CN110124029B - Application of IL-6R antibody and amniotic fluid stem cells in the preparation of drugs for treating NEC - Google Patents

Abstract

The invention discloses an application of an IL-6R antibody and amniotic fluid stem cells in preparing a medicament for treating NEC, and belongs to the technical field of biological medicines.

Description

Application of IL-6R antibody and amniotic fluid stem cells in the preparation of drugs for treating NEC

technical field

The invention belongs to the technical field of biomedicine, in particular to the application of IL-6R antibody and amniotic fluid stem cells in the preparation of medicines for treating NEC.

Background technique

Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in newborns. NEC is more common in premature infants, and its incidence and mortality are as high as 10% and 50% in extremely low birth weight (ELBW) infants, respectively. Survivors are often accompanied by serious gastrointestinal complications such as intestinal stenosis, intestinal fistula, cholestasis, and short bowel syndrome, and even affect the long-term development of the nervous system, which will bring a heavy burden to the family and society. At present, it is generally believed that the abnormal colonization of intestinal flora, ischemia and hypoxia, formula feeding and other factors synergistically lead to immature intestinal mucosal damage, and prompting inflammatory cells to release a large number of pro-inflammatory factors to cause inflammatory necrosis is the basic mechanism of its occurrence .

At present, more and more abnormal proliferations of immune cells such as monocytes/macrophages, γδT cells, Th17 cells (T helper17cell, Th17), regulatory T cells (regulatory T cells, Treg) have been confirmed to be closely related to NEC. relevant.

Therefore, it is urgent to provide a theoretical basis for strengthening and promoting the prevention and treatment of NEC through in-depth research on the molecular mechanism of cells closely related to NEC inflammation and necrosis.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies in the prior art, and provide the application of IL-6R antibody (IL-6 specific binding receptor) and amniotic fluid stem cells in the preparation of drugs for the treatment of NEC. The present invention combines IL-6R Antibodies and amniotic fluid stem cells can treat neonatal necrotizing enterocolitis: reverse Treg/Th17 cell imbalance, increase Foxp3 protein expression level, decrease RORγt protein expression level, increase STAT5 phosphorylation level and decrease STAT3 phosphorylation level.

In order to achieve the above object, the technical solution adopted by the present invention is: the application of an antibody that specifically binds to a receptor for IL-6 in the preparation of a drug for reversing Treg/Th17 cell imbalance.

In addition, the present invention also provides the application of the antibody specifically binding to the receptor of IL-6 in the preparation of drugs for increasing the expression level of Foxp3 protein and reducing the expression level of RORγt protein.

In addition, the present invention also provides the application of the antibody specifically binding to the receptor of IL-6 in the preparation of a drug for increasing the phosphorylation level of STAT5 and decreasing the phosphorylation level of STAT3.

In addition, the present invention also provides the application of the antibody specifically binding to the receptor of IL-6 in the preparation of a drug for treating neonatal necrotizing enterocolitis.

In addition, the present invention also provides a medicament for treating neonatal necrotizing enterocolitis, which includes an antibody specifically binding to a receptor of IL-6.

As an improvement of the above technical solution, the medicament further includes pharmaceutically acceptable auxiliary materials.

In addition, the present invention also provides the application of the antibody specifically binding to the receptor of IL-6 and the application of amniotic fluid stem cells in the preparation of medicine for treating neonatal necrotizing enterocolitis.

In addition, the present invention also provides a medicament for treating neonatal necrotizing enterocolitis, which comprises an antibody specifically binding to the receptor of IL-6 and amniotic fluid stem cells.

As an improvement of the above technical solution, the medicament further includes pharmaceutically acceptable auxiliary materials.

Beneficial effects of the present invention: the present invention provides the application of IL-6R antibody and amniotic fluid stem cells in the preparation of drugs for the treatment of NEC, and the present invention can treat neonatal necrotizing enterocolitis by combining IL-6R antibody and amniotic fluid stem cells: reverse Treg/ Th17 cell imbalance, increased Foxp3 protein expression level, decreased RORγt protein expression level, increased STAT5 phosphorylation level and decreased STAT3 phosphorylation level.

Description of drawings

Figure 1 shows that the expression of plasma IL-1β and IL-6 in NEC patients was significantly increased; among them, the ELISA detection and statistical analysis showed that the levels of plasma IL-1β (left in the figure) and IL-6 (right in the figure) in NEC patients were significantly higher than those in the CTRL group Elevated; CTRL: control group; NEC: disease group;

Figure 2 shows the effect of inflammatory factors on the polarization of Treg cells into IL-17 ⁺ Treg cells; among them, Figure 2A is a representative flow cytometry scattergram, showing that Treg cells are in the presence of IL-1β, IL-6 and the two together Under the induction conditions, the polarization of Treg cells to IL-17 ⁺ Treg cells; Figure 2B is a statistical analysis of the IL-17 ⁺ Treg cells induced by the above different conditions, the presence of IL-6 combined with IL-1β or IL-6 alone The polarization of IL-17 ⁺ Treg cells can be significantly induced under the induction conditions; CTRL: control group; NEC: disease group; ns: no significant difference between the two groups; *: P<0.05, **: P<0.01;

