WO2016127503A1 - 来源于粒细胞样髓源性抑制细胞的exosomes及其应用 - Google Patents

来源于粒细胞样髓源性抑制细胞的exosomes及其应用 Download PDF

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WO2016127503A1
WO2016127503A1 PCT/CN2015/077902 CN2015077902W WO2016127503A1 WO 2016127503 A1 WO2016127503 A1 WO 2016127503A1 CN 2015077902 W CN2015077902 W CN 2015077902W WO 2016127503 A1 WO2016127503 A1 WO 2016127503A1
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mdsc
exo
exosomes
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王胜军
王运刚
田洁
芮棵
马洁
马斌
汤新逸
许化溪
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Jiangsu University
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Definitions

  • the invention relates to a granulocyte-like myeloid-derived cell-derived exosomes and an application thereof, and belongs to the field of cell biology, molecular biology and clinical application, and particularly relates to the preparation and organism of G-MDSC exo for spleen isolation of tumor-bearing mice. Study of functional studies and its application in the treatment of autoimmune diseases in mice.
  • Myeloid-derived suppressor cells are a group of bone marrow-derived heterogeneous cells that are extensively expanded in pathological conditions such as tumors, inflammation, and pathogen infection (Gabrilovich DI, Nagaraj S. Myeloid-derived suppressor) Cells as regulators of the immune system. Nat Rev Immunol. 2009; 9(3): 162-74.).
  • the MDSCs of mice are Gr-1 (composed of Ly6G and Ly6C) and CD11b double positive cells. According to the cell morphology and the expression of Ly6G and Ly6C, they can be divided into CD11b + Ly6G + Ly6C low granulocyte-like MDSCs (G-MDSCs).
  • M-MDSCs monocyte-like MDSCs
  • G-MDSCs inhibit T cell-mediated adaptive immune responses and natural killer cells (NK) by expressing arginase 1, Arg-1 and reactive oxygen species (ROS). , a macrophage-mediated natural anti-tumor immune response.
  • NK natural killer cells
  • ROS reactive oxygen species
  • MDSCs can also down-regulate the function of effector T cells by inducing Treg cell differentiation through interferon gamma (interferon gamma) (INF-gamma) and interleukin 10 (IL-10)-dependent pathways.
  • interferon gamma interferon gamma
  • IL-10 interleukin 10
  • Inflammatory bowel disease includes ulcerative colitis (UC) and Crohn's disease (CD), mainly due to abnormal immunity of the body to the normal flora that is tolerant.
  • Non-specific inflammatory bowel disease caused by response, the clinical manifestations of the disease are abdominal pain, diarrhea, mucous pus and bloody stools, and cancer can occur after a long time.
  • Rheumatoid arthritis is a clinically common systemic autoimmune disease. The main clinical manifestations are symmetrical small joint lesions, characterized by persistent inflammation of multiple joints.
  • Treg cells play an important role in maintaining the immune stability of the intestinal tract.
  • the imbalance of Treg cells is often one of the causes of autoimmune diseases, and studies have confirmed that they are immune. Patients with epidemic disease have lower Treg cells than normal people.
  • the incidence of autoimmune diseases in China is on the rise.
  • the main focus is on symptomatic treatment, and there is no effective treatment. Therefore, it is urgent to explore a fundamental cure. Methods.
  • Exosomes is a membranous vesicle that is released into the extracellular environment by fusion of intracellular polysomes with the plasma membrane. Studies have shown that almost all living cells can secrete exosomes, and they are widely present in various body fluids (Beninson LA, M.Fleshner. Exosomes: an emerging factor in stress-induced immunomodulation [J]. Semin Immunol, 2014.26(5) :394-401.). Exosomes carries the protein, mRNA and miRNA components of the source cells, making them less susceptible to degradation by the external environment, which facilitates the biological functions of the relevant active ingredients. Exosomes are internalized by receptor cells by means of macrocytosis, lipid rafts and receptor-mediated endocytosis.
  • G-MDSC exo has a diameter of 40-100 nm and carries bioactive components. Compared with other vesicles secreted by G-MDSC, the biological characteristics of G-MDSC exo are clearer.
  • G-MDSC exo inhibits CD4 + T cell proliferation and attenuates the degree of footpad swelling in DTH mice, and promotes TGF- ⁇ -induced expansion of CD4 + T cells to Treg cells.
  • DTH delayed-type hypersensitivity
  • IBD IBD
  • CIA CIA
  • exosomes In recent years, studies on exosomes in disease treatment have focused on denosomes derived from dendritic cells and stem cells. In terms of disease treatment, exosomes has the advantages of easier storage and transportation, no cytotoxicity in itself, and less biosafety problems when applied; and the complex molecules on the surface of exosomes provide potential for targeting specific tissues and microenvironments. Homing mechanism (Tian Xinrui, Xu Wenbiao, Niu Bo, et al.
  • G-MD SC exo The application of G-MD SC exo in the treatment of diseases has not been reported. We found that G-MDSC exo can be maintained for a long time under low temperature conditions, and its biological activity is not affected. G-MDSC exo has the same effect as G-MDSC to inhibit the proliferation of CD4 + T cells and promote TGF- ⁇ . The induced CD4 + T cells are expanded to Treg cells.
  • G-MDSC exo is a natural immune modulator, which is of great significance for the treatment of autoimmune diseases and has a good clinical application value.
  • G-MDSC exo According to the structure and functional characteristics of G-MDSC exo, it can be applied to the treatment of autoimmune diseases, which can achieve the goal of “low input, high output” and has good clinical application prospects. It is expected to play a role in maintaining the body's immune balance. Actively, it provides a new therapeutic approach for the treatment of autoimmune diseases.
  • the present invention provides an exosomes derived from G-MDSCs, which have non-specific components of G-MDSCs and special proteins and nucleic acids such as nucleic acids in G-MDSCs, which are sub- A composite informative body of cell structure; it has both the immunosuppressive properties of G-MDSCs and its own surface molecule CD63; and its role in the treatment of autoimmune diseases, in order to seek a fundamental treatment of diseases.
  • the G-MDSC exo in the present invention is a membranous vesicle containing a lipid component of a cell membrane derived from a cell, and has a protective effect on a biologically active component thereof; G-MDSC exo can exert an immunosuppressive function similar to G-MDSCs, and It has many advantages that the cells themselves do not have, and is a subcellular structural information complex that transmits information between cells.
  • the invention discloses a simple method for preparing G-MDSC exo derived from a tumor individual or an autoimmune disease individual, and the whole process is short in time, high in yield, and proved to be easy to preserve and biological activity is not affected, for treatment
  • the purpose of mass production and production of G-MDSC exo lays the foundation. That is, a tumor-bearing mouse model or an autoimmune disease (such as CIA) model is constructed, the spleen G-MDSCs are isolated, and the culture supernatant is collected; and the G-MDSC exo is separated and extracted by using ultracentrifugation and microfiltration membrane method.
  • the collected G-MDSCs culture suspension was centrifuged at 4 ° C, and the supernatant was collected and repeated three times.
  • the collected supernatant was transferred to an MWCO 100 kDa ultrafiltration centrifuge tube, and the concentrate in the inner tube was collected by centrifugation;
  • the ExoQuick-TCTM Exosome reagent was mixed at a volume ratio of 5:1, shaken, and after standing at 4 ° C, the supernatant was centrifuged at 4 ° C, and the precipitate was collected to obtain exosomes.
  • the G-MDSC exo described in the present invention has an effect of inhibiting T cell proliferation and promoting Treg polarization, and the in vivo application may exert an intensity of immunosuppressive action, and is important for the treatment of autoimmune diseases.
  • the G-MDSC exo described in the present invention can effectively alleviate the clinical progression of mouse DTH, IBD and CIA, reduce pathological damage, and provide a new way to treat autoimmune diseases by restoring the body's immune balance.
  • the present invention also demonstrates that G-MDSC exo exerts an immunosuppressive effect by inhibiting CD4 + T cell proliferation and promoting Treg cell expansion, and has a protective effect on autoimmune diseases, and provides a new way for the treatment of diseases.
  • the present invention is the first to prepare G-MDSC exo derived from a tumor individual or an autoimmune disease individual, and lays a foundation for its application and related biological function research.
  • the preparation of the G-MDSC exo of the present invention is short in time, easy to operate, convenient for large dose extraction, and complete in morphology and structure.
  • the components contained in G-MDSC exo are stable in activity and easy to store, and the storage time at -80 ° C is more than 1 year; the structure and active ingredients are stable at room temperature, and easy to transport; no cryopreservation is required, G-MDSC
  • the exo clinical application provides the technical foundation.
  • G-MDSC exo is an information complex of nano-level subcellular structure, which is itself cytotoxic and has high safety in clinical application.
  • G-MDSC exo has a protective effect on autoimmune diseases, and its preparation and application provide a new way for the treatment of autoimmune diseases.
  • G-MDSC exo has a function of inhibiting proliferation of CD4 + T cells and promoting expansion of Treg cells, and provides a new idea for the treatment of autoimmune diseases.
  • FIG. 1 Flow cytometry (FACS) detects the purity of G-MDSCs sorted by immunomagnetic beads.
  • FIG. 1 Identification results of G-MDSC exo, where A is the morphology of G-MDSC exo; B is the frequency distribution of G-MDSC exo; C is the Western blot to detect the expression of G-MDSC exo.
  • FIG. 3 G-MDSC exo inhibits the action of CD4 + T cells.
  • A is the effect of G-MDSC exo on the proliferation of CD4 + T cells by 3 H-TdR incorporation method
  • B is the in vivo observation of G-MDSC exo The result of the impact on the DTH model.
  • FIG. 4 G-MDSC exo promotes TGF- ⁇ -induced expansion of CD4 + T cells to Treg cells in a dose-dependent manner.
  • A is G-MDSC exo promotes different concentrations of TGF- ⁇ -induced CD4 + T cell orientation. Treg cells were expanded;
  • B is G-MDSC exo to promote TGF- ⁇ -induced expansion of CD4 + T cells to Treg cells in a dose-dependent manner.
  • FIG. 5 Protective effect of G-MDSC exo on IBD mice, where A is the effect of G-MDSC exo on disease progression in IBD mice; B is the effect of G-MDSC exo on colon tissue in IBD mice; C is G-MDSC exo Effect on colonic inflammation in IBD mice.
  • Figure 6 Protective effect of G-MDSC exo on CIA mice, in which A is a flow chart of the experimental method of G-MDSC exo intervention in CIA mice; B is the effect of G-MDSC exo on the degree of paw swelling in CIA mice. C is the effect of G-MDSC exo on disease progression in CIA mice.
  • Arg-1 carried by G-MDSC exo plays an important role in slowing down autoimmune diseases.
  • A is the result of Arg-1 activity in G-MDSC exo and the Nor-NHOA inhibitor of Arg-1 inhibitor.
  • the inhibitory effect of Arg1 activity B is the effect of G-MDSC exo after inhibiting Arg-1 activity on the progression of IBD mice;
  • C is the effect of G-MDSC exo on colon tissue after inhibiting Arg-1 activity;
  • D is inhibition Effect of G-MDSC exo after Arg-1 activity on colonic inflammation.
  • Example 1 Sorting of G-MDSCs and preparation of culture supernatant
  • mice were sacrificed by eyeball exsanguination, and the spleens were aseptically removed; the spleens were ground in a 0.22 ⁇ m sieve, filtered and the suspension was 4 ° C. Centrifuge at 500g for 5min, discard the supernatant; add 5mL ACK to lyse the red blood cells in the cell pellet, mix and let stand for 5min; add RPMI-1640 medium to 10mL, centrifuge at 5°C, 500g for 5min, discard the supernatant; calculate the number of cells.
  • Magnetic bead sorting G-MDSCs After every 10 8 splenocytes were resuspended in 350 ⁇ L of PBE, 50 ⁇ L of FcR Blocking Reagent was added; placed on ice for 10 min; and anti-Ly-6G was added in an amount of 40 ⁇ L/10 8 splenocytes.
  • the MACS sorting column was placed on a VarioMACS sorter and rinsed once with 3 mL of PBE; the cell suspension was added, and after the first drop of the suspension was discharged, the sorting column was washed 3 times with 9 mLPBE; the sorting column was withdrawn.
  • the sorter was added with 5 mL of PBE, the column plug was pushed, the cells bound to the separation column were pressed out, and the cell suspension was collected, which was G
  • G-MDSCs Collect 1 ⁇ 10 6 G-MDSCs in EP tubes, resuspend in 1 mL PBS, centrifuge at 500°C for 5 min at 4°C, discard the remaining 100L of PBS, and add 0.5 ⁇ L after resuspension.
  • Anti-Ly-6G antibody and ⁇ L anti-CD11b antibody were incubated at 4 °C for 30 min; resuspended in 1 mL PBS, centrifuged at 500 g for 5 min at 5 °C, and the supernatant was discarded; resuspended in 200 ⁇ L PBS, and the expression of cell surface molecules was detected by flow cytometry. As shown in Fig.
  • G-MDSCs are CD11b and Ly-6G double positive cells.
  • G-MDSCs are sorted from the spleens of tumor-bearing mice by immunomagnetic beads method, and the purity of sorted G-MDSCs is determined by FACS, G- MDSCs are as pure as 95% or more.
  • G-MDSCs culture supernatant preparation The sorted G-MDSCs were resuspended in RPMI-1640 medium containing 10% calf serum (pre-equivalently centrifuged at 100000 g ⁇ 16 h), 1.5 ⁇ 10 6 /well The cells were seeded in a 24-well plate at a total volume of 1 mL/well; after 24 hours of incubation at 37 ° C under 5% CO 2 , the cells were harvested. The collected G-MDSCs culture suspension was centrifuged at 300 g for 20 min at 4 ° C, and the supernatant was collected.
  • the collected G-MDSCs supernatant was centrifuged at 1000 °C for 30 min at 4 ° C, and the supernatant was collected; the supernatant was centrifuged at 10000 g for 30 min at 4 ° C; the supernatant was transferred to an ultrafiltration centrifuge tube of MWCO 100 kDa, and centrifuged at 1500 g for 30 min. , collect the concentrate in the inner tube.
  • step (2) Extracting G-MDSC exo with ExoQuick-TCTM Exosome kit purchased from SBI:
  • the concentrate obtained in step (1) is mixed with ExoQuick-TCTM Exosome reagent at a volume ratio of 5:1, shaken, and allowed to stand at 4 °C. More than 12h; centrifuged at 1000g for 30min at 4°C, discard the supernatant, and collect the precipitate, which is G-MDSC exo.
  • the prepared exosomes were dissolved in PBS, dispensed into EP tubes, and stored at -80 ° C for subsequent experiments.
  • G-MDSC exo is a circular or elliptical micro-sac structure, with envelope, low electron density component in cavity The particle size is 30-150 nm, and the results are shown in FIGS.
  • A is a form of G-MDSC exo, and a microcapsule structure in which a G-MDSC exo is circular or elliptical can be observed under a transmission electron microscope.
  • the chamber has a low electron density component;
  • B is the frequency distribution of the particle size of G-MDSC exo, and the particle size of the G-MDSC exo photographed by transmission electron microscopy is mainly distributed at 30-150 nm.
  • Example 4 G-MDSC exo inhibits T cell proliferation and mouse DTH response
  • G-MDSC exo affects the proliferation of CD4 + T cells: CD4 + T cells isolated. 6-8w male C57BL/6 mice were sacrificed by cervical dislocation. The spleen cells were aseptically extracted and ground to prepare spleen cell suspension. The suspension was centrifuged at 5 °C, 500 g for 5 min, and the supernatant was discarded. 5 mL ACK lysed red blood cells were added to the cell pellet.
  • CD4 + T cells were seeded in 96-well plates at a concentration of 5 ⁇ 10 5 cells/well, and the total volume per well was 200 ⁇ L. In the presence of anti-CD3 and anti-CD28, different concentrations of G-MDSC exo were added, and the content was 10% small.
  • DTH mouse model was constructed by 6-8w male C57BL/6 mice, which were divided into normal control group (NC) and DTH group. , Neu exo treatment group, G-MDSC exo treatment group, 6 per group.
  • NRC normal control group
  • N-MDSC exo treatment group 6 per group.
  • 200 ⁇ L of OVA preparation with a final concentration of 1 mg/mL emulsified in complete Freund's adjuvant was injected intradermally into the tail and back of the mouse for initial immunization. After 7 days, 30 ⁇ L of 20 mg/mL OVA solution was used to administer the right mouse.
  • the footpad was injected and stimulated, and the thickness of the footpad was measured at 24h, 48h and 72h after challenge.
  • the degree of swelling of the foot pads of each group was calculated according to the judgment standard of the degree of DTH reaction. The results showed that the swelling of the foot pads of the G-MDSC exo treatment group was lighter than that of the DTH group and the Neu exo treatment group (Fig. 3B), and there were statistics. Academic significance (P ⁇ 0.05). It is suggested that G-MDSC exosomes can play a role in inhibiting CD4 + T cells in vivo.
  • the degree of swelling of the foot pad (thickness of the foot pad after 24 hours of OVA injection - thickness of the foot pad before injection of OVA) - (thickness of the foot pad after injection of PBS for 24 hours - thickness of the anterior foot pad of PBS injection)
  • Figure 3 is a graph showing the effect of G-MDSC exo on CD4 + T cells, in which: A, 3 H-TdR incorporation was used to observe the effect of G-MDSC exo on the proliferation of CD4 + T cells in vitro, and the results showed that G-MDSC exo was The proliferation of CD4 + T cells was inhibited in a dose-dependent manner; B. The effect of G-MDSC exo on delayed type hypersensitivity was observed in vivo using the DTH model. The degree of swelling of the footpad of each group was measured at 24h, 36h and 72h after OVA challenge. The results showed that the degree of swelling of the footpad of G-MDSC exo treatment group was significantly lower than that of other groups, indicating that G-MDSC exo can inhibit in vivo. DTH reaction.
  • Example 5 G-MDSC exo promoted TGF- ⁇ -induced expansion of CD4 + T cells to Treg cells in a dose-dependent manner.
  • the magnetic beads were sorted into CD4 + T cells (same as in Example 4), and 2 ⁇ 10 6 /well was seeded in a 24-well plate, and the total system was 1 mL.
  • TGF- ⁇ was used to induce polarization of CD4 + T cells to Treg cells.
  • Different amounts of G-MDSC exo were added to the culture system, and CD25 + Foxp3 + T cells were detected by FACS after 3 days of culture. The results show that G-MDSC exo promotes TGF- ⁇ -induced expansion of CD4 + T cells to Treg cells ( FIG. 4A ) in a dose-dependent manner ( FIG. 4B ).
  • Figure 4 is a graphical representation of the results of G-MDSC exo dose-dependently promoting TGF- ⁇ -induced expansion of CD4 + T cells to Treg cells.
  • A is G-MDSC exo to promote the expansion of CD4 + T cells induced by different concentrations of TGF- ⁇ to Treg cells.
  • the results show that G-MDSC exo can promote the expansion of CD4 + T cells to Treg cells at different concentrations of TGF- ⁇ .
  • Increased B G-MDSC exo dose-dependently promoted TGF- ⁇ -induced expansion of CD4 + T cells to Treg cells, and the results showed that TGF- ⁇ induced Treg cell expansion with increasing concentration of G-MDSC exo Enhanced.
  • NC Normal control group
  • IBD group Drink 2.5% DSS without any treatment
  • Neu exo treatment group induction of IBD on the 2nd, 4d and 6d, respectively, intraperitoneal injection of Neu exo at a dose of 30 ⁇ g / only;
  • G-MDSC exo treatment group Induction of IBD on the 2nd, 4th and 6th, respectively, at a dose of 30g ⁇ g / only by intraperitoneal injection of G-MDSC exo;
  • the day of drinking DSS is the 0th day.
  • Weight change ⁇ 1% gets 0 points, 1-5% gets 1 point, 5-10% gets 2 points, 10-15% gets 3 points, >15% gets 4 points;
  • Stool condition 0 points for normal, 2 points for loose formation, and 4 points for diarrhea;
  • the scores of each group are added and divided by 3 as the final score.
  • mice On the 9th day after IBD induction, the mice were sacrificed by eyeball bleeding, the mice were opened, and the colon tissue between the rectum of the anus and the distal cecum of each group was taken out. The degree and length of swelling of the mouse colon.
  • mice in each group were observed by changes in body weight, fecal traits and bloody stools; colonic specimens were observed and colonic histopathological staining was used to observe colonic injury in each group.
  • the results showed that the clinical disease scores of the G-MDSCexo treatment group were significantly reduced (Fig. 5A), and the degree of colon swelling and shortening was reduced (Fig. 5B).
  • the degree of histological damage and inflammatory cell infiltration and the extent of the inflammatory lesions were relatively light (Fig. 5). 5C).
  • FIG. 5 is a graphical representation of the protective effect of G-MDSC exo on IBD mice.
  • A is the effect of G-MDSC exo on disease progression in IBD mice.
  • the disease scores of mice in each group were observed by weight changes, fecal traits and bloody stools.
  • the results showed that the G-MDSC exo treatment group had clinical scores.
  • the group was lower than the IBD group and the neutrophil-derived exosomes (Neu exo) treatment group, and the difference was statistically significant (P ⁇ 0.05).
  • B is the effect of G-MDSC exo on colon tissue of IBD mice.
  • Example 8 Arg-1 carried by G-MDSC exo plays an important role in slowing down autoimmune diseases
  • G-MDSC group G-MDSC was cultured alone, and cells were collected.
  • G-MDSC + DMSO group G-MDSC was cultured by adding 7 ⁇ l of DMSO, and the cells were collected.
  • G-MDSC+NN group G-MDSC was cultured by adding 7 ⁇ l of nor-NOHA solution, and the cells were collected.
  • G-MDSC exo group G-MDSC was cultured alone, and the cell culture supernatant was collected.
  • G-MDSC + DMSO exo group G-MDSC was cultured by adding 7 ⁇ l of DMSO, and the cell culture supernatant was collected.
  • G-MDSC+NN exo group: G-MDSC was cultured by adding 7 ⁇ l of nor-NOHA solution, and the cell culture supernatant was collected.
  • Fig. 7A The result of the assay is shown in Fig. 7A, which indicates that Arg-1 is contained in G-MDSC exo, and the activity of this Arg-1 can be inhibited by NN.
  • G-MDSC exo participates in the protective effect of G-MDSC exo on autoimmune diseases through Arg-1:
  • This example uses the IBD model to observe the role of Arg-1 in G-MDSC exo-mediated IBD protective effects.
  • IBD mice were divided into Neu exo treatment group, G-MDSC exo treatment group, (G-MDSC+NN) exo treatment.
  • the treatment group and (G-MDSC + DMSO) exo treatment group, specific modeling methods, treatment methods and observation indicators are the same as in Example 5.
  • FIG. 7B shows the effect of (G-MDSC+NN) exo group on disease progression in IBD mice. Disease scores were obtained for each group of mice by weight change, fecal traits and bloody stools. The results showed (G-MDSC+NN). The clinical scores of the mice in the exo group were significantly higher than those in the G-MDSC exo group, and the difference was statistically significant.
  • 7C is the effect of (G-MDSC+NN) exo on colon tissue in IBD mice. The results showed that the degree of colon swelling and shortening of the mice treated with (G-MDSC+NN) exo were significantly higher than those of the G-MDSC exo treatment group.

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Abstract

提供了一种来源于粒细胞样髓源性抑制细胞的exosomes及其应用，该exosomes被命名为G-MDSC exo，提供了其在制备用于抑制自身免疫性疾病药物中的用途。G-MDSC exo在体外能有效抑制CD4+T细胞增殖，促进调节性T细胞诱导扩增，并且减轻迟发性超敏反应模型小鼠的足垫肿胀，抑制小鼠炎症性肠病(IBD)和胶原诱导性关节炎(CIA)的发病。

Description

一种粒细胞样髓源性抑制细胞来源exosomes及其应用 技术领域
本发明涉及一种粒细胞样髓源性抑制细胞来源exosomes及其应用，属细胞生物学、分子生物学领域及临床应用领域，具体涉及荷瘤小鼠脾脏分离的G-MDSC exo的制备、生物学功能研究及其在小鼠自身免疫性疾病治疗中的应用。
背景技术
骨髓来源的抑制性细胞(myeloid-derived suppressor cells，MDSCs)是一群骨髓来源的异质性细胞，在肿瘤、炎症及病原体感染等病理情况下大量扩增(Gabrilovich DI,Nagaraj S.Myeloid-derived suppressor cells as regulators of the immune system.Nat Rev Immunol.2009；9(3):162-74.)。小鼠的MDSCs是Gr-1(由Ly6G和Ly6C组成)和CD11b双阳性的细胞，按细胞形态及Ly6G、Ly6C表达量高低，可分为CD11b+Ly6G+Ly6Clow粒细胞样MDSCs(G-MDSCs)和CD11b+Ly6G-Ly6Chi单核细胞样MDSCs(M-MDSCs)两个亚型。在肿瘤及感染等疾病状态下，G-MDSCs和M-MDSCs均会扩增，而G-MDSCs明显多于M-MDSCs。G-MDSCs主要通过表达精氨酸酶1(arginase 1，Arg-1)和活性氧(reactive oxygen species,ROS)抑制T细胞介导的适应性免疫应答和自然杀伤细胞(natural killer cell，NK)、巨噬细胞介导的天然抗肿瘤免疫应答。研究表明MDSCs也可以通过干扰素γ(interferonγ，INF-γ)和白细胞介素10(interlenkin 10,IL-10)依赖的途径诱导Treg细胞分化从而下调效应性T细胞的功能。近年来相关研究表明MDSCs在自身免疫性疾病的治疗方面有巨大的潜能。因此，人们对MDSCs的治疗价值有了新的认识，已有实验利用MDSCs治疗小鼠胶原诱导性关节炎(collagen-induced arthritis，CIA)，并取得一定疗效。但是，由于MDSCs来源不足、储存不便及成分复杂等缺点限制了其应用。
自身免疫性疾病是因机体免疫系统对自身成分发生免疫应答而导致的疾病，在人群中有较高的发病率。炎症性肠病(inflammatory bowel disease，IBD)包括溃疡性结肠炎(ulcerative colitis，UC)和克罗恩病(Crohn’s disease，CD)，主要是由于机体对本处于耐受状态的正常菌群发生异常免疫应答所致的非特异性炎性肠道疾病，该病临床表现为腹痛、腹泻、黏液脓血便，病久可发生癌变。类风湿关节炎(rheumatoid arthritis,RA)是一种临床常见系统性自身免疫性疾病，临床主要表现为对称性小关节病变，以多关节持续性炎症为特征。大量研究表明促炎性细胞Th1和Th17及其来源的炎症因子在自身免疫性疾病发生发展过程中发挥重要作用。Treg细胞在维持肠道机体免疫稳定等方面发挥重要作用，Treg细胞作用失衡往往是自身免疫学疾病发生的原因之一，并且研究证实自身免 疫性疾病患者体内Treg细胞比正常人低。近年来自身免疫性疾病在我国的发病率呈上升趋势，针对该类疾病，临床上主要以对症治疗为主，尚没有一种行之有效的治疗手段，因此迫切需要探索一种从根本上治愈的方法。
Exosomes是细胞内多胞体与质膜融合后释放到细胞外环境中的一种膜性囊泡。研究表明几乎所有的活细胞都能分泌exosomes，并且其广泛存在于各种体液中(Beninson LA,M.Fleshner.Exosomes:an emerging factor in stress-induced immunomodulation[J].Semin Immunol,2014.26(5):394-401.)。exosomes携带来源细胞的蛋白质、mRNA及miRNA等成分，使之不易受外界环境降解，利于相关活性成分发挥生物学功能。通过巨胞饮作用，脂质筏和受体介导的内吞作用等方式，exosomes被受体细胞内化，内化的exosomes可以调控受体细胞的多种生物学功能，在细胞间交流过程中发挥重要作用。exosomes在免疫应答、凋亡、血管生成、炎症反应及肿瘤发生发展等过程中的作用均有报道。研究显示对疾病进行干预时，exosomes比其来源细胞更有优势。利用树突状细胞来源的exosomes治疗肿瘤的报道较多，并显示出良好疗效(Pitt JM,Charrier M,Viaud S,et al.Dendritic cell-derived exosomes as immunotherapies in the fight against cancer[J].J Immunol,2014；193(3):1006-11.)。但是有关G-MDSC exo的功能研究，以及在自身免疫性疾病中的应用尚未见报道。
我们发现G-MDSC exo直径为40-100nm，携带生物活性成分，与G-MDSC分泌的其他囊泡相比，G-MDSC exo的生物学特性更清晰。G-MDSC exo能抑制CD4+T细胞增殖并减轻DTH小鼠足垫肿胀程度，并且能促进TGF-β诱导的CD4+T细胞向Treg细胞扩增。体内应用G-MDSC exo能减缓小鼠迟发型超敏反应(DTH)、IBD和CIA的病情,并进一步证实，并且这种作用一定程度上依赖于G-MDSC exo携带的Arg-1。
近年来有关exosomes在疾病治疗方面的研究多集中在树突状细胞和干细胞来源的exosomes上。疾病治疗方面，相对于来源细胞，exosomes具有更容易保存和运输，本身无细胞毒性，应用时生物安全问题较少等优点；并且exosomes表面的复合分子为其靶向特定组织及微环境提供潜在的归巢机制(田新瑞,徐文蜻,牛勃,等.神经干细胞来源Exosomes制备及在神经系统疾病中的应用:中国,CN103740645A[P].2014-04-23.)；exosomes对治疗性的蛋白质和核酸还具有保护作用，减少降解(Marcus ME,Leonard JN.FedExosomes:Engineering Therapeutic Biological Nanoparticles that Truly Deliver[J].Pharmaceuticals(Basel), 2013,6(5):659-680.)。G-MD SC exo在疾病治疗中的应用尚未见报道。我们实验发现G-MDSC exo在低温条件下能长期保持，并且生物学活性不受影响；G-MDSC exo既具有与G-MDSC类似的抑制CD4+T细胞增殖的作用，又能促进TGF-β诱导的CD4+T细胞向Treg细胞扩增。G-MDSC exo是一种天然免疫调节剂，对自身免疫性疾病的治疗具有重要意义，在临床上具有较好的应用价值。
依据G-MDSC exo的结构和功能特性，将其应用于自身免疫性疾病的治疗，可达到“低投入、高产出”的目的，具有良好的临床应用前景，有望在维持机体免疫平衡方面发挥积极作用，为自身免疫性疾病的治疗提供一种全新的治疗途径。
发明内容
本发明为克服现有技术中存在的不足，提供一种G-MDSCs来源的exosomes，该exosomes具有G-MDSCs的非特异性成分和G-MDSCs内特殊的蛋白质和核酸等活性物质，是一种亚细胞结构的复合信息体；其既具有G-MDSCs所具备的免疫抑制特性又包含有自身表面分子CD63；并观察其在自身免疫性疾病治疗中的作用，以期寻求从根本上治疗疾病的方法。
本发明中的G-MDSC exo是含有来源细胞胞膜脂质成分的膜性囊泡，对其携带的生物活性成分具有保护作用；G-MDSC exo可发挥类似G-MDSCs的免疫抑制功能，并且具有细胞本身所不具备的众多优点，是细胞间信息传递的亚细胞结构信息复合体。
本发明公开了一种简便的制备来源于肿瘤个体或自身免疫性疾病个体G-MDSC exo的方法，整个过程耗时短，产量高，并且证实其易于保存且生物活性不受影响，为进行治疗目的规模生产化生产G-MDSC exo奠定基础。即构建荷瘤小鼠模型或者自身免疫性疾病(如CIA)模型，分离脾脏G-MDSCs，收集培养上清；联合使用超速离心与微孔滤膜法，分离提取G-MDSC exo。
其具体步骤如下：
将收集的G-MDSCs培养悬液于4℃离心后收集上清，重复三次，将收集到的上清转移至MWCO 100kDa的超滤离心管中，离心收集内管中的浓缩液；将浓缩液与ExoQuick-TCTM Exosome试剂按体积比5:1混合，震荡，4℃静置后于4℃离心弃上清，收集沉淀物，得exosomes。
本发明中所述的G-MDSC exo具有抑制T细胞增殖的作用及促进Treg极化的作用，体内应用可能发挥强度的免疫抑制作用，对自身免疫性疾病的治疗具有重要意义。
本发明中所述的G-MDSC exo能有效缓小鼠DTH、IBD和CIA的临床进程，减少病理损伤，提供一种通过恢复机体免疫平衡的方式来治疗自身免疫性疾病的新途径。
本发明还证实G-MDSC exo通过抑制CD4+T细胞增殖及促进Treg细胞扩增等方式发挥免疫抑制作用，并且对自身免疫性疾病有保护性作用，为疾病的治疗提供新的途径。
本发明的优点：
(1)本发明率先制备来源于肿瘤个体或自身免疫性疾病个体G-MDSC exo，为其应用及相关生物学功能研究奠定基础。
(2)本发明制备G-MDSC exo与传统的差速离心法制备exosomes相比，本专利的制备过程耗时短，操作方便，便于大剂量提取，并且形态结构完整。
(3)本发明中G-MDSC exo所含成分活性稳定，易于保存，-80℃保存时间达1年以上；室温下结构及活性成分都稳定，易于运输；无需冻存复苏，为G-MDSC exo临床应用提供技术基础。
(4)本发明中G-MDSC exo是一种纳米级亚细胞结构的信息复合体，本身无细胞毒性，临床应用时具有较高的安全性。
(5)本发明中G-MDSC exo对自身免疫性疾病具有保护性作用，其制备和应用为自身免疫性疾病的治疗提供了新的途径。
(6)本发明中G-MDSC exo具有抑制CD4+T细胞增殖和促进Treg细胞扩增的功能，为自身免疫性疾病的治疗提供新的思路。
附图说明:
图1：流式细胞术(FACS)检测免疫磁珠法分选的G-MDSCs的纯度。
图2：G-MDSC exo的鉴定结果，图中A为G-MDSC exo的形态；B为G-MDSC exo的粒径大小频数分布；C为Westem blot检测G-MDSC exo表达结果。
图3：G-MDSC exo抑制CD4+T细胞的作用，图中A为3H-TdR掺入法体外观察G-MDSC exo对CD4+T细胞增殖的影响结果；B为体内观察G-MDSC exo对DTH模型影响的结果。
图4：G-MDSC exo呈剂量依赖性促进TGF-β诱导的CD4+T细胞向Treg细胞扩增图示，图中A为G-MDSC exo促进不同浓度TGF-β诱导的CD4+T细胞向Treg细胞扩增；B为G-MDSC exo呈剂量依赖性促进TGF-β诱导的CD4+T细胞向Treg细胞扩增。
图5：G-MDSC exo对IBD小鼠的保护性作用图示，图中A为G-MDSC exo对IBD小鼠疾病进展的影响；B为G-MDSC exo对IBD小鼠结肠组织的影响；C为G-MDSC exo 对IBD小鼠结肠炎症的影响。
图6：G-MDSC exo对CIA小鼠的保护性作用，图中A为G-MDSC exo干预CIA小鼠的实验方法流程图；B为G-MDSC exo对CIA小鼠疾病评分爪子肿胀程度影响；C为G-MDSC exo对CIA小鼠疾病进程的影响。
图7：G-MDSC exo携带的Arg-1在其减缓自身免疫性疾病病情中发挥重要作用，图中A为G-MDSC exo中Arg-1活性检测结果和Arg-1抑制剂Nor-NHOA对Arg1活性的抑制效果；B为抑制Arg-1活性后的G-MDSC exo对IBD小鼠病情进展的影响；C为抑制Arg-1活性后的G-MDSC exo对结肠组织的影响；D为抑制Arg-1活性后的G-MDSC exo对结肠炎症的影响。
具体实施方式
下面结合具体实施例对本发明的技术方案作进一步描述，但本发明并不限于这些实施例。
实施例1：G-MDSCs的分选及培养上清制备
(1)构建Lewis肺癌移植瘤小鼠模型：本实验室保存的小鼠Lewis肺腺癌细胞于37℃、5％CO2条件下，用含10％小牛血清、pH 7.2的DMEM培养液培养，以细胞生长密度至培养皿底面积的85％为标准，0.25％胰蛋白酶消化传代。用对数生长期细胞，按每只小鼠3.0×106个细胞的量于右侧腹部皮下注射至6-8w雄性C57BL/6小鼠，肿瘤种植后观察肿瘤的生长情况。
(2)CIA模型建立：取等体积牛Ⅱ型胶原(CⅡ)与弗氏完全佐剂1∶1混合，充分研磨至混合物完全乳化，以乳化物滴入水中不松散为度(冰浴中操作)。取乳化后的CⅡ于小鼠尾根部皮内注射(0.1mL/只)，并使乳化物吸收完全。免疫当日为0天，于免疫后第21天，利用CⅡ与弗氏不完全佐剂的乳化物加强免疫。
(3)分离肿瘤小鼠或CIA小鼠脾细胞：造模成功后，眼球放血法处死小鼠，无菌摘取脾脏；在0.22μm的筛网中磨碎脾脏，过滤并将悬液4℃、500g离心5min，弃上清；细胞沉淀中加入5mL ACK裂解红细胞，混匀，静置5min；加RPMI-1640培养液至10mL，4℃、500g离心5min，弃上清；计算细胞数量。
(4)磁珠分选G-MDSCs：每108个脾细胞用350μL PBE重悬后加入50μL FcR Blocking Reagent；冰上放置10min；按照40μL/108个脾细胞的量加入anti-Ly-6G Biotin，混匀，冰上放置30min，每10min混匀一次；加入10mL PBE液，混匀，将悬液4℃、500g离心5min，弃上清；按照50μL/108个脾细胞的量加入anti-Ly-6G-Biotin Beads，混匀， 冰上放置30min，每10min混匀一次；加入10mL PBE液，混匀，将悬液4℃、500g离心5min，弃上清；加入500μLPBE，混匀；将MACS分选柱置于VarioMACS分选器上，用3mL PBE润洗1次；加入细胞悬液，待第一滴悬液流出后，用9mLPBE洗涤分选柱3次；将分选柱撤移出分选器，加入5mL PBE，推动柱栓，将结合于分离柱上的细胞压出，收集细胞悬液，即为G-MDSCs。
(5)G-MDSCs的纯度鉴定：收集1×106个G-MDSCs于EP管中，1mL PBS重悬，4℃、500g离心5min，弃上清剩余100L的PBS；重悬后加0.5μL抗Ly-6G抗体和μL抗CD11b抗体，4℃孵育30min；1mL PBS重悬后4℃、500g离心5min，弃上清；加入200μLPBS重悬，流式细胞仪检测细胞表面分子表达情况，结果如图1所示，G-MDSCs为CD11b和Ly-6G双阳性细胞，本发明利用免疫磁珠法从荷瘤小鼠脾脏分选G-MDSCs，通过FACS测定分选的G-MDSCs纯度，G-MDSCs纯度高达95％以上。
(6)G-MDSCs培养上清制备：将分选的G-MDSCs重悬于含10％小牛血清(预先经过100000g×16h超速离心)的RPMI-1640培养液中，1.5×106/孔种于24孔板，总体积为1mL/孔；在37℃、5％CO2条件下培养24h后，收集细胞。将收集的G-MDSCs培养悬液于4℃、300g离心20min，收集上清。
实施例2：G-MDSC exo制备及蛋白浓度测定
(1)将收集的G-MDSCs上清于4℃、1000g离心30min，收集上清；将上清4℃、10000g离心30min；将上清转移至MWCO 100kDa的超滤离心管中，1500g离心30min，收集内管中的浓缩液。
(2)用购自SBI公司的ExoQuick-TCTM Exosome试剂盒提取G-MDSC exo：步骤(1)中所得的浓缩液与ExoQuick-TCTM Exosome试剂按体积比5:1混合，震荡，4℃静置12h以上；4℃、1000g离心30min，弃上清，收集沉淀物，即为G-MDSC exo。将制备的exosomes用PBS溶解，分装至EP管中，-80℃保存，用于后续试验。
(3)用BCA蛋白定量试剂盒测定G-MDSC exo蛋白浓度：G-MDSC exo悬液与等体积的裂解液(RIPA:PMSF＝250：1)混匀后，冰上放置1h，每10min震荡一次；4℃、12000g离心15min，收集上清。按照BCA蛋白定量试剂盒说明书操作步骤测定exosomes裂解上清中的蛋白浓度。
实施例3：G-MDSC exo的鉴定
(1)透射电镜观察G-MDSC exo形态：取20μL G-MDSC exo悬液滴加于直径3mm的载样铜网上，室温静置2min；用滤纸轻轻吸干液体，滴加pH 6.8的2％磷钨酸溶液于 铜网上，负染lmin；滤纸吸干染液，白炽灯下烤干，透射电镜下观察到G-MDSC exo为圆形或椭圆形微囊状结构，有包膜，腔内为低电子密度成分，粒径为30-150nm，结果如图2A和2B所示，其中A为G-MDSC exo的形态，透射电子显微镜下，可观察到G-MDSC exo为圆形或椭圆形的微囊结构，有完整包膜，腔内为低电子密度成分；B为G-MDSC exo的粒径大小频数分布，透射电镜拍摄的G-MDSC exo的粒径主要分布在30-150nm。
(2)Western blot检测exosomes包含的蛋白分子CD63和线粒体包含的蛋白分子Calnexin：配制5％的浓缩胶和12％分离胶，将变性的G-MDSC exo按250μg蛋白总量上样，经100V恒压电泳后；再经350mA恒流电转90min后，5％脱脂牛奶封闭PVDF膜lh；用抗CD63单克隆抗体和抗Calnexin单克隆抗体4℃孵育过夜；取出后用TBS/T洗膜，10min×3次，用辣根过氧化物酶标记的二抗，37℃孵育30min；取出后用TBS/T洗膜，10min×3次；ImageQuant LAS 4000凝胶成像系统曝光显色，结果如图2C所示，图2C为Westem blot检测G-MDSC exo表达CD63分子，并且不表达线粒体相关分子Calnexin。
实施例4：G-MDSC exo抑制T细胞增殖和小鼠DTH反应
(1)3H-TdR掺入法观察G-MDSC exo对CD4+T细胞增殖的影响：CD4+T细胞的分离。断颈处死6-8w雄性C57BL/6小鼠，无菌摘取并研磨脾脏，制得脾细胞悬液；将悬液4℃、500g离心5min，弃上清；细胞沉淀中加入5mL ACK裂解红细胞，混匀，静置5min；加RPMI-1640培养液至10mL，4℃、500g离心5min，弃上清；10mLPBE重悬，4℃、500g离心5min，弃上清；按欲得到107个CD4+T细胞加入15μLanti-CD4-MicroBeads抗体，混匀后，置于冰上，孵育30min，每10min混匀1次；加10mLPBE，混匀，4℃、500g离心5min，弃上清，加500μL PBE，制得细胞悬液；将分选柱置于VarioMACS分选器上，用3mL的PBE润洗；加入细胞悬液，待第1滴悬液流出后用9mL PBE洗涤分选柱3次；将分选柱撤移出分选器，加入5mLPBE液，推动柱栓，收集流出的细胞悬液，即为CD4+T细胞。CD4+T细胞按5×105个细胞/孔，接种于96孔板，每孔总体积为200μL；抗CD3、抗CD28存在的条件下，加入不同浓度G-MDSC exo，用含10％小牛血清(经过100000g×16h离心)、pH 7.2的RPMI-1640培养液，37℃、5％CO2培养72h后加入3H-TdR(1μCi/孔)，16h后LS6500Multi-Purpose Scintillation Counter测定各孔cpm值。
(2)利用小鼠DTH模型体内观察G-MDSC exo对CD4+T细胞的抑制作用：取6-8w雄性C57BL/6小鼠构建DTH小鼠模型，分为正常对照组(NC)、DTH组、Neu exo处理组、G-MDSC exo处理组，每组6只。根据文献用乳化在氟氏完全佐剂中的终浓度为 1mg/mL的OVA制剂200μL于小鼠尾根部及背部皮内注射，进行初次免疫，7d后用30μL20mg/mL的OVA溶液于小鼠右侧足垫部注射激发，激发后的第24h、48h、72h测小鼠足垫厚度。按照DTH反应程度的判断标准计算各组小鼠脚垫肿胀度，结果显示G-MDSC exo处理组小鼠的脚垫肿胀度较DTH组和Neu exo处理组轻(图3B)，并且均有统计学意义(P<0.05)。提示G-MDSC exosomes在体内能发挥抑制CD4+T细胞的作用。
DTH反应程度的判断(以24h为例)：
脚垫肿胀程度＝(注射OVA 24h后脚垫厚度-注射OVA前脚垫厚度)-(注射PBS 24h后脚垫厚度-注射PBS前脚垫厚度)
图3为G-MDSC exo抑制CD4+T细胞的作用结果图，其中：A、3H-TdR掺入法体外观察G-MDSC exo对CD4+T细胞增殖的影响，结果显示G-MDSC exo呈剂量依赖性方式抑制CD4+T细胞增殖；B、利用DTH模型体内观察G-MDSC exo对迟发型超敏反应的影响。分别于OVA激发后的第24h、36h和72h测各组小鼠足垫肿胀程度，结果显示G-MDSC exo处理组小鼠足垫肿胀程度明显低于其他组，说明G-MDSC exo能够体内抑制DTH反应。
实施例5：G-MDSC exo呈剂量依赖性促进TGF-β诱导的CD4+T细胞向Treg细胞扩增。
磁珠分选CD4+T细胞(同实施例4)，2×106/孔种于24孔板中，总体系为1mL。在抗CD3和抗CD28存在情况下，用TGF-β诱导CD4+T细胞向Treg细胞极化，在培养体系中加入不同量的G-MDSC exo，培养3d后FACS检测CD25+Foxp3+T细胞。结果显示G-MDSC exo能促进TGF-β诱导的CD4+T细胞向Treg细胞扩增(图4A)，并且呈剂量依赖性(图4B)。
图4为G-MDSC exo呈剂量依赖性促进TGF-β诱导的CD4+T细胞向Treg细胞扩增的结果图示。图中A为G-MDSC exo促进不同浓度TGF-β诱导的CD4+T细胞向Treg细胞扩增，结果显示G-MDSC exo在不同浓度TGF-β时均能促进CD4+T细胞向Treg细胞扩增；B为G-MDSC exo呈剂量依赖性促进TGF-β诱导的CD4+T细胞向Treg细胞扩增，结果显示在随着G-MDSC exo浓度增加，TGF-β诱导的Treg细胞扩增程度增强。
实施例6：G-MDSC exo对IBD小鼠的治疗作用
(1)2.5％DSS的配制及炎症性肠病的诱导：将25g葡聚糖硫酸钠盐(Dextran Sulfate Sodium，DSS)溶于1L ddH2O中，高压灭盐后冷却，6-8w龄雄性C57BL/6小鼠连续自由饮用DSS溶液9d，诱导IBD的发病。
(2)实验共分4组，每组6只小鼠，分别为：
正常对照组(NC)：自由饮用ddH2O；
IBD组：饮用2.5％DSS而不做任何处理；
Neu exo处理组：诱导IBD的第2d、第4d和第6d分别以30μg/只的剂量经腹腔注射Neu exo；
G-MDSC exo处理组：诱导IBD的第2d、第4d和第6d分别以30gμg/只的剂量分别经腹腔注射G-MDSC exo；
注：以饮用DSS当天计为第0天。
(3)IBD进展程度的评价：每天监测各组小鼠体重，大便性状及便血情况。进行DAI评分，评分标准如下：
体重变化：<1％得0分，1-5％得1分，5-10％得2分，10-15％得3分，>15％得4分；
大便情况：正常得0分，成形松散得2分，腹泻不成形得4分；
便血情况：无得0分，肉眼血便得4分；
每组得分相加后除以3为最后得分。
(4)各组小鼠结肠大体标本观察：诱导IBD的第9d，眼球放血处死小鼠，打开小鼠腹腔，取出各组小鼠肛门末端直肠至远端盲肠间的结肠组织，观察各组小鼠结肠肿胀程度及长度。
(5)各组小鼠结肠组织病理学分析：取各组小鼠肛门末端直肠至远端盲肠间的肠组织，经10％福尔马林固定后，石腊包埋做HE染色。镜下观察各结肠组织病理损坏程度和炎症细胞浸润情况及累及范围。
(6)实验结果：通过小鼠体重变化、粪便性状及血便情况对各组小鼠进行疾病评分；结肠大体标本观察及结肠组织病理染色观察各组小鼠结肠损伤情况。结果显示G-MDSCexo处理组小鼠临床疾病评分明显减少(图5A)，结肠肿胀程度及缩短程度减少(图5B)，组织学损坏程度和炎症细胞浸润情况及累及范围炎症损伤均较轻(图5C)。
图5为G-MDSC exo对IBD小鼠的保护性作用图示。图中A为G-MDSC exo对IBD小鼠疾病进展的影响，通过小鼠体重变化、粪便性状及血便情况对各组小鼠进行疾病评分，结果显示G-MDSC exo处理组小鼠临床评分明显低于IBD组和中性粒细胞来源exosomes(Neu exo)处理组，并且差异具有统计学意义(P<0.05)。B为G-MDSC exo对IBD小鼠结肠组织的影响。结果显示G-MDSC exo处理组小鼠的结肠肿胀程度及缩短程度都明显低于IBD组和中性粒细胞来源exosomes(Neu exo)处理组。C为G-MDSC exo 对IBD小鼠结肠炎症的影响。通过各组小鼠结肠组织病理染色观察，结果显示G-MDSC exo处理组小鼠结肠组织炎症细胞损伤程度及炎性浸润情况较IBD组和中性粒细胞来源exosomes(Neu exo)处理组明显减轻。这些结果说明G-MDSC exo对小鼠IBD具有保护性作用。
实施例7：G-MDSC exo对CIA小鼠的治疗作用
G-MDSC exo对CIA小鼠的治疗作用：在第一次免疫后第18天和第24天对小鼠进行G-MDSC exo尾静脉处理，流程图如6A图。我们发现，经G-MDSC exo处理后，小鼠的关节炎发病程度明显减轻(图6B)，且发病时间显著推迟(图6C)。如图6B所示，未处理组在初次免疫后27天即开始出现红肿，而G-exo处理组至33天才开始出现红肿。另外，初次免疫后42天，未处理组关节评分高达10分，而G-exo处理组则仅有4分左右。
实施例8：G-MDSC exo携带的Arg-1在其减缓自身免疫性疾病病情中发挥重要作用
(1)G-MDSC exo中Arg1活性检测及nor-NOHA(NN)对G-MDSC exo中Arg1活性的抑制效果：分选G-MDSC(同实施例1)，按2×106/孔种于24孔板中，加入1mlRPMI-1640培养液，37℃、5％CO2的条件下培养16h，收集培养上清，制备G-MDSC exo(同实施例2),裂解G-MDSC exo(同实施例2)，用eBioscience公司的精氨酸酶测定试剂盒测定G-MDSC exo的活性。用1ml DMSO溶解5mg的nor-NOHA粉末，配制成5mg/mlnor-NOHA溶液。各实验组做如下处理：
G-MDSC组：单纯培养G-MDSC，收集细胞。
G-MDSC+DMSO组：加入7μl DMSO培养G-MDSC，收集细胞。
G-MDSC+NN组：加入7μl nor-NOHA溶液培养G-MDSC，收集细胞。
G-MDSC exo组：单纯培养G-MDSC，收集细胞培养上清。
(G-MDSC+DMSO)exo组：加入7μl DMSO培养G-MDSC，收集细胞培养上清。
(G-MDSC+NN)exo组：加入7μl nor-NOHA溶液培养G-MDSC，收集细胞培养上清。
测定结果如图7A，说明G-MDSC exo中包含Arg-1，并且此Arg-1的活性可以被NN抑制。
(2)G-MDSC exo通过Arg-1参与G-MDSC exo对自身免疫性疾病的保护作用：本实施例利用IBD模型观察Arg-1在G-MDSC exo介导的IBD保护性作用的角色。依据实验需求，将IBD小鼠分为Neu exo治疗组、G-MDSC exo治疗组、(G-MDSC+NN)exo治 疗组及(G-MDSC+DMSO)exo治疗组，具体造模方法、治疗方法及观察指标同实施例5。
结果表明Arg-1参与G-MDSC exo对自身免疫性疾病的保护作用。图7B为(G-MDSC+NN)exo组对IBD小鼠疾病进展的影响，通过小鼠体重变化、粪便性状及血便情况对各组小鼠进行疾病评分，结果显示(G-MDSC+NN)exo组处理组小鼠临床评分明显高于G-MDSC exo处理组，并且差异具有统计学意义。7C为(G-MDSC+NN)exo对IBD小鼠结肠组织的影响。结果显示(G-MDSC+NN)exo处理组小鼠的结肠肿胀程度及缩短程度都明显高于G-MDSC exo处理组。7D为(G-MDSC+NN)exo对IBD小鼠结肠炎症的影响。结果显示(G-MDSC+NN)exo处理组小鼠结肠组织炎症细胞损伤程度及炎性浸润情况较G-MDSC exo处理组明显增强。这些结果说明Arg-1参与G-MDSC exo对自身免疫性疾病的保护作用。

Claims (10)

  1. 一种粒细胞样髓源性抑制细胞(G-MDSC)来源的exosomes，其特征在于，所述exosomes是含有来源细胞胞膜脂质成分的膜性囊泡，具有G-MDSCs的非特异性成分和G-MDSCs内特殊的蛋白质和核酸等活性物质，是一种细胞间信息传递的亚细胞结构的复合信息体。
  2. 根据权利要求1所述的一种粒细胞样髓源性抑制细胞来源的exosomes，其特征在于，所述exosomes既具有G-MDSCs所具备的免疫抑制特性又包含有自身表面分子CD63。
  3. 根据权利要求1和2所述的一种粒细胞样髓源性抑制细胞来源的exosomes，其特征在于，所述exosomes中包含精氨酸酶-1(Arg-1)，发挥免疫抑制特性。
  4. 根据权利要求1-3任意一项权利要求所述的一种粒细胞样髓源性抑制细胞来源的exosomes，其特征在于，所述exosomes来源于肿瘤个体或自身免疫性疾病个体。
  5. 一种权利要求1-4任意一项权利要求所述的粒细胞样髓源性抑制细胞来源的exosomes的制备方法，其特征在于，所述制备方法为首先构建荷瘤小鼠模型或胶原诱导性关节炎模型，分离脾脏G-MDSCs，收集培养上清；联合使用超速离心与微孔滤膜法，分离提取G-MDSC exo。
  6. 根据权利要求5所述的一种粒细胞样髓源性抑制细胞来源的exosomes的制备方法，其特征在于，具体操作如下：
    将收集的G-MDSCs培养悬液于4℃离心后收集上清，重复三次，将收集到的上清转移至MWCO 100kDa的超滤离心管中，离心收集内管中的浓缩液；将浓缩液与ExoQuick-TCTM Exosome试剂按体积比5:1混合，震荡，4℃静置后于4℃离心弃上清，收集沉淀物，得exosomes。
  7. 如权利要求1-4任意一项权利要求所述的一种粒细胞样髓源性抑制细胞来源的exosomes在制备治疗自身免疫性疾病药物中的应用。
  8. 根据权利要求7所述的一种粒细胞样髓源性抑制细胞来源的exosomes在制备治疗自身免疫性疾病药物中的应用，其特征在于，所述自身免疫性疾病为炎症性肠病。
  9. 根据权利要求7所述的一种粒细胞样髓源性抑制细胞来源的exosomes在制备治疗自身免疫性疾病药物中的应用，其特征在于，所述自身免疫性疾病为胶原诱导性关节炎。
  10. 根据权利要求7所述的一种粒细胞样髓源性抑制细胞来源的exosomes在制备治疗自身 免疫性疾病药物中的应用，其特征在于，所述自身免疫性疾病为迟发型超敏反应。
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