WO2016184427A1 - 低氧处理的间充质干细胞及其应用 - Google Patents

低氧处理的间充质干细胞及其应用 Download PDF

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WO2016184427A1
WO2016184427A1 PCT/CN2016/082787 CN2016082787W WO2016184427A1 WO 2016184427 A1 WO2016184427 A1 WO 2016184427A1 CN 2016082787 W CN2016082787 W CN 2016082787W WO 2016184427 A1 WO2016184427 A1 WO 2016184427A1
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mesenchymal stem
cells
stem cells
hypoxic
treatment
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French (fr)
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时玉舫
杜黎明
林良宇
王莹
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中国科学院上海生命科学研究院
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Priority to US15/576,091 priority Critical patent/US20180200301A1/en
Priority to EP16795914.7A priority patent/EP3299023A4/en
Publication of WO2016184427A1 publication Critical patent/WO2016184427A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/30—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/02—Atmosphere, e.g. low oxygen conditions

Definitions

  • the present invention belongs to the field of biomedicine, and more particularly, the present invention relates to hypoxic treated mesenchymal stem cells and uses thereof.
  • MSCs Mesenchymal stem/stromal cells
  • tissue stem cells In almost all tissues of the body, MSCs are self-renewing and differentiate into specific tissue cells to repair tissue damage.
  • MSCs have been used to treat a variety of immune-related animal disease models or clinical diseases, and their effectiveness and safety have been confirmed. However, different studies have different explanations for their mechanisms of therapeutic effects. . It has been reported that MSCs can secrete a series of growth factors such as hepatocyte growth factor (HGF), epidermal growth factor (EGF) and transforming growth factor (TGF- ⁇ ), and the role of these factors in the treatment of diseases by MSCs still needs further research. Discussion.
  • HGF hepatocyte growth factor
  • EGF epidermal growth factor
  • TGF- ⁇ transforming growth factor
  • mesenchymal stem cells are considered to have broad application prospects, considering the complexity of the disease microenvironment in the body and the emerging cases of ineffective and failed MSCs treatment, it is still necessary to further explore the interaction and regulation of disease microenvironment and MSCs. Mechanisms to better allow MSCs-mediated stem cell therapy to truly move from the laboratory to the clinic.
  • a hypoxic treated mesenchymal stem cell or cell culture or culture supernatant for the manufacture of a medicament for preventing, ameliorating or treating an inflammatory disease.
  • the inflammatory disease comprises: multiple sclerosis, or inflammatory bowel disease (IBD).
  • IBD inflammatory bowel disease
  • hypoxia is from 1 to 15% by volume, preferably from 1 to 13%, more preferably from 5 to 10% by volume of oxygen.
  • the hypoxic-treated mesenchymal stem cell or cell culture or culture supernatant means: at a volume ratio of 1 to 15%, preferably 1 to 13%, more preferably 5
  • the obtained mesenchymal stem cells or cell cultures or culture supernatants are continuously cultured under conditions of ⁇ 10% oxygen.
  • the culture is carried out for 2 generations or more, and more preferably for 3 generations or more, such as 3 to 20 generations, 3 to 10 generations, 3 to 5 generations, and the like.
  • the medicament is further used to:
  • Treg cells regulatory T cells
  • a hypoxic-treated mesenchymal stem cell or cell culture or culture supernatant obtained by the following method: at a volume ratio of 1 to 15%, preferably 1 to Mesenchymal stem cells are continuously cultured under conditions of ⁇ 13%, more preferably 5-10% oxygen.
  • the culture is continued for one generation or more, and more preferably three generations or more, such as 3 to 20 generations, 3 to 10 generations, 3 to 5 generations, and the like.
  • the condition of 1 to 15% by volume, preferably 1 to 13%, more preferably 5 to 10% of oxygen means: culturing in air having a low oxygen concentration; that is, culturing thereof
  • the content of other substances in the air is consistent with the air content in the conventional atmospheric oxygen incubator.
  • a pharmaceutical composition for preventing, ameliorating or treating an inflammatory disease comprising an effective amount of said hypoxic treated mesenchymal stem cell or cell culture or culture supernatant And a pharmaceutically acceptable carrier.
  • a method of preparing a hypoxic treated mesenchymal stem cell or cell culture or culture supernatant comprising: at a volume ratio of 1 to 15%, preferably 1 to Mesenchymal stem cells are continuously cultured under conditions of 13%, more preferably 5 to 10% oxygen.
  • a method for preventing, ameliorating or treating inflammatory disease function of a mesenchymal stem cell or a cell culture or a culture supernatant comprising: hypoxia treatment of mesenchymal stem cells .
  • a method for promoting secretion of insulin-like growth factor-2 by mesenchymal stem cells comprising: subjecting mesenchymal stem cells to hypoxia treatment.
  • the hypoxia is from 1 to 15% by volume, preferably from 1 to 13%, more preferably from 5 to 10% by volume of oxygen.
  • IGF-2 insulin-like growth factor-2
  • the inflammatory disease comprises: multiple sclerosis, or inflammatory bowel disease (IBD).
  • the insulin-like growth factor-2 comprises: having or containing an insulin-like Active fragment of amino acid sequence 25 to 91 of growth factor-2.
  • the medicament is further used to:
  • a pharmaceutical composition for preventing, ameliorating or treating an inflammatory disease comprising: an effective amount of insulin-like growth factor-2, and pharmaceutically acceptable a.
  • the insulin-like growth factor-2 comprises an active fragment having or containing the amino acid sequence of the 25th to 91st insulin-like growth factor-2.
  • kits for preventing, alleviating or treating an inflammatory disease comprising: the hypoxic-treated mesenchymal stem cell or a cell culture or culture supernatant; or a pharmaceutical composition as described.
  • mice Treatment of experimental autoreactive encephalomyelitis with normoxia-msenchymal stem cells (N-MSCs) or hypoxia-mesenchymal stem cells (H-MSCs) (experimental autoimmune encephalomyelitis, EAE).
  • N-MSCs normoxia-msenchymal stem cells
  • H-MSCs hypoxia-mesenchymal stem cells
  • Mice were given a tail vein infusion of MSCs (2 ⁇ 10 5 /unit/time) on days 9, 12, and 15 after EAE induction, and the clinical scores of the mice were recorded every day. On the 15th day of the general course of EAE disease, the mice were sacrificed and the spinal cord was harvested.
  • C Proliferation of spleen cells in vitro in different treatment groups.
  • spleen cells from control, N-MSCs or H-MSCs treated EAE mice were isolated and stimulated with MOG35-55 (20 ⁇ g/ml) for 72 hours using 3 H-labeled thymidine incorporation.
  • the proliferation of spleen cells in each treatment group was tested experimentally.
  • the spleen cell activation index (SI) of each group was calculated by the number of proliferation of MOG35-55 activated spleen cells (MOG group)/proliferation number of spleen cells (Ctrl group) without MOG35-55.
  • the serum of EAE mice of the control group, N-MSCs or H-MSCs treatment group was isolated and the expression levels of IL-17 and IFN- ⁇ were detected by ELISA.
  • the spleen cells of the mice were isolated and stimulated with MOG35-55 (20 ⁇ g/ml) for 72 hours, and the contents of IL-17 and IFN- ⁇ in the culture supernatant were measured. The results showed that the serum and spleen cells of the mice were induced after EAE.
  • N-MSCs Both express high levels of IL-17 and IFN- ⁇ , and injection of N-MSCs can partially inhibit their expression levels. More importantly, H-MSCs can completely inhibit IL-17 and IFN- ⁇ in serum and spleen cells. Secretion and expression, Ctrl group was normal mice; MOG group was EAE induction group; N-MSCs showed EAC mice with oxygen-treated mesenchymal stem cell treatment group; H-MSCs indicated EAE mice treated with hypoxia treatment Stem cell treatment group. N.S., no significant difference; *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001.
  • N-MSCs normoxia-msenchymal stem cells
  • H-MSCs hypoxia-mesenchymal stem cells
  • N-MSCs normoxia-msenchymal stem cells
  • EAE Encephalomyelitis
  • C Proliferation of splenocytes in vitro in different treatment groups.
  • spleen cells of the control group (media-treated group), N-sup or H-sup treatment group EAE mice were isolated and stimulated with MOG35-55 (20 ⁇ g/ml) for 72 hours using 3 H Labeled thymidine incorporation assay was used to detect the proliferation of spleen cells in each treatment group.
  • the spleen cell activation index (SI) of each group was calculated by the number of proliferation of spleen cells (MOG group) activated by MOG35-55/the number of proliferation of spleen cells (Ctrl group) without MOG35-55.
  • D mRNA and protein levels of insulin-like growth factor-2 (IGF-2) in mesenchymal stem cells under normoxia or hypoxia.
  • MSCs were cultured in normoxia and hypoxia, respectively. After three generations of continuous culture, 6-well plates were plated at a cell density of 50%. On the second day after plating, fresh culture medium was replaced. After 48 hours, cells and supernatant were taken to detect insulin. Expression of the growth factor-2 gene level and protein level. Hypoxic treatment of MSCs expressed high levels of IGF-2 both at the protein level and mRNA; N-MSCs indicated that the mesenchymal stem cells were treated with normoxia; and H-MSCs indicated that the mesenchymal stem cells were treated with hypoxia. **p ⁇ 0.01, ***p ⁇ 0.001.
  • Figure 3 The role of insulin-like growth factor-2 and its Ala25-Glu91 fragment in the treatment of experimental autoreactive encephalomyelitis.
  • Neutralizing antibodies against insulin-like growth factor-2 can restore the therapeutic effect of hypoxia-treated mesenchymal stem cells (MSCs) cell culture supernatant on EAE.
  • MSCs mesenchymal stem cells
  • N-MSCs mesenchymal stem cells
  • hypoxic treated mesenchymal stem cells hypoxic treated mesenchymal stem cells
  • mice were injected with human IGF2 (hIGF2).
  • the neutralizing antibody or isotype control antibody blocked the function of IGF2, and the results showed that the neutralizing antibody of IGF2 can inhibit the therapeutic effect of hypoxia-treated MSCs culture supernatant on EAE.
  • Ctrl indicates 5% FBS medium treatment group
  • Nor-IgG indicates normal cultured MSCs culture supernatant group
  • Hyp-IgG indicates hypoxia cultured MSCs culture supernatant group
  • anti-hIGF2 indicates insulin-like growth factor-2 Neutralizing antibody
  • IgG A control antibody representing an insulin-like growth factor-2 neutralizing antibody. *p ⁇ 0.05.
  • EAE mice On day 8 of EAE induction, EAE mice were injected with Ala25-Glu91 fragment (5 ng/head) of human recombinant IGF2 daily to observe disease progression. The results showed that the injection of Ala25-Glu91 fragment of IGF2 can effectively inhibit the progression of EAE.
  • the PBS (phosphate buffer) injection group was the control group, and the IGF-2 was the insulin-like growth factor-2 Ala25-Glu91 fragment treatment group. *p ⁇ 0.05, **p ⁇ 0.01.
  • EAE was treated with Ala25-Glu91 fragment of IGF2.
  • mononuclear cells of spinal cord were isolated and counted by percoll.
  • the results showed that Ala25-Glu91 fragment of IGF2 can effectively inhibit the infiltration of mononuclear cells in the spinal cord of EAE mice.
  • the PBS (phosphate buffer) injection group was the control group, and the IGF-2 was the insulin-like growth factor-2 Ala25-Glu91 fragment treatment group. ***p ⁇ 0.001.
  • Ala25-Glu91 fragment of insulin-like growth factor-2 inhibits proliferation of antigen-specific T cells.
  • EAE was treated with Ala25-Glu91 fragment of IGF-2.
  • mouse spleen cells were isolated, stimulated with MOG35-55 (20 ⁇ g/ml), and spleen was detected by 3 H-labeled thymidine incorporation assay. Cell proliferation.
  • the spleen cell activation index (SI) of each group was calculated by the number of proliferation of spleen cells (MOG group) activated by MOG35-55/the number of proliferation of spleen cells (Ctrl group) without MOG35-55, and the results showed that IGF-2
  • the Ala25-Glu91 fragment can effectively inhibit the proliferation of MOG-specific T cells in EAE mice; the PBS (phosphate buffer) injection group is the control group, and the IGF-2 is the insulin-like growth factor-2 Ala25-Glu91 fragment treatment group. ***p ⁇ 0.001.
  • E Ala25-Glu91 fragment of insulin-like growth factor-2 up-regulates regulatory T cells.
  • EAE was treated with Ala25-Glu91 fragment of IGF-2, and mononuclear cells in the spinal cord of mice were isolated on the 15th day of EAE disease progression, and the expression of CD4 + Foxp3 + T cells was detected by flow cytometry. It was found that in the CD4 + cells of spinal cord mononuclear cells, the proportion of Foxp3 + cells increased significantly after treatment with Ala25-Glu91 fragment of IGF-2; the PBS (phosphate buffer) injection group was the control group, IGF- 2 is the Ala25-Glu91 fragment treatment group of insulin-like growth factor-2. ***p ⁇ 0.001.
  • mice with inflammatory bowel disease at different treatments A model of inflammatory bowel disease induced by aqueous solution of 3% DSS (dextran sodium sulfate, DSS) was prepared. On the 1st, 3rd, and 5th day after the induction of the disease model, mice were injected with normoxia-treated mesenchymal stem cells (normoxia). -mesenchymal stem cells (N-MSCs) or hypoxia-mesenchymal stem cells (H-MSCs) (1 ⁇ 10 6 /unit/time) were used to observe the survival of the mice.
  • N-MSCs normoxia-treated mesenchymal stem cells
  • H-MSCs hypoxia-mesenchymal stem cells
  • N-MSCs represent the group of oxidative enteritis mice treated with oxygen-treated mesenchymal stem cells
  • H-MSCs represent oxidative enteritis mice treated with hypoxic-treated mesenchymal stem cells.
  • mice Changes in body weight in mice receiving inflammatory bowel disease at different treatments.
  • N-MSCs normoxia-mesenchymal stem cells
  • hypoxic-treated mesenchymal stem cells hypoxic-treated mesenchymal stem cells (hypoxia-mesenchymal) by intraperitoneal injection.
  • Stem cells, H-MSCs (1 ⁇ 10 6 / only / time). The body weight of the mice was weighed daily, and the average body weight of each group of mice on the first day was taken as a baseline to calculate the change in body weight.
  • N-MSCs represent the group of oxidative enteritis mice treated with oxygen-treated mesenchymal stem cells
  • H-MSCs represent oxidative enteritis mice treated with hypoxic-treated mesenchymal stem cells. *p ⁇ 0.05.
  • mice Clinical scores of mice with inflammatory bowel disease at different treatments.
  • N-MSCs normoxia-msenchymal stem cells
  • hypoxic-treated mesenchymal stem cells hypoxic-treated mesenchymal stem cells
  • N-MSCs represent the group of oxidative enteritis mice treated with oxygen-treated mesenchymal stem cells
  • H-MSCs represent oxidative enteritis mice treated with hypoxic-treated mesenchymal stem cells. *p ⁇ 0.05.
  • mice Colon length of inflammatory bowelitis mice at different treatments.
  • N-MSCs normoxia-mesenchymal stem cells
  • H-MSCs hypoxic mesenchymal stem cells
  • Figure 5 Effect of Ala25-Glu91 fragment of insulin-like growth factor-2 in the treatment of inflammatory bowel disease.
  • FIG. 6 Effect of interfering with the expression of insulin-like growth factor-2 in MSCs on EAE.
  • sh-RNA770 and sh-RNA1526 interfered with the expression of insulin-like growth factor-2 (IGF-2) in MSCs.
  • IGF-2 insulin-like growth factor-2
  • MSCs were induced by EAE after oxygen culture or hypoxia culture for more than 3 generations.
  • mice were given a tail vein injection (2*10 5 /piece).
  • the interference of IGF-2 expression on MSCs could destroy the therapeutic effect of hypoxic-treated MSCs on EAE. *p ⁇ 0.05.
  • the inventors have intensively studied for the first time that after hypoxic treatment of mesenchymal stem cells, it is possible to significantly promote the remission or therapeutic effect of mesenchymal stem cells on inflammatory diseases.
  • the invention also discloses a hypoxic zone Mesenchymal stem cells are capable of producing insulin-like growth factor-2, a central role played by hypoxic-treated mesenchymal stem cells in the treatment of inflammatory diseases.
  • the present inventors selected an experimental autoreactive encephalomyelitis (EAE) animal model (an animal model of multiple sclerosis), a mesenchymal stem cell cultured under normoxia (human umbilical cord source) during cell therapy. It showed a certain therapeutic effect, and the effect of mesenchymal stem cells was further enhanced after hypoxic preconditioning.
  • EAE autoreactive encephalomyelitis
  • the present invention provides an MSCs which are hypoxic-treated MSCs, which are simple in preparation, do not require transgenic manipulation, do not involve insertion of foreign genes, and do not have safety problems during administration. .
  • the hypoxic treatment refers to a low oxygen environment in which oxygen is from 1 to 15% by volume; preferably from 1 to 13%; more preferably from 5 to 10% by volume.
  • oxygen is from 1 to 15% by volume; preferably from 1 to 13%; more preferably from 5 to 10% by volume.
  • the content of the elements in the other air is the same as the air content in the conventional atmospheric oxygen incubator.
  • a hypoxic treated MSC for the preparation of a medicament for the prevention, alleviation or treatment of an inflammatory disease.
  • the inflammatory diseases include, for example, experimental autoreactive encephalomyelitis (EAE) or inflammatory bowel disease (IBD). It is also used to: increase the proportion of regulatory T cells (Treg cells) in diseased tissues; reduce the ratio of Th1 and Th17 cells in diseased tissues; or inhibit IFN- ⁇ and IL-17 factors in serum.
  • the present invention also provides a composition (drug) comprising an effective amount (e.g., 0.000001 to 50% by weight; preferably 0.00001 to 20% by weight; more preferably 0.0001-10% by weight) of the hypoxic treated MSCs. And a pharmaceutically acceptable carrier.
  • an effective amount e.g., 0.000001 to 50% by weight; preferably 0.00001 to 20% by weight; more preferably 0.0001-10% by weight
  • a pharmaceutically acceptable carrier e.g., 0.000001 to 50% by weight; preferably 0.00001 to 20% by weight; more preferably 0.0001-10% by weight
  • the term “contains” means that the various ingredients can be used together in the mixture or composition of the invention. Therefore, the terms “consisting essentially of” and “consisting of” are encompassed by the term “contains.”
  • the term "effective amount” or “effective amount” refers to a composition or activity that is capable of functioning or active on a human and/or animal and is acceptable for use by humans and/or animals.
  • a "pharmaceutically acceptable” ingredient is one that is suitable for use in humans and/or mammals without excessive adverse side effects (eg, toxicity, irritation, and allergies), ie, having a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents.
  • the cells can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium.
  • the pH is usually from about 5 to about 8, preferably, the pH is from about 6 to about 8.
  • IGF-2 insulin-like growth factor-2
  • the present inventors used the supernatant of mesenchymal stem cells for the treatment of experimental autoreactive encephalomyelitis, and the results showed that only the supernatant of hypoxic cells can be used.
  • the therapeutic effect is exerted, which indicates that the hypoxic pretreated mesenchymal stem cells function as a disease treatment through the factors secreted by them.
  • the present inventors examined the difference in expression of mesenchymal stem cell factors under normoxia and hypoxia, and the results showed The expression level of insulin-like growth factor-2 was significantly improved after hypoxia treatment.
  • Insulin-like growth factor-2 is a growth factor secreted mainly by the liver and abundantly present in the blood. It has anti-apoptosis, growth regulation, insulin-like and mitogenic functions. It is generally believed that insulin-like growth factor-2 plays an important role in embryonic development and can promote embryonic development and organ formation. It has also been reported to be related to memory and reproduction. Through studies on genetically deficient mice, insulin-like growth factor- Loss of signal 2 can cause brain development to be imperfect. However, there have been no reports of the association of insulin-like growth factor-2 with the treatment of inflammatory diseases.
  • the present inventors used neutralizing antibodies to neutralize insulin-like growth factors in hypoxic supernatants. 2, the supernatant after neutralization disappeared in the treatment of disease. Afterwards, the present inventors attempted to directly treat the experimental autoreactive encephalomyelitis using the Ala25-Glu91 fragment of insulin-like growth factor-2, and the results showed that the use of the Ala25-Glu91 fragment of insulin-like growth factor-2 can be effectively treated.
  • Experimental autoreactive encephalomyelitis In the site of inflammation, i.e., the spinal cord, the inventors found that the proportion of regulatory T cells (Tregs) was significantly increased, while the ratio of Th1 and Th17 cells was significantly decreased.
  • hypoxic preconditioning can effectively improve the effect of treating experimental autoreactive encephalomyelitis compared with mesenchymal stem cells under normoxic culture.
  • the improvement of this therapeutic effect is dependent on insulin-like growth factor- 2
  • the increase in expression is achieved, and the Ala25-Glu91 fragment using insulin-like growth factor-2 alone can also achieve a good therapeutic effect on experimental autoreactive encephalomyelitis.
  • the present invention also provides the use of insulin-like growth factor-2 and its Ala25-Glu91 fragment for the preparation of a composition (drug) for preventing, alleviating or treating an inflammatory disease.
  • the insulin-like growth factor-2 comprises full length insulin-like growth factor-2 or a biologically active fragment thereof.
  • the full-length amino acid sequence of the insulin-like growth factor-2 can be substantially the same as the sequence shown by SEQ ID NO: 8 (NCBI protein database number P01344.1).
  • Insulin-like growth factor-2 formed by substitution, deletion or addition of one or more amino acid residues is also included in the present invention.
  • Insulin-like growth factor-2 or a biologically active fragment thereof comprises a substitution sequence of a portion of a conserved amino acid that does not affect its activity or retains its partial activity.
  • Proper replacement of amino acids is a technique well known in the art that can be readily implemented and ensures that the biological activity of the resulting molecule is not altered. These techniques have taught one in the art that, in general, altering a single amino acid in a non-essential region of a polypeptide does not substantially alter biological activity. See Watson et al, Molecular Biology of The Gene, Fourth Edition, 1987, The Benjamin/Cummings Pub. Co. P224.
  • the biologically active fragment of insulin-like growth factor-2 means a polypeptide which still retains all or part of the function of full-length insulin-like growth factor-2.
  • the biologically active fragment retains at least 50% of full length insulin-like growth factor-2 activity.
  • the active fragment is capable of maintaining 60%, 70%, 80%, 90%, 95%, 99%, or 100% activity of full length insulin-like growth factor-2.
  • the present invention may also employ modified or modified insulin-like growth factor-2 or an active fragment based on its 25th to 91st amino acid sequences, for example, in order to promote its half-life, effectiveness, metabolism, and/or An inflammatory disease modified or modified by the potency of the protein.
  • the present invention also provides a composition (drug) comprising an effective amount (e.g., 0.000001 to 50% by weight; preferably 0.00001 to 20% by weight; more preferably 0.0001-10% by weight) of the insulin-like growth factor- 2, and a pharmaceutically acceptable carrier.
  • an effective amount e.g., 0.000001 to 50% by weight; preferably 0.00001 to 20% by weight; more preferably 0.0001-10% by weight
  • a pharmaceutically acceptable carrier e.g., 0.000001 to 50% by weight; preferably 0.00001 to 20% by weight; more preferably 0.0001-10% by weight
  • hypoxic-treated mesenchymal stem cells have a therapeutic effect on inflammatory diseases under normoxia. Cultured mesenchymal stem cells are more desirable. It has also been found that the use of insulin-like growth factor-2 alone can also achieve a good therapeutic effect on experimental autoreactive encephalomyelitis. The present inventors believe that the above findings provide a new idea for elucidating the mechanism of mesenchymal stem cells for treating immune-related diseases such as autoimmune poliomyelitis, and for further understanding the value of insulin-like growth factor-2 in the treatment of immune-related diseases. New information is provided.
  • Ala25-Glu91 fragment of recombinant human IGF-2, IGF-2 neutralizing antibody, and control IgG antibody were purchased from R&D Systems.
  • Myelin oligodendrocyte glycoprotein (MOG 35-55 ) was purchased from Jill Biochemical Co., Ltd. (Shanghai, China).
  • Incomplete Freund's adjuvant (IFA) Mycobacterium tuberculosis (TB) was purchased from Sigma-Aldrich (MO, USA).
  • Pertusis toxin (PT) was purchased from List Biological Laboratories, USA.
  • Dextran sodium sulfate (DSS) was purchased from MP Biomedicals.
  • the human IGF-2 ELISA kit was purchased from (Mediagnost, Germany), and the mouse IFN ⁇ , IL-17 ELISA identification reagent was purchased from R&D Systems.
  • Flow cytometry antibody anti-mouse CD4 PerCP-Cy5.5, anti-mouse CD4 PerCP-Cy5.5, anti-mouse IL-17A PE, anti-mouse IL-17A PE was purchased from Ebioscience.
  • C57BL/6 mice were purchased from Shanghai Slack Laboratory Animals Co., Ltd. and were raised in the Department of Laboratory Animal Science of Shanghai Jiao Tong University School of Medicine.
  • Isolation and culture of human umbilical cord-derived mesenchymal stem cells After evacuating the blood vessels in the umbilical cord, the remaining umbilical cord tissue is cut into pieces and placed in a petri dish, allowing the MSCs to naturally climb out and change the liquid every two days. After the cells in the culture dish reach a certain density, they are digested and passaged for subsequent experiments.
  • hypoxic treatment of human mesenchymal stem cells The mesenchymal stem cells are cultured in a hypoxic incubator, and the reagents used are the same as those in the normoxic culture except for the change of the incubator. Hypoxic conditions: 10% O 2 by volume.
  • Construction of cells with knockdown of IGF-2 expression level stable knockdown by lentiviral infection using the control sequence: Ctrl-sh-RNA (5'-ttctccgaacgtgtcacgt-3' (SEQ ID NO: 1)); The sequences of IGF2 are: sh-RNA-770 (5'-gaagtcgatgctggtgcttct-3' (SEQ ID NO: 2)); sh-RNA-1526 (5'-gctttaaacacccttcacata-3' (SEQ ID NO: 3)).
  • the lentivirus is screened for a resistance gene with a GFP-tagged gene and puromycin, and the cells exhibit green fluorescence after infection.
  • the virus was added when the cell density reached 60%, and the normal culture solution was changed after 24 hours.
  • the infection efficiency was estimated under the microscope and the fluorescence efficiency was estimated.
  • puromycin was added, and the cells were used for passage after the cells were overgrown.
  • EAE autoimmune encephalomyelitis
  • CFA Complete Freund's Adjuvant
  • Emulsification of antigen Two glass needles were connected through a tee, an equal volume of antigen solution (300 ⁇ g MOG in 100 ⁇ l PBS) and 100 ⁇ l of complete Freund's adjuvant were added to the needle tube, and the bubbles in the needle tube were removed and pushed back and forth to obtain an emulsion. It takes about 500 pushes, and the resistance is gradually increased. At this time, the ingredients can be fully mixed into an emulsified state.
  • Pertussis toxin (PT) solution preparation Pertussis toxin was dissolved in PBS to a working concentration of 1 ng/ ⁇ l.
  • the supernatant injection time was from day 9 to day 13.
  • the Ala25-Glu91 fragment of insulin-like growth factor 2 was used, it was injected daily from the 8th day after the induction of the experiment, and the dose was 5 ng/head, which was administered intraperitoneally.
  • Preparation of hypoxia-treated MSCs continuous culture for more than 3 generations in a volume ratio of 10% O 2 .
  • Establishment of a 10% O 2 environment The establishment of a low oxygen environment was achieved by adjustment of the nitrogen content using a Forma TM Series II 3110 Water-Jacketed CO 2 Incubator.
  • DSS Sodium dextran sulfate
  • the C57BL/6 female rats of 8-10 weeks were used to replace the drinking water induction model with the prepared DSS solution.
  • the body weight of the mice was weighed daily and the feces were observed.
  • the DSS solution was changed or supplemented every other day.
  • Normal or hypoxic MSCs were intraperitoneally injected on day 1, day 3, and day 5, respectively, and each mouse was injected with 2 x 10 6 cells.
  • the Ala25-Glu91 fragment of insulin-like growth factor-2 was administered in an amount of 50 ng per mouse per day, administered intraperitoneally.
  • mice After the EAE mice were sacrificed, the spleens of the mice were taken out and a single cell suspension was obtained.
  • the spleen cells were plated in a 96-well plate of U-bottom according to the number of 3-5 ⁇ 10 5 cells per well, and MOG 35-55 was added. Up to 20 ⁇ g/ml.
  • the primer sequences used are as follows:
  • Reverse primer 5'-GGTCGTGCCAATTACATTTCA-3' (SEQ ID NO: 5);
  • Reverse primer 5'-AGGTGGACAGCGAGGCCAGGAT-3' (SEQ ID NO: 7).
  • Example 1 Hypoxic preconditioning enhances the therapeutic effect of mesenchymal stem cells on experimental autoreactive encephalomyelitis (EAE)
  • MSCs mesenchymal stem cells
  • hypoxic treatment significantly enhances the therapeutic effect of MSCs on EAE.
  • Pathological analysis of the spinal cord showed that the demyelinating and immune cell infiltration of the spinal cord of the mice treated with hypoxic MSCs were significantly inhibited, and the number of mononuclear cells infiltrated in the spinal cord was also significantly reduced, as shown in Figures 1A-B.
  • MSCs have a certain effect in the treatment of EAE, and hypoxic preconditioning can significantly enhance this effect, and all disease indicators have been significantly relieved.
  • Example 2 Culture supernatant of hypoxic mesenchymal stem cells is effective for the treatment of experimental autoreactive encephalomyelitis (EAE)
  • the present inventors In order to confirm whether the secreted factors of mesenchymal stem cells (MSCs) play a key role in the treatment of EAE, the present inventors directly used the culture supernatant of normoxia MSCs or hypoxic treated MSCs for the treatment of EAE (injection time). For the model from the 9th day to the 13th day after induction, the method is abdominal cavity).
  • MSCs mesenchymal stem cells
  • hypoxic MSCs Similar to the direct treatment of MSCs with hypoxia, the supernatant of hypoxic MSCs significantly inhibited demyelination and immune cell infiltration in the spinal cord of mice, as shown in Figures 2A-B, and significantly inhibited MOG-specific T cell proliferation in the spleen, such as Figure 2C.
  • hypoxic MSCs achieve therapeutic effects on EAE through secreted factors.
  • the present inventors used a gene chip to analyze the expression profiles of MSCs cultured in normoxia and hypoxia, and found that insulin growth factor-2 (IGS) in hypoxic MSCs compared with normoxia MSCs.
  • IGF-2 insulin growth factor-2
  • the expression level of IGF-2) was significantly increased, and mRNA and protein levels were verified by Real time-PCR and ELISA, as shown in Fig. 2D.
  • insulin-like growth factor-2 plays a key role in the treatment of experimental autoreactive encephalomyelitis (EAE) in hypoxic cultured mesenchymal stem cells
  • IGF-2 Insulin Like Growth Factor-2
  • MSCs Mesenchymal Stem Cells
  • the present inventors used sh-RNA770 (purchased from the gamma gene) and sh-RNA1526 (purchased from the gamma gene) to interfere with the expression level of IGF-2 in MSCs, as shown in Fig. 6, and the MSCs treated with the control group sh-RNA.
  • sh-RNA770 purchased from the gamma gene
  • sh-RNA1526 purchased from the gamma gene
  • the expression of interfering IGF-2 on MSCs can disrupt the therapeutic effect of hypoxic treated MSCs on EAE. This suggests that IGF-2 is a key factor in the treatment of EAE by hypoxic MSCs.
  • EAE EAE
  • Ala25-Glu91 fragment of IGF-2 purchased from R&D. It was found that the Ala25-Glu91 fragment of IGF-2 can effectively inhibit EAE after injection, which is mainly manifested in the improvement of clinical score in mice, as shown in Fig. 3B; the number of mononuclear cell infiltration in the spinal cord is significantly decreased, as shown in Fig. 3C; MOG-specific T Cell proliferation was significantly reduced, as shown in Figure 3D.
  • Example 4 hypoxic pretreated mesenchymal stem cells and insulin-like growth factor-2 Ala25-Glu91 fragments can be used for the treatment of inflammatory bowel disease
  • hypoxic treated mesenchymal stem cells Similar to the process of treating experimental autoreactive encephalomyelitis (EAE), hypoxic treated mesenchymal stem cells (MSCs) also show up in the treatment of inflammatory bowel disease (IBD). Significant treatment effect. Studies have found that hypoxic treatment of MSCs can significantly improve the survival of IBD mice, as shown in Figure 4A; improve the body weight of mice during the induction of IBD induction, as shown in Figure 4B; improve the clinical score of IBD mice, as shown in Figure 4C; The length of the colon, hypoxic treatment of MSCs can significantly inhibit the intestinal damage of IBD mice, as shown in Figure 4D;
  • the present inventors also analyzed the components of lymphocytes of the disease site, including mesenteric lymph nodes and intestinal infiltrating cells, and the present inventors have found that, like EAE, IGF-2 Ala25-Glu91 fragment treatment group, CD4 + Foxp3 The proportion of Tregs increased significantly, while the proportion of Th1 and Th17 cells decreased significantly, as shown in Figure 5C. These results indicate that the Ala25-Glu91 fragment of IGF-2 can play a role in the treatment of specific diseases by modulating immunity.

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Abstract

一种低氧处理的间充质干细胞及其应用，所述干细胞可缓解或治疗炎症性疾病，产生胰岛素样生长因子-2，该因子在低氧处理的间充质干细胞治疗炎症性疾病中起核心作用。

Description

低氧处理的间充质干细胞及其应用 技术领域
本发明属于生物医药领域，更具体地，本发明涉及低氧处理的间充质干细胞及其应用。
背景技术
间充质干细胞(mesenchymal stem/stromal cells，MSCs)具有自我更新和多向分化能力，也可称为组织干细胞。在机体内几乎所有的组织中，MSCs都能自我更新并且分化为特定的组织细胞进而修复组织损伤。
目前，体外分离培养的MSCs已经被用于治疗多种免疫相关的动物疾病模型或者临床疾病，其有效性和安全性得到了证实，但是，不同的研究对于其发挥治疗作用的机制有不同的阐述。有文章报道，MSCs可以分泌肝细胞生长因子(HGF)、表皮生长因子(EGF)、转化生长因子(TGF-β)等一系列生长因子，而这些因子在MSCs治疗疾病过程中的作用仍需要进一步的探讨。
虽然间充质干细胞被认为具有广泛的应用前景，但是考虑到体内疾病微环境的复杂性和不断涌现的MSCs治疗无效和失败案例，仍然需要更深层的探究疾病微环境和MSCs的相互作用及调控机制，以更好的让MSCs介导的干细胞治疗真正的从实验室走向临床。
发明内容
本发明的目的在于提供低氧处理的间充质干细胞及其应用。
在本发明的第一方面，提供低氧处理的间充质干细胞或细胞培养物或培养上清在制备预防、缓解或治疗炎症性疾病的药物中的用途。
在一个优选例中，所述的炎症性疾病包括：多发性硬化，或炎症性肠病(IBD)。
在另一优选例中，所述的低氧是按照体积1～15％，较佳地1～13％，更佳地5～10％的氧气。
在另一优选例中，所述的低氧处理的间充质干细胞或细胞培养物或培养上清是指：在按照体积比1～15％，较佳地1～13％，更佳地5～10％氧气的条件下持续培养获得的间充质干细胞或细胞培养物或培养上清。较佳地，培养2代以上，更佳地培养3代以上，如3～20代，3～10代，3～5代等。
在另一优选例中，所述的药物还用于：
升高患病组织中调节性T细胞(Treg细胞)比例；
降低患病组织中Th1和Th17细胞比例；或
抑制血清中IFN-γ，IL-17因子。
在本发明的另一方面，提供一种低氧处理的间充质干细胞或细胞培养物或培养上清，其是通过以下方法获得的：在按照体积比1～15％，较佳地1～～13％，更佳地5～10％氧气的条件下持续培养间充质干细胞。较佳地，持续培养1代以上，更佳地培养3代以上，如3～20代，3～10代，3～5代等。
在另一优选例中，按照体积比1～15％，较佳地1～～13％，更佳地5～10％氧气的条件是指：在氧浓度低的空气中培养；也即其培养环境中除了氧气含量降低且氮气含量上升以外，其它空气中应有物质的含量与常规常氧培养箱中空气含量一致。
在本发明的另一方面，提供一种用于预防、缓解或治疗炎症性疾病的药物组合物，其包括有效量的所述的低氧处理的间充质干细胞或细胞培养物或培养上清；以及药学上可接受的载体。
在本发明的另一方面，提供一种制备低氧处理的间充质干细胞或细胞培养物或培养上清的方法，所述方法包括：在按照体积比1～15％，较佳地1～13％，更佳地5～10％氧气的条件下持续培养间充质干细胞。
在本发明的另一方面，提供一种提高间充质干细胞或细胞培养物或培养上清的预防、缓解或治疗炎症性疾病功能的方法，该方法包括：对间充质干细胞进行低氧处理。
在本发明的另一方面，提供一种促进间充质干细胞分泌胰岛素样生长因子-2的方法，该方法包括：对间充质干细胞进行低氧处理。较佳地，所述的低氧是按照体积1～15％，较佳地1～13％，更佳地5～10％的氧气。
在本发明的另一方面，提供一种胰岛素样生长因子-2(IGF-2)的用途，用于制备预防、缓解或治疗炎症性疾病的药物。较佳地，所述的炎症性疾病包括：多发性硬化，或炎症性肠炎(IBD)。
在一个优选例中，所述的胰岛素样生长因子-2包括：具有或含有胰岛素样 生长因子-2第25～91位氨基酸序列的活性片段。
在一个优选例中，所述的药物还用于：
调节免疫反应；
升高患病组织中调节性T细胞比例；
降低患病组织中Th1和Th17细胞比例；或
减少炎症组织中炎症细胞的浸润。
在本发明的另一方面，提供一种用于预防、缓解或治疗炎症性疾病的药物组合物，所述的药物组合物中包括：有效量的胰岛素样生长因子-2，以及药学上可接受的载体。所述的胰岛素样生长因子-2包括：具有或含有胰岛素样生长因子-2第25～91位氨基酸序列的活性片段。
在本发明的另一方面，提供一种药用于预防、缓解或治疗炎症性疾病的药盒，其特征在于，所述的药盒中包括：所述的低氧处理的间充质干细胞或细胞培养物或培养上清；或所述的药物组合物。
本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
图1、低氧预处理增强了间充质干细胞对实验性自身反应性脑脊髓炎(EAE)中的治疗效果。
(A)应用常氧处理间充质干细胞(normoxia-msenchymal stem cells，N-MSCs)或低氧处理间充质干细胞(hypoxia-mesenchymal stem cells，H-MSCs)治疗实验性自身反应性脑脊髓炎(experimental autoimmune encephalomyelitis，EAE)。在EAE诱导后的第9、12、15天给予小鼠尾静脉输注MSCs(2×105/只/次)，每天记录小鼠的临床评分。在EAE疾病总进程的第15天，处死小鼠并获取脊髓，以Fast blue染色和苏木素-伊红染色检测对照组(PBS组)、N-MSCs或H-MSCs治疗组EAE小鼠脊髓部位的免疫细胞浸润和髓鞘脱失情况，结果表明低氧处理MSCs表现出了更好的疗效，脊髓部位炎性浸润和髓鞘脱失均明显下降；PBS为对照组，即EAE小鼠注射PBS，N-MSCs表示常氧处理间充质干细胞治疗EAE组；H-MSCs表示低氧处理间充质干细胞治疗EAE组。***p<0.001。
(B)不同处理组脊髓单个核细胞浸润情况。在EAE疾病进程第15天，处死 小鼠并取脊髓样本，以percoll分离单个核细胞，分析不同处理组小鼠脊髓部位浸润单个核细胞的数量；PBS为对照组，即EAE小鼠注射PBS，N-MSCs表示常氧处理间充质干细胞治疗EAE组；H-MSCs表示低氧处理间充质干细胞治疗EAE组。***p<0.001。
(C)不同处理组脾脏细胞体外增殖情况。在EAE疾病进程第15天，分离对照组、N-MSCs或H-MSCs治疗组EAE小鼠的脾脏细胞，以MOG35-55(20μg/ml)刺激72小时，利用3H标记的胸腺嘧啶掺入实验检测各个处理组脾脏细胞增殖情况。以MOG35-55活化脾脏细胞(MOG组)的增殖数/未经MOG35-55活化脾脏细胞(Ctrl组)的增殖数计算各组脾细胞激活指数(stimulation index，SI)，结果表明H-MSCs治疗后MOG35-55特异性T细胞增殖明显下降；MOG组为EAE诱导组；N-MSCs表示EAE小鼠经常氧处理的间充质干细胞治疗组；H-MSCs表示EAE小鼠经低氧处理的间充质干细胞治疗组。*p<0.05。
(D)不同处理组小鼠血清以及脾细胞培养上清中IL-17和IFN-γ的含量。在EAE疾病进程第15天，分离获得对照组、N-MSCs或H-MSCs治疗组EAE小鼠的血清，以ELISA方法检测IL-17和IFN-γ的表达水平。同时，分离小鼠的脾脏细胞，以MOG35-55(20μg/ml)刺激72小时后，检测培养上清中IL-17和IFN-γ的含量，结果表明，诱导EAE后小鼠血清及脾脏细胞均表达高水平的IL-17和IFN-γ，N-MSCs的注射可以部分地抑制它们的表达水平，更重要的是H-MSCs可以完全抑制IL-17和IFN-γ在血清和脾脏细胞中分泌及表达，Ctrl组为正常小鼠；MOG组为EAE诱导组；N-MSCs表示EAE小鼠经常氧处理的间充质干细胞治疗组；H-MSCs表示EAE小鼠经低氧处理的间充质干细胞治疗组。N.S.，无显著性差异；*p<0.05，**p<0.01，***p<0.001。
图2、低氧间充质干细胞的培养上清可以治疗实验性自身反应性脑脊髓炎。
(A)应用常氧处理间充质干细胞(normoxia-msenchymal stem cells，N-MSCs)或低氧处理间充质干细胞(hypoxia-mesenchymal stem cells，H-MSCs)的培养上清治疗实验性自身反应性脑脊髓炎(EAE)。MSCs分别培养于常氧和低氧环境中，待细胞密度达70％换5％FBS的培养液继续培养48小时后，收取上清，进行浓缩保留3KD以上所有蛋白，浓缩倍数为10倍。EAE治疗时，从第9天至第13天，以腹腔注射的方式每天给予每只小鼠注射200μl浓缩上清，每天记录小鼠的临床评分。在EAE疾病进程第15天，以Fast blue染色和苏木素-伊红染色检测对照组(培养基处理组)、N-sup或H-sup治疗组EAE小鼠脊髓部位的免疫细胞浸润和髓鞘脱失情况，结果发现，H-MSCs培养上清表现出了显著的治疗效果； Ctrl-sup表示5％FBS培养基治疗组，N-sup表示常氧间充质干细胞的上清治疗组；H-sup表示低氧处理的间充质干细胞的上清治疗组。**p<0.01。
(B)不同处理组脊髓单个核细胞浸润情况。在EAE疾病进程的第15天，处死小鼠并取得脊髓组织，利用percoll获取单个核细胞，并检测不同处理组小鼠脊髓部位浸润单个核细胞的数量，结果表明H-sup治疗后脊髓浸润单个核细胞数明显抑制；Ctrl-sup表示5％FBS培养基治疗组，N-sup表示常氧间充质干细胞的上清治疗组；H-sup表示低氧处理的间充质干细胞的上清治疗组。***p<0.001。
(C)不同处理组脾细胞体外增殖情况。在EAE疾病进程第15天，分离对照组(培养基处理组)、N-sup或H-sup治疗组EAE小鼠的脾脏细胞，以MOG35-55(20μg/ml)刺激72小时，利用3H标记的胸腺嘧啶掺入实验检测各个处理组脾脏细胞增殖情况。以MOG35-55活化脾脏细胞(MOG组)的增殖数/未经MOG35-55活化脾脏细胞(Ctrl组)的增殖数计算各组脾细胞激活指数(stimulation index，SI)，结果表明H-sup治疗后EAE小鼠MOG35-55特异性T细胞增殖明显下降；MOG组为EAE诱导组；N-sup表示EAE小鼠经常氧间充质干细胞的上清治疗组；H-sup表示EAE小鼠经低氧处理的间充质干细胞的上清治疗组。**p<0.01。
(D)间充质干细胞在常氧或者低氧下胰岛素样生长因子-2(insulin like growth factor-2，IGF-2)的mRNA和蛋白水平。MSCs分别培养于常氧和低氧环境中，连续培养三代后，铺6孔板，细胞密度为50％，铺板后第二天，更换新鲜培液，48小时后取细胞和上清检测其中胰岛素样生长因子-2基因水平和蛋白水平的表达情况。低氧处理MSCs无论在蛋白水平还是mRNA上均表达高水平的IGF-2；N-MSCs表示常氧处理间充质干细胞；H-MSCs表示低氧处理间充质干细胞。**p<0.01，***p<0.001。
图3、胰岛素样生长因子-2及其所含的Ala25-Glu91片段在治疗实验性自身反应性脑脊髓炎中的作用。
(A)胰岛素样生长因子-2的中和抗体可以回复低氧处理间充质干细胞(mesenchymal stem cells，MSCs)细胞培养上清对EAE的治疗效果。应用常氧处理间充质干细胞(normoxia-MSCs，N-MSCs)或低氧处理间充质干细胞(hypoxia-MSCs，H-MSCs)的培养上清治疗EAE时，给予小鼠注射人IGF2(hIGF2)的中和抗体或同型对照抗体以阻断IGF2功能，结果表明，IGF2的中和抗体可以抑制低氧处理MSCs培养上清对EAE的治疗作用。Ctrl表示5％FBS培养基治疗组，Nor-IgG表示常氧培养MSCs的培养上清治疗组，Hyp-IgG表示低氧培养MSCs的培养上清治疗组；anti-hIGF2表示胰岛素样生长因子-2中和抗体，IgG 表示胰岛素样生长因子-2中和抗体的对照抗体。*p<0.05。
(B)胰岛素样生长因子-2的Ala25-Glu91片段治疗实验性自身反应性脑脊髓炎。在EAE诱导第8天起，每天给予EAE小鼠注射人重组IGF2的Ala25-Glu91片段(5ng/只)，观察疾病进展情况。结果表明，IGF2的Ala25-Glu91片段注射可以有效抑制EAE的进展。PBS(磷酸盐缓冲液)注射组为对照组，IGF-2为胰岛素样生长因子-2的Ala25-Glu91片段治疗组。*p<0.05，**p<0.01。
(C)胰岛素样生长因子-2的Ala25-Glu91片段抑制实验性自身反应性脑脊髓炎病灶部位的单个核细胞浸润。以IGF2的Ala25-Glu91片段治疗EAE，在EAE疾病进程第15天，利用percoll分离脊髓的单个核细胞并计数，结果表明IGF2的Ala25-Glu91片段可以有效抑制EAE小鼠脊髓中单个核细胞的浸润；PBS(磷酸盐缓冲液)注射组为对照组，IGF-2为胰岛素样生长因子-2的Ala25-Glu91片段治疗组。***p<0.001。
(D)胰岛素样生长因子-2的Ala25-Glu91片段抑制抗原特异性T细胞的增殖。以IGF-2的Ala25-Glu91片段治疗EAE，在EAE疾病进程的第15天，分离小鼠脾脏细胞，以MOG35-55(20μg/ml)刺激，利用3H标记的胸腺嘧啶掺入实验检测脾脏细胞增殖情况。以MOG35-55活化脾脏细胞(MOG组)的增殖数/未经MOG35-55活化脾脏细胞(Ctrl组)的增殖数计算各组脾细胞激活指数(stimulation index，SI)，结果表明IGF-2的Ala25-Glu91片段可以有效抑制EAE小鼠中MOG特异性T细胞的增殖；PBS(磷酸盐缓冲液)注射组为对照组，IGF-2为胰岛素样生长因子-2的Ala25-Glu91片段治疗组。***p<0.001。
(E)胰岛素样生长因子-2的Ala25-Glu91片段上调调节性T细胞。以IGF-2的Ala25-Glu91片段治疗EAE，在EAE疾病进程的第15天，分离小鼠脊髓中的单个核细胞，利用流式细胞分析技术检测CD4+Foxp3+T细胞的表达情况。结果发现，在脊髓单个核细胞的CD4+的细胞中，Foxp3+的细胞比例在使用IGF-2的Ala25-Glu91片段治疗后显著上升；PBS(磷酸盐缓冲液)注射组为对照组，IGF-2为胰岛素样生长因子-2的Ala25-Glu91片段治疗组。***p<0.001。
(F)胰岛素样生长因子-2的Ala25-Glu91片段治疗后小鼠脊髓中Th1和Th17细胞比例显著下降。以IGF-2的Ala25-Glu91片段治疗EAE，在EAE疾病进程的第15天，分离小鼠脊髓中的单个核细胞，利用流式细胞分析技术检测CD4+IL-17+以及CD4+IFN-γ+细胞的表达情况。结果发现，IGF-2的Ala25-Glu91片段治疗显著抑制脊髓CD4+细胞中IFN-γ+细胞和IL-17+细胞的比例；PBS(磷酸盐缓冲液)注射组为对照组，IGF-2为胰岛素样生长因子-2的Ala25-Glu91片段治疗组。*p<0.05，**p<0.01。
图4、低氧预处理的间充质干细胞在炎症性肠炎中具有更好的治疗效果。
(A)炎症性肠炎小鼠在接受不同治疗时的存活曲线。配制3％DSS(dextran sodium sulfate，DSS)水溶液诱导小鼠的炎症性肠炎模型，在疾病模型诱导开始的第1，3，5天，通过腹腔给小鼠注射常氧处理间充质干细胞(normoxia-mesenchymal stem cells，N-MSCs)或低氧处理间充质干细胞(hypoxia-mesenchymal stem cells，H-MSCs)(1×106/只/次)，观察小鼠的存活情况。结果显示，低氧处理MSCs对炎症性肠炎的治疗效果明显好于常氧细胞治疗组和PBS处理的对照组。N-MSCs表示炎症性肠炎小鼠经常氧处理间充质干细胞治疗组；H-MSCs表示炎症性肠炎小鼠经低氧处理间充质干细胞治疗组。
(B)炎症性肠炎小鼠在接受不同治疗的体重变化。在炎症性肠炎诱导的第1，3，5天，通过腹腔给小鼠注射常氧处理间充质干细胞(normoxia-mesenchymal stem cells，N-MSCs)或低氧处理间充质干细胞(hypoxia-mesenchymal stem cells，H-MSCs)(1×106/只/次)。每天称取小鼠的体重，以第一天各组小鼠的平均体重为基线，计算其体重变化情况。结果显示，低氧处理MSCs在治疗肠炎小鼠中的效果明显好于常氧细胞治疗组和PBS处理的对照组。N-MSCs表示炎症性肠炎小鼠经常氧处理间充质干细胞治疗组；H-MSCs表示炎症性肠炎小鼠经低氧处理间充质干细胞治疗组。*p<0.05。
(C)炎症性肠炎小鼠在接受不同治疗时的临床评分。在肠炎诱导过程的第1，3，5天，通过腹腔给小鼠注射常氧处理间充质干细胞(normoxia-msenchymal stem cells，N-MSCs)或低氧处理间充质干细胞(hypoxia-mesenchymal stem cells，H-MSCs)(1×106/只/次)。每天称取小鼠的体重，并记录小鼠稀便和血便的情况，并给予相应的临床评分，计算体重时，以第1天各自组小鼠的平均体重为基线。结果显示，H-MSCs在治疗肠炎小鼠中的效果明显好于N-MSCs组和PBS处理的对照组。N-MSCs表示炎症性肠炎小鼠经常氧处理间充质干细胞治疗组；H-MSCs表示炎症性肠炎小鼠经低氧处理间充质干细胞治疗组。*p<0.05。
(D)炎症性肠炎小鼠在接受不同治疗时的结肠长度。在肠炎诱导过程的第1，3，5天，通过腹腔给小鼠注射常氧处理间充质干细胞(normoxia-mesenchymal stem cells，N-MSCs)或低氧处理间充质干细胞(hypoxia-mesenchymal stem cells，H-MSCs)(1×106/只/次)。在疾病进程的第8天，处死小鼠并取得结肠组织，量取结肠组织的长度。结果显示，H-MSCs在治疗肠炎小鼠中的效果明显好于N-MSCs组和PBS处理的对照组，Ctrl组为正常小鼠。N-MSCs表示炎症性肠炎小鼠经常氧处理间充质干细胞治疗组；H-MSCs表示炎症性肠炎小鼠经低氧处理间充质干 细胞治疗组。*p<0.05。
图5、胰岛素样生长因子-2的Ala25-Glu91片段在治疗炎症性肠炎中的作用。
(A)配制含有3％葡聚糖硫酸钠(dextran sodium sulfate，DSS)的水溶液诱导炎症性肠炎，从第1天起每天腹腔注射胰岛素样生长因子-2的Ala25-Glu91片段(50ng/只)治疗炎症性肠炎，每天称取小鼠体重。以各自组小鼠第一天的平均体重为基数，结果表明使用IGF-2的Ala25-Glu91片段治疗后小鼠体重降低得到缓解；PBS(磷酸盐缓冲液)组为对照组，IGF-2为胰岛素样生长因子-2的Ala25-Glu91片段治疗组。***p<0.001。
(B)使用胰岛素样生长因子-2的Ala25-Glu91片段治疗后第8天，处死小鼠并取得其结肠，量取结肠长度，结果表明，使用IGF-2的Ala25-Glu91片段治疗后，肠炎小鼠结肠长度的缩减得到缓解；利用percoll分离结肠组织中的单个核细胞并进行计数，结果显示使用IGF-2的Ala25-Glu91片段治疗后，肠炎小鼠结肠中的单个核细胞浸润数下降；PBS(磷酸盐缓冲液)组为对照组，IGF-2为胰岛素样生长因子-2的Ala25-Glu91片段治疗组。***p<0.001。
(C)胰岛素样生长因子-2的Ala25-Glu91片段治疗后第8天，处死小鼠并取得其肠系膜淋巴结和结肠组织，对于结肠组织，利用percoll分离其中的单个核细胞，并利用流式细胞术分析其中Treg，Th1和Th17的细胞比例。结果表明，使用IGF-2的Ala25-Glu91片段治疗后，小鼠结肠(LP)和肠系膜淋巴结(MLN)中，在CD4+的细胞中，Treg(Foxp3+)细胞比例显著上升，Th1(IFN-γ+)和Th17(IL-17+)细胞比例显著下降。PBS(磷酸盐缓冲液)组为对照组，IGF-2为胰岛素样生长因子-2的Ala25-Glu91片段治疗组。*p<0.05，**p<0.01。
图6、干扰胰岛素样生长因子-2在MSCs中的表达对EAE的影响。利用sh-RNA770、sh-RNA1526干扰胰岛素样生长因子-2(insulin like growth factor-2，IGF-2)在MSCs中的表达水平，MSCs经常氧培养或低氧培养3代以上后，在EAE诱导第8天给予小鼠尾静脉注射(2*105/只)。与对照组sh-RNA处理MSCs相比较，干扰IGF-2在MSCs上的表达可以破坏低氧处理MSCs对EAE的治疗作用。*p<0.05。
具体实施方式
本发明人经过深入的研究，首次揭示了低氧处理间充质干细胞后，能够显著地促进间充质干细胞对于炎症性疾病的缓解或治疗作用。本发明还揭示了低氧处 理间充质干细胞能够产生胰岛素样生长因子-2，该因子在低氧处理的间充质干细胞治疗炎症性疾病中起到的核心作用。
低氧处理的间充质干细胞及其用途及药物组合物
本发明人选用了实验性自身反应性脑脊髓炎(EAE)这一动物模型(为多发性硬化的动物模型)，在细胞治疗过程中，常氧下培养的间充质干细胞(人脐带来源)表现出了一定的治疗效果，而经过低氧预处理之后，间充质干细胞的疗效得到了进一步的增强。
基于本发明人的新发现，本发明提供了一种MSCs，其是低氧处理的MSCs，其制备方法简单，无需转基因操作，不涉及外源基因的插入，给药时不会存在安全性问题。
作为本发明的优选方式，所述的低氧处理是指一种低氧环境，该环境中氧气为按照体积1～15％；较佳地1～13％；更佳地5～10％。较佳地，也即其培养环境中除了氧气含量降低且氮气含量上升以外，其它空气中应有的元素的含量与常规常氧培养箱中空气含量一致。
基于本发明的新发现，还提供了一种低氧处理的MSCs的用途，用于制备预防、缓解或治疗炎症性疾病的药物。所述的炎症性疾病例如包括：实验性自身反应性脑脊髓炎(EAE)或炎症性肠炎(IBD)。其还用于：升高患病组织中调节性T细胞(Treg细胞)比例；降低患病组织中Th1和Th17细胞比例；或抑制血清中IFN-γ，IL-17因子等。
本发明还提供了一种组合物(药物)，它含有有效量(如0.000001-50wt％；较佳的0.00001-20wt％；更佳的，0.0001-10wt％)的所述的低氧处理的MSCs，以及药学上可接受的载体。
如本文所用，术语“含有”表示各种成分可一起应用于本发明的混合物或组合物中。因此，术语“主要由...组成”和“由...组成”包含在术语“含有”中。
如本文所用，术语“有效量”或“有效剂量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的如本文所用。
如本文所用，“药学上可接受的”的成分是适用于人和/或哺乳动物而无过度不良副反应(如毒性、刺激和变态反应)的，即具有合理的效益/风险比的物质。术语“药学上可接受的载体”指用于治疗剂给药的载体，包括各种赋形剂和稀释剂。
通常，可将所述细胞配制于无毒的、惰性的和药学上可接受的水性载体介质 中，其中pH通常约为5-8，较佳地，pH约为6-8。
胰岛素样生长因子-2(IGF-2)在间充质干细胞治疗中的作用
为了验证间充质干细胞是不是通过其分泌的因子发挥作用，本发明人将间充质干细胞的上清用于实验性自身反应性脑脊髓炎的治疗，结果显示只有低氧细胞的上清可以发挥治疗效果，这就说明低氧预处理的间充质干细胞是通过其分泌的因子发挥了疾病治疗的功能。为了进一步确定是哪种或哪几种因子在低氧预处理的间充质干细胞治疗过程中发挥作用，本发明人检测了常氧和低氧培养下间充质干细胞因子表达的差异，结果显示胰岛素样生长因子-2的表达量在低氧处理后有了明显的提高。
胰岛素样生长因子-2是一种主要由肝脏分泌并在血液中大量存在的生长因子，它具有抗凋亡、生长调节、类胰岛素和促有丝分裂等功能。普遍认为胰岛素样生长因子-2在胚胎发育中具有重要的作用，可以促进胚胎发育和器官形成；也有报道称其与记忆和生殖有关，通过对基因缺陷小鼠的研究发现，胰岛素样生长因子-2的信号缺失会造成脑发育不健全。但是，至今尚无将胰岛素样生长因子-2与炎症性疾病的治疗相关联的报道。
为了验证胰岛素样生长因子-2在间充质干细胞治疗实验性自身反应性脑脊髓炎过程中是否发挥作用，本发明人使用了中和抗体中和掉低氧上清中的胰岛素样生长因子-2，中和后的上清在治疗疾病时疗效消失了。之后，本发明人尝试直接使用胰岛素样生长因子-2的Ala25-Glu91片段治疗实验性自身反应性脑脊髓炎，实验结果显示，胰岛素样生长因子-2的Ala25-Glu91片段的使用可以有效的治疗实验性自身反应性脑脊髓炎。在炎症部位，即脊髓中，本发明人发现调节性T细胞(Tregs)的比例有了显著提高，与此同时，Th1和Th17细胞的比例显著下降。
可见，相比于常氧培养下的间充质干细胞，低氧预处理可以有效的提高其治疗实验性自身反应性脑脊髓炎的效果，这种治疗效果的提高是依赖于胰岛素样生长因子-2表达的上升实现的，单独使用胰岛素样生长因子-2的Ala25-Glu91片段也可以实现对实验性自身反应性脑脊髓炎的良好治疗效果。
因此，本发明还提供了胰岛素样生长因子-2及其Ala25-Glu91片段的用途，用于制备预防、缓解或治疗炎症性疾病的组合物(药物)。
所述的胰岛素样生长因子-2包括全长的胰岛素样生长因子-2或其生物活性片段。优选的，所述的胰岛素样生长因子-2全长的氨基酸序列可以与SEQ ID NO:8(NCBI protein database编号P01344.1)所示的序列基本上相同。
经过一个或多个氨基酸残基的取代、缺失或添加而形成的胰岛素样生长因子-2的氨基酸序列也包括在本发明中。胰岛素样生长因子-2或其生物活性片段包括一部分保守氨基酸的替代序列，所述经氨基酸替换的序列并不影响其活性或保留了其部分的活性。适当替换氨基酸是本领域公知的技术，所述技术可以很容易地被实施并且确保不改变所得分子的生物活性。这些技术使本领域人员认识到，一般来说，在一种多肽的非必要区域改变单个氨基酸基本上不会改变生物活性。见Watson等，Molecular Biology of The Gene，第四版，1987，The Benjamin/Cummings Pub.Co.P224。
任何一种胰岛素样生长因子-2及含有其第25-91位氨基酸序列的生物活性片段都可以应用到本发明中。在这里，胰岛素样生长因子-2的生物活性片段的含义是指作为一种多肽，其仍然能保持全长的胰岛素样生长因子-2的全部或部分功能。通常情况下，所述的生物活性片段至少保持50％的全长胰岛素样生长因子-2的活性。在更优选的条件下，所述活性片段能够保持全长胰岛素样生长因子-2的60％、70％、80％、90％、95％、99％、或100％的活性。
本发明也可采用经修饰或改良的胰岛素样生长因子-2或以其第25位至91位氨基酸序列为基础的活性片段，比如，可采用为了促进其半衰期、有效性、代谢、和/或蛋白的效力而加以修饰或改良的炎症性疾病。
本发明还提供了一种组合物(药物)，它含有有效量(如0.000001-50wt％；较佳的0.00001-20wt％；更佳的，0.0001-10wt％)的所述的胰岛素样生长因子-2，以及药学上可接受的载体。
在本领域对低氧处理的间充质干细胞在免疫相关疾病治疗时的表现还缺乏认识的情况下，本发明首次揭示低氧处理的间充质干细胞对于炎症性疾病的治疗效果比常氧下培养的间充质干细胞更为理想。并且还发现，单独使用胰岛素样生长因子-2也可以实现对实验性自身反应性脑脊髓炎的良好治疗效果。本发明人相信，上述发现为阐述间充质干细胞治疗自体免疫性脊髓灰质炎等免疫相关疾病的机制提供的新的思路，也为进一步认识胰岛素样生长因子-2在免疫相关疾病治疗中的价值提供了新的信息。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
材料和方法
除非另外说明，本发明所用的试剂和实验材料均可从市售获得，其中生物材料来源如下：
重组人源的IGF-2的Ala25-Glu91片段，IGF-2中和抗体，对照IgG抗体购自R&D Systems。髓鞘少突胶质细胞糖蛋白(MOG35-55)购自吉尔生化有限公司(上海，中国)。不完全弗氏佐剂(IFA)，结核分支杆菌(TB)购自Sigma-Aldrich(MO，USA)。百日咳毒素(pertusis toxin，PT)购自List Biological Laboratories，USA。葡聚糖硫酸钠(dextran sodium sulfate，DSS)购自MP Biomedicals。人IGF-2 ELISA试剂盒购自(Mediagnost，德国)，小鼠IFNγ，IL-17 ELISA鉴定试剂购自R&D Systems。流式细胞术抗体anti-mouse CD4 PerCP-Cy5.5，anti-mouse CD4 PerCP-Cy5.5，anti-mouse IL-17A PE，anti-mouse IL-17A PE购自Ebioscience。
C57BL/6小鼠购自上海斯莱克实验动物有限公司，饲养于上海交通大学医学院实验动物科学部。
1、细胞制备方法
人脐带来源间充质干细胞的分离培养：抽离脐带中的血管后，将剩余脐带组织切成碎块并置于培养皿中，让MSCs自然爬出，每两天换液一次。待培养皿中细胞达到一定密度后，进行消化传代，用于后续实验。
人间充质干细胞的低氧处理方法：将间充质干细胞置于低氧培养箱中培养，除培养箱的改变外，其余所用试剂均与常氧培养时保持一致。低氧条件：按照体积10％O2。
构建IGF-2表达水平敲低的细胞：利用慢病毒感染的方法进行稳定敲除，使用的对照序列为：Ctrl-sh-RNA(5’-ttctccgaacgtgtcacgt-3’(SEQ ID NO:1))；IGF2的序列分别为：sh-RNA-770(5’-gaagtcgatgctggtgcttct-3’(SEQ ID NO:2))；sh-RNA-1526(5’-gctttaaacacccttcacata-3’(SEQ ID NO:3))。慢病毒据带有GFP-标记基因和puromycin筛选抗性基因，感染后细胞呈现绿色荧光。基因敲出实验中，待细胞密度达到60％时加入病毒，24小时后换正常培液，镜下观察并根据荧光比例推测感染效率，稳定24小时后加入puromycin，等待细胞长满后传代用于后续实验。
2、实验性自身免疫性脑脊髓炎(EAE)模型的建立及治疗
(1)实验前准备过程
制备完全弗氏佐剂(CFA)：将热灭活的结核分支杆菌加入不完全弗氏佐剂中直至终浓度达到5mg/ml，使用前充分颠倒混匀。
抗原的乳化：将两支玻璃针管通过三通管连接，将等体积的抗原溶液(300μgMOG于100μl PBS中)和100μl完全弗氏佐剂加入针管中，排除针管内气泡并来回推动获得乳化液。大约需要推动约500次，推动阻力会逐渐增加，此时各成分可充分混匀成乳化状态。
百日咳毒素(PT)溶液配制：将百日咳毒素溶解于PBS至工作浓度1ng/μl。
(2)实验性自身反应性脑脊髓炎(EAE)模型的建立和治疗
第0天，将200μl乳化好的抗原皮下注射于C57BL/6小鼠背部进行抗原免疫，胸部对应的背部两侧每侧100μl；同时通过尾静脉注射PT，200μl/只。
第2天，再次通过尾静脉注射PT，200μl/只。
第8天，第11天和第14天，尾静脉注射200μl 2×105个常氧培养MSCs；或者同等数目低氧培养MSCs；200μl生理盐水尾静脉注射组作为对照。
使用细胞上清治疗时，上清注射时间为第9天至第13天。
使用胰岛素样生长因子2的Ala25-Glu91片段治疗时则从实验诱导后第8天每天注射，剂量为5ng/只，腹腔给药。
低氧处理MSCs制备：在按照体积比10％O2的条件下持续培养3代以上。10％O2环境的建立：使用FormaTM Series II 3110 Water-Jacketed CO2Incubator，通过氮气含量的调节实现低氧环境的建立。
(3)EAE临床评分标准
0分：无异常表现；
0.5分：尾尖下垂；
1分：整条尾巴瘫痪；
2分：后肢无力；
3分：一后肢瘫痪；
4分：双后肢瘫痪；
5分：死亡。
3、炎症性肠炎IBD模型的建立和治疗
葡聚糖硫酸钠(DSS)按照质量体积比2.5:100配成溶液，使用22μm的筛网过滤以保证无菌，用封口膜封口备用。
选用8-10周的C57BL/6雌鼠，用配好的DSS溶液替换饮用水诱导模型，每天称取小鼠体重并观察粪便情况，DSS溶液每隔1天换液或补加。
常氧或低氧的MSCs分别在第1天，第3天，第5天腹腔注射，每次每只小鼠注射2×106个细胞。
胰岛素样生长因子-2的Ala25-Glu91片段治疗时则为每只小鼠50ng，每天腹腔注射给药。
4、脾细胞体外增殖实验
处死EAE小鼠后，取出小鼠的脾并获取单细胞悬液，按照每孔3-5×105个细胞的数量将脾细胞铺在U型底的96孔板中，加MOG35-55至20μg/ml。
37℃温箱中培养72小时后加入3H标记的胸腺嘧啶，6小时之后将孔板经反复冻融后上机通过真空吸附到专用滤膜上，Wallac MicroBeta液闪计数仪上读取cpm值，检测3H的掺入量。
5、实时荧光定量PCR(Real time PCR)检测基因表达
参照TIANGEN细胞RNA抽提试剂盒说明书抽提RNA。参考TIANGEN TIANScript cDNA第一链生成试剂盒说明将RNA反转为cDNA。采用TAKARA荧光定量PCR试剂，参考其说明书进行荧光定量PCR。以β-actin作为内参，结果分析处理采用2-△△CT法，所有基因表达都换算为β-actin的倍数。所用引物序列如下：
人IGF-2：
正向引物：5’-CTTGGACTTTGAGTCAAATTGG-3’(SEQ ID NO:4)；
反向引物：5’-GGTCGTGCCAATTACATTTCA-3’(SEQ ID NO:5)；
β-actin：
正向引物：5’-TTGCCGACAGGATGCAGAAGGA-3’(SEQ ID NO:6)；
反向引物：5’-AGGTGGACAGCGAGGCCAGGAT-3’(SEQ ID NO:7)。
6、细胞内细胞因子或Foxp3的免疫荧光染色
(1)所有细胞(约5～10×105)用PBS洗一遍后，都用抗小鼠CD16/CD32抗体封闭10分钟，冰上，每管30μl体系。
(2)先进行细胞表面分子的染色，取细胞1×106重悬于100μl FACS缓冲液中用于染色。加入荧光标记的抗体后，4℃避光孵育30分钟。
(3)FACS缓冲液洗一遍后，每管各加入Fixation/Permeabilization buffer 100μl，放4℃固定过夜。
(4)400×g，离心5min，加入1×Permeabilization buffer 200μl，混匀。
(5)400×g，离心5min，加入相应的细胞因子抗体或者Foxp3抗体(抗体用1×Permeabilization buffer稀释)，每管50μl体系，放冰上避光染色1小时。
(6)直接加入1×Permeabilization buffer 200μl，混匀，400×g，离心5min。
(7)弃上清后，再加入1×Permeabilization buffer 200μl，混匀，400×g离心5min。
(8)弃上清后，再加入FACS buffer 200μl，混匀，400×g，离心5min。细胞沉淀用FACS buffer 200μl重悬，即可用流式细胞仪检测分析。
7、数据处理和统计学分析
所示实验结果中每个处理组都设置有3个或3个以上的重复，通过Graphpad Prism 5软件作图，图中所示数据以平均数±标准差表示。数据的统计分析采用Student’s t检验分析。显著性水准定为α＝0.05，*代表p<0.05；**代表p<0.01；***代表p<0.001。
实施例1、低氧预处理增强了间充质干细胞对实验性自身反应性脑脊髓炎(EAE)的治疗效果
为了验证低氧预处理是否可以影响间充质干细胞(mesenchymal stem cells，MSCs)对EAE的治疗效果，本发明人将MSCs培养在10％O2的条件下持续培养3代以上，然后将处理好的细胞用于EAE治疗(疾病诱导后第9天、第12天、第15天注射细胞)。
本发明人发现，低氧处理显著增强MSCs对EAE的治疗效果。脊髓部位病理分析显示，低氧MSCs治疗组小鼠脊髓部位的脱髓鞘和免疫细胞浸润均明显抑制，同时脊髓部位单个核细胞浸润数量也显著减少，如图1A～B。
体外脾细胞增殖实验显示，低氧处理MSCs治疗EAE后，MOG特异T细胞的增殖反应显著下降，如图1C。
对小鼠血清的检测发现，低氧处理MSCs治疗EAE后，小鼠血清中IFN-γ，IL-17等炎症因子被显著抑制，如图1D。
综合而言，MSCs在治疗EAE时有一定的效果，而低氧预处理可以很显著地增强这一效果，各项疾病指标都得到了明显缓解。
实施例2、低氧间充质干细胞的培养上清有效治疗实验性自身反应性脑脊髓炎(EAE)
为了确认是不是间充质干细胞(mesenchymal stem cells，MSCs)分泌的因子在EAE治疗中发挥关键作用，本发明人直接使用常氧MSCs或低氧处理MSCs的培养上清用于治疗EAE(注射时间为模型诱导后第9天至第13天，方式为腹腔)。
结果显示，在使用上清进行治疗时，常氧MSCs上清没有表现出治疗效果，而低氧MSCs的上清呈现显著的治疗效果，如图2A。
与直接使用低氧处理MSCs类似，低氧MSCs的上清可以显著抑制小鼠脊髓中的脱髓鞘和免疫细胞浸润，如图2A～B，并且明显抑制脾脏中MOG特异性T细胞增殖，如图2C。
这些结果说明，低氧MSCs是通过分泌因子实现对EAE的治疗作用。
因此，本发明人使用基因芯片对常氧和低氧培养的MSCs的表达谱进行分析，研究发现，与常氧MSCs相比较，低氧MSCs中胰岛素样生长因子-2(insulin growth factor-2，IGF-2)的表达量明显升高，并利用Real time-PCR和ELISA对其mRNA和蛋白水平进行验证，如图2D。
实施例3、胰岛素样生长因子-2在低氧培养的间充质干细胞治疗实验性自身反应性脑脊髓炎(EAE)过程中发挥关键作用
为了验证胰岛素样生长因子-2(Insulin Like growth Factor-2，IGF-2)在间充质干细胞(Mesenchymal Stem Cells，MSCs)治疗EAE过程中的关键作用，本发明人使用了IGF-2的中和抗体(购自R&D)阻断MSCs分泌上清中IGF-2的功能。在应用低氧处理MSCs上清治疗EAE时，给予小鼠腹腔注射中和抗体5ng/天/只后，低氧上清的治疗效果消失了，如图3A。此外，本发明人利用sh-RNA770(购自吉玛基因)、sh-RNA1526(购自吉玛基因)干扰IGF-2在MSCs中的表达水平，如图6，与对照组sh-RNA处理MSCs相比较，干扰IGF-2在MSCs上的表达可以破坏低氧处理MSCs对EAE的治疗作用。这就说明，IGF-2是低氧MSCs发挥治疗EAE的关键因子。
为了进一步确定IGF-2的作用，本发明人直接使用购自R&D的IGF-2的Ala25-Glu91片段治疗EAE(自模型诱导后第8天起每天给药，5ng/只)。结果发现，IGF-2的Ala25-Glu91片段注射后可以有效抑制EAE，主要表现在小鼠临床分数明显改善，如图3B；脊髓中单个核细胞浸润数目明显下降，如图3C；MOG特异性T细胞增殖明显下降，如图3D。
值得注意的是：IGF-2的Ala25-Glu91片段治疗EAE后，小鼠脊髓中 CD4+Foxp3+调节性T细胞比例明显上升，如图3E；而Th1和Th17细胞比例显著下降，如图3F。这也表明IGF-2的Ala25-Glu91片段具有显著的免疫调节作用，从而起到治疗EAE的作用。
实施例4、低氧预处理的间充质干细胞和胰岛素样生长因子-2Ala25-Glu91片段均可用于炎症性肠炎的治疗
类似于治疗实验性自身反应性脑脊髓炎(EAE)的过程，低氧处理的间充质干细胞(mesenchymal stem cells，MSCs)同样在治疗炎症性肠炎(inflammatory bowel disease，IBD)的过程中表现出显著的治疗效果。研究发现，低氧处理MSCs可以显著提高IBD小鼠存活，如图4A；改善IBD诱导过程中小鼠体重的显著下降，如图4B；改善IBD小鼠的临床评分，如图4C；通过计算小鼠结肠的长度，低氧处理MSCs可以显著抑制IBD小鼠肠道的损伤，如图4D；
进一步实验表明，胰岛素样生长因子-2(insulin like growth factor-2，IGF-2)的Ala25-Glu91片段在治疗IBD中的作用，单纯IGF-2的Ala25-Glu91片段的使用就可以使小鼠的体重降低得到显著缓解，如图5A；其结肠的长度减低，以及结肠中炎症细胞的浸润得到了有效抑制，如图5B。本发明人也分析了其疾病部位的淋巴细胞，包括肠系膜淋巴结和肠浸润细胞的成分，本发明人发现，和治疗EAE一样，IGF-2的Ala25-Glu91片段治疗组小鼠中，CD4+Foxp3+Tregs比例明显上升，而Th1和Th17细胞比例显著下降，如图5C。这些结果说明，IGF-2的Ala25-Glu91片段可以通过调节免疫发挥治疗特定疾病的作用。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (17)

  1. 低氧处理的间充质干细胞或细胞培养物或培养上清在制备预防、缓解或治疗炎症性疾病的药物中的用途。
  2. 如权利要求1所述的用途，其特征在于，所述的炎症性疾病包括：多发性硬化，或炎症性肠病。
  3. 如权利要求1所述的用途，其特征在于，所述的低氧是按照体积1～15％的氧气。
  4. 如权利要求1所述的用途，其特征在于，所述的药物还用于：
    升高患病组织中调节性T细胞比例；
    降低患病组织中Th1和Th17细胞比例；或
    抑制血清中IFN-γ，IL-17因子。
  5. 一种低氧处理的间充质干细胞或细胞培养物或培养上清，其特征在于，其是通过以下方法获得的：在按照体积比1～15％氧气的条件下持续培养间充质干细胞。
  6. 如权利要求5所述的低氧处理的间充质干细胞或细胞培养物或培养上清，其特征在于，持续培养1代以上，更佳地培养3代以上。
  7. 一种用于预防、缓解或治疗炎症性疾病的药物组合物，其特征在于，其包括有效量的权利要求5或6所述的低氧处理的间充质干细胞或细胞培养物或培养上清；以及药学上可接受的载体。
  8. 一种制备低氧处理的间充质干细胞或细胞培养物或培养上清的方法，其特征在于，所述方法包括：在按照体积比1～15％氧气的条件下持续培养间充质干细胞。
  9. 一种提高间充质干细胞或细胞培养物或培养上清的预防、缓解或治疗炎症性疾病功能的方法，其特征在于，该方法包括：对间充质干细胞进行低氧处理。
  10. 一种促进间充质干细胞分泌胰岛素样生长因子-2的方法，其特征在于，该方法包括：对间充质干细胞进行低氧处理。
  11. 如权利要求9或10所述的方法，其特征在于，所述的低氧是按照体积1～15％的氧气。
  12. 一种胰岛素样生长因子-2的用途，用于制备预防、缓解或治疗炎症性疾病的药物。
  13. 如权利要求12所述的用途，其特征在于，所述的胰岛素样生长因子-2包括：包含胰岛素样生长因子-2第25～91位氨基酸序列的活性片段。
  14. 如权利要求12所述的用途，其特征在于，所述的炎症性疾病包括：多发性硬化，或炎症性肠炎。
  15. 如权利要求12所述的用途，其特征在于，所述的药物还用于：
    调节免疫反应；
    升高患病组织中调节性T细胞比例；
    降低患病组织中Th1和Th17细胞比例；或
    减少炎症组织中炎症细胞的浸润。
  16. 一种用于预防、缓解或治疗炎症性疾病的药物组合物，其特征在于，所述的药物组合物中包括：有效量的胰岛素样生长因子-2，以及药学上可接受的载体。
  17. 一种用于预防、缓解或治疗炎症性疾病的药盒，其特征在于，所述的药盒中包括：权利要求5～6任一所述的低氧处理的间充质干细胞或细胞培养物或培养上清；或权利要求7所述的药物组合物；或权利要求16所述的药物组合物。
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