WO2021013214A1 - Use of neutrophilic granulocytes in preparation of drugs for treating and/or preventing autoimmune hepatitis - Google Patents

Abstract

The present invention relates to the field of medicine, and relates to use of neutrophilic granulocytes in preparation of drugs for treating and/or preventing autoimmune hepatitis. On one hand, the present invention provides the use of neutrophilic granulocytes in preparation of drugs for treating and/or preventing autoimmune hepatitis and use in preparation of drugs for clearing apoptotic hepatocytes. On the other hand, the present invention also provides an auxiliary diagnostic kit for the autoimmune hepatitis. The application of the present invention can provide a novel option for the clinic diagnosis and treatment of the autoimmune hepatitis; the pain caused by the autoimmune hepatitis on a patient is relieved; important social benefits and economic benefits are realized.

Description

Use of neutrophils in preparing medicine for treating and/or preventing autoimmune hepatitis Technical field

The invention relates to the use of neutrophils in the preparation of drugs for treating and/or preventing autoimmune hepatitis, and belongs to the field of medicine.

Background technique

Apoptosis is the process of programmed cell death, which removes aging or damaged cells to maintain internal tissue homeostasis. In adults, hundreds of billions of cells die every day. These cells must be processed quickly and efficiently so that the cytoplasm does not leak or cause inflammation. Clearance of apoptotic cell defects is often associated with a variety of inflammatory reactions and autoimmune diseases. It is generally believed that apoptotic cells are eliminated by professional phagocytic cells (such as macrophages, mature dendritic cells) or nearby non-professional phagocytic cells (such as epithelial cells, endothelial cells, and fibroblasts). Although the morphology of apoptotic cells can be easily distinguished in histological sections, due to the efficient clearance of phagocytes, apoptotic cells are rarely observed in normal human tissue sections. Therefore, the types of phagocytes responsible for clearing apoptotic cells in the steady state are not yet clear, and it is unknown whether they are tissue-specific.

Neutrophils account for about 50%-70% of human white blood cells. They are the highest proportion of white blood cells in the blood and are the body's first line of defense against infection and tissue damage. Neutrophils are a type of terminally differentiated and short-lived cells. They play the role of inflammatory cells responsible for killing bacteria and fighting infections. They are not considered to play a major role in eliminating apoptotic cells. Peripheral blood neutrophils recruit to the site of infection and release toxic mediators (for example, reactive oxygen species and proteases) at the site of infection, which can kill bacteria, but also have a certain toxic effect on tissues. To prevent inflammation, apoptotic cells release "forbidden" signals, such as lactoferrin, which prevents neutrophils from recruiting to the site of apoptosis.

Autoimmune disease (Autoimmune disease, abbreviated as AID) is an abnormal immune response in the human body that attacks normal cells. At least 80 kinds of autoimmune diseases are known to affect human health. Autoimmune hepatitis is a chronic liver inflammatory disease mediated by autoimmune response. Its clinical features are elevated serum transaminase, hypergamma-globulinemia, autoantibody positive, and histological features are lymphocytes, plasma Interface hepatitis dominated by cell infiltration can rapidly progress to cirrhosis and liver failure in severe cases. Therefore, early diagnosis and treatment of autoimmune hepatitis is an important issue that needs to be resolved urgently.

So far, there is no report about neutrophil phagocytosis of apoptotic hepatocytes, let alone the use of neutrophils to treat autoimmune hepatitis.

Summary of the invention

The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, one of the objectives of the present invention is to provide the use of neutrophils in the preparation of drugs for treating and/or preventing autoimmune hepatitis. Another object of the present invention is to provide an auxiliary diagnosis kit for autoimmune hepatitis.

The invention provides the use of neutrophils in the preparation of medicines for treating and/or preventing autoimmune hepatitis.

The invention provides the use of neutrophils in preparing medicines for clearing apoptotic liver cells.

Further, the medicine is a preparation prepared by adding pharmaceutically acceptable excipients or auxiliary components with neutrophils as active ingredients.

Further, the preparation is intravenous injection.

Further, the neutrophils are bone marrow neutrophils and/or peripheral blood neutrophils.

The present invention provides an auxiliary diagnosis kit for autoimmune hepatitis, which contains a reagent for detecting the number of neutrophils in apoptotic liver cells.

The present invention provides the use of the following reagents in preparing an auxiliary diagnosis kit for autoimmune hepatitis: the reagent is a reagent for detecting the number of neutrophils in apoptotic liver cells.

The present invention provides an auxiliary diagnosis kit for autoimmune hepatitis. The kit contains neutrophil markers, apoptotic liver cell markers, cell membrane markers and nuclear markers.

Further, the auxiliary diagnosis kit contains neutrophil elastase antibody, TUNEL stain, E-cadherin antibody and DAPI.

For liver tissue samples, use TUNEL to label apoptotic cells, neutrophil elastase antibody (NE) to specifically label neutrophils, E-cadherin antibody to label cell membranes, and DAPI to label cell nuclei. Perform immunofluorescence staining to observe neutrality. The phagocytosis of apoptotic hepatocytes by granulocytes is counted, which reflects the neutrophil phagocytic defect in patients with autoimmune hepatitis and provides a basis for the diagnosis of autoimmune hepatitis.

The present invention provides the use of the following reagents in the preparation of an auxiliary diagnosis kit for autoimmune hepatitis: the reagents contain neutrophil markers, apoptotic liver cell markers, cell membrane markers and cell nuclear markers.

Further, the reagent contains neutrophil elastase antibody, TUNEL stain, E-cadherin antibody and DAPI.

The present invention provides an auxiliary diagnostic instrument for autoimmune hepatitis, which contains a device for detecting the number of neutrophils in apoptotic liver cells.

The present invention provides the use of the following reagents in the preparation of an auxiliary diagnosis kit for autoimmune hepatitis: the reagents contain reagents for detecting IL-1β and/or IL-8 serum levels.

Further, the auxiliary diagnostic kit is an Elisa kit.

The present invention provides the use of the following device in the manufacture of an auxiliary diagnostic instrument for autoimmune hepatitis: the device is a device for detecting serum levels of IL-1β and/or IL-8.

The present invention provides the use of an active ingredient that up-regulates the serum level of IL-1β and/or IL-8 in the preparation of a medicine for treating and/or preventing autoimmune hepatitis.

The present invention provides neutrophils for the treatment and/or prevention of autoimmune hepatitis.

The present invention provides neutrophils for removing apoptotic liver cells.

Further, the neutrophils are bone marrow neutrophils and/or peripheral blood neutrophils.

The present invention provides an auxiliary diagnosis kit for autoimmune hepatitis, which contains reagents for detecting IL-1β and/or IL-8 serum levels.

Further, the auxiliary diagnostic kit is an Elisa kit.

The present invention provides drugs for the treatment and/or prevention of autoimmune hepatitis. The drugs up-regulate the serum levels of IL-1β and/or IL-8.

The present invention provides a method for treatment and/or prevention of autoimmune hepatitis: neutrophils are administered to patients in need.

The present invention provides a method for eliminating apoptotic liver cells: neutrophils are administered to patients in need.

Further, the neutrophils are bone marrow neutrophils and/or peripheral blood neutrophils.

The invention provides an auxiliary diagnosis method for autoimmune hepatitis: detecting the number of neutrophils in apoptotic liver cells.

The present invention provides an auxiliary diagnosis method for autoimmune hepatitis: detecting IL-1β and/or IL-8 serum levels.

The present invention provides a method for the treatment and/or prevention of autoimmune hepatitis: an active ingredient that up-regulates the serum level of IL-1β and/or IL-8 is administered to patients in need.

The present invention proves through experiments that IL-1β and IL-8 secreted by apoptotic L02 cells are the key signals to attract neutrophils and induce subsequent phagocytosis, and the down-regulation of IL-1β and IL-8 may be induced by autoimmune hepatitis One of the factors. Based on the results of this experiment, it is possible to develop corresponding clinical diagnosis methods for autoimmune hepatitis, routinely detect the serum levels of IL-1β and IL-8 in patients with autoimmune hepatitis to determine the severity of the disease, so as to give corresponding treatment in time. The method has stable technical performance, strong reproducibility, reliable results, and can quickly determine the condition of autoimmune hepatitis, which is of great significance to the development of clinical diagnosis and treatment of autoimmune hepatitis. In addition, the above experimental results also uncovered new therapeutic targets for autoimmune hepatitis, providing another effective way for autoimmune hepatitis drug screening.

The present invention provides a new use of neutrophils in addition to killing bacteria and fighting infections—clearing apoptotic liver cells, and finding the reason for the clearance defects of apoptotic liver cells in patients with autoimmune hepatitis, that is, the existence of patients with autoimmune hepatitis Neutrophils drill the defect of apoptotic hepatocytes. Based on this, the present invention proposes a more targeted diagnosis and treatment method for autoimmune hepatitis, which helps to alleviate the pain caused by autoimmune hepatitis to patients, and has important social and economic benefits. The present invention further explores the key steps for neutrophils to clear apoptotic hepatocytes, and makes an important contribution to the pathogenesis of autoimmune diseases and early diagnosis and treatment.

Description of the drawings

Figure 1 is a schematic diagram of neutrophils phagocytosis of apoptotic hepatocytes in a specific embodiment;

Figure 2 is a diagram of neutrophils clearing apoptotic hepatocytes in human normal liver tissue in Example 1;

Figure 3 is a statistical diagram of cell counts of neutrophils and Kupffer cells related to apoptotic liver cells in Example 1;

4 is a 3D reconstruction diagram of neutrophils phagocytosis of apoptotic liver cells in human normal liver tissue in Example 1;

Figure 5 is a diagram of immunostaining of liver sections with anti-Ly-6G and anti-E-cadherin antibodies in Example 1;

Figure 6 is an electron micrograph of neutrophil-related apoptotic hepatocytes in Example 1;

Fig. 7 is a CD68 staining diagram of a human liver sample with normal or AIL disease in Example 1;

Fig. 8 is a diagram showing the results of neutrophil-mediated clearance of apoptotic cells in tumor non-cancerous liver tissue and normal liver tissue of hemangioma in Example 1;

Fig. 9 is a picture of normal liver tissue stained and labeled with KCS using CD68 antibody in Example 1;

Figure 10 is a diagram of normal liver tissue stained and labeled with CD34 antibody in Example 1;

Figure 11 is a fluorescence image of the interaction between pkh67-labeled HL-60 cells and pkh26-labeled L02 cells in Example 2 at a specified time point;

Figure 12 is a diagram showing the quantitative detection results of in vitro phagocytosis of apoptotic L02 cells by neutrophils isolated from patients with normal or AIL diseases in Example 2;

Figure 13 is a diagram showing the in vitro phagocytosis of apoptotic L02 cells by neutrophils isolated from patients with normal or AIL disease in Example 2;

14 is a flow cytometric analysis of the results of HL-60 cells phagocytosing non-apoptotic or apoptotic L02 cells in Example 2;

15 is a comparison diagram of the phagocytic ability of apoptotic L02 cells and non-apoptotic L02 cells in Example 2;

16 is a diagram showing the analysis results of extracellular DNA, SOD and ROS during the phagocytosis of non-apoptotic or apoptotic L02 cells by HL60 cells in Example 2;

17 is a diagram of neutrophils burrowing into apoptotic hepatocytes under an intravital microscope in Example 3 and a diagram of Imaris analysis results;

18 is a diagram showing the peripheral blood neutrophil count of the control group and neutrophil-deficient mice in Example 3, and the result of the peripheral neutrophil count at a specified time after antibody injection;

Figure 19 shows the H&E and TUNEL staining images of the livers of mice in the neutrophil-deficient group and the control group in Example 3, and the results of cell counts of neutrophils in or related to apoptotic liver cells in tissue samples;

20 is a graph showing the cell count and apoptotic cell count results of macrophages in or related to apoptotic liver cells in the tissue sample of Example 3;

21 is a diagram of liver tissue stained with anti-CD11b antibody in a control group or a neutrophil-deficient mouse in Example 3 and a diagram of quantitative detection results;

Figure 22 is a graph showing the results of detection of liver function indexes of AST, ALT, ALP/AST ratio, DBIL and TBIL of the control group or neutrophil-deficient mice in Example 3;

Figure 23 is a diagram showing the detection result of neutrophil defect in Mrp8cre/DTR mice in Example 3;

24 is a graph showing the expression results of antinuclear antigen, smooth muscle actin, liver and kidney microsomes and total IgG in the serum of control group and neutrophil deficient mice in Example 3;

Figure 25 shows the H&E and TUNEL staining diagrams of the liver of the neutrophil-deficient group, ConA-induced AIL model mouse group, and control group in Example 3;

Figure 26 shows the expression of autoantibodies in the serum of the control group, ConA treatment group, and normal neutrophils intravenously injected into the ConA treatment group in Example 3: antinuclear antigen, smooth muscle actin, liver and kidney microsomes and total IgG expression Result graph

Figure 27 shows the phagocytosis of HL-60 cells on non-apoptotic L02 cells in Example 3, the phagocytosis of HL60 cells on apoptotic L02 cells, and the phagocytosis of HL60 cells on apoptotic L02 cells after treatment with ConA or Annexin v Test result diagram;

Fig. 28 is a TUNEL and HE staining image of a mouse liver tissue sample treated with ConA or ConA+PMN in Example 3 and a cell count result of apoptotic hepatocytes;

Figure 29 is a fluorescence image of the interaction of pkh67-labeled HL-60 cells with pkh26-labeled HUVEC and HEK293 cells at a specified time point in Example 4;

Figure 30 is a diagram showing the detection result of the clearance effect of macrophages on apoptotic hepatocytes in Example 4;

Figure 31 is a diagram of the migration of NE-specifically labeled neutrophils to apoptotic cells in Example 5;

Fig. 32 is a diagram showing the migration of MPO-specifically labeled neutrophils to apoptotic cells in the human normal liver tissue section of Example 5;

Figure 33 is a graph showing the expression results of cytokines IL-1β, IL-6, IL-8 and IL-12 in apoptotic L02 cells in Example 5;

Figure 34 is a graph showing the expression results of cytokines IFN-γ, TNF-α, IL-1α, IL-2, IL-4, IL-10 and GM-CSF in apoptotic L02 cells in Example 5;

Figure 35 is a graph showing the results of phagocytosis of non-apoptotic L02 cells by HL60 cells in Example 5, and phagocytosis of apoptotic L02 cells by HL60 cells without treatment or RNAi knockout of various cytokine receptors.

Detailed ways

Neutrophils (PMNs) can eliminate invading bacteria through phagocytosis, degranulation, release of reactive oxygen species (ROS), and formation of extracellular germicidal networks (NETs). They are responsible for killing bacteria and inflammatory cells that fight infection effect. It is generally believed that neutrophils do not play a major role in eliminating apoptotic cells. The inventors of the present invention discovered in the clinicopathological section that neutrophils phagocytosis of apoptotic liver cells. Discover the new functions of neutrophils in addition to killing bacteria and fighting infections, explore the mechanism of neutrophils phagocytosis of apoptotic cells, supplement and improve the immune function of neutrophils, remove apoptotic cells, maintain tissue homeostasis, and The treatment of autoimmune diseases is of great significance.

The present invention finds that neutrophils infiltrate in a non-inflammatory manner and clear apoptotic hepatocytes through phagocytosis (Figure 1), which is confirmed from the following five aspects: 1) Neutrophils in normal human liver tissues Cell phagocytic apoptotic hepatocytes, neutrophils in autoimmune hepatitis cases have phagocytic defects; 2) Neutrophils can eliminate apoptotic hepatocytes in vitro; 3) Establishing a mouse model of autoimmune hepatitis, under study The phagocytic effect of neutrophils; 4) The phagocytic effect of neutrophils on apoptotic hepatocytes is selective; 5) The mechanism of neutrophils to clear apoptotic hepatocytes.

The solution of the present invention will be explained below in conjunction with examples. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the examples, the procedures shall be carried out in accordance with the techniques or conditions described in the literature in the field or in accordance with the product specification. The reagents or instruments used without the manufacturer's indication are all conventional products that are commercially available.

Example 1 Neutrophils phagocytosis of apoptotic hepatocytes in normal human liver tissues, patients with autoimmune hepatitis have neutrophil phagocytosis defects

This example mainly compares normal human liver tissue samples and autoimmune hepatitis liver tissue samples to study the clearance effect of neutrophils on apoptotic liver cells and the neutrophil phagocytic defects in autoimmune liver cases, thereby revealing neutrality The new function of granulocytes-clear apoptotic cells.

1. Experimental design

(1) The phagocytosis of apoptotic liver cells by neutrophils in normal human liver tissues

In this part of the experiment, the non-cancerous liver normal tissues adjacent to the cancer were obtained from patients with liver cancer. 1) Tissue sections were performed on them, and HE staining and TUNEL staining were used to observe the degree of nuclear pyknosis and count the number of neutrophils and apoptotic cells. 2) Tissue sectioning of normal non-cancerous liver tissue, using TUNEL to label apoptotic cells, neutrophil elastase antibody (NE) to specifically label neutrophils, E-cadherin antibody to label cell membranes, and DAPI to label cell nuclei. Immunofluorescence staining was used to observe the phagocytosis of apoptotic cells by neutrophils and count the number; 3) Ultrathin sectioning of normal non-cancerous liver tissues, and transmission electron microscopy to observe the phagocytosis of neutrophils on apoptotic liver cells, Further clarify the effect of neutrophils on apoptotic liver cells.

(2) Phagocytosis of apoptotic liver cells by neutrophils in liver tissues of autoimmune hepatitis

In this part of the experiment, liver tissues were obtained from patients with autoimmune hepatitis, and tissue sections were performed. 1) HE staining and TUNEL staining were used to observe the degree of nuclear pyknosis and count the number of neutrophils and apoptotic cells; 2) TUNEL to mark apoptosis Cells, neutrophil elastase antibody (NE) specifically label neutrophils, E-cadherin antibody label cell membrane, DAPI label cell nucleus, perform immunofluorescence staining, observe the phagocytosis of apoptotic cells by neutrophils and make statistics Quantity. Clarify that patients with autoimmune hepatitis have neutrophil drilling defects.

(3) Phagocytosis of apoptotic liver cells by other cells in human normal liver tissue

In this part of the experiment, we obtained normal non-cancerous liver tissues from patients with hepatocellular carcinoma and sliced them. 1) KCs was specifically labeled with CD68 antibody; 2) Endothelial cells were stained with CD34 antibody and immunofluorescent stained. Observe the degree of nuclear pyknosis and count the numbers of Kupffer cells (KCs) and endothelial cells, revealing that neutrophils are the main cells related to phagocytosis of apoptotic liver cells.

2. Experimental method

(1) The phagocytosis of apoptotic liver cells by neutrophils in normal human liver tissues

Paraffin embedding and sectioning

Fix the non-cancerous normal liver tissue samples of patients with liver cancer in freshly prepared 4% PFA, paraffin-embed the sections, HE stain and TUNEL stain, and observe the changes of apoptotic liver cell nucleus with an Olympus DP80 upright fluorescence microscope. The polymorphonuclear morphology of neutrophils, and the number and distribution of neutrophils and apoptotic cells in liver tissue sections.

Frozen section and immunofluorescence

OCT-embedded non-cancerous liver normal tissue samples from patients with liver cancer, frozen sections, and laser confocal microscope to detect apoptotic liver cells (TUNEL label) and neutrophils (NE specific label), and observe neutrophils The phagocytosis of apoptotic liver cells and count the number.

Ultrathin section

Obtain normal tissue samples of non-cancerous liver adjacent to liver cancer patients, fix them with 0.1M cacodylate buffer containing 2.5% glutaraldehyde, slice them, make ultrathin sections, and observe the morphology of apoptotic liver cell nuclei with transmission electron microscope Changes, the polymorphonuclear morphology of neutrophils and the distribution of neutrophils and apoptotic liver cells.

(2) Phagocytosis of apoptotic liver cells by neutrophils in liver tissues of autoimmune hepatitis

Paraffin embedding and sectioning

The liver tissue samples of autoimmune hepatitis patients were fixed in freshly prepared 4% PFA, paraffin-embedded sections, HE stained and TUNEL stained, and Olympus DP80 upright fluorescence microscope was used to observe the morphological changes of apoptotic liver cells and neutrophils. Cell polymorphonuclear morphology, and detect the number and distribution of neutrophils and apoptotic cells in liver tissue sections.

Frozen section and immunofluorescence

The liver tissue samples of patients with autoimmune hepatitis were embedded in OCT, frozen and sectioned, apoptotic liver cells (TUNEL label) and neutrophils (NE specific label) were detected by laser confocal microscope, and the apoptosis of neutrophils was observed Phagocytosis of hepatocytes and count the number.

(3) Phagocytosis of apoptotic liver cells by other cells in human normal liver tissue

Frozen section and immunofluorescence

The non-cancerous liver normal tissue samples of patients with liver cancer were embedded in OCT, frozen and sectioned, and apoptotic liver cells (TUNEL labeled), KCs (specifically labeled with CD68 antibody), and endothelial cells (CD34 antibody stained) were detected by laser confocal microscope. Mark), observe the location of KCs and endothelial cells around apoptotic liver cells and count the numbers.

3. Experimental results

(1) Neutrophils in normal human liver tissues phagocytosis of apoptotic liver cells

In this experiment, it was found in the non-cancerous liver tissues obtained from patients with hepatocellular carcinoma that a large number of neutrophils are present in liver cells. The liver cells occupied by neutrophils are apoptotic, and their chromatin is pyknotic (Figure 2) . Importantly, apoptotic liver cells are rarely found in the human body, and the reason may be the rapid clearance of apoptotic cells by phagocytes. Among a total of 241 apoptotic hepatocytes derived from 32 liver samples, each apoptotic hepatocyte was filled with up to 22 neutrophils (Figure 2, Figure 3, Figure 4, Figure 5, Figure 4 E-cadherin is a red-labeled cell membrane dye, and DAPI is a blue-labeled nuclear dye). Ly6G antibody immunofluorescence staining (Figure 5, the image shows Ly-6G-positive neutrophils in apoptotic liver cells, and the white arrow indicates the divided apoptotic nucleus), and electron microscope (Figure 6, the apoptotic nucleus with membrane deformation) (AN) is indicated by a white arrow) It is confirmed that neutrophils are located inside apoptotic liver cells. The number of neutrophils (PMN) present in apoptotic liver cells in human liver tissues (two-dimensional images are taken from conventional tissue sections (5μm thick), three-dimensional images are taken from 45μm thick tissue sections, reconstructed with a confocal microscope) see below table:

Table 1 The number of neutrophils (PMN) present in apoptotic liver cells in human liver tissue

(2) Neutrophil phagocytosis deficiency in patients with autoimmune hepatitis

Autoimmune diseases are usually associated with impaired clearance of apoptotic cells. In order to determine whether patients with autoimmune hepatitis have neutrophil defects, biopsy samples from patients diagnosed with AIL disease were examined, and normal human liver tissues and tissues from autoimmune hepatitis patients were stained with HE and TUNEL or immunofluorescence. In contrast to the normal population of the control group, a total of 22 AIL patient samples contained apoptotic hepatocytes that were not phagocytosed or invaded by neutrophils, and there were more apoptotic cells related to macrophages (Figure 5). Since the number of neutrophils in the peripheral blood of patients with AIL is in the normal range, it is speculated that the neutrophils from patients with AIL disease may have phagocytic defects. Compared with the normal neutrophil group, the phagocytic ability of neutrophils from AIL patients on apoptotic L02 cells was significantly reduced (Figure 7 and Figure 8, the green arrow in Figure 7 indicates macrophages, and the black arrow indicates apoptotic body , The white arrow indicates neutrophils; in Figure 8, Tunel labeled apoptotic cells is green, NE labeled neutrophils is purple, E-cad labeled cell membrane is red, and DAPI labeled nucleus is blue).

(3) Other immune cells are not significantly related to apoptotic liver cells

KCs are phagocytes derived from monocytes residing in the liver, and their role is thought to be to eliminate apoptotic cells. Using CD68 antibody to specifically label KCs and perform immunofluorescence staining, there was almost no KCs invasion or phagocytosis of apoptotic liver cells (Figure 3, Figure 9). In addition, almost no endothelial cells (labeled by CD34 antibody staining) surround or engulf apoptotic liver cells (Figure 10).

Example 2 The effect of neutrophils on apoptotic liver cells in vitro

This part of the study mainly observes the phagocytosis of neutrophils on apoptotic hepatocytes and other cells in vitro, as well as the leakage of cell contents of neutrophils in the process of phagocytosis, so as to reveal the effect of neutrophils on apoptotic liver cells The clearing effect is achieved by drilling and phagocytosis.

1. Experimental design

(1) Study the phagocytic effect of neutrophils on apoptotic liver cells at the cellular level

In this part of the in vitro experimental research, the human PMN cell line HL-60, human liver cell L02, HL-60-GFP, and L02-Tomato that have been established by the research group will be used. Puromycin or TGF-β was used to induce human liver cells L02, 1) L02 was stained with red membrane dye (PKH-26), and differentiated HL60 was stained with green membrane dye (PKH-67). Add differentiated HL60 to normal L02 and apoptotic L02; 2) Add differentiated HL-60-GFP to normal L02-Tomato and apoptotic L02-Tomato respectively, and observe the effects of differentiated HL60 on apoptosis using a confocal microscope. The phagocytosis of dead L02 cells clarifies that neutrophils eliminate apoptotic liver cells through phagocytosis.

Isolate mouse polymorphonuclear neutrophils (PMN) from C57BL/6 mouse bone marrow, induce human liver cell L02 with puromycin or TGF-β, and stain L02 with red membrane dye (PKH-26), The PMN was dyed with green membrane dye (PKH-67). PMN was added to normal L02 and apoptotic L02 respectively, and the phagocytosis of apoptotic L02 cells by PMN was observed with a confocal microscope, which clarified that mouse neutrophils cleared apoptotic liver cells through phagocytosis.