Figure 3 shows the effects of inflammatory factors on the expression of the transcription factor RORγt in co-cultured cells; among them, Figure 3A is a representative flow histogram, showing that Treg cells were co-cultured for 4 The expression of RORγt after two days; Figure 3B statistically analyzes the expression of RORγt under the above-mentioned different induction conditions, and the expression of RORγt can be significantly induced under the induction conditions of IL-6 combined with IL-1β or IL-6 alone; MFI: mean fluorescence Intensity; ns: no statistically significant difference; *: P<0.05;

Figure 4 shows the effect of blocking inflammatory signaling on the polarization of Treg cells to IL-17 ⁺ Treg cells; among them, Figure 4A is a representative flow cytometry scatter plot showing that Treg cells were induced by the combination of IL-1β and IL-6 Under the following conditions, anti-IL-1β antibody and anti-IL-6 antibody were added separately or jointly to block, Treg cells polarized to IL-17 ⁺ Treg cells; Figure 4B shows the generation of IL-17 ⁺ Treg cells under the above different blocking conditions Statistical analysis showed that anti-IL-6 antibody combined with anti-IL-1β antibody or anti-IL-6 antibody alone could significantly inhibit the polarization of Treg cells to IL-17 ⁺ Treg cells; IL-1β antibody, aIL-6 means anti-IL-6 antibody, None means no antibody added; ns: no statistically significant difference; *: P<0.05, **: P<0.01;

Figure 5 shows that anti-IL-6 antibody can significantly inhibit the polarization of Treg cells induced by pro-inflammatory factors; among them, Figure 5A is a representative flow scattergram, showing that Treg cells are under the condition of the co-existence of IL-1β and IL-6 , adding or not adding anti-IL-6 antibody for induction for 8 days, and observing its transdifferentiation into IL-17 ⁺ Treg ^and Th17 cells; Statistical analysis of Th17 cells showed that the addition of anti-IL-6 antibody blocking conditions not only significantly inhibited the polarization of Treg cells to IL-17 ⁺ Treg cells, but also effectively inhibited the polarization of Th17 cells;

Figure 6 shows that anti-IL-6R antibody can effectively reduce the mortality of NEC; Figure 6A shows the schematic diagram of NEC model induction and antibody treatment; Figure 6B shows the incidence of death in the three groups; CTRL: neonatal mice and mother mice were fed in the same cage as the control group; NEC+cIgG: NEC induction group was treated with neutralizing antibody; NEC+aIL6R: NEC induction group was treated with anti-IL-6R antibody; 3 independent experiments were carried out, CTRL group 5 rats each time, NEC+cIgG and NEC +aIL6R each 10; *: P<0.05, **: P<0.01;

Figure 7 shows that the anti-IL-6R antibody can effectively reduce the degree of tissue damage and the incidence of NEC; among them, Figure 7A is a HE staining (HE×100) image of the terminal ileum tissue, showing that the NEC+aIL6R treatment group can significantly reduce the degree of tissue damage ; Figure 7B is the double-blind scoring chart, 0 points for intact intestinal mucosal villi and normal tissue structure; 1 points for mild submucosal and/or lamina propria swelling and separation, 2 points for moderate submucosa and/or lamina propria Separation, submucosal and (or) muscular layer edema, 3 points for severe submucosa and (or) lamina propria separation, submucosal and (or) muscular layer edema, local villi loss, 4 points for disappearance of intestinal villi with intestinal necrosis, Statistical analysis showed that the NEC+aIL6R treatment group could significantly reduce the degree of tissue damage; Figure 7C statistical analysis showed that the NEC+aIL6R treatment group could significantly reduce the incidence of NEC; CTRL: neonatal mice were fed in the same cage as the control group; NEC+cIgG: NEC-induced group was treated with neutralizing antibody and control group; NEC+aIL6R: NEC-induced group was treated with anti-IL-6R antibody; three independent experiments were carried out, CTRL group with 5 rats each time, NEC+cIgG and NEC+aIL6R each time 10 each; *: P<0.05, ***: P<0.001;

Figure 8 shows that anti-IL-6R antibody can effectively reverse the Treg/Th17 cell imbalance in NEC tissue; Among them, Figure 8A is a representative flow histogram showing Treg and Th17 cell-specific transcription factors Foxp3 and The expression of RORγt; Figure 8B is a statistical analysis of the expression of Foxp3 and RORγt under the above different conditions. The NEC+aIL6R treatment group can significantly increase the expression level of Foxp3 and effectively reduce the expression level of RORγt; CTRL (n=7): newborn Mice and female mice were fed in the same cage as the control group; NEC+cIgG (n=6): NEC induction group was treated with neutralizing antibody; NEC+aIL6R (n=8): NEC induction group was treated with anti-IL-6R antibody ;Each sample was merged from 2-3 5cm intestinal tissues of the terminal ileum; *: P<0.05, ***: P<0.001; MFI: mean fluorescence intensity; ns: no statistically significant difference;

Figure 9 shows that anti-IL-6R antibody treatment can significantly increase the expression level of Foxp3 and reduce the expression level of RORγt; wherein, Figure 9A is the effect of Western Blot detection of anti-IL-6R antibody treatment on the protein expression level of Foxp3 and RORγt in tissues; 9B Calculate the gray value of the Western Blot conditions and then perform statistical analysis, showing that anti-IL-6R antibody treatment can significantly increase the expression level of Foxp3 in the tissue while reducing the expression level of RORγt; CTRL: Newborn mice and mother mice were fed in the same cage as the control group; NEC+cIgG: NEC induction group was treated with neutralizing antibody and control group; NEC+aIL6R: NEC induction group was treated with anti-IL-6R antibody; Western Blot was carried out in 3 independent experiments, and each group loaded 2 samples; β-actin: Internal reference protein; ***: P<0.001;

Figure 10 shows that anti-IL-6R antibody treatment can significantly increase the phosphorylation level of STAT5 and reduce the phosphorylation level of STAT3; wherein, the upper figure in Figure 10 shows the effect of anti-IL-6R antibody treatment on tissue STAT3 and STAT5 protein phosphorylation by Western Blot The impact of the level of phospholipidization; the lower figure in Figure 10 is calculated by gray value and then statistically analyzed for the Western Blot conditions, showing that anti-IL-6R antibody treatment can significantly increase the phosphorylation level of STAT5 in the tissue while reducing the phosphorylation level of STAT3; CTRL: Newborn mice and mother mice were fed in the same cage as the control group; NEC+cIgG: NEC induction group was treated with neutralizing antibody; NEC+aIL6R: NEC induction group was treated with anti-IL-6R antibody; Western Blot conducted three independent experiments , 2 samples per group; β-actin: internal reference protein; **: P<0.01, ***: P<0.001; ns: no statistically significant difference;

Figure 11 The combination therapy of anti-IL-6R antibody and amniotic fluid stem cells can prolong the survival rate of NEC model mice; among them, *: P<0.05; **: P<0.01.

Detailed ways

In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings.

It should be noted that: in the invention, the anti-IL-6R antibody, the IL-6R antibody and the antibody specifically binding to the receptor of IL-6 are the same substance, but the expression method is changed; others can be deduced by analogy.

Materials and Methods

research object

Case group: Selected from March 2016 to September 2017 in the Department of Neonatology of the Sixth Affiliated Hospital of Sun Yat-sen University, the Department of Neonatology of Guangdong Maternal and Child Health Hospital (including neonatal surgery), the Department of Neonatology of the Fifth People's Hospital of Dongguan, and the Child Health Care of Foshan City. Seventy-seven patients with NEC who were hospitalized in the Neonatology Ward of our hospital were divided into 3 groups according to the final diagnosis results according to the revised Bell staging criteria of NEC in "Practical Neonatology" (Fourth Edition): Suspected NEC (28 cases of NEC stage I), Diagnosed NEC (NEC stage II, 30 cases) and progressive NEC (NEC stage III, 19 cases), and suspected NEC all had symptoms of feeding intolerance (abdominal distension, gastric retention), and all cases underwent imaging examinations.

Control group: 80 non-NEC neonates who were hospitalized at the same time and matched with the case at the same gestational age, gender, birth weight, mode of delivery, Apgar score at 5 minutes after birth, and blood collection date (excluding systemic inflammatory response syndrome, sepsis, etc.) , gastrointestinal malformations and genetic metabolic diseases) as the control group; there was no significant difference in basic clinical data between the two groups. This study has passed the review of the Ethics Committee of the Sixth Affiliated Hospital of Sun Yat-sen University, conforming to the ethical standards set by the Chinese Ethics Committee, and obtaining the informed consent of the guardians of the research subjects.

Experimental animals: SPF freshmen C57/BL6 8-10 days old, male or female, purchased from the Experimental Animal Center of Southern Medical University. This study has been approved by the Experimental Animal Ethics Committee of Sun Yat-sen University.

main reagent

FITC-labeled anti-CD3 (HIT3a), BD Company, USA; APC/Cy7-labeled anti-CD4 (RPA-T4), BD Company, USA; PE-Cy7-labeled anti-CD25 (BC96), BioLegend Company, USA; PE-labeled anti-Foxp3 (259D/C7), BD Company, the United States; PE-labeled anti-RORγt (Q21-559), BD Company, the United States; PerCP-Cy5.5 labeled anti-IL-17A (N49-653), BD Company, The U.S.; FITC marked anti-CD3 (17A2), BD Company, the United States; APC/Cy7 marked anti-CD4 (GK1.5), BioLegend Company, the United States; PE-Cy7 marked anti-CD25 (PC61), BioLegend Company, the United States; PE marker anti-Foxp3 (MF23), BD Company, USA; PE marker anti-RORγt (Q31-378), BD Company, USA; BD Cytofix/Cytoperm ^TM Fixation/Permeabilization Kit, BD Company, USA; LaminaPropria Dissociation Kit, Miltenyi Biotec, Germany; Phosphate buffered saline (PBS), Invitrogen, USA; Fetal Bovine Serum (FBS), Invitrogen, USA; PMA, sigma, USA; ionomycin, sigma, USA; Brefeldin A, sigma, USA ; RPMI-1640, GIBCO, USA; IL-6 ELISA kit, Wuhan Huamei Bioengineering Co., Ltd. (Cat. No.: CSB-E04638h), China; IL-1β ELISA kit, Wuhan Huamei Bioengineering Co., Ltd. (Product No.: CSB-E08053h), China; Anti-IL-6(6078), RD Company, USA; Anti-IL-1β(8516), RD Company, USA; Anti-IL-6R(17506 ), RD Company, USA; Anti-RORγt (ab207082, 58kDa), abcam Company, UK; Anti-Foxp3 (ab10901, 47kDa), abcam Company, UK; Anti-STAT5 (ab16276, 90kDa), abcam Company, UK; -P-STAT5 (phospho Y694) (ab32364, 90kDa), abcam, UK; Anti-STAT3 (ab68153, 88kDa), abcam, UK; Anti-STAT3 (phospho S727) (ab32143, 88kDa), abcam, UK ; Anti-β-Actin (ab179467, 42kDa), abcam, UK; Goat anti-rabbit IgG-HRP (ab6721), abcam, UK; Goat anti-mouse IgG-HRP (ab6789), abcam, UK; PageRuler Prestained Protein Ladder (26616) (10-170KDa), Thermo scientific company, USA; reduced SDS-PAGE 5×Loading Buffer, Kangwei Century Biotechnology Co., Ltd., China; human CD4+CD25+CD127 ^dim/ -regulatory T Cell isolation kit, Miltenyi Biotec, Germany.

experimental method

Human Specimen Collection and Processing

The samples of the case group were collected within 4 hours after the clinical diagnosis of NEC, and 2 ml of peripheral venous blood was collected using sterile, heparin anticoagulant tubes. The control group selected blood collection at the same age as the case group. After collection, they were thoroughly mixed and separated by density gradient centrifugation to obtain peripheral blood mononuclear cells (PBMCs).

Magnetic bead sorting of Treg cells

1) Firstly, magnetically labeled non-CD4 ⁺ and CD127 ^hi T cells were labeled by the combined antibody indirect method, and the labeled PBMCs cell suspension was entered into the magnetic bead automatic sorter;

2) Collect the outflowing CD4 ⁺ CD127 ^dim/- T cells, add the collected cells to the magnetic bead antibody labeled with anti-CD25, and then enter the magnetic bead automatic sorter, then the magnetic field will select CD4 ⁺ CD25 ⁺ CD127 ^dim/- Treg cells Sex stays.

3) Mark CD3, CD4, CD25 and CD127, and detect the purity of CD4 ⁺ CD25 ⁺ CD127 ^dim/- Treg cells by PFC.

Induced polarized culture of Treg cells

The CD4 ⁺ CD25 ⁺ CD127 ^dim/- Treg cells sorted by magnetic beads were resuspended in supplemented with L-glutamine (2 mM), 2-mercaptoethanol (50 μM), recombinant human IL-2 (100 IU/ml) and In the TexMACS medium of double antibodies against penicillin and streptomycin (100 U/ml and 100 μg/ml respectively), adjust the cell density to 1×10 ⁶ /ml, add 100 μl/well to a 96-well culture plate, and add Immunomagnetic beads coated with anti-CD3/CD28 monoclonal antibody, add polarization factor IL-6 (10 μg/ml), or IL-1β (10 μg/ml) or IL-6 (10 μg/ml) as required IL-1β (10 μg/ml), induced for 4 days or 8 days. For antibody blocking experiments, anti-IL-6 antibody (10 μg/ml) or anti-IL-1β Antibody (10 μg/ml) or anti-IL-6 antibody (10 μg/ml) combined with anti-IL-1β antibody (100 ng/ml) was also induced and cultured for 4 days or 8 days.

ELISA test

The concentration of plasma IL-6 and IL-1β was determined by enzyme-linked immunosorbent assay kit (Wuhan Huamei).

Animal experiment

Establishment of Necrotizing Enterocolitis Model in Neonatal Rats

The specific steps of the neonatal mouse NEC model are as follows: newborn 8-10-day-old C57/BL6 mice were separated from the mother mice, and reared in the neonatal mouse incubator. According to the amount of 5g body weight and 200μl, clean and sterile silicone tube was used to oral Intubation gave formula milk gavage, time controlled at 2 to 3 minutes/time, gavage 5 times a day. At 8 o'clock and 20 o'clock every day, hypoxia (5% O ₂ , 95% N ₂ ) and low temperature (put in a refrigerator at 4°C) treatment were given, each for 10 minutes. At the same time, during the second gavage every day, add LPS (the amount of LPS is calculated according to 10 mg per kg body weight).

Animal grouping and handling

Normal control group (CTRL): mother mice were fed in the same cage; control antibody treatment group (NEC+cIgG): when NEC was induced, control antibody (100ng/kg/day) was given intraperitoneally; anti-IL6R antibody treatment group (NEC + aIL6R ): When NEC was induced, an anti-IL6R antibody (100 ng/kg/day) was administered intraperitoneally at the same time.

Specimen collection and processing

12 hours after the last hypoxia combined with low temperature stimulation treatment, they were sacrificed by cervical dislocation, and then the abdominal skin was cleaned and disinfected under aseptic conditions, and the abdominal cavity was opened along the midline of the abdomen with ophthalmic scissors, and the intestinal tube from the lower end of the duodenum to the ileocecal was removed , 0.5 cm of intestinal tube near the ileocecal portion was retained and fixed in 4% formaldehyde solution.

Observation indicators and methods

1) General condition and body weight of newborn mice

General conditions and body weight changes of mice: General conditions include spirit, reaction, activity status, food intake, abdominal distension, vomiting, gastric retention, changes in stool properties, etc.; weigh the body weight before the first feeding every day, and record the death Condition.

2) Gross shape change of intestinal tissue

Gross shape changes of intestinal tissue: After opening the abdominal cavity, the color change of intestinal tissue, pneumatosis in the intestinal cavity, necrosis and bleeding were observed and recorded with the naked eye.

3) Histopathological scoring

The fixed ileal tissue was stained with HE, and scored by two pathologists in a double-blind way. The scoring criteria are shown in Table 1. NEC was determined with a histological score ≥ 2.

Table 1 Intestinal histopathological scoring criteria

Preparation of Paraffin Sections of Intestinal Tissues of Neonatal Rats

1) Material collection: Fix the cut fresh intestinal tissue samples with 4% paraformaldehyde for no less than 24 hours; take the fixed tissue, use a scalpel in the fume hood to smooth the target site, and then trim the Good tissues and corresponding labels are placed in the dehydration box;

2) Dehydration: Put the dehydration box into the hanging basket of the dehydration machine, and dehydrate in the following order: 75% alcohol dehydration for 4 hours, 85% alcohol dehydration for 2 hours, 90% alcohol dehydration for 2 hours, 95% alcohol dehydration for 1 hour , dehydration with absolute ethanol for 30 minutes, second dehydration with absolute ethanol for 30 minutes, alcohol benzene dehydration for 5-10 minutes, xylene dehydration for 5-10 minutes, second xylene dehydration for 5-10 minutes, soft soaking wax for 1h, Hard wax dipping 1h;

3) Embedding: Use an embedding machine to embed the wax-soaked sample; put the melted wax into the embedding frame, take the sample out of the dehydration box before the wax solidifies, put it into the embedding frame and label it; Freeze on a -20° freezer, take out and trim the wax block after solidification;

4) Slicing: Place the trimmed wax block on a microtome with a slice thickness of 4 μm; then flatten the slice on 40°C warm water in the slide spreader, transfer it to a glass slide, test the slice in a 60°C oven for about 6 hours, and place it at room temperature Save for later.

HE staining

1) Dewaxing and hydration of paraffin sections: first put the sections in xylene for 20 minutes, repeat once, then put them in absolute ethanol for 10 minutes, repeat once, then put 95% alcohol for 5 minutes, 90% alcohol for 5 minutes, 80% alcohol for 5 minutes, 70% alcohol for 5 minutes, and finally wash with distilled water;

2) Hematoxylin staining of cell nuclei: put the section in Harris hematoxylin staining solution for about 5 minutes, then rinse with tap water, then differentiate with 1% hydrochloric acid alcohol differentiation solution for a few seconds, rinse with tap water immediately, and finally return to 0.6% ammonia water Blue, wash with tap water;

3) Cytoplasmic staining with eosin: put the slices into the eosin staining solution and stain for about 2 minutes;

4) Dehydration and sealing: first put the slice in 95% alcohol for 5 minutes, repeat once, then put it in absolute ethanol for 5 minutes, repeat once, then put it in xylene for 5 minutes, repeat once, to make it dehydrated Transparent, at this time, take the slice out of xylene to dry slightly, seal the slice with neutral gum, and pay attention to avoid the generation of air bubbles;

5) Use a microscope for image acquisition and analysis.

Western blot detection

Extraction of total protein

Take an appropriate amount of tissue in the grinder, and add the corresponding amount of lysate according to the proportion, and then grind it with a grinder to fully lyse; after lysing, centrifuge the lysate at 12000×g for 5 minutes, and absorb the supernatant to obtain The total protein extract was quantified using the BCA protein quantification kit; after quantification, 4 times the volume of 5×SDS protein loading buffer was added and boiled at 100°C for 10 minutes; then, it was stored at -20°C for later use.

Detection of protein expression by Western blotting

1) Make SDS-PAGE glue: clamp the aligned glass plates and fix them vertically on the shelf; prepare the separation gel solution, after adding TEMED at the end, shake it up immediately, pour the glue and seal it with water; wait for the water, glue When there is a refraction line between them, wait another 5 minutes for the gel to fully solidify, discard the water in the upper layer; prepare the concentrated gel, the process is the same as the preparation of the separating gel, fill the remaining space with the concentrated gel, insert the comb into the concentrated gel; concentrate After the gel is solidified, gently pull out the comb; rinse it with deionized water, then put it into the electrophoresis tank, and add enough electrophoresis buffer to start the sample electrophoresis;

2) Protein loading: use a micro-sampler to absorb 30ug of sample and slowly add it to the sample well, reserve one well and add 5μl of protein marker;

3) Separation electrophoresis: turn on the power, first adjust the voltage to 80V, and run the electrophoresis for about 30 minutes. After running the stacking gel, adjust the voltage to 120V, and run the electrophoresis for about 60 minutes (bromofin blue runs to about 1cm from the bottom of the separation gel, stop the electrophoresis);

4) Transfer membrane: cut off the target protein strip according to the position of the protein marker; then prepare PVDF membrane: first soak in methanol for 1-5 minutes, then equilibrate with transfer solution for 10 minutes, and put the filter paper and sponge into the transfer membrane at the same time After soaking, start from the positive electrode, put sponge, filter paper, PVDF membrane, gel, filter paper, sponge in sequence, then close the negative electrode, put it into the electrotransfer tank, turn on the power to transfer the membrane; according to the target protein Select the appropriate transfer current and time (300mA, 60 minutes for RORγt, Foxp3; 300mA, 90 minutes for STAT5, P-STAT5, STAT3, P-STAT3);

5) Blocking: After the membrane transfer, remove the membrane, gently rinse once in TBST, seal all proteins with 5% skimmed milk powder, and block overnight at 4°C;

6) Primary antibody incubation: discard the blocking solution, wash the membrane with TBST for 5 minutes, and repeat 6 times; incubate the primary antibody, and dilute it with TBST according to the desired antibody concentration (the concentration used for the target protein is 1 :1000; 1:5000 for STAT3, 1:200000 for P-STAT3); incubate overnight at 4°C;

7) Secondary antibody incubation: wash the membrane with TBST for 5 minutes, repeat 6 times, incubate the secondary antibody, dilute with TBST according to the required antibody concentration, and incubate at room temperature for 2 hours (the concentration of the secondary antibody used is 1:4000);

8) Color development: wash the membrane with TBST for 5 minutes, repeat 6 times, use the ECL color development kit to develop color, according to the size of the membrane, take the appropriate amount of A and B solution, mix well, use filter paper to wash the membrane from one corner of the membrane Drain the liquid, put it on a clean plastic wrap, spread the mixed solution, develop the color for 1-5 minutes, absorb the color developing solution, fold the plastic wrap so that the film is sandwiched in the middle of the plastic wrap, and place it in a cassette; Observe the brightness of the fluorescence in the darkroom to determine the exposure time, develop and fix the film;

9) Take photos (or scans) and record the analysis results.

Isolation of mononuclear cells (LPMCs) from the lamina propria of the small intestine

1) Merge the remaining small intestinal tissue into one sample according to the groups of 2 to 3 mice, and wash it with HBSS (w/o) for 2 to 3 times, and remove the adhering fat tissue at the same time. Cut in the direction and cut into small pieces of about 0.5cm;

2) Put the above-treated tissue block into a 50ml centrifuge tube containing 20ml lamina propria tissue pre-digestion solution, cover the tube cover and digest in a shaking incubator at 37°C for 20 minutes;

3) After digestion, vortex on the vortex for 10 seconds, then filter with a 100μm cell sieve, discard the filtrate, transfer the upper tissue piece to a new 50ml centrifuge tube again, add 20ml tissue pre-digestion solution, cover Digest the caps of the tubes in a shaking incubator at 37°C for 20 minutes;

4) After digestion, vortex on a vortex for 10 seconds, then filter with a 100 μm cell sieve, discard the filtrate, transfer the upper layer tissue piece to a new 50ml centrifuge tube, add HBSS (w/o) to wash solution, cover the tube and incubate in a shaking incubator at 37°C for 20 minutes;

5) After incubation, vortex on the vortex for 10 seconds, then filter with a 100μm cell sieve, discard the filtrate, at this time the upper tissue becomes white and transparent, and then place it in 3ml lamina propria tissue digestion solution , cover the tube and digest in a shaking incubator at 37°C for 60 minutes;

6) After digestion, centrifuge at 300×g for 5 minutes at room temperature, discard the supernatant, add 5ml of PBS buffer, and wash once;

7) Add 10ml of PBS buffer solution, vortex for 10 seconds on a vortex, then filter with a 100 μm cell sieve, collect the filtrate to obtain small intestinal tissue lamina propria mononuclear cells (lamina propria mononuclear cells, LPMCs).

flow detection

1) Collect stimulated cultured Treg cells or separate LPMCs obtained from small intestine tissue, add PMA at a final concentration of 50ng/ml, 1μM ionomycin, and 10μg/ml BFA, mix well and store at 37°C, containing 5% Incubate for 5 hours in a CO2 cell incubator;

2) Collect the cells, centrifuge at 500g, 4°C for 5 minutes, discard the supernatant;

3) Resuspend the cells, add 2ml 1× staining buffer, vortex gently to wash the cells; repeat step 2), discard the supernatant, and resuspend the cells;

4) Add 100 μl 1× staining buffer, add the corresponding amount of surface-labeled antibody according to the antibody use requirements, and keep in an ice bath for 20 minutes in the dark; add 1ml 1× staining buffer, 500g, 4°C, centrifuge for 5 minutes, wash twice, discard clear;

5) Add 500 μl of pre-cooled fixation/permeabilization agent, vortex gently, and incubate at room temperature in the dark for 20 minutes; centrifuge at 500 g, 4°C for 5 minutes, and discard the supernatant;

6) Add 2ml of BD Perm/Wash ^TM buffer, incubate at room temperature in the dark for 10 minutes; centrifuge at 500g, 4°C for 5 minutes, and discard the supernatant;

7) Add 100 μl BD Perm/Wash ^TM buffer, add the corresponding amount of intracellular labeled antibody according to the antibody use requirements, and keep it in the dark for 30 minutes; add 2ml BD Perm/Wash ^TM buffer, 500g, 4°C, centrifuge for 5 minutes, and wash 2 times, discard the supernatant;

8) Add 300 μl ~ 500 μl 1× staining buffer to resuspend the cells, and test on the machine.

Data Statistics and Analysis

Unless otherwise stated, all data are expressed as mean ± standard deviation, and Mann-Whitney U test is used for statistics, and Spearman rank correlation is used for Bell stage and CCR9 ⁺ IL-17 ⁺ Treg cell correlation analysis.

result

IL-6 Promotes the Polarization of Peripheral Blood Treg Cells

The plasma pro-inflammatory factors IL-1β and IL-6 were detected in NEC and CTRL groups, as shown in Figure 1, the levels of IL-1β and IL-6 in the plasma of NEC patients were significantly increased.

Peripheral blood Treg cells of newborns in NEC and CTRL groups were obtained respectively. In the basic medium containing anti-CD3, CD28 antibody-coated magnetic beads and IL-2, IL-1β and IL-6 were added separately or simultaneously under induction conditions, Co-cultured for 4 days, and observed the polarization of Treg cells to IL-17 ⁺ Treg cells; as shown in Figure 2, it was found that: 1) only a small amount of IL-17 cells were produced under the basic medium culture conditions; 2) In the presence of the pro-inflammatory factor IL-1β, although there was a slight increase compared with the basic medium culture group, the difference was not statistically significant; 3) Under the induction condition of IL-6 or IL-6 combined with IL-1β, Th17 cells were significantly increased, although the combined induction was higher than that induced by IL-6 alone, but the difference was not statistically significant; 4) Treg cells in NEC patients were polarized to IL-17 ⁺ The proportion of Treg cells was significantly higher than that of CTRL group.

At the same time, the Th17 cell-specific transcription factor RORγt was detected, as shown in Figure 3, and the above-mentioned consistent change rules were found.

Furthermore, under the condition of joint induction of pro-inflammatory factors IL-6 and IL-1β, anti-IL-6 antibody, anti-IL-1β antibody or joint addition was added, as shown in Figure 4, it was found that although anti-IL-1β antibody could lighten the The frequency of Th17 cells was significantly reduced, but the difference was not statistically significant. On the contrary, anti-IL-6 antibody combined with anti-IL-1β antibody or anti-IL-6 antibody alone could significantly reduce the proportion of Th17 cells, and there was no difference between the two Statistical significance.

The above studies have shown that IL-6 can promote the polarization of Treg cells in the peripheral blood of neonates, especially NEC patients, to IL-17 ⁺ Treg cells. The IL-6 and IL-1β combined group was cultured for 8 days, as shown in Figure 5, it was found that: 1) Treg cells could be polarized into Th17 cells by prolonging the induction culture time, 2) when anti-IL-6 antibody was added, It was found that not only the proportion of IL-17 ⁺ Treg cells can be reduced, but also the proportion of Th17 cells can be reduced.

Anti-IL-6R antibody can reduce the incidence of NEC and the degree of tissue damage in neonatal mice

Through the NEC model of neonatal mice, anti-IL-6R antibody or control antibody IgG was injected intraperitoneally to observe its therapeutic effect. As shown in Figure 6, it was found that injection of anti-IL-6R antibody can effectively reduce the mortality of neonatal mice.

As shown in Figure 7, histological examination found that anti-IL-6R antibody treatment can also significantly reduce the degree of tissue damage and the incidence of NEC.

Anti-IL-6R antibody can reverse Treg/Th17 cell imbalance

As shown in Figure 8, the status of Treg/Th17 cell subsets in tissues was detected by PFC, and it was found that compared with anti-IgG antibodies, anti-IL-6R antibody treatment can significantly increase the frequency of Treg cells while reducing the frequency of Th17 cells.

In fact, by Western Blot technology, as shown in Figure 9, the detection of Treg and Th17 cell-specific transcription factors Foxp3 and RORγt also found that anti-IL-6R antibody treatment can significantly increase the protein level of Foxp3 and reduce the protein level of RORγt.

Since STAT3 and STAT5 play an important role in the transcriptional regulation of Th17 and Treg cells, respectively. It was further evaluated whether anti-IL-6R antibody treatment reversed the unbalanced Treg/Th17 cells by regulating the phosphorylation level of STAT3/STAT5. Using Western Blot technology, as shown in Figure 10, it was found that compared with the anti-IgG antibody treatment group, anti-IL-6R antibody treatment can significantly increase the phosphorylation level of STAT5 and reduce the phosphorylation level of STAT3.

There are two types of IL-6 receptors, one is specific binding receptor IL-6R, the other is gp130, IL-6R is divided into membrane-bound type and free type, membrane-bound IL-6R participates in classical signaling Transduction pathway, free IL-6R participates in the trans-signal transduction pathway. Compared with anti-IL-6 antibody and anti-gp130 antibody, the present invention unexpectedly found that anti-IL-6R antibody can reduce the frequency of Th17 cells, thereby exerting a therapeutic effect.

Combination therapy of anti-IL-6R antibody and amniotic fluid stem cells can prolong the survival rate of NEC model mice

The amniotic fluid samples were obtained from the amniotic fluid extracted from pregnant women in the second trimester (16-24 weeks of pregnancy) undergoing prenatal examination and amniocentesis. Under sterile conditions, about 5 mL of amniotic fluid was extracted by B-ultrasound positioning and amniocentesis. Preparation of MSCs medium: Low-sugar DMEM medium (containing 10% fetal bovine serum by volume) and MESEN PRO medium were prepared in a volume ratio of 1:1 (Gibco, USA).

Isolation and culture steps

Centrifuge the amniotic fluid in the second trimester (1500r/min, 7min), remove the supernatant, resuspend the pellet in PBS, centrifuge (1000r/min, 5min) 3 times, remove 4mL of PBS amniotic fluid medium, resuspend the pellet and mix well, then move to ^25cm2 for culture cultured in a bottle at 37°C in an incubator with a volume fraction of 5% CO ₂ and saturated humidity, observe after 7 days and change the medium for the first time, replace the culture medium with MSCs medium, and then change the medium every 3 days. When the confluence is about 80%, it is recorded as the first generation P1. After that, when the cell confluency reaches more than 85%, it is subcultured at a ratio of 1:2 to 1:3; the cultured P3 cells are collected and used.

As shown in Figure 11, the survival rates of amniotic fluid stem cells (hAFMSC) combined with anti-IL-6R antibody (aIL6R) treatment group, anti-IL-6R antibody treatment group alone, and control antibody treatment group were 85%, 70%, and 37.5%, respectively; Thus, the combined treatment of anti-IL-6R antibody and amniotic fluid stem cells can prolong the survival rate of NEC model mice.

Finally, it should be noted that the above embodiments are used to illustrate the technical solutions of the present invention rather than limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can The technical solution of the present invention shall be modified or equivalently replaced without departing from the spirit and scope of the technical solution of the present invention.

Claims (4)

1. The application of an antibody specifically binding to a receptor of IL-6 in the preparation of a drug for treating neonatal necrotizing enterocolitis, characterized in that the drug is used to increase the expression level of Foxp3 protein and reduce the level of RORγt protein expression , or for increasing the phosphorylation level of STAT5 and decreasing the phosphorylation level of STAT3; the antibody specifically binding to the receptor of IL-6 is Anti-IL-6R 17506. 2. The application according to claim 1, wherein the medicine further comprises pharmaceutically acceptable auxiliary materials. 3. The application of IL-6 specifically binding receptor antibody and amniotic fluid stem cells in combination in the preparation of medicines for treating neonatal necrotizing enterocolitis, characterized in that the IL-6 specifically binding receptor antibody For Anti-IL-6R17506. 4. The application according to claim 3, characterized in that, the medicine further comprises pharmaceutically acceptable auxiliary materials.