WO2017012226A1 - 间质干细胞、其克隆源性扩增的方法、其分离方法及其应用 - Google Patents

间质干细胞、其克隆源性扩增的方法、其分离方法及其应用 Download PDF

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WO2017012226A1
WO2017012226A1 PCT/CN2015/094825 CN2015094825W WO2017012226A1 WO 2017012226 A1 WO2017012226 A1 WO 2017012226A1 CN 2015094825 W CN2015094825 W CN 2015094825W WO 2017012226 A1 WO2017012226 A1 WO 2017012226A1
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mesenchymal stem
stem cells
igf1r
cells
cell
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PCT/CN2015/094825
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English (en)
French (fr)
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徐伟成
林振寰
李玮
谢佳宏
许重义
蔡长海
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中国医药大学
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Priority to EP15898782.6A priority Critical patent/EP3321356B1/en
Priority to ES15898782T priority patent/ES2900819T3/es
Priority to JP2017567682A priority patent/JP6766082B2/ja
Publication of WO2017012226A1 publication Critical patent/WO2017012226A1/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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • 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
    • C12N5/0018—Culture media for cell or tissue culture

Definitions

  • the present invention relates to a stem cell, and in particular to a mesenchymal stem cell which expresses a specific receptor.
  • Stem Cell is a non-differentiated primary cell in an organism that has the ability to continuously replicate, renew, and differentiate into mature cells with specific patterns and functions.
  • Stem cells can be mainly divided into embryonic stem cells (ESCs) and adult stem cells.
  • the embryonic stem cells are taken from the inner cell mass in the blastocyst, and the adult stem cells are derived from various tissues. .
  • Stem cells can be divided into three major categories according to their differentiation ability. One is totipotent stem cells.
  • the pluripotent stem cells have complete ability to differentiate into intact embryos or organisms.
  • the other is pluripotent stem cells.
  • pluripotent stem cells have the ability to differentiate into three germ layers, but cannot develop into complete embryos or organisms, but can form all cells of a certain tissue or organ; three are multipotent stem cells. Including stem cells of specific tissues, such as neural stem cells, blood stem cells, liver stem cells, skin stem cells, and the like.
  • MSCs Mesenchymal stem cells
  • MSCs Mesenchymal stem cells
  • a variety of interstitial tissues such as cells or bone cells.
  • Mesenchymal stem cells can be obtained from interstitial tissues such as bone marrow, fat, pulp or umbilical cord. Depending on the source, there is a tendency to differentiate into specific tissues. When tissues in the body are damaged, mesenchymal stem cells can Repair directly or indirectly.
  • Mesenchymal stem cells can be applied to the repair of nerve, heart, liver, lung, kidney, bone, cartilage, and retinal injuries.
  • mesenchymal stem cells have been found to have immunomodulatory functions, which may help treat many immune abnormal diseases. Because the antigenicity of mesenchymal stem cells is smaller than that of other stem cells, it is not a good source of cell therapy.
  • hematopoietic stem cells must be strictly matched before transplantation, and there is no ethical consideration when embryonic stem cells are used. .
  • the ability of self-renewal and pluripotent differentiation of cytoplasmic stem cells is crucial. Therefore, cell surface receptors related to the maintenance of pluripotency of mesenchymal stem cells have become one of the main topics in the development of stem cell medical related technologies.
  • an isolated mesenchymal stem cell expressing an insulin-like growth factor 1 receptor is provided.
  • IGF1R insulin-like growth factor 1 receptor
  • the isolated mesenchymal stem cells are pluripotent.
  • the isolated mesenchymal stem cells may be human cells, and more preferably umbilical cord mesenchymal stem cells.
  • the isolated mesenchymal stem cells of the present invention express the insulin-like growth factor-1 receptor on the cell surface, which has the characteristics of self-renewal and pluripotent differentiation.
  • Another embodiment of the present invention provides a method for cloning a mesenchymal stem cell clone comprising culturing the aforementioned isolated mesenchymal stem cells in a medium containing human umbilical cord blood serum, wherein Isolated mesenchymal stem cells express the insulin-like growth factor-1 receptor.
  • the method according to the aforementioned isolated mesenchymal stem cell clone-derived amplification, wherein the concentration of the human cord blood serum of the medium may be 1 to 10% (v/v), preferably 2% (v/v). .
  • the culture medium uses the human umbilical cord blood serum as a raw material, and the cord blood serum is rich in growth factors, and the mesenchymal stem cells can express the insulin-like growth factor in a large amount. 1 receptor, and maintain pluripotent differentiation ability, and the use of human cord blood serum culture medium is convenient to obtain, and can avoid allergic reactions caused by the use of non-human serum and the risk of infection with viruses or pathogens.
  • a further embodiment of the invention provides a method of isolating pluripotent mesenchymal stem cells comprising providing a mixture of cells from a mammalian tissue for performing a separation step.
  • the separation step is to isolate cells positive for the insulin-like growth factor-1 receptor from the cell mixture to obtain pluripotent mesenchymal stem cells.
  • the separating step further comprises isolating cells positive for interleukin 22 receptor alpha 1, IL22RA1.
  • the mammalian tissue is selected from the group consisting of bone marrow, pulp, placenta, umbilical cord, cord blood, and fat.
  • the method for isolating pluripotent mesenchymal stem cells of the present invention can screen cells positive for IGF1R from a mixture of cells derived from mammalian tissue, and more preferably, cells which are positive for IL22RA1 can be screened.
  • the mesenchymal stem cells with pluripotent differentiation ability can be used to purify pluripotent mesenchymal stem cells quickly and exclusively.
  • Yet another embodiment of the present invention is the use of the aforementioned isolated mesenchymal stem cells for the preparation of a medicament for treating ischemic heart disease.
  • the ischemic heart disease is a myocardial infarction.
  • the drug for ischemic heart disease may be a drug for reducing fibrosis caused by myocardial infarction or a drug for reducing an immune response.
  • the isolated mesenchymal stem cells of the present invention are used for cell therapy, the dysfunction of the left ventricle after myocardial infarction can be reduced, the infarct size can be reduced, the fibrosis caused by myocardial infarction can be reduced, and the immune response can be reduced. It can be used to treat ischemic heart disease in an individual.
  • Yet another embodiment of the present invention is the use of the aforementioned isolated mesenchymal stem cells for the preparation of a medicament for treating brain tissue damage in an individual.
  • brain tissue damage is caused by a cerebral ischemic disease, such as a stroke.
  • brain tissue damage is caused by a neurodegenerative disease, such as Parkinson's disease.
  • the drug for treating brain tissue damage in an individual may be a drug that increases glucose metabolic activity, a drug that promotes angiogenesis, or a drug that promotes neurite regeneration.
  • the isolated mesenchymal stem cells of the present invention can be used for cell therapy, which can increase the metabolic activity of glucose, promote angiogenesis, and enhance neurite regeneration, and can be used to treat individuals with brain tissue damage.
  • Figure 1A is a photomicrograph of the primary culture of umbilical cord mesenchymal stem cells
  • IGF1R insulin-like growth factor 1 receptor
  • Figure 1C is a diagram showing the results of expression of interleukin 22 receptor alpha 1, IL22RA1 in isolated mesenchymal stem cells of the present invention
  • FIG. 1D and FIG. 1E are diagrams showing the results of specific cell surface molecule expression of cells in the Wharton's gel of the isolated mesenchymal stem cells of the present invention.
  • Figure 1F is a graph showing the results of pluripotency marker expression of isolated mesenchymal stem cells of the present invention.
  • Figure 2 is a graph showing the amplification index of the isolated mesenchymal stem cells of the present invention.
  • 3A is a human cytokine wafer analysis diagram of human cord blood serum (hUCS) and fetal calf serum (FCS);
  • Figure 3B is an ELISA analysis diagram of hUCS and FCS
  • 4A is a graph showing the expression level of IGF1R after the introduction of shRNA by isolated mesenchymal stem cells of the present invention.
  • Figure 4B is a graph showing the amplification trend of the isolated mesenchymal stem cells of the present invention.
  • Figure 4C is a diagram showing the cell proliferation analysis of the isolated mesenchymal stem cells of the present invention.
  • 5A is a flow cytometry double staining analysis diagram of the isolated mesenchymal stem cells of the present invention.
  • 5B is a photomicrograph of the immunofluorescence staining double staining method of the isolated mesenchymal stem cells of the present invention.
  • Figure 5C is a graph showing the results of quantitative RT-PCR of mesenchymal stem cell expression pluripotency markers
  • Figure 6A is a photomicrograph of the differentiated mesenchymal stem cells of the present invention differentiated into different tissue cells
  • Figure 6B is a photomicrograph showing the differentiation of isolated mesenchymal stem cells into neural cells of the present invention.
  • Figure 7A is a graph showing the expression levels of IGF1R and chemokine CXCR4 after treatment of different doses of insulin-like growth factor-1 (IGF1) to the isolated mesenchymal stem cells of the present invention
  • Figure 7B is a graph showing the expression levels of IGF1R and chemokine CXCR4 after treatment of different doses of platelet-derived growth factor-BB (PDGF-BB) to the isolated mesenchymal stem cells of the present invention. ;
  • PDGF-BB platelet-derived growth factor-BB
  • Figure 7C is a graph showing the analysis of the amount of IGF1R expression after simultaneous treatment of different doses of IGF1 and PDGF-BB to the isolated mesenchymal stem cells of the present invention
  • Figure 7D shows phosphorylation of protein kinase B (p-Akt) and phosphorylation signaling transcriptional activation gene-3 (p-Stat3) after simultaneous treatment of different doses of IGF1 and PDGF-BB to isolated mesenchymal stem cells of the invention.
  • p-Akt protein kinase B
  • p-Stat3 phosphorylation signaling transcriptional activation gene-3
  • Figure 8A is a graph showing the results of a rat body swing test
  • Figure 8B is a graph showing the results of a rat body swing test of the blocking test.
  • Figure 9A is a graph showing the results of a rat vertical activity test
  • Figure 9B is a graph showing the results of a rat vertical activity time test
  • Figure 9C is a graph showing the results of the rat vertical motion quantity test
  • Figure 10 is a graph showing the results of a rat fore limb gripping force test
  • Figure 11A is a [ 18 F]fluoro-2-deoxyglucose positron angiography (FDG-PET) image of a rat after cell treatment;
  • Figure 11B is a quantogram of [ 18 F]FDG-PET images of rats after cell treatment
  • Figure 12 is a graph showing the expression of anti-surgical protein in damaged brain tissue of rats after cell treatment
  • Figure 13A is a photomicrograph of co-localization of diphenylimide-labeled nuclei and human nuclear antigens in rat brain tissue after cell treatment;
  • Figure 13B is a photomicrograph of the number of isolated mesenchymal stem cells of the present invention implanted in rat brain tissue after cell treatment;
  • Figure 14A is a photomicrograph showing co-expression of the isolated mesenchymal stem cell GFAP of the present invention and IGF1R or CXCR4 in a damaged brain tissue of a stroke rat;
  • Figure 14B is a photomicrograph showing the co-expression of the isolated mesenchymal stem cell MAP-2 of the present invention and IGF1R or CXCR4 in the injured brain tissue of a stroke rat;
  • Figure 14C is a photomicrograph showing co-expression of the isolated mesenchymal stem cell NeuN of the present invention implanted in a damaged brain tissue of a stroke rat with IGF1R or CXCR4;
  • Figure 15A is a photomicrograph showing co-expression of the isolated mesenchymal stem cell vWF of the present invention and IGF1R or CXCR4 in a damaged brain tissue of a stroke rat;
  • Figure 15B is a graph showing the results of perfusion of FITC-dextran in a stroke rat with the isolated mesenchymal stem cells of the present invention.
  • Figure 15C shows the results of blood vessel density determination of isolated rat mesenchymal stem cells in the present invention
  • Figure 16 is a graph showing the results of cerebral blood flow of an ischemic brain of a stroke rat transplanted with the isolated mesenchymal stem cells of the present invention.
  • Figure 17A is a graph showing the results of a neurite regenerative test
  • Figure 17B is a graph showing the results of an in vitro test of neurite regeneration
  • Figure 18 is a diagram showing the area of myocardial infarction in acute myocardial infarction rats transplanted with the isolated mesenchymal stem cells of the present invention
  • Figure 19A is a cardiac ultrasound map of an acute myocardial infarction rat transplanted with the isolated mesenchymal stem cells of the present invention.
  • Figure 19B is a quantitative diagram of cardiac ultrasound in acute myocardial infarction rats transplanted with isolated mesenchymal stem cells of the present invention.
  • Figure 20 is a diagram showing the results of immunohistochemical staining of acute myocardial infarction rats after transplantation of the isolated mesenchymal stem cells of the present invention for 3 days;
  • Figure 21 is a diagram showing the results of immunofluorescence staining of acute myocardial infarction rats after transplantation of the isolated mesenchymal stem cells of the present invention for 3 days;
  • Figure 22 is a graph showing the expression of pro-inflammatory cytokines in acute myocardial infarction rats after transplantation of the isolated mesenchymal stem cells of the present invention for 3 days;
  • Fig. 23 is a graph showing the results of the plum three-color staining method of acute myocardial infarction rats after transplantation of the isolated mesenchymal stem cells of the present invention for 28 days.
  • the present disclosure proposes an isolated mesenchymal stem cell that expresses a specific cell surface receptor and has self-renewal ability and pluripotent differentiation ability.
  • the present specification further provides a method for cloning a mesenchymal stem cell clone, which allows the isolated mesenchymal stem cells to express a large amount of specific cell surface receptors and maintain pluripotent differentiation ability.
  • the present disclosure also provides a method of isolating pluripotent mesenchymal stem cells that can rapidly and specifically screen for mesenchymal stem cells with pluripotent differentiation capabilities from a mixture of cells derived from mammalian tissues.
  • the present disclosure provides a use of the aforementioned isolated mesenchymal stem cells, which can be applied to the preparation of a medicament for treating ischemic heart disease or for preparing a medicament for treating brain tissue damage in an individual.
  • the present invention provides an isolated mesenchymal stem cell which expresses an insulin-like growth factor 1 receptor (IGF1R), hereinafter referred to as an isolated compartment representing the present invention.
  • IGF1R + mesenchymal stem cells are pluripotent and can be derived from human cells, more preferably umbilical mesenchymal stem cells.
  • the method for cloning the mesenchymal stem cell clone of the present invention is to culture the IGF1R + mesenchymal stem cells in a medium containing human cord blood serum (hUCS), wherein the concentration of hUCS is 1 to 10 % (v/v), more preferably, the concentration of hUCS is 2% (v/v).
  • hUCS human cord blood serum
  • the method for isolating pluripotent mesenchymal stem cells of the present invention is to screen cells positive for IGF1R from a mixture of cells derived from mammalian tissue, and more preferably, to rescreen for interleukin-22 receptor.
  • interleukin-22 receptor alpha 1, IL22RA1 interleukin 22 receptor alpha 1, IL22RA1 positive cells, which can separate pluripotent mesenchymal stem cells from a mixture of cells, wherein the mammalian tissue is selected from the group consisting of bone marrow, pulp, placenta, umbilical cord, cord blood and fat. Among the constituents of the group.
  • the IGF1R + mesenchymal stem cells of the present invention can be applied to the preparation of a medicament for treating ischemic heart disease and for preparing a medicament for treating brain tissue damage in an individual. Furthermore, the IGF1R + mesenchymal stem cells of the present invention can reduce left ventricular dysfunction after myocardial infarction, reduce infarct size, reduce fibrosis due to myocardial infarction, and reduce immune response, so that it can be used to treat ischemia in an individual. Sexual heart disease, in which ischemic heart disease can be myocardial infarction.
  • the IGF1R + mesenchymal stem cells of the invention can increase the metabolic activity of glucose, promote angiogenesis and enhance neurite regeneration, and have the effect of nerve remodeling by the interaction of IGF1R and the chemokine receptor CXCR4, so that it can be used
  • An individual for treating brain tissue damage wherein the brain tissue damage may be a cerebral ischemic disease or a neurodegenerative disease, and the cerebral ischemic disease may be a stroke, and the neurodegenerative disease may be Parkinson's disease.
  • IGF1 insulin-like growth factor 1 receptor
  • IL22 interleukin 22
  • IL-22 a cell surface receptor of interleukin 22
  • IL-22 its ligand IL-22 is a cytokine having both anti-inflammatory and pro-inflammatory properties and can be secreted by various immune cells.
  • the aforementioned is a specific receptor for stromal cell-derived factor-1a (SDF-1), which is expressed in most tissues and organs in the body, and is composed of 352 amines.
  • SDF-1 stromal cell-derived factor-1a
  • a G protein-coupled receptor (GPCR) consisting of a base acid has a seven-pass membrane structure. Its ligand SDF-1 has a strong chemotactic effect on lymphocytes.
  • the mammalian tissue used in this experimental example was human umbilical cord tissue, while the human umbilical cord mesenchymal stem cells in human umbilical cord tissue were derived from Wharton's jelly.
  • the collected human umbilical cord tissue was washed three times with PBS (DPBS, Life Technology) without Ca 2+ and Mg 2+ , and the human umbilical cord tissue was cut with scissors in the midline direction and extracted from Wharton's gel.
  • Umbilical arteries, umbilical veins, and adventitia were used in this experimental example.
  • the interstitial tissue of Wharton's gel was then cut into cubes of less than 0.5 cm 3 , treated with collagenase 1 (Sigma), and cultured at 37 ° C for 14-18 hours at 5% CO 2 , 95% saturated humidity. .
  • the explants were respectively treated with DMEM containing 2% hUCS or 10% fetal calf serum (FCS) as cell culture medium, and antibiotics were added to the cell culture medium at 37 ° C in 5% CO 2 , 95% saturated humidity was allowed to stand for 5-7 days. Explant culture of human umbilical cord tissue will be maintained until fusiform adherent cells are found to grow outward from the explant.
  • Fig. 1A a photomicrograph of the primary culture of umbilical cord mesenchymal stem cells, wherein the black arrow indicates the explant, and after 4 to 8 passages, the cells migrate from the explant and the cell morphology changes. It is a uniform fusiform.
  • the cell surface molecules of the primary cultured umbilical cord mesenchymal stem cells were analyzed by flow cytometry.
  • the cell surface molecules analyzed included specific cell surface molecules of cells in IGF1R, IL22RA1, and Wharton's gel, as well as pluripotency markers.
  • Specific cell surface molecules of cells in Wharton's gel contain CD13, CD29, CD34, CD44, CD45, CD73, CD90, CD105, CD117, CD166, HLA-ABC and HLA-DR.
  • the pluripotency markers include Oct-4, Sox-2, Nanog, and SSEA-4.
  • FIG. 1B is an isolated mesenchymal stem cell IGF1R expression of the present invention
  • FIG. 1C is a diagram showing the results of IL22RA1 expression of the isolated mesenchymal stem cells of the present invention
  • FIG. 1D and FIG. 1E are specific cell surfaces of cells of the isolated mesenchymal stem cells of the invention
  • Fig. 1F is a graph showing the results of pluripotency marker expression of the isolated mesenchymal stem cells of the present invention.
  • FIG. 1B IGF1R expression of the primary cultured umbilical cord mesenchymal stem cells was first analyzed by flow cytometry, and IGF1R expression of mesenchymal stem cells from different sources was analyzed by Western blotting.
  • the analyzed cell types included human fibroblasts and humans.
  • Dental pulp stem cells, adipose-derived mesenchymal stem cells, umbilical cord mesenchymal stem cells, and bone marrow mesenchymal stem cells the results of Figure 1B show that IGF1R expression can be seen in mesenchymal stem cells from different sources.
  • FIG. 1C to 1F analyze the expression of IL22R1A in the primary cultured umbilical cord mesenchymal stem cells, the specific cell surface molecule expression of cells in the Wharton's gel, and the pluripotency marker expression by flow cytometry. From the results of Fig. 1C, the expression of IL22R1A was observed in the primary cultured umbilical cord mesenchymal stem cells, and about 45.4% of the umbilical cord mesenchymal stem cells expressed IL22R1A compared with the control group. From the results of Fig. 1D and Fig.
  • IGF1R + mesenchymal stem cells were further isolated from the cells analyzed by flow cytometry.
  • the purification method was as follows: after mixing the cells with the anti-IGF1 antibody, the cells stained with the anti-IGF1 antibody were sorted by FACSTAR + flow cytometry (Becton Dickinson), and then analyzed by trypan blue exclusion test. The sorted cells had a cell viability of approximately 96%.
  • the selected IGF1R + mesenchymal stem cells were treated with DMEM containing 2% hUCS or 10% FCS as the cell culture medium, and antibiotics were added to the cell culture medium at 37 ° C in 5% CO 2 , 95% saturated humidity. Environmental training. The growth kinetics of IGF1R + mesenchymal stem cells cultured with hUCS and FCS were analyzed separately.
  • FIG. 2 there is an amplification index map of IGF1R + mesenchymal stem cells. Shown by the results in FIG. 2, cultured in a medium containing hUCS in IGF1R + interphase mesenchymal stem cells speed than cultured in medium containing FCS in IGF1R + mesenchymal stem cells faster, doubling generation time was 22 Hours and can be expanded for more than 150 days without signs of aging and spontaneous differentiation.
  • a human cytokine wafer analysis map of hUCS and FCS where P represents a positive control and N represents a negative control.
  • the expression levels of five cytokines in hUCS were significantly higher than those of FCS, namely epidermal growth factor (EGF; box 1) and angiogenin (ANGogen). 2) macrophage inflammatory protein (MIP-1 ⁇ ; box 3), regulated T-cell expressed and presumably secreted (RANTES; box 4) and platelet-derived The growth factor-BB (platelet-derived growth factor BB, PDGF-BB), the multiple differences in the expression levels of the five cytokines are 2, 3, 3, 2 and 4 times. In contrast, the expression levels of insulin-like growth factor 1 (IGF-1) were similar in hUCS and FCS.
  • IGF-1 insulin-like growth factor 1
  • this experiment used hUCS and FCS as samples, and analyzed the concentration of PDGF-BB and IGF-1 by ELISA (enzyme-linked immunosorbent assay).
  • Fig. 3B which is an ELISA analysis chart of hUCS and FCS
  • the results of Fig. 3B show that the expression levels of IGF1 in the two serums are similar, but in the PDGF-BB portion, the concentration of PDGF-BB contained in hUCS is significantly higher. Content in FCS (p ⁇ 0.05).
  • lentiviruses were used to introduce shRNA targeting IGF1R (LV-IGF1R-sh, sc-29358-V, Santa Cruz Biotechnology) to hUCS. Or IGF1R + mesenchymal stem cells cultured in FCS medium to reduce the expression of IGF1R in IGF1R + mesenchymal stem cells.
  • the lentivirus was also introduced into the control group shRNA (LV-control-sh, Santa Cruz Biotechnology). IGF1R + mesenchymal stem cells cultured in a medium containing hUCS or FCS was used as an experimental control group.
  • IGF1R + mesenchymal stem cells after shRNA introduction was analyzed by Western blotting.
  • the growth kinetics of IGF1R + mesenchymal stem cells after introduction of shRNA were also analyzed.
  • FIG. 4A is an analysis diagram of IGF1R expression amount after introduction of shRNA
  • FIG. 4B is an amplification trend diagram of IGF1R + mesenchymal stem cells.
  • FIG There are four groups 4A respectively lentiviral introduced LV-IGF1R-sh and introduced IGF1R LV-control-sh are + mesenchymal stem cells, and not introduced the shRNA IGF1 R + mesenchymal stem cells, in FIG. 4A
  • the four groups of IGF1R + mesenchymal stem cells were cultured in a medium containing hUCS. From the results of Fig.
  • Fig. 4A the expression of IGF1R was decreased in the group into which LV-IGF1R-sh was introduced, and the amount of IGF1R in the other group was lower than that in the group into which LV-control-sh was introduced, 48 hours after infection with lentivirus. The amount of expression is equivalent.
  • Fig. 4B there are four groups, respectively, which are cultured with hUCS-containing medium and introduced into LV-control-sh IGF1R + mesenchymal stem cells (LV-control-sh U-IGF1R + mesenchymal stem cells) to contain FCS.
  • the medium was cultured and introduced into LV-control-sh IGF1R + mesenchymal stem cells (LV-control-sh F-IGF1R + mesenchymal stem cells), cultured in hUCS-containing medium and introduced into LV-IGF1R-sh IGF1R + Qualified stem cells (LV-IGF1R-sh-U-IGF1R + mesenchymal stem cells), and IGF1R + mesenchymal stem cells (LV-IGF1R-sh-F-IGF1R + ) cultured in FCS-containing medium and introduced into LV-IGF1R-sh Mesenchymal stem cells). Growth kinetics displayed by the results of FIG. 4B, or whether by hUCS FCS containing culture medium IGF1R + mesenchymal stem cells, after introduction LV-IGF1R-sh, both slow down the proliferation of mesenchymal stem cells between IGF1R +.
  • the DNA was calibrated using Bromodeoxyuridine (BrdU) and detected by the cell proliferation assay (BrdU proliferation assay) to investigate the proliferative potential of IGF1R + mesenchymal stem cells.
  • BrdU proliferation assay the cell proliferation assay
  • IGF1R + mesenchymal stem cells were cultured in the medium without supplements for 4 to 6 hours, and then IGF1R + mesenchymal stem cells were cultured separately with 2% hUCS, 10% FCS or 100 ng/mL SDF-1.
  • the medium was introduced for 2 days and introduced into LV-control-sh or LV-IGF1R-sh, respectively, and the proliferation of IGF1R + mesenchymal stem cells was analyzed by BrdU chemiluminescence immunoassay kits (Roche).
  • FIG. 4C there is a graph of cell proliferation analysis of IGF1R + mesenchymal stem cells.
  • IGF1R + mesenchymal stem cells cultured in a medium supplemented with SDF-1 as a positive control group, cultured in a medium containing hUCS and introduced into IGF1R + interstitial of LV-control-sh.
  • IGF1R + mesenchymal stem cells in medium containing FCS culture and introduced into the LV-control-sh of IGF1R + mesenchymal stem cells to medium containing hUCS culture and introduced IGF1R LV-IGF1R-sh mesenchymal stem cells, and containing the culture + FCS,
  • the IGF1R + mesenchymal stem cells of LV-IGF1R-sh were cultured and introduced. From the results of Fig. 4C, it was revealed that IGF1R + mesenchymal stem cells cultured in a medium containing hUCS showed significantly more BrdU insertion than IGF1R + mesenchymal stem cells cultured in a medium containing FCS.
  • IGF1R + mesenchymal stem cells were double stained with IGF1R/Oct-4 antibody, IGF1R/Sox-2 antibody, IGF1R/Nanog antibody, IGF1R/SSEA4 antibody, respectively.
  • IGF1R and pluripotency markers were analyzed by flow cytometry and immunofluorescence staining, respectively.
  • FIG. 5A is a flow cytometry double staining analysis diagram of IGF1R + mesenchymal stem cells
  • FIG. 5B is a photomicrograph of IGF1R + interstitial cell immunofluorescence staining double staining method, wherein IGF1R + Mesenchymal stem cells are derived from 5 independent samples. From the results of Fig. 5A and Fig. 5B, IGF1R in IGF1R + mesenchymal stem cells was co-expressed with pluripotency markers Oct-4, Sox-2, Nanog and SSEA-4, while the results of Fig. 5B showed that IGF1R was also associated with CXCR4. Co-expression.
  • a quantitative RT-PCR results of expression of pluripotency markers for IGF1R + mesenchymal stem cells and IGF1R - mesenchymal stem cells wherein the cells tested were IGF1R + mesenchymal stem cells and IGF1R - mesenchymal stem cells, respectively, and human Fibroblasts were used as a negative control group, and induced pluripotent stem cells (iPS) were used as a positive control group.
  • iPS induced pluripotent stem cells
  • IGF1R + mesenchymal stem cells in a medium containing hUCS or FCS would affect its pluripotent differentiation ability.
  • IGF1R + mesenchymal stem cells cultured in hUCS or FCS medium for 5 to 10 passages were separately cultured in a cell culture dish at a density of 5 ⁇ 10 3 cells/cm 2 and contained 10% FCS, respectively.
  • IGF1R + mesenchymal stem cells cultured in different differentiation media of 2% hUCS, and induced to differentiate into adipocytes, chondrocytes, osteoblasts, angiogenesis and nerve cells, and observe the cell type under the microscope, and further staining Confirm the cell type after differentiation.
  • FIG. 6A is a photomicrograph of differentiation of IGF1R + mesenchymal stem cells into different tissue cells
  • FIG. 6B is a photomicrograph of differentiation of IGF1R + mesenchymal stem cells into neural cells.
  • the differentiated cells were identified as fat cells by oil red O stain, and the differentiated cells were confirmed to be osteoblasts by Alizarin red S stain, and stained with azadi blue. (Alican blue stain) It was confirmed that the differentiated cells were chondrocytes, and whether the IGF1R + mesenchymal stem cells formed blood vessels was confirmed by the cell type in the bright field. From the results of Fig. 6A, it was revealed that IGF1R + mesenchymal stem cells can be differentiated into adipocytes, chondrocytes, osteoblasts, and blood vessel formation under the culture of differentiation medium.
  • the microscopic photograph of the bright field in Fig. 6B shows that IGF1R + mesenchymal stem cells cultured in a neural cell differentiation medium can be seen to expand the cytoplasmic cell type, indicating that the differentiated cells are nerve cells.
  • the differentiated cells express mature neural markers, and the mature neural markers include GFAP (glial fibrillary acidic protein), MAP-2 (microtubule-associated protein 2), O4 and Tuj-1 (Neuron). -specific class III beta-tubulin). From the results of Fig. 6B, it was revealed that the differentiated cells were expressed by four mature neural markers, and it was confirmed that the differentiated cells were indeed nerve cells.
  • the expression ratios of the four mature neural cell markers were IGF1R + mesenchymal stem cells cultured in 2% hUCS differentiation medium or 10% FCS differentiation medium.
  • the results showed that the expression ratio of the IGF 1R + mesenchymal stem cells cultured in the 2% hUCS differentiation medium was higher than that of the culture medium containing 10% FCS differentiation medium, indicating that it was cultured with hUCS.
  • the base cultured IGF1R + mesenchymal stem cells are more likely to differentiate into nerve cells.
  • IGF1R + CXCR4 expression levels of IGF1R + mesenchymal stem cells are regulated by IGF1 alone or by IGF1 and PDGF-BB (a key component of hUCS and FCS differences).
  • IGF1R + mesenchymal stem cells were treated with different doses of IGF1 or different doses of PDGF-BB, and the expression levels of IGF1R and CXCR4 were detected by Western blotting.
  • FIG. 7A is an analysis diagram of the expression levels of IGF1R and CXCR4 after treatment of different doses of IGF1 to IGF1R + mesenchymal stem cells.
  • Figure 7B is a graph showing the expression levels of IGF1R and CXCR4 after treatment of different doses of PDGF-BB to IGF1R + mesenchymal stem cells.
  • Figure 7C is a graph showing the expression of IGF1R expression after simultaneous treatment of different doses of IGF1 and PDGF-BB to IGF1R + mesenchymal stem cells.
  • IGF1R + mesenchymal stem cells are treated with different concentrations of IGF1 or different concentrations of PDGF-BB (in serum-free conditions) and Western The expression levels of phosphorylated signal transducers and activator of transcription 3 (p-Stat3) were detected by blotting.
  • FIG. 7D an analysis of the expression levels of p-Akt and p-Stat3 after simultaneous treatment of different doses of IGF1 and PDGF-BB to IGF1R + mesenchymal stem cells, wherein * indicates the experimental group (adding different doses of IGF1 or/and P ⁇ 0.05 for the group of PDGF-BB) and the control group (group without IGF1 and PDGF-BB).
  • the results in Figure 7D show that the addition of PDGF-BB significantly increased the expression levels of p-Akt and p-Stat3, but the addition of IGF1 had no effect on increasing the expression levels of p-Akt and p-Stat3.
  • p-Akt inhibitor (LY294002)
  • p-Stat3 inhibitor (AG490) were added to IGF1R + mesenchymal stem cells, respectively, and it was found that phosphorylation of Akt and Stat3 induced by PDGF-BB was completely inhibited (results not shown). .
  • the above results show that PDGF-BB activates downstream important information transduction pathways more efficiently than IGF1, and also shows that hUCS-containing medium is more suitable as a medium for mesenchymal stem cells than FCS-containing medium.
  • IGF1R + mesenchymal stem cells for the treatment of brain tissue damage
  • IGF1R + mesenchymal stem cell transplantation improves neurological behavior in a rat model of stroke
  • This experimental example is to evaluate the self-renewal ability and nerve regeneration potential of IGF1R + mesenchymal stem cells in a rat model of stroke, and to detect the neurological behavior of rats before and after stroke through three neurological deficit models to assess whether neurological function is restored. .
  • the stroke rat model was subjected to an ischemia-reperfusion model to simulate the symptoms of transient local cerebral ischemia in rats.
  • the test animals used were 250-300 g male SD (Sprague-Dawley). Rat.
  • the cerebral ischemia/reperfusion model in rats is induced by local carotid arteries (CCA) and the middle cerebral artery (MCA). After a minute, the patient was reperfused, and after 1 hour, the cells were intravenously transplanted into the rats, and the amount of cells injected was 2 ⁇ 10 6 .
  • the rats were divided into 5 groups, and 5 different cells or vectors were injected for cell treatment.
  • the cells of group 1 were cultured in IGF1R + mesenchymal stem cells (U-IGF1R + mesenchymal stem cells) containing hUCS medium.
  • the cells of group 2 were cultured in IGF1R + mesenchymal stem cells (F-IGF1R + mesenchymal stem cells) containing FCS medium, and the cells of group 3 were mesenchymal stem cells (U-mesenchymal stem cells) cultured in hUCS-containing medium.
  • the cells of group 4 were mesenchymal stem cells (F-interstitial stem cells) cultured in a medium containing FCS, and the group 5 was injected with PBS (vehicle) into a rat as a control group.
  • CXCR4 antibody R&D system
  • IGF1R inhibitor PPP, Santa Cruz Biotechnology
  • the time points of detection of neurobehavioral were 5 days before cerebral ischemia/reperfusion, 1st day, 7th day, 14th day and 28th day after cell transplantation.
  • the three neurological deficit models were used to assess the left and right asymmetry, mobility, and forelimb grip of the rats.
  • the left and right asymmetry of the rat's body was evaluated by the body swing test.
  • the experiment was first placed on the bottom of the feeding cage 10 cm above the tail, and the number of lateral movements of the rat was recorded, especially on the opposite side of the ischemic side.
  • the head swing frequency is counted in 20 consecutive rows and normalized by baseline score.
  • Figure 8A is a graph showing the results of a rat body swing test
  • Figure 8B is a graph showing the results of a rat body swing test of the block test. From the results of Fig. 8A, it was revealed that regardless of which mesenchymal stem cells were transplanted, significant neurological recovery was observed as compared with the control group.
  • the group of mesenchymal stem cells cultured in hUCS-containing medium (group 1 and group 3) was transplanted, and the recovery of neurological function in rats was compared with that of mesenchymal stem cells cultured in FCS-containing medium (group).
  • Rats' ability to move was detected by the VersaMax Animal Activity Monitoring System (Accuscan Instruments) for 2 hours.
  • the VersaMax animal activity monitoring system consists of a 16-level infrared sensor and 8 vertical infrared sensors. The vertical sensors are located 10 cm above the bottom of the chamber.
  • the rat's mobility is interrupted by the movement of the rats in the chamber. The number of times is quantified.
  • the three vertical motion parameters measured are: vertical activity, vertical activity time, and number of vertical motions.
  • Figure 9A is a graph showing the results of a rat vertical activity test
  • Figure 9B is a graph showing the results of a rat vertical activity time test
  • Figure 9C is a graph showing the results of a rat vertical motion number test. From the results of Figs. 9A to 9C, it was revealed that rats transplanted with mesenchymal stem cells showed significant improvement in activity compared to the control group. The group of transplanted U-IGF1R + mesenchymal stem cells was more effective than the group of transplanted F-IGF1R + mesenchymal stem cells, but the CXCR4 antibody or PPP blocked CXCR4 or IGF1R blocking test group. Specifically, the results of the activity ability of the rats were comparable to those of the control group, indicating that blocking CXCR4 or IGF1R inhibited the therapeutic effect of transplanting U-IGF1R + mesenchymal stem cells.
  • the fore limb grip test of rats was detected using a TEE-Systems.
  • the gripping force of each forelimb of the rat was measured separately in the experiment, and the average value of the gripping force of the rat was calculated for 20 times, and the ratio of the gripping force of the ipsilateral side was calculated.
  • this experimental example also calculates the ratio of rat grip strength before and after cell treatment to evaluate changes in rat grip strength after cell treatment.
  • Fig. 10 there is shown a graph of the results of the rat fore limb gripping force test. From the results of Fig. 10, it was revealed that the rat for IGF1R + mesenchymal stem cells significantly improved the forelimb grip strength of the rat after the cell treatment compared with the control group.
  • the group of transplanted U-IGF1R + mesenchymal stem cells was more effective than the group of transplanted F-IGF1R + mesenchymal stem cells, but the blocking ability of CXCR4 or IGF1R was blocked by adding CXCR4 antibody or PPP.
  • the forelimb grip strength of the rats was comparable to that of the control group, indicating that blocking CXCR4 or IGF1R inhibited the therapeutic effect of transplanting U-IGF1R + mesenchymal stem cells.
  • IGFR-1 + mesenchymal stem cells can improve the neurobehavioral behavior of stroke rats, while U-IGFR-1 + mesenchymal stem cells are more stable than F-IGFR-1 + Stem cells have the potential for nerve regeneration, and this nerve regeneration is through the IGF1R and CXCR4 receptor pathways.
  • FIG. 11A is a [ 18 F]FDG-PET image of a rat after cell treatment
  • FIG. 11B is a quantitative image of a [ 18 F]FDG-PET image of a rat after cell treatment.
  • the results of Fig. 11A show that rats transplanted with IGF1R + mesenchymal stem cells can increase glucose metabolism in damaged brain tissue (right brain in the figure), while groups transplanted with U-IGF1R + mesenchymal stem cells improve damaged brain tissue. The extent of glucose metabolism is greater than that of the transplanted F-IGF1R + mesenchymal stem cells.
  • FIG. 11B is a semi-quantitative image of [ 18 F]FDG-PET, and the results of Fig. 11B show that the transplanted U-IGF1R + mesenchymal stem cells have increased glucose metabolism activity, and CXCR4 or IGF1R is blocked by injection of PPP or CXCR4 antibody. After that, the glucose metabolism activity of the rat in the damaged brain tissue was comparable to that of the control group, indicating that blocking CXCR4 or IGF1R inhibited the effect of transplanting U-IGF1R + mesenchymal stem cells to increase glucose metabolism activity.
  • IGF1R + mesenchymal stem cell therapy increases neural differentiation in vivo
  • IGF1R + mesenchymal stem cells can differentiate into glial cells.
  • the stroke of 28 days after cell treatment was experimentally confirmed by immunofluorescence staining using a conjugated-focus laser scanning microscope.
  • the number of IGF1R + mesenchymal stem cells in the damaged brain tissue of the rat, and the differentiated cell type of the implanted IGF1R + mesenchymal stem cells were determined by immunofluorescence double staining.
  • Figure 13A is a photomicrograph of co-localization of diphenylimide-labeled nuclei and human nuclear antigens in rat brain tissue after cell treatment
  • Figure 13B is a cell therapy. Photomicrographs of the number of IGF1R + mesenchymal stem cells implanted in rat brain tissue. From the results of Fig. 13A, all of the IGF1R + mesenchymal stem cells labeled with bisphthalimide expressed human nuclear antigen (hNA), and it was confirmed that the source of the cells was human tissue. From the results of Fig.
  • specific cell type markers IGF1R + mesenchymal stem cells differentiated
  • CXCR4 CXCR4-specific cell type markers
  • the specific cell type marker contains glial fibrillary acidic protein and neuron specificity. Microtubule-associated protein 2 and neuron nuclear antigen.
  • FIG. 14A is a photomicrograph showing the co-expression of U-IGF1R + mesenchymal stem cells GFAP and IGF1R or CXCR4 in damaged brain tissue of a rat with stroke
  • FIG. 14B is a damaged rat implanted with stroke.
  • Fig. 14C shows U-IGF1R + mesenchymal stem cells NeuN and IGF1R or CXCR4 implanted in damaged brain tissue of stroke rats Photomicrographs of co-expression.
  • Figs. 14A to 14C From the results of Figs. 14A to 14C, in the damaged brain tissue region of a stroke rat implanted with U-IGF1R + mesenchymal stem cells, some cells of bisbenzimide-labeled and expressed CXCR4 were co-expressed. Neural cell markers (GFAP, MAP-2 and NeuN). In addition, cells in which bisbenzimide was labeled and expressed IGF1R were also observed, and these cells also co-expressed neural cell markers (GFAP, MAP-2, and NeuN), respectively.
  • GFAP Neural cell markers
  • MAP-2 MAP-2
  • NeuN neural cell markers
  • IGF1R + mesenchymal stem cell transplantation promotes angiogenesis in vivo
  • vascular endothelial cell markers vWF Von Willebrand factor
  • IGF1R and CXCR4 were calibrated by immunofluorescence double staining to confirm the co-expression of vascular endothelial cell markers and IGF1R, and the co-expression of vascular endothelial cell markers and CXCR4.
  • the nuclei were labeled with diphenylimide.
  • Figure 15A a photomicrograph showing the co-expression of U-IGF1R + mesenchymal stem cells vWF and IGF1R or CXCR4 in damaged brain tissue of a rat with stroke, the results of Fig.
  • the vascular endothelial cell marker CD31 in the ischemic brain tissue section of stroke rats was further calibrated by immunofluorescence staining to calculate the vascular density.
  • Fig. 15C the results of blood vessel density measurement of a rat rat with IGF1R + mesenchymal stem cells, the results of Fig.
  • FIG. 15C show the vascular density of a rat with U-IGF1R + mesenchymal stem cells in ischemic brain tissue.
  • the transplanted U-IGF1R + mesenchymal stem cells were p ⁇ 0.05 compared with transplanted F-IGF1R + mesenchymal stem cells, and the transplanted U-IGF1R + mesenchymal stem cells were p ⁇ 0.01 compared with the control group.
  • transplantation of IGF1R + mesenchymal stem cells into a stroke rat for cell therapy can increase angiogenesis in ischemic brain tissue of stroke rats, and transplantation of IGF1R + mesenchymal stem cells cultured in hUCS-containing medium is more It has the potential to increase angiogenesis in ischemic brain tissue.
  • CBF cerebral blood flow
  • the rats were anesthetized with chloral hydrate.
  • the local blood perfusion condition of the ischemic brain tissue of rats with stroke was monitored by laser micro-flow instrument (Moor Instrutments).
  • the cerebral blood flow results of ischemic brain of a rat with IGF1R + mesenchymal stem cells were transplanted.
  • the results of Figure 16 show that transplantation of IGF1R + mesenchymal stem cells can increase the ischemic brain tissue of stroke rats.
  • Cerebral blood flow, and transplantation of U-IGF1R + mesenchymal stem cells increased the cerebral blood flow in ischemic brain tissue of stroke rats, in which U-IGF1R + mesenchymal stem cells were transplanted with F-IGF1R + mesenchymal stem cells.
  • the transplanted U-IGF1R + mesenchymal stem cells were p ⁇ 0.01 compared with the control group.
  • IGF1R + mesenchymal stem cells were intravenously transplanted into stroke rats.
  • the transplanted cell groups were U-IGF1R + mesenchymal stem cells, F-IGF1R + mesenchymal stem cells, blocking test group and control. group, wherein the blocking test group is introduced into the transplanted LV-IGF1R-sh of the U-IGF1R + mesenchymal stem cells, and introduced into the LV-CXCR4-sh U-IGF1R + mesenchymal stem cells into the body of stroke in rats, the control group injected intravenously places PBS to stroke rats.
  • the rats in the stroke were sacrificed for 28 days after transplantation of IGF1R + mesenchymal stem cells.
  • the brain tissue sections were subjected to immunofluorescence staining to calibrate the neuron-specific marker III-tubulin, and the length of the neurites was calculated by the image analysis software (SigmaScan). Cells in which nerve cells are more than twice the diameter of the cell body are counted as neurite-bearing cells.
  • FIG. 17A is a graph showing the results of a neurite regenerative test in vivo
  • the results of Fig. 17A show that transplantation of IGF1R + mesenchymal stem cells can improve neurite regeneration in ischemic brain tissue of a rat, while transplanting U-IGF1R + interstitial In stroke rats with stem cells, the number of neurite regeneration in ischemic brain tissue was significantly higher than that in other groups, and the neurite length was also significantly longer than other groups.
  • the results of the detachment test group showed that blocking IGF1R or CXCR4 inhibited the increased neurite length and neurite cell number of U-IGF1R + mesenchymal stem cells.
  • this experimental example further co-cultures primary cerebral cortical cells with IGF1R + mesenchymal stem cells in oxygen and glucose.
  • IGF1R + mesenchymal stem cells contain U-IGF1R + mesenchymal stem cells, F-IGF1R + mesenchymal stem cells and blocking test group, and cells in the blocking test group are U-IGF1R introduced into LV-IGF1R-sh + Mesenchymal stem cells and U-IGF1R + mesenchymal stem cells into which LV-CXCR4-sh was introduced, and primary cerebral cortical cells cultured separately in an environment where oxygen and glucose supply were insufficient were used as a control group.
  • the co-cultured cells were labeled with immunofluorescence staining for III-tubulin, and neurite regeneration and neuronal survival were measured by the aforementioned methods.
  • FIG 17B is a graph showing the results of an in vitro test for neurite regeneration
  • the results of Figure 17B show that co-culture with IGF1R + mesenchymal stem cells increases the number of neurite cells and neurites in primary cerebral cortical cells in the absence of oxygen and glucose supply.
  • the length indicates that IGF1R + mesenchymal stem cells can help neurite regeneration in primary cerebral cortical cells with insufficient supply of oxygen and glucose, while the number of neurite regeneration in co-cultured U-IGF1R + mesenchymal stem cells is significantly higher than in other groups. And its neurite length is also significantly longer than other groups.
  • the results of the test group showed that blocking IGF1R or CXCR4 inhibited the increased neurite length and neurite cell number of U-IGF1R + mesenchymal stem cells.
  • IGF1R + mesenchymal stem cells can promote neurite regeneration of hypoxic ischemic brain tissue/brain cells, and transplantation of IGF1R + mesenchymal stem cells cultured in hUCS-containing medium is more It has the potential to increase neurite regeneration in ischemic and hypoxic brain/brain cells, and this neurite regeneration is through the IGF1R and CXCR4 receptor pathways.
  • IGF1R + mesenchymal stem cells are known to have self-renewal ability and pluripotent differentiation ability, and IGF1R + mesenchymal stem cells have the effect of treating brain tissue damage individuals.
  • the experimental example of this part will discuss IGF1R + mesenchymal stem cells. It is used to treat the effects of ischemic heart disease.
  • This experimental example is to evaluate whether IGF1R + mesenchymal stem cell transplantation improves myocardial function recovery after myocardial infarction in a rat model of acute myocardial infraction (AMI).
  • the rat model of acute myocardial infarction is a left anterior descending (LAD) coronary artery that blocks the symptoms of transient myocardial ischemia in rats.
  • the test animals used in this experiment were male SD rats weighing 250-300 g.
  • the rats were anesthetized with 2% isoflurane (in 100% oxygen), and placed on the operating table after general anesthesia.
  • the respirator (SN-480-7) was inserted and artificially ventilated at a tidal volume of 1 mL/100 g and a respiratory rate of 80 beats/min.
  • the rib retractor (MY-9454S) was used to cut longitudinally along the 4th and 5th rib gaps of the left sternal border, and the left lung was deflated with gauze soaked in physiological saline. Cut the happy bag, and then use the 6-0-polyethylene suture thread (Ethicon) to block the left anterior descending coronary artery. The obstructed area was whitened and the electrocardiogram T wave was high. The lungs were re-inflated before the chest was sutured, that is, the acute myocardial infarction was completed. Rat model.
  • the acute myocardial infarction rats were divided into three groups, and the number of mesenchymal stem cells or IGF1R + mesenchymal stem cells was 2 ⁇ 10 6 , and the normal saline was injected as a control group.
  • a sham-operated group was also subjected to the same surgical procedure as described above, but did not block the left anterior descending coronary artery of the sham operation group.
  • rats with acute myocardial infarction 28 days after transplantation of mesenchymal stem cells were sacrificed.
  • the heart tissue sections were immersed in triphenyltetrazolium chloride solution, and then dehydrogenase was immersed to reveal necrosis.
  • the area is reddish blue, and the area without infarction is brick red.
  • transplantation of mesenchymal stem cells can improve the myocardial infarct size of rats with acute myocardial infarction, and also thicken the arterial wall of the infarcted area, and the myocardium of acute myocardial infarction rats transplanted with IGF1R+ mesenchymal stem cells.
  • the extent of infarct reduction and the extent of arterial wall thickening in the infarct area were higher than those of mesenchymal stem cells.
  • FIG. 19A is a cardiac ultrasonogram of acute myocardial infarction rats transplanted with mesenchymal stem cells and IGF1R+ mesenchymal stem cells
  • FIG. 19B is a cardiac ultrasonogram of acute myocardial infarction rats transplanted with mesenchymal stem cells and IGF1R+ mesenchymal stem cells
  • FIG. 19B is an acute myocardial infarction rat heart transplanted with mesenchymal stem cells and IGF1R+ mesenchymal stem cells. A quantized map of the ultrasound. From the results of Fig. 19A and Fig. 19B, the group of transplanted mesenchymal stem cells and IGF1R+ mesenchymal stem cells can detect a lower left ventricular end-systolic diameter (left ventricular end) in cardiac ultrasound analysis compared with the control group.
  • This experimental example will further investigate whether transplantation of mesenchymal stem cells and IGF1R+ mesenchymal stem cells into acute myocardial infarction rats can inhibit the inflammatory response after myocardial infarction.
  • rats with acute myocardial infarction 3 days after transplantation of mesenchymal stem cells and IGF1R+ mesenchymal stem cells were sacrificed.
  • the heart tissue sections were examined by immunohistochemical staining to detect the degree of inflammation, and immunofluorescence staining was used to detect macrophages. Dahe and Quantitative RT-PCR were used to detect the expression level of pro-inflammatory factors.
  • FIG. 20 there are shown results of immunohistochemical staining of acute myocardial infarction rats 3 days after transplantation of mesenchymal stem cells and IGFIR + mesenchymal stem cells.
  • the upper panel is a photomicrograph of the heart tissue section of an acute myocardial infarction rat with hematoxylin-eosin staining (H&E stain).
  • H&E stain hematoxylin-eosin stain staining
  • the inflammation of the heart tissue is divided into 0-5 grades, and the higher the number of grades. The more severe the degree of inflammation, the results of different groups were classified and counted according to the above criteria.
  • the statistical results are shown in the lower figure of Figure 20.
  • the results in Figure 20 show that transplantation of mesenchymal stem cells and IGF1R+ mesenchymal stem cells can improve the degree of inflammation of heart tissue in rats with acute myocardial infarction, and transplantation of IGF1R+ mesenchymal stem cells can improve the inflammation of heart tissue in rats with acute myocardial infarction.
  • the stem cell group was p ⁇ 0.05 compared with the control group, and the IGF1R + mesenchymal stem cell group was p ⁇ 0.01 compared with the control group.
  • Fig. 21 the results of immunofluorescence staining of acute myocardial infarction rats after transplantation of mesenchymal stem cells and IGFIR + mesenchymal stem cells for 3 days, the results of Fig. 21 show that groups of mesenchymal stem cells and IGF1R + mesenchymal stem cells were transplanted.
  • the infiltration of inflammatory cells in the ischemic myocardium usually increases the expression of pro-inflammatory cytokines.
  • This test further detects the expression of pro-inflammatory cytokines in the heart tissue of rats 3 days after myocardial infarction by quantitative RT-PCR.
  • the pro-inflammatory factors include IL-1 ⁇ , IL-6, TNF- ⁇ , and IFN- ⁇ , and the expression level of the anti-inflammatory factor IL-10 is additionally detected in this experimental example.
  • Fig. 22 there is shown an analysis chart of the amount of inflammatory factor expression in rats with acute myocardial infarction 3 days after transplantation of mesenchymal stem cells and IGFIR + mesenchymal stem cells. From the results of Fig.
  • the groups of mesenchymal stem cells and IGF1R + mesenchymal stem cells were compared with the control group, and the pro-inflammatory factors IL-1 ⁇ , IL-6, and TNF were found in the heart tissues of rats 3 days after myocardial infarction.
  • the expression levels of ⁇ - and IFN- ⁇ were all decreased, and the expression levels of the pro-inflammatory factors IL-1 ⁇ , IL-6, TNF- ⁇ and IFN- ⁇ were decreased to a greater extent in the group of IGF1R+ mesenchymal stem cells.
  • the expression level of anti-inflammatory factors is opposite to that of pro-inflammatory factors.
  • the combination of mesenchymal stem cells and IGF1R + mesenchymal stem cells increases the expression of anti-inflammatory factor IL-10, which is transplanted with IGF1R + mesenchymal stem cells.
  • the increase in the expression of anti-inflammatory factor IL-10 was greater, and the results also showed that transplantation of IGF1R + mesenchymal stem cells can significantly improve the inflammation of heart tissue in rats with acute myocardial infarction.
  • IGF1R + mesenchymal stem cell therapy reduces fibrosis caused by myocardial infarction
  • the muscle fibers of the heart are different from normal muscle fibers and can work for a long time to get blood to every part of the body.
  • the myocardium produces irreversible necrosis, and the necrotic part is replaced by fibrous tissue, which is fibrotic after several weeks.
  • This experimental example will investigate whether transplantation of IGF1R + mesenchymal stem cells can reduce fibrosis caused by myocardial infarction. Rats with acute myocardial infarction 28 days after transplantation of mesenchymal stem cells were sacrificed. Heart tissue sections were observed by Masson's trichrome staining. The collagen fibers were blue. The muscle fibers were red.
  • the analyzed groups included acute myocardial infarction rats transplanted with mesenchymal stem cells, acute myocardial infarction rats transplanted with IGF1R + mesenchymal stem cells, control rats, and sham-operated rats.
  • FIG. 23 there is a graph of the results of the plum three-color staining of acute myocardial infarction rats after transplantation of mesenchymal stem cells and IGFIR + mesenchymal stem cells for 28 days.
  • the above figure is a photomicrograph of the heart tissue section of rats with acute myocardial infarction stained by Meisheng trichrome staining. The fibrosis in the heart tissue is divided into 0-5 grades. The more severe the degree of fibrosis, the results of different groups were classified and counted according to the above criteria. The statistical results are shown in the lower figure of Fig. 23.
  • the results in Figure 23 show that transplantation of mesenchymal stem cells and IGF1R + mesenchymal stem cells can improve the fibrosis of heart tissue in rats with acute myocardial infarction, and the ability of transplantation of IGF1R + mesenchymal stem cells to improve cardiac tissue fibrosis in rats with acute myocardial infarction is more
  • the mesenchymal stem cell group was p ⁇ 0.05 compared with the control group
  • the IGF1R + mesenchymal stem cell group was p ⁇ 0.01 compared with the control group.
  • the isolated mesenchymal stem cells of the present invention express the insulin-like growth factor-1 receptor on the cell surface, which has the characteristics of self-renewal and pluripotent differentiation.
  • the medium is made of hUCS, and hUCS is rich in growth factors, especially PDGF-BB, which can cause mesenchymal stem cells to express insulin-like growth factor-1 in large amounts.
  • PDGF-BB growth factor-BB
  • the method for isolating pluripotent mesenchymal stem cells of the present invention can screen cells positive for IGF1R from a mixture of cells derived from mammalian tissue, and more preferably, can be rescreened for
  • the cells with positive IL22RA1 are selected as mesenchymal stem cells with pluripotent differentiation ability, so the pluripotent mesenchymal stem cells can be purified quickly and specifically.
  • the isolated mesenchymal stem cells of the present invention are used for cell therapy, they are useful for treating brain tissue damage in an individual and ischemic heart disease in an individual.
  • the isolated mesenchymal stem cells of the present invention can increase glucose metabolic activity, promote angiogenesis, and enhance neurite regeneration in a portion of brain tissue damage of an individual, and by interaction of IGF1R and CXCR4, It has the effect of nerve remodeling.
  • the isolated mesenchymal stem cells of the present invention can alleviate left ventricular dysfunction after myocardial infarction, reduce infarct size, reduce fibrosis due to myocardial infarction, and reduce immune response. .

Abstract

提供一种间质干细胞、其克隆源性扩增的方法、其分离方法及其应用，所述间质干细胞表达胰岛素样生长因子-1受体，且具有自我更新能力和多能性分化的能力。

Description

间质干细胞、其克隆源性扩增的方法、其分离方法及其应用 技术领域
本发明涉及一种干细胞，特别是一种表现特殊受体的间质干细胞。
背景技术
干细胞(Stem Cell)是生物体内尚未分化的原生细胞，其具有可以长时间地不断复制、更新，并分化衍生成具有特殊型态和功能的成熟细胞的能力。干细胞依其来源主要可分成胚胎干细胞(embryonic stem cells，ESCs)及成体干细胞(adult stem cell)两种，胚胎干细胞取自囊胚里的内细胞团，而成体干细胞则来自各式各样的组织。干细胞又依其分化能力，主要可分成三大类，一为全能性干细胞(totipotent stem cells)，全能性干细胞具有完全能力，可分化发育成为完整胚胎或生物体；二为多能性干细胞(pluripotent stem cells)，多能性干细胞具有分化为三个胚层的能力，但无法发育成为完整胚胎或生物体，而可形成某种组织或器官的所有细胞；三为复效性干细胞(multipotent stem cells)，包括特定组织的干细胞，如神经干细胞、血球干细胞、肝脏干细胞、皮肤干细胞等。
间质干细胞(mesenchymal stem cells，MSCs)为一种成体干细胞，属于多能性干细胞，具有干细胞增生及多向分化的能力，可分化成身体所需的神经细胞、血管细胞、胶质细胞、脂肪细胞或骨头细胞等多种间质组织。间质干细胞可由骨髓、脂肪、牙髓或脐带等间质组织当中取得，依据取得来源的不同，也会有倾向分化为某些特定组织的能力，而当体内组织受损时，间质干细胞能直接或间接进行修复。
间质干细胞可应用于神经、心脏、肝脏、肺脏、肾脏、骨胳、软骨、视网膜受伤的修复，近年来更发现间质干细胞具有免疫调整功能，可能有助于治疗许多免疫异常的疾病。由于间质干细胞的抗原性比其他干细胞小，临床运用时不像造血干细胞在移植前必须先经过严格配对，也不像胚胎干细胞在使用时有伦理上的考量，因此是很好的细胞治疗来源。在临床的应用上，间 质干细胞的自我更新和多能性分化能力至为关键，因此与维持间质干细胞多能性相关的细胞表面受体，已成为干细胞医疗相关技术研发的主要课题之一。
发明内容
依据本发明的一实施例是在提供一种经分离的间质干细胞，其表达胰岛素样生长因子-1受体(insulin-like growth factor 1 receptor，IGF1R)。
依据前述经分离的间质干细胞，其中经分离的间质干细胞为多能性。
依据前述经分离的间质干细胞，其中经分离的间质干细胞可为人类细胞，更佳地可为脐带间质干细胞。
借此，本发明的经分离的间质干细胞是于细胞表面表达胰岛素样生长因子-1受体，其具有自我更新和多能性(pluripotent)分化的特性。
本发明的另一实施例是在提供一种间质干细胞克隆源性扩增(clonogenic expansion)的方法，包含于含人类脐带血血清的培养基中培养前述的经分离的间质干细胞，其中经分离的间质干细胞表达胰岛素样生长因子-1受体。
依据前述经分离的间质干细胞克隆源性扩增的方法，其中培养基的人类脐带血血清的浓度可为1～10％(v/v)，较佳地可为2％(v/v)。
借此，本发明的间质干细胞克隆源性扩增的方法中，培养基是使用人类脐带血血清为原料，脐带血血清中富含生长因子，可使间质干细胞大量表达胰岛素样生长因子-1受体，并维持多能性分化能力，而使用人类脐带血血清的培养基方便获得，且可避免因使用非人类血清所引起的过敏反应及感染病毒或病原菌的危险。
本发明的再一实施例是在提供一种分离多能性的间质干细胞的方法，包含提供来自哺乳动物组织的细胞混合物，进行分离步骤。分离步骤是自细胞混合物分离对于胰岛素样生长因子-1受体呈阳性的细胞，以获得多能性的间质干细胞。
依据前述分离多能性的间质干细胞的方法，其中分离步骤更包含分离对于介白素-22受体(interleukin 22 receptor alpha 1，IL22RA1)呈阳性的细胞。
依据前述分离多能性的间质干细胞的方法，其中哺乳动物组织是选自骨髓、牙髓、胎盘、脐带、脐带血及脂肪所组成的族群中。
借此，本发明的分离多能性间质干细胞的方法，可自哺乳动物组织来源的细胞混合物中，筛选对于IGF1R呈阳性的细胞，更佳地，可再筛选对于IL22RA1呈阳性的细胞，筛选出的即为具多能性分化能力的间质干细胞，故可快速且专一的纯化多能性的间质干细胞。
本发明的又一实施例是在提供一种前述的经分离的间质干细胞的用途，其是用于制备治疗缺血性心脏病的药物。
依据前述经分离的间质干细胞的用途，其中缺血性心脏病为心肌梗塞。
依据前述经分离的间质干细胞的用途，其中缺血性心脏病的药物可为减少因心肌梗塞引起的纤维化的药物或减少免疫反应的药物。
借此，将本发明的经分离的间质干细胞用于细胞治疗时，其可以减轻心肌梗塞后左心室的功能障碍、降低梗塞面积、减少因心肌梗塞引起的纤维化的以及减少免疫反应，故可用以治疗个体的缺血性心脏病。
本发明的又一实施例是在提供一种前述的经分离的间质干细胞的用途，其是用于制备治疗个体脑组织损伤的药物。
依据前述经分离的间质干细胞的用途，其中脑组织损伤是由脑缺血疾病造成，例如中风。
依据前述经分离的间质干细胞的用途，其中脑组织损伤是由神经退化性疾病造成，例如帕金森氏症。
依据前述经分离的间质干细胞的用途，其中治疗个体脑组织损伤的药物可为增加葡萄糖代谢活性的药物、促进血管新生的药物或促进神经突再生的药物。
借此，本发明的经分离的间质干细胞用于细胞治疗时，其可以增加葡萄糖的代谢活性、促进血管新生和加强神经突再生，故可用以治疗脑组织损伤的个体。
上述发明内容旨在提供本公开内容的简化摘要，以使阅读者对本公开内容具备基本的理解。此发明内容并非本公开内容的完整概述，且其用意并非在指出本发明实施例的重要/关键元件或界定本发明的范围。
附图说明
为使本发明的上述和其他目的、特征、优点与实施例能更明显易懂，提 供附图，所附附图的说明如下：
图1A为脐带间质干细胞初代培养的显微照片图；
图1B为本发明的经分离的间质干细胞的胰岛素样生长因子-1受体(insulin-like growth factor 1 receptor，IGF1R)表达的结果图；
图1C为本发明的经分离的间质干细胞的介白素-22受体(interleukin 22 receptor alpha 1，IL22RA1)表达的结果图；
图1D和图1E为本发明的经分离的间质干细胞的华顿氏胶中细胞的特异性细胞表面分子表达的结果图；
图1F为本发明的经分离的间质干细胞的多能性标记表达的结果图；
图2为本发明的经分离的间质干细胞的扩增指数图；
图3A为人类脐带血血清(human cord blood serum，hUCS)和胎牛血清(Fetal Calf serum，FCS)的人类细胞激素晶片分析图；
图3B为hUCS和FCS的ELISA分析图；
图4A为本发明的经分离的间质干细胞导入shRNA后IGF1R表达量分析图；
图4B为本发明的经分离的间质干细胞的扩增趋势图；
图4C为本发明的经分离的间质干细胞的细胞增生分析图；
图5A为本发明的经分离的间质干细胞的流式细胞仪双染法分析图；
图5B为本发明的经分离的间质干细胞的免疫荧光染色双染法的显微照片图；
图5C为间质干细胞表达多能性标记的定量RT-PCR结果图；
图6A为本发明的经分离的间质干细胞分化为不同组织细胞的显微照片图；
图6B为本发明的经分离的间质干细胞分化为神经细胞的显微照片图；
图7A为处理不同剂量的胰岛素样生长因子-1(insulin-like growth factor 1，IGF1)至本发明的经分离的间质干细胞后，其IGF1R和趋化因子CXCR4表达量的分析图；
图7B为处理不同剂量的血小板衍生的生长因子-BB(platelet-derived growth factor BB，PDGF-BB)至本发明的经分离的间质干细胞后，其IGF1R和趋化因子CXCR4表达量的分析图；
图7C为同时处理不同剂量的IGF1和PDGF-BB至本发明的经分离的间质干细胞后，其IGF1R表达量的分析图；
图7D为同时处理不同剂量的IGF1和PDGF-BB至本发明的经分离的间质干细胞后，其磷酸化蛋白质激酶B(p-Akt)和磷酸化信号传递转录活化基因-3(p-Stat3)表达量的分析图；
图8A为大鼠身体摆动测试的结果图；
图8B为阻断试验的大鼠身体摆动测试的结果图；
图9A为大鼠垂直活动测试的结果图；
图9B为大鼠垂直活动时间测试的结果图；
图9C为大鼠垂直运动数量测试的结果图；
图10为大鼠前肢抓握力测试的结果图；
图11A为经细胞治疗后大鼠的[18F]氟-2-脱氧葡萄糖正子造影(FDG-PET)影像图；
图11B为经细胞治疗后大鼠的[18F]FDG-PET影像的量化图；
图12为经细胞治疗后大鼠受损脑组织的抗雕亡蛋白表达量的分析图；
图13A为经细胞治疗后大鼠脑组织中双苯酰亚胺标定的细胞核和人类细胞核抗原共同表达(co-localization)的显微照片图；
图13B为经细胞治疗后大鼠脑组织中植入的本发明的经分离的间质干细胞数量的显微照片图；
图14A为植入中风大鼠受损脑组织的本发明经分离的间质干细胞GFAP与IGF1R或CXCR4共同表达的显微照片图；
图14B为植入中风大鼠受损脑组织的本发明经分离的间质干细胞MAP-2与IGF1R或CXCR4共同表达的显微照片图；
图14C为植入中风大鼠受损脑组织的本发明经分离的间质干细胞NeuN与IGF1R或CXCR4共同表达的显微照片图；
图15A为植入中风大鼠受损脑组织的本发明经分离的间质干细胞vWF与IGF1R或CXCR4共同表达的显微照片图；
图15B为移植本发明经分离的间质干细胞的中风大鼠FITC-葡聚醣灌流的结果图；
图15C为本发明经分离的间质干细胞的中风大鼠血管密度测定的结果 图；
图16为移植本发明经分离的间质干细胞的中风大鼠缺血性脑的脑血流量结果图；
图17A为神经突再生体内试验的结果图；
图17B为神经突再生体外试验的结果图；
图18为移植本发明经分离的间质干细胞的急性心肌梗塞大鼠的心肌梗塞面积图；
图19A为移植本发明经分离的间质干细胞的急性心肌梗塞大鼠的心脏超音波图；
图19B为移植本发明经分离的间质干细胞的急性心肌梗塞大鼠心脏超音波的量化图；
图20为移植本发明经分离的间质干细胞3天后的急性心肌梗塞大鼠的免疫组织化学染色法结果图；
图21为移植本发明经分离的间质干细胞3天后的急性心肌梗塞大鼠的免疫荧光染色法结果图；
图22为移植本发明经分离的间质干细胞3天后的急性心肌梗塞大鼠促发炎因子表达量的分析图；以及
图23为移植本发明经分离的间质干细胞28天后的急性心肌梗塞大鼠的梅生三色染色法结果图。
具体实施方式
本说明书公开内容提出一种经分离的间质干细胞，其表达特定的细胞表面受体，且具自我更新能力和多能性(pluripotent)分化能力。本说明书公开内容另提供一种间质干细胞克隆源性扩增(clonogenic expansion)的方法，其可使前述的经分离的间质干细胞大量表达特定的细胞表面受体，并维持多能性分化能力。本说明书公开内容也提供一种分离多能性的间质干细胞的方法，其可由来自哺乳类动物组织的细胞混合物中，快速且专一性的筛选出具多能性分化能力的间质干细胞。此外，本说明书公开内容提供一种前述的经分离的间质干细胞的用途，其可应用于制备治疗缺血性心脏病的药物或制备治疗个体脑组织损伤的药物。
更进一步说，本发明提供一种经分离的间质干细胞，其表达胰岛素样生长因子-1受体(insulin-like growth factor 1 receptor，IGF1R)，下文中将以表示本发明的经分离的间质干细胞。IGF1R+间质干细胞为多能性，其来源可为人类细胞，更佳地为脐带间质干细胞。而本发明的间质干细胞克隆源性扩增的方法，是将IGF1R+间质干细胞培养于含有人类脐带血血清(human cord blood serum，hUCS)的培养基中，其中hUCS的浓度为1～10％(v/v)，更佳地，hUCS的浓度为2％(v/v)。而本发明的分离具多能性的间质干细胞的方法，是自哺乳动物组织来源的细胞混合物中，筛选对于IGF1R呈阳性的细胞，更佳地，可再筛选对于介白素-22受体(interleukin 22 receptor alpha 1，IL22RA1)呈阳性的细胞，即可自细胞混合物中分离多能性的间质干细胞，其中哺乳动物组织是选自骨髓、牙髓、胎盘、脐带、脐带血及脂肪所组成的族群中。
本发明的IGF1R+间质干细胞可应用于制备治疗缺血性心脏病的药物和制备治疗个体脑组织损伤的药物。更进一步地说，本发明的IGF1R+间质干细胞可以减轻心肌梗塞后左心室的功能障碍、降低梗塞面积、减少因心肌梗塞引起的纤维化的以及减少免疫反应，故可用以治疗个体的缺血性心脏病，其中缺血性心脏病可为心肌梗塞。另本发明的IGF1R+间质干细胞可以增加葡萄糖的代谢活性、促进血管新生和加强神经突再生，并藉由IGF1R和趋化因子受体CXCR4的交互作用，具有神经重塑的效果，故可用以治疗脑组织损伤的个体，其中脑组织损伤可为脑缺血疾病或神经退化性疾病，而脑缺血疾病可为中风，神经退化性疾病可为帕金森氏症。
以下为本说明书中所用特定名词的说明：
前述的是胰岛素样生长因子1(insulin-like growth factor 1 receptor，IGF1)的细胞表面受体，属于酪氨酸激酶受体家族。其配体IGF1，是一种在分子结构上与多肽类激素胰岛素类似的激素，在生长发育和成人的合成代谢中有重要作用。
前述的是介白素-22(interleukin 22，IL22)的细胞表面受体，其配体IL-22是一种同时具有抗炎和促炎双重特性的细胞因子，可由多种免疫细胞分泌。
前述的是基质细胞衍生因子1(stromal cell-derived factor-1a，SDF-1)的特异受体，CXCR4在体内大部分组织和器官上都有表达，是由352个胺 基酸组成的G蛋白偶联受体(G protein-coupled receptor，GPCR)，具有七次穿膜结构。其配体SDF-1对淋巴细胞有强烈的趋化作用。
兹以下列具体实验例进一步示范说明本发明，用以有利于本发明所属技术领域技术人员，可在不需过度解读的情形下完整利用并实践本发明，而不应将这些实验例视为对本发明范围的限制，但用于说明如何实施本发明的材料及方法。
<实验例>
第一部分：本发明的IGF1R+间质干细胞
1.1：IGF1R+间质干细胞制备
为制备IGF1R+间质干细胞，于本实验例中所使用的哺乳动物组织为人类脐带组织，而人类脐带组织中的人类脐带间质干细胞源自华顿氏胶(Wharton′s jelly)中。将所收集的人类脐带组织先以不含Ca2+和Mg2+的PBS(DPBS，Life Technology)洗涤三次后，将人类脐带组织在中线方向以剪刀剪开，并自华顿氏胶中抽出脐动脉血管、脐静脉血管和外膜。再将华顿氏胶的间质组织切割成小于0.5cm3的立方体，以胶原蛋白酶1型(Sigma)处理，并在37℃下于5％CO2、95％饱和湿度培养14-18小时后。再将外植体分别以含有2％hUCS或10％胎牛血清(Fetal Calf serum，FCS)的DMEM为细胞培养基，并在细胞培养基中加入抗生素，在37℃下于5％CO2、95％饱和湿度静置培养5-7天。人类脐带组织的外植体培养，将维持到发现有梭形贴壁细胞自外植体中向外生长。
参照图1A，为脐带间质干细胞初代培养的显微照片图，其中黑色箭头所指的处为外植体，在培养4至8代后，可见细胞自外植体中迁移，且细胞形态变为均匀的梭状。
后续再以流式细胞仪分析前述初代培养的脐带间质干细胞的细胞表面分子。分析的细胞表面分子包含IGF1R、IL22RA1、华顿氏胶中细胞的特异性细胞表面分子以及多能性标记。华顿氏胶中细胞的特异性细胞表面分子包含CD13、CD29、CD34、CD44、CD45、CD73、CD90、CD105、CD117、CD166、HLA-ABC和HLA-DR。多能性标记包含Oct-4、Sox-2、Nanog和SSEA-4。
参照图1B至图1F，图1B为本发明的经分离的间质干细胞IGF1R表达 的结果图，图1C为本发明的经分离的间质干细胞的IL22RA1表达的结果图，图1D和图1E为本发明的经分离的间质干细胞的华顿氏胶中细胞的特异性细胞表面分子表达的结果图，图1F为本发明的经分离的间质干细胞的多能性标记表达的结果图。
图1B中先以由流式细胞仪分析前述初代培养的脐带间质干细胞的IGF1R表达，再以Western印迹法分析不同来源的间质干细胞的IGF1R表达，分析的细胞种类包含人类纤维母细胞、人类牙髓干细胞、脂肪间质干细胞、脐带间质干细胞以及骨髓间质干细胞，图1B的结果显示，不同来源的间质干细胞皆可见IGF1R的表达。
图1C至图1F以流式细胞仪分析前述初代培养的脐带间质干细胞的IL22R1A表达、华顿氏胶中细胞的特异性细胞表面分子表达以及多能性标记表达。由图1C的结果显示，前述初代培养的脐带间质干细胞可见IL22R1A的表达，与对照组相比约有45.4％的脐带间质干细胞表达IL22R1A。由图1D和图1E的结果显示，前述初代培养的脐带间质干细胞可见CD13、CD29、CD44、CD73、CD90、CD105、CD166和HLA-ABC的表达，而未见CD34、CD45、CD117和HLA-DR的表达，显示前述初代培养的脐带间质干细胞并非造血干细胞表达型。而由图1F的结果显示，前述初代培养的脐带间质干细胞皆表达Oct-4、Sox-2、Nanog和SSEA-4代表多能性分化能力的多能性标记。
实验上再自前述经流式细胞仪分析的细胞中分选出IGF1R+间质干细胞。纯化的方法如下：将细胞与抗IGF1抗体混合后，使用FACSTAR+流式细胞仪(Becton Dickinson)分选出染上抗IGF1抗体的细胞，再以锥蓝质排除测试(trypan blue exclusion test)分析分选出的细胞，细胞存活率大约为96％。
1.2：IGF1R+间质干细胞培养
分选出的IGF1R+间质干细胞分别以含有2％hUCS或10％FCS的DMEM为细胞培养基，并在细胞培养基中加入抗生素，于37℃下于5％CO2、95％饱和湿度的环境培养。并分别分析培养于含hUCS和FCS的IGF1R+间质干细胞的生长动力学。
参照图2，为IGF1R+间质干细胞的扩增指数图。由图2的结果显示， 培养于含hUCS的培养基中的IGF1R+间质干细胞分裂的速度，较培养于含FCS的培养基中的IGF1R+间质干细胞还快，其倍增一代的时间为22小时，并可扩增超过150天而没有衰老和自发分化的迹象。
为评估使用含hUCS的培养基培养IGF1R+间质干细胞的优点，实验上以人类细胞激素晶片(RayBiotechTM)分析比较hUCS和FCS中多个特定的细胞激素。于本实验例中，共分析42种细胞激素，并透过光密度测定hUCS或FCS中细胞激素的表达水平。
参照图3A，为hUCS和FCS的人类细胞激素晶片分析图，其中P表示正对照，N表示负对照。由图3A的结果显示，在hUCS中有5种细胞激素的表达水平显著高于FCS，分别为表皮生长因子(epidermal growth factor，EGF；方框1)、血管生成素(angiogenin，ANG；方框2)、巨噬细胞炎性蛋白(macrophage inflammatory protein，MIP-1δ；方框3)、趋化激素RANTES(regulated on activation，normal T-cell expressed and presumably secreted，RANTES；方框4)和血小板衍生的生长因子-BB(platelet-derived growth factor BB，PDGF-BB)，此5种细胞激素表达水平相差的倍数分别为2倍、3倍、3倍、2倍和4倍。相较之下，在hUCS和FCS中胰岛素样生长因子1(insulin-like growth factor 1，IGF-1)的表达水平相似。
为了更准确地定量测定hUCS和FCS中PDGF-BB和IGF-1的浓度，本实验例以hUCS和FCS为样品，利用ELISA(enzyme-linked immunosorbent assay)进行PDGF-BB和IGF-1浓度分析。
参照图3B，为hUCS和FCS的ELISA分析图，由图3B的结果显示，2种血清中的IGF1表达水平相似，但在PDGF-BB的部分，hUCS中含有的PDGF-BB浓度显著的高于FCS中的含量(p＜0.05)。
1.3：IGF1R+间质干细胞的自我更新能力
为了探讨IGF1R信息途径是否有助于间质干细胞自我更新的调控，实验上使用慢病毒分别导入靶向IGF1R的shRNA(LV-IGF1R-sh，sc-29358-V，Santa Cruz Biotechnology)至以含hUCS或FCS的培养基培养的IGF1R+间质干细胞中，以降低IGF1R+间质干细胞中IGF1R的表达，实验上也使用慢病毒分别导入控制组的shRNA(LV-control-sh，Santa Cruz Biotechnology)至以含 hUCS或FCS的培养基培养的IGF1R+间质干细胞中做为实验对照组。并在感染慢病毒48小时后，利用Western印迹法分析导入shRNA后IGF1R+间质干细胞的IGF1R的表达量。另分析导入shRNA后IGF1R+间质干细胞的生长动力学。
参照图4A至图4B，图4A为导入shRNA后IGF1R表达量分析图，图4B为IGF1R+间质干细胞的扩增趋势图。图4A中共有4个组别，分别为使用慢病毒分别导入LV-IGF1R-sh和导入LV-control-sh的IGF1R+间质干细胞，以及未导入shRNA的IGF1R+间质干细胞，在图4A中4个组别的IGF1R+间质干细胞皆是以含hUCS的培养基培养。由图4A的结果显示，在感染慢病毒48小时后，导入LV-IGF1R-sh的组别相较于导入LV-control-sh的组别，其IGF1R的表达量降低，而其它组别的IGF1R表达量相当。而图4B中共有4个组别，分别为以含hUCS的培养基培养并导入LV-control-sh的IGF1R+间质干细胞(LV-control-sh U-IGF1R+间质干细胞)、以含FCS的培养基培养并导入LV-control-sh的IGF1R+间质干细胞(LV-control-sh F-IGF1R+间质干细胞)、以含hUCS的培养基培养并导入LV-IGF1R-sh的IGF1R+间质干细胞(LV-IGF1R-sh-U-IGF1R+间质干细胞)，以及以含FCS的培养基培养并导入LV-IGF1R-sh的IGF1R+间质干细胞(LV-IGF1R-sh-F-IGF1R+间质干细胞)。由图4B的生长动力学结果显示，不论是以含hUCS或FCS的培养基培养的IGF1R+间质干细胞，导入LV-IGF1R-sh后，皆会减缓IGF1R+间质干细胞的增生。
本实验例进一步利用尿密啶类似物(Bromodeoxyuridine，BrdU)标定DNA，以细胞增生分析法(BrdU proliferation assay)进行检测，探讨IGF1R+间质干细胞的增殖潜力。实验上先将IGF1R+间质干细胞培养于未添加补充物的培养基中4至6小时，再将IGF1R+间质干细胞分别培养于含2％hUCS、10％FCS或100ng/mL SDF-1的培养基中2天，并分别导入LV-control-sh或LV-IGF1R-sh，再以BrdU chemiluminescence immunoassay kits(Roche)分析IGF1R+间质干细胞的增生情况。
参照图4C，为IGF1R+间质干细胞的细胞增生分析图。图4C中共有5个组别，分别为以添加SDF-1的培养基培养的IGF1R+间质干细胞作为正控制组、以含hUCS的培养基培养并导入LV-control-sh的IGF1R+间质干细 胞、以含FCS的培养基培养并导入LV-control-sh的IGF1R+间质干细胞、以含hUCS的培养基培养并导入LV-IGF1R-sh的IGF1R+间质干细胞，以及以含FCS的培养基培养并导入LV-IGF1R-sh的IGF1R+间质干细胞。由图4C的结果显示，以含hUCS的培养基培养的IGF1R+间质干细胞，显著地较以含FCS的培养基培养的IGF1R+间质干细胞有更多的BrdU嵌入。相较之下，不论是以含hUCS或FCS的培养基培养的IGF1R+间质干细胞，导入LV-IGF1R-sh后，BrdU的嵌入量将大幅下降，显示IGF1R为间质干细胞增值时不可或缺的细胞表面受体。
1.4：IGF1R+间质干细胞的多能性
为探讨IGF1R与IGF1R+间质干细胞的多能性是否有关，实验上分别将IGF1R+间质干细胞双染IGF1R/Oct-4抗体、IGF1R/Sox-2抗体、IGF1R/Nanog抗体、IGF1R/SSEA4抗体，再分别以流式细胞仪和免疫荧光染色法分析IGF1R和多能性标记共表达的情况。
参照图5A和图5B，图5A为IGF1R+间质干细胞的流式细胞仪双染法分析图，图5B为IGF1R+间质于细胞的免疫荧光染色双染法的显微照片图，其中IGF1R+间质干细胞的细胞来自5个独立的样本。由图5A和图5B的结果显示，IGF1R+间质干细胞中IGF1R与多能性标记Oct-4、Sox-2、Nanog和SSEA-4共表达，而图5B的结果更显示，IGF1R也与CXCR4共表达。
本实验例进一步以定量RT-PCR(qRT-PCR)探讨IGF1R+间质干细胞和不表达IGF1R的间质干细胞(IGF1R-间质干细胞)其多能性标记的表达差异。参照图5C，为IGF1R+间质干细胞和IGF1R-间质干细胞表达多能性标记的定量RT-PCR结果图，其中试验的细胞分别为IGF1R+间质干细胞和IGF1R-间质干细胞，并以人类纤维母细胞作为负向控制组，人类诱导性多功能干细胞(induced pluripotent stem cell，iPS)作为正向控制组。结果显示，不论Oct-4、Sox-2、SSEA-4或Nanog的表达，IGF1R+间质干细胞皆较IGF1R-间质干细胞的表达量高，显示IGF1R为间质干细胞维持多能性重要的细胞表面受体。
1.5：IGF1R+间质干细胞的分化能力
本实验例以体外分化试验进一步探讨以含hUCS或FCS的培养基培养IGF1R+间质干细胞，是否会影响其多能性分化能力。实验上分别将培养于含hUCS或FCS培养基继代5至10代的IGF1R+间质干细胞，以5×103细胞/cm2的密度种于细胞培养皿中，并分别以含有10％FCS或2％hUCS不同的分化培养基培养IGF1R+间质干细胞，引导其分化为脂肪细胞、软骨细胞、成骨细胞、血管形成和神经细胞，并在显微镜下观察细胞型态，以及进一步以染色法确认分化后的细胞种类。
参照图6A和图6B，图6A为IGF1R+间质干细胞分化为不同组织细胞的显微照片图，图6B为IGF1R+间质干细胞分化为神经细胞的显微照片图。
图6A中利用油红组织染色(oil red O stain)确认分化后的细胞为脂肪细胞，利用茜素红染色(Alizarin red S stain)确认分化后的细胞为成骨细胞，以及利用阿新蓝染色(Alican blue stain)确认分化后的细胞为软骨细胞，并以明视野下的细胞型态确认IGF1R+间质干细胞是否会血管形成。由图6A的结果显示，IGF1R+间质干细胞在分化培养基的培养下，可以分化为脂肪细胞、软骨细胞、成骨细胞以及血管形成。
图6B中明视野的显微照片显示，培养于神经细胞分化培养基的IGF1R+间质干细胞，可见扩展神经突状结构细胞型态，显示分化后的细胞为神经细胞。本实验例更进一步以免疫荧光染色确认分化后的细胞是否表达成熟神经标记，成熟神经标记包含GFAP(glial fibrillary acidic protein)、MAP-2(microtubule-associated protein 2)、O4和Tuj-1(Neuron-specific class III beta-tubulin)。由图6B的结果显示，分化后的细胞4种成熟神经标记皆有表达，证明分化后的细胞确实为神经细胞。
另参照下表一，为以含有2％hUCS分化培养基或10％FCS分化培养基培养的IGF1R+间质干细胞，4种成熟神经细胞标记的表达比例。结果显示，以含2％hUCS分化培养基培养的IGF 1R+间质干细胞，不论是哪一种成熟神经标记的表达比例皆较以含10％FCS分化培养基培养的高，显示以含hUCS培养基培养的IGF1R+间质干细胞更容易分化为神经细胞。
表一
血清 GFAP MAP-2 O4 Tuj-1
2％hUCS 15.2±3.1％ 12.1±3.1％ 9.4±2.1％ 10.2±1.7％
10％FCS 8.6±2.2％ 7.1±2.7％ 5.8±1.6％ 6.1±1.5％
1.6：IGF1R+间质干细胞的调控机制
本实验例将探讨IGF1R+间质干细胞的IGF1R和CXCR4表达水平是否单独受IGF1调控，或由IGF1和PDGF-BB(hUCS和FCS差异的关键成分)调控。实验上先分别加入不同剂量的IGF1或不同剂量的PDGF-BB处理IGF1R+间质干细胞，并以Western印迹法侦测IGF1R和CXCR4的表达量。
参照图7A至图7C，图7A为处理不同剂量的IGF1至IGF1R+间质干细胞后，其IGF1R和CXCR4表达量的分析图。图7B为处理不同剂量的PDGF-BB至IGF1R+间质干细胞后，其IGF1R和CXCR4表达量的分析图。图7C为同时处理不同剂量的IGF1和PDGF-BB至IGF1R+间质干细胞后，其IGF1R表达量的分析图。其中*表示实验组(加入不同剂量IGF1或/和PDGF-BB的组别)和控制组(未加入IGF1和PDGF-BB的组别)相比P＜0.05，**表示实验组和控制组相比P＜0.01。由图7A的结果显示，加入IGF1会降低IGF1R+间质干细胞的IGF1R表达，且两者之间的关系呈现剂量依赖性，但加入IGF1会增加CXCR4的表达，且在加入IGF1浓度为5nM时，CXCR4的表达量最高。而由图7B的结果显示，加入PDGF-BB会同时提高IGF1R和CXCR4的表达，且存在剂量依赖性。但由图7C的结果显示，加入PDGF-BB至IGF1R+间质干细胞中，可以有效抑制因IGF1引导的IGF1R表达降低。
为了进一步确定PDGF-BB是否比IGF1更有效地激活控制细胞增殖的信息途径，IGF1R+间质干细胞分别处理不同浓度的IGF1或不同浓度的PDGF-BB(在无血清的条件下)，并以Western印迹法侦测磷酸化蛋白质激酶(phosphorylated Akt，p-Akt)和磷酸化信号传递转录活化基因-3(phosphorylated signal transducers and activator of transcription 3，p-Stat3)的表达水平。
参照图7D，为同时处理不同剂量的IGF1和PDGF-BB至IGF1R+间质干细胞后，其p-Akt和p-Stat3表达量的分析图，其中*表示实验组(加入不同剂量IGF1或/和PDGF-BB的组别)和控制组(未加入IGF1和PDGF-BB的组 别)相比P＜0.05。图7D的结果显示，加入PDGF-BB显著地增加p-Akt和p-Stat3的表达量，但加入IGF1则对增加p-Akt和p-Stat3表达量没有效果。本试验另分别加入p-Akt抑制剂(LY294002)和p-Stat3抑制剂(AG490)至IGF1R+间质干细胞中，发现由PDGF-BB引导的Akt和Stat3磷酸化完全被抑制(结果未显示)。上述结果显示，PDGF-BB比IGF1更有效地激活下游重要的信息传导途径，也显示含hUCS的培养基比含FCS的培养基更适于作为间质干细胞的培养基。
第二部分：IGF1R+间质干细胞用于治疗脑组织损伤
根据前述已知IGF1R+间质干细胞具有自我更新能力及多能分化能力，此部份的实验例将探讨将IGF1R+间质干细胞用于治疗脑组织损伤个体的效果。
2.1：IGF1R+间质干细胞移植改善中风大鼠模型的神经行为
本实验例是以中风大鼠模型评估IGF1R+间质干细胞在体内自我更新能力和的神经再生潜力，并透过3种神经功能缺损模式检测中风前后大鼠的神经行为，以评估神经功能是否恢复。
中风大鼠模型采取脑缺血/再灌注模型(ischemia-reperfusion model)来模拟大鼠中暂时性局部脑缺血的症状，所使用的试验动物为体重250-300克雄性SD(Sprague-Dawley)大鼠。大鼠的脑缺血/再灌注模型是以阻塞大鼠的双侧颈总动脉(common carotid arteries；CCA)及右侧大脑中动脉(middle cerebral artery；MCA)诱发局部性脑缺血，阻塞90分钟后解除以进行再灌注，1小时后再静脉移植细胞至大鼠中，注射的细胞量为2×106。实验上先将大鼠分成5组，分别注射5种不同细胞或载体进行细胞治疗，组别1的细胞为培养于含hUCS培养基的IGF1R+间质干细胞(U-IGF1R+间质干细胞)，组别2的细胞为培养于含FCS培养基的IGF1R+间质干细胞(F-IGF1R+间质干细胞)，组别3的细胞为培养于含hUCS培养基的间质干细胞(U-间质干细胞)，组别4的细胞为培养于含FCS的培养基的间质干细胞(F-间质干细胞)，组别5则为注射PBS(载体)至大鼠体内作为对照组。而组别1的大鼠将另进行阻断试验，分别注射CXCR4抗体(R&D system)和IGF1R抑制剂(PPP，Santa Cruz Biotechnology)以阻断CXCR4和IGF1R。CXCR4抗体以腹腔注 射至大鼠体内2周，注射频率为每周2次。PPP以腹腔注射至大鼠体内3天，注射量为20mg/kg/day。
神经行为的检测时间点为脑缺血/再灌注前5天、细胞移植后第1天、第7天、第14天和第28天。3种神经功能缺损模式分别为评估大鼠的身体左右不对称、活动能力和前肢抓握力。
i.身体摆动测试
大鼠的身体左右不对称是利用身体摆动测试评估，实验上先将大鼠悬空放置在尾巴上方10公分的饲养笼底板上，并记录大鼠的横向移动次数，特别是缺血侧对侧的头部摆动频率是以连续20计数，并以基线评分进行标准化。
参照图8A和图8B，图8A为大鼠身体摆动测试的结果图，图8B为阻断试验的大鼠身体摆动测试的结果图。由图8A的结果显示，不论移植何种间质干细胞的大鼠，相较于对照组有显著的神经功能恢复。而移植以含hUCS培养基培养的间质干细胞的组别(组别1和组别3)，大鼠神经功能恢复的状况又较以含FCS培养基培养的间质干细胞的组别(组别2和组别4)更好，其中又以组别1(移植U-IGF1R+UMSCs)比其它组别有显著的治疗效果。而由图8B的结果显示，加入CXCR4抗体或PPP阻断CXCR4或IGF1R后，阻断试验组大鼠的神经功能恢复结果和对照组相当，显示阻断CXCR4或IGF1R会抑制移植U-IGF1R+间质干细胞的治疗效果。
ii.活动能力测试
大鼠的活动能力是使用VersaMax动物活动监测系统(Accuscan Instruments)侦测大鼠2小时。VersaMax动物活动监测系统包含16水平红外线感测器和8个垂直红外感测器，垂直感测器分别位于腔室底板上方10cm处，大鼠的活动能力运动由腔室内大鼠因运动打断光束的次数进行定量。测量的3个垂直运动参数为：垂直活动、垂直活动时间以及垂直运动数量。
参照图9A至图9C，图9A为大鼠垂直活动测试的结果图，图9B为大鼠垂直活动时间测试的结果图，图9C为大鼠垂直运动数量测试的结果图。由图9A至图9C的结果显示，移植间质干细胞的大鼠相较于对照组有显著的活动能力改善。而移植U-IGF1R+间质干细胞的组别又较移植F-IGF1R+间质干细胞的组别改善活动能力的程度又更大，但加入CXCR4抗体或PPP阻 断CXCR4或IGF1R的阻断试验组别，大鼠的活动能力的结果和对照组相当，显示阻断CXCR4或IGF1R会抑制移植U-IGF1R+间质干细胞的治疗效果。
iii.前肢抓握力测试
大鼠的前肢抓握力测试是使用握力仪(TSE-Systems)侦测。实验上分开测量大鼠每只前肢的抓握力，计算大鼠拉20次抓握力的平均值，并计算同侧：对侧的抓握力比率。此外，本实验例也计算细胞治疗前和细胞治疗后大鼠抓握力的比率，以评估细胞治疗后大鼠抓握力的改变。
参照图10，为大鼠前肢抓握力测试的结果图。由图10的结果显示，移植IGF1R+间质干细胞的大鼠相较于对照组的大鼠，在细胞治疗后大鼠的前肢抓握力大幅度的改善。而移植U-IGF1R+间质干细胞的组别又较移植F-IGF1R+间质干细胞的组别改善前肢抓握力的程度又更大，但加入CXCR4抗体或PPP阻断CXCR4或IGF1R的阻断试验组别，大鼠的前肢抓握力和对照组相当，显示阻断CXCR4或IGF1R会抑制移植U-IGF1R+间质干细胞的治疗效果。
上述3个神经功能缺损模式试验呈现相似的结果，显示IGFR-1+间质干细胞可改善中风大鼠的神经行为，而U-IGFR-1+间质干细胞又较F-IGFR-1+间质干细胞更具神经再生的潜力，且此神经再生是透过IGF1R和CXCR4受体途径。
2.2：IGF1R+间质干细胞移植提高葡萄糖代谢
为了探讨移植U-IGF1R+间质干细胞是否影响脑缺血大鼠的葡萄糖代谢活性，本实验例是使用小型动物专用正子扫描仪(microPET)以[18F]氟-2-脱氧葡萄糖正子造影(fluoro-2-deoxyglucose positron emission tomography，FDG-PET)检测细胞治疗后大鼠脑组织的葡萄糖代谢。
参照图11A和图11B，图11A为经细胞治疗后大鼠的[18F]FDG-PET影像图，图11B为经细胞治疗后大鼠的[18F]FDG-PET影像的量化图，检测的大鼠为经细胞治疗后4周的大鼠，检测的组别共5组，分别为移植U-IGF1R+间质干细胞的大鼠(N＝6)、移植F-IGF1R+间质干细胞的大鼠(N＝6)、移植U-IGF1R+间质干细胞并注射PPP的大鼠(N＝6)、移植U-IGF1R+间质干 细胞并注射CXCR4抗体的大鼠(N＝6)以及对照组大鼠(注射PBS，N＝6)。由图11A的结果显示，移植IGF1R+间质干细胞的大鼠可增加受损脑组织(图中右侧脑)的葡萄糖代谢，而移植U-IGF1R+间质干细胞的组别改善受损脑组织葡萄糖代谢的程度又较移植F-IGF1R+间质干细胞的组别更大。图11B是将[18F]FDG-PET的影像图进行半定量，由图11B的结果显示，移植U-IGF1R+间质干细胞增加的葡萄糖代谢活性，在注射PPP或CXCR4抗体阻断CXCR4或IGF1R后，大鼠在受损脑组织的葡萄糖代谢活性与对照组相当，显示阻断CXCR4或IGF1R会抑制移植U-IGF1R+间质干细胞增加葡萄糖代谢活性的效果。
2.3：静脉移植IGF1R+间质干细胞增加抗雕亡蛋白的表达
为探讨以U-IGF1R+间质干细胞治疗中风大鼠是否是藉由增加存活因子的表达改善的神经功能，实验上使用Western印迹法检测经细胞治疗后大鼠受损脑组织中抗雕亡蛋白的表达，检测的抗雕亡蛋白为Bcl-2(B-cell lymphoma 2)和Bcl-xL(B-cell lymphoma-extra large)。本实验例也检测经细胞治疗后大鼠受损脑组织中促雕亡蛋白的表达，检测的促雕亡蛋白为Bax(BCL2-associated X protein)和Bad(Bcl-2-associated death promoter)。
参照图12，为经细胞治疗后大鼠受损脑组织的抗雕亡蛋白表达量的分析图。由图12的结果显示，经IGF1R+间质干细胞治疗的大鼠，在受损脑组织中抗雕亡蛋白Bcl-2和Bcl-xL的表达量增加，且以U-IGF1R+间质干细胞治疗的大鼠Bcl-2和Bcl-xL表达量增加更为显著，其中F-IGF1R+间质干细胞组别(N＝6)与对照组(N＝6)相比p＜0.05；U-IGF1R+间质干细胞组别(N＝6)与对照组(N＝6)相比p＜0.01。相较之下，经IGF1R+间质干细胞(U-IGF1R+间质干细胞和F-IGF1R+间质干细胞)治疗的大鼠，在受损脑组织中的促雕亡蛋白Bax和Bad的表达量和对照组相当。
2.4：IGF1R+间质干细胞治疗增加体内的神经分化
为分析移植的IGF1R+间质干细胞是否可以分化为神经胶质细胞，实验上将经细胞治疗后28天的中风大鼠，利用共轭焦雷射扫描显微镜，以免疫荧光染色法确认植入中风大鼠受损脑组织中IGF1R+间质干细胞的数量，和 以免疫荧光双染法确定植入的IGF1R+间质干细胞其分化的细胞型态。
参照图13A和图13B，图13A为经细胞治疗后大鼠脑组织中双苯酰亚胺标定的细胞核和人类细胞核抗原共同表达(co-localization)的显微照片图，图13B为经细胞治疗后大鼠脑组织中植入的IGF1R+间质干细胞数量的显微照片图。由图13A的结果显示，所有以双苯酰亚胺标定的IGF1R+间质干细胞皆表达人类细胞核抗原(hNA)，确认细胞的来源为人类组织。由图13B的结果显示，移植U-IGF1R+间质干细胞的组别(N＝8)相较移植F-IGF1R+间质干细胞的组别(N＝8)，在受损脑组织中有更多的植入IGF1R+间质干细胞(p＜0.05)。但由阻断试验组别(同时移植U-IGF1R+间质干细胞和注射PPP或同时移植U-IGF1R+间质干细胞和注射CXCR4抗体)的结果显示，植入IGF1R+间质干细胞数量的差异会因注射PPP或CXCR4抗体而消失。
为进一步确定植入的IGF1R+间质干细胞分化的细胞型态，实验上以免疫荧光双染法标定特定细胞型态标记、IGF1R和CXCR4，以确认特定细胞型态标记与IGF1R共同表达的情况，以及特定细胞型态标记与CXCR4共同表达的情况，并以双苯酰亚胺(bisbenzimide)标定细胞核，特定细胞型态标记包含星形胶质细胞标记GFAP(glial fibrillary acidic protein)、神经元特异性细胞骨架蛋白MAP-2(microtubule-associated protein 2)和神经核标记NeuN(neuronal nuclear antigen)。
参照图14A至图14C，图14A为植入中风大鼠受损脑组织的U-IGF1R+间质干细胞GFAP与IGF1R或CXCR4共同表达的显微照片图，图14B为植入中风大鼠受损脑组织的U-IGF1R+间质干细胞MAP-2与IGF1R或CXCR4共同表达的显微照片图，图14C为植入中风大鼠受损脑组织的U-IGF1R+间质干细胞NeuN与IGF1R或CXCR4共同表达的显微照片图。
由图14A至图14C的结果显示，在植入U-IGF1R+间质干细胞的中风大鼠的受损脑组织区域中，一些双苯酰亚胺标定且表达CXCR4的细胞，这些细胞分别共同表达神经细胞标记(GFAP、MAP-2和NeuN)。此外，也可见双苯酰亚胺标定且表达IGF1R的细胞，这些细胞也分别共同表达神经细胞标记(GFAP、MAP-2和NeuN)。
另参照下表二，为以免疫荧光双染法标定植入的U-IGF1R+间质干细胞的中风大鼠脑组织(N＝8)或F-IGF1R+间质干细胞的中风大鼠脑组织(N＝8)， 并计算免疫荧光双染法中被双苯酰亚胺标定的细胞与GFAP、MAP-2和NeuN共同表达的比例。由表二的结果显示，植入的U-IGF1R+间质干细胞的中风大鼠脑组织细胞，双苯酰亚胺与GFAP、MAP-2或NeuN共同表达的比例(分别为9.5％、12％和10％)皆高于植入的F-IGF1R+间质干细胞的中风大鼠脑组织细胞(分别为4％、5％和4％)，显示以U-IGF1R+间质干细胞治疗中风大鼠，植入的IGF1R+间质干细胞较易分化为神经细胞。
表二
Figure PCTCN2015094825-appb-000001
2.5：IGF1R+间质干细胞移植促进体内的血管新生
本试验以免疫荧光双染法、FITC-葡聚醣灌流和血管密度测定，探讨植入U-IGF1R+间质干细胞增加中风大鼠大脑缺血区域的血管生成。
实验上以免疫荧光双染法标定血管内皮细胞标记vWF(Von Willebrand factor)、IGF1R和CXCR4，以确认血管内皮细胞标记与IGF1R共同表达的情况，以及血管内皮细胞标记与CXCR4共同表达的情况，并以双苯酰亚胺标定细胞核。参照图15A，为植入中风大鼠受损脑组织的U-IGF1R+间质干细胞vWF与IGF1R或CXCR4共同表达的显微照片图，由图15A的结果显示，在中风大鼠的缺血性脑半球的血管周围细胞及内皮区域，可见一些被双苯甲亚胺标定的细胞共同表达IGF1R或CXCR4，且这些细胞共同表达血管内皮细胞标记(vWF)。
本试验进一步以FITC-葡聚醣灌流评估中风大鼠缺血脑组织中的微循环，以探讨中风大鼠缺血组织中血管新生的状况，评估的组别为移植U-IGF1R+间质干细胞的中风大鼠(N＝6)、移植F-IGF1R+间质干细胞的中风大鼠(N＝6)以及注射PBS的对照组中风大鼠(N＝6)。参照图15B，为移植IGF1R+间质干细胞的中风大鼠FITC-葡聚醣灌流的结果图，由图15B的结果显示，移植U-IGF1R+间质干细胞的中风大鼠与其他组别(移植F-IGF1R+间质干细 胞或对照组)相较，显著的增加脑微血管灌流。
本实验例再进一步以免疫荧光染色法标定中风大鼠缺血脑组织切片中的血管内皮细胞标记CD31，以计算血管密度，评估的组别为移植U-IGF1R+间质干细胞的中风大鼠(N＝6)、移植F-IGF1R+间质干细胞的中风大鼠(N＝6)以及注射PBS的对照组中风(N＝6)。参照图15C，为移植IGF1R+间质干细胞的中风大鼠血管密度测定的结果图，由图15C的结果显示，移植U-IGF1R+间质干细胞的中风大鼠于缺血脑组织中的血管密度高于其他组别，其中移植U-IGF1R+间质干细胞与移植F-IGF1R+间质干细胞相比p＜0.05，移植U-IGF1R+间质干细胞与对照组相比p＜0.01。
由上述结果显示，移植IGF1R+间质干细胞至中风大鼠体内进行细胞治疗，可以增加中风大鼠缺血性脑组织中的血管新生，且移植以含hUCS培养基培养的IGF1R+间质干细胞更具有增加缺血性脑组织中血管新生的潜力。
2.6：植入以含hUCS的培养基培养的IGF1R+间质干细胞增加缺血性脑的脑血流量
增加血管密度往往会增加脑血流量(cerebral blood flow，CBF)，进而可以更有效地提供氧气、营养物质以及提高神经元的存活。因此本实验例将进一步监测中风大鼠缺血性脑组织中的脑血流量。实验上先将大鼠以水合氯醛(chloral hydrate)麻醉，以雷射杜卜勒微流仪(Moor Instrutments)监测中风大鼠缺血性脑组织局部血液灌流状况，实验的组别为移植U-IGF1R+间质干细胞的中风大鼠(N＝6)、移植F-IGF1R+间质干细胞的中风大鼠(N＝6)以及注射PBS的对照组中风(N＝6)。参照图16，为移植IGF1R+间质干细胞的中风大鼠缺血性脑的脑血流量结果图，由图16的结果显示，移植IGF1R+间质干细胞可增加中风大鼠缺血脑组织中的脑血流量，且移植U-IGF1R+间质干细胞增加中风大鼠缺血脑组织中的脑血流量的幅度更大，其中移植U-IGF1R+间质干细胞与移植F-IGF1R+间质干细胞相比p＜0.05，移植U-IGF1R+间质干细胞与对照组相比p＜0.01。
2.7：CXCR4和IGF1R的交互作用调节IGF1R+间质干细胞引导的神经再生
本实验例将定量中风大鼠缺血脑组织的神经突再生，以及间质干细胞与初代大脑皮质细胞(primary cortical cells，PCC)共培养(co-culture)后的神经突再生，以探讨IGF1R+间质干细胞和神经组织的交互作用是否刺激体内和体外的神经突生长。
在体内试验的部分，实验上以静脉移植IGF1R+间质干细胞至中风大鼠体内，移植的细胞组别为U-IGF1R+间质干细胞、F-IGF1R+间质干细胞、阻断试验组和对照组，其中阻断试验组是移植导入LV-IGF1R-sh的U-IGF1R+间质干细胞和导入LV-CXCR4-sh的U-IGF1R+间质干细胞至中风大鼠体内，对照组则以静脉注射PBS至中风大鼠体内。牺牲在移植IGF1R+间质干细胞后28天的中风大鼠，将其脑组织切片进行免疫荧光染色以标定神经细胞特异性标记III-tubulin，并以影像分析软体(SigmaScan)计算神经突的长度，并将神经细胞大于细胞体直径2倍以上的细胞计算为神经突细胞(neurite-bearing cells)。
参照图17A，为神经突再生体内试验的结果图，由图17A的结果显示，移植IGF1R+间质干细胞可改善中风大鼠缺血脑组织中的神经突再生，而移植U-IGF1R+间质干细胞的中风大鼠，于缺血脑组织中的神经突再生数量显著的高于其他组别，且其神经突长度也显著的长于其他组别。但由组断试验组的结果显示，阻断IGF1R或CXCR4会抑制移植U-IGF1R+间质干细胞所增加的神经突长度和神经突细胞数量。
为了评估IGF1R+间质干细胞是否能影响初代大脑皮质细胞氧气和葡萄糖供应不足(oxygen glucose deprivation，OGD)的反应，本实验例进一步将初代大脑皮质细胞与IGF1R+间质干细胞共培养于氧气和葡萄糖供应不足的环境，其中IGF1R+间质干细胞包含U-IGF1R+间质干细胞、F-IGF1R+间质干细胞和阻断试验组，阻断试验组的细胞为导入LV-IGF1R-sh的U-IGF1R+间质干细胞和导入LV-CXCR4-sh的U-IGF1R+间质干细胞，实验上另以于氧气和葡萄糖供应不足的环境单独培养的初代大脑皮质细胞作为对照组。再将共培养后的细胞以免疫荧光染色标定III-tubulin，依前述的方法测定神经突的再生和神经元的存活。
参照图17B，为神经突再生体外试验的结果图，图17B的结果显示，与IGF1R+间质干细胞共培养可增加初代大脑皮质细胞在氧气和葡萄糖供应 不足情况下的神经突细胞数量和神经突长度，显示IGF1R+间质干细胞可帮助氧气和葡萄糖供应不足下初代大脑皮层细胞的神经突再生，而共培养U-IGF1R+间质干细胞的组别神经突再生数量显著的高于其他组别，且其神经突长度也显著的长于其他组别。但由组断试验组的结果显示，阻断IGF1R或CXCR4会抑制U-IGF1R+间质干细胞所增加的神经突长度和神经突细胞数量。
由上述体内和体外神经突再生试验的结果显示，IGF1R+间质干细胞可帮助缺血缺氧性脑组织/脑细胞的神经突再生，且移植以含hUCS培养基培养的IGF1R+间质干细胞更具有增加缺血缺氧性脑组织/脑细胞中神经突再生的潜力，且此神经突再生是透过IGF1R和CXCR4受体途径。
第三部分：IGF1R+间质干细胞用于治疗缺血性心脏病
根据前述实验例已知IGF1R+间质干细胞具有自我更新能力及多能分化能力，且IGF1R+间质干细胞具有治疗脑组织损伤个体的效果，此部份的实验例将探讨将IGF1R+间质干细胞用于治疗缺血性心脏病的效果。
3.1：施用IGF1R+间质干细胞降低心肌梗塞后的左室功能不全以及减少心肌梗塞后的梗塞面积
本实验例是以急性心肌梗塞(acute myocardial infraction，AMI)大鼠模型，评估IGF1R+间质干细胞移植是否改善心肌梗塞后的心肌功能恢复。
急性心肌梗塞大鼠模型是阻塞大鼠的左前下行(left anterior descending，LAD)冠状动脉来模拟大鼠暂时性心脏缺血的症状。本试验中所使用的试验动物为体重250-300克雄性SD大鼠，实验上先将大鼠以2％异氟醚(于100％氧气中)麻醉，全麻后仰置手术台上，口腔插入呼吸器(SN-480-7)，以1mL/100g的潮气量以及80次/分钟的呼吸频率人工通气。再使用肋骨牵开器(MY-9454S)沿胸骨左缘4、5肋间隙纵向切开，并将用左肺以浸泡生理食盐水的纱布放气。剪开心包，然后使用6-0-聚乙烯缝合针线(Ethicon)阻塞左前下行冠状动脉，肉眼观察到阻塞区变白、心电图T波高耸，将胸部缝合前将肺重新充气，即完成急性心肌梗塞大鼠模型。在将急性心肌梗塞大鼠分成3组，分别静脉移植细胞数为2×106的间质干细胞或IGF1R+间质干细胞，另注射生理盐水作为对照组。本试验中另有假手术组(sham)的大鼠，其 为经历上述相同的手术步骤，但未阻塞假手术组的左前下行冠状动脉。
参照图18，为移植间质干细胞和IGF1R+间质干细胞的急性心肌梗塞大鼠的心肌梗塞面积图。实验上牺牲移植间质干细胞后28天的急性心肌梗塞大鼠，将心脏组织切片浸泡于三苯基四氮唑氯化物(triphenyltetrazolium chloride)溶液中，再浸泡去氢酶(dehydrogenase)，可发现坏死区域呈现红蓝色，没有梗塞的区域呈现砖红色。由图18的结果显示，移植间质干细胞可改善急性心肌梗塞大鼠的心肌梗塞面积，也可使梗塞区域的动脉血管壁增厚，而移植IGF1R+间质干细胞的急性心肌梗塞大鼠，其心肌梗塞面积减少的幅度和梗塞区域动脉血管壁增厚的幅度更高于移植间质干细胞的组别。
本实验例进一步藉由M模式的心脏超音波分析心肌梗塞后28天大鼠的左心室功能，以评估IGF1R+间质干细胞治疗缺血性心脏病的效果，分析的组别包含移植间质干细胞的急性心肌梗塞大鼠、移植IGF1R+间质干细胞的急性心肌梗塞大鼠、对照组大鼠以及假手术组大鼠。参照图19A和图19B，图19A为移植间质干细胞和IGF1R+间质干细胞的急性心肌梗塞大鼠的心脏超音波图，图19B为移植间质干细胞和IGF1R+间质干细胞的急性心肌梗塞大鼠心脏超音波的量化图。由图19A和图19B的结果显示，移植间质干细胞和IGF1R+间质干细胞的组别相较于对照组，在心脏超音波分析中可侦测到较低的左心室收缩末期内径(left ventricular end systolic diameter，LVESD)/左心室舒张末期内径(left ventricular end diastolic diameter，LVEDD)，以及较高的射出分率(ejection fraction，EF)/部分缩短(fraction shortening，FS)，但不影响急性心肌梗塞大鼠的心跳速率，显示移植间质干细胞和IGF1R+间质干细胞可改善急性心肌梗塞大鼠的左心室功能。其中又以移植IGF1R+间质干细胞的急性心肌梗塞大鼠，其左心室功能改善的幅度更高于其他组别。
3.2：IGF1R+间质干细胞对缺血心肌的抗发炎影响
本实验例将进一步探讨移植间质干细胞和IGF1R+间质干细胞至急性心肌梗塞大鼠中，是否能抑制心肌梗塞后的炎症反应。实验上牺牲移植间质干细胞和IGF1R+间质干细胞后3天的急性心肌梗塞大鼠，将心脏组织切片以免疫组织化学染色法侦测发炎的程度、以免疫荧光染色法侦测巨噬细胞的表 达和以定量RT-PCR侦测促发炎因子的表达量。
参照图20，为移植间质干细胞和IGFIR+间质干细胞3天后的急性心肌梗塞大鼠的免疫组织化学染色法结果图。在图20中，上图为以苏木素-伊红染色(H&E stain)的急性心肌梗塞大鼠心脏组织切片的显微照片图，将心脏组织发炎的情形分为0-5级，级数越高表示发炎程度越严重，并将不同组别所拍摄的结果，依上述标准进行分类和统计，统计的结果如图20的下图所示。图20的结果显示，移植间质干细胞和IGF1R+间质干细胞可改善急性心肌梗塞大鼠心脏组织的发炎程度，且移植IGF1R+间质干细胞改善急性心肌梗塞大鼠心脏组织发炎的能力更好，其中间质干细胞组别与对照组相比p＜0.05，IGF1R+间质干细胞组别与对照组相比p＜0.01。
为进一步侦测移植间质干细胞和IGF1R+间质干细胞后3天的急性心肌梗塞大鼠于心脏组织中巨噬细胞的表达，实验上将急性心肌梗塞大鼠心脏组织切片以免疫荧光染色法标定巨噬细胞标记CD68，并以荧光显微镜观察结果。参照图21，为移植间质干细胞和IGFIR+间质干细胞3天后的急性心肌梗塞大鼠的免疫荧光染色法结果图，由图21的结果显示，移植间质干细胞和IGF1R+间质干细胞的组别相较于对照组，心肌梗塞后3天大鼠的心脏组织中，有较少的CD68+细胞浸润在心肌梗塞周围区域，其中移植IGF1R+间质干细胞的组别，CD68+细胞表达量显著少于其他组别。显示移植IGF1R+间质干细胞可以大幅改善急性心肌梗塞大鼠心脏组织的发炎程度。
在缺血心肌观察到发炎细胞的浸润通常会增加促发炎因子的表达，本试验进一步以定量RT-PCR侦测心肌梗塞后3天大鼠的心脏组织中促发炎因子的表达量，侦测的促发炎因子包含IL-1β、IL-6、TNF-α和IFN-γ，本实验例中另侦测抗发炎因子IL-10的表达量。参照图22，为移植间质干细胞和IGFIR+间质干细胞3天后的急性心肌梗塞大鼠促发炎因子表达量的分析图。由图22的结果显示，移植间质干细胞和IGF1R+间质干细胞的组别相较于对照组，心肌梗塞后3天大鼠的心脏组织中，促发炎因子IL-1β、IL-6、TNF-α和IFN-γ的表达量皆降低，其中移植IGF1R+间质干细胞的组别，促发炎因子IL-1β、IL-6、TNF-α和IFN-γ的表达量降低的幅度更大。而在抗发炎因子的表达量则与促发炎因子的表达量相反，移植间质干细胞和IGF1R+间质干细胞的组别增加抗发炎因子IL-10的表达量，其中移植IGF1R+间质干 细胞的组别，抗发炎因子IL-10表达量增加的幅度更大移植，此结果也显示移植IGF1R+间质干细胞可以大幅改善急性心肌梗塞大鼠心脏组织的发炎程度。
3.3：IGF1R+间质干细胞治疗减少心肌梗塞引起的纤维化
心脏的肌纤维与一般的肌纤维不同，能长时间工作把血液打到身体的每一部分。在心肌梗塞时心肌会产生不可逆的坏死，且坏死的部份会由纤维组织替代，在数周后纤维化。本实验例将探讨移植IGF1R+间质干细胞是否能减少心肌梗塞引起的纤维化现象。实验上牺牲移植间质干细胞后28天的急性心肌梗塞大鼠，将心脏组织切片以梅生三色染色法(Masson′s trichrome staining)观察大鼠心脏组织纤维化的情况，其中胶原纤维呈现蓝色，肌肉纤维呈现红色，分析的组别包含移植间质干细胞的急性心肌梗塞大鼠、移植IGF1R+间质干细胞的急性心肌梗塞大鼠、对照组大鼠以及假手术组大鼠。
参照图23，为移植间质干细胞和IGFIR+间质干细胞28天后的急性心肌梗塞大鼠的梅生三色染色法结果图。在图23中，上图为以梅生三色染色法染色的急性心肌梗塞大鼠心脏组织切片的显微照片图，将心脏组织中纤维化的情形分为0-5级，级数越高表示纤维化程度越严重，并将不同组别所拍摄的结果，依上述标准进行分类和统计，统计的结果如图23的下图所示。图23的结果显示，移植间质干细胞和IGF1R+间质干细胞可改善急性心肌梗塞大鼠心脏组织的纤维化情况，且移植IGF1R+间质干细胞改善急性心肌梗塞大鼠心脏组织纤维化的能力更好，其中间质干细胞组别与对照组相比p＜0.05，IGF1R+间质干细胞组别与对照组相比p＜0.01。
综合上述，本发明的经分离的间质干细胞是于细胞表面表达胰岛素样生长因子-1受体，其具有自我更新和多能性(pluripotent)分化的特性。本发明之间质干细胞克隆源性扩增的方法中，培养基是使用hUCS为原料，hUCS中富含生长因子，特别是PDGF-BB，可使间质干细胞大量表达胰岛素样生长因子-1受体，并维持多能性分化能力，而使用人类脐带血血清的培养基获得方便，且可避免因使用非人类血清所引起的过敏反应及感染病毒或病原菌的危险。本发明的分离多能性间质干细胞的方法，可自哺乳动物组织来源的细胞混合物中，筛选对于IGF1R呈阳性的细胞，更佳地，可再筛选对于 IL22RA1呈阳性的细胞，筛选出的即为具多能性分化能力的间质干细胞，故可快速且专一的纯化多能性的间质干细胞。将本发明的经分离的间质干细胞用于细胞治疗时，其可用于治疗个体的脑组织损伤和个体的缺血性心脏病。进一步地说，在治疗个体的脑组织损伤的部份，本发明的经分离的间质干细胞可以增加葡萄糖的代谢活性、促进血管新生和加强神经突再生，并藉由IGF1R和CXCR4的交互作用，具有神经重塑的效果。而在治疗个体的缺血性心脏病的部分，本发明的经分离的间质干细胞可以减轻心肌梗塞后左心室的功能障碍、降低梗塞面积、减少因心肌梗塞引起的纤维化的以及减少免疫反应。虽然本发明已以实施方式公开如上，然而其并非用以限定本发明，任何本领域技术人员，在不脱离本发明的精神和范围内，当可作各种的更动与润饰，因此本发明的保护范围当视权利要求所界定者为准。

Claims (18)

  1. 一种经分离的间质干细胞，其表达胰岛素样生长因子-1受体。
  2. 如权利要求1所述的经分离的间质干细胞，其特征在于，所述经分离的间质干细胞为多能性。
  3. 如权利要求1所述的经分离的间质干细胞，其特征在于，所述经分离的间质干细胞为人类细胞。
  4. 如权利要求1所述的经分离的间质干细胞，其特征在于，所述经分离的间质干细胞为脐带间质干细胞。
  5. 一种间质干细胞克隆源性扩增的方法，包含：
    在含有人类脐带血血清的培养基中培养如权利要求1所述的经分离的间质干细胞，所述经分离的间质干细胞表达胰岛素样生长因子-1受体。
  6. 如权利要求5所述的间质干细胞克隆源性扩增的方法，其特征在于，所述培养基的人类脐带血血清的浓度为1～10％(v/v)。
  7. 如权利要求5所述的间质干细胞克隆源性扩增的方法，其特征在于，所述培养基的人类脐带血血清的浓度为2％(v/v)。
  8. 一种分离具多能性的间质干细胞的方法，包含：
    提供来自一哺乳动物组织的细胞混合物；以及进行分离步骤，所述分离步骤是自所述细胞混合物分离对于胰岛素样生长因子-1受体呈阳性的细胞，以获得所述多能性的间质干细胞。
  9. 如权利要求8的分离具多能性的间质干细胞的方法，其特征在于，所述分离步骤还包含分离对于介白素22受体呈阳性的细胞。
  10. 如权利要求8的分离具多能性的间质干细胞的方法，其特征在于，所述哺乳动物组织是选自骨髓、牙髓、胎盘、脐带、脐带血及脂肪所组成的族群中。
  11. 一种如权利要求1所述的经分离的间质干细胞的用途，其特征在于，所述是用于制备治疗缺血性心脏病的药物。
  12. 如权利要求11所述的经分离的间质干细胞的用途，其特征在于，所述缺血性心脏病为心肌梗塞。
  13. 如权利要求11所述的经分离的间质干细胞的用途，其特征在于，所述治疗缺血性心脏病的药物包含减少因心肌梗塞引起的纤维化的药物或减少免疫反应的药物。
  14. 一种如权利要求1所述的经分离的间质干细胞的用途，所述其用于制备治疗个体脑组织损伤的药物。
  15. 如权利要求14所述的经分离的间质干细胞的用途，其特征在于，所述脑组织损伤是由脑缺血疾病造成。
  16. 如权利要求15所述的经分离的间质干细胞的用途，其特征在于，所述脑缺血疾病为中风。
  17. 如权利要求14所述的经分离的间质干细胞的用途，其特征在于，所述脑组织损伤是由神经退化性疾病造成。
  18. 如权利要求17所述的经分离的间质干细胞的用途，其特征在于，所述神经退化性疾病为帕金森氏症。19.如权利要求14所述的经分离的间质干细胞的用途，其特征在于，所述治疗个体脑组织损伤的药物包含增加葡萄糖代谢活性的药物、促进血管新生的药物或促进神经突再生的药物。
PCT/CN2015/094825 2015-07-22 2015-11-17 间质干细胞、其克隆源性扩增的方法、其分离方法及其应用 WO2017012226A1 (zh)

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