CN116083362B - 一种人脂肪干细胞转分化为神经干细胞的方法及应用 - Google Patents
一种人脂肪干细胞转分化为神经干细胞的方法及应用 Download PDFInfo
- Publication number
- CN116083362B CN116083362B CN202310258868.0A CN202310258868A CN116083362B CN 116083362 B CN116083362 B CN 116083362B CN 202310258868 A CN202310258868 A CN 202310258868A CN 116083362 B CN116083362 B CN 116083362B
- Authority
- CN
- China
- Prior art keywords
- stem cells
- adipose
- neural
- medium
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 96
- 210000001178 neural stem cell Anatomy 0.000 title claims abstract description 66
- 238000000034 method Methods 0.000 title claims abstract description 27
- 230000006698 induction Effects 0.000 claims abstract description 16
- 239000002609 medium Substances 0.000 claims description 29
- 210000002569 neuron Anatomy 0.000 claims description 21
- 238000012258 culturing Methods 0.000 claims description 19
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 15
- 239000001963 growth medium Substances 0.000 claims description 13
- 230000001537 neural effect Effects 0.000 claims description 12
- 210000002966 serum Anatomy 0.000 claims description 10
- CDOVNWNANFFLFJ-UHFFFAOYSA-N 4-[6-[4-(1-piperazinyl)phenyl]-3-pyrazolo[1,5-a]pyrimidinyl]quinoline Chemical compound C1CNCCN1C1=CC=C(C2=CN3N=CC(=C3N=C2)C=2C3=CC=CC=C3N=CC=2)C=C1 CDOVNWNANFFLFJ-UHFFFAOYSA-N 0.000 claims description 6
- 239000011159 matrix material Substances 0.000 claims description 5
- 102000004219 Brain-derived neurotrophic factor Human genes 0.000 claims description 4
- 108090000715 Brain-derived neurotrophic factor Proteins 0.000 claims description 4
- 102000034615 Glial cell line-derived neurotrophic factor Human genes 0.000 claims description 4
- 108091010837 Glial cell line-derived neurotrophic factor Proteins 0.000 claims description 4
- 230000029087 digestion Effects 0.000 claims description 4
- 230000008569 process Effects 0.000 claims description 4
- 108010019160 Pancreatin Proteins 0.000 claims description 3
- 230000003203 everyday effect Effects 0.000 claims description 3
- 229940055695 pancreatin Drugs 0.000 claims description 3
- 238000004115 adherent culture Methods 0.000 claims description 2
- 210000004027 cell Anatomy 0.000 abstract description 33
- 230000004069 differentiation Effects 0.000 abstract description 28
- 108090000623 proteins and genes Proteins 0.000 abstract description 15
- 210000003061 neural cell Anatomy 0.000 abstract description 9
- 208000028389 Nerve injury Diseases 0.000 abstract description 7
- 230000008764 nerve damage Effects 0.000 abstract description 7
- 230000009466 transformation Effects 0.000 abstract description 7
- 230000008672 reprogramming Effects 0.000 abstract description 4
- 239000000126 substance Substances 0.000 abstract description 4
- 208000012902 Nervous system disease Diseases 0.000 abstract description 3
- 238000006243 chemical reaction Methods 0.000 abstract description 3
- 239000000203 mixture Substances 0.000 abstract description 3
- 230000008439 repair process Effects 0.000 abstract description 3
- 230000002452 interceptive effect Effects 0.000 abstract description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 15
- 210000004556 brain Anatomy 0.000 description 13
- 238000002347 injection Methods 0.000 description 11
- 239000007924 injection Substances 0.000 description 11
- 238000004458 analytical method Methods 0.000 description 10
- 238000002474 experimental method Methods 0.000 description 9
- 230000014509 gene expression Effects 0.000 description 9
- 238000011580 nude mouse model Methods 0.000 description 9
- 241000699660 Mus musculus Species 0.000 description 8
- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 description 8
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 6
- 210000005036 nerve Anatomy 0.000 description 6
- 241000282553 Macaca Species 0.000 description 5
- 241000700605 Viruses Species 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 238000004445 quantitative analysis Methods 0.000 description 5
- -1 small molecule compounds Chemical class 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 101001038339 Homo sapiens LIM homeobox transcription factor 1-alpha Proteins 0.000 description 4
- 101000979001 Homo sapiens Methionine aminopeptidase 2 Proteins 0.000 description 4
- 101000969087 Homo sapiens Microtubule-associated protein 2 Proteins 0.000 description 4
- 102100040290 LIM homeobox transcription factor 1-alpha Human genes 0.000 description 4
- 102100023174 Methionine aminopeptidase 2 Human genes 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 229960003638 dopamine Drugs 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 238000003384 imaging method Methods 0.000 description 4
- 210000004263 induced pluripotent stem cell Anatomy 0.000 description 4
- 230000001939 inductive effect Effects 0.000 description 4
- 241000282693 Cercopithecidae Species 0.000 description 3
- 101150009249 MAP2 gene Proteins 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 102000001435 Synapsin Human genes 0.000 description 3
- 108050009621 Synapsin Proteins 0.000 description 3
- 208000027418 Wounds and injury Diseases 0.000 description 3
- 210000000577 adipose tissue Anatomy 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 210000003491 skin Anatomy 0.000 description 3
- 238000011222 transcriptome analysis Methods 0.000 description 3
- 208000024827 Alzheimer disease Diseases 0.000 description 2
- 208000005623 Carcinogenesis Diseases 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 102100037241 Endoglin Human genes 0.000 description 2
- 102000006354 HLA-DR Antigens Human genes 0.000 description 2
- 108010058597 HLA-DR Antigens Proteins 0.000 description 2
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 2
- 101000881679 Homo sapiens Endoglin Proteins 0.000 description 2
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 2
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 2
- 101000800116 Homo sapiens Thy-1 membrane glycoprotein Proteins 0.000 description 2
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 2
- 108700005075 Regulator Genes Proteins 0.000 description 2
- 101100424636 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) TUB3 gene Proteins 0.000 description 2
- 206010039966 Senile dementia Diseases 0.000 description 2
- 101150026222 TUBB3 gene Proteins 0.000 description 2
- 102100033523 Thy-1 membrane glycoprotein Human genes 0.000 description 2
- 102000013814 Wnt Human genes 0.000 description 2
- 108050003627 Wnt Proteins 0.000 description 2
- 206010052428 Wound Diseases 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- 208000029028 brain injury Diseases 0.000 description 2
- 230000036952 cancer formation Effects 0.000 description 2
- 231100000504 carcinogenesis Toxicity 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 229960003692 gamma aminobutyric acid Drugs 0.000 description 2
- 239000003102 growth factor Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000010166 immunofluorescence Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 230000000977 initiatory effect Effects 0.000 description 2
- 210000000265 leukocyte Anatomy 0.000 description 2
- 238000007443 liposuction Methods 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 206010033675 panniculitis Diseases 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 230000019491 signal transduction Effects 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 210000004003 subcutaneous fat Anatomy 0.000 description 2
- 238000002054 transplantation Methods 0.000 description 2
- OGNSCSPNOLGXSM-UHFFFAOYSA-N (+/-)-DABA Natural products NCCC(N)C(O)=O OGNSCSPNOLGXSM-UHFFFAOYSA-N 0.000 description 1
- 102100022464 5'-nucleotidase Human genes 0.000 description 1
- 102100026605 Aldehyde dehydrogenase, dimeric NADP-preferring Human genes 0.000 description 1
- 206010003210 Arteriosclerosis Diseases 0.000 description 1
- AQGNHMOJWBZFQQ-UHFFFAOYSA-N CT 99021 Chemical compound CC1=CNC(C=2C(=NC(NCCNC=3N=CC(=CC=3)C#N)=NC=2)C=2C(=CC(Cl)=CC=2)Cl)=N1 AQGNHMOJWBZFQQ-UHFFFAOYSA-N 0.000 description 1
- 102000019034 Chemokines Human genes 0.000 description 1
- 108010012236 Chemokines Proteins 0.000 description 1
- 102000029816 Collagenase Human genes 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- 208000035984 Colonic Polyps Diseases 0.000 description 1
- 101100193652 Dictyostelium discoideum rapA gene Proteins 0.000 description 1
- 101100351026 Drosophila melanogaster ey gene Proteins 0.000 description 1
- 102100039289 Glial fibrillary acidic protein Human genes 0.000 description 1
- 101710193519 Glial fibrillary acidic protein Proteins 0.000 description 1
- 241001481828 Glyptocephalus cynoglossus Species 0.000 description 1
- 101000678236 Homo sapiens 5'-nucleotidase Proteins 0.000 description 1
- 101000717964 Homo sapiens Aldehyde dehydrogenase, dimeric NADP-preferring Proteins 0.000 description 1
- 101000977643 Homo sapiens Immunoglobulin superfamily containing leucine-rich repeat protein 2 Proteins 0.000 description 1
- 101000959028 Homo sapiens Mitochondrial 10-formyltetrahydrofolate dehydrogenase Proteins 0.000 description 1
- 101001132698 Homo sapiens Retinoic acid receptor beta Proteins 0.000 description 1
- 101000651309 Homo sapiens Retinoic acid receptor responder protein 1 Proteins 0.000 description 1
- 101001111656 Homo sapiens Retinol dehydrogenase 10 Proteins 0.000 description 1
- 102100023540 Immunoglobulin superfamily containing leucine-rich repeat protein 2 Human genes 0.000 description 1
- 206010060820 Joint injury Diseases 0.000 description 1
- YQEZLKZALYSWHR-UHFFFAOYSA-N Ketamine Chemical compound C=1C=CC=C(Cl)C=1C1(NC)CCCCC1=O YQEZLKZALYSWHR-UHFFFAOYSA-N 0.000 description 1
- 102000043136 MAP kinase family Human genes 0.000 description 1
- 108091054455 MAP kinase family Proteins 0.000 description 1
- 102100039076 Mitochondrial 10-formyltetrahydrofolate dehydrogenase Human genes 0.000 description 1
- 102000008109 Mixed Function Oxygenases Human genes 0.000 description 1
- 108010074633 Mixed Function Oxygenases Proteins 0.000 description 1
- 102000008730 Nestin Human genes 0.000 description 1
- 108010088225 Nestin Proteins 0.000 description 1
- XNOPRXBHLZRZKH-UHFFFAOYSA-N Oxytocin Natural products N1C(=O)C(N)CSSCC(C(=O)N2C(CCC2)C(=O)NC(CC(C)C)C(=O)NCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(CCC(N)=O)NC(=O)C(C(C)CC)NC(=O)C1CC1=CC=C(O)C=C1 XNOPRXBHLZRZKH-UHFFFAOYSA-N 0.000 description 1
- 101800000989 Oxytocin Proteins 0.000 description 1
- 102100031951 Oxytocin-neurophysin 1 Human genes 0.000 description 1
- 102000007354 PAX6 Transcription Factor Human genes 0.000 description 1
- 101150081664 PAX6 gene Proteins 0.000 description 1
- 208000002500 Primary Ovarian Insufficiency Diseases 0.000 description 1
- 102100027682 Retinoic acid receptor responder protein 1 Human genes 0.000 description 1
- 102100023918 Retinol dehydrogenase 10 Human genes 0.000 description 1
- 108091005735 TGF-beta receptors Proteins 0.000 description 1
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 1
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 1
- 102000016715 Transforming Growth Factor beta Receptors Human genes 0.000 description 1
- 102000013127 Vimentin Human genes 0.000 description 1
- 108010065472 Vimentin Proteins 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 210000001789 adipocyte Anatomy 0.000 description 1
- 230000002424 anti-apoptotic effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 208000011775 arteriosclerosis disease Diseases 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000002449 bone cell Anatomy 0.000 description 1
- 210000002798 bone marrow cell Anatomy 0.000 description 1
- 210000003321 cartilage cell Anatomy 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000010307 cell transformation Effects 0.000 description 1
- 230000008668 cellular reprogramming Effects 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 229960002424 collagenase Drugs 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000003412 degenerative effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000001339 epidermal cell Anatomy 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 238000011331 genomic analysis Methods 0.000 description 1
- 210000005046 glial fibrillary acidic protein Anatomy 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 238000003125 immunofluorescent labeling Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000004957 immunoregulator effect Effects 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 229960003299 ketamine Drugs 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004660 morphological change Effects 0.000 description 1
- 208000031225 myocardial ischemia Diseases 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 210000005055 nestin Anatomy 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- XNOPRXBHLZRZKH-DSZYJQQASA-N oxytocin Chemical compound C([C@H]1C(=O)N[C@H](C(N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CSSC[C@H](N)C(=O)N1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(C)C)C(=O)NCC(N)=O)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 XNOPRXBHLZRZKH-DSZYJQQASA-N 0.000 description 1
- 229960001723 oxytocin Drugs 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 206010036601 premature menopause Diseases 0.000 description 1
- 208000017942 premature ovarian failure 1 Diseases 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000002331 protein detection Methods 0.000 description 1
- 239000000700 radioactive tracer Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000008054 signal transmission Effects 0.000 description 1
- 210000004927 skin cell Anatomy 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 208000020431 spinal cord injury Diseases 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000011476 stem cell transplantation Methods 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 208000006379 syphilis Diseases 0.000 description 1
- 101150056532 terf2ip gene Proteins 0.000 description 1
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 230000005740 tumor formation Effects 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 0.000 description 1
- 210000005048 vimentin Anatomy 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
Classifications
-
- 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
- C12N5/0618—Cells of the nervous system
- C12N5/0623—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
- A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
- A61P25/16—Anti-Parkinson drugs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/13—Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/70—Enzymes
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/70—Enzymes
- C12N2501/72—Transferases [EC 2.]
- C12N2501/727—Kinases (EC 2.7.)
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/70—Enzymes
- C12N2501/73—Hydrolases (EC 3.)
-
- 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
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
- C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
- C12N2506/1384—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from adipose-derived stem cells [ADSC], from adipose stromal stem cells
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Neurology (AREA)
- Neurosurgery (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- General Chemical & Material Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Zoology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- General Engineering & Computer Science (AREA)
- Epidemiology (AREA)
- Virology (AREA)
- Immunology (AREA)
- Hospice & Palliative Care (AREA)
- Psychiatry (AREA)
- Ophthalmology & Optometry (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Psychology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明涉及干细胞医学转化应用技术领域，尤其涉及一种人脂肪干细胞转分化为神经干细胞的方法及应用。本发明将人原代脂肪干细胞经过化学的小分子和因子诱导分3步法转化为神经干细胞，分化过程中没有利用到细胞重编程技术，没有外源性的干扰细胞的基因组成，保证了转化所得神经细胞的功能性和安全性。本发明提供的转分化方法及获得的神经干细胞有望成为运用自体干细胞的转化应用在临床工作中进行神经损伤修复及相关神经系统疾病的治疗的一种新手段。
Description
技术领域
本发明涉及干细胞医学转化应用技术领域，尤其涉及一种人脂肪干细胞转分化为神经干细胞的方法及应用。
技术背景
脂肪间充质干细胞是目前在干细胞转化应用领域中最受关注的“种子细胞”。脂肪干细胞移植入患者体内后，可生长分化出许多新的正常细胞，如骨细胞，软骨细胞，脂肪细胞，血管内皮细胞，神经细胞等，替代体内病变细胞，修复组织器官损伤，达到治疗疾病的作用。脂肪干细胞还具有免疫调节作用，能够分泌多种细胞因子以及抗凋亡因子、炎性趋化因子等，具有抗氧化、抗炎症能力。脂肪干细胞具有较低的免疫原性，无论宿主与捐献者的人类白细胞抗原(human leukocyte antigen，HLA)是否相匹配，移植均可进行。因为脂肪间充质干细胞可以实施自体的干细胞移植应用，在临床转化应用上受到科学家们的青睐，目前已经进入临床实验II期应用于治疗糖尿病，关节损伤，膝关节炎，结肠性息肉，改善心肌缺血，卵巢早衰等。
与自体的骨髓干细胞比较，骨髓细胞的分离、培养和体外扩增难度很大，细胞培养过程中需要表皮细胞专用培养基，费用昂贵，细胞生长速度缓慢。脂肪干细胞则易培养，在体外培养时生长迅速，分化能力不随培养时间增加而降低，能在低温环境下存活；脂肪源性的神经干细胞体外扩增和分化移植技术具有更高的安全性，而且吸脂术相比骨髓穿刺损伤小、患者痛苦小。
对脂肪间充质干细胞在治疗神经系统疾病如帕金森，老年痴呆，神经损伤上的应用，发现脂肪细胞可以大量快速的体外扩增，而后将脂肪细胞诱导成为神经细胞回植于患者自体，可以进行有效的治疗。在诱导脂肪干细胞朝向神经方向分化过程中，需要小分子化合物作用于细胞信号传导通路，使细胞重编程，再定向分化。Park等[Park J,Lee N,Lee J,ET Al.Small molecule-based lineages witch of human adipose-derived stem cellsinto neural stem cells and functional GABA corgi neurons.Sci Rep,2017,7(1):10166.]运用TGF-β受体抑制剂SB431542和选择性BMP信号通路抑制剂LDN 193289分三步进行诱导。使用小分子化合物，将人脂肪干细胞转分化为神经干细胞。最终超过56％的细胞诱导为神经元，而功能性γ氨基丁酸能神经元的诱导效率超过45％。小分子化合物的使用，为脂肪干细胞朝向神经方向分化提供了可重复且有效的方法，但存在一定安全风险。
到目前为止，未曾有将人原代脂肪干细胞转分化为神经细胞与神经元且无致癌作用的研究报道。
发明内容
为了解决现有技术中存在的问题，本发明的目的之一在于提供一种人脂肪干细胞转分化为神经干细胞的方法。
本发明采用的技术方案如下：
一种人脂肪干细胞转分化为神经干细胞的方法，该方法将人原代脂肪干细胞培养至第4代，第4代脂肪干细胞在贴壁培养第二天后，加入培养基A培养，培养3天后，细胞形态改变，用NIM神经元诱导培养基培养，每2天更换一次所述NIM神经元诱导培养基；诱导12天后，用NM培养基培养2天，得到所需神经干细胞；
所述的培养基A配方为：DMEM/F12，体积比10％人的血清替代物、0.5mM I.B.M.X、0.1μM LDN-193189、20ng/ml FGF、20ng/ml EGF和体积比2％B27；
所述NIM神经元诱导培养基配方为：DMEM/F12，体积比2％ B27，体积比1％ N2，10μM Foskolin，3μM CHIR99021，0.1μM LDN-193189，10μMSB431542；
所述NM培养基配方为：无血清神经基质培养基，体积比2％ B27，体积比1％ N2，10-50ng/ml BDNF，10ng/ml GDNF。
优选的，人原代脂肪干细胞培养至第4代的过程为：将人原代脂肪干细胞培养于含10％血清替代物的DMEM培养基中，胰酶消化传代。
优选的，所述人原代脂肪干细胞培养至第4代的培养条件为：37℃，5％CO2培养，每3～5天更换所述含体积比10％血清替代物的DMEM培养基，并按照培养基体积比例1:3进行传代。
优选的，所述无血清神经基质培养基StemPro NSC SFM。
优选的，NM培养基培养2天后，得到的神经干细胞使用NM培养基进行维持培养，每天更换所述NM培养基。
本发明的目的之二在于提供一种诱导得到的神经干细胞，所述的神经干细胞是采用如上所述的方法由人脂肪干细胞转分化得到。
本发明的目的之二在于提供一种如上所述的神经干细胞在制备神经损伤修复的药物或制剂中的应用。
优选的，所述神经损伤修复是指促进神经损伤的创面愈合和/或改善创面神经系统功能和/或改善退行性神经疾病。
本发明利用人原代脂肪干细胞组织扩增培养3代，对获取的人脂肪干细胞采用化学小分子和细胞因子相结合进行分步诱导，使细胞基因重编程逐步向神经细胞方向转化。本发明提供的由脂肪干细胞转化为神经干细胞的过程不包含外源基因整合的细胞重编程、如iPS(induced pluripotent stem cells)的诱导技术采用了含病毒质粒介导的转染等改变细胞基因组成的操作。转录组基因测序结果表明所得神经细胞不仅保留了部分脂肪间充质干细胞的基因组型，而且具有神经干细胞的特性，没有病毒介入细胞，是一种安全，有效的方法。
通过动物试验对本发明制备的神经干细胞进行安全性鉴定，本发明神经干细胞注射到裸鼠皮下，经过45天观察，未发现有肿瘤形成，注射产生的细胞肿块，45天后完全吸收与消失，结果证明我们的干细胞安全，无致瘤性。注射经过荧光标记的本发明神经干细胞至猴脑内，两个月后能够检测到标记的神经干细胞信号，表明分化的细胞可以在脑内长期存活，发生效益。
本发明的有益效果在于：
1)本发明提供的由脂肪干细胞转分化而来的神经干细胞可以在临床上进行应用或制成干细胞因子等药物。制备神经干细胞过程中，转录组分析中的分化产物中的一些尚未完全分化成神经干细胞的脂肪干细胞，通过PCR定量分析可能随时间推移在体内逐渐进一步转分化为具有更好分化功能性的神经元。
2)本发明在进行脂肪干细胞转化为神经细胞过程中，未采用任何动物源性的因子成分，而是采用化学小分子改变细胞信号传递通路的方式进行基因重编程技术。诱导其向神经细胞相关通路上转化。通过对分化后细胞进行的基因转录组分析，多巴胺检测，神经干细胞的基因与相关蛋白检测，能够证明本发明方法的可靠性。
3)目前神经干细胞的获取与培养以及体外扩增难度均很大，培养基昂贵且神经干细胞生长缓慢。经典的在采用胚胎干细胞或iPS技术诱导出来的神经干细胞，因其具有致瘤风险和外源性病毒进入人体的风险而限制了其应用，而脂肪干细胞的来源比较广泛，可以采用自体的脂肪组织进行培养和扩增，其操作的技术相对比较成熟。人体脂肪组织的含量占体重的15～20％，人体可以通过吸脂肪手术获得1000ml的脂肪，每ml脂肪可以分离出106个原代干细胞，脂肪干细胞即可以自体应用，也可以异体应用，且无免疫排斥反应，在国际上已经批准的干细胞药物中，脂肪干细胞占1/5。
本专利使用脂肪干细胞诱导得到的神经干细胞具有神经干细胞的特点和功能，即可以分泌多巴胺，并表达神经干细胞的关键基因和关键蛋白，这一方法即从自体脂肪组织分离脂肪干细胞再分化为神经干细胞有希望解决对神经干细胞的需求难题，进一步可以开发成细胞药物批量生产。
4)本发明将人原代脂肪干细胞经过化学的小分子和因子诱导分3步法转化为神经干细胞，分化过程中没有利用病毒介导的细胞重编程技术(iPS技术方法)，没有外源性的干扰细胞的基因组成，保证了转化所得神经细胞的功能性和安全性。本发明提供的转分化方法及获得的神经干细胞有望成为运用自体干细胞的转化应用在临床工作中进行神经损伤修复及相关神经系统疾病(帕金森，老年痴呆，脑损伤，脑萎缩，侧索动脉硬化症，脊髓损伤等系列相关神经疾病)治疗的一种新手段。
附图说明
图1为本发明中人脂肪干细胞形态，左侧为分离的脂肪干细胞贴壁的形态，右侧为三代分化前细胞的形态。
图2为流式技术鉴定脂肪干细胞表达各种标志物：脂肪干细胞干性标志物细胞表面分子HLA-dr、CD34、CD45、CD90、CD105、CD73的流式检测结果。
图3为脂肪干细胞向神经分化的形态与时间记录：细胞在不同的神经分化培养基中分化与不同分化时间3天，6天，10天，18天何28天(时间在图左下角标注)的细胞形态变化。
图4为分化的干细胞经过免疫荧光鉴定表达神经干细胞和神经元主要标志物的分析；其中A：GAFP+DAPI；B：Vimentin+DAPI；C：Map2+DAPI；D：Nestin+DAPI；E：Tuj1+DAPI；F：Neun3+DAPI；G：LMX1A+DAPI；H：TH+DAPI；I：HNF3+DAPI；J：Olig2+DAPI；K：Tub3/LMX1A/DAPI；L：neuron sphere TH/Map2/DAPI；M：Synapsin(mchery)；N：MAP2(FITC)；O：Synapsin/MAP2。
图5为采用高压液相色谱鉴定分析分化的神经干细胞分泌的多巴胺的结果。
图6为分化产生的神经球形态(左图)及神经球再贴壁生长的形态(右图)。
图7为分化过程中表达早期不同时间，不同培养基中神经干细胞的重要基因的定量PCR分析；其中A：为ADSC神经分化初始与3天时干细胞相关干性基因表达的定量分析B：为ADSC神经分化12天时三组不同培养基神经干细胞相关干性基因表达的定量分析(DMEM为对照组，NM为现在市场商业培养基，NIM为我们设计培养基)C：为ADSC神经分化初始与4天时神经干细胞相关干性基因表达的定量分析D：为ADSC神经分化初始与9天时神经干细胞相关干性基因表达的定量分析。
图8为分化前后的脂肪干细胞与神经干细胞转录组对比测序分析。
图9为转录组分析中信号通路表达的分析。
图10为裸鼠皮下致瘤性实验，左图为刚注射细胞，右图为45天后的皮肤。
图11为猕猴注射脂肪干细胞源性分化的神经干细胞GFP与YFP荧光标记的立体定位注射后一周的双光子成像分析；经过荧光标记的干细胞进行双光子检测和经过纳米Fe标记的干细胞经过立体定位注射到脑内一个月和2个月的细胞情况。其中A：GFP荧光标记的立体定位注射后一周的双光子成像分析结果。B：荧光标记的立体定位注射后一周的双光子成像分析显示注射的干细胞减少或消失。C：纳米Fe标记的干细胞经过立体定位注射到脑内一个月MRI图像。D：纳米Fe标记的干细胞经过立体定位注射到脑内二个月MRI图像。
具体实施方式
为了便于理解，下面结合实施例对本发明的技术方案做出更为具体的说明，除非另有说明，本文中所使用的术语均具有本领域技术人员常规理解的含义。
实施例1
脂肪干细胞的制备和培养
1、脂肪干细胞的获取
经医院伦理批准，在患者知情同意下，选择肝炎病毒抗体、梅毒抗体及HIV均为阴性的医美患者的吸取脂肪手术剥离下来的皮下脂肪组织，再用PBS清洗5min 3遍，无菌条件下剪碎脂肪组织为0.5×0.5cm3大小的组织块，将脂肪收集起来，加入0.075％ I型胶原酶，37℃消化1h，1000g离心10min，组织过75目过滤器过滤，离心后获得人原代的脂肪干细胞。
2、人原代脂肪干细胞的培养及扩增及鉴定
将人原代脂肪干细胞培养于含10％人血清替代物(HR)的DMEM中，37℃、5％ CO2培养箱中培养，每3～5天换液，根据细胞的生长速度和细胞培养皿中成纤维细胞密度进行1:3传代。人脂肪干细胞贴壁形态如图1左所示，传代培养中(未分化)形态如图1右所示。采用流式细胞技术检测脂肪干细胞表面标志物，结果参见图2。通过HLA-dr和CD双阴性，CD34，CD45，CD73，CD90，CD105阳性证明分离所得细胞为脂肪干细胞。
实施例2
1、脂肪干细胞向神经细胞的转化
实施例1中人原脂肪干细胞培养于含10％血清替代物的DMEM培养基中，常规细胞培养方法用胰酶消化传代至第3代，然后第4代细胞开始神经细胞分化。第4代人脂肪干细胞培养于含10％血清替代物的DMEM培养基中，平板用LM511预先包被处理，37℃、5％ CO2培养箱中培养，第2天换液，加入DMEM/F1，10％人的血清替代物、0.5mM I.B.M.X、0.1μM LDN-193189、FGF(20ng/ml)、EGF(20ng/ml)和2％ B27，3天后，细胞形态改变，用NIM神经元诱导培养基B(DMEM/F2，2％ B27，1％ N2，10μM Foskolin，3μM CHIR99021，0.1μM LDN-193189，10μM SB431542)，每2天更换一次培养基。诱导12天后，改为NM(神经基质培养基，StemProNSC SFM，GIBCO)，2％ B27，1％ N2，10-50ng/ml BDNF，10ng/ml GDNF)，2天每天更换NM，细胞在分化3、6、10、18、28天的形态变化如图3所示。
2、分化的神经细胞的鉴定分析
如图4所示，分化在细胞在第12天时采用免疫荧光染色技术进行国常用的神经干细胞的早期标志物染色，结果表达了早期与中期的神经干细胞和神经元在标志蛋白如GFAP(图中A)，VIMINTIN(图中B)，MAP2(图中C)，NESTIN(图中D)，TUB3(Tuj1，图中E)，中期和成熟神经元的标准物Neun3(图中F)，LMX1A(图中G)，洛氨酸羟化酶TH(图中H)，HNF3(图中I)，Olig2(图中J)，Tub3/LMX1A/DAPI三染(图中K)，Map2加DAPI,HNF3(图中L)，联会蛋白Synapsin(图中M)，神经球染色发现表达了标志蛋白TH/Map2/DAPI(图中L)，再继续培养至28天，在28天时，采用高压液相色谱对分化的神经干细胞进行多巴胺测定，发现能够检测到有神经元产生的多巴胺(如图5所示)。
3、细胞成球实验检测诱导所得神经干细胞的多能性
将脂肪干细胞诱导所得神经干细胞在第12天后消化离散，加入第三阶段的培养基(含BDNF生长因子、GDNF生长因子)，8天后(分化的第20天)显微镜观察每组细胞悬浮球形态，发现神经细胞成球体积增大而且数量增多。图6左侧列显示了分化产生的神经球形态，图6右侧列显示了神经球再贴壁生长的形态。采用免疫荧光技术，利用晚期神经干细胞特异性标志物TH/Map2/DAPI两种抗体同时染色单标悬浮球，发现表达双阳性(图4中L)。
4、在人脂肪干细胞第4代分化早期，对不同培养基的神经干细胞进行定量PCR，检测结果如图7所示。参见图7中A，在分化发育三天时干细胞分化的重要调控基因Nanog,Pax6具有高表达的状态，而且在相继加入分化诱导培养基NDM，NIM后，一些神经干细胞的早期重要的发育调控基因开始表达并增高，同时，检测到了神经干细胞和神经元的相关基因的表达，参见图7中B(DMEM为对照组，NM为市场商业培养基，NIM为本发明设计培养基)、C(4天)、D图(9天)。
如图8为分化前后的脂肪干细胞与神经干细胞转录组对比测序分析，第四代的分化细胞的转录组分析结果表明了一些神经发育的早期基因RARB，RARRES1，ISLR2，ALDH1L2，ALDH3A1，RDH10与Wnt信号表达的高调，如图9所示，信号通路分析中还确认了与神经干细胞的信号相关通路的WNT，TGF-β，CGMP-PKG and MAPK，PI3K-Akt，Ras，Rap1，Hippo，Oxytocin，Notch，P53，and cAMP表达。
实施例3：动物实验
1、裸鼠荷瘤实验检测诱导所得细胞的安全性
进行裸鼠皮下致瘤性实验，取雄性裸鼠10只(由北京—华富康实验动物有限公司提供)，将由脂肪干细胞诱导所得神经干细胞用流式细胞仪计数为1x107/ml，选取裸鼠右侧背部皮下注射。结果如图10，裸鼠注射细胞处皮肤可见点状突起(图10左)，40天后，注射到裸鼠背部皮肤细胞已经全部吸收，无突起(图10右)，两组裸鼠均未见肿瘤组织，表明本发明制备的神经干细胞安全风险低。
2、诱导所得神经干细胞修复猕猴脑损伤实验
在北京大学实验动物中心使用猕猴进行干细胞示踪观察实验。猕猴用氯胺酮麻醉，用立体定位仪固定猴的脑部，电钻颅顶开口，用立体定位注射器向脑内注射用GFP与YFP荧光标记的干细胞，细胞组均将0.2ml的1×107/ml单细胞悬液经过立体定位在损伤脑内上，空白对照组创面不做细胞注射处理。一周后通过双光子成像分析检测猴脑(图11中A)，发现注射实验组猴脑部干细胞减少，细胞基本吸收，信号消失(图11中B)，说明注射干细胞可以在大脑中很快被吸收与代谢。
将纳米Fe标记的本发明制备的脂肪干细胞分化的神经干细胞经过立体定位注射到猴脑内进行MRI检测，注射后一个月结果如图11中C所示，注射后二个月结果如图11中D所示，发现经过Fe纳米标记的脂肪干细胞分化的干细胞仍然存在很强的信号，提示大脑中仍然有大量在干细胞存在，可以持续地对损伤的大脑进行修复。
通过以上实验表明，本发明制备的脂肪干细胞分化的神经干细胞可能有望成为临床工作中神经损伤修复的一种安全、高效的新手段。
以上实施方式仅用以说明本发明的技术方案，而并非对本发明的限制；尽管参照前述实施方式对本发明进行了详细的说明，本领域的普通技术人员应当理解：凡在本发明创造的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明创造的保护范围之内。
Claims (5)
1.一种人脂肪干细胞转分化为神经干细胞的方法，其特征在于，方法具体为：将人原代脂肪干细胞培养至第4代，第4代脂肪干细胞在贴壁培养第二天后，加入培养基A培养，培养3天后，细胞形态改变，用NIM神经元诱导培养基培养，每2天更换一次所述NIM神经元诱导培养基；诱导12天后，用NM培养基培养2 天，得到所需神经干细胞；
所述的培养基A配方为：DMEM/F12，体积比10%人的血清替代物、0.5 mM I.B.M.X、0.1μMLDN-193189、20 ng / ml FGF、20 ng / ml EGF和体积比2% B27；
所述NIM神经元诱导培养基配方为：DMEM/F12，体积比2% B27，体积比1% N2，10μMFoskolin，3μM CHIR99021，0.1μM LDN-193189，10μM SB431542；
所述NM培养基配方为：无血清神经基质培养基，体积比2% B27，体积比1% N2，10-50 ng/ ml BDNF，10 ng / ml GDNF。
2.如权利要求1所述的一种人脂肪干细胞转分化为神经干细胞的方法，其特征在于，人原代脂肪干细胞培养至第4代的过程为：将人原代脂肪干细胞培养于含体积比10%血清替代物的DMEM培养基中，胰酶消化传代。
3.如权利要求2所述的一种人脂肪干细胞转分化为神经干细胞的方法，其特征在于，所述人原代脂肪干细胞培养至第4代的培养条件为：37℃，5% CO2培养，每3~5天更换所述含10%血清替代物的DMEM培养基，并按照培养基体积比例1:3进行传代。
4.如权利要求1所述的一种人脂肪干细胞转分化为神经干细胞的方法，其特征在于，所述无血清神经基质培养基为StemPro NSC SFM。
5.如权利要求1所述的一种人脂肪干细胞转分化为神经干细胞的方法，其特征在于，NM培养基培养2 天后，得到的神经干细胞使用NM培养基进行维持培养，每天更换所述NM培养基。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310258868.0A CN116083362B (zh) | 2023-03-17 | 2023-03-17 | 一种人脂肪干细胞转分化为神经干细胞的方法及应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310258868.0A CN116083362B (zh) | 2023-03-17 | 2023-03-17 | 一种人脂肪干细胞转分化为神经干细胞的方法及应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116083362A CN116083362A (zh) | 2023-05-09 |
| CN116083362B true CN116083362B (zh) | 2024-05-10 |
Family
ID=86187115
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310258868.0A Active CN116083362B (zh) | 2023-03-17 | 2023-03-17 | 一种人脂肪干细胞转分化为神经干细胞的方法及应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116083362B (zh) |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20120022409A (ko) * | 2010-09-02 | 2012-03-12 | 부산대학교 산학협력단 | ｍｉＲＮＡ 조절을 이용한 중간엽줄기세포의 증식, 분화 촉진 및 노화 억제용 조성물 |
| WO2015089228A2 (en) * | 2013-12-11 | 2015-06-18 | The Regents Of The University Of California | Compositions and methods for producing and administering brown adipocytes |
| CN105331580A (zh) * | 2015-12-10 | 2016-02-17 | 厦门大学 | 增强间充质干细胞趋化能力和趋化因子ccl5表达方法 |
| CN105567636A (zh) * | 2016-03-14 | 2016-05-11 | 厚朴生物科技（苏州）有限公司 | 一种人源脂肪干细胞来源的神经元细胞的制备方法及其应用 |
| WO2017179767A1 (ko) * | 2016-04-12 | 2017-10-19 | 서울대학교 산학협력단 | 지방줄기세포에서 신경줄기세포, 신경세포 및 가바성 신경세포로의 분화 유도 방법, 및 인간 골수 유래 중간엽 줄기세포로부터 성장인자를 다량 분비하는 인간 줄기세포의 분화 유도 방법 |
| KR20190109234A (ko) * | 2018-03-16 | 2019-09-25 | (주)메디노 | 신경줄기세포의 신경분화 방법 |
| CN111518757A (zh) * | 2020-04-08 | 2020-08-11 | 重庆福美干细胞生物科技发展有限公司 | 一种免疫抑制或抗炎功能增强型pd-l1阳性间充质干细胞、诱导试剂盒及应用 |
| CN111621525A (zh) * | 2020-06-18 | 2020-09-04 | 山东如戴生物科技有限公司 | Stx1b基因在促进人脂肪间充质干细胞生长和分化中的用途 |
| CN113444689A (zh) * | 2021-07-21 | 2021-09-28 | 东莞十度生物科技有限公司 | 人源自体脂肪干细胞与脐带间充质干细胞共培养诱导分化成神经样干细胞的方法 |
| CN115944978A (zh) * | 2022-12-28 | 2023-04-11 | 深圳市一路有你健康管理有限公司 | 一种医疗用干细胞的过滤装置及其过滤方法 |
| CN116286638A (zh) * | 2022-11-16 | 2023-06-23 | 晟造源生物科技有限公司 | 促进cd34+细胞向cd16+巨噬细胞分化并向m2极化的方法 |
-
2023
- 2023-03-17 CN CN202310258868.0A patent/CN116083362B/zh active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20120022409A (ko) * | 2010-09-02 | 2012-03-12 | 부산대학교 산학협력단 | ｍｉＲＮＡ 조절을 이용한 중간엽줄기세포의 증식, 분화 촉진 및 노화 억제용 조성물 |
| WO2015089228A2 (en) * | 2013-12-11 | 2015-06-18 | The Regents Of The University Of California | Compositions and methods for producing and administering brown adipocytes |
| CN105331580A (zh) * | 2015-12-10 | 2016-02-17 | 厦门大学 | 增强间充质干细胞趋化能力和趋化因子ccl5表达方法 |
| CN105567636A (zh) * | 2016-03-14 | 2016-05-11 | 厚朴生物科技（苏州）有限公司 | 一种人源脂肪干细胞来源的神经元细胞的制备方法及其应用 |
| WO2017179767A1 (ko) * | 2016-04-12 | 2017-10-19 | 서울대학교 산학협력단 | 지방줄기세포에서 신경줄기세포, 신경세포 및 가바성 신경세포로의 분화 유도 방법, 및 인간 골수 유래 중간엽 줄기세포로부터 성장인자를 다량 분비하는 인간 줄기세포의 분화 유도 방법 |
| KR20190109234A (ko) * | 2018-03-16 | 2019-09-25 | (주)메디노 | 신경줄기세포의 신경분화 방법 |
| CN111518757A (zh) * | 2020-04-08 | 2020-08-11 | 重庆福美干细胞生物科技发展有限公司 | 一种免疫抑制或抗炎功能增强型pd-l1阳性间充质干细胞、诱导试剂盒及应用 |
| CN111621525A (zh) * | 2020-06-18 | 2020-09-04 | 山东如戴生物科技有限公司 | Stx1b基因在促进人脂肪间充质干细胞生长和分化中的用途 |
| CN113444689A (zh) * | 2021-07-21 | 2021-09-28 | 东莞十度生物科技有限公司 | 人源自体脂肪干细胞与脐带间充质干细胞共培养诱导分化成神经样干细胞的方法 |
| CN116286638A (zh) * | 2022-11-16 | 2023-06-23 | 晟造源生物科技有限公司 | 促进cd34+细胞向cd16+巨噬细胞分化并向m2极化的方法 |
| CN115944978A (zh) * | 2022-12-28 | 2023-04-11 | 深圳市一路有你健康管理有限公司 | 一种医疗用干细胞的过滤装置及其过滤方法 |
Non-Patent Citations (5)
| Title |
|---|
| Calcium: A novel and efficient inducer of differentiation of adipose‐derived stem cells into neuron‐like cells;Farjam Goudarzi等;《J Cell Physiol》;20180605;全文 * |
| Neuron-like differentiation of adipose tissue-derived stromal cells and vascular smooth muscle cells;Hongxiu Ning等;《Differentiation》;20061231;全文 * |
| 人脐带间充质干细胞在组织损伤修复中的研究进展;高小月;张玉泉;杨晓清;;生物医学工程与临床;20180313(第02期);全文 * |
| 干细胞与转化医学研究进展浅析;高舒平;曲春辉;周瑞;秦川;;中国实验动物学报;20160830(第04期);全文 * |
| 脂肪组织来源的基质细胞向神经细胞分化及其在脑缺血疾病治疗中的应用进展;董静;刘斌;;中国康复医学杂志;20071126(第11期);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116083362A (zh) | 2023-05-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Lou et al. | The effect of bone marrow stromal cells on neuronal differentiation of mesencephalic neural stem cells in Sprague–Dawley rats | |
| Krampera et al. | Induction of neural-like differentiation in human mesenchymal stem cells derived from bone marrow, fat, spleen and thymus | |
| KR101686722B1 (ko) | 신경줄기세포 제조를 위한 배지와 그의 용도 | |
| CN104419661B (zh) | 神经干细胞的制备方法 | |
| WO2010069204A1 (zh) | 亚全能干细胞、其制备方法及其用途 | |
| KR20100065338A (ko) | 인간 또는 동물 배아에서 중간엽 줄기세포를 추출 및 그 분비물을 추출하는 방법 | |
| KR100825864B1 (ko) | 피부 유래 줄기세포를 함유하는 신경질환 치료용세포치료제 | |
| CN104946590A (zh) | 成人骨髓中Muse细胞诱导为神经前体细胞的方法 | |
| Azevedo-Pereira et al. | Superparamagnetic iron oxide nanoparticles as a tool to track mouse neural stem cells in vivo | |
| JP5388297B2 (ja) | アディポクラスター | |
| WO2009069991A2 (en) | Cellular therapeutic agent for incontinence of urine comprising stem cells originated from decidua or adipose | |
| KR101389851B1 (ko) | 신경능선줄기세포의 배양방법 및 그 용도 | |
| Jin et al. | Analysis of the long-term effect of bone marrow mononuclear cell transplantation for the treatment of cerebral infarction | |
| CN116083362B (zh) | 一种人脂肪干细胞转分化为神经干细胞的方法及应用 | |
| CN116694567A (zh) | 将人诱导性多能干细胞间接诱导为间充质干细胞的培养液和方法 | |
| CN116970557A (zh) | 将人诱导性多能干细胞直接诱导为间充质干细胞的培养液和方法 | |
| Mou et al. | Static pressure-induced neural differentiation of mesenchymal stem cells | |
| US20180171291A1 (en) | Derivation of endothelial cells from mammalian pluirpotent stem cells | |
| JP2011211956A (ja) | 幹細胞の未分化維持剤及び増殖促進剤 | |
| JP7038782B2 (ja) | 高速かつ効率的にインビトロ増幅ヒト間葉系幹細胞的方法および使用 | |
| CN113801843B (zh) | 一种增强人尿源性干细胞干性的方法 | |
| CN110628712B (zh) | 基于诱导多能干细胞的治疗级间充质干细胞制备方法及应用 | |
| CN111304167B (zh) | 人源脂肪干细胞来源的神经元前体细胞及其制备方法和应用 | |
| CN104130975B (zh) | 人源脂肪干细胞来源的神经元样细胞及其制备方法和应用 | |
| Barzilay et al. | Adult stem cells for neuronal repair |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |