Example 1.

In addition, ROC (Receiver Operating Characteristic) curve analysis was performed for each protein to calculate specificity, sensitivity, and accuracy (AUC).

As a result, as shown in FIG. 1 , the AUC value of ANGPTL-4 was 0.998; As shown in FIG. 2 , the AUC value of C5a was also 0.998, indicating that all of them were significant.

In addition, the present inventors applied a logistic regression analysis model to link protein quantitative analysis data and patient clinical information with cut-off values to investigate the possibility of diagnosing mild acute pancreatitis.

At this time, since the ANGPTL-4 and C5a levels are continuous measurement values, in order to obtain the optimal cut-off value, which is judged to be highly likely to be mild acute pancreatitis, Youden's J statistic (Youden's index = sensitivity) +specificity-1) was used.

As a result, in the case of ANGPTL-4, the optimal cut-off value by Youden index was 40.98 ng/ml, and the sensitivity and specificity were 97% and 97%, respectively; For C5a, the optimal cut-off value by Youden index was 5.44 ng/ml, with sensitivity and specificity of 100% and 96%, respectively.

That is, it can be said that the accuracy (AUC), specificity, and sensitivity all increased.

These results show that when ANGPTL-4 and C5a are used in combination, they can be particularly usefully used for the early diagnosis and mild diagnosis of acute pancreatitis.

Therefore, if the cut-off values for diagnosing a patient with mild acute pancreatitis are ANGPTL-4 40.98 ng/ml and C5a 5.44 ng/ml, it is possible to diagnose a patient with mild acute pancreatitis.

Claims (7)

A method of providing information for diagnosing the severity of acute pancreatitis, comprising the steps of:
(a) the expression level of angiopoietin like 4 (ANGPTL-4) protein in a biological sample isolated from a subject; and measuring the expression level of the C5a (complement component 5a) protein, respectively; and
(b) the cut-off value calculated as a result of logistic regression analysis by comparing the expression level of each protein in the biological sample isolated from the subject with the expression level of each protein in the normal group sample is ANGPTL-4 40.98 ng/ml for C5a and 5.4 ng/ml for C5a, predicting or diagnosing the subject as having mild acute pancreatitis.
delete According to claim 1,
When the calculated cut-off value in step (b) is 40.98 ng/ml for ANGPTL-4 and 5.4 ng/ml for C5a, the subject has a sensitivity of 97 to 100% and a specificity (specificity) ) is 96 to 98%, characterized in that it is diagnosed as mild acute pancreatitis,
An informational method for diagnosing the severity of acute pancreatitis.
According to claim 1,
If the cut-off value calculated in step (b) is less than 40.98 ng/ml for ANGPTL-4 and less than 5.4 ng/ml for C5a, the subject is normal or does not have mild acute pancreatitis characterized by diagnosing
An informational method for diagnosing the severity of acute pancreatitis.
According to claim 1,
The biological sample is characterized in that at least one sample selected from the group consisting of whole blood, serum, plasma, saliva, urine, sputum, lymph and cells isolated from a subject,
An informational method for diagnosing the severity of acute pancreatitis.
According to claim 1,
Methods for measuring the expression level of the protein include Western blot, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion method, rocket ( rocket) 1 selected from the group consisting of immunoelectrophoresis, tissue immunostaining, Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Cell Sorter (FACS) and protein chip characterized in that it is a method of more than one species,
An informational method for diagnosing the severity of acute pancreatitis.
A diagnostic kit that provides information on the severity of acute pancreatitis by the method according to any one of claims 1 and 3 to 6.

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