CN116473991A - miR-143-3p在制备治疗周围神经损伤药物或材料中的应用 - Google Patents
miR-143-3p在制备治疗周围神经损伤药物或材料中的应用 Download PDFInfo
- Publication number
- CN116473991A CN116473991A CN202310661001.XA CN202310661001A CN116473991A CN 116473991 A CN116473991 A CN 116473991A CN 202310661001 A CN202310661001 A CN 202310661001A CN 116473991 A CN116473991 A CN 116473991A
- Authority
- CN
- China
- Prior art keywords
- mir
- nerve
- nerve injury
- regeneration
- motor neuron
- 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
- 108091073532 miR-143 stem-loop Proteins 0.000 title claims abstract description 62
- 208000010886 Peripheral nerve injury Diseases 0.000 title claims abstract description 15
- 239000000463 material Substances 0.000 title claims abstract description 13
- 239000003814 drug Substances 0.000 title claims abstract description 9
- 238000002360 preparation method Methods 0.000 title claims abstract description 6
- 230000008439 repair process Effects 0.000 claims abstract description 11
- 229940079593 drug Drugs 0.000 claims abstract description 6
- 210000005036 nerve Anatomy 0.000 claims description 11
- 210000003497 sciatic nerve Anatomy 0.000 claims description 7
- 239000013543 active substance Substances 0.000 claims description 6
- 230000014509 gene expression Effects 0.000 claims description 6
- 230000000694 effects Effects 0.000 claims description 5
- 230000001737 promoting effect Effects 0.000 claims description 5
- 239000013604 expression vector Substances 0.000 claims description 4
- 210000000578 peripheral nerve Anatomy 0.000 claims description 4
- 230000001537 neural effect Effects 0.000 claims description 3
- 210000003461 brachial plexus Anatomy 0.000 claims description 2
- 210000001617 median nerve Anatomy 0.000 claims description 2
- 230000003387 muscular Effects 0.000 claims description 2
- 210000002979 radial nerve Anatomy 0.000 claims description 2
- 210000002161 motor neuron Anatomy 0.000 abstract description 34
- 210000003050 axon Anatomy 0.000 abstract description 22
- 230000008929 regeneration Effects 0.000 abstract description 21
- 238000011069 regeneration method Methods 0.000 abstract description 21
- 238000001890 transfection Methods 0.000 abstract description 13
- 208000028389 Nerve injury Diseases 0.000 abstract description 8
- 230000008764 nerve damage Effects 0.000 abstract description 8
- 230000002018 overexpression Effects 0.000 abstract description 7
- 230000012010 growth Effects 0.000 abstract description 6
- 238000000338 in vitro Methods 0.000 abstract description 5
- 238000011160 research Methods 0.000 abstract description 4
- 230000028600 axonogenesis Effects 0.000 abstract description 3
- 238000001727 in vivo Methods 0.000 abstract description 3
- 230000001105 regulatory effect Effects 0.000 abstract description 3
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 238000002474 experimental method Methods 0.000 abstract description 2
- 230000003278 mimic effect Effects 0.000 abstract description 2
- 108091070501 miRNA Proteins 0.000 abstract 1
- 239000002679 microRNA Substances 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 15
- 210000001519 tissue Anatomy 0.000 description 11
- 241000700159 Rattus Species 0.000 description 9
- 241000699666 Mus <mouse, genus> Species 0.000 description 8
- 108700011259 MicroRNAs Proteins 0.000 description 6
- 230000001605 fetal effect Effects 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 210000002569 neuron Anatomy 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 229930040373 Paraformaldehyde Natural products 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- 230000000903 blocking effect Effects 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 230000007547 defect Effects 0.000 description 4
- 238000007865 diluting Methods 0.000 description 4
- 230000002055 immunohistochemical effect Effects 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 229920002866 paraformaldehyde Polymers 0.000 description 4
- 238000010008 shearing Methods 0.000 description 4
- 239000000741 silica gel Substances 0.000 description 4
- 229910002027 silica gel Inorganic materials 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 208000024827 Alzheimer disease Diseases 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 238000001543 one-way ANOVA Methods 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 241000283074 Equus asinus Species 0.000 description 2
- 239000012098 Lipofectamine RNAiMAX Substances 0.000 description 2
- 239000012124 Opti-MEM Substances 0.000 description 2
- 108010019160 Pancreatin Proteins 0.000 description 2
- 239000006143 cell culture medium Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000003125 immunofluorescent labeling Methods 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 230000002427 irreversible effect Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 108010082117 matrigel Proteins 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 229940055695 pancreatin Drugs 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 230000001172 regenerating effect Effects 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 210000000278 spinal cord Anatomy 0.000 description 2
- 210000003594 spinal ganglia Anatomy 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 239000012096 transfection reagent Substances 0.000 description 2
- 230000035899 viability Effects 0.000 description 2
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 108091032955 Bacterial small RNA Proteins 0.000 description 1
- 102000000131 Beta tubulin Human genes 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 206010028289 Muscle atrophy Diseases 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108091066608 Rattus norvegicus miR-143 stem-loop Proteins 0.000 description 1
- 206010039670 Sciatic nerve injury Diseases 0.000 description 1
- 108091081021 Sense strand Proteins 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 208000024770 Thyroid neoplasm Diseases 0.000 description 1
- 102000004357 Transferases Human genes 0.000 description 1
- 108090000992 Transferases Proteins 0.000 description 1
- 108090000704 Tubulin Proteins 0.000 description 1
- 108091023045 Untranslated Region Proteins 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 229960004373 acetylcholine Drugs 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 230000003376 axonal effect Effects 0.000 description 1
- 208000036815 beta tubulin Diseases 0.000 description 1
- 230000002146 bilateral effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 210000005013 brain tissue Anatomy 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 210000003414 extremity Anatomy 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 230000007954 hypoxia Effects 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 238000003365 immunocytochemistry Methods 0.000 description 1
- 238000011532 immunohistochemical staining Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 230000030214 innervation Effects 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 108091056924 miR-124 stem-loop Proteins 0.000 description 1
- 108091092839 miR-124-1 stem-loop Proteins 0.000 description 1
- 108091045380 miR-124-2 stem-loop Proteins 0.000 description 1
- 108091048120 miR-124-3 stem-loop Proteins 0.000 description 1
- 108091047546 miR-124-4 stem-loop Proteins 0.000 description 1
- 108091034147 miR-124-5 stem-loop Proteins 0.000 description 1
- 108091028854 miR-124-6 stem-loop Proteins 0.000 description 1
- 238000000845 micromoulding in capillary Methods 0.000 description 1
- 210000004925 microvascular endothelial cell Anatomy 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000020763 muscle atrophy Effects 0.000 description 1
- 201000000585 muscular atrophy Diseases 0.000 description 1
- 230000019581 neuron apoptotic process Effects 0.000 description 1
- 230000007514 neuronal growth Effects 0.000 description 1
- 230000009689 neuronal regeneration Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 210000001428 peripheral nervous system Anatomy 0.000 description 1
- 210000004345 peroneal nerve Anatomy 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 201000000980 schizophrenia Diseases 0.000 description 1
- 210000004116 schwann cell Anatomy 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 206010041823 squamous cell carcinoma Diseases 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 201000002510 thyroid cancer Diseases 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 210000000689 upper leg Anatomy 0.000 description 1
- 230000002618 waking effect Effects 0.000 description 1
Classifications
-
- 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
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
-
- 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
- A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Neurology (AREA)
- Neurosurgery (AREA)
- Biochemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Dermatology (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Transplantation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明公开了miR‑143‑3p在制备周围神经损伤修复药物或材料中的应用。本发明研究结果显示过表达miR‑143‑3p可以显著促进神经损伤后轴突再生。本发明利用微流控装置体外培养运动神经元，转染miR‑143‑3p mimic可以显著促进原代培养的运动神经元轴突生长与再生。在体实验结果显示miR‑143‑3p mimic促进周围神经损伤后轴突再生。miR‑143‑3p可以通过调节运动神经元轴突生长参与周围神经损伤修复，有助于更好地理解miRNA在神经损伤修复过程中的重要作用，并为神经损伤后的治疗提供新的靶点。
Description
技术领域
本发明属于生物医药领域。具体地，涉及miR-143-3p在制备治疗周围神经损伤药物或材料中的应用。
背景技术
周围神经损伤的修复一直是神经科学领域的研究重点和难点。临床上常见神经损伤后由于再生速度有限，患者肌肉在失神经再支配之前已经出现不可逆性肌萎缩，最终导致肢体功能障碍。因此，神经损伤后促进轴突快速再生，在靶器官失神经不可逆损伤前完成神经再支配是修复的关键因素。
MicroRNA(miRNA)是一类长度约为20-24个核苷酸的内源性非编码的小RNA，通过与靶基因3’端的非翻译区完全或不完全结合，抑制靶基因的翻译或直接降解靶基因来发挥作用。miRNA在周围神经系统中不仅能够促进神经元轴突的再生，抑制神经元的凋亡，还能通过调节施万细胞的增殖和迁移，巨噬细胞的表型促进周围神经再生。
miR-143-3p是miRNA家族的一种高度保守成员。研究报道，miR-143-3p在肺癌、口腔鳞癌、肝癌、胃癌、甲状腺癌、大肠癌中异常表达，参与肿瘤细胞恶性进展。此外，miR-124-3p是神经元特异性miRNA。现有技术研究显示，miR-143-3p还可表达于脑组织中，与阿尔茨海默症、精神分裂症等神经系统疾病发生发展关系密切。体外实验证实干扰miR-143-3p表达，可缓解脑血管内皮细胞缺糖、低氧性损伤及凋亡(陈伯勇,高庆春，王玉周,等.MicroRNA-143-3p对缺糖缺氧的大鼠脑微血管内皮细胞的保护作用及机制[J.卒中与神经疾病,2020,27(5):561-566.)。Sun等人发现，抑制miR-143-3p表达可促进阿尔茨海默症神经元再生(Sun C,Jia N,Li R,et al.miR-143-3p inhibition promotesneuronalsurvival in an Alzheimer's disease cell model by targetingneuregulin-1[J].Folia Neuropathol,2020,58(1):10-21.)。
目前尚无miR-143-3p在周围神经损伤修复领域的研究。
发明内容
本发明首次明确了miR-143-3p在周围神经损伤修复过程中对运动神经元的调控作用。
本发明具体技术方案如下：
miR-143-3p在制备治疗周围神经损伤药物或材料中的应用。
miR-143-3p在人和鼠高度保守，人源和小鼠的miR-143-3p核苷酸序列完全一致，NCBI Gene ID：406935(人)，387161(小鼠)，大鼠的miR-143-3p NCBI Gene ID：100314035。
所述周围神经为坐骨神经、臂丛神经、桡神经、腋神经、肌皮神经、正中神经、尺神经、股神经、腓总神经。
具体的，所述应用以miR-143-3p为靶点，设计或筛选具有促进miR-143-3p表达作用的活性物质，过表达miR-143-3p或外源性给予miR-143-3p或其表达载体。
本发明一个具体的示例，所述活性物质为miR-143-3p mimics。
本发明所述的应用，所述材料为组织工程神经材料。
本发明另一目的在于提供一种周围神经损伤修复药物或材料，含有促进miR-143-3p表达作用的活性物质，或者miR-143-3p或其表达载体。
本发明优点：
本发明以miR-143-3p作为分子干预靶点，过表达miR-143-3p可以显著促进神经损伤后轴突再生。本发明利用微流控装置体外培养运动神经元，转染miR-143-3p mimic可以显著促进原代培养的运动神经元轴突生长与再生。在体实验结果显示miR-143-3p mimic促进周围神经损伤后轴突再生。本发明所述miR-143-3p可以通过调节运动神经元轴突生长参与周围神经损伤修复，有助于更好地理解miRNA在神经损伤修复过程中的重要作用，并为神经损伤后的治疗提供新的靶点。
附图说明
图1为实施例1运动神经元光镜及免疫细胞化学染色图。A：运动神经元光镜图(Bar＝50μm)；B：运动神经元免疫细胞染色图(Bar＝25μm)；E：纯度鉴定统计图。
图2为实施例1体外过表达miR-143-3p显著促进运动神经元生长。A：ICC结果显示转染miR-143-3p促进运动神经元突起生长(Bar＝100μm)，β-TublinⅢ标记神经元突起；B：miR-143-3p促运动神经元突起生长长度统计图(**P＜0.01vs.Control,One-way ANOVA)。
图3为实施例2转染miR-143-3p促运动神经元轴突离断后再生。A：ICC结果显示转染miR-143-3p促进运动神经元突起离断后再生(Bar＝75μm)，β-TublinⅢ标记神经元再生轴突；B：miR-143-3p促运动神经元突起离断再生长度统计图(**P＜0.01vs.Control，One-way ANOVA)。
图4为实施例3miR-143-3p构建的组织工程神经促坐骨神经损伤后再生。A：免疫组化结果显示不同组别再生轴突，右侧一列为左侧图像白色方框区域局部放大(Bar＝500μm,25μm)；A：NF200标记再生轴突；B：再生轴突长度统计图(**P＜0.01vs PBS组，##P＜0.01vsNC agomir，One-way ANOVA)。
具体实施方式
以下通过实施例说明本发明的具体步骤，但不受实施例的限制。
在本发明中所使用的术语，除非另有说明，一般具有本领域普通技术人员通常理解的含义。
下面结合具体实施例并参照数据进一步详细描述本发明。应理解，这些实施例只是为了举例说明本发明，而非以任何方式限制本发明的范围。
在以下实施例中，未详尽描述的各种过程和方法是本领域中公知的常规方法。
下面结合具体实施例对本发明进一步说明。
实施例1体外过表达miR-143-3p促进运动神经元轴突生长。
1.运动神经元的培养：将孕13.5d的SD大鼠颈椎脱臼处死，取出胎鼠，于解剖镜下打开椎管，去除背根神经节和被膜，分离出胎鼠腹侧脊髓，置于盛有冰冷解剖液的培养皿中。用显微剪剪成0.5cm3大小的组织块后转移至15mL离心管中，加入1ml 0.125％胰酶将组织吹散，37℃消30min，加入DMEM+10％FBS终止消化，1000r/min离心5min，弃上清。细胞沉淀用L15吹散，400目筛网过滤。将过滤后的细胞缓慢加入5ml梯度离心液中使其分层，2000rpm离心20min。加入5mL 9％ Optiprep梯度分离液，2000rpm离心20min，离心管中的液体分为3层，其中中间层含有脊髓运动神经元。小心将中间层吸出，加入3mL培养基1000r/min离心5min，弃上清以去除细胞碎片。加入DMEM+10％FBS重悬后接种细胞，4h后观察细胞贴壁情况以及活力，换预热的神经元培养基(97％ Neurobasal+2％B27+1％ GluMAX)，之后每3d半量换液，β-Tubulin III和乙酰胆碱转移酶(ChAT)标记运动神经元纯度鉴定见图1。
2.神经元细胞mimics转染
运动神经元接种于微流控装置后，待其贴壁，先用Opti-MEM减血清培养基(Gibco，货号：31985070)稀释Lipofectamine RNAiMAX转染试剂(Thermo Fisher Scientific，货号：13778075)获得转染液，然后采用无PS的运动神经元培养基稀释转染液(比例为1440μl+160μl转染液)后转染miR-143-3p mimics(rno-miR-143-3p MIMIC序列：正义链(5'->3')：UGAGAUGAAGCACUGUAGCUCA，反义链(5'->3'):UGAGCUACAGUGCUUCAUCUCA)及阴性对照(终浓度100nM，广州锐博生物公司)，8h后换为运动神经元培养基。
3.细胞免疫荧光染色及轴突生长长度测量
运动神经元细胞转染4d后弃掉细胞培养基，预温的PBS润洗一遍，加入4％多聚甲醛，室温固定20min。弃掉多聚甲醛后，用PBS洗三遍，每遍5min。加入免疫组化封闭液，室温封闭1h。加入一抗mouse anti-β-TublinⅢantibody(1：400，abcam)4℃孵育过夜。弃掉一抗，PBS洗3遍，每遍5min。加入荧光二抗Donkey anti-mouse 488(1：400，Sigma)，避光室温孵育2h。弃掉二抗，PBS洗3遍，每遍5min。加入适量PBS，ZEISS正置荧光显微镜下观察，拍照。每个视野选取最长的15条突起，采用Image J软件计量长度并统计。结果显示，过表达miR-143-3p(miR-143-3p mimics)可以显著促进运动神经元轴突的生长(图2)。
实施例2体外过表达miR-143-3p促进运动神经元轴突再生。
1.运动神经元的培养：将孕13.5d的SD大鼠颈椎脱臼处死，取出胎鼠，于解剖镜下打开椎管，去除背根神经节和被膜，分离出胎鼠腹侧脊髓，置于盛有冰冷解剖液的培养皿中。用显微剪剪成0.5cm3大小的组织块后转移至15mL离心管中，加入1ml 0.125％胰酶将组织吹散，37℃消30min，加入DMEM+10％FBS终止消化，1 000r/min离心5min，弃上清。细胞沉淀用L15吹散，400目筛网过滤。将过滤后的细胞缓慢加入5ml梯度离心液中使其分层，2000rpm离心20min。加入5mL 9％ Optiprep梯度分离液，2000rpm离心20min，离心管中的液体分为3层，其中中间层含有脊髓运动神经元。小心将中间层吸出，加入3mL培养基1000r/min离心5min，弃上清以去除细胞碎片。加入DMEM+10％FBS重悬后接种细胞，4h后观察细胞贴壁情况以及活力，换预热的神经元培养基(97％ Neurobasal+2％B27+1％ GluMAX)，之后每3d半量换液。
2.神经元细胞mimics转染
运动神经元接种于微流控装置后，待其贴壁，先用Opti-MEM减血清培养基(Gibco，货号：31985070)稀释Lipofectamine RNAiMAX转染试剂(Thermo Fisher Scientific，货号：13778075)获得转染液，然后采用无PS的运动神经元培养基稀释转染液(比例为1440ul+160ul转染液)后转染miR-143-3p mimics及阴性对照(终浓度100nM，广州锐博生物公司)，8h后换为运动神经元培养基。
3.细胞免疫荧光染色及再生轴突长度测量
运动神经元细胞转染4d后采用真空泵进行轴突离断，将抽吸压力设置大于0.06Mpa抽吸15-30s后加入相应培养基。培养24h后弃掉细胞培养基，预温的PBS润洗一遍，加入4％多聚甲醛，室温固定20min。弃掉多聚甲醛后，用PBS洗三遍，每遍5min。加入免疫组化封闭液，室温封闭1h。加入一抗mouse anti-β-TublinⅢantibody(1：400，abcam)4℃孵育过夜。弃掉一抗，PBS洗3遍，每遍5min。加入荧光二抗Donkey anti-mouse 488(1：400，Sigma)，避光室温孵育2h。弃掉二抗，PBS洗3遍，每遍5min。加入适量PBS，ZEISS正置荧光显微镜下观察，拍照。每个视野选取最长的15条突起，采用Image J软件计量长度并统计。结果显示，过表达miR-143-3p(miR-143-3p mimics)可以显著促进运动神经元轴突的再生(图3)。
实施例3本发明所述miR-143-3p构建的组织工程神经在修复神经缺损中的应用
1.制备miR-143-3p组织工程神经：首先将硅胶管灭菌处理后置于无菌培养皿中，根据实验需求截取7mm长硅胶管(内径2mm，外径3mm)，分别将PBS、miR-143-3p agomir或NCagomir与温敏性Matrigel基质胶两者以1:1体积比混合，所有操作均在冰上进行。将混合均匀的PBS、miR-143-3p agomir或NC agomir和Matrigel基质胶混合物约18μl注入硅胶导管中，将导管置于37℃静置5min后取出，混合物凝固成凝胶状，可用于修复周围神经缺损。
2.在体建立大鼠坐骨神经5mm缺损桥接模型，观察miR-143-3p对坐骨神经再生的作用。SD大鼠随机分为3组：PBS组、miR-143-3p agomir组和NC agomir组。大鼠深度麻醉后，右侧大腿术区备皮、消毒后切开皮肤，钝性分离肌肉，暴露坐骨神经。横断坐骨神经，并切除3mm，经过两侧回缩后形成5mm的缺损并拍照记录。使用8-0带线缝合针，一端使用外科结连接并固定神经残端，一端连接预先制好的组织工程神经，将神经残端与硅胶管内壁固定，缝合外皮，将大鼠放置到保温的桌垫上等待苏醒。术后大鼠常规喂食饲养，术后10d进行灌注取材、切片、免疫组织化学染色，使用NF-200标记再生轴突，统计再生轴突长度。免疫组化结果表明与PBS和NC agomir组相比，miR-143-3p agomir组可以显著增加再生轴突长度。表明miR-143-3p可以促进坐骨神经损伤后轴突再生(图4)。
Claims (6)
1.miR-143-3p在制备治疗周围神经损伤药物或材料中的应用。
2.如权利要求1所述的应用，其特征在于所述周围神经为坐骨神经、臂丛神经、桡神经、腋神经、肌皮神经、正中神经、尺神经、股神经、腓总神经。
3.如权利要求1所述的应用，其特征在于以miR-143-3p为靶点，设计或筛选具有促进miR-143-3p表达作用的活性物质，过表达miR-143-3p或外源性给予miR-143-3p或其表达载体。
4.如权利要求4所述的应用，其特征在于所述活性物质为miR-143-3p mimics。
5.如权利要求1所述的应用，其特征在于所述材料为组织工程神经材料。
6.一种周围神经损伤修复药物或材料，其特征在于含有促进miR-143-3p表达作用的活性物质，或者miR-143-3p或其表达载体。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310661001.XA CN116473991A (zh) | 2023-06-06 | 2023-06-06 | miR-143-3p在制备治疗周围神经损伤药物或材料中的应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310661001.XA CN116473991A (zh) | 2023-06-06 | 2023-06-06 | miR-143-3p在制备治疗周围神经损伤药物或材料中的应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116473991A true CN116473991A (zh) | 2023-07-25 |
Family
ID=87225377
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310661001.XA Pending CN116473991A (zh) | 2023-06-06 | 2023-06-06 | miR-143-3p在制备治疗周围神经损伤药物或材料中的应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116473991A (zh) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106474549A (zh) * | 2016-11-21 | 2017-03-08 | 南通大学 | MicroRNA基因介导的新型组织工程化神经的构建及其在修复神经缺损的应用 |
| US20200165617A1 (en) * | 2017-05-09 | 2020-05-28 | New York University | Micrornas for treatment of neuropathic pain |
| CN115181724A (zh) * | 2022-06-10 | 2022-10-14 | 浙江工业大学 | 一种间充质干细胞来源的外泌体及其制备方法与应用 |
-
2023
- 2023-06-06 CN CN202310661001.XA patent/CN116473991A/zh active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106474549A (zh) * | 2016-11-21 | 2017-03-08 | 南通大学 | MicroRNA基因介导的新型组织工程化神经的构建及其在修复神经缺损的应用 |
| US20200165617A1 (en) * | 2017-05-09 | 2020-05-28 | New York University | Micrornas for treatment of neuropathic pain |
| CN115181724A (zh) * | 2022-06-10 | 2022-10-14 | 浙江工业大学 | 一种间充质干细胞来源的外泌体及其制备方法与应用 |
Non-Patent Citations (1)
| Title |
|---|
| NORCINI， MONICA ET AL.: "Intrathecal Injection of miR-133b-3p or miR-143-3p Prevents the Development of Persistent Cold and Mechanical Allodynia Following a Peripheral Nerve Injury in Rats", NEUROSCIENCE, vol. 386, 21 August 2018 (2018-08-21), pages 223 - 239 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Jahromi et al. | The advances in nerve tissue engineering: From fabrication of nerve conduit to in vivo nerve regeneration assays | |
| RU2619221C2 (ru) | Иммортализованная стволовая клетка и лекарственная композиция и лекарственный препарат, содержащие ее в качестве активного ингредиента | |
| AU2018100795A4 (en) | Construction of microrna gene-mediated novel tissue engineered nerve and applications thereof in repairing nerve defect | |
| JP7235509B2 (ja) | 植え込み可能な生きた電極およびその製造方法 | |
| La Noce et al. | Neural crest stem cell population in craniomaxillofacial development and tissue repair | |
| US20060247195A1 (en) | Method of altering cell properties by administering rna | |
| Li et al. | KLF7 overexpression in bone marrow stromal stem cells graft transplantation promotes sciatic nerve regeneration | |
| Li et al. | Laminin‑chitosan‑PLGA conduit co‑transplanted with Schwann and neural stem cells to repair the injured recurrent laryngeal nerve | |
| Tu et al. | The legacy effects of electromagnetic fields on bone marrow mesenchymal stem cell self-renewal and multiple differentiation potential | |
| CN113940949B (zh) | 一种负载外泌体的GelMA水凝胶微针及其制备方法和应用 | |
| DE69531712T2 (de) | Hybridgel, das eine biologisch aktive Substanz sekretiert | |
| BR112020015616B1 (pt) | Composição de biotinta para folha de regeneração de derme, método para fabricar folha de regeneração de derme customizada usando a mesma e folha de regeneração de derme customizada fabricada usando o método de fabricação | |
| Singh et al. | Evaluation of polymeric aligned NGCs and exosomes in nerve injury models in diabetic peripheral neuropathy condition | |
| Liu et al. | Self-assembly of gelatin microcarrier-based MSC microtissues for spinal cord injury repair | |
| Zhou et al. | Bone marrow stromal and Schwann cells from adult rats can interact synergistically to aid in peripheral nerve repair even without intercellular contact in vitro | |
| Kamei et al. | Axonal supercharged interpositional jump‐graft with a hybrid artificial nerve conduit containing adipose‐derived stem cells in facial nerve paresis rat model | |
| CN116473991A (zh) | miR-143-3p在制备治疗周围神经损伤药物或材料中的应用 | |
| KR101635148B1 (ko) | 신경세포 및 엘라스틴 유사 폴리펩타이드를 포함하는 파킨슨병 치료 촉진용 약학적 조성물 | |
| KR102632692B1 (ko) | 3차원 스페로이드형 세포 응집체 유래 세포외소포를 포함하는 신경 재생 촉진 조성물 | |
| Wang et al. | Nerve implants with bioactive interfaces enhance neurite outgrowth and nerve regeneration in vivo | |
| CN112043721B (zh) | miR-132-5p在制备神经再生药物或材料中的应用 | |
| Zhang et al. | Transplantation of human telomerase reverse transcriptase gene-transfected Schwann cells for repairing spinal cord injury | |
| Lai et al. | Combining Piezoelectric Stimulation and Extracellular Vesicles for Cartilage Regeneration | |
| Fortuna et al. | Three-dimensional Collagen Scaffolds in Cultures of Olfactory Ensheathing Cells Used for Severed Spinal Cord Regeneration | |
| Ahmadi et al. | European patent law framework regarding nanotechnology applications in stem cells |
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 |