CD53 as biomarker for diagnosis and prognosis of acute pancreatitis, CD53 neutralizing antibody and application of CD53 neutralizing antibody

Abstract

The invention belongs to the field of biological medicine, and particularly relates to CD53 serving as a biomarker for diagnosis and prognosis of acute pancreatitis, a CD53 neutralizing antibody and application of the CD53 neutralizing antibody. The research proves that the expression level of the CD53 plays a key role in acute pancreatitis, and the CD53 can be used as a marker of the acute pancreatitis for the first time and is related to the prognosis of a patient with the acute pancreatitis, so that the CD53 can be used as a biomarker for diagnosis and prognosis judgment of the acute pancreatitis. The research proves that the CD53 neutralizing antibody can obviously reduce inflammatory injury in pancreatic tissues of mice and formation of NETs in the tissues. CD53 is expected to be developed into an effective biomarker for early diagnosis or prognosis judgment of acute pancreatitis, and the CD53 neutralizing antibody can be used as an effective medicine for preventing or treating acute pancreatitis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to CD53 as a biomarker for the diagnosis and prognosis of acute pancreatitis, CD53 neutralizing antibodies and applications thereof. Background Art

[0002] Acute pancreatitis (AP) is an inflammatory disease of the pancreas characterized by pathological activation of digestive enzymes and self-digestion of pancreatic acinar cells. Most AP patients have mild symptoms and can resolve on their own. Repeated or excessive acinar cell damage causes pancreatic necrosis and releases a large number of proinflammatory mediators and inflammatory chemokines, which can lead to pancreatic necrosis infection, systemic inflammatory response, and multiple organ failure, and can progress to SAP, which is a serious disease with a mortality rate of up to 20-40%. Dysregulation of immune system activation is crucial in the progression and pathogenesis of SAP. Studies have shown that in the early stages of AP, activated immune cells capture microorganisms and release bactericidal enzymes to relieve pancreatic inflammatory responses; if the inflammatory response related to immune cells is not cleared in time, it will aggravate pancreatic damage and cause systemic inflammatory responses. Therefore, it is urgent to find new targets for targeting immune cell infiltration and inflammatory mediator release to alleviate the severity of AP.

[0003] Neutrophils are the most numerous immune cells in the blood circulation and the final effector cells of acute inflammatory responses. They play a major role in clearing extracellular pathogens. During AP pancreatic tissue damage, neutrophils are the first to migrate from the circulating blood to the site of injury, and activated neutrophils form neutrophile extracellular traps (NETs). NETs play a vital role in the development of AP, widely mediating AP inflammatory responses and aggravating the severity of AP. The level of NETs formation is also positively correlated with the clinical prognosis of AP. Targeting NETs formation and its key components can significantly alleviate pancreatic tissue damage and inflammatory responses in AP mice, reduce damage to distant organs, and improve the survival rate of mice.

[0004] CD53 (also known as MOX44 or TSPAN25) is a member of a new family of molecules with four transmembrane domains. It belongs to the pan-leukocyte antigen of the tetraspanin superfamily and is widely expressed in leukocytes, including B cells, T cells, monocytes and granulocytes. At present, CD53 has important functions in the immune system and is related to B cell development, lymphocyte trafficking, inflammatory diseases and tumor diseases. Studies have shown that CD53 plays a role in early B cell development and lymphocyte recirculation by stabilizing L-selectin on the surface of lymphocytes. In addition, CD53 can affect cell activation by inhibiting α3 integrin, resulting in increased cell adhesion, promoting leukocyte adhesion, extravasation and aggregation to the endothelium at the site of inflammation.

[0005] So far, there is no report on the function of CD53 in releasing NETs to promote AP, and there is no existing technology to show its related prevention and treatment effects in acute pancreatitis. Therefore, if an effective marker for diagnosing AP can be found, it will play a positive role in promoting the prevention and treatment of AP. Summary of the invention

[0006] In view of the deficiencies of the prior art, the present invention provides CD53 as a biomarker for the diagnosis and prognosis of acute pancreatitis, CD53 neutralizing antibodies and their applications. The present invention studies and confirms the key role of CD53 expression level in acute pancreatitis. It is the first time that CD53 is found to be a marker for acute pancreatitis and is related to the prognosis of patients with acute pancreatitis. Therefore, it can be used as a biomarker for the diagnosis and prognosis of acute pancreatitis.

[0007] The technical solution provided by the present invention is as follows:

[0008] The present invention provides use of CD53 as a diagnostic marker in preparing a product for diagnosing acute pancreatitis. The product includes a test paper or a test strip, a detection chip or a test kit.

[0009] Further, the diagnosis of acute pancreatitis comprises the following steps: (1) Collect samples from test subjects and control samples; (2) detecting and comparing the expression level of CD53 in the test subject sample and the control sample; If the expression level of CD53 in the sample of the test subject is increased compared with the expression level of CD53 in the control sample, the test subject is diagnosed as having acute pancreatitis or being at risk of having acute pancreatitis.

[0010] The present invention provides use of CD53 as a prognostic marker in the preparation of a product for prognosis judgment of acute pancreatitis. The product comprises a detection test paper or a test strip, a detection chip or a test kit.

[0011] Furthermore, the prognosis of acute pancreatitis comprises the following steps: (1) Collect samples from patients with acute pancreatitis as the test group, and samples from patients with acute pancreatitis before the diagnosis as the control group; (2) Detect and compare the expression levels of CD53 in the samples of the test group and the control group; If the expression level of CD53 in the test group samples is lower than the expression level of CD53 in the control group samples, it is judged that the prognosis of the test group is good.

[0012] Furthermore, the samples suitable for detecting the prognosis and pre-prognosis samples of patients with acute pancreatitis are blood, plasma or serum from peripheral blood.

[0013] Furthermore, the samples suitable for detection of the samples from patients with acute pancreatitis are neutrophils or serum from peripheral blood.

[0014] The present invention provides use of a CD53 neutralizing antibody in preparing a medicament for preventing and / or treating acute pancreatitis. The CD53 neutralizing antibody Anti-CD53 downregulates the expression level of CD53.

[0015] Furthermore, the drug is a drug that reduces the inflammatory response by down-regulating the expression of serum inflammatory factors.

[0016] Furthermore, the drug is a drug that improves pancreatic tissue damage by inhibiting the formation of neutrophil extracellular traps.

[0017] Furthermore, the drug is a drug for reducing serum amylase and serum lipase levels.

[0018] Beneficial Effects

[0019] 1. The present invention confirms the key role of CD53 expression level in acute pancreatitis. It is the first time that CD53 can be used as a marker for acute pancreatitis. It conducts in-depth research on the entire process of acute pancreatitis, thereby diagnosing acute pancreatitis early and / or taking measures to prevent the occurrence of acute pancreatitis. It has excellent accuracy, specificity and sensitivity, and has important scientific significance and clinical value.

[0020] 2. The present invention has complete follow-up data of acute pancreatitis, and has initially established a blood sample library of patients with acute pancreatitis, currently totaling more than 200 cases; and has skillfully constructed an animal model of acute pancreatitis. Through experimental verification, the diagnostic marker CD53 of the present invention is highly expressed in the peripheral blood of patients with acute pancreatitis and in the neutrophils of the pancreatic tissue of mice, and is positively correlated with the clinical prognosis of patients with acute pancreatitis.

[0021] 3. The present study confirmed that CD53 neutralizing antibodies can significantly inhibit inflammatory damage and NETs formation in mouse pancreatic tissue.

[0022] Therefore, CD53 is expected to be developed as an effective biomarker for early diagnosis or prognosis of acute pancreatitis, and CD53 neutralizing antibodies can be used as effective drugs for the preparation of prevention or treatment of acute pancreatitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a study on the expression of CD53 in the PMA-induced in vitro NETs model;

[0024] Figure 1 (A) is a schematic diagram of the analysis of two sets of transcriptome sequencing data after PMA (100 nM) stimulation for 4 h using a Venn diagram;

[0025] Figure 1 (B) is a schematic diagram of the analysis of two groups of transcriptome sequencing data stimulated by PMA (100 nM) for 4 hours using volcano plots and heat maps;

[0026] Figure 1 (C&D) are schematic diagrams of the results of detecting the protein level of CD53 in neutrophils by Western blotting and the corresponding semi-quantitative analysis;

[0027] Figure 1 (E) is a schematic diagram of the results of detecting the proportion of neutrophils and the expression level of CD53 by flow cytometry;

[0028] Figure 1 (F) is a schematic diagram showing the changes in the mRNA level of CD53 after neutrophils were stimulated with PMA (100 nM) for different time gradients (1h, 2h, 3h and 4h) by RT-PCR;

[0029] Figure 2 This is a study on the expression of CD53 in peripheral blood neutrophils of AP patients;

[0030] Figure 2 (A) is a schematic diagram showing the results of detecting the expression level of CD53 in human peripheral blood neutrophils by flow cytometry;

[0031] Figure 2 (BD) Graphpad prism was used to detect CD53 in different groups. + Schematic representation of neutrophil percentage;

[0032] Figure 2 (E) is a schematic diagram of the correlation coefficient heat map analysis of neutrophil CD53 levels and other clinical indicators in AP patients;

[0033] Figure 2 (F) ROC curve analysis of CD53 + Schematic diagram of the diagnostic value of PMN, PMN, and CRP levels for MODS;

[0034] Figure 3 It is a study on the expression of CD53 in AP mice;

[0035] Figure 3 (A) is a schematic diagram of the results of immunofluorescence detection of CD53 and MPO in mouse pancreatic tissue in the caerulein-induced AP mouse model;

[0036] Figure 3 (B) is a schematic diagram of the densitometric analysis of CitH3 and MPO fluorescence by ImageJ;

[0037] Figure 4 is the application of CD53 neutralizing antibody (Anti-CD53) in PMA-induced NETs;

[0038] Figure 4 (A) is a schematic diagram of the results of flow cytometry detection of MPO and ROS expression levels in neutrophils stimulated with PMA (100 nM) and incubated with different doses of Anti-CD53 (10, 20 and 40 ng / ml);

[0039] Figure 4 (B) is a schematic diagram of the detection of CitH3 and MPO fluorescence in neutrophils and the magnification of neutrophils by immunofluorescence and scanning electron microscopy;

[0040] Figure 4 (C) Schematic diagram of densitometric analysis of CitH3 and MPO fluorescence by ImageJ.

[0041] Figure 4 (D) is a schematic diagram of the results of detecting supernatant IL-6, TNF-α and MCP-1 levels by ELISA;

[0042] Figure 5 is the application of CD53 neutralizing antibody (Anti-CD53) in PMA-induced NETs;

[0043] Figure 5 (A) is a schematic diagram of the results of detecting the protein level of PAD4 in neutrophils by Western blotting;

[0044] Figure 5 (B) is a histogram of the relative protein expression of PAD4 analyzed by ImageJ;

[0045] Figure 6 The application of CD53 neutralizing antibody (Anti-CD53) in the prevention / treatment of acute pancreatitis;

[0046] Figure 6 (A) is a schematic diagram of detecting the pancreas of AP mice by HE staining and immunofluorescence staining;

[0047] Figure 6 (B) is a schematic diagram of the results of detecting serum amylase and lipase levels using an ELISA reader;

[0048] Figure 6 (C) Schematic diagram of densitometric analysis of CitH3 and MPO fluorescence by ImageJ.

[0049] Figure 6 (D) is a schematic diagram of the analysis of pathological scores of pancreatic tissues using Graphpad prism;

[0050] Figure 6 (E) is a schematic diagram of the results of ELISA detection of supernatant IL-6, TNF-α and MCP-1 levels. DETAILED DESCRIPTION

[0051] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods. The provided embodiments are only for illustrating the method of the present invention, and are not intended to limit the rest of the contents disclosed by the present invention in any way.

[0052] The experimental methods used in the examples of the present invention are conventional methods unless otherwise specified. The reagents and materials used in the examples can be purchased conventionally, and the quantitative experiments involved in the examples are repeated at least three times, and the results are averaged.

[0053] The C57BL / 6 mice used were purchased from Nanjing Jicui Yaokang Biotechnology Co., Ltd.

[0054] The caerulein used was purchased from NJPeptide Company with the product number 22-09-26.

[0055] The phorbol ester (PMA) used was purchased from MCE Company with the product number HY-18739.

[0056] The CD53 neutralizing antibody (Anti-CD53) used was purchased from Biolend with catalog number 124701.

[0057] Example 1 Study on the expression of CD53 in PMA-induced in vitro NETs model

[0058] PMA-induced mouse bone marrow-derived neutrophil NETs model.

[0059] Neutrophils were extracted from the bone marrow of C57BL / 6J male mice. Freshly isolated bone marrow neutrophils were divided into 1×10 7 / ml were inoculated in a 12-well plate and cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) at 37°C and 5% CO2 incubator for 30 minutes. Then 100nM PMA was given to establish an in vitro NETs model, and the control group was given an equal amount of PBS. The cells were collected 4 hours after modeling, and the mRNA of the mouse bone marrow neutrophil group before and after PMA induction was extracted for RNA-Seq analysis. In order to reduce batch differences, we jointly analyzed the two batches of NETs model RNA-Seq databases of the research group and found that there were 16,118 differentially expressed genes overlapping in the two sets of data. Further analysis revealed significant changes in multiple cell surface molecules. We combined the P value and Log2FoldChange changes, and finally screened through volcano plots and heat map analysis and found that CD53 increased significantly after PMA-induced NETs formation ( Figure 1 AB). To further verify the expression changes of CD53 in NETs formation, we extracted proteins for WB analysis, flow cytometry analysis and RT-PCR experimental verification. Figure 1 As shown in (CD), CD53 protein level increased significantly 4 hours after PMA modeling. Mouse bone marrow-derived neutrophils were stimulated with PMA (100 nM) for 4 hours and then stained for flow cytometry analysis. Figure 1 (E) As shown, bone marrow neutrophils (CD45.2 + CD11b + 6G + Representative flow cytometry gating of NETs. Representative flow cytometry plots and bar graphs depicting the proportion of neutrophils and CD53 expression levels. CD53 is significantly highly expressed in the NETs network structure. Figure 1 As shown in (F), after stimulation with PMA (100 nM) for different time gradients (1h, 2h, 3h and 4h), RT-PCR analysis showed that the mRNA level of CD53 was significantly increased. The results showed that the expression level of CD53 in PMA-induced NETs was significantly upregulated compared with the control group.

[0060] Example 2 Study on the expression of CD53 in peripheral blood neutrophils of AP patients.

[0061] Blood samples were collected from patients with acute pancreatitis in the Affiliated Hospital of Yangzhou University and healthy controls for isolation of neutrophils.

[0062] Anticoagulated blood samples were extracted from patients with acute pancreatitis (AP) and healthy controls in the Affiliated Hospital of Yangzhou University. Neutrophils were separated using a separation kit and analyzed by flow cytometry. Figure 2 As shown in (A), the level of CD53 on peripheral blood neutrophils in AP patients was significantly increased compared with that in the healthy control group. According to the modified Marshall scoring criteria, AP patients were divided into AP without MODS (Non-MODS AP) group and AP with MODS (MODS AP) group. According to whether local complications or systemic inflammatory response occurred, AP patients were divided into the group without local complications (Non-LC) group and the group with local complications (LC), the group without systemic inflammatory response (Non-SIRS) group and the group with systemic inflammatory response (SIRS). Figure 2 As shown in (BD), we found that the expression level of CD53 on peripheral blood neutrophils in AP patients with poor prognosis was significantly higher than that in the control group, and was correlated with the severity of the disease. Figure 2 As shown in (E), variable correlation heat map analysis showed that the level of CD53 in peripheral blood neutrophils of AP patients was positively correlated with the level of C-reactive protein (CRP). Figure 2 (F) We found that CD53 + Neutrophils are more accurate and sensitive in predicting the progression of AP patients with MODS. + Neutrophils have diagnostic value for MODS. The results showed that CD53 expression level was significantly upregulated in patients with AP and was correlated with the severity of the disease.

[0063] Example 3 Study on the expression of CD53 in AP mice

[0064] A caerulein-induced acute pancreatitis model in mice.

[0065] Experimental animals were 6-8 week old male C57 / BL6J mice (average weight 25 g).

[0066] The model group mice were intraperitoneally injected with caerulein (100ug / kg, 1 hour interval, 10 injections in a row) to establish an acute pancreatitis model. The control group mice were given a strict PBS control intraperitoneal injection. Twelve hours after the first injection of caerulein, the mice were anesthetized and killed with ZOLETIL (Virbac). The pancreatic tissue was harvested and the samples were fixed with 4% paraformaldehyde for 48 hours, embedded in paraffin blocks, sectioned, and then immunofluorescence staining was performed. Figure 3 As shown in (AB), immunofluorescence detection showed that the co-localization of CD53 and MPO in the damaged pancreatic tissue of the AP group was significantly higher than that of the control group, indicating that CD53 was mainly highly expressed in neutrophils after AP. The results showed that CD53 was significantly highly expressed in the pancreatic tissue of AP mice.

[0067] Example 4 Application of CD53 neutralizing antibody (Anti-CD53) in PMA-induced NETs

[0068] PMA-induced mouse bone marrow-derived neutrophil NETs model.

[0069] Neutrophils from bone marrow of C57BL / 6J male mice were extracted and inoculated into 12-well plates at 1×107 / ml. The cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) at 37°C and 5% CO2 in an incubator for 30 minutes. The following grouping treatments were performed: Control group: cultured for 4 hours; PMA group: 100 nM PMA was added to each well and cultured for 4 hours; PMA group + Anti-CD53 low, medium and high concentration groups: 100 nM PMA and 10, 20, 40 ng / mL Anti-CD53 were added to each well and cultured for 4 hours.

[0070] Cells from each group were collected, and the production of MPO and ROS in mouse bone marrow neutrophils was detected by flow cytometry; the results are shown in Figure 4 As shown in (A), after PMA stimulation, the MPO and ROS production in the Anti-CD53 40ng / mL group was more significantly protected than in other groups. Figure 4 As shown in (BC), inhibition of CD53 can effectively reduce the release of NETs. The cell supernatants of each group were collected and the levels of inflammatory cytokines were detected. Figure 4 As shown in (D), the levels of inflammatory factors (IL6, TNF-α, MCP-1) in the Anti-CD53 40ng / mL group were also significantly lower than those in the PMA group. Figure 5 As shown in (AB), we extracted proteins for WB analysis. The protein level of PAD4 in neutrophils was significantly increased in the PMA group, and the protein level of PAD4 decreased after administration of Anti-CD53. The results showed that inhibition of CD53 significantly reduced the formation of NETs in vitro.

[0071] Example 5 Application of CD53 neutralizing antibody (Anti-CD53) in the prevention / treatment of acute pancreatitis

[0072] 1. Ceruin-induced acute pancreatitis model in mice

[0073] Experimental animals: 6-8 week old male C57BL / 6J mice (average weight 25 g).

[0074] The model group mice were injected with caerulein (100ug / kg, 1 hour interval, 10 injections in a row) to establish an acute pancreatitis model. The control group mice were given a strict PBS control intraperitoneal injection. The mice were perfused with the heart in vivo to remove the blood in the circulation. The pancreatic tissue was quickly extracted, part of which was used for protein extraction for WB experiment, and part of which was fixed and dehydrated, and paraffin sections and eosin staining were made.

[0075] 2. Intraperitoneal injection of Anti-CD53

[0076] Mice were randomly divided into NC group, NC+Anti-CD53 (160μg / kg) treatment group, AP group (Cae), AP+Anti-CD53 with different dose gradients (7 mice in each group). Anti-CD53 (40μg / kg, 80μg / kg, 160μg / kg) was injected intraperitoneally in the Anti-CD53 with different dose gradients groups after the first injection of caerulein, the Control group was injected intraperitoneally with the same dose of phosphate buffered saline (PBS) as in the other groups, and the AP group and the Control+Anti-CD53 (160μg / kg) treatment group were injected intraperitoneally with the same amount of IgG.

[0077] All mice were killed 12 hours after the first intraperitoneal injection of caerulein, and the mouse serum was collected for serum enzyme level detection. Subsequently, the mice underwent in vivo cardiac perfusion to remove the circulating blood, and the pancreatic tissue was quickly extracted for fixation and dehydration, and paraffin sections stained with HE and eosin were prepared.

[0078] The results are as follows Figure 6 As shown in (A) and (D), Anti-CD53 can significantly improve pancreatic tissue damage (pathological damage). Figure 6 As shown in (A) and (C), immunofluorescence detection revealed that the formation of NETs (Cith3 / MPO co-localization) in the pancreatic tissue of mice treated with Anti-CD53 was significantly reduced compared with that in the AP group. Figure 6 (B) Anti-CD53 can reduce the levels of serum amylase and lipase in AP. Figure 6 As shown in (E), Anti-CD53 also significantly reduced the levels of serum inflammatory factors (IL-6, TNF-α, MCP-1) in AP mice. The results showed that Anti-CD53 can protect AP mice from pancreatic damage.

[0079] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. Use of CD53 as a diagnostic marker in the preparation of a product for the diagnosis of acute pancreatitis, characterized in that: The products include test papers or test strips, test chips or test kits.

2. The use according to claim 1, characterized in that: The diagnosis of acute pancreatitis includes the following steps: (1) Collect samples from test subjects and control samples; (2) detecting and comparing the expression level of CD53 in the test subject sample and the control sample; If the expression level of CD53 in the sample of the test subject is increased compared with the expression level of CD53 in the control sample, the test subject is diagnosed as having acute pancreatitis or being at risk of having acute pancreatitis.

3. The use of CD53 as a prognostic marker in the preparation of a product for the prognosis judgment of acute pancreatitis, characterized in that: The products include test papers or test strips, test chips or test kits.

4. The use according to claim 3, characterized in that: The prognosis of acute pancreatitis comprises the following steps: (1) Collect samples from patients with acute pancreatitis as the test group, and samples from patients with acute pancreatitis before the diagnosis as the control group; (2) Detect and compare the expression levels of CD53 in the samples of the test group and the control group; If the expression level of CD53 in the test group samples is lower than the expression level of CD53 in the control group samples, it is judged that the prognosis of the test group is good.

5. The use according to claim 4, characterized in that: The samples suitable for detecting the prognosis and pre-prognosis samples of acute pancreatitis patients are blood, plasma or serum from peripheral blood.

6. The use according to claim 5, characterized in that: The samples suitable for detecting the prognosis and pre-prognosis samples of patients with acute pancreatitis are neutrophils or serum from peripheral blood.

7. Use of a CD53 neutralizing antibody in the preparation of a medicament for preventing and / or treating acute pancreatitis, characterized in that: The CD53 neutralizing antibody Anti-CD53 downregulates the expression level of CD53.

8. The use according to claim 7, characterized in that: The drug is a drug that reduces the expression of serum inflammatory factors to alleviate inflammatory response.

9. The use according to claim 7, characterized in that: The drug is a drug for improving pancreatic tissue damage by inhibiting the formation of neutrophil extracellular traps.

10. The use according to claim 7, characterized in that: The drug is a drug for reducing serum amylase and serum lipase levels.

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