CN115806615A

CN115806615A - Antibody and mesenchymal stem cell exosome preparation method and application of antibody and mesenchymal stem cell exosome in combined treatment of diseases

Info

Publication number: CN115806615A
Application number: CN202210866141.6A
Authority: CN
Inventors: 张化杰; 裴舜
Original assignee: Beijing Yiyuan Biotechnology Co ltd
Current assignee: Shenzhen Betis Biotechnology Co ltd
Priority date: 2022-07-22
Filing date: 2022-07-22
Publication date: 2023-03-17
Anticipated expiration: 2042-07-22
Also published as: CN115806615B

Abstract

The application provides a pharmaceutical composition for treating inflammatory bowel diseases and application thereof, wherein the pharmaceutical composition comprises a cell exosome and an anti-IL-17 antibody, and the cell exosome is a stem cell exosome induced by TGF-beta, IL-2, IGF-1 and bFGF. The IBD is treated by combining the stem cell exosome and the anti-IL-17 antibody, so that IBD symptoms can be obviously relieved, and the stability of the immune environment in vivo is maintained; the anti-IL-17 antibody with medium affinity is selected, so that the transient immune stress reaction can be inhibited, the normal physiological function of an organism can be maintained, and the IBD symptom is prevented from being worsened; the exosome derived from the umbilical cord mesenchymal stem cells is selected, the ethical risk is overcome, the source is stable and wide, and the clinical large-scale application is facilitated; the exosome generated by the umbilical cord mesenchymal stem cells after being induced by various immune factors can obviously improve the proliferation capacity of colon mucosal epithelial cells, effectively resist oxidative stress and maintain the normal physiological function of the digestive tract.

Description

Antibody and mesenchymal stem cell exosome preparation method and application of antibody and mesenchymal stem cell exosome in combined treatment of diseases

The technical field is as follows:

the invention belongs to the field of medicine research, and particularly provides a preparation method of an antibody and a mesenchymal stem cell exosome and application of the antibody and the mesenchymal stem cell exosome in combined treatment of diseases.

Background art:

inflammatory Bowel Disease (IBD) is a long-term recurrent Inflammatory disease that affects all parts of the gastrointestinal tract, IBD is widely distributed around the world, there are about 500 million patients worldwide, and the prevalence of this disease has increased significantly in young people in recent years. IBD includes two common diseases, crohn's Disease (CD) and Ulcerative Colitis (UC), both of which have the same clinical course, with differences limited to the location and nature of the inflammatory lesions, a transmural disease affecting the entire intestinal wall, which may be intermittently related to any part of the intestine, but most cases are primarily limited to the distal end of the small intestine; ulcerative colitis is primarily a disease of the colon that extends continuously from the rectum to the proximal end of the large intestine, and unlike CD, UC lesions are usually confined to the epithelial mucosa.

Clinically, IBD shows various clinical symptoms including abdominal discomfort, diarrhea, bloody stool, fever, fatigue, weight loss and the like, IBD patients often show parenteral complications possibly involving skin diseases (erythema nodosum and pyoderma gangrenosum), eye diseases (conjunctivitis, uveitis), rheumatic diseases (ankylosing spondylitis and peripheral arthritis), hepatobiliary diseases (primary sclerosing cholangitis) and urinary system diseases (kidney stones) and the like, in addition to the above frequently reported symptoms, which seriously affect the quality of life of patients and also cause a great economic burden.

The exact pathogenesis of IBD is not well defined and is often considered to be the result of a persistent inflammatory process against endogenous microorganisms in genetically predisposed individuals. This disease has been shown to be a genetic disease and twin studies have shown a consistent incidence of IBD, especially in the same 25% to 30% of cases with a positive family history in the same pair. In addition to genetic factors, IBD is mediated by a variety of environmental factors, including smoking, stress, oral contraceptives, non-steroidal anti-inflammatory drugs (NSAIDs), vitamin D deficiency, pollution, diet, and early antibiotic use, among others. It has also been reported that the gut microbiome is clearly essential for the development of IBD, and that people with less microbial diversity are predisposed to the disease, in which case a drastic change in gut microbial composition is associated with increased susceptibility to IBD. IBD is also attributed to an uncontrolled inflammatory state triggered by a dysregulated immune response at the level of intestinal epithelial cells, which occurs due to up-regulation of various pro-inflammatory pathways and parallel down-regulation of several regulatory pathways, which can be attributed firstly to an impaired integrity of the intestinal barrier and secondly to the diffusion of luminal pathogens into the intestinal wall making the innate immune cells unable to recognize and eventually eliminate the invading bacteria, and to an increased expression of potent inflammatory molecules such as IL-1, IL-6, IL-12, IL-23 and TNF- α, leading to delayed clearance of microbial agents and subsequent excessive inflammatory response.

The therapeutic means of IBD have been advanced to a certain extent, and for mild IBD, aminosalicylate drugs represented by mesalamine are the first choice therapeutic drugs, and mesalamine is rectally administered to relieve UC or CD symptoms; for moderate disease patients with mescaline resistance, oral administration of glucocorticoids or immunomodulators such as TNF- α monoclonal antibody (infliximab) is a common treatment regimen; steroid drugs such as budesonide are also an alternative therapeutic drug, but are prone to systemic adverse effects upon prolonged administration, and therefore steroid drugs are generally blocked within 6 weeks. In addition to drug therapy, surgical therapy is also a routine treatment, patients with severe fistulas are treated with shunt ostomy, patients with severe UC are treated with total colectomy, but surgical therapy poses a severe economic burden and life inconvenience, along with a risk of recurrence. Therefore, there is an urgent clinical need to develop novel therapeutic drugs or therapeutic techniques to effectively and chronically alleviate the symptoms of IBD. The main action mechanism of IBD therapy is to correct immune disorder and suppress inflammatory reaction in intestinal mucosa, and extracellular secretion is one of the research hotspots in cell biology in recent years, and this kind of substance can participate in the body immune regulation through multiple ways, thus becoming a new breakthrough in the field of IBD therapy.

In 1983, pan and Johnstone discovered for the first time that transferrin receptor is released into the extracellular space during maturation of sheep reticulocytes and is associated with a vesicle, the first report on extracellular secretion (Pan BT, johnstone RM.title of the transduction receptor reduction of skin microorganisms in vitro: selective expression of the receptor. Cell1983; 33; in 1989, johnstone defined this functional vesicle as an exosome (Johnstone RM, bianchini A, long K.regenerative vesicle formation and exosome release: transfer receiver ligation exosomes show multiple vesicles membrane functions. Blood 1989. In the last three decades, research on exosomes was in a state of stasis, and no unified concept of extracellular vesicles was proposed, which are referred to as "microvesicles", "exosomes", "microparticles" and appear in different research reports; researchers now classify extracellular vesicles into two broad categories, depending on the manner in which they are secreted. One class is called microvesicles, which are shed directly from the cell membrane; the other, called exosomes, release vesicles by exocytosis when they fuse with the plasma membrane. Exosomes can be released from many cell types, such as blood cells, endothelial cells, immune cells, platelets and smooth muscle cells, and it is widely believed that exosomes, which play an important role in immune responses, tumor progression and neurodegenerative diseases, can modulate the biological activity of recipient cells by transporting lipids, proteins and nucleic acids when circulating in the extracellular space.

Exosomes are abundant and complex in composition, mainly comprising proteins, micro-RNAs (mirnas), messenger RNAs (mrnas), and lipids, and exosome proteins that have been found include the Ras superfamily of monomeric G proteins (Rab), adhesion molecules, cytokines, heat shock proteins (e.g., HSC73, HSC 90), annexins I, II, V, and VI, cytoskeletal proteins (e.g., actin, moesin, albumin), tetraspanins (CD 9, CD63, CD81, CD 82), and GTPases, among others; exosome lipids include sphingolipids such as cholesterol, phospholipids (e.g., phosphatidic acid, phosphatidylserine), diglycerides, glycerophospholipids, arachidonic acid (polyunsaturated omega-6 fatty acids), ceramides, and sphingomyelins; exosome RNAs are quite abundant in species, and exosome RNA databases report 18333 mrnas, 58330 circular RNAs (circrnas), and 15501 long non-coding RNAs (lncrnas), which are involved in regulating gene expression associated with stem cell differentiation, hematopoiesis, organogenesis, tumorigenesis, and tumor metastasis, and play a key role in the protein transport process.

Numerous studies have shown that exosomes can participate in the physiological processes of IBD, exerting immunomodulatory functions including: (1) Participating in cellular immunomodulation, such as the systemic administration of exosomes obtained from bone marrow mesenchymal stem cells, which cause M2b macrophages to polarize without causing intestinal fibrosis, maintain intestinal barrier integrity and down regulate inflammatory responses, may greatly reduce colitis in various models of IBD, and are also involved in the regulation of a variety of proteins with anti-inflammatory bowel disease activity, particularly metallothionein-2, which is involved in the suppression of inflammatory responses; exosome-induced immune system cell regulation; administration of exosomes to Inflammatory Bowel Disease (IBD) environment can promote anti-inflammatory responses to polarize macrophages to M2 phenotype, suppress dendritic cell activation and induce their immune tolerance, trigger regulatory T cell (Treg) activation while suppressing T helper type 1 (Th 1) cells; (2) Participating in gut microbiome regulation, exosomes may use gram-negative receptors (e.g. Toll like receptor 4, tlrr 4) and gram-positive receptors (e.g. TLR 2) to stimulate pro-inflammatory responses, and the role of exosome HSP72 in IEC function, and protein components of cow's milk-derived EV have also been studied and reported for a total of 1963 proteins, including common EV-associated proteins such as CD9, flotillin-1 and Annexin A5, and 633 unidentified proteins, this active EV component may be involved in gut microbiome regulation in normal and IBD mucosal microenvironments, altering the diversity and composition of the gut microbiome in mice; (3) Participating in intestinal barrier regulation, exosomes derived from mesenchymal stem cells of bone marrow remarkably restore abnormal intestinal barrier function by decreasing intestinal permeability and reducing infiltration of intestinal microbiota into the lamina propria, and the mRNA levels of colonic antimicrobial proteins such as lysozyme 1 (Lyz 1), angiopoietin-4 and defensins Defa29 and Defa20 are also restored.

Stem cell-derived exosomes, which elicit their therapeutic activity by delivering a cargo consisting of potentially therapeutic proteins and RNAs to recipient cells, are increasingly considered as potent bioregulators. A great deal of research shows that the stem cell-derived exosomes can relieve IBD symptoms, such as exosomes obtained from human umbilical cord Mesenchymal Stem Cells (MSCs) can remarkably relieve IBD by up-regulating the levels of IL10 and IFN-gamma induced protein-10 (IP 10) and simultaneously down-regulating the levels of IL-1 beta, TNF-alpha, IL-6 and ubiquitin-binding enzyme (E2M); decreased expression levels of proteins such as K48, K63 and FK2 in exosome-treated IBD mice indicate that they are involved in modulating ubiquitin modification; MSC-derived exosome therapy may also down-regulate inflammatory responses, maintain gut mucosal barrier integrity and polarize macrophages to the M2b phenotype, inhibiting intestinal fibrosis; bone marrow MSC-derived exosomes also prevent IBD by lowering the mRNA and protein levels of nuclear factors kappa B (NF-. Kappa.B), p65, iNOS, COX2, TNF-. Alpha.and IL-1. Beta., but increasing IL-10 levels, oxidation-inducing factors such as MPO and Malondialdehyde (MDA) are down-regulated, while antioxidant factors such as Glutathione (GSH) and superoxide dismutase (SOD) are increased. Although the stem cell exosome plays an important role in the treatment process of IBD, the preparation process and the structural components of the stem cell exosome are complex and have high instability, so that the clinical application is limited.

The occurrence and development of IBD are also related to in vivo inflammatory factor imbalance, IL-17 is one of the important inflammatory factors, IL-17 is the main cytokine secreted by Th17 cells, and is divided into 6 subtypes, such as IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, etc., wherein IL-17A is the most common, and IL-17 refers to IL-17A unless otherwise specified in the prior art. The clinical research shows that the content of Th17 cells and IL-17 in colon mucosa of inflammatory bowel diseases including UC and CD patients is much higher than that of healthy people, and the research shows that the RNA expression of IL-17A and IL-17F in the intestinal tract of inflammatory bowel diseases patients is also much higher than that of normal people, and the results all indicate that the expression of IL-17 is increased to be closely related to the onset of IBD. However, the present research results show that IL-17 seems to have dual effects of anti-inflammatory and pro-inflammatory, and contradictory results appear in clinical and animal experiments, on one hand, the research shows that IL-17 neutralizing antibody or IL-17 inhibitor can relieve IBD symptoms, if researchers administer IL-17 antibody to T cell transformed mouse colitis model to obtain similar therapeutic effect with IL-6 antibody, and IL-17 and IL-6 antibody together can obtain additive therapeutic effect; inhibition of IL-17A or IL-17F alone, blocking the signaling pathway of both cytokines, was able to inhibit CD4/CD25 metastasis induced colitis (Wedebye Schmidt EG, larsen HL, kristensen NN, et al. TH17 cell indication and effects of IL-17A and IL-17F block in experimental colitis. Inflamm Bowel Dis2013; on the other hand, overuse of IL-17 inhibitors also appears to exacerbate the colonic validation response, disrupting intestinal mucosal structure, as in clinical trials using approved marketed anti-IL-17 antibodies ixekizumab and brodalumab for the treatment of psoriasis, symptoms of IBD are observed in some patients; in IL-17 knockout mice, symptoms of knockout mice were aggravated by using a dextran sulfate nano tube (DDS) induced colitis model. The above evidence makes it difficult to give a clear mechanism of action of IL-17 in IBD, making IL-17 antibodies challenging to treat IBD.

In order to solve the above-mentioned difficulties encountered in the development of IBD drugs, the invention provides a pharmaceutical composition, which comprises exosomes derived from mesenchymal stem cells and anti-IL-17 antibody with medium affinity, and can effectively alleviate IBD devices, inhibit the over-expression of inflammatory factors, inhibit the apoptosis of intestinal mucosal epithelial cells, resist oxidative stress, maintain the normal immune microenvironment of intestinal tract, and improve intestinal tract function.

Disclosure of Invention

In order to solve the technical problems, the invention provides a pharmaceutical composition for treating inflammatory bowel disease, which is characterized by comprising a cell exosome and an anti-IL-17 antibody, wherein the cell exosome is a stem cell exosome induced by TGF-beta, IL-2, IGF-1 and bFGF; the heavy chain variable region of the anti-IL-17 antibody comprises HCDR1 shown in SEQ ID NO. 1, HCDR2 shown in SEQ ID NO. 2 and HCDR3 shown in SEQ ID NO. 3, and the light chain variable region of the anti-IL-17 antibody comprises LCDR1 shown in SEQ ID NO. 4, LCDR2 shown in SEQ ID NO. 5 and LCDR3 shown in SEQ ID NO. 6.

On one hand, in the invention, the IBD is treated by using the mesenchymal stem cell exosome, the mesenchymal stem cell has multidirectional differentiation capacity, the exosome contains various active ingredients such as abundant regulatory protein, cell factor, immune factor, lipid component, long-chain non-coding RNA (ribonucleic acid), miRNA (micro ribonucleic acid) and the like, and the exosome secreted by the umbilical cord-derived stem cell can effectively relieve IBD symptoms; in addition, when the mesenchymal stem cell exosome is prepared, the exosome is induced by immunoregulatory factors such as TGF-beta, IL-2, IGF-1, bFGF and the like, so that the cell proliferation promoting capacity and the immunoregulatory capacity of the exosome are enhanced.

On the other hand, in the invention, the anti-IL-17 antibody is used for treating IBD, IL-17 is an immune factor with dual functions of anti-inflammation and proinflammatory, and the excessive or underexpression of the IL-17 content in the organism has adverse effects on IBD patients, so that the anti-IL-17 antibody with medium affinity is selected in the invention, not only can neutralize the over-expressed IL-17, but also can prevent the over-inhibition of an IL-17 signal channel, thereby effectively relieving IBD symptoms.

Furthermore, the amino acid sequence of the heavy chain variable region of the anti-IL-17 antibody is shown in SEQ ID NO. 7.

Furthermore, the amino acid sequence of the variable region of the light chain of the anti-IL-17 antibody is shown as SEQ ID NO. 8.

Further, the stem cell exosome is selected from at least one of bone marrow mesenchymal stem cell exosome, umbilical cord mesenchymal stem cell exosome, adipose mesenchymal stem cell exosome, endothelial progenitor cell exosome and neural stem cell exosome.

Further, the stem cell exosome is an umbilical cord mesenchymal stem cell exosome, and the preparation method comprises the following steps: culturing umbilical cord mesenchymal stem cells, adding exosome induction medium when the cell fusion degree reaches more than 80%, 5% ₂ Culturing in an incubator at 37 ℃ for 48 hours; collecting stem cell exosome by gradient centrifugation, collecting cell supernatant after induction culture, filtering with a 0.22 μm needle filter, centrifuging at low temperature of 300 Xg for 10min, and collecting supernatant; centrifuging at 2000 Xg for 10min, and collecting supernatant; centrifuging at 10000 Xg for 30min, and collecting supernatant; centrifuging at 100000 Xg for 1h, and resuspending the precipitate with sterile PBS solution to obtain stem cell exosome.

In the invention, the mesenchymal stem cells from the umbilical cord are used, and the exosome of the stem cells is obtained through induced expression, so that the stem cells have strong multidirectional differentiation capability and rich sources, and have no ethical risk and limitation, thereby being convenient for large-scale clinical use.

Further, the exosome-inducing medium is an α MEM medium containing 10% FBS, 50 μ g/mL TGF- β, 10 μ g/mL IL-2, 10 μ g/mL IGF-1, 10 μ g/mLbFGF.

The invention combines the physiological characteristics of mesenchymal stem cells and the pathogenesis of IBD, creatively proposes that four factors such as TGF-beta, IL-2, IGF-1, bFGF and the like are induced, and the applicable proportion is optimized so as to achieve the optimal combination effect.

Further, the pharmaceutical composition also comprises a pharmaceutically acceptable carrier.

Provides the application of the pharmaceutical composition in preparing a medicament for treating inflammatory bowel disease.

Further, the inflammatory bowel disease includes crohn's disease and ulcerative colitis.

Advantageous effects

The application provides a pharmaceutical composition for treating inflammatory bowel disease and application thereof, and has the following advantages:

(1) The stem cell exosome is combined with the anti-IL-17 antibody to treat IBD, so that IBD symptoms can be relieved obviously, and the stability of the immune environment in vivo is maintained;

(2) The anti-IL-17 antibody with medium affinity is selected, so that the transient immune stress reaction can be inhibited, the normal physiological function of an organism can be maintained, and the IBD symptom is prevented from being worsened;

(3) The exosome derived from the umbilical cord mesenchymal stem cells is selected, the ethical risk is overcome, the source is stable and wide, and the clinical large-scale application is facilitated;

(4) The exosome generated by the umbilical cord mesenchymal stem cells after being induced by various immune factors can obviously improve the proliferation capacity of the epithelial cells of the colon mucosa, effectively resist oxidative stress and maintain the normal physiological function of the digestive tract.

Drawings

FIG. 1: NCM460 cell proliferation capacity;

FIG. 2: rat DAI score;

FIG. 3: serum TNF- α expression levels;

FIG. 4: serum IL-1 β expression level;

FIG. 5: serum IL-6 expression levels.

Detailed Description

The following non-limiting examples enable those of ordinary skill in the art to more fully understand the present invention and are not intended to limit the invention in any way. All the technologies implemented based on the above-mentioned contents of the present invention should fall within the scope of the claims of the present application.

The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagent biomaterials, test kits, if not specifically indicated, are commercially available.

EXAMPLE 1 anti-IL-17 antibody preparation

Fully emulsified recombinant human IL-17 protein (Peprotech, USA) with complete Freund's adjuvant, BALB/c mice were injected subcutaneously in multiple immunizations at a dose of 50. Mu.g/mouse, once every two weeks, three immunizations in total. Taking blood from tail veins 10 days after the third immunization, determining the antibody titer, and selecting the mouse tail vein with the highest antibody titer for boosting immunization once, wherein the antibody titer is up to 1; killing the mice after 3 days, removing spleen, separating spleen lymphocytes by pressure water injection, centrifuging and washing cells, resuspending with DMEM culture solution, collecting mouse myeloma cells SP2/0 cells, culturing with 10% bovine serum DMEM culture medium, and 5% CO ₂ SP2/0 myeloma cells were primary-cultured in a 37 ℃ incubator, and cell fusion was performed after the cells had entered the logarithmic growth phase. Adding mouse spleen lymphocytes and mouse myeloma cells SP2/0 to the cell culture plate at a ratio of 10 ₂ Culturing in an incubator at 37 ℃, and replacing a cell culture medium according to the growth condition of cells; after 10 days of culture, positive clones were screened by ELISA.

Culturing positive hybridoma cell until the cell grows to logarithmic growthAfter a long period, 8-10 weeks of BALB/C healthy mice were selected and inoculated with 5X 10 of BALB/C per abdomen ⁶ The abdomen of the mouse is obviously enlarged after the positive hybridoma cell PBS buffer solution is inoculated with the cell for 7-10 days, the abdomen symptoms of the health condition of the mouse are closely observed, and the ascites of the mouse is collected when the ascites is as much as possible. The monoclonal antibody in ascites is purified by an affinity purification method (protein G agar gel), and the purity of the obtained antibody is determined to be more than 95 percent and meets the requirement of subsequent experiments.

The affinity of the antibody obtained by screening and the target protein human IL-17 is detected by using a molecular interaction analysis platform Biacore, and the result is shown in Table 1. The prior research shows that the IL-17 factor has double effects of anti-inflammation and proinflammatory, and the over-expression or the under-expression of the IL-17 factor can aggravate the pathogenesis symptoms of inflammatory bowel diseases and cause intestinal immune dysfunction, and the expression level of IL-17 in an IBD organism is observed to be increased compared with that of a normal individual in the existing human or animal experiments, so that the inventor realizes that the maintenance of the normal level of IL-17 is crucial to the maintenance of the immune balance of the organism, and can not excessively inhibit or excessively promote the expression of IL-17, so that the 3F7 antibody with medium affinity is selected for subsequent experiments in the invention so as to maintain the immune balance of the organism while effectively inhibiting the high expression of IL-17, thereby providing a favorable immune environment for treating IBD.

TABLE 1 antibody affinity assay

Antibodies	K _D Value (M)
		2A1	15.24E-11
3E5	25.87E-10
		3F7	35.21E-9
4D2	18.64E-9
		5E3	58.36E-8
5E9	35.47E-11

Through analysis and determination, the heavy chain variable region of the anti-IL-17 antibody comprises HCDR1 shown in SEQ ID NO. 1, HCDR2 shown in SEQ ID NO. 2 and HCDR3 shown in SEQ ID NO. 3, and the light chain variable region of the anti-IL-17 antibody comprises LCDR1 shown in SEQ ID NO. 4, LCDR2 shown in SEQ ID NO. 5 and LCDR3 shown in SEQ ID NO. 6; furthermore, the heavy chain variable region amino acid sequence of the anti-IL-17 antibody is shown as SEQ ID NO. 7, and the light chain variable region amino acid sequence is shown as SEQ ID NO. 8.

Example 2 preparation of exosomes of umbilical cord mesenchymal stem cells

2.1 Primary culture of umbilical cord mesenchymal Stem cells

Taking umbilical cord specimen of healthy newborn, washing umbilical cord with sterile PBS solution, removing blood stain and other impurities, stripping umbilical artery and umbilical vein, separating umbilical cord Wharton's jelly tissue, cleaning, and cutting into pieces of 0.5-1mm ³ Digesting the tissue mass with 0.2% (v/v) type II collagenase and hyaline collagenase at 37 deg.C for 1-2h, centrifuging at 500g for 5min, and collecting cells; the cell pellet was resuspended in α -MEM medium containing 10% by weight of FBS +1% streptomycin, inoculated into a culture flask, cultured at 37 ℃ in a 5% CO2 incubator, and the culture medium was replaced with fresh medium every 2 to 3 days depending on the cell growth, and subculture was carried out when the cell confluence reached 80% or more.

2.2 preparation of exosomes of umbilical cord mesenchymal Stem cells

And (3) mixing the obtained product with the obtained product.Subculturing umbilical cord mesenchymal stem cells for 3-5 passages in section 1, adding an exosome-inducing medium comprising 10% FBS, 50. Mu.g/mL TGF-beta, 10. Mu.g/mL IL-2, 10. Mu.g/mL IGF-1, 10. Mu.g/mL mLbFGF and alpha MEM containing 5% CO when the degree of cell fusion is 80% or more ₂ Culturing in an incubator at 37 ℃ for 48 hours; collecting the stem cell exosomes by adopting a gradient centrifugation method, which comprises the following specific steps: collecting cell supernatant after induction culture, filtering with 0.22 μm needle filter, centrifuging at 300 × g for 10min, and collecting supernatant; centrifuging the supernatant at 2000 Xg for 10min, and collecting the supernatant; centrifuging at 10000 Xg for 30min, and collecting supernatant; centrifuging at a low temperature of 100000 Xg for 1h, resuspending the precipitate by adopting sterile PBS solution, recording the stem cell exosome as in-EXO, and placing the stem cell exosome in a refrigerator at the temperature of-80 ℃ for long-term storage.

Non-induced umbilical cord mesenchymal stem cell exosomes were prepared in a similar manner using α MEM medium containing 10% fbs, the same procedure was followed, and the obtained exosomes were designated as EXO, resuspended in PBS and stored in a refrigerator at-80 ℃ for a long period of time.

2.3 umbilical cord mesenchymal stem cell exosome cell proliferation promotion experiment

In order to verify the proliferation promoting effect of the stem cell exosome on human intestinal cells, the human colorectal mucosal epithelial cell line NCM460, which is preserved by the inventor, is selected as a research object in the embodiment.

CCK8 is adopted to detect the cell proliferation promoting capacity of the stem cell exosome, and 1 x 10 cells are inoculated on a 96-well plate ⁵ Each well of NCM460,5% CO2 was cultured at 37 ℃ for 12 hours, then 20. Mu.g/mL of EXO, 20. Mu.g/mL of in-EXO and the same amount of DMEM medium (control) were added thereto, and after 24 hours, 10ul of CCK8 detection solution was added to each well, the plate was placed in an incubator and incubated for 3 hours, and the OD value of each well was measured using a microplate reader at a wavelength of 450 nm.

As shown in fig. 1, the stem cell exosomes provided in the present invention can effectively promote NCM460 cell proliferation, while the stem cell exosomes induced without adding cytokines have limited proliferation-promoting ability, which is only slightly improved compared with the control group; the invention relates to a method for inducing umbilical cord mesenchymal stem cells by using cytokines such as TGF-beta, IL-2, IGF-1, bFGF and the like, wherein the cell proliferation promoting capacity is remarkably improved, the cytokines can mediate various physiological responses in vivo, for example, the TGF-beta is reported to be a main regulatory factor for driving fibrosis of all organs including intestinal tracts, expression of the TGF-beta and a receptor thereof is increased in intestinal cells of IBD patients, particularly CD patients, but blocking a TGF-beta signal path by using an inhibitor is quite dangerous, because the TGF-beta is involved in various immune regulation and cell proliferation and differentiation promoting effects besides inducing tissue fibrosis, and factors such as IL-2, IGF-1, bFGF and the like are necessary for maintaining normal life activities.

Example 3 treatment of animal models of inflammatory bowel disease

3.1 preparation of animal models of inflammatory bowel disease

Taking clean SD rats with half male and half female, the body weight of 200 +/-20 g, the room temperature of 20-24 ℃, the relative humidity of 40-60 percent, carrying out molding IBD experiment after cage-adaptive feeding for 1 week. The SD rats were anesthetized by intraperitoneal injection with 2% sodium pentobarbital, a rubber catheter with a diameter of 2mm was inserted into the colon approximately 6cm from the anal orifice, and a TNBS solution with a concentration of 30mg/mL (5% TNBS: absolute ethanol = 3: 2) was slowly introduced in an administration volume of 3 mL/kg. Weighing the body weight and observing the stool character every day, wherein the IBD rats on the 3 rd day of model building have the conditions of loose stool, bloody stool, weight reduction and poor mobility, which indicates that the model building is successful.

3.2 animal grouping and dosing treatment

After successful modeling, the IBD model rats were randomly divided into 4 groups of 10 rats each, and treated as follows: in-EXO group: injecting 50mg/Kg in-EXO into tail vein every two days; the anti-IL-17 antibody group is injected with 50mg/Kg of anti-IL-17 antibody every two days in tail vein; combination, 50mg/Kg in-EXO and 50mg/Kg anti-IL-17 antibody were injected every two days of tail vein injection; the control group was treated with the same amount of sterile saline every two days for 10 days.

3.3 DAI score

During the administration, the mice were observed every 2 days for weight change, eating and drinking, hair gloss, mental state, activity, etc., and rat DAI scores were made according to the criteria established by Ganta et al, with DAI scores on the basis shown in Table 2. As shown in FIG. 2, although the DAI score of the model group was fluctuating, but was consistently maintained at a higher level, the symptoms of IBD were reduced after treatment with in-EXO or anti-IL-17 antibody, which appeared to be more effective than anti-IL-17 antibody; the in-EXO and anti-IL-17 antibodies together showed a significant increase in efficacy late in treatment and a significant decrease in the DAI score in the combination from day 8.

TABLE 2 DAI Scoring benchmark

Remarking: DAI = weight loss rate score + fecal occult blood/macroscopic bloody stool score.

3.4 oxidative stress assay

The intestinal epithelium, which has organ-specific and immune functions, has been thought to play a central role in the pathogenesis of IBD, once excess Reactive Oxygen Species (ROS) are produced in the intestinal tract, oxidative stress accelerates cell damage by altering protein function and causing lipid peroxidation. During mucosal inflammation, neutrophils and macrophages produce superoxide and nitric oxide by activating Nitric Oxide (NOX) and Inducible Nitric Oxide Synthase (iNOS), respectively, both of which are induced by Inflammatory cytokines, producing more ROS/RNS through NOX and iNOS activation, which ROS overload destroys cytoskeletal proteins and causes changes in tight junctions and epithelial permeability in IEC, ultimately leading to barrier disruption and thus IBD (Tian Tian, ziling Wang, and Jinhua Zhuang, patholomenses of Oxidative Stress in Inflammatory bone diseases and topical inflammation therapeutics, oxidative Med Cell change.2017; 2017H 4535194). SOD is the only antioxidant enzyme which can specifically remove superoxide radical, MDA is the final product of lipid peroxidation, and the SOD and the MDA can comprehensively reflect oxidative stress conditions in organisms or tissues.

In this example, rats were sacrificed 10 days after treatment, colon tissues of the rats were taken, 9 times of cold physiological saline was added to prepare homogenate, freeze thawing was repeated 3 times after completion of the homogenate to break cells, and the supernatant was taken to measure the contents of SOD and MDA according to a kit (purchased from Nanjing, biochemical Co., ltd.). As a result, as shown in Table 3, the SOD expression levels were increased in each treatment group, while the MDA levels were decreased to various degrees, and the effect of the stem cell exosomes appeared to be stronger against oxidative stress than the anti-IL-17 antibody, and the ability against oxidative stress was maintained and strengthened in the combination group.

TABLE 3 levels of SOD and MDA in colon tissue of various groups of rats

3.5 detection of TNF-alpha, IL-1 beta, IL-6 in serum

Inflammatory factors such as TNF-alpha, IL-1 beta, IL-6 and the like play an important role in the process of inducing IBD, and the secretion of these proinflammatory cytokines can not only cause tissue damage, but also activate the adaptive immune system, resulting in chronic inflammation. In clinical trials, researchers have observed varying degrees of up-regulation of the expression of the above inflammatory factors, and administration of related inhibitors can alleviate the symptoms of IBD to some extent. For example, anti-TNF- α therapies that have been used in the clinical treatment of IBD, using TNF- α antagonists such as infliximab, adalimumab, golimumab, certolizumab, etanercept, etc., produce a sustained anti-inflammatory response that promotes mucosal healing and restoration of intestinal epithelial barrier function. In the embodiment, ELISA is adopted to detect the expression levels of inflammatory factors such as TNF-alpha, IL-1 beta, IL-6 and the like in rat serum so as to investigate whether the medicament provided by the invention can effectively inhibit inflammatory reaction.

After 10 days of treatment, the rat orbit was bled, centrifuged at 3500g at 4 ℃ for 5min to collect serum, and the contents of TNF-alpha, IL-1 beta and IL-6 in the rat serum were determined according to the ELISA kit instructions (purchased from Kyoto Ching Biotech Co., ltd.). The results are shown in FIGS. 3-5, wherein the expression of TNF-alpha and IL-1 beta are similar, the therapeutic effects of the in-EXO and anti-IL-17 antibody are similar, the expression levels of TNF-alpha and IL-1 beta are reduced, the therapeutic effects of the TNF-alpha and IL-1 beta are obviously weaker than those of the combined group, and the expression levels of TNF-alpha and IL-1 beta are greatly reduced under the condition of combining the in-EXO and anti-IL-17 antibody; similar results were not seen in the IL-6 assay, and in this example, the IL-6 expression levels were similar in the three treatment groups, although all were lower than in the control group, but did not appear to show significant differences between the three groups.

While this invention has been particularly shown and described with references to exemplary embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

1. An anti-IL-17 antibody characterized by: the heavy chain variable region of the anti-IL-17 antibody comprises HCDR1 shown in SEQ ID NO. 1, HCDR2 shown in SEQ ID NO. 2 and HCDR3 shown in SEQ ID NO. 3, and the light chain variable region of the anti-IL-17 antibody comprises LCDR1 shown in SEQ ID NO. 4, LCDR2 shown in SEQ ID NO. 5 and LCDR3 shown in SEQ ID NO. 6.

2. The antibody of claim 1, wherein: the heavy chain variable region amino acid sequence of the anti-IL-17 antibody is shown as SEQ ID NO. 7, and the light chain variable region amino acid sequence of the anti-IL-17 antibody is shown as SEQ ID NO. 8.

3. A pharmaceutical composition for the treatment of inflammatory bowel disease, characterized in that it comprises an extracellular exosome and an anti-IL-17 antibody according to any one of claims 1-2.

4. The pharmaceutical composition of claim 3, wherein the stem cell exosomes are selected from at least one of bone marrow mesenchymal stem cell exosomes, umbilical cord mesenchymal stem cell exosomes, adipose mesenchymal stem cell exosomes, endothelial progenitor cell exosomes, neural stem cell exosomes.

5. The pharmaceutical composition of claim 4, wherein the stem cell exosome is an umbilical cord mesenchymal stem cell exosome, and the preparation method comprises the following steps: culturing umbilical cord mesenchymal stem cells, adding an exosome-inducing medium when the cell fusion degree reaches 80% or more, 5% ₂ Culturing in an incubator at 37 ℃ for 48 hours; collecting stem cell exosome by gradient centrifugation, collecting cell supernatant after induction culture, filtering with a 0.22 μm needle filter, centrifuging at low temperature of 300 Xg for 10min, and collecting supernatant; centrifuging at 2000 Xg for 10min, and collecting supernatant; centrifuging at 10000 Xg for 30min, and collecting supernatant; centrifuging at 100000 Xg for 1h, and resuspending the precipitate with sterile PBS solution to obtain stem cell exosome.

6. The pharmaceutical composition of claim 5, wherein the exosome-inducing medium is an α MEM medium containing 10% fbs, 50 μ g/mL TGF- β, 10 μ g/mL IL-2, 10 μ g/mL IGF-1, 10 μ g/mL bFGF.

7. The pharmaceutical composition according to any one of claims 3-6, further comprising a pharmaceutically acceptable carrier.

8. Use of a pharmaceutical composition according to any one of claims 3 to 7 in the manufacture of a medicament for the treatment of inflammatory bowel disease.

9. The use of claim 8, wherein the inflammatory bowel disease comprises crohn's disease and ulcerative colitis.