CN109439631A - A kind of interstital stem cell being overexpressed CCR2 is for treating acute ischemic cerebral apoplexy and preparation method thereof - Google Patents
A kind of interstital stem cell being overexpressed CCR2 is for treating acute ischemic cerebral apoplexy and preparation method thereof Download PDFInfo
- Publication number
- CN109439631A CN109439631A CN201811328565.7A CN201811328565A CN109439631A CN 109439631 A CN109439631 A CN 109439631A CN 201811328565 A CN201811328565 A CN 201811328565A CN 109439631 A CN109439631 A CN 109439631A
- Authority
- CN
- China
- Prior art keywords
- ccr2
- msc
- stem cell
- overexpressed
- plasmid
- 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.)
- Granted
Links
- 101710149815 C-C chemokine receptor type 2 Proteins 0.000 title claims abstract description 57
- 102100031151 C-C chemokine receptor type 2 Human genes 0.000 title claims abstract description 55
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 27
- 230000002490 cerebral effect Effects 0.000 title abstract description 15
- 230000000302 ischemic effect Effects 0.000 title abstract description 12
- 206010008190 Cerebrovascular accident Diseases 0.000 title abstract description 11
- 208000006011 Stroke Diseases 0.000 title abstract description 11
- 230000001154 acute effect Effects 0.000 title abstract description 8
- 238000002360 preparation method Methods 0.000 title abstract description 8
- 239000013612 plasmid Substances 0.000 claims abstract description 30
- 238000000034 method Methods 0.000 claims abstract description 24
- 230000014509 gene expression Effects 0.000 claims abstract description 23
- 230000002018 overexpression Effects 0.000 claims abstract description 9
- 239000003814 drug Substances 0.000 claims abstract description 6
- 229940079593 drug Drugs 0.000 claims abstract description 4
- 108090000623 proteins and genes Proteins 0.000 claims description 14
- 238000003786 synthesis reaction Methods 0.000 claims description 13
- 230000015572 biosynthetic process Effects 0.000 claims description 12
- 238000001890 transfection Methods 0.000 claims description 10
- 238000011144 upstream manufacturing Methods 0.000 claims description 9
- 230000029087 digestion Effects 0.000 claims description 7
- 239000002299 complementary DNA Substances 0.000 claims description 5
- 239000012634 fragment Substances 0.000 claims description 5
- 206010008092 Cerebral artery thrombosis Diseases 0.000 claims description 4
- 238000010839 reverse transcription Methods 0.000 claims description 4
- 210000004976 peripheral blood cell Anatomy 0.000 claims 1
- 208000032382 Ischaemic stroke Diseases 0.000 abstract description 25
- 206010056292 Androgen-Insensitivity Syndrome Diseases 0.000 abstract description 23
- 108020004999 messenger RNA Proteins 0.000 abstract description 21
- 230000008685 targeting Effects 0.000 abstract description 8
- 230000004069 differentiation Effects 0.000 abstract description 6
- 238000001727 in vivo Methods 0.000 abstract description 5
- 230000001225 therapeutic effect Effects 0.000 abstract description 5
- 230000003902 lesion Effects 0.000 abstract description 4
- 101150083327 CCR2 gene Proteins 0.000 abstract description 3
- 238000012239 gene modification Methods 0.000 abstract description 2
- 230000007646 directional migration Effects 0.000 abstract 1
- 231100000025 genetic toxicology Toxicity 0.000 abstract 1
- 230000001738 genotoxic effect Effects 0.000 abstract 1
- 230000007365 immunoregulation Effects 0.000 abstract 1
- 238000002626 targeted therapy Methods 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 37
- 239000007788 liquid Substances 0.000 description 18
- 230000006378 damage Effects 0.000 description 15
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 14
- 239000000243 solution Substances 0.000 description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 12
- 210000005013 brain tissue Anatomy 0.000 description 11
- 210000001519 tissue Anatomy 0.000 description 10
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 9
- 210000004556 brain Anatomy 0.000 description 9
- 201000010099 disease Diseases 0.000 description 9
- 230000006698 induction Effects 0.000 description 9
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 8
- 238000001514 detection method Methods 0.000 description 8
- 230000008499 blood brain barrier function Effects 0.000 description 7
- 210000001218 blood-brain barrier Anatomy 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 239000000499 gel Substances 0.000 description 7
- 239000003292 glue Substances 0.000 description 7
- 238000000338 in vitro Methods 0.000 description 7
- 238000011534 incubation Methods 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 239000003963 antioxidant agent Substances 0.000 description 6
- 230000003078 antioxidant effect Effects 0.000 description 6
- 235000006708 antioxidants Nutrition 0.000 description 6
- 210000004271 bone marrow stromal cell Anatomy 0.000 description 6
- 239000000975 dye Substances 0.000 description 6
- 230000006872 improvement Effects 0.000 description 6
- 102100022464 5'-nucleotidase Human genes 0.000 description 5
- 102100024210 CD166 antigen Human genes 0.000 description 5
- 102100032912 CD44 antigen Human genes 0.000 description 5
- 102100037241 Endoglin Human genes 0.000 description 5
- 101000678236 Homo sapiens 5'-nucleotidase Proteins 0.000 description 5
- 101000980840 Homo sapiens CD166 antigen Proteins 0.000 description 5
- 101000868273 Homo sapiens CD44 antigen Proteins 0.000 description 5
- 101000881679 Homo sapiens Endoglin Proteins 0.000 description 5
- 101000935043 Homo sapiens Integrin beta-1 Proteins 0.000 description 5
- 101000800116 Homo sapiens Thy-1 membrane glycoprotein Proteins 0.000 description 5
- 102100025304 Integrin beta-1 Human genes 0.000 description 5
- 102100033523 Thy-1 membrane glycoprotein Human genes 0.000 description 5
- 230000017531 blood circulation Effects 0.000 description 5
- 230000003399 chemotactic effect Effects 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 230000003647 oxidation Effects 0.000 description 5
- 238000007254 oxidation reaction Methods 0.000 description 5
- 230000002980 postoperative effect Effects 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 210000003462 vein Anatomy 0.000 description 5
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 4
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 238000010253 intravenous injection Methods 0.000 description 4
- 238000013508 migration Methods 0.000 description 4
- 210000005087 mononuclear cell Anatomy 0.000 description 4
- 210000002569 neuron Anatomy 0.000 description 4
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 230000008439 repair process Effects 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 238000012546 transfer Methods 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 102100031658 C-X-C chemokine receptor type 5 Human genes 0.000 description 3
- 102000009410 Chemokine receptor Human genes 0.000 description 3
- 108050000299 Chemokine receptor Proteins 0.000 description 3
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 3
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 3
- 241000282414 Homo sapiens Species 0.000 description 3
- 101000897480 Homo sapiens C-C motif chemokine 2 Proteins 0.000 description 3
- 101000922405 Homo sapiens C-X-C chemokine receptor type 5 Proteins 0.000 description 3
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 3
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 3
- 239000006180 TBST buffer Substances 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 230000006907 apoptotic process Effects 0.000 description 3
- 230000004888 barrier function Effects 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 210000000269 carotid artery external Anatomy 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 206010008118 cerebral infarction Diseases 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 239000012894 fetal calf serum Substances 0.000 description 3
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 230000005012 migration Effects 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 210000005259 peripheral blood Anatomy 0.000 description 3
- 239000011886 peripheral blood Substances 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 102000005962 receptors Human genes 0.000 description 3
- 108020003175 receptors Proteins 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 206010002091 Anaesthesia Diseases 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 102100022718 Atypical chemokine receptor 2 Human genes 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 101800004637 Communis Proteins 0.000 description 2
- 208000005156 Dehydration Diseases 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 101000678892 Homo sapiens Atypical chemokine receptor 2 Proteins 0.000 description 2
- 101001090047 Homo sapiens Peroxiredoxin-4 Proteins 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 102000004877 Insulin Human genes 0.000 description 2
- 108090001061 Insulin Proteins 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- NPGIHFRTRXVWOY-UHFFFAOYSA-N Oil red O Chemical compound Cc1ccc(C)c(c1)N=Nc1cc(C)c(cc1C)N=Nc1c(O)ccc2ccccc12 NPGIHFRTRXVWOY-UHFFFAOYSA-N 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 102100034768 Peroxiredoxin-4 Human genes 0.000 description 2
- 102000006382 Ribonucleases Human genes 0.000 description 2
- 108010083644 Ribonucleases Proteins 0.000 description 2
- 210000001744 T-lymphocyte Anatomy 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 230000002293 adipogenic effect Effects 0.000 description 2
- 230000037005 anaesthesia Effects 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 230000000740 bleeding effect Effects 0.000 description 2
- 230000036772 blood pressure Effects 0.000 description 2
- 210000004204 blood vessel Anatomy 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 230000012292 cell migration Effects 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 208000026106 cerebrovascular disease Diseases 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- 238000002591 computed tomography Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 230000018044 dehydration Effects 0.000 description 2
- 238000006297 dehydration reaction Methods 0.000 description 2
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 description 2
- 229960003957 dexamethasone Drugs 0.000 description 2
- 230000003073 embolic effect Effects 0.000 description 2
- 210000002889 endothelial cell Anatomy 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000012997 ficoll-paque Substances 0.000 description 2
- 210000003194 forelimb Anatomy 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 238000001415 gene therapy Methods 0.000 description 2
- 238000011194 good manufacturing practice Methods 0.000 description 2
- 230000011132 hemopoiesis Effects 0.000 description 2
- CGIGDMFJXJATDK-UHFFFAOYSA-N indomethacin Chemical compound CC1=C(CC(O)=O)C2=CC(OC)=CC=C2N1C(=O)C1=CC=C(Cl)C=C1 CGIGDMFJXJATDK-UHFFFAOYSA-N 0.000 description 2
- 230000002757 inflammatory effect Effects 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 208000014674 injury Diseases 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 208000037906 ischaemic injury Diseases 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 210000002751 lymph Anatomy 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 230000000116 mitigating effect Effects 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 230000004072 osteoblast differentiation Effects 0.000 description 2
- 230000002188 osteogenic effect Effects 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 210000002381 plasma Anatomy 0.000 description 2
- 229920002401 polyacrylamide Polymers 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 230000001376 precipitating effect Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 230000004224 protection Effects 0.000 description 2
- 238000012797 qualification Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 239000011535 reaction buffer Substances 0.000 description 2
- 239000003642 reactive oxygen metabolite Substances 0.000 description 2
- 238000004064 recycling Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 230000001732 thrombotic effect Effects 0.000 description 2
- 238000012549 training Methods 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- JKYKXTRKURYNGW-UHFFFAOYSA-N 3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2-sulfonic acid Chemical compound O=C1C2=CC=CC=C2C(=O)C2=C1C(O)=C(O)C(S(O)(=O)=O)=C2 JKYKXTRKURYNGW-UHFFFAOYSA-N 0.000 description 1
- 208000019901 Anxiety disease Diseases 0.000 description 1
- 241000180579 Arca Species 0.000 description 1
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 101001011741 Bos taurus Insulin Proteins 0.000 description 1
- 201000006474 Brain Ischemia Diseases 0.000 description 1
- 208000003174 Brain Neoplasms Diseases 0.000 description 1
- 208000014644 Brain disease Diseases 0.000 description 1
- 206010048962 Brain oedema Diseases 0.000 description 1
- 102000004219 Brain-derived neurotrophic factor Human genes 0.000 description 1
- 108090000715 Brain-derived neurotrophic factor Proteins 0.000 description 1
- 102100031172 C-C chemokine receptor type 1 Human genes 0.000 description 1
- 101710149814 C-C chemokine receptor type 1 Proteins 0.000 description 1
- 102100037853 C-C chemokine receptor type 4 Human genes 0.000 description 1
- 101710149863 C-C chemokine receptor type 4 Proteins 0.000 description 1
- 102100036301 C-C chemokine receptor type 7 Human genes 0.000 description 1
- 102100025074 C-C chemokine receptor-like 2 Human genes 0.000 description 1
- 102100031650 C-X-C chemokine receptor type 4 Human genes 0.000 description 1
- 102100025618 C-X-C chemokine receptor type 6 Human genes 0.000 description 1
- 108090000835 CX3C Chemokine Receptor 1 Proteins 0.000 description 1
- 102100039196 CX3C chemokine receptor 1 Human genes 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 206010008089 Cerebral artery occlusion Diseases 0.000 description 1
- 206010008120 Cerebral ischaemia Diseases 0.000 description 1
- 108010012236 Chemokines Proteins 0.000 description 1
- 102000019034 Chemokines Human genes 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 208000005189 Embolism Diseases 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 208000004929 Facial Paralysis Diseases 0.000 description 1
- 102000009123 Fibrin Human genes 0.000 description 1
- 108010073385 Fibrin Proteins 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 206010019468 Hemiplegia Diseases 0.000 description 1
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 1
- 101000777558 Homo sapiens C-C chemokine receptor type 10 Proteins 0.000 description 1
- 101000716068 Homo sapiens C-C chemokine receptor type 6 Proteins 0.000 description 1
- 101000716065 Homo sapiens C-C chemokine receptor type 7 Proteins 0.000 description 1
- 101000716070 Homo sapiens C-C chemokine receptor type 9 Proteins 0.000 description 1
- 101000922348 Homo sapiens C-X-C chemokine receptor type 4 Proteins 0.000 description 1
- 101000856683 Homo sapiens C-X-C chemokine receptor type 6 Proteins 0.000 description 1
- 101001046686 Homo sapiens Integrin alpha-M Proteins 0.000 description 1
- 101000946889 Homo sapiens Monocyte differentiation antigen CD14 Proteins 0.000 description 1
- 206010061216 Infarction Diseases 0.000 description 1
- 102100022338 Integrin alpha-M Human genes 0.000 description 1
- 239000002211 L-ascorbic acid Substances 0.000 description 1
- 235000000069 L-ascorbic acid Nutrition 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102100035877 Monocyte differentiation antigen CD14 Human genes 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 239000012124 Opti-MEM Substances 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 101710124239 Poly(A) polymerase Proteins 0.000 description 1
- 108091036407 Polyadenylation Proteins 0.000 description 1
- 238000012181 QIAquick gel extraction kit Methods 0.000 description 1
- 238000003559 RNA-seq method Methods 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 101100219913 Rattus norvegicus Ccl2 gene Proteins 0.000 description 1
- 229940122208 Ribonuclease inhibitor Drugs 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 102000040739 Secretory proteins Human genes 0.000 description 1
- 108091058545 Secretory proteins Proteins 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 208000010513 Stupor Diseases 0.000 description 1
- 230000006052 T cell proliferation Effects 0.000 description 1
- 101710137500 T7 RNA polymerase Proteins 0.000 description 1
- 206010052779 Transplant rejections Diseases 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 208000036826 VIIth nerve paralysis Diseases 0.000 description 1
- 206010070995 Vascular compression Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000006909 anti-apoptosis Effects 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 210000000612 antigen-presenting cell Anatomy 0.000 description 1
- 230000036506 anxiety Effects 0.000 description 1
- 201000007201 aphasia Diseases 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 210000001367 artery Anatomy 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 210000001130 astrocyte Anatomy 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000003542 behavioural effect Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000008081 blood perfusion Effects 0.000 description 1
- 239000001045 blue dye Substances 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- IXIBAKNTJSCKJM-BUBXBXGNSA-N bovine insulin Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H]1CSSC[C@H]2C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3C=CC(O)=CC=3)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3NC=NC=3)NC(=O)[C@H](CO)NC(=O)CNC1=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O)=O)CSSC[C@@H](C(N2)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)C(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(C)C)C1=CN=CN1 IXIBAKNTJSCKJM-BUBXBXGNSA-N 0.000 description 1
- 210000004781 brain capillary Anatomy 0.000 description 1
- 210000004958 brain cell Anatomy 0.000 description 1
- 208000006752 brain edema Diseases 0.000 description 1
- 229940077737 brain-derived neurotrophic factor Drugs 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000002975 chemoattractant Substances 0.000 description 1
- 239000005482 chemotactic factor Substances 0.000 description 1
- 210000002987 choroid plexus Anatomy 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 210000000795 conjunctiva Anatomy 0.000 description 1
- 208000010247 contact dermatitis Diseases 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 235000019628 coolness Nutrition 0.000 description 1
- 238000013211 curve analysis Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000006837 decompression Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 229960000935 dehydrated alcohol Drugs 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000003511 endothelial effect Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 229960004756 ethanol Drugs 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 210000003414 extremity Anatomy 0.000 description 1
- 210000003191 femoral vein Anatomy 0.000 description 1
- 229950003499 fibrin Drugs 0.000 description 1
- 239000003527 fibrinolytic agent Substances 0.000 description 1
- 238000010304 firing Methods 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 230000009760 functional impairment Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 125000003051 glycosyloxy group Chemical group 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 102000046768 human CCL2 Human genes 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 229960000905 indomethacin Drugs 0.000 description 1
- 230000007574 infarction Effects 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 239000004026 insulin derivative Substances 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- 210000004731 jugular vein Anatomy 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 230000002101 lytic effect Effects 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 1
- 210000003657 middle cerebral artery Anatomy 0.000 description 1
- 201000007309 middle cerebral artery infarction Diseases 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 210000004498 neuroglial cell Anatomy 0.000 description 1
- 230000004112 neuroprotection Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 238000000879 optical micrograph Methods 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 239000004417 polycarbonate Substances 0.000 description 1
- 229920000515 polycarbonate Polymers 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000003716 rejuvenation Effects 0.000 description 1
- 230000010410 reperfusion Effects 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 239000003161 ribonuclease inhibitor Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 210000002027 skeletal muscle Anatomy 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- AVPCPPOOQICIRJ-UHFFFAOYSA-L sodium glycerol 2-phosphate Chemical compound [Na+].[Na+].OCC(CO)OP([O-])([O-])=O AVPCPPOOQICIRJ-UHFFFAOYSA-L 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000009168 stem cell therapy Methods 0.000 description 1
- 238000009580 stem-cell therapy Methods 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 210000002536 stromal cell Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000009182 swimming Effects 0.000 description 1
- 230000002537 thrombolytic effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 230000010474 transient expression Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 210000001364 upper extremity Anatomy 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 238000001262 western blot Methods 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
- C12N5/0652—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
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
- C07K14/7158—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for chemokines
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- 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
- C12N2510/00—Genetically modified cells
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Developmental Biology & Embryology (AREA)
- Molecular Biology (AREA)
- Public Health (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Urology & Nephrology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Virology (AREA)
- Rheumatology (AREA)
- Hematology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Epidemiology (AREA)
- Vascular Medicine (AREA)
- Cardiology (AREA)
- Heart & Thoracic Surgery (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
Abstract
The invention discloses a kind of interstital stem cell and preparation method thereof for being overexpressed CCR2, purposes of the interstital stem cell for being overexpressed CCR2 in the drug for treating acute ischemic cerebral apoplexy (Acute ischemic stroke, AIS) is also disclosed.Present invention optimizes the treatment methods of acute ischemic cerebral apoplexy, more have targeting and validity by being treated the MSC of CCR2 gene modification, can more significantly improve the therapeutic effect of MSC.The preparation method of the interstital stem cell of overexpression CCR2 of the invention is by the plasmid of building expression CCR2, and the method transfected using mRNA can make MSC be overexpressed receptor CCR2, while this method does not influence MSC phenotype itself, differentiation capability and immunoregulation capability；With lower genotoxicity, make MSCCCR2In vivo the purpose of targeted therapy can be more effectively played with directional migration to lesions position.
Description
Technical field
The present invention relates to stem-cell therapy technical field more particularly to a kind of interstital stem cells for being overexpressed CCR2
(Mesenchymal stromal cells, MSC) and its preparation method and application.
Background technique
Caused by acute ischemic cerebral apoplexy (Acute ischemic stroke, AIS) is a kind of blood circulation disorder because of brain
Sudden local cerebral functional impairment disease, clinically show as the symptom and body of transient or permanent brain disorder
Sign, is the common disease and frequently-occurring disease of Neurology, accounts for about the 60%~80% of whole cerebral apoplexies.AIS mainly include thrombotic with
Two kinds of embolic, occasionally in vasopasm or brain tumor vascular compression.Thrombotic is cerebrovascular wall because atherosis is cured lumen of vessels
Come it is smaller, it is even completely blocking；Embolic is to occur embolus suddenly in blood vessel, is stuck in the lesser blood vessel of bore with blood flow, hinders
Blood circulation.Brain tissue will lose rapidly its function after lacking blood perfusion, generate nervous centralis disease, when morbidity with
Hemiplegia, facial paralysis, aphasia, or even stupor suddenly, senseless is primary symptom.Chinese Center for Disease Control and Prevention was once
Carry out epidemiological survey to AIS, data show: 2011, China 40 years old or more crowd's AIS disease incidence was about 2,30/,100,000,
New hair number is about 133.4 ten thousand；Crowd's accounting is about 50% within 65 years old or less, and rejuvenation trend is presented.
Currently, being clinically intravenous injection thrombolytic drug " recombinant tissue-type's fibrin to the common method for the treatment of of AIS
Dissolved preferment activator (tPA) ", intracerebral blood circulation is rebuild by thrombolysis, to play treatment and improve the work of prognosis
With.However, there are more limiting factors for the performance of tPA therapeutic effect: window is narrow when 1. treating；2. there is no processing and decompression
In the case where the slight blood pressure lowering of medicine, its blood pressure need≤185/110mmHG when patient treats；3. being needed before being treated
Guarantee there is no bleeding in patient's brain by computed tomography (CT)；4. patient itself cannot be easy bleeding constitution；⑤
Do not confirm be cerebral apoplexy patient cannot use tPA treat；6. tPA is treated there are more side effect, for example encephalic goes out
Mass formed by blood stasis shape etc..In recent years, window plays positive effect when the implementation of intravenous injection is for expanding tPA treatment, can be by the time
It is expanded to after AIS occurs 12 hours, has pushed the clinical application of tPA, however use the disability rate after tPA treatment successful rescue still
It is so higher.Statistics shows in the AIS patient of survival that about 3/4 loses labour capacity to some extent, wherein serious disabled person
About 40%.
Due to drawbacks described above, the clinical application of tPA is not perhaps therefore the best means for the treatment of AIS compel to be essential at this stage
Explore a kind of new method for treating AIS.
Interstital stem cell (mesenchymal stem cells, MSC) is a kind of non-hematopoiesis found from marrow earliest
The stem cell (Friedenstein, A.J., et al, 1974) of class, is distributed widely in each tissue of whole body and organ, removes marrow
Outside, it is also present in gum, in the tissue such as skeletal muscle, fat, the injury repair and stable state for participating in tissue are maintained.MSC lacks special
Property marker, mainly express the interstitials marker such as CD29, CD44, CD73, CD90, CD105 and CD166, do not express CD11b,
The hematopoiesis Research of predicting markers such as CD14, CD19, CD34, CD45；It does not express or low expression HLA-I class molecule, does not express HLA-II class
Molecule.MSC has multi-lineage potential.Experiment in vitro proves that MSC can break up as neuron and Deiter's cells, in vivo
Experiment also turns out that the MSC transplanted after AIS can express the marker of neuron and Deiter's cells in central nervous system,
This directly repairs damage field for MSC and provides the foundation；The MSC transplanted after AIS can transfer endogenous neural member precursor
It is proliferated, MSC can also promote proliferation factor and anti-apoptosis factor by secretion to promote the survival and neuroglia of neuron
The proliferation of cell, reduces the phenomena of apoptosis in AIS, and MSC can also activate astroglia, generate some neuroprotections
Property molecule to promote to repair and restore, such as brain-derived neurotrophic factor；MSC has immunoloregulation function, can cause after AIS
Strong inflammatory reaction, and MSC is then able to carry out negativity adjusting.MSC can be by inhibiting T cell proliferation, T being promoted to adjust cell
It generates, inhibit CD4+ and CD8+T cell to inhibit the function of T cell, B cell and other antigen presenting cells can also be adjusted；
In addition, the HLA molecule of MSC oneself expression reduced levels, avoids graft-rejection；MSC can secrete Angiogensis
The factor, and survival and regeneration important role of the angiogenesis for neuron in AIS.Have benefited from the above-mentioned of interstital stem cell
Advantage, many different preclinical laboratories all consistently demonstrate MSC nervous function and having on behavioral function after improving AIS
Effect property.
In the animal model of experimental middle cerebral artery occlusion for simulating people AIS, research finds only fraction
The MSC of intravenous injection moves to damage field by the blood-brain barrier of defect, and most MSC are blocked in the organs such as lung
(Paul Lin.et al.,2013).A large amount of stem cells can bring security risk in the aggregation of non-target tissue；MSC in marrow simultaneously
Storage level only account for the 0.001-0.01% of cell total amount, source is rare；Therefore targeting of the MSCs to disease damage tissue is improved
It is the critical issue of the clinical conversion of MSC treatment.Chemotactic factor (CF)-chemoattractant receptor axis is considered as mediating MSCs chemotactic to disease damage group
The chemokine receptors of the important mechanisms knitted, MSC surface expression has CCR1, CCR4, CCR6, CCR7, CCR9, CCR10, CXCR4,
CXCR5, CXCR6, CX3CR1 etc., but these expression of receptor amounts are lower, and in vitro in incubation, the expression quantity of receptor by
Gradually decline (Sarkar, D., et al., 2011).
For many research workers using the targeting of raising MSC treatment as research direction, many scholars throw sight at present
To gene therapy, it such as is overexpressed CXCR5 with the method for virus transfection, MSC is promoted to move to contact hypersensitivity inflammation part
It moves (Xiaoran, Z., et al., 2017).The overexpression of chemokine receptors plays core in the targeting chemotactic process for improving cell
Heart effect, however the viral vectors currently used for gene therapy all has potential security risk；On the other hand, non-virus carrier
Transfection efficiency is relatively low, this to take a kind of more safely and effectively transport agent.
Recent studies suggest that thering is specific chemokine expression to compose, wherein there is 3 to become in the cerebral tissue of AIS sufferer
Change axis and plays major function, respectively SDF1-CXCR4 chemotactic axis, CX3CL1-CX3CR1 chemotactic axis, CCL2-CCR2 chemotactic axis
(Bonaventura A.,et al.,2016)。
A large amount of active oxygens are generated in patient's AIS disease damage tissue, induce cell apoptosis destruction blood-brain barrier, and then lead to inflammatory
Cellular infiltration to brain tissue, discharge inflammatory factor and be further exacerbated by active oxygen generation (Obermeier B., et al.,
2013).Blood-brain barrier refer to barrier between blood plasma and brain cell that brain capillary wall and Deiter's cells are formed and by
The barrier between blood plasma and cerebrospinal fluid that choroid plexus is formed, these barriers can prevent harmful substance from entering brain tissue by blood,
The destruction of blood-brain barrier is one of important indication of AIS poor prognosis (Khatri R., et al., 2012).
Summary of the invention
Based on this, plasmid of the present invention by building expression CCR2, the method transfected using mRNA makes MSC is instantaneous to cross table
Up to receptor CCR2, the ability of MSC targeting AIS disease damage tissue is improved, the therapeutic effect of MSC can be effectively improved.
On the one hand, the present invention provides a kind of interstital stem cells for being overexpressed CCR2.
On the other hand, the present invention also provides the above-described interstital stem cells for being overexpressed CCR2 to prepare for treating
Purposes in the drug of cerebral arterial thrombosis.
In another aspect, the present invention also provides a kind of for treating the drug of cerebral arterial thrombosis, it includes the above
Overexpression CCR2 interstital stem cell.
Another aspect, the present invention also provides a kind of sides for preparing the above-described interstital stem cell for being overexpressed CCR2
Method comprising following steps:
(1) plasmid of building expression CCR2；
(2) CCR2mRNA is synthesized using the plasmid of step (1) building；
(3) by the CCR2mRNA transfection interstital stem cell of step (2) synthesis to get the interstital stem cell for being overexpressed CCR2.
Pass through experimental observation, it has been found that the MSC for transfecting overexpression CCR2 using mRNA is safe and effective, can determine in vivo
To migrating to lesion, and the MSC that further discovery is overexpressed CCR2 in mechanism can secrete it is higher levels of anti-oxidant
Albumen PRDX4 is to reduce reactive oxygen species, to repair blood-brain barrier structure and reach the better curative effect to AIS.
As further improvement to above-mentioned technical proposal, in the step (1), the plasmid of building expression CCR2 is used to be carried
Body is 3.1 carrier of pcDNA.
As further improvement to above-mentioned technical proposal, the step (1) is specifically included: it is thin to extract the single core of peripheral blood
Born of the same parents RNA, reverse transcription amplifies overall length CCR2cDNA using CCR2 Specific PCR primers at cDNA, by PCR product and plasmid
PcDNA 3.1 is purified after Kpn I and Sfu I double digestion, and CCR2 target gene fragment is connected to 3.1 plasmid of pcDNA,
Obtain the plasmid of expression CCR2.
As further improvement to above-mentioned technical proposal, the CCR2 Specific PCR primers are as follows:
Upstream primer: 5 '-TTGGTACCTACGGTGCTCCCTGTCATAAA-3 ',
Downstream primer: 5 '-TTTTTCGAATAAGATGAGGACGACCAGCAT-3 '.
As further improvement to above-mentioned technical proposal, in the step (2), the CCR2mRNA is with 5 ' end caps
The CCR2mRNA of minor structure and 3 ' end poly A tracts.
As further improvement to above-mentioned technical proposal, the step (2) is specifically included:
(21) plasmid linearization for constructing step (1), obtains linear plasmid DNA；
(22) for the linear plasmid DNA obtained using step (21) as template, synthesis has the CCR2mRNA of 5 ' end cap minor structures；
(23) tailing is carried out to get CCR2mRNA to the CCR2mRNA with 5 ' end cap minor structures of step (22) synthesis.
MRNA connects the intermediate bridge of DNA and protein as courier, can eliminate virus to cell as carrier
Toxic effect and DNA are integrated into brought security risk in genome.However mRNA is more unstable, seriously inhibits its conduct
The transfection efficiency and feasibility of carrier.Result of study shows degeneration-resistant to turn cap analog (Anti-reverse cap
Analogues, ARCA), additional nontranscribed domain, poly (A) tail can be improved the translation efficiency of mRNA in the cell, realize
The transient expression of protein in the cell；And mRNA has the advantages such as low, the pharmacology safety of immunogenicity, can be used as albumen and crosses table
The effective tool reached.Although mRNA transfection is only capable of instantaneously being overexpressed destination protein, since the MSC of venoclysis reaches disease damage portion
Position only needs 1~2h, expresses chemokine receptors in short-term and is enough to improve the targeting of MSC.
Another aspect, the present invention also provides a kind of CCR2 Specific PCR primers sequences comprising:
Upstream primer: 5 '-TTGGTACCTACGGTGCTCCCTGTCATAAA-3 ',
Downstream primer: 5 '-TTTTTCGAATAAGATGAGGACGACCAGCAT-3 '.
Compared with existing scheme, the invention has the following advantages:
Present invention optimizes the treatment methods of acute ischemic cerebral apoplexy, by controlling the MSC of CCR2 gene modification
Treatment more has targeting and validity, can more significantly improve the therapeutic effect of MSC；
The preparation method of the interstital stem cell of overexpression CCR2 of the invention is utilized by the plasmid of building expression CCR2
The method of mRNA transfection, can make MSC be overexpressed receptor CCR2, while this method does not influence MSC phenotype itself, differentiation capability and exempts from
Epidemic disease regulating power；
MSC made from the preparation method of the interstital stem cell of overexpression CCR2 through the inventionCCR2, there is lower base
Because of toxicity, MSC not only can be improvedCCR2The ability of ischemic tissue of brain is arrived in field planting of going back to the nest in vivo, makes MSCCCR2It can determine in vivo
To migrating to lesions position, and MSC obtained has higher safety.Ischemic brain is treated with this interstital stem cell
Stroke (AIS) will more have targeting and validity, can effectively improve the therapeutic effect of MSC.
Detailed description of the invention
Fig. 1 is MSCCCR2With MSC expression CCR2 figure；
Fig. 2 is MSCCCR2Phenotype itself and differentiation capability testing result figure；
Fig. 3 is the external and vivo detection result figure for being overexpressed the transfer ability of hBMSCs trend CCL2 of CCR2；
Fig. 4 shows MSCCCR2The expression and its oxidation resistance of middle antioxidant genes；
Fig. 5 is intravenous injection MSCsCCR2The testing result for mitigating cerebral ischemic injury and nervous function being promoted to restore
Figure.
Specific embodiment
To facilitate the understanding of the present invention, a more comprehensive description of the invention is given in the following sections with reference to the relevant attached drawings.In attached drawing
Give presently preferred embodiments of the present invention.But the invention can be realized in many different forms, however it is not limited to this paper institute
The embodiment of description.On the contrary, purpose of providing these embodiments is keeps the understanding to the disclosure more thorough
Comprehensively.
Unless otherwise defined, all technical and scientific terms used herein and belong to technical field of the invention
The normally understood meaning of technical staff is identical.Term as used herein in the specification of the present invention is intended merely to description tool
The purpose of the embodiment of body, it is not intended that in the limitation present invention.Term as used herein "and/or" includes one or more phases
Any and all combinations of the listed item of pass.
Embodiment 1Establish the interstital stem cell (MSC for being overexpressed CCR2CCR2)
1.MSC culture and phenotypic evaluation:
Healthy volunteer marrow 20ml is taken, is diluted with 1 × PBS by 1:1, using Ficoll-Paque lymph separating liquid with close
Degree gradient centrifugation is isolated mononuclearcell (2000rpm, 30min) from marrow, the mononuclearcell being collected by 1 ×
105/ cm2 density is inoculated into 75cm2 culture bottle and is cultivated.It is cultivated 3 days under the conditions of 37 DEG C, 5%CO2 with L-DMEM culture medium
Afterwards, suspension cell is removed, liquid is changed and continues to cultivate.After cell length to 80% density, culture medium is sucked, is washed 2 times with PBS, used
0.125% pancreatin digests 1-ni72min, and passage ratio is 1:3.MSC is isolated by the marrow that health donors are contributed, and clinic is used
The separation of MSC expands, freezes, recovering etc. in the condition for meeting GMP (good manufacturing practice) standard
Lower progress.Observe primary and growing state and morphological feature of passage cell, film making record daily under inverted microscope.Take body
The MSC of outer culture is digested to single cell suspension, is washed one time, is discarded with the PBS (pH 7.4) containing 0.1%BSA+0.05%NaN3
Supernatant, adjustment cell density are 106/ ml in streaming pipe, using streaming antibody CD29, CD34, CD44, CD45, CD73, CD90,
CD105, CD166 mark MSCs, and mixing fullys shake, 4 DEG C, is protected from light and is incubated for 30min, then with containing 0.1%BSA+0.05%
The PBS (pH 7.4) of NaN3 is washed twice, to remove Excess antibody；Supernatant is abandoned, cell, streaming inspection is resuspended with 200ul 1%PFA
It surveys MSCs cell phenotype (CD29+, CD34-, CD44+, CD45-, CD73+, CD90+, CD105+, CD166+), it was demonstrated that external training
It supports on MSC cell phenotype without influence.
P2 is reached into six orifice plates for cell, length to 60% or so is for use.
2. the acquisition of peripheral blood mononuclear cells:
Healthy volunteer fresh peripheral blood 20ml is taken, is diluted with 1 × PBS by 1:1, is separated using Ficoll-Paque lymph
Liquid is separated by density-gradient centrifugation method, collects whole tunica albuginea layer mononuclearcells, is diluted with sterile PBS by 1:4.2000rpm,
It is centrifuged 10min, abandons supernatant.Again plus enough PBS are washed twice.With RPMI-1640 complete medium suspension cell, that is, obtain outside people
All blood mononuclear cells (PBMC).
3.Trizol method extracts peripheral blood mononuclear cells RNA:
1ml Trizol solution is added in the human peripheral blood single nucleus cell (PBMC) of acquisition, piping and druming mixes, and fills cell
Division solution, stands 5min；200 μ l chloroforms are added, shakes vigorously and mix well 20s, comes into full contact with water phase and organic phase, be stored at room temperature
15min；At 4 DEG C, 12000rpm is centrifuged 15min, it is seen that is divided into three layers, it is new carefully to move to another in upper strata aqueous phase by RNA
RNase free EP pipe；0.5ml isopropanol is added, gently mixes well, in being stored at room temperature 10min, precipitates RNA；At 4 DEG C,
12000g is centrifuged 10min, collects RNA precipitate, removes supernatant；Tube wall is washed twice with 75% ethyl alcohol, and super-clean bench air-dries；50 μ l are added
DEPC water dissolution precipitating, NanoDrop ultramicrospectrophotometer measure concentration.
4. reverse transcription:
Removal genomic DNA: RNA (1 μ g)+DNase I (1 μ l)+Buffer DNase I with MgCl2 (1 μ l)+
DEPC water, 10 μ l systems, 37 DEG C of incubation 30min；It is added EDTA (1 μ l), 65 DEG C of incubation 10min；It is added Oligo (dT) (1 μ l),
65 DEG C of incubation 10min；Finally, 5 × Reaction Buffer (5 μ l) is added, RNase-Ribonuclease Inhibitor (1
μ l), 10mM dNTP Mix (2 μ l), M-MLV RT (1 μ l), RNase Free Water totally 25 μ l system, 42 DEG C of incubations
60min collects cDNA product.
5.PCR reaction and recycling CCR2cDNA segment:
PCR reaction: 2 × Star mix (DNAPolymerase containing Taq, dNTPs, Mg2+, reaction buffer and stabilizer
Deng) 10 μ l, 7 μ l of DEPC water, 1 μ l of upstream primer, 1 μ l of downstream primer, cDNA1 μ l, total system 20ul；CXCR5 clip size
1119bp.Glue recycling: sharp scalpel cuts target fragment band, uses Ago-Gel DNA QIAquick Gel Extraction Kit gel extraction
The segment of PCR target gene CCR2.
Wherein, the segment for obtaining target gene CCR2 is reacted above by PCR, used primer sequence is as follows:
Upstream primer: TTTGGTACCTACGGTGCTCCCTGTCATAAA (SEQ ID NO:1),
Downstream primer: TTTTTCGAATAAGATGAGGACGACCAGCAT(SEQ ID NO:2)。
Wherein, restriction enzyme site Kpn I is shown in the underscore part of upstream primer sequence and downstream primer sequence respectively
With Sfu I.
The building of 6.pcDNA3.1/CCR2 plasmid
Double digestion is carried out to PCR product with Kpn I and Sfu I, with the universal DNA purification and recovery kit of Tiangen into
Row purification and recovery.Double digestion is carried out with Kpn I and Sfu I simultaneously, carries out purification and recovery with mentioned reagent box.Connected using T4DNA
Enzyme is connect to connect target gene fragment at 16 DEG C with pcDNA3.1 linear plasmid overnight.After connection product transfects competence T1 bacterium,
After being coated on 37 DEG C of culture 12h of culture plate containing Kanamycin, picking single colonie carries out PCR identification.Benefit after Preliminary Identification success
Plasmid is extracted with the small extraction reagent kit of Tiangen plasmid, and Sangon Biotech (Shanghai) Co., Ltd. is sent to carry out sequencing mirror
It is fixed.
The external synthesis of 7.CCR2mRNA
1. the linearisation of plasmid: using the restriction enzyme site of the Pme I of CCR2 downstream of gene in plasmid, making through Pme I digestion
PcDNA3.1/CCR2 plasmid linearization.The digestive juice of plasmid after Proteinase K and SDS processing, then by phenol/chloroform and
The pcDNA3.1/CCR2 linear plasmid DNA of purifying can be obtained in ethanol precipitation.
2. the external synthesis of cap sequence mRNA: containing cap sequence with the synthesis of mMESSAGE rna transcription kit
mRNA.Key step are as follows: using the DNA of linearisation as template, using 4 kinds of NTP and cap analogs as raw material, in t7 rna polymerase
Under catalysis, synthesis has the mRNA of 5 ' end cap minor structures.Above-mentioned synthesis reaction solution digests (remove template DNA) through DNase I
Afterwards, mRNA is separated and is purified by LiC l precipitating.
3. the poly A tailing of mRNA: using ATP as raw material, under the catalysis of yeast poly (A) polymerase, to 5 ' end caps
The CCR2mRNA of structure carries out tailing, it is made to generate the mRNA of structural integrity, i.e., with 5 ' end cap minor structures and 3 ' end poly A tracts
CCR2mRNA.Finally by the size of Denaturing Agarose Gel electrophoretic examinations mRNA.Measure the absorbance at wavelength 260nm
(A) concentration of value estimation mRNA.
The CCR2mRNA in-vitro transfection of 8.MSC
It is transfected using CCR2mRNA of the TransIT-mRNA kit to synthesis.Concrete operations are as follows:
1. being resuspended with fresh complete culture solution to density is 1 × 10 after growing to the MSC digestion in 3-4 generation5/ mL, and
It is seeded to 12 orifice plates, every hole 1mL.
2. second day, 1h replaced fresh complete culture solution before transfecting after cell is adherent.
3. 1 μ g CCR2mRNA is taken to be added in 100 μ L Opti-MEM culture solutions, gently piping and druming is mixed.
4. each 1 μ L of BOOSTreagent and TransIT-mRNA is added in a step mixture then up.
5. mixture is slowly uniformly added into MSC culture solution, slowly shaking makes mRNA be evenly distributed, 37 DEG C, 5%CO2Training
It supports and is cultivated in case.
9. detecting the expression of CCR2mRNA and albumen
The cell in 12 orifice plates after mRNA transfection is collected after 72 hours, purifies to obtain transgenosis by selected by flow cytometry apoptosis
Cell line simultaneously expands；
MSC expression after real-time fluorescence quantitative PCR (Real-time Quantitative PCR, RT-PCR) detection transfection
The content of CCR2mRNA:
1. extracting RNA and reverse transcription: step 3 in step reference implementation example 1,4；
2. pcr amplification reaction system:
20 μ l of total system；
Reaction condition: 95 DEG C of 10min；3 footworks, 40 circulations: 95 DEG C of 15s, 60 DEG C of 30s, 72 DEG C of 15s；Melt curve analysis: 55
It DEG C -95 DEG C, reads 1 time per minute.
Wherein, primer used in pcr amplification reaction system is as follows:
GAPDH:
Upstream primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO:3)
Downstream primer: GAAGATGGTGATGGGATTTC (SEQ ID NO:4)
CCR2:
Upstream primer: TACGGTGCTCCCTGTCATAAA (SEQ ID NO:5)
Downstream primer: TAAGATGAGGACGACCAGCAT (SEQ ID NO:6)
Western Blot detects the protein level of metainfective MSC expression CCR2:
1. extracting albumen: taking the MSC (MSC of the overexpression CCR2 in cultureCCR2) and control group MSC, it is placed on ice, removal
After washing twice with the PBS of pre-cooling, 1 × SDS sample-loading buffer (62.5mM Tris-HCl containing 5%DTT is added in culture solution
(pH6.8), 2% (w/v) SDS, 10%glycerol, 50mM DTT, 0.1% (w/v) bromjophenol blue), the liquid-transfering gun of 1ml is used rapidly
(about 10 times) are blown and beaten back and forth, abundant lytic cell.It draws liquid after piping and druming to be put into the Eppendorf centrifuge tube of 1.5mL, 4 DEG C
Ultrasonication 3 times, every time 1 second；100 DEG C are boiled 5 minutes, are set 4 DEG C of coolings and in 4 DEG C with 15000g centrifugation 5 minutes, are prepared electrophoresis
Or -80 DEG C save backup.
2. gel electrophoresis separation sample: denaturing polyacrylamide gel (denatured polyacrylamide gel,
SDS-PAGE): adding separation gel 4.1mL, with deionized water sealing, after sharp interface to appear, incline and fall water seal layer, addition has been matched
The concentration glue made is to the top of short glass blocks.Comb is inserted, when irregular shape occurs in glue surface, shows that glue is aggregated good, it can
To pull out comb, loading.
In order, 100 DEG C of 15 μ L of sample for boiling 5 minutes are added in every swimming lane, with constant pressure 120V in SDS electrophoretic buffer
Electrophoresis about 45 minutes.
3. transferring film: while electrophoresis, material required for transferring film, such as sponge, filter paper, pvdf membrane being immersed in transferring film
In buffer (25mM Tris base, 0.2M glycine, 20%methanol pH8.5).After electrophoresis, gel is removed,
Remove concentration glue part above.Glue is placed in transferring film liquid and is balanced 15~30 minutes, to remove the SDS of glue surface attachment.So
After start fill transferring film sandwich mold, according to sponge, one layer of filter paper, gel, pvdf membrane, one layer of filter paper and sponge from cathode to anode
Sequence unit sandwich mold is put in transfer groove after fixed, and pvdf membrane faces toward positive extreme direction.Sandwich mold and ice chest are placed in
In transfer groove, injection 600mL transfering buffering liquid, constant current 200mA2 hours.
4. antigen-antibody reaction:
After transferring film, pvdf membrane is removed, is washed in the TBS (50mM Tris-HCl pH7.4,150mMNaCl) of 25mL
It 10 minutes, then goes in 20mL confining liquid [1 × TBST (0.05%Tween-20in TBS) containing 5% defatted milk], room temperature
Lower oscillation is closed 1 hour, is added accordingly with containing the 5% diluted primary antibody dilution of (w/v) defatted milk.4 DEG C of mild shaken overnights.
Secondary daily 1 × TBST is washed film 3 times, and 5 minutes every time, then addition confining liquid was diluted by horseradish peroxidase
Secondary antibody (1:2000) 15mL of (horseradish peroxidase, HRP) label, shaken at room temperature 1 hour.Later with 1 ×
TBST is washed film 3 times, 10 minutes every time, is developed using the Chemiluminescence Apparatus of bio-rad.
As a result as shown in Figure 1, Fig. 1 is shown: streaming counting detection discovery is overexpressed CCR2 efficiency and is up to 83.3%；Fluorescence is fixed
CCR2 gene level significantly increases in amount PCR detection MSC；Protein immunoblot experiment prove MSC in CCR2 protein overexpression at
Function.
Embodiment 2The identification of MSC biology performance
The identification of 1.MSC phenotype
The MSC of in vitro culture is taken to be digested to single cell suspension, with the PBS (pH 7.4) containing 0.1%BSA+0.05%NaN3
Washing one time, discards supernatant, and adjustment cell density is 106/ml in streaming pipe, using streaming antibody CD29, CD34, CD44,
CD45, CD73, CD90, CD105, CD166 mark MSCs, fully shake mixings, 4 DEG C, are protected from light incubation 30min, then with containing
The PBS (pH 7.4) of 0.1%BSA+0.05%NaN3 is washed twice, to remove Excess antibody；Supernatant is abandoned, with 200ul 1%PFA
Be resuspended cell, flow cytometer detection MSCs cell phenotype (CD29+, CD34-, CD44+, CD45-, CD73+, CD90+, CD105+,
CD166+), it was demonstrated that in vitro culture on MSC cell phenotype without influence, as a result as shown in Figure 2 A.The mirror of 2.MSC Multidirectional Differentiation ability
It is fixed
(1) Osteoblast Differentiation: the induction of Osteoblast Differentiation is carried out to the MSC after modification, MSC is with 4 × 103/cm2Cell density connects
Kind in 6 orifice plates, after cell reach 50% converge after change self-bone grafting culture solution into and induced, induction liquid is that DMEM is added
100ml/L fetal calf serum, 50mg/L ascorbic acid, 0.1 μm of ol/L dexamethasone, 0.5mmol/L sodium β-glycerophosphate.To the 5th,
8,10 generation cells sampling in 6,9,12,15 days after induction liquid is added is dyed and carries out quantitative analysis to its osteogenic ability.It lures simultaneously
It leads the 14th, 21 day, sucks osteogenic induction liquid, washed 3 times with PBS, appropriate alizarin red S dye liquor drop is added dye 3-5 minutes, Jing Xiaguan
Examine calcium tubercle coloration result.
(2) break up at rouge: carrying out the induction broken up at rouge to the MSC after modification, MSC is with 4 × 103/cm2The density of cell
It is inoculated in 6 orifice plates, reaches to cell and change rouge induction broth into after converging completely and induced, adipogenic induction liquid A is added
(L-DMEM basic culture solution contains 10% fetal calf serum, the dexamethasone of 1 μm of ol/L, the IBMX of 0.5mmol/L, the ox of 10 μ g/mL
Insulin, 0.2mmol/L indomethacin), it induces 3 days；With adipogenic induction liquid B, (HDMEM basic culture solution contains 10% again
Fetal calf serum, 10 μ g/ml bovine insulins) processing 1 day.So circulation 3 times, control group addition bovine insulin containing 10mg/L, 10%
FCSL-DMEM was changed liquid 1 time every 3-4 days.The variation of microscopically observation cellular morphology and growing state.Cell after induction
It is washed 3 times with PBS, 10% paraformaldehyde fixes 10 minutes, and oil red O stain 5-10 minutes, 60% isopropanol slightly washed away extra dye
Liquid, distillation washing, optical microphotograph microscopic observation oil red O stain result.The qualification result of MSC Multidirectional Differentiation ability such as Fig. 2 B-2CC
Shown, show in figure: the MSC for modifying front and back has normal skeletonization and lipoblast differentiation capability.
Embodiment 3MCAO model construction
(1) MCAO model construction
1. rat pre-operative anxiety 12h, free water.10% chloraldurate 350mg/kg induced anesthesia is injected intraperitoneally, lies on the back
Position is fixed, takes neck median incision, and separation right carotid, vena jugularis externa and jugular vein, coagulation blow External Carotid Artery Branch,
It ligatures and the external carotid artery trunk that dissociates, cuts an osculum in free section, it will be dynamic outside the 4-0 nylon wire merging neck of end firing round end
Arteries and veins, through arteria carotis communis bifurcated such as internal carotid, depth counts about 18~20mm by arteria carotis communis crotch, until there is light resistance
Until sense.Line bolt is retreated into external carotid artery stump when rat cerebral ischemia 1.5h, forms Reperfu- sion.
2. Sham group is inserted into bolt line only about 10mm, blood flow of middle cerebral artery is not blocked to supply.
(2) animal is divided into 5 groups, every group 8:
Sham-operation 1. (Sham) group；
2. PBS treats (PBS) group；
3. MSC treats (MSC) group；
④MSCCCR2Treat (MSCdTomato) group.
Embodiment 4b valence is overexpressed the migration of the MSC of CCR2
Experiment in vitro carries out in the micro- migration plate in 48 holes of built-in 8 μm of aperture polycarbonate membranes, migrates and distinguishes in room under plate
Blank control, recombined human CCL2 and recombinant rat CCL2, MSC is addedCCR2And MSCdtomatoIt is added separately to upper chamber, observes cell
Migration situation calculates cell migration index.As a result as shown in Figure 3, wherein Fig. 3 A shows MSCCCR2Group has stronger than control group
Shift function, Fig. 3 B shows that the CCL2 ligand in rat and people source can effectively attract MSCCCR2Cell migration.Fig. 3 C is shown
The MSC for being overexpressed CCR2 can more effectively navigate to headstroke damage location (white arrow indicates MSC cell).Fig. 3 D is indicated
With the increase of number of days after modeling, MSC navigates to lesion location increasing number.
Embodiment 5It is overexpressed MSC antioxidant genes expression and the oxidation resistance detection of CCR2
1. antioxidant genes expression
MSC after mRNA transcription process is added Trizol and is resuspended, and -80 DEG C are transferred to after liquid nitrogen flash freezer and is saved for RNA-
seq。
RNA-seq is done by Shanghai Ou Yi biomedicine Science and Technology Ltd., and briefly steps are as follows: being digested using DNase
It is enriched with RNA after DNA, with interrupting reagent for mRNA and being broken into short-movie section and as templated synthesis cDNA, uses kits
Double-strand cDNA carries out end reparation plus A tail again, selects suitable clip size to carry out PCR amplification after jointing, builds
After the Agilent 2100Bioanalyzer quality inspection qualification of library, surveyed using 2500 sequenator of Illumina HiSeqTM
Sequence.
After comparison result passes through QC, expression analysis is carried out to gene, gene expression amount uses FPKM (Fragments per
Kb per Million reads) method calculates and analyzes.
2. oxidation resistance detects
The Mice Inoculated brain endothelial cell in 24 orifice plates, density guarantee that second day degrees of fusion reaches 80% or so.Then
It carries out glycosyloxy and deprives (OGD model is a kind of model of the research endothelial injuries of in-vitro simulated internal MCAO).Culture medium before detecting
It is changed to fresh containing 5 μm of ol/Loxidative stress reagents(Life Technologies)
Complete medium, cultivate 30min in 37 DEG C of cell incubators, then discard culture medium with PBS and wash cell 5min × 3
It is secondary, remove remaining dyestuff；It is discarded after trypsin digestion cell 1min, is terminated and digested with FBS, blown and beaten into single cell suspension, it will be thin
Born of the same parents are collected into 1.5mL EP pipe, and centrifugation removal FBS adds 300 μ L PBS and cell, upper machine testing is resuspended.Testing result is used
Fluorescence intensity indicates, it should be noted that avoiding stronger light direct irradiation cell during whole operation.
MSCCCR2The expression of middle antioxidant genes and its testing result of oxidation resistance are as shown in figure 4, Fig. 4 is shown: OGD
The endothelial cell of group generates a large amount of active oxygen, and co-cultures MSCCCR2And MSCdtomatoEndothelial cell (i.e. MSCCCR2Group and
MSC dtomatoGroup), reactive oxygen species are remarkably decreased, and MSC is prompted to have powerful oxidation resistance.Further pass through RNA-
All in seq, analysis MSC to express to anti-oxidant relevant secretory protein, PRDX4 expresses highest as the result is shown, prompts it may
Antioxidation is generated by secreting the albumen.
Embodiment 6Migration after evaluating MSC vein transplantation and the curative effect to acute ischemic cerebral apoplexy disease damage tissue
1. healthy adult male SD rat is taken to be divided into four groups: 1. sham-operation group；2. the postoperative tail vein injection PBS for 24 hours of MCAO
Group；3. the postoperative MSC group of tail vein injection for 24 hours of MCAO；4. the postoperative tail vein injection MSC for 24 hours of MCAOCCR2Group.
Infarction of brain area size is measured after 2.TTC dyeing
Brain tissue is taken within postoperative 4 days, 20min in -20 DEG C of refrigerators carries out 1mm coronal section from back to front, is placed in 2%TTC dye
In color liquid, 37 DEG C are protected from light incubation 30min, and the fixed 6h of 4% paraformaldehyde observes cerebral infarction stove staining conditions and simultaneously measured with ImageJ
Cerebral infarction volume.
3. detecting the variation of each group rat nerve function (Menzies) scoring
Rat MCAO is 1 day postoperative, 4 days, 7 days personnel for not knowing about grouping by one carry out nervous function to experimental rat and lack
Damage scoring, using Menzies standards of grading: impassivity function damage, double forelimbs symmetrically stretch (0 point) to the ground；Opposite side forelimb
(1 point) is received in continuing；Opposite side forelimb grip declines (2 points)；Minimal irritation rat stomach turn-takes (3 points) to opposite side；Independently persistently turn
It encloses (4 points).
4. detecting blood-brain barrier (BBB) integrality
(1) degree of cerebral edema is detected
Dry and wet weight method measures brain water content.10% chloraldurate intraperitoneal injection of anesthesia, quickly broken end takes brain, takes ischemic
Side brain tissue, filter paper suck brain surface moisture, and measurement brain tissue dry weight is placed on 95 DEG C of dehydrations for 24 hours, dry weight is measured after dehydration.Brain
Tissue water content=(weight in wet base-dry weight)/weight in wet base * 100%.
(2) EB dyestuff seepage discharge is detected
3h injects 2%EB normal saline solution (6ml/kg) through femoral vein before rat is put to death, rat eye conjunctiva after several seconds,
Four limbs etc. are displayed in blue.It takes brain tissue to be placed in the solution of trichloroacetic acid of 1ml 5% to be homogenized, homogenate is through being centrifuged
Supernatant is taken after (15000g, 15min), 4 times are diluted in dehydrated alcohol.Using sepectrophotofluorometer (excitation wavelength 620nm,
Launch wavelength 680nm, bandwidth 10nm) detection fluorescent value.It is drawn out between EB and fluorescent value according to the external standard of EB solution
Standard curve (in a linear relationship).Sample concentration is calculated according to standard curve by the fluorescent value measured, acquires brain tissue extracting
EB content in liquid, then compared with the quality of brain tissue, obtain EB content in every gram of weight in wet base brain tissue.
It is injected intravenously MSCCCR2The testing result such as Fig. 5 institute for mitigating cerebral ischemic injury and nervous function being promoted to restore
Show, shown in figure: being injected intravenously MSC after MCAO modelingCCR2Isosorbide-5-Nitrae afterwards collects pattern detection in 7 days, by Neuroscore,
Evans Blue dye test experience proves, MSCCCR2It can contribute to promote nervous function resulted in ischemic cerebral apoplexy
Restore.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Sequence table
<110>Zhongshan University
<120>a kind of interstital stem cell for being overexpressed CCR2 is for treating acute ischemic cerebral apoplexy and preparation method thereof
<130> 2018
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 30
<212> DNA
<213>artificial sequence (Artificial)
<400> 1
tttggtacct acggtgctcc ctgtcataaa 30
<210> 2
<211> 30
<212> DNA
<213>artificial sequence (Artificial)
<400> 2
tttttcgaat aagatgagga cgaccagcat 30
<210> 3
<211> 19
<212> DNA
<213>artificial sequence (Artificial)
<400> 3
gaaggtgaag gtcggagtc 19
<210> 4
<211> 20
<212> DNA
<213>artificial sequence (Artificial)
<400> 4
gaagatggtg atgggatttc 20
<210> 5
<211> 21
<212> DNA
<213>artificial sequence (Artificial)
<400> 5
tacggtgctc cctgtcataa a 21
<210> 6
<211> 21
<212> DNA
<213>artificial sequence (Artificial)
<400> 6
taagatgagg acgaccagca t 21
Claims (10)
1. a kind of interstital stem cell for being overexpressed CCR2.
2. the interstital stem cell according to claim 1 for being overexpressed CCR2 is preparing the medicine for treating cerebral arterial thrombosis
Purposes in object.
3. a kind of for treating the drug of cerebral arterial thrombosis, which is characterized in that include overexpression CCR2 described in claim 1
Interstital stem cell.
4. a kind of method for preparing the interstital stem cell described in claim 1 for being overexpressed CCR2, which is characterized in that including as follows
Step:
(1) plasmid of building expression CCR2；
(2) CCR2mRNA is synthesized using the plasmid of step (1) building；
(3) by the CCR2mRNA transfection interstital stem cell of step (2) synthesis to get the interstital stem cell for being overexpressed CCR2.
5. according to the method described in claim 4, it is characterized in that, building is expressed used in the plasmid of CCR2 in the step (1)
Carrier is 3.1 carrier of pcDNA.
6. according to the method described in claim 4, it is characterized in that, the step (1) specifically includes: extracting the single core of peripheral blood
Cell RNA, reverse transcription amplifies overall length CCR2cDNA using CCR2 Specific PCR primers at cDNA, by PCR product and plasmid
PcDNA 3.1 is purified after Kpn I and Sfu I double digestion, and CCR2 target gene fragment is connected to 3.1 plasmid of pcDNA,
Obtain the plasmid of expression CCR2.
7. according to the method described in claim 6, it is characterized in that, the CCR2 Specific PCR primers are as follows:
Upstream primer: 5 '-TTGGTACCTACGGTGCTCCCTGTCATAAA-3 ',
Downstream primer: 5 '-TTTTTCGAATAAGATGAGGACGACCAGCAT-3 '.
8. according to the method described in claim 4, it is characterized in that, the CCR2mRNA is with 5 ' ends in the step (2)
The CCR2mRNA of cap sequence and 3 ' end poly A tracts.
9. according to the method described in claim 4, it is characterized in that, the step (2) specifically includes:
(21) plasmid linearization for constructing step (1), obtains linear plasmid DNA；
(22) for the linear plasmid DNA obtained using step (21) as template, synthesis has the CCR2mRNA of 5 ' end cap minor structures；
(23) tailing is carried out to get CCR2mRNA to the CCR2mRNA with 5 ' end cap minor structures of step (22) synthesis.
10. a kind of CCR2 Specific PCR primers sequence characterized by comprising
Upstream primer: 5 '-TTGGTACCTACGGTGCTCCCTGTCATAAA-3 ',
Downstream primer: 5 '-TTTTTCGAATAAGATGAGGACGACCAGCAT-3 '.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811328565.7A CN109439631B (en) | 2018-11-09 | 2018-11-09 | Mesenchymal stem cells overexpressing CCR2 for treating acute ischemic stroke and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811328565.7A CN109439631B (en) | 2018-11-09 | 2018-11-09 | Mesenchymal stem cells overexpressing CCR2 for treating acute ischemic stroke and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109439631A true CN109439631A (en) | 2019-03-08 |
| CN109439631B CN109439631B (en) | 2021-02-09 |
Family
ID=65552054
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811328565.7A Active CN109439631B (en) | 2018-11-09 | 2018-11-09 | Mesenchymal stem cells overexpressing CCR2 for treating acute ischemic stroke and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109439631B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110946877A (en) * | 2019-12-30 | 2020-04-03 | 深圳爱生再生医学科技有限公司 | Stem cell biological product for treating liver cirrhosis and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105734075A (en) * | 2016-02-04 | 2016-07-06 | 南京大学 | Carrier for interfering with expression of gene ABCB5 and application of carrier in tumor stem cell treatment |
| CN106011074A (en) * | 2016-06-12 | 2016-10-12 | 中山大学 | Mesenchymal stem cells of high-expression CXCR5 and preparation and application of mesenchymal stem cells |
-
2018
- 2018-11-09 CN CN201811328565.7A patent/CN109439631B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105734075A (en) * | 2016-02-04 | 2016-07-06 | 南京大学 | Carrier for interfering with expression of gene ABCB5 and application of carrier in tumor stem cell treatment |
| CN106011074A (en) * | 2016-06-12 | 2016-10-12 | 中山大学 | Mesenchymal stem cells of high-expression CXCR5 and preparation and application of mesenchymal stem cells |
Non-Patent Citations (4)
| Title |
|---|
| BONAVENTURA A.ET AL: "Update on Inflammatory Biomarkers and Treatments in Ischemic Stroke", 《INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES》 * |
| YINONG HUANG ET AL: "Targeted homing of CCR2-overexpressing mesenchymal stromal cells to ischemic brain enhances post-stroke recovery partially through PRDX4- mediated blood-brain barrier preservation", 《THERANOSTICS》 * |
| 刘翔等: "归巢受体与TLR4在溃疡性结肠炎小鼠肠道中的相关性研究", 《中国现代医学杂志》 * |
| 王涛等: "CCR2抑制剂RS504393对小鼠短暂性局灶性脑缺血的保护作用研究", 《中国临床神经科学》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110946877A (en) * | 2019-12-30 | 2020-04-03 | 深圳爱生再生医学科技有限公司 | Stem cell biological product for treating liver cirrhosis and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109439631B (en) | 2021-02-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Chang et al. | Hypoxic preconditioning enhances the therapeutic potential of the secretome from cultured human mesenchymal stem cells in experimental traumatic brain injury | |
| Zhang et al. | Transplantation of expanded endothelial colony-forming cells improved outcomes of traumatic brain injury in a mouse model | |
| CN106636210B (en) | Transcription factor combines the method that induced fibroblast transdifferentiation is class interstitial glands | |
| TW201130977A (en) | Mesenchymal stem cells (MSCs) isolated from mobilized peripheral blood | |
| CN109718250B (en) | Methods and compositions for treating traumatic brain injury and for modulating neurogenic cell migration | |
| Shipounova et al. | Analysis of results of acute graft-versus-host disease prophylaxis with donor multipotent mesenchymal stromal cells in patients with hemoblastoses after allogeneic bone marrow transplantation | |
| CN106038597B (en) | Application of mesenchymal stem cells in preparation of preparation for treating acute lung injury | |
| CN108728410A (en) | The preparation method of source for mesenchymal stem cells excretion body based on medical preconditioning | |
| Jin et al. | Angiogenic characteristics of human stromal vascular fraction in ischemic hindlimb | |
| EP1807509B1 (en) | Multipotent stem cells isolated from umbilical cord blood and the cellular therapeutic agent comprising the same for treating ischemic disease | |
| CN110777120B (en) | Application of TGFBI as marker for regulating and controlling mesenchymal stem cell adipogenic differentiation | |
| CN106754680A (en) | A kind of separation method of placenta derived stem cells and its application | |
| CN113274411A (en) | Application of genetically modified bone marrow mesenchymal stem cell-derived microvesicles in preparation of medicines for treating renal injury | |
| CN106434564A (en) | Establishing method of CD36 mutant gene stable eukaryotic expression cell line causing CD36 deletion | |
| CN107088223A (en) | The application of Metrnl albumen or gene in treatment Endothelial dysfunction | |
| US20190201456A1 (en) | Alleviation and treatment of ischemia reperfusion-induced lung injury using pluripotent stem cells | |
| Lohmann-Matthes et al. | Tumor metastases and cell-mediated immunity in a model system in DBA/2 mice. VII. Interaction of metastasizing and nonmetastasizing tumors with normal tissue in vitro | |
| EP4151719A1 (en) | Method for promoting and improving properties of adipose tissue, tissue and cells obtained by said method | |
| CN109439631A (en) | A kind of interstital stem cell being overexpressed CCR2 is for treating acute ischemic cerebral apoplexy and preparation method thereof | |
| US20200215117A1 (en) | Mesenchymal stem cell over-expressing cxcr5, preparation method and use thereof | |
| KR20100074386A (en) | Mesenchymal stem cell producing human hepatic growth factor (hhgf), method for preparing the same and therapeutic agent of liver diseases | |
| CN106065401A (en) | Lentivirus mediated CXCR7 high expressed through engineering approaches endothelial progenitor cells treatment use in ischemic diseases | |
| Balaji et al. | Pluripotent lineage of CD133 stem cells isolated from human skin samples | |
| JP2021512654A (en) | Methods for Isolating Stem Cells from Tissues Under Thermal Conditions and Their Use | |
| KR101228626B1 (en) | Culture medium for mononuclear cell derived from human, cultured mononuclear cell, and composition for treating ischemic diseases comprising epithelial progenitor cells differentiated from the mononuclear cell |
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 | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20201110 Address after: 510700 8/F, Building C3, Accelerator of Science and Technology Enterprise, 11 Kaiyuan Avenue, Huangpu District, Guangzhou City, Guangdong Province Applicant after: GUANGZHOU SAIJUN BIOLOGICAL TECHNOLOGY Co.,Ltd. Address before: 510275 Xingang West Road, Guangdong, Guangzhou, No. 135, No. Applicant before: SUN YAT-SEN University |
|
| TA01 | Transfer of patent application right | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |