Application of reagent for detecting expression quantity of HINT2 protein

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to application of a reagent for detecting the expression quantity of HINT2 protein. A new predictive factor which has clinical value and can be used for judging the state of the acute pancreatitis is obtained from a blood sample of a patient with the acute pancreatitis at the early stage. Clinical tests find that HINT2 can be used as an excellent factor for early prediction of SAP conditions, also shows a specific pre-judgment value for organ function failure, especially for prediction of respiratory failure, and has a certain prediction capability for local complications. The invention provides an accurate, effective and easy-to-obtain new method for predicting and evaluating the early severity of the SAP. The development of the SAP early warning diagnosis marker is helpful for early recognition of non-mild disease patients, realization of early intervention treatment and reduction of the case fatality rate of acute pancreatitis, and has a wide application prospect in clinical diagnosis and treatment of acute pancreatitis.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and particularly relates to the use of a reagent for detecting the expression level of HINT2 protein. Background Art

[0002] Acute pancreatitis (AP) is a disease characterized by the activation of pancreatic enzymes caused by various pathological factors in vivo and in vitro, followed by an inflammatory reaction in the pancreas itself and its surrounding or the whole body, with or without damage to other extrapancreatic organ functions. AP is a common acute abdominal disease in clinical practice, with a global incidence rate of 13-45 per 100,000, which is the pancreatic disease with the highest incidence rate. Approximately 70-80% of patients with AP are diagnosed with mild acute pancreatitis (MAP), which has a mild condition, a short course, and a good prognosis. Generally, it can gradually recover within 1-2 weeks after systematic and regular treatment, and the overall mortality rate is less than 1%. However, another approximately 20% of patients develop into moderately severe acute pancreatitis (MSAP) or severe acute pancreatitis (SAP), accompanied by local or systemic complications, single or multiple organ system function damage, a critical condition, a poor prognosis, and a mortality rate as high as 36-50%. It can be seen that the prognosis of AP is closely related to the severity of the disease. Timely, rapid, and accurate prediction of the disease severity is a key link in the effective treatment of AP. It not only helps doctors formulate treatment plans early and allocate medical resources reasonably, which is crucial for reducing the incidence of AP complications and lowering the mortality rate of patients, but also helps improve the prognosis of patients, shorten the hospital stay, and reduce the economic burden.

[0003] Early prediction and assessment of the severity of SAP are currently hot topics and difficult problems in the research field of AP. Currently, the prediction and assessment of the severity of AP include single factors such as CRP, PCT, MCP-1, and pro-inflammatory cytokines, as well as scoring systems that comprehensively consider multiple clinical indicators. Therefore, the importance of early disease assessment is self-evident. Among these prediction and assessment systems, the Ranson score is the oldest and most well-known scoring system, which includes 5 indicators at the time of admission and 6 indicators 48 hours after admission. Some studies have shown that when the score ≥ 3, it indicates ABP, and the sensitivity and specificity are 96.4% and 96.7% respectively. Of course, the Ranson scoring standard also has certain limitations: it is usually difficult to observe 11 indicators within 48 hours of admission because AP has an acute onset and a rapid progression, and observing the condition for a long time will surely delay the treatment opportunity; and there is only one score within 48 hours after admission, so dynamic assessment cannot be carried out, which is not conducive to the early prediction of high-risk patients with SAP.

[0004] In addition, APACHE II, CTSI and BISAP scoring systems are also relatively common and frequently used evaluation systems. APACHE II can collect patient clinical data relatively comprehensively, but it has many observation indicators and takes a long time. Some indicators are not routine clinical indicators, and its results are non-specific for the occurrence and progression of AP. Therefore, its clinical application is relatively limited. The CTSI scoring system is a modified imaging evaluation system that takes the area of pancreatic necrosis into consideration on the basis of CT grading. It is the most important scoring system for AP imaging evaluation and is the most widely used clinically. However, the morphological structure, necrosis and abnormalities of the pancreas itself and adjacent tissues and organs are often not obvious in the early stages of the disease. Abdominal CT cannot judge the severity of AP in a timely and accurate manner. Therefore, when to conduct CT examinations and dynamic reexaminations are difficult problems in imaging evaluation; and contrast agents may damage microcirculation, increase the burden on the kidneys, and aggravate the condition.

[0005] This demonstrates that various scoring systems have their own strengths and weaknesses, and relying solely on a single scoring system cannot accurately assess and predict the severity and prognosis of AP. Therefore, further research is needed to identify optimal factors for the early prognosis of SAP. The search for new acute pancreatitis markers, particularly those for early warning diagnosis, is crucial for identifying patients with more severe symptoms, enabling rigorous testing, early intervention, and reducing the mortality rate of acute pancreatitis. Summary of the Invention

[0006] In order to solve the above problems existing in the prior art, the object of the present invention is to provide a use of a reagent for detecting the expression level of HINT2 protein.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention provides use of a reagent for detecting HINT2 protein expression in preparing a kit for screening severe acute pancreatitis.

[0009] The present invention also provides use of a reagent for detecting the expression level of HINT2 protein in preparing a kit for screening acute pancreatitis organ failure.

[0010] Furthermore, the organ failure caused by acute pancreatitis is one or more of respiratory failure, renal failure, and cardiovascular system failure.

[0011] Furthermore, the organ failure caused by acute pancreatitis is respiratory failure.

[0012] The present invention also provides use of a reagent for detecting the expression level of HINT2 protein in preparing a kit for screening local complications of acute pancreatitis.

[0013] Furthermore, the local complications of acute pancreatitis are one or more of acute peripancreatic fluid collection, pancreatic pseudocyst, acute necrotic collection, and walled-off necrosis.

[0014] Furthermore, the local complications of acute pancreatitis are one or both of acute peripancreatic fluid collection and acute necrotic collection.

[0015] Furthermore, the reagent for detecting the expression level of HINT2 protein is a reagent for detecting the expression level of HINT2 protein in peripheral blood.

[0016] Furthermore, the peripheral blood is the peripheral blood of patients with acute pancreatitis.

[0017] Furthermore, the reagent is a reagent for immunoblotting, enzyme-linked immunosorbent assay, immunofluorescence, mass spectrometry, or immunoprecipitation.

[0018] The present invention has achieved the following beneficial effects:

[0019] The present invention obtains a new clinically valuable predictor from the blood samples of patients with acute pancreatitis in the early stage of onset, which can be used to judge the severity of acute pancreatitis. Through clinical trials, the present invention finds that HINT2 can be an excellent factor for early prediction of the severity of SAP. The present invention provides an accurate, effective, and easily accessible new method for the early prediction and evaluation of the severity of SAP. Developing markers for early warning diagnosis of SAP helps to identify non-mild patients at an early stage, achieve early intervention and treatment, reduce the mortality rate of acute pancreatitis, and has broad application prospects in the clinical diagnosis and treatment of acute pancreatitis.

[0020] The present invention finds that HINT2 also shows specific predictive value for organ failure in AP, especially for the prediction of respiratory failure, and the predictive efficacy for respiratory failure is significantly better than that for renal failure.

[0021] The present invention finds that HINT2 can also be used as a predictor for local complications of AP.

[0022] The key of the present invention is that it is determined that the content of HINT2 in the blood samples of patients with acute pancreatitis is significantly correlated with the severity of patients with acute pancreatitis, significantly correlated with the risk of organ failure in AP, and significantly correlated with the risk of local complications of AP. Therefore, the risk can be judged by detecting the content of HINT2 in human blood. As for the specific means of detecting HINT2 in human blood, various means disclosed in the prior art can be adopted. In the embodiments of the present invention, immunoblotting is specifically used for detection, but it is not limited to this means. Any method capable of detecting the content of HINT2 can be used for the above screening.

[0023] Obviously, based on the above content of the present invention, according to the common general technical knowledge and conventional means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions or changes can also be made.

[0024] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. Description of the Drawings

[0025] Figure 1 Comparison of the expression of HINT2 in the peripheral blood of AP patients in the MAP, MSAP, and SAP groups.

[0026] Figure 2 ROC curves for predicting SAP with different indicators.

[0027] Figure 3 ROC curves for predicting organ failure of AP with different indicators.

[0028] Figure 4 ROC curves for predicting respiratory failure and renal failure of AP by HINT2.

[0029] Figure 5 ROC curves for predicting local complications of AP with different indicators. Detailed Description of the Invention

[0030] The reagents and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

[0031] The following is an explanation of the grading criteria for the severity of acute pancreatitis, the definition of organ failure and local complications:

[0032] 1. Grading criteria for the severity of AP, according to the revised 2012 Atlanta criteria:

[0033] 1) Mild acute pancreatitis (MAP): Diagnosed as acute pancreatitis, without organ failure, and without local or systemic complications;

[0034] 2) Mild severe acute pancreatitis (MSAP): Diagnosed as acute pancreatitis, with transient organ failure (the duration of organ failure ≤ 48 h), or accompanied by local complications of the pancreas;

[0035] 3) Severe acute pancreatitis (SAP): Diagnosed as acute pancreatitis with persistent organ failure (organ failure > 48 hours).

[0036] 2. Diagnostic criteria for local complications:

[0037] 1) Acute peripancreatic fluid collection (APFC)

[0038] Occurs in interstitial edematous pancreatitis; the fluid collection has a uniform density; is confined within the normal peripancreatic fascia; has no cyst wall encapsulation; is adjacent to the pancreas (not extending from within the pancreas);

[0039] 2) Pancreatic pseudocyst

[0040] Well-defined, often round or oval; the fluid has a uniform density; contains no non-fluid components; has a distinct cyst wall completely encapsulating it; usually forms 4 weeks after the onset of acute pancreatitis and occurs in interstitial edematous pancreatitis;

[0041] 3) Acute necrotic collection (ANC)

[0042] Occurs only in acute necrotizing pancreatitis; shows varying degrees of non-uniform non-fluid density shadows in different locations (partially uniform in the early stage of the disease course); the collection has no cyst wall encapsulation; location - within or outside the pancreas;

[0043] 4) Walled-off necrosis (WON)

[0044] Heterogeneous fluid and non-fluid density shadows (partially uniform), with varying degrees of small cavity formation; has a distinct cyst wall completely encapsulating it; location - within and / or outside the pancreas; usually forms 4 weeks after the onset of acute necrotizing pancreatitis.

[0045] 3. Diagnostic criteria for AP organ failure:

[0046] The 2012 revised Atlanta criteria for judging organ failure (OF) follow the modified Marshall score table.

[0047] A score of ≥ 2 in any one organ system is defined as OF. If the duration > 48 hours, it is defined as persistent OF. If the duration of OF is less than 48 hours, it is defined as transient OF.

[0048] Table 1 Marshall Scoring Criteria for Organ Failure in Acute Pancreatitis

[0049]

[0050] The protein determination method involved in the examples is Western blotting, and the specific operation is as follows:

[0051] 1. Collect peripheral blood specimens, centrifuge at 3000 rpm for 5 min, separate the serum and store it in a -20°C refrigerator for future measurement.

[0052] 2. Measure the total protein concentration by the BCA method: Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance value at A562 nm, and calculate the total protein concentration in the tissue according to the obtained standard curve;

[0053] 3. Specific operation steps of WB:

[0054] 1) Prepare the sample: Take out the sample, add 5× loading buffer, and heat it at 100°C for 10 min for denaturation;

[0055] 2) Prepare the gel and load the sample: Prepare a 12% SDS-PAGE separating gel and a 5% stacking gel according to a certain ratio. After about half an hour, use a pipette to load 10 μl of the sample and 5 μl of the prestained protein marker;

[0056] 3) Electrophoresis: Place the prepared gel in the electrophoresis apparatus, pour in the prepared electrophoresis buffer, and make the electrophoresis buffer cover the top of the gel plate. First, run the stacking gel at 80 V for 30 min. When the stacking gel contacts the separating gel, adjust the voltage to a constant voltage of 120 V for 60 min;

[0057] 4) Transfer: Prepare the transfer buffer, open the gel plate, cut out the gel containing the target protein and transfer it to the transfer sandwich containing the transfer buffer. According to the length of the tape, cut a 0.22 PVDF membrane. Immerse the PVDF membrane in formaldehyde solution for 1 minute, then take it out and cover it on the target protein gel. Arrange the soaked filter paper and sponge in a "sandwich" arrangement of black sponge, filter paper, protein gel, PVDF membrane, filter paper, black sponge from the negative electrode to the positive electrode, and clamp it in the transfer plate (note to remove air bubbles). Place it in a 4°C chromatography cabinet and perform transfer at a constant current of 250 mA for 60 min;

[0058] 5) Blocking: Take out the successfully transferred PVDF membrane with forceps and put it into the prepared blocking solution, and block it on a shaker at room temperature for 1 hour;

[0059] 6) Primary antibody incubation: After blocking, rinse the PVDF membrane taken out from the blocking solution 3 times with TBST, 10 min each time. Add the primary antibody diluted 1:1000 and the internal reference antibody diluted 1:2000, and incubate overnight on a shaker in the chromatography cabinet;

[0060] 7) Secondary antibody incubation: After the primary antibody incubation is completed, wash the PVDF membrane taken out from the primary antibody incubation solution 3 times with TBST for 10 minutes each time. Select a secondary antibody diluted at 1:5000 and incubate it at room temperature for 60 minutes;

[0061] 8) Exposure and analysis: After the secondary antibody incubation is completed, wash the PVDF membrane taken out from the secondary antibody incubation solution 2 times with TBST for 10 minutes each time. After preparing the ECL luminescent A / B solution at a ratio of 1:1, drop it on the PVDF membrane placed in the automatic exposure machine and then scan and expose immediately. Use ImageJ software for band analysis, and represent the relative expression of the target protein by the ratio of the gray values of the target protein and the internal reference protein.

[0062] Select AP patients for the study of the present invention according to the following criteria:

[0063] 1. Inclusion criteria

[0064] 1) Meet the AP diagnostic criteria (revised 2012 Atlanta criteria), within 48 hours from the onset to admission;

[0065] 2) Voluntarily serve as a research subject and sign an informed consent form;

[0066] P) Aged 18 to 80 years old;

[0067] 2. Exclusion criteria

[0068] 1) Recurrent acute pancreatitis, with complications not yet healed after the last attack;

[0069] 2) History of chronic pancreatitis;

[0070] 3) AP caused by trauma;

[0071] 4) Complicated with pregnancy;

[0072] 5) Having a history of other infectious, autoimmune or hematological diseases in the past;

[0073] 6) Having a history of tumors;

[0074] 7) Complicated with severe disorders of multiple organs such as the heart, liver, lungs, kidneys, brain, etc. and / or mental disorders.

[0075] 3. Exclusion criteria

[0076] 1) Patients with a data integrity < 85%;

[0077] 2) Patients who are discharged automatically.

[0078] A total of 74 eligible AP patients were finally included in this study, including 50 males (67.6%) and 24 females (32.4%), with a male-to-female ratio of 2.08:1. The average age of the patients was 47.2 years. Among the AP patients, there were 27 cases (36.5%) of MAP, 26 cases (35.1%) of MSAP, and 21 cases (28.4%) of SAP. The differences in Example 1, HINT2 among the MAP, MSAP, and SAP groups

[0079] According to the revised 2012 Atlanta criteria, AP patients were divided into MAP, MSAP, and SAP groups according to the severity of the disease. The level of HINT2 protein (histidine triad nucleotide-binding protein 2) in the peripheral blood of AP patients after admission was detected by Western blotting and standardized with MAP (mild acute pancreatitis) as the benchmark. The results are as Figure 1 shown. Compared with the MAP group, the level of HINT2 in the peripheral blood of patients in the MSAP group was significantly decreased (p < 0.05); compared with the MSAP group, the level of HINT2 in the SAP group was significantly decreased (p < 0.05).

[0080] It is proved that the level of HINT2 in the peripheral blood of AP patients is negatively correlated with the severity of the disease and decreases with the aggravation of the AP disease degree.

[0081] Example 2, Evaluation of the predictive efficacy of different indicators for SAP

[0082] The inventors found that the level of HINT2 protein in the peripheral blood of AP patients after admission was significantly correlated with the risk of SAP and verified it through the following results: ROC curves were made with HINT2 (the level of HINT2 protein in the peripheral blood of AP patients after admission), Ranson score, and CTSI score as the predictive indicators for SAP respectively. The results are as Figure 2 shown. The AUC values of the above indicators were 0.820, 0.772, and 0.783 in turn. The higher the AUC value, the better the predictive value. The AUC value of HINT2 was above 0.8, indicating that it was an excellent predictor.

[0083] The above results indicate that screening for SAP can be achieved by detecting the level of HINT2 protein in the peripheral blood of AP patients.

[0084] Example 3, Evaluation of the predictive efficacy of different indicators for AP organ failure

[0085] The inventors found that the level of HINT2 protein in the peripheral blood of AP patients after admission was significantly correlated with the risk of AP organ failure and verified it through the following results: ROC curves were made with HINT2 and PCT as the predictive indicators for AP organ failure (respiratory failure, renal failure, and cardiovascular system failure) respectively. The results are as Figure 3As shown, the AUC values of the above indicators are 0.749 and 0.682 in sequence. Among them, the AUC value of HINT2 > 0.7, which is an effective factor for predicting AP organ failure.

[0086] The above results indicate that screening for AP organ failure can be achieved by detecting the HINT2 protein level in the peripheral blood of AP patients.

[0087] Example 4. Evaluation of the predictive efficacy of HINT2 for respiratory failure and renal failure

[0088] The inventors' research found that the HINT2 protein level in the peripheral blood of AP patients after admission was significantly correlated with the risk of AP respiratory failure, and the following results were used for verification: Given that the above research results showed the predictive role of HINT2 in AP organ failure, further predictions were made for respiratory failure and renal failure respectively to evaluate its organ specificity. The results are as Figure 4 shown. The AUC values of HINT2 for predicting respiratory failure and renal failure are 0.721 and 0.575 respectively. It shows that the predictive sensitivity and specificity of HINT2 for renal failure are inferior to those for respiratory failure, and it can be used as an effective predictive factor for evaluating respiratory failure.

[0089] The above results indicate that screening for AP organ failure by detecting the HINT2 protein level in the peripheral blood of AP patients has obvious specificity, and the predictive efficacy for respiratory failure is significantly better than that for renal failure.

[0090] Example 5. Evaluation of the predictive efficacy of different indicators for local complications of AP

[0091] The inventors' research found that the HINT2 protein level in the peripheral blood of AP patients after admission was significantly correlated with the risk of local complications of AP, and the following results were used for verification: ROC curves were made with HINT2, CRP, PCT, and Ranson score as the predictive indicators for local complications of AP (acute peripancreatic fluid collection, acute necrotic collection) respectively. The results are as Figure 5 shown. The AUC values of the above indicators are 0.774, 0.711, 0.713, and 0.704 in sequence. It can be seen that when predicting local complications of AP, the AUC value of HINT2 is the highest, and the sensitivity and specificity are the best, which is an excellent predictive factor for local complications of AP.

[0092] The above results indicate that screening for local complications of AP can be achieved by detecting the HINT2 protein level in the peripheral blood of AP patients.

[0093] In summary, the present invention obtains a new clinically valuable predictor from the blood samples of acute pancreatitis patients in the early stage of onset, which can be used to judge the severity of acute pancreatitis. Through clinical trials, the present invention finds that HINT2 can be an excellent factor for early prediction of SAP condition, and at the same time also shows specific predictive value for organ failure, especially for the prediction of respiratory failure, and has a certain predictive ability for local complications. The present invention provides an accurate, effective and easily accessible new method for predicting and evaluating the early severity of SAP. Developing markers for early warning diagnosis of SAP helps to early identify non-mild patients, achieve early intervention and treatment, reduce the mortality rate of acute pancreatitis, and has broad application prospects in the clinical diagnosis and treatment of acute pancreatitis.

Claims

1. Use of a reagent for detecting the expression level of HINT2 protein in the preparation of a kit for screening severe acute pancreatitis.

2. Use of a reagent for detecting the expression level of HINT2 protein in the preparation of a kit for screening organ failure in acute pancreatitis.

3. The use according to claim 2, characterized in that: The organ failure in acute pancreatitis is one or more of respiratory failure, renal failure, and cardiovascular system failure.

4. The use according to claim 3, characterized in that: The organ failure in acute pancreatitis is respiratory failure.

5. Use of a reagent for detecting the expression level of HINT2 protein in the preparation of a kit for screening local complications of acute pancreatitis.

6. The use according to claim 5, characterized in that: The local complications of acute pancreatitis are one or more of acute peripancreatic fluid collection, pancreatic pseudocyst, acute necrotic collection, and walled-off necrosis.

7. Use according to claim 6, characterized in that: The local complications of acute pancreatitis are one or both of acute peripancreatic fluid collection and acute necrotic collection.

8. The use according to any one of claims 1-7, characterized in that: The reagent for detecting the expression level of HINT2 protein is a reagent for detecting the expression level of HINT2 protein in peripheral blood.

9. The use according to claim 8, wherein: The peripheral blood is the peripheral blood of a patient with acute pancreatitis.

10. Use according to any one of claims 1 - 7, characterized in that: The reagent is a reagent for immunoblotting, enzyme-linked immunosorbent assay, immunofluorescence, mass spectrometry, or immunoprecipitation.

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