CN116270713B - miR-1246在制备促进腱骨愈合药物中的应用 - Google Patents
miR-1246在制备促进腱骨愈合药物中的应用 Download PDFInfo
- Publication number
- CN116270713B CN116270713B CN202310281099.6A CN202310281099A CN116270713B CN 116270713 B CN116270713 B CN 116270713B CN 202310281099 A CN202310281099 A CN 202310281099A CN 116270713 B CN116270713 B CN 116270713B
- Authority
- CN
- China
- Prior art keywords
- mir
- tendon
- healing
- macrophages
- bone
- 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
- 108091043184 miR-1246 stem-loop Proteins 0.000 title claims abstract description 54
- 210000002435 tendon Anatomy 0.000 title claims abstract description 47
- 210000000988 bone and bone Anatomy 0.000 title claims abstract description 44
- 230000035876 healing Effects 0.000 title claims abstract description 35
- 239000003814 drug Substances 0.000 title claims abstract description 23
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 230000001737 promoting effect Effects 0.000 title claims abstract description 11
- 210000001074 muscle attachment cell Anatomy 0.000 claims abstract description 20
- 230000008439 repair process Effects 0.000 claims abstract description 19
- 208000027418 Wounds and injury Diseases 0.000 claims abstract description 10
- 208000014674 injury Diseases 0.000 claims abstract description 10
- 230000006378 damage Effects 0.000 claims abstract description 9
- 229940079593 drug Drugs 0.000 claims abstract description 9
- 230000005012 migration Effects 0.000 claims abstract description 8
- 238000013508 migration Methods 0.000 claims abstract description 8
- 230000035755 proliferation Effects 0.000 claims abstract description 8
- 210000000513 rotator cuff Anatomy 0.000 claims description 15
- 238000002347 injection Methods 0.000 claims description 9
- 239000007924 injection Substances 0.000 claims description 9
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 4
- 239000002105 nanoparticle Substances 0.000 claims description 4
- 210000003041 ligament Anatomy 0.000 claims description 3
- 239000003937 drug carrier Substances 0.000 claims description 2
- 210000001808 exosome Anatomy 0.000 claims description 2
- 239000002502 liposome Substances 0.000 claims description 2
- 239000002184 metal Substances 0.000 claims description 2
- 229920000642 polymer Polymers 0.000 claims description 2
- 239000002096 quantum dot Substances 0.000 claims description 2
- 239000000377 silicon dioxide Substances 0.000 claims description 2
- 230000000699 topical effect Effects 0.000 claims 1
- 210000002540 macrophage Anatomy 0.000 abstract description 34
- 230000014509 gene expression Effects 0.000 abstract description 20
- 238000000034 method Methods 0.000 abstract description 14
- 238000011282 treatment Methods 0.000 abstract description 14
- 230000000770 proinflammatory effect Effects 0.000 abstract description 10
- 102000010400 1-phosphatidylinositol-3-kinase activity proteins Human genes 0.000 abstract description 9
- 206010061218 Inflammation Diseases 0.000 abstract description 9
- 108091007960 PI3Ks Proteins 0.000 abstract description 9
- 230000003110 anti-inflammatory effect Effects 0.000 abstract description 9
- 230000004054 inflammatory process Effects 0.000 abstract description 9
- 102000013530 TOR Serine-Threonine Kinases Human genes 0.000 abstract description 7
- 108010065917 TOR Serine-Threonine Kinases Proteins 0.000 abstract description 7
- 230000008569 process Effects 0.000 abstract description 6
- 230000010354 integration Effects 0.000 abstract description 5
- 230000002829 reductive effect Effects 0.000 abstract description 4
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 230000007012 clinical effect Effects 0.000 abstract description 3
- 230000008929 regeneration Effects 0.000 abstract description 3
- 238000011069 regeneration method Methods 0.000 abstract description 3
- 230000035479 physiological effects, processes and functions Effects 0.000 abstract description 2
- 238000011160 research Methods 0.000 abstract description 2
- 230000009466 transformation Effects 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 23
- 210000001519 tissue Anatomy 0.000 description 20
- 230000000694 effects Effects 0.000 description 9
- 108090000623 proteins and genes Proteins 0.000 description 8
- 210000002758 humerus Anatomy 0.000 description 7
- 108091070501 miRNA Proteins 0.000 description 7
- 210000003205 muscle Anatomy 0.000 description 6
- 241000700159 Rattus Species 0.000 description 5
- 208000024288 Rotator Cuff injury Diseases 0.000 description 5
- 238000003501 co-culture Methods 0.000 description 5
- 239000002158 endotoxin Substances 0.000 description 5
- 230000002757 inflammatory effect Effects 0.000 description 5
- 229920006008 lipopolysaccharide Polymers 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 108090000978 Interleukin-4 Proteins 0.000 description 4
- 108090001005 Interleukin-6 Proteins 0.000 description 4
- 102000004889 Interleukin-6 Human genes 0.000 description 4
- 108700012920 TNF Proteins 0.000 description 4
- 239000012620 biological material Substances 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 239000002679 microRNA Substances 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 102100027456 Cytochrome c oxidase subunit 2 Human genes 0.000 description 3
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 3
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 3
- 101000725401 Homo sapiens Cytochrome c oxidase subunit 2 Proteins 0.000 description 3
- 101000605127 Homo sapiens Prostaglandin G/H synthase 2 Proteins 0.000 description 3
- -1 PAKT Proteins 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 210000002744 extracellular matrix Anatomy 0.000 description 3
- 239000000835 fiber Substances 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 230000028709 inflammatory response Effects 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 230000037431 insertion Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000003287 optical effect Effects 0.000 description 3
- 210000000323 shoulder joint Anatomy 0.000 description 3
- 230000019491 signal transduction Effects 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- 102000008186 Collagen Human genes 0.000 description 2
- 108010035532 Collagen Proteins 0.000 description 2
- 102000003777 Interleukin-1 beta Human genes 0.000 description 2
- 108090000193 Interleukin-1 beta Proteins 0.000 description 2
- 208000034693 Laceration Diseases 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 206010062575 Muscle contracture Diseases 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 210000003690 classically activated macrophage Anatomy 0.000 description 2
- 229920001436 collagen Polymers 0.000 description 2
- 210000004439 collateral ligament Anatomy 0.000 description 2
- 208000006111 contracture Diseases 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000010899 nucleation Methods 0.000 description 2
- 239000012188 paraffin wax Substances 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 230000010287 polarization Effects 0.000 description 2
- 230000002980 postoperative effect Effects 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- 101150071146 COX2 gene Proteins 0.000 description 1
- 101100114534 Caenorhabditis elegans ctc-2 gene Proteins 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 206010015866 Extravasation Diseases 0.000 description 1
- 108010002352 Interleukin-1 Proteins 0.000 description 1
- 102000000589 Interleukin-1 Human genes 0.000 description 1
- 241000735234 Ligustrum Species 0.000 description 1
- 210000004322 M2 macrophage Anatomy 0.000 description 1
- 102000009571 Macrophage Inflammatory Proteins Human genes 0.000 description 1
- 108010009474 Macrophage Inflammatory Proteins Proteins 0.000 description 1
- 206010073713 Musculoskeletal injury Diseases 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- 101150000187 PTGS2 gene Proteins 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 206010039227 Rotator cuff syndrome Diseases 0.000 description 1
- 206010061363 Skeletal injury Diseases 0.000 description 1
- 208000021945 Tendon injury Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 210000003423 ankle Anatomy 0.000 description 1
- 210000001264 anterior cruciate ligament Anatomy 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000001516 cell proliferation assay Methods 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 239000012050 conventional carrier Substances 0.000 description 1
- 230000005786 degenerative changes Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000036251 extravasation Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000002695 general anesthesia Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 210000001503 joint Anatomy 0.000 description 1
- 238000009940 knitting Methods 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 230000011987 methylation Effects 0.000 description 1
- 238000007069 methylation reaction Methods 0.000 description 1
- 238000000465 moulding Methods 0.000 description 1
- 210000000651 myofibroblast Anatomy 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 230000036407 pain Effects 0.000 description 1
- 206010033675 panniculitis Diseases 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 210000002967 posterior cruciate ligament Anatomy 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 210000001991 scapula Anatomy 0.000 description 1
- 239000004017 serum-free culture medium Substances 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 210000004872 soft tissue Anatomy 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 210000004304 subcutaneous tissue Anatomy 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000009864 tensile test Methods 0.000 description 1
- 229940098465 tincture Drugs 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- 238000005406 washing Methods 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Physical Education & Sports Medicine (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Rheumatology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Biochemistry (AREA)
- Epidemiology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Immunology (AREA)
- Pain & Pain Management (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明属于生物医药技术领域，具体涉及miR‑1246在制备促进腱骨愈合药物中的应用。研究证明，所述miR‑1246可以促进腱骨界面接近生理性的愈合，并增加其生物力学强度，同时促进了肌腱细胞的增殖和迁移。miR‑1246通过作用于巨噬细胞，激活巨噬细胞的PI3K/AKT/mTOR通路，使巨噬细胞发生极化，调控促炎因子和抗炎因子的表达，从而减轻损伤修复早期阶段巨噬细胞诱导的炎症反应，有效促进腱骨的修复和再生，避免移植体带来的整合不良、免疫排斥等不良临床效果和较高的复发率，能够解决腱骨愈合不良的临床难题。并且，采用miR‑1246进行治疗时，安全、高效，更有利于该新药的临床转化过程。
Description
技术领域
本发明属于生物医药技术领域。更具体地，涉及miR-1246在制备促进腱骨愈合药物中的应用。
背景技术
肩袖撕裂(Rotator cuff tears，RCT)是最常见的肌肉骨骼损伤类型之一。由外伤、退化变性等因素导致，常引起严重的疼痛，甚至残疾。肩袖撕裂损伤约占肩关节疾病的50％，在我国肩袖撕裂损伤患病率逐年递增，给社会和患者带来沉重的负担。目前，修复肩袖损伤有多种治疗方式，包括肌腱-骨固定技术、自体移植物、同种异体移植物和异种移植物等。如中国专利申请CN107496052A公开了一种肩袖补片，经编工艺编织而成，生物力学强度好，能够为肩袖关节提供足够的力学支撑；中国专利申请CN211534754U公开了一种用于加快肩袖腱骨愈合的自动PRF植入铆钉，所具有的储药槽内储存有能够加快肩袖腱骨连接处愈合的生物材料，通过铆钉主体直接植入到腱骨连接处，减少后期植入生物材料的步骤，利用生物材料在术后初期加快肩袖腱骨连接处的愈合。
但是上述治疗方案大部分是依靠移植材料提供力学支撑，不可避免的存在移植体整合不良、免疫排斥反应等缺陷，没有从根本上解决腱骨愈合不良的问题，后续移植部位撕裂的复发率仍可高达90％。可见，现有技术仍无法提供一种能够真正解决腱骨愈合失败问题的有效方法。
发明内容
本发明要解决的技术问题是克服现有腱骨损伤依靠移植材料治疗过程中存在的移植体整合不良、免疫排斥反应发生、易复发和力学性能差等缺陷和不足，提供一种miR-1246在制备促进腱骨愈合药物中的应用，通过诱导巨噬细胞极化，调控炎症因子释放，促进自身肌腱细胞的增殖和迁移，从而促进腱骨界面接近生理的愈合，增强其生物力学强度，从损伤修复的早期阶段对炎症进行精细调控，即使不依靠移植材料也能达到显著促进腱骨愈合的效果。
本发明上述目的通过以下技术方案实现：
现有技术研究表明，巨噬细胞驱动的炎症是肌腱修复早期阶段的关键特征，而过度炎症则是导致不良临床效果的关键因素。动物实验研究显示，由于促炎型M1巨噬细胞可导致炎症、细胞外基质(ECM)降解和细胞死亡，阻碍了肌腱的愈合。体外证据也支持这一观点，即肌腱愈合不良很大程度上是由于M1巨噬细胞分泌的促炎因子的有害作用。但相反的，抗炎型M2巨噬细胞则倾向于抵抗炎症反应、调节ECM平衡，从而促进组织愈合。现有技术大多数的技术对于改善腱骨修复过程的尝试主要是针对愈合的后期阶段，但是都收效甚微。因此，本发明在损伤修复的早期阶段对炎症进行精细调节以改善腱骨的不良愈合。所采用的miRNA是一类由内源性基因编码的单链小分子RNA，广泛存在生物体中参与转录后基因表达调控，能够调节多种重要的生物学功能。本发明实验研究首次发现，miR-1246可以显著促进腱骨愈合。
因此，本发明要求保护miR-1246在制备促进腱骨愈合药物中的应用，所述miR-1246的序列为：AAUGGAUUUUUGGAGCAGG。
进一步地，所述miR-1246促进腱骨界面愈合，提高生物力学强度。
更进一步地，所述miR-1246促进肌腱细胞的增殖和迁移。
进一步地，所述腱骨愈合为人体的肌腱、韧带在骨止点损伤的修复，包括肩袖撕裂修复、肩锁关节韧带重建术后修复、肘关节侧副韧带修复、前后交叉韧带重建术后修复、踝关节侧副韧带修复。
更进一步地，所述药物还包括药学上可接受的载体。miR-1246通过载体的递送能更高效的进入组织和细胞内，以促进腱骨愈合。
优选地，所述载体选自脂质体、外泌体、聚合物、介孔二氧化硅纳米颗粒、量子点和金属基纳米颗粒中的一种或多种。
优选地，所述药物的剂型为注射剂或外用药剂。更优选地，所述注射剂包括注射液、粉针剂；所述外用药剂包括凝胶、膏剂、粉剂、酊剂、喷剂。
另外的，本发明还提供了miR-1246在制备诱导巨噬细胞极化试剂中的应用，所述miR-1246的序列为：AAUGGAUUUUUGGAGCAGG。
进一步地，所述miR-1246作用于巨噬细胞，抑制促炎因子的表达。
更进一步地，所述促炎因子包括TNFα、IL-1β、IL-6和COX2。
进一步地，所述miR-1246作用于巨噬细胞，促进抗炎因子的表达。
更进一步地，所述抗炎因子包括IL-1RA、IL-4、IL-10。
本发明具有以下有益效果：
本发明首次提供了miR-1246在制备促进腱骨愈合药物中的应用，研究证明，所述miR-1246可以促进腱骨界面接近生理的愈合，并增加其生物力学强度，同时促进了肌腱细胞的增殖和迁移。miR-1246通过作用于巨噬细胞，激活巨噬细胞的PI3K/AKT/mTOR通路，使巨噬细胞发生极化，调控促炎因子和抗炎因子的表达，从而减轻损伤修复早期阶段巨噬细胞诱导的炎症反应，有效促进腱骨界面的修复和再生，避免了移植体带来的整合不良、免疫排斥等不良临床效果和较高的复发率，能够解决腱骨愈合不良的临床难题。并且，采用miR-1246进行治疗时，安全、高效，更有利于该新药的临床转化过程。
附图说明
图1为实施例1中大鼠肩袖撕裂伤模型手术步骤及损伤部位注射药物的图。
图2为实施例1中AgomiR-1246治疗组(A)和AgomiR-NC对照组(B)腱骨结合部愈合情况的大体观察图。
图3为实施例1中腱骨结合部肌腱组织的生物力学测试示意图(A)和各组腱骨界面肌腱的极限失效载荷(B)、刚度(C)比较数据统计图；其中，数据以均数±SEM表示，*P<0.05，**P<0.01，****P<0.0001。
图4为实施例1中各组肩袖腱骨结合部的病理组织学图。
图5为实施例2中由LPS激活的巨噬细胞和肌腱细胞的Transwell共培养体系示意图(A)和经miR-1246mimics处理后肌腱细胞在光学显微镜下20倍(B)和40倍(C)的形态和数量照片。
图6为实施例2中各组miR1246的表达水平数据统计图(A)和各组PI3K、PAKT、PS6相关蛋白表达的蛋白印迹图(B)。
图7为实施例2中各组炎症因子的表达数据统计图(A)和各组间炎症因子的蛋白表达数据统计图(B)。
图8为实施例3中划痕实验检测各组肌腱细胞的迁移能力显微镜下照片(A)、各组肌腱细胞的形态和数量显微镜下照片(B)和各组肌腱细胞的增殖能力数据统计图(C)。
图9为miR 1246促进肩袖撕裂伤后腱骨愈合的作用及机制示意图。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
RAW264.7巨噬细胞系购自ATCC细胞库。动物体内注射应用的miR 1246模拟物AgomiR 1246(利用甲基化+胆固醇修饰，由厂家提供，为常规技术手段)，对照AgomiR-NC以及体外细胞模型实验中应用的miR 1246模拟物miR 1246mimics，对照miR 1246mimics NC以及转染试剂盒(为纳米材料载体，由厂家提供，为常规技术手段，其他常用载体也能达到相同效果)均可购自广州锐博生物技术有限公司。
除非特别说明，以下实施例所用试剂和材料均为市购。
实施例1miR-1246对RCT后腱骨愈合的影响
(1)构建大鼠RCT模型：
大鼠全身麻醉后，上肢肩部备皮并消毒。采用长约3cm的经大结节纵行皮肤切口，钝性分离皮下组织、肌肉，暴露冈上肌，沿肱骨止点锐性离断冈上肌，剥离肱骨大结节残存组织直到骨质。为了消除大结节上残余冈上肌止点组织对组织学观察的影响，术中需彻底切除大结节上的止点组织。取距冈上肌止点约0.3cm、距边缘1/4处两点为进针点，使用两根2-0不可吸收普理灵缝线对肌腱残端行褥式缝合，一侧进针点由内向外，另一侧则有外向内，两根普理灵尾线留取同等长度。将两根PDS线穿入直径1mm的空针针芯，并使用空针针头于肱骨大结节下缘缓慢钻开两组平行骨隧道，出口定位足印区下界，拔出针头将两根PDS线留于骨隧道中。将骨两组隧道中的PDS引线分别与向对应的肌腱残端的两根普理灵缝线拉出骨隧道，将冈上肌腱牵拉锚定缝合在足印区上，建立大鼠单侧冈上肌损伤修复模型，具体如图1中的A～B所示。
(2)药物治疗：
AgomiR 1246治疗组：在肩袖腱骨结合部缓慢注射5nmol AgomiR 1246溶液约200μl，避免药物溢出，每周注射2次。AgomiR-NC对照组：在肩袖腱骨结合部予以注射AgomiR-NC约200μl，注射方法和频次同前。操作过程参见图1中的C。
(3)取材腱骨结合部的组织：
于造模后1周，即修复早期阶段取材腱骨结合部组织。以手术区域冈上肌肌腱止点为中心，完整取下包含完整肩关节的肱骨近端组织及完整冈上肌，剔除皮肤肌肉和软组织，保留完整冈上肌肌腱与肱骨大结节交界区域，即冈上肌-肱骨复合体。
(4)大体观察：
给予药物后1周，观察手术区域切口愈合情况(有无感染等炎症反应)、有无因组织挛缩或粘连而导致的肩关节活动度受限。并取材腱骨结合部组织，观察各组标本手术区有无炎症反应、周围组织粘连情况、肌腱与肱骨之间的间隙及肌腱和腱骨止点外形变化。
实验结果如图2所示，由图可见，给予AgomiR 1246注射后1周，大体观察可见，腱骨界面愈合良好，伤口区域未见感染征及明显炎症反应，腱骨界面周围组织未见因组织挛缩或粘连而导致的肩关节活动度明显受限。而对照组给予AgomiR-NC注射后1周，可见腱骨界面愈合不良，周围组织有粘连，肌腱表面不光滑。
(5)生物力学评估：
根据生物力学检测仪的操作规范，将冈上肌-肱骨复合体固定在AGX-S拉力测试试验机上。设定传感器范围100N，拉伸速度为5mm/min。将肩胛骨与肱骨分别固定在试验机的拉力臂和底座上，调整固定方向，保证拉力的方向与冈上肌纤维附着走行一致。开启拉力测试，精确记录拉力曲线变化，测试最大拉伸载荷冈上肌组织。分为NC：正常组，RCT：未给予任何处理的模型对照组，RCT+AgomiR-NC对照组，RCT+AgomiR 1246治疗组。所有试样均以恒定拉伸速度加载至肌腱拉伸断裂。详细记录其最大断裂载荷(ultimate failure load，N)，载荷-位移曲线的线性斜率为作为刚度(stiffness，N/mm)。
示意图及生物力学性能指标如图3所示。由图可见，和AgomiR-NC对照组相比，AgomiR 1246治疗组肩袖腱骨结合部的极限失效载荷明显提高；AgomiR 1246治疗组表现出刚度性能明显增强。
(6)组织病理学检测：
将腱骨结合部组织在4％多聚甲醛中固定，5％硝酸进行完全脱钙，用石蜡包埋，制成约20μm厚的石蜡切片，并用HE、阿尔新蓝进行染色。
病理结果如图4所示，可见RCT+AgomiR 1246治疗组腱骨界面周围的组织未见明显免疫炎症反应，肌腱和骨质之间呈现出了连续性，肌成纤维细胞向骨质内的生长，导致了更有序排列的肌腱胶原纤维和细胞形成，显示出了组织特征的改善。而在对照组样本中，可见肌腱胶原纤维和细胞失去了其特有的平行方向，肌腱与骨质之间生长不连续性。
实施例2miR-1246对巨噬细胞的影响
(1)肌腱细胞和巨噬细胞系共培养体系的建立
将正常大鼠肌腱组织进行消化，体外分离肌腱细胞，将P5代肌腱细胞接种于transwell的下室内在12孔板中以5×104个/孔的密度扩增培养；在肌腱细胞培养24h后与RAW264.7共培养，制备共培养体系：RAW264.7细胞接种于transwell的上室内，以5×104细胞/室的密度接种，RAW264.7细胞接种6小时后，加入脂多糖(LPS，1μg/mL)刺激8小时，以构建巨噬细胞炎症模型。肌腱细胞和巨噬细胞系共培养体系和经miR-1246处理后肌腱细胞在光学显微镜下的数量和形态照片，具体可参见图5。
(2)转染miR1246 mimics进入巨噬细胞中
根据制造商的说明书，利用转染试剂盒将miR1246 mimics，miRNA mimics NC递送进入巨噬细胞内，在37℃细胞培养箱中孵育24h，24h后更换为新鲜培养基继续培养24h，取细胞获得转染后的细胞样本，分为miR1246 mimics+LPS组；miRNA mimics NC+LPS对照组。
(3)RT-qPCR检测巨噬细胞中miR1246的表达水平
应用TRIzol分别从miR1246 mimics+LPS组，miRNA mimics NC+LPS对照组的巨噬细胞中提取总RNAs，使用Bulge-Loop miRNA特异性逆转录引物(RiboBio)进行逆转录，并使用针对靶向miRNAs合成的特异性引物(RiboBio)进行qPCR，U6作为内源性对照。将表达数据统一标准化，计算巨噬细胞中miR1246的相对表达量的差异。
实验统计结果如图6A所示，由图可见，与miRNA mimics NC组相比，miR1246mimics组的巨噬细胞中miR1246的表达量明显升高。
(4)Westernblot检测miR1246对巨噬细胞中PI3K/AKT/mTOR信号通路的调控作用
PI3K/AKT/mTOR信号通路与巨噬细胞极化密切相关，应用Westernblot方法比较各组PI3K/AKT/mTOR信号通路相关蛋白表达水平的差异。
检测结果如图6B所示，miR1246的过表达使得巨噬细胞中PI3K/AKT/mTOR通路中的PI3K、PAKT、PS6相关蛋白因子的表达显著上调。
(5)RT-qPCR和ELISA检测巨噬细胞相关下游炎症因子
为了进一步探究miR1246的作用机制，根据试剂盒说明书，应用qRT-PCR法和ELISA试剂盒定量共培养体系中细胞上清液中炎症因子的基因和蛋白表达水平，以评估miR1246对M1和M2型巨噬细胞释放的促炎和抗炎因子的影响，包括促炎因子TNFα、IL-1β、IL-6、COX2；抗炎因子IL-1RA、IL-4、IL-10。
实验结果如图7所示，由图可见，miR1246 mimics组中M1型巨噬细胞分泌的促炎因子TNFα、IL-1β、IL-6、COX2的表达量显著下调；M2型巨噬细胞分泌的抗炎因子IL-1RA、IL-4、IL-10的表达量显著上调。
实施例3miR-1246对炎症环境下肌腱细胞的影响
利用划痕实验检测肌腱细胞的迁移能力：在96孔板后均匀划横线，大约每隔0.5～1cm一道，横穿过孔，每孔至少穿过5条线；孔中加入一定数量的细胞，接种原则为过夜后融合率达到100％；用枪头比着直尺，垂直于细胞平面，沿着平板背面的线在细胞层上进行划痕；用PBS洗细胞3次，去除划下的细胞，加入无血清培养基；放入37度5％CO2培养箱培养。按0h、24h，48h取样，拍照。
应用细胞增殖实验(CCK8)检测各组肌腱细胞的增殖能力：消化两组细胞、离心、计数和重悬后制备细胞悬液，按300个/孔的接种密度种于96孔板中，细胞培养箱中培养24h后开始观察1～7天细胞的生长状况。紫外分光光度计上(450nm)测溶液的OD值，以培养时间为横坐标，以细胞光密度值为纵坐标，绘制细胞增殖曲线。
实验结果如图8所示，由图可见，划痕实验显示miR1246表达上调后不仅可以显著促进肌腱细胞的迁移，而且可以促使肌腱细胞的增殖能力明显增强。
综上所述，miR1246通过作用于巨噬细胞，激活巨噬细胞的PI3K/AKT/mTOR通路，促使巨噬细胞发生极化，调控促炎因子(TNFα、IL-1β、IL-6、COX2)和抗炎因子(IL-1RA、IL-4、IL-10)的表达，以减轻损伤修复早期阶段巨噬细胞诱导的炎症反应，从而有效促进了腱骨的修复和再生；同时miR-1246还促进了自身肌腱细胞的增殖和迁移。相关机制说明具体参见图9。在上述共同作用下达到了促使腱骨界面接近生理的愈合，增加了其生物力学强度的治疗效果，避免了移植体带来的整合不良、免疫排斥等不良临床效果和较高的复发率，解决了腱骨愈合不良的临床难题，并且实验过程中未出现任何毒副反应，安全性高。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
Claims (6)
1.miR-1246在制备促进腱骨愈合药物中的应用，其特征在于，所述miR-1246的序列为：AAUGGAUUUUUGGAGCAGG；
所述腱骨愈合为人体的肌腱、韧带在骨止点损伤的修复，具体为肩袖撕裂修复。
2.根据权利要求1所述应用，其特征在于，所述miR-1246促进腱骨界面愈合，提高生物力学强度。
3.根据权利要求1所述应用，其特征在于，所述miR-1246促进肌腱细胞的增殖和迁移。
4.根据权利要求1所述应用，其特征在于，所述药物还包括药学上可接受的载体。
5.根据权利要求4所述应用，其特征在于，所述载体选自脂质体、外泌体、聚合物、介孔二氧化硅纳米颗粒、量子点和金属基纳米颗粒中的一种或多种。
6.根据权利要求1所述应用，其特征在于，所述药物的剂型为注射剂或外用药剂。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310281099.6A CN116270713B (zh) | 2023-03-20 | 2023-03-20 | miR-1246在制备促进腱骨愈合药物中的应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310281099.6A CN116270713B (zh) | 2023-03-20 | 2023-03-20 | miR-1246在制备促进腱骨愈合药物中的应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116270713A CN116270713A (zh) | 2023-06-23 |
| CN116270713B true CN116270713B (zh) | 2023-11-07 |
Family
ID=86795643
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310281099.6A Active CN116270713B (zh) | 2023-03-20 | 2023-03-20 | miR-1246在制备促进腱骨愈合药物中的应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116270713B (zh) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108653328A (zh) * | 2018-03-26 | 2018-10-16 | 杭州市萧山区中医院 | 间充质干细胞联合富血小板血浆在制备用于促进肩袖损伤腱骨复合体愈合的药物中的应用 |
| WO2021105404A1 (en) * | 2019-11-29 | 2021-06-03 | Novadip Biosciences | Biomaterials for the prevention and the treatment of tissue disorders |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20190000886A1 (en) * | 2016-03-16 | 2019-01-03 | Duncan ROSS | Method of treatment of osteochondral defects |
| JP2019514416A (ja) * | 2016-05-16 | 2019-06-06 | エグゾステム バイオテック リミテッド | 間葉系幹細胞と筋損傷及び筋肉関連疾患の治療のための間葉系幹細胞の使用 |
-
2023
- 2023-03-20 CN CN202310281099.6A patent/CN116270713B/zh active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108653328A (zh) * | 2018-03-26 | 2018-10-16 | 杭州市萧山区中医院 | 间充质干细胞联合富血小板血浆在制备用于促进肩袖损伤腱骨复合体愈合的药物中的应用 |
| WO2021105404A1 (en) * | 2019-11-29 | 2021-06-03 | Novadip Biosciences | Biomaterials for the prevention and the treatment of tissue disorders |
Non-Patent Citations (4)
| Title |
|---|
| M2型巨噬细胞在腱骨愈合中作用和机制研究;施又兴;中国博士学位论文全文数据库医药卫生科技(第1期);摘要 * |
| stem cell-derived exosomal transcriptomes for wound healing;Guiling Chen等;frontiers;第9卷;1-12 * |
| 非编码RNA调控巨噬细胞极化的研究进展;魏文燕;陈建英;;山东医药(第29期);摘要、第91页右栏倒数第1段 * |
| 魏文燕 ; 陈建英 ; .非编码RNA调控巨噬细胞极化的研究进展.山东医药.2018,(29),摘要、第91页右栏倒数第1段. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116270713A (zh) | 2023-06-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US10639399B2 (en) | Construction of MicroRNA gene-mediated novel tissue engineered nerve and applications thereof in repairing nerve defect | |
| Gu et al. | Application of marrow mesenchymal stem cell‐derived extracellular matrix in peripheral nerve tissue engineering | |
| Goto et al. | A rolled sheet of collagen gel with cultured Schwann cells: model of nerve conduit to enhance neurite growth | |
| US20120114755A1 (en) | Methods and materials for tissue repair | |
| Sun et al. | Improving nerve regeneration of acellular nerve allografts seeded with SCs bridging the sciatic nerve defects of rat | |
| US20040013652A1 (en) | Treatments with autologous fibroblast | |
| de Souza Lucena et al. | Experimental considerations concerning the use of stem cells and tissue engineering for facial nerve regeneration: a systematic review | |
| BR112020022667A2 (pt) | método para gerar uma população de células precursoras do músculo humano derivadas do músculo esquelético, população de células precursoras do músculo humano derivado do músculo esquelético, meio de crescimento celular para produção de uma população de células precursoras do músculo humano, método para a produção de um meio de crescimento celular, método para a produção de uma composição e composição | |
| US8496956B2 (en) | Implantable nerve regeneration conduit | |
| Zhang et al. | Bone marrow mesenchymal stem cells accelerate the morphological and functional recovery of neovaginas | |
| Zhang et al. | Transplantation of olfactory ensheathing cells combined with chitosan down-regulates the expression of P2X7 receptor in the spinal cord and inhibits neuropathic pain | |
| CN104736709A (zh) | 基于钙粘蛋白调节的组合物和治疗 | |
| Song et al. | Three-dimensional printing of microfiber-reinforced hydrogel loaded with oxymatrine for treating spinal cord injury | |
| Merguerian et al. | Grafting of cultured uroepithelium and bladder mucosa into de-epithelialized segments of colon in rabbits | |
| CN116270713B (zh) | miR-1246在制备促进腱骨愈合药物中的应用 | |
| CN115252893B (zh) | 一种抗炎抗氧化性神经导管的制备 | |
| WO2020102590A1 (en) | Methods of bioengineering internal anal sphincter constructs | |
| Pan et al. | Exosomes from Hair Follicle Epidermal Neural Crest Stem Cells Promote Acellular Nerve Allografts to Bridge Rat Facial Nerve Defects | |
| Shrestha et al. | Bladder reconstruction using stem cells seeded on multilayered scaffolds in a mucosa preserving partial cystectomy model | |
| Ma | Collagen nanomaterial in the recovery of motor ability of patients with peripheral nerve transection injury | |
| CN115192775B (zh) | 一种复合POU6F1-SCs的脱细胞神经支架及其制备方法与应用 | |
| KR102334886B1 (ko) | 성장판 재생용 조성물 | |
| CN115975929A (zh) | Kat2a抑制剂在治疗脊髓及坐骨神经损伤中的应用 | |
| Wang et al. | Outcome of a novel porcine-derived UBM/SIS composite biological mesh in a rabbit vaginal defect model | |
| Kim et al. | Engineered regenerative isolated peripheral nerve interface for targeted reinnervation |
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 |