Obtain blood samples from normal people and autoimmune liver patients, separate neutrophils, and stain them with green membrane dye (PKH-67); use puromycin or TGF-β to induce apoptosis of human liver cells L02, use Red membrane dye (PKH-26) dyes L02. Add neutrophils to apoptotic L02, use a confocal microscope to photograph the phagocytic effect of neutrophils on apoptotic L02 in real time, clarify that human neutrophils clear apoptotic liver cells through phagocytosis, and It further shows that patients with autoimmune hepatitis have neutrophil drilling defects.

(2) Study on the phagocytosis of apoptotic liver cells by neutrophils using specific markers of apoptosis

This part of the experiment uses differentiated HL60 cells, human liver cells L02. Puromycin or TGF-β was used to induce apoptosis of human liver cells L02, differentiated HL60 cells were stained with a pH-sensitive dye-PHrodo, and L02 was stained with green membrane dye (PKH-67). The differentiated HL60 cells were added to normal L02 and apoptotic L02, and the double-positive part of PHrodo dye and PKH-67 dye was analyzed by flow cytometry to further quantify the phagocytic effect of neutrophils on apoptotic L02.

(3) Leakage of cell contents during neutrophil drilling and phagocytosis

This part of the experiment uses differentiated HL60 cells, human liver cells L02. Puromycin or TGF-β was used to induce apoptosis of human liver cells L02. The differentiated HL60 cells were added to normal L02 and apoptotic L02 co-cultured for 30 min-1h, and the co-culture supernatant was taken to detect dsDNA, SOD, ROS The release status of neutrophils clarifies that no content is leaked during the process of neutrophil phagocytosis, and further proves that neutrophils eliminate apoptotic liver cells through phagocytosis.

2. Experimental method

(1) Study the phagocytic effect of neutrophils on apoptotic liver cells at the cellular level

Induce apoptosis

Treatment with 2.5μg/ml puromycin for 12h or 5ng/ml TGF-β treatment for 18h induced L02 apoptosis.

Extraction of PMN from mouse bone marrow

The mice were euthanized, the femurs and tibias were removed, washed with HBSS plus 0.1% BSA, and then the cells were centrifuged and resuspended in HBSS. After filtering through a 40μm filter, 3mL HBSS cells were added to the pre-prepared gradient solution (3 mL of NycoPrep at the top, 3 mL of 72% Percoll at the bottom). The sample was centrifuged at 2,400 rpm for 20 minutes at room temperature, the middle layer was collected and washed once in HBSS. Add sterile distilled water to remove red blood cells, collect the cells and resuspend them in HBSS or medium.

Extraction of neutrophils from human peripheral blood

Extract with peripheral blood neutrophil extraction kit, add sterile distilled water to remove red blood cells, collect cells and resuspend in HBSS or culture medium.

In vitro drilling test

1) Membrane staining

Stain L02 with red membrane dye (PKH-26), stain the extracted neutrophils, mouse PMN or HL-60 with green membrane dye (PKH-67) and add them to the apoptotic cells with confocal Observe the penetration of neutrophils into apoptotic cells under a microscope and count the penetration of neutrophils.

2) Construction of cell lines

In the preliminary work of this group, stable HL-60-GFP and L02-Tomato cells have been established using lentiviral expression vectors pLVX-IRES-ZsGreen1 and pLVX-IRES-tdTomato. The differentiated HL-60-GFP was added to the apoptotic L02, and the penetration of neutrophils into apoptotic cells was observed with a confocal microscope and the penetration of neutrophils was counted.

(2) Study on the phagocytosis of apoptotic liver cells by neutrophils using specific markers of apoptosis

Streaming technical analysis

Treatment with 2.5μg/ml puromycin for 12h or 5ng/ml TGF-β treatment for 18h induces L02 apoptosis, apoptotic L02 is stained with PHrodo-red, and HL-60 is stained with green membrane dye (PKH-67) Add the apoptotic L02 cells and incubate them at 37°C for 1 h, and quantify the phagocytosis with a flow cytometer.

(3) Leakage of cell contents during neutrophil drilling and phagocytosis

ROS detection

Treat L02 with 2.5μg/ml puromycin for 12h or 5ng/ml TGF-β for 18h to induce apoptosis of L02. Add HL-60 to apoptotic L02 and incubate at 37℃ for 30min-1h. Use MitoSOXTM kit to detect mitochondrial ROS production .

dsDNA detection

Treat L02 with 2.5μg/ml puromycin for 12h or 5ng/ml TGF-β for 18h to induce L02 apoptosis, add HL-60 to apoptotic L02 and incubate at 37℃ for 30min-1h, analyze with Quant-iT ^TM PicoGreen ^TM dsDNA The kit detects the production of dsDNA.

SOD detection

Treat L02 with 2.5μg/ml puromycin for 12h or 5ng/ml TGF-β for 18h to induce apoptosis of L02. Add HL-60 to apoptotic L02 and incubate at 37℃ for 30min-1h. Superoxide dismutase (SOD) assay The kit detects the production of SOD.

3. Experimental results

(1) The phagocytic effect of neutrophils on apoptotic liver cells

In order to study the phagocytosis of living cells mediated by neutrophils, this experiment used puromycin or TGF-β to induce apoptosis of human hepatocytes L02, and then added human PMN cell line HL-60, using red membrane dye (PKH-26 ) Dye L02 and dye HL60 with green membrane dye (PKH-67). The green-labeled HL60 did not respond to normal L02 cells (Figure 11). However, when L02 cells apoptotic, the green-labeled HL60 cells either polarize and invade dead cells or surround and bite the dead cells little by little (Figure 11).

Since the number of neutrophils in the peripheral blood of patients with AIL is in the normal range, it is speculated that neutrophils from patients with AIL disease may have defects in phagocytic ability. Compared with the normal neutrophil group, the phagocytic ability of neutrophils from AIL patients on apoptotic L02 cells was significantly reduced (Figure 12). Normal human neutrophils burrow into apoptotic L02 and begin to phagocytically, while AIL disease neutrophils have no response to apoptotic L02 cells (Figure 13). The above data confirms the existence of defective neutrophil-mediated apoptotic hepatocyte clearance defects in human AIL disease samples.

(2) Quantify the number of neutrophils in apoptotic liver cells

Count the number of neutrophils that entered apoptotic L02 or use PHrodo (a pH-sensitive dye) staining flow cytometry technique to quantify the phagocytosis. It was found that the number of HL-60 phagocytosed apoptotic liver cells was much greater than that of normal non-apoptotic The number of hepatocytes drilled (Figure 14, Figure 15, pkh67 in Figure 14 is marked in green).

(3) No leakage of cell contents during neutrophil drilling

The detection of extracellular DNA levels, SOD, and ROS showed that there was almost no cytoplasm or DNA leakage during the phagocytosis of apoptotic L02 cells by HL60 (Figure 16).

Example 3 Establishing a mouse model of autoimmune hepatitis to study the phagocytic effect of neutrophils in vivo

This part studies 1) the phagocytic effect of neutrophils in a mouse autoimmune hepatitis model; 2) the degree of apoptotic cell clearance defects after knocking out neutrophils in mice; 3) restoration of neutrophils Post-cell clearance of apoptotic cells and relief of AIL disease symptoms. Clarify that neutrophils clear apoptotic cells and maintain tissue homeostasis.

1. Experimental design

(1) The single/two-photon intravital microscopy imaging system studies the phagocytic effect of neutrophils on apoptotic liver cells under physiological conditions

In this part of the study, the existing C57BL/6 mice in the research group were used. After the mice were anesthetized, they were injected intravenously with anti-Ly6G 1A8 FITC, anti-F4/80 PE and Annexin V, and observed with a single/two-photon intravital imaging system Under physiological conditions, the effect of neutrophils on apoptotic hepatocytes was analyzed. Imaris was used to analyze intracellular particles, and 3D reconstruction was used to further prove that neutrophils can clear apoptotic hepatocytes through phagocytosis.

(2) Establish a neutrophil defect mouse model to study the neutrophil defect-mediated apoptosis and hepatocyte clearance defect

This part of the study used two methods to knock out neutrophils: 1) using intraperitoneal injection of anti-mouse Ly6G 1A8 antibody to deplete neutrophils; 2) using genetic neutrophil ablation to express diphtheria toxin receptor (DTR) Rosa26i DTR mice were bred on the Mrp8 ^- Cre genetic background. Cre expression in cells expressing MRP (mainly granulocytes) will activate DTR expression only in MRP expressing cells. Rosa26iDTR/Mrp8 ^- Cre mice and C57BL/6 mice were treated with diphtheria toxin to induce Mrp8 ⁺ cell depletion. Using the above-mentioned neutrophil deficient mouse model, after the mice were anesthetized, 1) Take the serum of C57BL/6 mice and model mice, and use the Elisa kit to detect anti-nuclear antigen (anti-ANA), smooth muscle actin (anti- -α-SMA), liver and kidney microsomes (anti-LKM) and total IgG antibody (total anti-lgG) expression; 2) Take the liver tissues of C57BL/6 mice and model mice for tissue sections, and use HE staining and TUNEL Stain, observe the apoptosis of mouse liver cells and count the number of neutrophils related to apoptotic cells; 3) Take the liver tissues of C57BL/6 mice and model mice for tissue sections, and use CD11b and DAPI for immunofluorescence staining , Observe the inflammatory infiltration of macrophages in the liver tissues of mice; 4) Take blood from C57BL/6 mice and model mice for biochemical tests of liver function, and analyze liver damage in neutrophil-deficient mice. Elucidate that neutrophil defect mediates apoptotic hepatocyte clearance defect and induces autoimmune hepatitis.

(3) Establish an autoimmune liver disease (AIL) model to study the phagocytic effect of neutrophils in the body

In this part of the study, the AIL model was constructed by drug interventions such as ConA, Fasl and α-Galcer. After the mice were anesthetized, 1) Take the serum of C57BL/6 mice and model mice, and use the Elisa kit to detect the expression of anti-nuclear antigen, smooth muscle actin, liver and kidney microsomes and total IgG antibodies to verify the successful model construction; 2 ) Take the liver tissues of C57BL/6 mice and model mice for tissue sections, use HE staining and TUNEL staining to observe the apoptosis of mouse liver cells and count the number of neutrophils related to apoptotic cells; 3) Take C57BL/ The liver tissues of 6 mice and model mice were frozen sectioned, apoptotic cells were specifically labeled with Cas3, neutrophils were specifically labeled with neutrophil elastase antibody (NE), cell membranes were labeled with E-cadherin antibody, and cell nuclei were labeled with DAPI. CD11b specifically labels inflammatory cells, performs immunofluorescence staining, observes the phagocytosis of apoptotic cells by neutrophils and counts the numbers, and analyzes inflammatory infiltration. Clarify that there are neutrophil defects in autoimmune liver diseases, and neutrophil defects mediate apoptotic liver cell clearance defects.

(4) Restore neutrophils in autoimmune liver disease models and study the role of neutrophils in AIL disease

In this part of the study, C57BL/6 mouse bone marrow neutrophils were taken, and autoimmune liver disease (AIL) model mice were injected intravenously (Rescue). 1) Rescue mouse serum was taken, and the Elisa kit was used to detect antinuclear antigen , Smooth muscle actin, liver and kidney microsomes, and total IgG antibody expression; 2) Take the liver tissue of Rescue mice for tissue section, use HE staining and TUNEL staining to observe the apoptosis of mouse liver cells, statistics and apoptosis The number of cell-related neutrophils. Clarify that autoimmune hepatitis can reduce disease indicators and the number of apoptotic cells by restoring the number of neutrophils.

2. Experimental method

(1) The single/two-photon intravital microscopy imaging system studies the phagocytic effect of neutrophils on apoptotic liver cells under physiological conditions

Intravital microscopy

The mice were anesthetized 1 hour before imaging, and the newly prepared anti-Ly6G 1A8 FITC antibody, anti-F4/80 PE antibody and Annexin V were injected intravenously into the mice. Before imaging, the mice were anesthetized again, a midline laparotomy was performed on the mice, all blood vessels in the skin were cauterized, and then the sickle ligament between the liver and the diaphragm was cut to expose the right lobe of the liver. Observe apoptotic cells (Annexin V, blue), Kupffer cells (anti-F4/80, red) and neutrophils (anti-Ly-6G 1A8, green) with the single/two-photon live microscopy imaging system, in analysis In the case of neutrophils burrowing into apoptotic liver cells, Imaris is used to analyze intracellular granules and 3D simulation is used to reconstruct.

(2) Establish a neutrophil deficient mouse model and study the neutrophil deficient mediation of apoptosis and hepatocyte elimination deficiency antibody to clear mouse PMN

Inject 500 μg of anti-mouse Ly6G antibody (1A8 clone) or negative control antibody (2A3 clone) intraperitoneally, once every 48 hours for one month.

Mouse PMN clearance in a genetic background

Rosa26i DTR mice expressing diphtheria toxin receptor (DTR) were bred on Mrp8 ^- Cre genetic background. Cre expression in cells expressing MRP (mainly granulocytes) will activate DTR expression only in MRP expressing cells. At 3 months, Rosa26iDTR/Mrp8-Cre mice and C57BL/6 mice were treated with diphtheria toxin (DT, 20ng/day for 3 days) to induce Mrp8 ⁺ cell clearance.

Elisa detects autoantigens

After the mice were anesthetized, the peripheral blood of the mice was taken and the serum was separated, and the Elisa kit was used to detect anti-nuclear antigen (anti-ANA), smooth muscle actin (anti-α-SMA), liver and kidney microsomes (anti-LKM) And total IgG antibody (total anti-lgG).

Paraffin embedding and sectioning

After the mice were anesthetized, the liver samples of neutrophil-deficient mice and C57BL/6 mice were fixed in freshly prepared 4% PFA, paraffin-embedded sections, HE stained and TUNEL stained, Olympus DP80 upright Fluorescence microscope was used to observe the morphological changes of apoptotic liver cell nuclei, the polymorphonuclear morphology of neutrophils, and detect the number and distribution of neutrophils and apoptotic cells in liver tissue sections.

Frozen section and immunofluorescence

After the mice were anesthetized, the liver samples of neutrophil-deficient mice and C57BL/6 mice were OCT-embedded, frozen and sectioned, and the inflammatory cells (CD11b labeled) and nuclei (DAPI labeled) were detected by laser confocal microscope. , Observe the inflammatory infiltration of macrophages in the liver tissues of mice.

Liver function biochemical test

After the mice were anesthetized, the peripheral blood of the mice was taken, and the samples were sent to the biotechnology company for liver function biochemical testing, and the liver function damage of the neutrophil-deficient mice was analyzed.

(3) Establish an autoimmune liver disease (AIL) model to study the phagocytic effect of neutrophils in the body

Concanavalin (ConA) induces autoimmune liver disease (AIL) model in mice

After the mice were anesthetized, concanavalin A (ConA, 20 mg/kg) was intravenously injected into the mice, and subsequent experiments were performed 8 hours later.

Fasl induces mouse autoimmune liver disease (AIL) model

After the mice were anesthetized, 0.25 mg/kg soluble fasl was injected intravenously, and the liver was taken one week later for follow-up experiments.

α-Galcer induces autoimmune liver disease (AIL) model in mice

Α-Galcer was dissolved in PBS containing 0.5% Tween20 at a final concentration of 0.2 mg/ml, and placed in a glass tube to be sonicated until completely dissolved. After the mice were anesthetized, α-Galcer was injected intraperitoneally, 2μg/mouse. After 10 days, the liver was taken for subsequent experiments.

Elisa detects autoantigens

After the mice were anesthetized, the peripheral blood of the aforementioned model mice and C57BL/6 mice were taken and the serum was separated, and the Elisa kit was used to detect anti-nuclear antigen (anti-ANA), smooth muscle actin (anti-α-SMA), liver Kidney microsomes (anti-LKM) and total IgG (total anti-lgG) were used to analyze whether drug intervention was successful in constructing an AIL model.

Paraffin embedding and sectioning

After the mice were anesthetized, the liver samples of the aforementioned model mice and C57BL/6 mice were fixed in freshly prepared 4% PFA, paraffin-embedded sections, HE stained and TUNEL stained, and observed by Olympus DP80 upright fluorescence microscope Mouse liver cell apoptosis and count the number of neutrophils related to apoptotic cells.

Frozen section and immunofluorescence

The non-cancerous liver normal tissue samples of patients with liver cancer were embedded in OCT, frozen and sectioned, and apoptotic liver cells (Cas3 specific marker), neutrophils (NE specific marker), and inflammation were detected by laser confocal microscope Cells (CD11b specific marker), observe the neutrophil phagocytosis of apoptotic hepatocytes and count the number, analyze the inflammatory infiltration.

(4) Restore neutrophils in autoimmune liver disease models and study the role of neutrophils in AIL disease

Restore neutrophils in mice with AIL disease

C57BL/6 mouse bone marrow PMN was obtained according to the aforementioned method, and the model mice were anesthetized and the PMN (2×10 ⁶ /mouse) was injected into the autoimmune liver disease (AIL) model mice through the canthal vein.

Elisa detects autoantigens

After intravenous injection of PMN for 12 hours, the mice were anesthetized, the peripheral blood of the mice was taken and the serum was separated, and the Elisa kit was used to detect anti-nuclear antigen (anti-ANA), smooth muscle actin (anti-α-SMA), liver and kidney microsomes (anti-LKM) and total IgG antibody (total anti-lgG).

Paraffin embedding and sectioning

After intravenous injection of PMN for 12 hours, the mice were anesthetized, and the mouse liver samples were fixed in freshly prepared 4% PFA, paraffin-embedded sections, HE stained and TUNEL stained, and the mouse liver was observed with an Olympus DP80 upright fluorescence microscope Cell apoptosis and count the number of neutrophils related to apoptotic cells.

3. Experimental results

(1) Neutrophils have a phagocytic effect on apoptotic liver cells under physiological conditions

Using a two-photon microscope (Figure 17, where neutrophils were labeled with intravenous anti-Ly6G antibody (green), KCs were labeled with anti-F4/80 antibody (red), and apoptotic cells were labeled with annexin V (blue) ), Ly6G antibody immunofluorescence staining (Figure 5), and electron microscope (Figure 6) confirm that neutrophils are located inside apoptotic liver cells. The 3D reconstructed image clearly shows neutrophils in apoptotic liver cells.

(2) In a mouse model of neutrophil deficiency, neutrophil deficiency mediates apoptotic hepatocyte clearance defects

Detect whether the use of antibodies or genetic methods to knock out neutrophils affects the clearance of apoptotic liver cells. Antibody knockout can make the neutrophil knockout rate reach more than 90% (Figure 18). In this experiment, liver samples from neutrophil knockout and control mice were analyzed and HE stained. Similar to human liver samples, apoptotic hepatocytes in control mice are occupied and swallowed by neutrophils (Figure 19, where the black arrows indicate apoptotic bodies, white arrows indicate neutrophils, and green arrows indicate macrophages) . After neutrophil knockout, apoptotic cells no longer correlate with neutrophils (Figure 20). However, this experiment found that in the neutrophil knockout samples, macrophages are related to apoptotic hepatocytes (Figure 20), which shows that macrophages play a compensatory role in the absence of neutrophils , Phagocytosis of apoptotic liver cells. Compared with the control group, the proportion of apoptotic liver cells in the neutrophil knockout samples was significantly increased (Figure 20). Therefore, in mouse liver, neutrophil knockout affects the clearance of apoptotic cells.

(3) In the neutrophil deficient mouse model, liver tissue has obvious inflammation and liver function damage

Compared with the untreated control group, the liver of neutrophil knockout mice showed obvious inflammation (Figure 21, green is anti-CD11b antibody staining) and liver function damage (aspartate aminotransferase and alanine). Acid aminotransferase activity increases, and total and direct bilirubin levels increase) (Figure 22). The same results were obtained in MRP8Cre/DTR knockout mice (Figure 23, where Figure 23A-Figure 23C shows the neutrophils in the peripheral blood of Mrp8cre/DTR mice and C57BL/6 mice (Figure 23A), lymph The counts of cells (Figure 23B) and monocytes (Figure 23C); Figure 23D-Figure 23E show aspartate aminotransferase (AST, D) and alanine aminotransferase (AST, D) in Mrp8cre/DTR mice and C57BL/6 mice ALT, E) liver function analysis results; Figure 23F-Figure 23H show the cell counts of neutrophils (F) and macrophages (G) in the liver samples of Mrp8cre/DTR mice and C57BL/6 mice Apoptotic hepatocytes (H) related cell count).

(4) Accumulation of autoantibodies in neutrophil-deficient mice

Autoimmune diseases are usually associated with impaired clearance of apoptotic cells. The purpose of this experiment is to determine whether defective neutrophil-mediated apoptotic cell clearance is related to AIL disease. Compared with the control group, 6 out of 16 neutrophil knockout mice were detected with an increase in autoantibodies (ie anti-nuclear antigen, smooth muscle actin, liver and kidney microsomes, and total IgG antibodies) of about 2- 3 times (Figure 24). Therefore, neutrophil knockout not only affects the clearance of apoptotic hepatocytes, but also leads to the production of autoantibodies, which indicates that in AIL disease, the lack of neutrophils mediates the clearance of apoptotic cell defects.

(5) Neutrophils in autoimmune liver disease (AIL) model mice have a phagocytic effect

Treatment of mice with concanavalin can cause acute liver damage in mice. In this experiment, concanavalin was used for drug intervention to simulate human AIL disease. After concanavalin treatment, the number of apoptotic hepatocytes in the liver increased significantly (Figure 20, Figure 25, the black arrow in Figure 25 indicates apoptotic bodies, the white arrow indicates neutrophils, and the green arrow indicates macrophages). Antibody expression increased (Figure 26), and at the same time, neutrophils associated with apoptotic cells were significantly reduced (Figure 19, Figure 25). In vitro phagocytosis analysis showed that concanavalin significantly inhibited neutrophil-mediated clearance of apoptotic hepatocytes (Figure 27).

Lipid phosphatidylserine (PtdSer) is the "eat me" signal for apoptosis clearance. This experiment found that using Annexin V to cover PtdSer would affect the neutrophil-mediated apoptotic hepatocyte clearance (Figure 27), which indicates that the drilled neutrophils can recognize PtdSer based on the inside of the liver cell membrane.

(6) Restore neutrophils in autoimmune liver disease models, neutrophils clear apoptotic liver cells

In order to study whether the phenotype caused by concanavalin can be alleviated by restoring the number of neutrophils. In this experiment, intravenous injection of normal neutrophils in ConA model mice restored the clearance of neutrophils on apoptotic hepatocytes (Figure 28, the black arrow indicates apoptotic bodies, the white arrow indicates neutrophils, green Arrows indicate macrophages) and reduce the level of autoantibodies in mice treated with concanavalin (Figure 26). The above indicates that the phenotype caused by concanavalin has alleviated.

Example 4 Neutrophils are selective for the phagocytosis of apoptotic hepatocytes

This experiment mainly observes the phagocytosis of other non-liver cells by neutrophils and the phagocytosis of apoptotic liver cells by other immune cells, and clarifies that neutrophils are selective for the phagocytosis of apoptotic liver cells.

1. Experimental design

(1) To study the selectivity of neutrophils to apoptotic hepatocytes at the cellular level

This part of the experiment uses the human PMN cell lines HL-60, HL-60-GFP, macrophages RAW264.7, RAW264.7-GFP, human liver cells L02, L02-Tomato, endothelial cells HUVEC, HUVEC that have been established by the research group -Tomato, epithelial cells HEK293, HEK293-Tomato, cardiomyocytes HCM, HCM-Tomato, pancreatic β cells, pancreatic β cells-Tomato. This experiment 1) Use puromycin or TGF-β to induce apoptosis of human liver cells L02, endothelial cells HUVEC, epithelial cells HEK293, cardiomyocytes HCM, and pancreatic islet β cells. Red membrane dye (PKH-26) was used to treat L02 and HUVEC respectively. , HEK293, HCM, pancreatic islet β cells, stain the differentiated HL60 with green membrane dye (PKH-67), add the differentiated HL60 to normal L02 and apoptotic L02, HUVEC, HEK293, HCM, pancreatic islet β Use a confocal microscope to observe the phagocytosis of apoptotic L02, HUVEC, HEK293, HCM, pancreatic islet β cells by differentiated HL60; 2) Use puromycin or TGF-β to induce human liver cells L02-Tomato and endothelial cells HUVEC -Tomato, epithelial cells HEK293-Tomato, cardiomyocytes HCM-Tomato, pancreatic β-cells-Tomato apoptosis, add differentiated HL60-GFP to normal L02-Tomato and apoptotic L02-Tomato, HUVEC-Tomato, HEK293- Tomato, HCM-Tomato, pancreatic islet β-cell-Tomato, using a confocal microscope to observe the phagocytosis of apoptotic L02, HUVEC, HEK293, HCM, pancreatic islet β cells by differentiated HL60; 3) Puromycin or TGF-β induces humans Liver cell L02 apoptosis, stain L02 with red membrane dye (PKH-26), stain RAW264.7 with green membrane dye (PKH-67), add RAW264.7 to normal L02 and apoptotic L02, Use a confocal microscope to observe the phagocytosis of apoptotic L02 by RAW264.7; 4) Puromycin or TGF-β induces apoptosis of human liver cells L02-Tomato, add RAW264.7-GFP to normal L02-Tomato and apoptosis The dead L02-Tomato was used to observe the phagocytosis of apoptotic L02 by RAW264.7 using a confocal microscope. Clarify that neutrophils are selective for the phagocytosis of apoptotic liver cells.

(2) To study the selectivity of neutrophils to apoptotic hepatocytes at the molecular level

This part of the experiment 1) Take blood samples from normal people and autoimmune hepatitis patients, and use gene chips to detect their total RNA expression; 2) Use puromycin or TGF-β to induce apoptosis of human liver cells L02, and compare normal L02 and apoptosis L02 Perform gene chip detection to analyze the total RNA expression; 3) Use puromycin or TGF-β to induce human liver cell L02, endothelial cell HUVEC, epithelial cell HEK293, cardiomyocyte HCM, pancreatic β cell apoptosis, and extract normal L02, HUVEC, HEK293, HCM, pancreatic β-cells and apoptosis L02, HUVEC, HEK293, HCM, pancreatic β-cell cDNA, for E-CAD, E-selectin, L-selectin, Pecam, P-selectin, ICAM1, IL-1β , IL-8, INTA2, INTA6, INTB1, INTB4, P-CAD and other genes were subjected to fluorescence quantitative PCR, and the two experiments were compared to observe the changes in gene expression. To clarify the phagocytic selectivity of neutrophils to apoptotic hepatocytes.

2. Experimental method

(1) To study the selectivity of neutrophils to apoptotic hepatocytes at the cellular level

In vitro drilling test

1) Membrane staining

Puromycin or TGF-β was used to induce apoptosis of human liver cells L02, endothelial cells HUVEC, epithelial cells HEK293, cardiomyocytes HCM, pancreatic β cells, and stained with red membrane dye (PKH-26), and HL- 60 or macrophages RAW264.7 were stained with green membrane dye (PKH-67) and then added to the apoptotic cells respectively, and the situation of neutrophils or macrophages drilling into the apoptotic cells was observed with a confocal microscope. Drill into neutrophils to count.

2) Construction of cell lines

In the previous work of this research group, the lentiviral expression vectors pLVX-IRES-ZsGreen1 and pLVX-IRES-tdTomato have been used to establish stable HL-60-GFP, RAW264.7-GFP, L02-Tomato, HUVEC-Tomato, HEK293-Tomato , HCM-Tomato, Pancreatic β-cell-Tomato. Add the differentiated HL-60-GFP or RAW264.7-GFP to the apoptotic L02-Tomato, HUVEC-Tomato, HEK293-Tomato, HCM-Tomato, pancreatic β-cell-Tomato, and observe the neutrophils with a confocal microscope Cells or macrophages drilled into apoptotic cells and counted the drilled neutrophils.

(2) To study the selectivity of neutrophils to apoptotic hepatocytes at the molecular level

Gene chip detection

Take blood samples of normal people and autoimmune hepatitis patients, use gene chip to detect their total RNA expression; use puromycin or TGF-β to induce apoptosis of human liver cell L02, and detect normal L02 and apoptotic L02 by gene chip to analyze the results Total RNA expression.

Fluorescence quantitative PCR

Extract normal L02, HUVEC, HEK293, HCM, pancreatic islet β cells and apoptotic L02, HUVEC, HEK293, HCM, pancreatic β cells cDNA, for E-CAD, E-selectin, L-selectin, Pecam, P-selectin, ICAM1 , IL-1β, IL-8, INTA2, INTA6, INTB1, INTB4, P-CAD and other genes were subjected to fluorescence quantitative PCR, and the two experiments were compared to observe the changes in gene expression.

3. Experimental results

In order to explore whether HL-60 engulfed other apoptotic cells, the phagocytic ability of HL-60 on apoptotic endothelial cells HUVEC and apoptotic epithelial cells HEK293 was tested. Compared with the control group, no phagocytosis of HL-60 on these cells was found (Figure 14, Figure 29), indicating that neutrophils are selective in the phagocytosis of apoptotic liver cells. Importantly, this experiment used human peripheral blood neutrophils or mouse bone marrow neutrophils to obtain the same results (Figure 30, respectively): normal mice and neutrophil-deficient mice liver sections, F4\ 80-red, NE-green, DAPI-blue; human normal liver tissue section (left) and autoimmune hepatitis patient liver tissue section (right); mouse neutrophils, HL-60 and macrophages affect apoptosis Results of quantitative detection of L02 cell phagocytosis). By comparing the phagocytic ability of HL-60 and macrophage RAW264.7 on apoptotic hepatocytes, it is found that the phagocytic ability of RAW cells on apoptotic LO2 cells is much lower than that of HL-60 (Figure 30). This result is similar to that of neutrophils. It plays a major role in mediating apoptotic liver cell clearance.

Example 5 The main mechanism of neutrophils to clear apoptotic liver cells

The purpose of this experiment is to confirm that neutrophils respond to cytokines and chemokines for non-inflammatory infiltration and migrate to apoptotic cells, and further clear the apoptotic cells through phagocytosis. To clarify the signal pathway of neutrophil migration to the apoptotic site and the main mechanism of neutrophils to clear apoptotic cells through phagocytosis, and to reveal the new function of neutrophils-apoptotic cell clearance on the pathogenesis and pathogenesis of autoimmune diseases. Treatment has an important role.

1. Experimental design

(1) Research on the signal pathway of granulocyte migration to apoptotic liver cells

This part of the study 1) Obtain liver tissue samples from patients with autoimmune hepatitis and non-cancerous liver tissues adjacent to cancer in patients with liver cancer, slice them, and use Cas 3 antibody to specifically label apoptotic cells and MPO antibody to specifically label neutral Granulocytes and DAPI label cell nuclei and perform immunofluorescence staining to observe the migration of neutrophils in response to apoptosis signals; 2) Take the peripheral blood of normal people and autoimmune hepatitis patients, and use gene chips to detect their total RNA expression; 3 ) According to the results of gene chip detection, puromycin or TGF-β was used to induce apoptosis of human liver cells L02, and Elisa was used to detect IL-1β, IL-6, IL-8, IL-12 in apoptotic L02 cells and normal L02 cells , GM-CSF, IFN-γ, TNF-α, IL-1α, IL-2, IL-4 and IL-10 expression changes; 4) Use puromycin or TGF-β to induce apoptosis of human liver cells L02 , Extract the cDNAs of normal L02 and apoptosis L02, for E-CAD, E-selectin, L-selectin, Pecam, P-selectin, ICAM1, IL-1β, IL-8, INTA2, INTA6, INTB1, INTB4, P- Carry out fluorescence quantitative PCR for CAD and other genes, compare the two experiments, observe changes in gene expression, and screen the signals that attract neutrophils to migrate to apoptotic liver cells.

(2) Neutrophils migrate to apoptotic liver cells through non-inflammatory infiltration

In this part of the study, liver tissue samples were obtained from autoimmune hepatitis patients and non-cancerous liver normal tissues of patients with liver cancer, and tissue sections were taken. 1) HE staining was used to observe the inflammatory infiltration; 2) CD11b antibody specific labeling Inflammatory cells, CD45RA antibody specifically labeled lymphocytes, MPO antibody specifically labeled neutrophils, E-cadherin antibody labeled cell membranes, and DAPI labeled cell nuclei. Perform immunofluorescence staining to observe inflammatory infiltration. Clarify that neutrophils migrate to apoptotic liver cells through non-inflammatory infiltration.

(3) IL-1β, IL-6, IL-8, IL-12, E-CAD, E-selectin, L-selectin, Pecam, P-selectin, ICAM1-deficient cells can phagocytose apoptotic liver by neutrophils Cell impact

In this part of the in vitro experimental research, the human PMN cell lines HL-60, IL-1β ^-/- HL-60, IL-6 ^-/- HL-60, IL-8 ^-/- HL- established by the research group will be used. 60, IL-12 ^-/- HL-60, SELE ^-/- HL-60, SELP ^-/- HL-60, SELL ^-/- HL-60, CDH1 ^-/- HL-60, CDH3 ^-/- HL- 60. PECAM ^-/- HL-60, ICAM1 ^-/- HL-60 cells, use puromycin or TGF-β to induce apoptosis of human liver cells L02, 1) stain L02 with red membrane dye (PKH-26) , Use green membrane dye (PKH-67) to HL-60, IL-1β ^-/- HL-60, IL-6 ^-/- HL-60, IL-8 ^-/- HL-60, IL-12 ^{-/ - HL-60, SELE - /} - HL-60, SELP - / - HL-60, SELL - / - HL-60, CDH1 - / - HL-60, CDH3 - / - HL-60, PECAM - / - HL -60, ICAM1 ^-/- HL-60 cells were stained. The above HL-60 cells were co-cultured with apoptotic L02 and normal L02, and the phagocytosis of HL60 on apoptotic L02 cells was observed and compared with a confocal microscope; 2) The pH-sensitive dye-PHrodo was used for HL-60 and IL- 1β ^-/- HL-60, IL-6 ^-/- HL-60, IL-8 ^-/- HL-60, IL-12 ^-/- HL-60, SELE ^-/- HL-60, SELP ^-/- HL-60, SELL ^-/- HL-60, CDH1 ^-/- HL-60, CDH3 ^-/- HL-60, PECAM ^-/- HL-60, ICAM1 ^-/- HL-60 cells are stained with green membrane Dye (PKH-67) dyes L02. HL-60, IL-1β ^-/- HL-60, IL-6 ^-/- HL-60, IL-8 ^-/- HL-60, IL-12 ^-/- HL-60, SELE ^-/- HL -60, SELP ^-/- HL-60, SELL ^-/- HL-60, CDH1 ^-/- HL-60, CDH3 ^-/- HL-60, PECAM ^-/- HL-60, ICAM1 ^-/- HL-60 Cells were added with normal L02 and apoptotic L02, and the overlap of PHrodo dye and PKH-67 dye was analyzed by flow cytometry to further quantify the phagocytic effect of neutrophils on apoptotic L02. It is clarified that IL-1β and IL-8 secreted by apoptotic L02 cells are the key signals to attract neutrophils and induce subsequent phagocytosis.

2. Experimental method

(1) The signal pathway of centrogranulocyte migration to apoptotic liver cells

Frozen section and immunofluorescence

The non-cancerous liver tissue samples of patients with liver cancer and autoimmune hepatitis liver tissue samples were embedded in OCT, frozen and sectioned, and apoptotic cells (Cas3 specific label) and neutrophils (MPO specific) were detected by laser confocal microscope. Mark), cell nucleus (DAPI mark), observe the migration of neutrophils in response to apoptosis signals. Gene chip detection

Take the peripheral blood of normal people and autoimmune hepatitis patients, and use gene chip to detect the total RNA expression.

ELisa cytokine screening

Puromycin or TGF-β was used to induce apoptosis of human liver cells L02, and the human multiple inflammatory cytokine Elisa analysis kit was used to detect IL-1β, IL-6, IL-8, IL in apoptotic L02 cells and normal L02 cells -12, GM-CSF, IFN-γ, TNF-α, IL-1α, IL-2, IL-4 and IL-10 expression changes.

Fluorescence quantitative PCR

Extract the cDNA of normal L02 and apoptotic L02 for E-CAD, E-selectin, L-selectin, Pecam, P-selectin, ICAM1, IL-1β, IL-8, INTA2, INTA6, INTB1, INTB4, P-CAD Fluorescence quantitative PCR was performed on isogenes, and the two experiments were compared to observe changes in gene expression and screen the signals that attract neutrophils to migrate to apoptotic liver cells.

(2) Neutrophils migrate to apoptotic liver cells through non-inflammatory infiltration

Paraffin embedding and sectioning

Fix the non-cancerous normal liver tissue samples and autoimmune hepatitis liver tissue samples of liver cancer patients in freshly prepared 4% PFA, paraffin-embedded sections, HE staining and TUNEL staining, and observing by Olympus DP80 upright fluorescence microscope Inflammatory infiltration of neutrophils.

Frozen section and immunofluorescence

The non-cancerous liver tissue samples of patients with liver cancer and autoimmune hepatitis liver tissue samples were embedded in OCT, frozen and sectioned, and laser confocal microscope was used to detect inflammatory cells (CD11b label) and lymphocytes (CD45RA antibody specific label) , Neutrophils (MPO antibody specific label), cell membrane (E-cad specific label), cell nucleus (DAPI label), observe the inflammatory infiltration of macrophages in human liver tissue.

(3) The effect of IL-1β, IL-6, IL-8, IL-12 deficient cells on neutrophil phagocytosis of apoptotic hepatocytes

Build cell lines

HL-60, IL-1β ^-/- HL-60, IL-6 ^-/- HL-60, IL-8 ^-/- HL-60, IL-12 ^-/- HL have been established in the preliminary work of this research group -60, SELE ^-/- HL-60, SELP ^-/- HL-60, SELL ^-/- HL-60, CDH1 ^-/- HL-60, CDH3 ^-/- HL-60, PECAM ^-/- HL-60 , ICAM1 ^-/- HL-60 cell line.

Membrane staining

Puromycin or TGF-β was used to induce apoptosis of human liver cell L02, the apoptotic L02 was stained with red membrane dye (PKH-26), and HL-60, IL-1β ^-/- HL-60, IL-6 ^-/- HL-60, IL-8 ^-/- HL-60, IL-12 ^-/- HL-60, SELE ^-/- HL-60, SELP ^-/- HL-60, SELL ^-/- HL-60 , CDH1 ^-/- HL-60, CDH3 ^-/- HL-60, PECAM ^-/- HL-60, ICAM1 ^-/- HL-60 are stained with green membrane dye (PKH-67) and then added to apoptotic L02 In, a confocal microscope was used to observe the phagocytosis of apoptotic L02 cells by HL60 silenced by different genes.

Streaming technical analysis

Treatment with 2.5μg/ml puromycin for 12h or 5ng/ml TGF-β treatment for 18h induces L02 apoptosis, apoptotic L02 was stained with PHrodo-red, HL-60, IL-1β ^-/- HL-60, IL -6 ^-/- HL-60, IL-8 ^-/- HL-60, IL-12 ^-/- HL-60, SELE ^-/- HL-60, SELP ^-/- HL-60, SELL ^-/- HL -60, CDH1 ^-/- HL-60, CDH3 ^-/- HL-60, PECAM ^-/- HL-60, ICAM1 ^-/- HL-60 are stained with green membrane dye (PKH-67) and then added to apoptosis The L02 cells were incubated at 37°C for 1 h, and the phagocytosis was quantified by a flow cytometer.

3. Experimental results

This experiment found that neutrophils migrated to apoptotic cells (Figure 31, Figure 32, the left image in Figure 31 shows a human normal liver tissue section, the right image shows a liver tissue section of an autoimmune hepatitis patient, NE-red, E-Cad -Purple, Tunel-green, DAPI-blue; MPO-red, Tunel-green, DAPI-blue in Fig. 32). In order to confirm the signal that attracts neutrophils to migrate to apoptotic liver cells, cytokines and chemokines were screened before and after LO2 apoptosis. The expressions of IL-1β, IL-6, IL-8 and IL-12 in apoptotic LO2 cells were significantly up-regulated (Figure 33). In contrast, GM-CSF, IFN-γ, TNF-α, IL-1α, IL-2, IL-4, and IL-10 did not change significantly (Figure 34).

In order to clarify the role of these up-regulated cytokines, knock down the IL-1β, IL-6, IL-8 and IL-12 receptors of HL60 and test its phagocytic ability. Knockdown of IL-1β receptor, HL60 loses its ability to phagocytic apoptotic LO2; knockdown of IL-8 receptor reduces phagocytic efficiency by 40%; downregulation of other cytokine receptors has no significant effect on HL-60 phagocytic ability (Figure 35 ). Therefore, IL-1β and IL-8 secreted by apoptotic L02 cells are key signals to attract neutrophils and induce subsequent phagocytosis.

The above experimental results show that IL-1β and IL-8 secreted by apoptotic L02 cells are the key signals to attract neutrophils and induce subsequent phagocytosis, and the down-regulation of IL-1β and IL-8 may be the predisposing factors of autoimmune hepatitis one.

Claims (16)

1. The use of neutrophils in preparing medicines for treating and/or preventing autoimmune hepatitis.
2. The use of neutrophils in preparing medicines for clearing apoptotic liver cells.
3. The use according to claim 1 or 2, characterized in that the medicine is a preparation prepared by adding pharmaceutically acceptable excipients or auxiliary components with neutrophils as an active ingredient.
4. The use according to claim 3, wherein the preparation is an intravenous injection.
5. The use according to any one of claims 1 to 4, wherein the neutrophils are bone marrow neutrophils and/or peripheral blood neutrophils.
6. The auxiliary diagnosis kit for autoimmune hepatitis is characterized in that it contains a reagent for detecting the number of neutrophils in apoptotic liver cells.
7. The use of the following reagents in the preparation of an auxiliary diagnosis kit for autoimmune hepatitis is characterized in that: the reagent is a reagent for detecting the number of neutrophils in apoptotic liver cells.
8. The auxiliary diagnosis kit for autoimmune hepatitis is characterized by: containing neutrophil markers, apoptotic liver cell markers, cell membrane markers and nuclear markers.
9. The auxiliary diagnostic kit according to claim 8, characterized in that it contains neutrophil elastase antibody, TUNEL stain, E-cadherin antibody and DAPI.
10. The use of the following reagents in the preparation of an auxiliary diagnostic kit for autoimmune hepatitis is characterized in that the reagents contain neutrophil markers, apoptotic liver cell markers, cell membrane markers and cell nuclear markers.
11. The use according to claim 10, wherein the reagent contains neutrophil elastase antibody, TUNEL stain, E-cadherin antibody and DAPI.
12. The auxiliary diagnostic instrument for autoimmune hepatitis is characterized in that it contains a device for detecting the number of neutrophils in apoptotic liver cells.
13. The use of the following reagents in the preparation of an auxiliary diagnostic kit for autoimmune hepatitis is characterized in that the reagents contain reagents for detecting IL-1β and/or IL-8 serum levels.
14. The use according to claim 12, wherein the auxiliary diagnostic kit is an Elisa kit.
15. The use of the following device in the manufacture of an auxiliary diagnostic instrument for autoimmune hepatitis is characterized in that: the device is a device for detecting serum levels of IL-1β and/or IL-8.
16. The use of an active ingredient that up-regulates the serum level of IL-1β and/or IL-8 in the preparation of a medicine for treating and/or preventing autoimmune hepatitis.

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