CN114073714A - 干细胞外泌体制剂及其应用 - Google Patents
干细胞外泌体制剂及其应用 Download PDFInfo
- Publication number
- CN114073714A CN114073714A CN202010801445.5A CN202010801445A CN114073714A CN 114073714 A CN114073714 A CN 114073714A CN 202010801445 A CN202010801445 A CN 202010801445A CN 114073714 A CN114073714 A CN 114073714A
- Authority
- CN
- China
- Prior art keywords
- exosome
- stem cells
- stem cell
- exosomes
- preparation
- 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.)
- Pending
Links
- 210000001808 exosome Anatomy 0.000 title claims abstract description 114
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 57
- 238000002360 preparation method Methods 0.000 title claims abstract description 24
- 238000011282 treatment Methods 0.000 claims abstract description 28
- 230000001965 increasing effect Effects 0.000 claims abstract description 6
- 235000015872 dietary supplement Nutrition 0.000 claims abstract description 5
- 239000003814 drug Substances 0.000 claims abstract description 5
- 239000003937 drug carrier Substances 0.000 claims abstract description 5
- 210000002379 periodontal ligament Anatomy 0.000 claims description 21
- 239000000203 mixture Substances 0.000 claims description 12
- 238000009472 formulation Methods 0.000 claims description 11
- 238000002347 injection Methods 0.000 claims description 6
- 239000007924 injection Substances 0.000 claims description 6
- 210000004271 bone marrow stromal cell Anatomy 0.000 claims description 2
- 239000002775 capsule Substances 0.000 claims description 2
- 210000001671 embryonic stem cell Anatomy 0.000 claims description 2
- 230000003511 endothelial effect Effects 0.000 claims description 2
- 210000003958 hematopoietic stem cell Anatomy 0.000 claims description 2
- 210000004185 liver Anatomy 0.000 claims description 2
- 239000002674 ointment Substances 0.000 claims description 2
- 239000008363 phosphate buffer Substances 0.000 claims description 2
- 239000002504 physiological saline solution Substances 0.000 claims description 2
- 239000000843 powder Substances 0.000 claims description 2
- 239000007921 spray Substances 0.000 claims description 2
- 239000003826 tablet Substances 0.000 claims description 2
- 239000002417 nutraceutical Substances 0.000 claims 2
- 235000021436 nutraceutical agent Nutrition 0.000 claims 2
- 230000036541 health Effects 0.000 abstract description 3
- 239000000047 product Substances 0.000 abstract description 3
- 210000002997 osteoclast Anatomy 0.000 description 27
- 238000000034 method Methods 0.000 description 15
- 230000004069 differentiation Effects 0.000 description 13
- 241000699666 Mus <mouse, genus> Species 0.000 description 12
- 102000007591 Tartrate-Resistant Acid Phosphatase Human genes 0.000 description 11
- 108010032050 Tartrate-Resistant Acid Phosphatase Proteins 0.000 description 11
- 210000004027 cell Anatomy 0.000 description 11
- 102000004169 proteins and genes Human genes 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- 241000699670 Mus sp. Species 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 102100024230 Dendritic cell-specific transmembrane protein Human genes 0.000 description 6
- 101710190014 Dendritic cell-specific transmembrane protein Proteins 0.000 description 6
- 238000010186 staining Methods 0.000 description 6
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 210000002540 macrophage Anatomy 0.000 description 5
- 230000003239 periodontal effect Effects 0.000 description 5
- 239000002953 phosphate buffered saline Substances 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 238000001262 western blot Methods 0.000 description 5
- 230000009471 action Effects 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 4
- 101000990902 Homo sapiens Matrix metalloproteinase-9 Proteins 0.000 description 3
- 102100030412 Matrix metalloproteinase-9 Human genes 0.000 description 3
- 210000000988 bone and bone Anatomy 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 210000001616 monocyte Anatomy 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 208000006386 Bone Resorption Diseases 0.000 description 2
- 102100025222 CD63 antigen Human genes 0.000 description 2
- 102100027221 CD81 antigen Human genes 0.000 description 2
- 101000934368 Homo sapiens CD63 antigen Proteins 0.000 description 2
- 101000914479 Homo sapiens CD81 antigen Proteins 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 230000024279 bone resorption Effects 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 230000012202 endocytosis Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 210000004283 incisor Anatomy 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 238000010253 intravenous injection Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 210000002050 maxilla Anatomy 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000010603 microCT Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 238000003757 reverse transcription PCR Methods 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- 238000005199 ultracentrifugation Methods 0.000 description 2
- GZCWLCBFPRFLKL-UHFFFAOYSA-N 1-prop-2-ynoxypropan-2-ol Chemical compound CC(O)COCC#C GZCWLCBFPRFLKL-UHFFFAOYSA-N 0.000 description 1
- 102000013563 Acid Phosphatase Human genes 0.000 description 1
- 108010051457 Acid Phosphatase Proteins 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 208000019901 Anxiety disease Diseases 0.000 description 1
- 101000577180 Aspergillus oryzae (strain ATCC 42149 / RIB 40) Neutral protease 2 Proteins 0.000 description 1
- 238000000035 BCA protein assay Methods 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 102000029816 Collagenase Human genes 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- 238000000116 DAPI staining Methods 0.000 description 1
- 208000004232 Enteritis Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241000581650 Ivesia Species 0.000 description 1
- 206010061274 Malocclusion Diseases 0.000 description 1
- 102000002274 Matrix Metalloproteinases Human genes 0.000 description 1
- 108010000684 Matrix Metalloproteinases Proteins 0.000 description 1
- 102000001776 Matrix metalloproteinase-9 Human genes 0.000 description 1
- 108010015302 Matrix metalloproteinase-9 Proteins 0.000 description 1
- 102000004067 Osteocalcin Human genes 0.000 description 1
- 108090000573 Osteocalcin Proteins 0.000 description 1
- 102000014128 RANK Ligand Human genes 0.000 description 1
- 108010025832 RANK Ligand Proteins 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 208000008617 Tooth Demineralization Diseases 0.000 description 1
- 206010072665 Tooth demineralisation Diseases 0.000 description 1
- 206010044046 Tooth malformation Diseases 0.000 description 1
- HZEWFHLRYVTOIW-UHFFFAOYSA-N [Ti].[Ni] Chemical compound [Ti].[Ni] HZEWFHLRYVTOIW-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000036506 anxiety Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004763 bicuspid Anatomy 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 230000010256 bone deposition Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 229910002091 carbon monoxide Inorganic materials 0.000 description 1
- 230000023402 cell communication Effects 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 229960002424 collagenase Drugs 0.000 description 1
- 238000007906 compression Methods 0.000 description 1
- 230000006835 compression Effects 0.000 description 1
- 238000002591 computed tomography Methods 0.000 description 1
- 238000001218 confocal laser scanning microscopy Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 230000001054 cortical effect Effects 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 208000002925 dental caries Diseases 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 201000002491 encephalomyelitis Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- HJUFTIJOISQSKQ-UHFFFAOYSA-N fenoxycarb Chemical compound C1=CC(OCCNC(=O)OCC)=CC=C1OC1=CC=CC=C1 HJUFTIJOISQSKQ-UHFFFAOYSA-N 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 210000002894 multi-fate stem cell Anatomy 0.000 description 1
- 229910001000 nickel titanium Inorganic materials 0.000 description 1
- 231100000957 no side effect Toxicity 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 210000000963 osteoblast Anatomy 0.000 description 1
- 229940094443 oxytocics prostaglandins Drugs 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 210000001778 pluripotent stem cell Anatomy 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 150000003180 prostaglandins Chemical class 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 210000003370 receptor cell Anatomy 0.000 description 1
- 230000007115 recruitment Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 208000006860 root resorption Diseases 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 229940095064 tartrate Drugs 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000004357 third molar Anatomy 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000004627 transmission electron microscopy Methods 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
Images
Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
-
- 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
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- 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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Genetics & Genomics (AREA)
- Animal Behavior & Ethology (AREA)
- Developmental Biology & Embryology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Wood Science & Technology (AREA)
- Virology (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Rheumatology (AREA)
- Epidemiology (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明提供了一种用于牙齿正畸的干细胞外泌体制剂,其包括干细胞来源的外泌体和药学上可接受的载体。本发明还提供了干细胞来源的外泌体或干细胞外泌体制剂在制备用于牙齿正畸或者加快正畸牙齿的移动速度的药物、保健品或营养补充剂中的用途。
Description
技术领域
本发明总体涉及生物医药领域,具体地涉及一种干细胞外泌体制剂及其用途。
背景技术
牙齿矫正,又称为牙齿正畸,是指通过一系列的方法矫正牙齿、解除各种错牙和畸形。通常,不拔牙患者的正畸疗程大概需要21-27个月,而拔牙患者的疗程更长,大概需要25-35个月。较长的正畸疗程不仅会引起患者的焦虑情绪,而且会引起牙龈炎症、牙齿脱矿、龋病、牙根吸收等风险。正畸牙齿移动速度大概是每个月0.8-1.2mm,加快正畸牙齿移动(OTM)是缩短正畸疗程的有效方法。
正畸牙齿移动的过程主要是牙槽骨改建的过程,表现为受压侧的牙周膜收缩,破骨细胞分化引起骨吸收;同时牵张侧的牙周膜拉伸,成骨细胞分化引起骨沉积,进而引起正畸牙齿移动。加速牙齿移动的过程中,破骨细胞的招募、分化、活化起着关键作用。牙周膜在正畸牙齿移动中受到机械力的作用,牙周膜干细胞(PDLSCs)是牙周膜中的多能干细胞,牙周膜干细胞受力后能够将机械信号转导为化学信号,并能释放各种生物调节分子以促进破骨细胞分化。
目前在临床上应用的加快正畸牙齿移动的方法主要是骨皮质切开术,但是这是一种有创的方法;非手术方法包括低强度脉冲超声、激光等,但目前的效果不确定。另外有很多生物调节分子可以促进动物模型的正畸牙齿移动如骨钙蛋白、前列腺素、H2S等,但这些分子在体内存留的时间不长。因此,寻找一种新的无创伤且高效的加快正畸牙齿移动的方法迫在眉睫。
外泌体是多种细胞在正常及病理状态下均可分泌到细胞外的包含复杂RNA和蛋白质的直径约为30-150nm的囊泡,其在细胞内以微囊泡的形式存在。研究发现,外泌体能够在细胞间转移各种生物调节分子进行细胞通讯,进而影响受体细胞的表型和功能。另外,多能干细胞来源的外泌体在动物模型中可以改善多种疾病的表现,如:自身免疫性脑脊髓炎、肠炎等。然而,外泌体在牙齿正畸治疗中的应用还未见报道。
发明内容
本发明的一个目的在于提供一种通过施用干细胞来源的外泌体来加快正畸牙齿移动的更稳定的、无创伤的生物学方法,从而为牙齿正畸提供新的途径。
本发明的另一目的在于提供一种用于牙齿正畸的干细胞来源的外泌体制剂,其中所述外泌体来自于各种来源的干细胞且所述外泌体制剂能够有效地矫正牙齿畸形。
本发明的又一目的在于提供干细胞来源的外泌体在牙齿正畸治疗,特别是加快正畸牙齿移动中的用途。
为了实现上述目的,根据本发明的一个方面,提供了一种用于牙齿正畸的干细胞外泌体制剂,包含干细胞来源的外泌体和药学上可接受的载体。
优选地,每100μg-1mL所述干细胞外泌体制剂包含100μg至300μg的外泌体。优选地,所述干细胞外泌体制剂包含120μg、140μg、160μg、180μg、200μg、220μg、240μg、260μg或280μg的所述外泌体。
优选地,所述外泌体的直径为40nm至150nm。优选地,所述外泌体的直径为50nm、60nm、70nm、80nm、90nm、100nm、110nm、120nm、130nm、140nm或者它们之间任何范围内的值。
优选地,所述干细胞选自胚胎干细胞、造血干细胞、生殖干细胞、间充质干细胞、神经干细胞、肝脏干细胞、胰腺干细胞和内皮干细胞中的一种或多种。
优选地,所述干细胞选自牙周膜干细胞。
可用于本发明的牙周膜可来源于临床因正畸治疗或阻生而拔除的双尖牙、切牙,或者完整拔除的智齿。
优选地,所述外泌体制剂能够加快正畸牙齿的移动速度。
优选地,所述外泌体制剂为注射剂、胶囊剂、片剂、粉末剂、软膏剂或喷雾剂的形式。
优选地,所述外泌体制剂可以通过静脉注射或者牙周膜注射方式施用。
优选地,所述药学上可接受的载体包括磷酸盐缓冲液、生理盐水或其组合。
根据本发明的另一方面,提供了干细胞来源的外泌体或者包含其的制剂在制备用于牙齿正畸的药物、保健品或营养补充剂中的应用。
根据本发明的又一方面,提供了干细胞来源的外泌体或者包含其的干细胞外泌体制剂在制备用于加快正畸牙齿的移动速度的药物、保健品或营养补充剂中的应用。
本发明的有益效果包括:本发明的干细胞来源的外泌体制剂制备简单、材料来源广泛、且相较于微米级的细胞,外泌体囊泡的提取方法更为简单灵活且容易保存。通过施用干细胞来源的外泌体,显著地加快正畸牙齿的移动速度,从而实现牙齿正畸治疗的目的。另外,本发明的外泌体制剂不具有副作用或副作用小、稳定性好且不会引起患者免疫排斥反应,因此能够有效地长期用于牙齿正畸治疗。
附图说明
图1示出牙周膜干细胞来源的外泌体的透射电子显微镜(TEM)图(a)以及该外泌体标记物的蛋白质印迹(Western blot)的结果(b);
图2示出施用外泌体的小鼠的正畸牙齿移动模式图;
图3示出对照、在加力作用下以及在加力和外泌体作用下小鼠正畸牙齿移动的效果图,其中图3(a)为体视镜图和显微-CT(微计算机断层扫描术)图且图3(b)为对照、在加力作用下以及在加力和外泌体作用下的正畸牙齿移动距离的点状图;
图4示出对照、在加力作用下以及在加力和外泌体作用下对小鼠破骨细胞分化的影响,其中图4(a)为进行HE染色和TRAP染色的破骨细胞组织形态学图且图4(b)为对照、在加力作用下以及在加力和外泌体作用下的TRAP+阳性的破骨细胞数的点状图;
图5示出对照和外泌体处理的小鼠RAW264.7单核/巨噬细胞内吞外泌体的共聚焦荧光显微镜图,其中图5(a)示出对照和外泌体处理的小鼠RAW264.7细胞的PKH26、肌动蛋白和DAPI染色的分布图以及它们的合并图且图5(b)示出对照和外泌体处理的荧光相对强度的点状图;
图6示出对照和外泌体处理的破骨细胞分化的效果图,其中图6(a)示出对照和外泌体处理的TRAP阳性的破骨细胞分布且图6(b)示出在注射外泌体1天和7天后TRAP阳性的破骨细胞数的点状图;和
图7示出对照和外泌体处理的小鼠RAW264.7单核/巨噬细胞中破骨分化相关分子树突状细胞-特异性跨膜蛋白(DC-STAMP)和基质金属蛋白酶9(MMP9)的表达的柱状图,其中图7(a)示出对照和外泌体处理的DC-STAMP的相对RNA水平和图7(b)示出对照和外泌体处理的MMP9的相对RNA水平。
具体实施方式
下面结合实施例,进一步阐述本发明。应理解,本发明的技术方案不限于以下所列举的具体实施方式,还包括各种具体实施方式的组合。
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1:牙周膜干细胞外泌体的制备
1、牙周膜干细胞的分离和培养
1)将牙周膜(PDL)从牙根表面分离出来,用含有200U/mL青霉素和200g/mL链霉素的无菌磷酸盐缓冲盐水(PBS)冲洗三次(Biofluids,Rockville,MD);
2)将冲洗后的PDL置于用PBS缓冲液溶解的3mg/ml 1型胶原酶(WorthingtonBiochem,Freehold,NJ,USA)和4mg/ml中性蛋白酶II(Roche,Mannheim,Germany)消化液中,在37℃水浴中孵育1小时。将孵育后的溶液通过70μm过滤器过滤,获得牙周膜干细胞(PDLSC)的单细胞悬液;
3)用含有15%胎牛血清(Gibco,Carlsbad,CA)、100U/mL青霉素和100g/mL链霉素(Biofluids,Rockville,MD)的α-MEM培养基,在CO2培养箱(37℃,100%湿度、5%CO2和95%空气)培养PDLSC,传代至第5代。期间每一代培养至70%-80%时,更换到含无外泌体血清(100,000g离心2h去除外泌体)的α-MEM培养基中培养48小时,收集培养上清液后传代。
其中,α-MEM培养基为常规的可商购的α-MEM培养基。
2、牙周膜干细胞外泌体提取与鉴定
采用外泌体提取试剂盒(System Biosciences)或超速离心的方法从PDLSC的培养上清液中提取外泌体。然后用透射电子显微镜(TEM)观察外泌体、蛋白质印迹(Westernblot)鉴定外泌体标记物CD81和CD63以及BCA蛋白浓度测定试剂盒(Thermo scientificPierceTM BCA Protein Assay)测量外泌体内蛋白含量。具体步骤如下:
(1)外泌体的提取
A.将牙周膜干细胞在无外泌体的培养基中扩增3天,收集培养基;
B.将收集到的细胞培养基在15℃以2000rpm离心30min去除培养基中的细胞碎片;
C.弃去沉淀,收集上清液;
D.对上清液进行系列超速离心以获得外泌体,具体地:1)300g离心10分钟,收集上清液;2)3,000g离心10分钟,收集上清液;3)20,000g离心30分钟,收集上清液;4)120,000g离心70分钟,收集沉淀,收集的沉淀部分即为分离的外泌体。
(2)外泌体的鉴定
通过透射电子显电镜(FEI Tecnai Spirit,120kv)观察所分离的牙周膜干细胞,发现细胞内存在大量的微囊泡样的外泌体,如图1(a)所示。
用常规方法提取外泌体蛋白,通过蛋白质印迹检测外泌体中表面标志物CD81和CD63的表达,如图1(b)所示。
根据以下BCA蛋白浓度测定方法测量外泌体内蛋白含量。
向外泌体中以1:1的质量比加入Ripa裂解液,vertex 15s,室温静置5min,获得外泌体蛋白。96孔板中加入18μl ddH2O+2μl外泌体蛋白,20μl标准BCA浓度试剂,再加入200μlBCA工作液(A液:B液=1:50),37℃,避光孵育30min。分光光度计测定563nm波长的吸光度值,换算成蛋白浓度为1μg/μl。
(3)外泌体直径的测定:
1)由培养基分离得到的细胞外囊泡重悬于PBS中;
2)经不同比例的稀释,利用NanoSight LM10显微镜系统,记录细胞外囊泡的形态,并进行直径大小的测量。其中,牙周膜干细胞分离得到的外泌体囊泡的尺寸集中在40-150nm范围内,尤其在100nm附近。
实施例2:牙周膜干细胞外泌体对正畸牙齿的作用
I-外泌体对正畸牙齿移动的影响
通过静脉注射/牙周膜注射的方法将适量的外泌体注射到患者体中以提高正畸牙齿移动的速度,缩短正畸治疗流程。
(1)小鼠正畸牙齿移动模型:取6-8周龄的雄性小鼠C57小鼠,体重20-25g,作为实验小鼠。实验小鼠均从北京维通利华购买。实验设计为3个处理:对照(未加力+未施用外泌体)、单纯的施加外力(加力)和加力+外泌体组合处理。每个处理随机采用5只小鼠作为实验对象,然后分别在加力和外泌体注射后7天选取其中3只外观表现基本一致的小鼠进行指标测试,其中破骨相关分子DC-STAMP和MMP9的含量为三次测量的平均值+SD。
如图2所示,在建模当天,对于加力处理,在小鼠上颌骨左侧第一磨牙与切牙之间放置一个能够产生大约30g的力量的镍钛拉簧(直径1mm,长度1mm),将该拉簧保持7天。对于加力+外泌体处理,在用拉簧加力的同时,在20分钟内通过小鼠尾静脉注射100μg的外泌体。建模后第7天处死小鼠,收集各个处理的样本进行牙齿和外泌体的形态学观察以及外泌体中的蛋白测定。
如图3所示,在体视镜下并测量第一磨牙(M1)远中边缘嵴切线(如方向向右的箭头所示)与第二磨牙(M2)近中边缘嵴切线(如方向向左的箭头所示)之间的距离。结果表明,加力和外泌体处理都导致第一磨牙的移动。对于牙齿之间移动的距离,加力和外泌体组合的处理>加力处理>对照处理。此外,用显微-CT扫描小鼠的上颌骨,进行了CT三维和二维重建,截取牙齿距离最小的纵切面图,从图3(a)中可以看到加力后第一磨牙出现了移动,而外泌体和加力组合的处理牙齿移动的距离最大。这些结果说明,牙周膜干细胞来源的外泌体能够显著地促进小鼠正畸牙齿的移动。分析其原因在于,外泌体促进破骨细胞的分化,该破骨细胞分化引起压力侧牙槽骨吸收更多,进而引起牙齿移动距离增大。
如图4所示,用光学显微镜(Nikon ECLIPSE 80i,20倍镜)对各个处理的小鼠第一磨牙远中颊根根中1/3处经由苏木精-伊红(HE)染色和抗酒石酸酸性磷酸酶(TRAP)染色进行组织学观察,分析破骨细胞的分布。其中,抗酒石酸酸性磷酸酶(TRAP)是破骨细胞的特异性标记酶,TRAP染色能够将破骨细胞染成红色(图中深色区域即为红色区域),AB表示牙槽骨(alveolar bone),R表示牙根(root),PDL表示牙周膜(periodontal ligament)。图中向上的黑色箭头方向是加力的方向,上方是压力侧,加力后压力侧的破骨细胞数量增加,而外泌体组破骨细胞的数量更多,说明外泌体可能通过诱导压力侧破骨细胞的分化进而促进正畸牙齿移动。
II-外泌体对破骨细胞分化的影响
利用小鼠RAW264.7单核/巨噬细胞系进行体外实验,来探究RAW细胞对外泌体的内吞作用。
PKH26(Sigma-Aldrich,MINI26-1KT)是一种细胞膜染料,用PKH26把外泌体染成红色后加入到RAW细胞中培养1小时或3小时。具体方法如下:新鲜提取的外泌体,用Diluent C将样品量增加到1ml。加6μl PKH26至液体中,混匀,室温静置5min。加入2ml 10%BSA,用培养基将样增加至8.5ml。加入1.5ml的0.971M的蔗糖至管底,190,000g,离心2h,PBS重悬沉淀。从图5(a)和图5(b)中可以看到加入外泌体1小时后,在细胞质中就可以观察到红色外泌体,加入外泌体3小时后,红色的亮度更高,说明RAW264.7可以内吞牙周膜干细胞来源的外泌体,而且时间越长,内吞细胞数目越多。
另外,如图6所示,采用TRAP染色分析了外泌体是否能够诱导破骨细胞的分化。具体方法如下:为了诱导破骨细胞分化,将Raw264.7巨噬细胞以每孔5×106细胞的密度接种在6孔培养皿中,培养过夜,然后用50ng/ml的可溶性RANKL处理。同期加入外泌体培养1天或7天,然后用TRAP染色观察破骨细胞。图6中深色区域即为TRAP染色阳性的破骨细胞,结果表明外泌体和加力处理组破骨细胞的数量明显多于加力组和对照组。
另外,用PCR探究RAW264.7中破骨相关分子DC-STAMP、MMP9的表达量。用TRIZOL试剂(Invitrogen)提取细胞的总RNA,使用SuperScript RT-PCR系统(Thermo FisherScientific)和Platinum Taq High Fidelity(Invitrogen)进行cDNA合成。使用2x SYBRGreen(Invitrogen Life Technologies,USA)在实时PCR系统(Applied Biosystems 7500)上进行定量RT-PCR。使用了以下引物:
GAPDH,5'-AACTTTGGCATTGTGGAAGG-3,
5'-ACACATTGGGGGTAGTAGGAACA-3';
DC-STAMP,5'-TCCTCCATGAACAAACAGTTCCAA-3',
5'-AGACGTGGTTTAGGAATGCAGCTC-3';
MMP9,5'-AGTTTGGTGTCGCGGAGCAC-3',
5'-TACATGAGCGCTTCCGGCAC-3'。
通过熔解曲线证实了设计的引物的扩增特异性。图7结果表明外泌体增加了RAW264.7中这两种分子的表达量。这些结果说明PDLSCs外泌体能够诱导破骨细胞的分化。
综上所述,牙周膜干细胞来源的外泌体能够被单核巨噬细胞内吞,并诱导其分化成破骨细胞,进而促进正畸牙齿移动。单纯的施加外力仅仅造成正畸牙齿微小移动,而外泌体的施用则显著地扩大了外力对牙齿移动的影响。
以上所述仅仅是本发明的优选实施方式。应当指出的是,在不脱离本发明的精神和实质的情况下,本领域技术人员可对本发明的细节进行各种修改、变更或替换。这些修改、变更或替换也应理解为包括在本发明要求保护的范围之内。
Claims (10)
1.一种用于牙齿正畸的干细胞外泌体制剂,包含干细胞来源的外泌体和药学上可接受的载体。
2.根据权利要求1所述的制剂,其中,每100μg-1mL所述干细胞外泌体制剂包含100μg至300μg的外泌体。
3.根据权利要求1所述的制剂,其中,所述外泌体的直径为40nm至150nm。
4.根据权利要求1所述的制剂,其中,所述干细胞选自胚胎干细胞、造血干细胞、生殖干细胞、间充质干细胞、神经干细胞、肝脏干细胞、胰腺干细胞和内皮干细胞中的一种或多种。
5.根据权利要求1所述的制剂,其中,所述干细胞选自牙周膜干细胞。
6.根据权利要求1所述的制剂,其中,所述干细胞外泌体制剂为注射剂、胶囊剂、片剂、粉末剂、软膏剂或喷雾剂的形式。
7.根据权利要求1所述的制剂,其中,所述药学上可接受的载体包括磷酸盐缓冲液、生理盐水或其组合。
8.干细胞来源的外泌体或者根据权利要求1-7中任一项所述的干细胞外泌体制剂在制备用于牙齿正畸的药物、保健品或营养补充剂中的用途。
9.干细胞来源的外泌体或根据权利要求1-7中任一项所述的干细胞外泌体制剂在制备用于加快正畸牙齿的移动速度的药物、保健品或营养补充剂中的用途。
10.根据权利要求8或9所述的用途,其中,所述干细胞选自牙周膜干细胞。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010801445.5A CN114073714A (zh) | 2020-08-11 | 2020-08-11 | 干细胞外泌体制剂及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010801445.5A CN114073714A (zh) | 2020-08-11 | 2020-08-11 | 干细胞外泌体制剂及其应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN114073714A true CN114073714A (zh) | 2022-02-22 |
Family
ID=80279880
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202010801445.5A Pending CN114073714A (zh) | 2020-08-11 | 2020-08-11 | 干细胞外泌体制剂及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114073714A (zh) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110403959A (zh) * | 2019-09-12 | 2019-11-05 | 北京大学口腔医学院 | 间充质干细胞外泌体制剂及其应用 |
WO2020061627A1 (en) * | 2018-09-26 | 2020-04-02 | CK Cell Technologies Pty Ltd | Mesenchymal stem cells and products therefrom |
-
2020
- 2020-08-11 CN CN202010801445.5A patent/CN114073714A/zh active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2020061627A1 (en) * | 2018-09-26 | 2020-04-02 | CK Cell Technologies Pty Ltd | Mesenchymal stem cells and products therefrom |
CN110403959A (zh) * | 2019-09-12 | 2019-11-05 | 北京大学口腔医学院 | 间充质干细胞外泌体制剂及其应用 |
Non-Patent Citations (4)
Title |
---|
BYOUNG-MOO SEO: "Investigation of multipotent postnatal stem cells from human periodontal ligament", 《THE LANCET》, 10 July 2004 (2004-07-10), pages 149 - 155 * |
吴晓楠: "不同干细胞来源外泌中国实用口腔科杂志", 《国际口腔医学杂志》, 1 March 2020 (2020-03-01), pages 148 * |
吴晓楠;马宁;侯建霞;: "不同干细胞来源外泌体在牙周再生领域的研究进展", 国际口腔医学杂志, no. 02, 1 March 2020 (2020-03-01) * |
朱斌: "骨髓间充质干细胞来源的外泌体促进牙周再生的体外研究体在牙周再生领域的研究进展", 《中国实用口腔科杂志》, 15 December 2016 (2016-12-15) * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Zhang et al. | Tooth repair and regeneration: potential of dental stem cells | |
de Souza Costa et al. | Human pulp response to resin cements used to bond inlay restorations | |
ES2767182T3 (es) | Células madre pluripotentes que inducen reparación y regeneración después de un infarto de miocardio | |
JP6188578B2 (ja) | 歯科用インプラントおよびその製造方法 | |
Chen et al. | Human umbilical cord mesenchymal stem cells: a new therapeutic option for tooth regeneration | |
JP2020505345A (ja) | 乏血小板血漿(ppp)に封入された実質的に純粋な多能性間質細胞集団を含む組成物 | |
Gao et al. | Effects of targeted delivery of metformin and dental pulp stem cells on osteogenesis via demineralized dentin matrix under high glucose conditions | |
Alamoudi et al. | Treatment of oral ulcers in dogs using adipose tissue-derived mesenchymal stem cells | |
Xi et al. | Nrf2 activation is involved in osteogenic differentiation of periodontal ligament stem cells under cyclic mechanical stretch | |
US8338174B2 (en) | Material for ameliorating skin tissue and method for producing the same | |
Shaikh et al. | Human umbilical cord mesenchymal stem cells: current literature and role in periodontal regeneration | |
WO2015110082A1 (zh) | 牙源性干细胞和基因修饰的牙源性干细胞的用途 | |
Chen et al. | Effects of restorative materials on dental pulp stem cell properties | |
Mallishery et al. | Regenerative endodontics–looking inward | |
US20210052699A1 (en) | Pharmaceutical composition for preventing or treating dentin-dental pulp diseases or periodontal disease including cpne4 protein | |
CN114073714A (zh) | 干细胞外泌体制剂及其应用 | |
Li et al. | Adult stem cell-based apexogenesis | |
TWI672147B (zh) | 以外源性粒線體爲有效成份之組合物、其用途及修復細胞之方法 | |
Kuntjoro et al. | The effect of advanced glycation end products (AGEs) on human umbilical cord mesenchymal stem cells (hUCMSCs) with regard to osteogenesis and calcification | |
Rodríguez-Lozano et al. | Stem cells for endodontic regeneration | |
Karimi et al. | Stem Cells in Dentistry | |
Salama et al. | Histo-pathological Evaluation of the Effect of Aloe Vera Gel as a Pulpotomy Agent in Young Permanent Teeth: A pilot Study | |
Baranwal | Regenerative Stem Cells for Endodontic Tissue Engineering: Past, Present and Future | |
Safwat et al. | Survival and Adherence of Apical Stem Cells to Root Canal Dentin after Conditioning with Apple Vinegar | |
Lee et al. | Effects of ethanol washing and storage duration on primary culture of stem cells from human exfoliated deciduous teeth |
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 |