CN111304171B - 烟碱乙酰胆碱受体靶向性的过表达circDYM的外泌体及其制备方法与应用 - Google Patents
烟碱乙酰胆碱受体靶向性的过表达circDYM的外泌体及其制备方法与应用 Download PDFInfo
- Publication number
- CN111304171B CN111304171B CN202010090487.2A CN202010090487A CN111304171B CN 111304171 B CN111304171 B CN 111304171B CN 202010090487 A CN202010090487 A CN 202010090487A CN 111304171 B CN111304171 B CN 111304171B
- Authority
- CN
- China
- Prior art keywords
- circdym
- cells
- acetylcholine receptor
- exosomes
- nicotinic acetylcholine
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 210000001808 exosome Anatomy 0.000 title claims abstract description 56
- 102000019315 Nicotinic acetylcholine receptors Human genes 0.000 title claims abstract description 18
- 108050006807 Nicotinic acetylcholine receptors Proteins 0.000 title claims abstract description 18
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 210000004027 cell Anatomy 0.000 claims abstract description 43
- 230000002018 overexpression Effects 0.000 claims abstract description 11
- 238000005119 centrifugation Methods 0.000 claims abstract description 10
- POPPVIRYGJQIOF-UHFFFAOYSA-N 2-acetyloxyethyl(trimethyl)azanium;3-(1-methylpyrrolidin-2-yl)pyridine Chemical compound CC(=O)OCC[N+](C)(C)C.CN1CCCC1C1=CC=CN=C1 POPPVIRYGJQIOF-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000001963 growth medium Substances 0.000 claims abstract description 8
- 239000013612 plasmid Substances 0.000 claims abstract description 8
- 108010009685 Cholinergic Receptors Proteins 0.000 claims abstract description 7
- 241000713666 Lentivirus Species 0.000 claims abstract description 7
- 239000003814 drug Substances 0.000 claims abstract description 7
- 238000012258 culturing Methods 0.000 claims abstract description 6
- 239000002244 precipitate Substances 0.000 claims abstract description 6
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims abstract description 5
- 239000006228 supernatant Substances 0.000 claims abstract description 4
- 239000012634 fragment Substances 0.000 claims abstract description 3
- 210000001178 neural stem cell Anatomy 0.000 claims abstract description 3
- 102000034337 acetylcholine receptors Human genes 0.000 claims abstract 2
- 239000002245 particle Substances 0.000 claims description 8
- 239000002609 medium Substances 0.000 claims description 4
- 238000001890 transfection Methods 0.000 claims description 4
- 208000015181 infectious disease Diseases 0.000 claims description 3
- 230000008685 targeting Effects 0.000 abstract description 7
- 230000007267 depressive like behavior Effects 0.000 abstract description 5
- 230000000694 effects Effects 0.000 abstract description 5
- 210000004556 brain Anatomy 0.000 abstract description 4
- 230000008499 blood brain barrier function Effects 0.000 abstract description 3
- 210000001218 blood-brain barrier Anatomy 0.000 abstract description 3
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 description 18
- 229960004373 acetylcholine Drugs 0.000 description 18
- 238000012360 testing method Methods 0.000 description 10
- 241000699666 Mus <mouse, genus> Species 0.000 description 6
- 241000699670 Mus sp. Species 0.000 description 6
- QZAYGJVTTNCVMB-UHFFFAOYSA-N serotonin Chemical compound C1=C(O)C=C2C(CCN)=CNC2=C1 QZAYGJVTTNCVMB-UHFFFAOYSA-N 0.000 description 6
- 102000009660 Cholinergic Receptors Human genes 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 239000000935 antidepressant agent Substances 0.000 description 4
- 229940005513 antidepressants Drugs 0.000 description 4
- 229910052802 copper Inorganic materials 0.000 description 4
- 239000010949 copper Substances 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 238000003753 real-time PCR Methods 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 102100025222 CD63 antigen Human genes 0.000 description 3
- 101000934368 Homo sapiens CD63 antigen Proteins 0.000 description 3
- 101000613251 Homo sapiens Tumor susceptibility gene 101 protein Proteins 0.000 description 3
- 102100040879 Tumor susceptibility gene 101 protein Human genes 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 206010067484 Adverse reaction Diseases 0.000 description 2
- 230000005653 Brownian motion process Effects 0.000 description 2
- 108091028075 Circular RNA Proteins 0.000 description 2
- 206010012374 Depressed mood Diseases 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- 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 2
- 229930006000 Sucrose Natural products 0.000 description 2
- 230000006838 adverse reaction Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000001430 anti-depressive effect Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000006399 behavior Effects 0.000 description 2
- 230000003542 behavioural effect Effects 0.000 description 2
- 238000005537 brownian motion Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 238000012048 forced swim test Methods 0.000 description 2
- 230000014509 gene expression Effects 0.000 description 2
- 238000005286 illumination Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 230000009182 swimming Effects 0.000 description 2
- 229940126585 therapeutic drug Drugs 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 239000002023 wood Substances 0.000 description 2
- SFLSHLFXELFNJZ-QMMMGPOBSA-N (-)-norepinephrine Chemical compound NC[C@H](O)C1=CC=C(O)C(O)=C1 SFLSHLFXELFNJZ-QMMMGPOBSA-N 0.000 description 1
- 208000017194 Affective disease Diseases 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 208000028698 Cognitive impairment Diseases 0.000 description 1
- 206010010144 Completed suicide Diseases 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 206010021137 Hypovolaemia Diseases 0.000 description 1
- 235000003332 Ilex aquifolium Nutrition 0.000 description 1
- 235000002296 Ilex sandwicensis Nutrition 0.000 description 1
- 235000002294 Ilex volkensiana Nutrition 0.000 description 1
- 208000032382 Ischaemic stroke Diseases 0.000 description 1
- 208000037490 Medically Unexplained Symptoms Diseases 0.000 description 1
- 229940123685 Monoamine oxidase inhibitor Drugs 0.000 description 1
- 208000019022 Mood disease Diseases 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 241000219000 Populus Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 101150063416 add gene Proteins 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000037326 chronic stress Effects 0.000 description 1
- 208000010877 cognitive disease Diseases 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000001493 electron microscopy Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000006353 environmental stress Effects 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 235000003642 hunger Nutrition 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000002025 microglial effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000002899 monoamine oxidase inhibitor Substances 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 108091027963 non-coding RNA Proteins 0.000 description 1
- 102000042567 non-coding RNA Human genes 0.000 description 1
- 239000002767 noradrenalin uptake inhibitor Substances 0.000 description 1
- 229960002748 norepinephrine Drugs 0.000 description 1
- SFLSHLFXELFNJZ-UHFFFAOYSA-N norepinephrine Natural products NCC(O)C1=CC=C(O)C(O)=C1 SFLSHLFXELFNJZ-UHFFFAOYSA-N 0.000 description 1
- 229940127221 norepinephrine reuptake inhibitor Drugs 0.000 description 1
- 238000003921 particle size analysis Methods 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- IYDGMDWEHDFVQI-UHFFFAOYSA-N phosphoric acid;trioxotungsten Chemical compound O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.OP(O)(O)=O IYDGMDWEHDFVQI-UHFFFAOYSA-N 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000003772 serotonin uptake inhibitor Substances 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 230000004037 social stress Effects 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 230000037351 starvation Effects 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 239000003029 tricyclic antidepressant agent Substances 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- 238000005303 weighing Methods 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
- C12N5/0618—Cells of the nervous system
- C12N5/0623—Stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/24—Antidepressants
-
- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- 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
- C12N15/86—Viral vectors
-
- 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
- C12N2510/02—Cells for production
-
- 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
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15041—Use of virus, viral particle or viral elements as a vector
- C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Neurosurgery (AREA)
- Physics & Mathematics (AREA)
- Neurology (AREA)
- Plant Pathology (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Cell Biology (AREA)
- Developmental Biology & Embryology (AREA)
- Virology (AREA)
- Pain & Pain Management (AREA)
- Psychiatry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种烟碱乙酰胆碱受体靶向性的过表达circDYM的外泌体的制备方法：使用DMEM高糖培养基培养HEK 293T细胞，采用pcDNA GNSTM‑3‑RVG‑10‑Lamp2b‑HA质粒和circDYM‑GFP‑LVs慢病毒转染HEK 293T细胞；收集细胞上清，通过梯度离心的方法依次离心去除漂浮细胞、死细胞、细胞碎片；将所获沉淀重悬，得到烟碱乙酰胆碱受体靶向性的过表达circDYM的神经干细胞外泌体。本发明制备得到的具有烟碱乙酰胆碱受体靶向性的过表达circDYM外泌体，其能够有效通过血脑屏障靶向大脑，显著减少抑郁症模型小鼠的抑郁样行为，具有抗抑郁效果，有望开发为治疗抑郁症的潜在药物。
Description
技术领域
本发明属于医药技术，具体涉及一种烟碱乙酰胆碱受体靶向性的过表达circDYM的外泌体及其制备方法与应用。
背景技术
抑郁症是一种以心境低落为主要特征的常见的情感性精神障碍综合症。抑郁症的主要临床特诊包括心境低落、思维迟缓、意志活动减退、认知功能损害和躯体症状等。抑郁症具有高复发率、高致残率和高自杀率的特征，给患者、家庭和社会带来沉重负担，已成为严重的社会问题。
长期以来，临床上一直缺乏理想的抑郁治疗药物。传统的三环类、四环类抗抑郁药物和单胺氧化酶抑制剂由于不良反应较大，目前应用明显减少，已退出临床一线用药范围。目前临床上一线的抗抑郁药主要包括选择性5-羟色胺再摄取抑制剂、5-羟色胺和去甲肾上腺素再摄取抑制剂、去甲肾上腺素和特异性5-羟色胺能抗抑郁药等。然而抑郁药物起效慢、不良反应多，加之抑郁症病因多样，病情复杂，部分抑郁症患者依然对治疗药物无反应，甚至产生耐药。因此，新型抗抑郁药物的研发迫在眉睫。
环状RNA是一种特殊的非编码RNA，已被证实在诸多疾病的发生发展中发挥重要作用，如抑郁症、阿尔茨海默症、缺血性脑卒中等。CircDYM具有调控脑内小胶质细胞活化，进而发挥改善抑郁样行为的作用。但是如何将环状RNA开发为核酸药物仍是科学界亟待的重大科学问题。
发明内容
发明目的：针对现有技术中存在的上述技术问题，本发明提供了一种烟碱乙酰胆碱受体靶向性的过表达circDY的外泌体及其制备方法，还提供了其制药应用。
技术方案：本发明所述的一种烟碱乙酰胆碱受体靶向性的过表达circDYM的外泌体的制备方法，包括以下步骤：
(1)培养HEK 293T细胞；
(2)采用circDYM-GFP-LVs慢病毒转染HEK 293T细胞；
(3)更换培养基，加入pcDNA GNSTM-3-RVG-10-Lamp2b-HA质粒转染；
(4)更换培养基继续培养后收集细胞上清，通过梯度离心的方法依次离心去除漂浮细胞、死细胞、细胞碎片；
(5)将所获沉淀重悬，离心得到烟碱乙酰胆碱受体靶向性的过表达circDYM的神经干细胞外泌体。
其中，步骤(1)、(3)、(4)中，培养HEK 293T细胞均采用DMEM高糖培养基。
步骤(2)中，调整HEK-293T细胞为1*10^5-1*10^6个，按照3MOL的感染比例转染circDYM-GFP-LVs慢病毒12-24小时。
步骤(3)中，所述的pcDNA GNSTM-3-RVG-10-Lamp2b-HA质粒转染条件为：每1*10^5-1*10^6个HEK 293T细胞，加入1-10ug质粒，转染12-24小时。
步骤(4)中，更换培养基继续培养48小时。所述的梯度离心条件为：300g转速离心5分钟，去除漂浮细胞；3000g转速离心30分钟，去除死细胞；10000g转速离心60分钟，去除细胞碎片；200000g转速离心120分钟，收集所得沉淀。
步骤(5)中，所述重悬溶液为PBS溶液。
根据上述方法制备所得烟碱乙酰胆碱受体靶向性的过表达circDYM的外泌体也在本发明的保护范围内。
本发明所述外泌体具有烟碱乙酰胆碱受体靶向性，粒径为40-100nm的盘状囊泡结构，circDYM较对照组外泌体的过表达倍数为7-9倍。
上述烟碱乙酰胆碱受体靶向性的过表达circDYM的外泌体在制备预防或治疗抑郁症药物中的应用也在本发明的保护范围内。
有益效果：本发明创新性的采用靶向中枢外泌体递送环状RNA的方法，一次性解决了环状RNA成药的三大难题：1)高稳定的环状RNA克服了核酸药物稳定性差的弊端；2)外泌体解决了慢病毒载体无法实现临床转化的劣势；3)外泌体的靶向性实现了无创递送外泌体至中枢的作用。本发明制备得到了一种具有烟碱乙酰胆碱受体靶向性的过表达circDYM外泌体，其能够有效通过血脑屏障靶向递送circDYM入脑，显著减少抑郁症模型小鼠的抑郁样行为，具有抗抑郁效果,有望开发为治疗抑郁症的潜在药物。
附图说明
图1为本发明实施例2对提供的烟碱乙酰胆碱受体靶向性的过表达circDYM外泌体的电镜鉴定结果图(50000倍)；
图2为本发明实施例3对提供的烟碱乙酰胆碱受体靶向性的过表达circDYM外泌体的表面标记蛋白鉴定结果图；
图3为本发明实施例4对提供的烟碱乙酰胆碱受体靶向性的过表达circDYM外泌体的粒径鉴定结果图；
图4为本发明实施例5对提供的烟碱乙酰胆碱受体靶向性的过表达circDYM外泌体的定量PCR检测结果；
图5为本发明实施例6对提供的烟碱乙酰胆碱受体靶向性的过表达circDYM外泌体的体内分布经红外结果图；
图6为本发明实施例7对提供的烟碱乙酰胆碱受体靶向性的过表达circDYM外泌体的抑郁症模型小鼠抑郁杨行为的改善作用结果图。
具体实施方式
下面结合具体实施例对本申请做出详细说明。
HEK-293T细胞购于中国科学研究院上海细胞库。
pcDNA GNSTM-3-RVG-10-Lamp2b-HA质粒购于美国addgene公司。
circDYM-GFP-LVs慢病毒购于汉恒生物科技(上海)有限公司。
胎牛血清购于浙江天杭生物有限公司(四季青胎牛血清)。
DMEM高糖培养基购于美国康宁公司(10-013-CVR)
实施例1烟碱乙酰胆碱受体靶向性的过表达circDYM外泌体的制备
一种烟碱乙酰胆碱受体靶向性的过表达circDYM外泌体的制备方法，包括以下步骤：
(1)使用胎牛血清和DMEM高糖培养基培养HEK-293T细胞；
(2)调整HEK-293T细胞为1*10^5-1*10^6个，按照3MOL的感染比例转染circDYM-GFP-LVs慢病毒，转染24小时；
(3)更换新鲜培养基，每1*10^5-1*10^6个细胞，加入1-10ug质粒，转染24小时；
(4)转染48小时后，更换新鲜培养基，继续培养48小时，然后收集细胞上清液；
(5)采用梯度离心的方式纯化所得外泌体：300g转速离心5分钟，去除漂浮细胞；3000g转速离心30分钟，去除死细胞；10000g转速离心60分钟，去除细胞碎片；200000g转速离心120分钟，收集所得沉淀；
(6)采用PBS溶液重悬上述步骤(5)中所得沉淀；
(7)200000g转速离心上述步骤(6)中的重悬溶液120分钟，得到烟碱乙酰胆碱受体靶向性的过表达circDYM外泌体。
将上述制备所得烟碱乙酰胆碱受体靶向性的过表达circDYM外泌体应用于实施例2-6中的检测和分析试验。
实施例2烟碱乙酰胆碱受体靶向性的过表达circDYM外泌体的电镜鉴定方法
准备烟碱乙酰胆碱受体靶向性的过表达circDYM外泌体20ul，充分混匀后滴加在直径为2毫米的载样铜网上，室温静置5分钟。用滤纸吸干铜网边缘的残余液体，然后将铜网倒扣在30g/L的磷钨酸(pH6.8)液滴上，室温下负染色5分钟。白炽灯下烘干铜网，通过透射电镜观察并拍照，烟碱乙酰胆碱受体靶向性的过表达circDYM外泌体的分离鉴定结果如图1所示，其中烟碱乙酰胆碱受体靶向性的过表达circDYM外泌体为直径约为100nm的盘状囊泡。
实施例3烟碱乙酰胆碱受体靶向性的过表达circDYM外泌体的表面标记蛋白鉴定方法
配制15％SDS-PAGE电泳胶，将烟碱乙酰胆碱受体靶向性的过表达circDYM外泌体及靶向性对照外泌体充分裂解后加入1/4体积的5×SDS上样缓冲液，煮沸5min，按200μg蛋白质总量上样，电转移(350mA，120min)将蛋白质转至PVDF膜上，用含50g/L脱脂牛奶的TBS/T室温封闭1h，分别与HA/Lamp2b/CD63/TSG101/GM130抗体(1:500)于4℃反应过夜，次日用TBS/0.5％Tween 20洗膜3次后，与HRP标记的二抗37℃温育1h)，TBS/0.5％Tween20洗膜3次后，加入预混HRP化学发光底物，并通过化学发光凝胶成像系统进行检测。图2为Westernblot检测结果，烟碱乙酰胆碱受体靶向性的过表达circDYM外泌体中HA/Lamp2b/CD63/TSG101/GM130的表达结果如图2所示，HA/Lamp2b/CD63/TSG101呈阳性表达，GM130呈阴性表达。
实施例4烟碱乙酰胆碱受体靶向性的过表达circDYM外泌体的粒径分析
纳米颗粒跟踪分析(NTA)是将一束能量集中的激光穿过玻璃棱镜对样品(悬浮外泌体的溶液)进行照射，通过装有摄像头的显微镜实现颗粒可视化，捕捉颗粒布朗运动的视频文件，并对每个颗粒的布朗运动进行追踪和分析，快速准确的计算出样本中哪里颗粒的流体力学半径和浓度。设置NTA仪器参数，测量时间为60秒。取外泌体，用水按照1:2500比例稀释成1ml，通过NTA进行测量分析，计算出外泌体的粒径和浓度。结果如图3所述，烟碱乙酰胆碱受体靶向性的过表达circDYM外泌体的峰值为104.32±1.93nm。
实施例5烟碱乙酰胆碱受体靶向性的过表达circDYM外泌体的定量PCR检测方法
定量PCR检测烟碱乙酰胆碱受体靶向性的过表达circDYM外泌体中circDYM的增高倍数，定量PCR检测结果如图4所示：烟碱乙酰胆碱受体靶向性的过表达circDYM外泌体中circDYM的表达量呈显著性的增高。定量检测circDYM的引物序列如下所示：
| 基因名称 | 引物方向 | 序列(5’-3’) |
| circDYM | Forward | GAAGAAAAGTCCCCCGGCAG |
| Reverse | AAGACCTTAGTTAGCGCAGCA |
实施例6烟碱乙酰胆碱受体靶向性的过表达circDYM外泌体的体内经红外分布
使用Dir(细胞膜红色荧光探针)标记烟碱乙酰胆碱受体靶向性的过表达circDYM外泌体。
通过小鼠尾静脉注射上述外泌体，分别于于0、6、12和24小时外泌体的体内分布。结果如图5所述，烟碱乙酰胆碱受体靶向性的过表达circDYM外泌体能够透过血脑屏障进入小鼠脑部，并均匀分布。
实施例7烟碱乙酰胆碱受体靶向性的过表达circDYM外泌体的对抑郁模型小鼠抑郁样行为的改善作用
1、实验动物8周龄C57BL/6J雄性小鼠，体重22～25g，购自南京大学模式动物中心。动物生产许可证：SCXK(苏)2015-0001。SPF级环境饲养，光照为12小时/12小时明暗交替，自由饮食。实验前，将动物置于实验环境适应7天。
2、实验分组实验共设6个分组：对照组、模型组、治疗组(100/200/300/400ug)。对照组不造模，模型组基于CUS造模，治疗组分别给与100/200/300/400ug烟碱乙酰胆碱受体靶向性的过表达circDYM外泌体治疗。
3、CUS小鼠抑郁模型模型建立
参考经典的抑郁动物模型CUS方案，诱导小鼠慢性应激抑郁模型。将小鼠随机暴露于低强度社会和环境压力源，每天2-3次，持续5周。压力源包括以下(1)食物剥夺24h，(2)缺水24h，(3)过夜照明，(4)笼子里没有木屑24h，(5)用水浸湿木屑24h，(6)在8℃下强行游泳5min，(7)夹住尾部(距离尾尖1cm)，(8)物理约束6h，以及(9)45°笼式倾斜垂直轴3h。4、小鼠行为学测试
在CUS诱导抑郁模型后进行行为学测试。所有测试均在9:00至17:00之间低强度光线下、保持安静室内进行，并由同一评估者进行评分。在测试前，小鼠在室内适应至少3h。通过位于测试设备前面的摄像机监视行为，随后由经验丰富的研究人员用Plexon软件系统(Plexon Inc，Dallas，TX，USA)分析图像，动物完成蔗糖偏好测试(Sucrose preferencetest，SPT)，尾部悬浮测试(Tail suspension test，TST)和强迫游泳测试(Forcedswimming test，FST)。
结果如图6所示，给与100/200/300/400ug烟碱乙酰胆碱受体靶向性的过表达circDYM外泌体治疗的小鼠抑郁样行为明显改善，100-400ug计量范围内的烟碱乙酰胆碱受体靶向性的过表达circDYM外泌体具有抗抑郁效果。
Claims (5)
1.一种烟碱乙酰胆碱受体靶向性的过表达circDYM的外泌体在制备预防或治疗抑郁症药物中的应用，其特征在于，所述烟碱乙酰胆碱受体靶向性的过表达circDYM的外泌体为粒径40-100nm的盘状囊泡结构，其制备方法为，包括以下步骤：
(1)培养HEK 293T细胞；
(2)采用circDYM-GFP-LVs慢病毒转染HEK 293T细胞，调整HEK-293T细胞为1*10^5-1*10^6个，按照3MOL的感染比例转染circDYM-GFP-LVs慢病毒12-24小时；
(3)更换培养基，加入pcDNA GNSTM-3-RVG-10-Lamp2b-HA质粒转染，所述的pcDNAGNSTM-3-RVG-10-Lamp2b-HA质粒转染条件为：每1*10^5-1*10^6个HEK 293T细胞，加入1-10ug质粒，转染12-24小时；
(4)更换培养基继续培养后收集细胞上清，通过梯度离心的方法依次离心去除漂浮细胞、死细胞、细胞碎片；
(5)将所获沉淀重悬，离心得到烟碱乙酰胆碱受体靶向性的过表达circDYM的神经干细胞外泌体。
2.根据权利要求1所述的应用，其特征在于，步骤(1)、(3)、(4)中，培养HEK 293T细胞均采用DMEM高糖培养基。
3.根据权利要求1所述的应用，其特征在于，步骤(4)中，更换培养基继续培养48小时。
4.根据权利要求1所述的应用，其特征在于，步骤(4)中，所述的梯度离心条件为：300g转速离心5分钟，去除漂浮细胞；3000g转速离心30分钟，去除死细胞；10000g转速离心60分钟，去除细胞碎片；200000g转速离心120分钟，收集所得沉淀。
5.根据权利要求1所述的应用，其特征在于，步骤(5)中，所述重悬溶液为PBS溶液。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010090487.2A CN111304171B (zh) | 2020-02-13 | 2020-02-13 | 烟碱乙酰胆碱受体靶向性的过表达circDYM的外泌体及其制备方法与应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010090487.2A CN111304171B (zh) | 2020-02-13 | 2020-02-13 | 烟碱乙酰胆碱受体靶向性的过表达circDYM的外泌体及其制备方法与应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111304171A CN111304171A (zh) | 2020-06-19 |
| CN111304171B true CN111304171B (zh) | 2023-11-28 |
Family
ID=71150963
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010090487.2A Active CN111304171B (zh) | 2020-02-13 | 2020-02-13 | 烟碱乙酰胆碱受体靶向性的过表达circDYM的外泌体及其制备方法与应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111304171B (zh) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112386679B (zh) * | 2020-11-18 | 2023-05-30 | 徐州医科大学 | 一种包载sigma-1受体的外泌体在制备抗抑郁药物中的应用 |
| CN113262212A (zh) * | 2021-04-26 | 2021-08-17 | 北京大学口腔医学院 | 一种靶向炎症区域的细胞膜微囊泡及其应用 |
| CN113215104A (zh) * | 2021-05-13 | 2021-08-06 | 西南医科大学 | 一种含有CD10-dm蛋白的外泌体及其制备方法和应用 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109837306A (zh) * | 2019-03-20 | 2019-06-04 | 江南大学附属医院（无锡市第四人民医院） | 包载miRNA-204-5p的外泌体及其制备方法和应用 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3472193A4 (en) * | 2016-06-20 | 2020-01-08 | The Board of Trustees of the Leland Stanford Junior University | CIRCULAR RNA AND THEIR USE IN IMMUNO MODULATION |
-
2020
- 2020-02-13 CN CN202010090487.2A patent/CN111304171B/zh active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109837306A (zh) * | 2019-03-20 | 2019-06-04 | 江南大学附属医院（无锡市第四人民医院） | 包载miRNA-204-5p的外泌体及其制备方法和应用 |
Non-Patent Citations (4)
| Title |
|---|
| "CircDYM ameliorates depressive-like behavior by targeting miR-9 to regulate microglial activation via HSP90 ubiquitination";Yuan Zhang, et al.;《Mol Psychiatry.》;20181109;第25卷(第6期);摘要，材料和方法部分，讨论部分 * |
| "Exosomes: Diagnostic Biomarkers and Therapeutic Delivery Vehicles for Cancer";Liangdi Jiang,et al;《Mol Pharm》;20190710;全文 * |
| "Extracellular vesicle-mediated delivery of circDYM alleviates CUS-induced depressive-like behaviours";Xiaoyu Yu,et al;《J Extracell Vesicles》;20220131;第11卷(第1期);全文 * |
| "The emerging role of exoxomes in mental disorders";Saumeh Saeedi, et al.;《Translational Psychiatry》;20190328;摘要，第5页右栏第2段，第6页右栏第1段，引用文献26 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111304171A (zh) | 2020-06-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Hepler et al. | Identification of functionally distinct fibro-inflammatory and adipogenic stromal subpopulations in visceral adipose tissue of adult mice | |
| CN111304171B (zh) | 烟碱乙酰胆碱受体靶向性的过表达circDYM的外泌体及其制备方法与应用 | |
| CN104703609B (zh) | 干细胞微粒 | |
| KR101913293B1 (ko) | 탈모 질환의 치료 방법 | |
| Kawai et al. | Long-term selective stimulation of transplanted neural stem/progenitor cells for spinal cord injury improves locomotor function | |
| US20210113641A1 (en) | Extraction of plant source "medicinal soup" and manual preparation of "herbal medicine" and related products | |
| Ruetz et al. | In vitro and in vivo CRISPR-Cas9 screens reveal drivers of aging in neural stem cells of the brain | |
| CN112899233B (zh) | 一种急性淋巴细胞性白血病小鼠模型的构建方法 | |
| Kauer et al. | Inositol Polyphosphate-5-phosphatase K (Inpp5k) enhances sprouting of corticospinal tract axons after CNS trauma | |
| Siddiqui et al. | Nerve growth factor receptor (Ngfr) induces neurogenic plasticity by suppressing reactive astroglial Lcn2/Slc22a17 signaling in Alzheimer’s disease | |
| Niu et al. | Follistatin-like protein 1 functions as a potential target of gene therapy in proliferative diabetic retinopathy | |
| Comer et al. | Detection of mRNA species in bulbospinal neurons isolated from the rostral ventrolateral medulla using single-cell RT–PCR | |
| CN113046301B (zh) | 抑瘤素m联合脂多糖诱导的人鼻粘膜上皮细胞炎症模型、制法以及应用 | |
| US20070128168A1 (en) | Novel genes regulated in the developing human ventral mesencephalon | |
| CN114672462B (zh) | 阿尔茨海默病模型及其建立方法和用途 | |
| WO2023216299A1 (zh) | 环状核糖核酸翻译蛋白在抑郁症诊断和治疗领域的应用 | |
| CN113209301B (zh) | γCamkⅡ表达促进剂或稳定剂在制备减缓衰老和抗神经退行性药物中的应用 | |
| Kalvala et al. | Air-liquid interface culture of midbrain organoids improves neuronal functionality and integration of microglia | |
| Thomasen et al. | SorCS2 binds progranulin to regulate motor neuron development | |
| CN115919848B (zh) | 死亡受体激动剂在促进毛细胞再生以治疗耳聋的新用途及一种验证该用途的模型 | |
| CN114134181B (zh) | 一种表达单元、重组慢病毒表达载体、重组慢病毒及其制备方法和应用 | |
| CN1161744A (zh) | 中枢神经系统(cns)功能和机能障碍的体外模型 | |
| Sebastian et al. | Single cell transcriptomic profiling of human brain organoids reveals developmental timing-and cell-type-specific vulnerabilities induced by NRXN1 CNVs in schizophrenia | |
| CN116492463B (zh) | Cd155分子在肝纤维化领域中的应用 | |
| Nomura et al. | Reciprocal differentiation via GABAergic components and ASD-related phenotypes in hES with 1q21. 1 CNV |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |