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Functional tissue engineering material for nerve repair, preparation method and use thereof

A functional organization and nerve repair technology, applied in the field of nerve function repair and inhibition of nerve re-injury, can solve the problems of excessive use of neurotrophic factors, low therapeutic effect, and inability to maintain effective therapeutic concentration

Inactive Publication Date: 2014-04-09
PLA NAVY GENERAL HOSIPTAL
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

However, the neurotrophic factors that are simply applied to the injury site cannot accumulate in the injury site for a long time, but will diffuse to the surrounding normal tissues with the body fluid, resulting in the loss of neurotrophic factors, which cannot maintain its effective therapeutic concentration, and the therapeutic effect is low; periodicity Although repeated administration can maintain the drug concentration at the injury site, it can easily lead to excessive use of neurotrophic factors, triggering inflammatory reactions and brain epilepsy
Moreover, when a more serious nerve injury occurs, different degrees of tissue defects will appear at the injury site. At this time, simply relying on in situ application of neurotrophic factors can no longer meet the needs of promoting nerve repair. reinjured calpain
[0004] At present, there are no products and treatments that specifically target the three major factors of central nervous system injury: tissue damage, presence of calpain, and lack of neurotrophic factors

Method used

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  • Functional tissue engineering material for nerve repair, preparation method and use thereof
  • Functional tissue engineering material for nerve repair, preparation method and use thereof
  • Functional tissue engineering material for nerve repair, preparation method and use thereof

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preparation example Construction

[0028] The preparation method of functional tissue engineering material is as follows: Traut's reagent and Sulfo-SMCC are dissolved in phosphate buffered saline (PBS, pH8.0, 4mM EDTA) and PBS (pH7.2, 4mM EDTA) respectively, will prepare Soak human amniotic membrane in PBS (pH8.0, 4mM EDTA), react Traut’s reagent with human amniotic membrane overnight at 4°C, remove excess cross-linking agent, and wash 3 times with PBS (pH7.2, 4mM EDTA) In addition, the calpain inhibitor and Sulfo-SMCC were reacted at room temperature in PBS (pH7.2, 4mM EDTA) for 30 minutes, then reacted with the human amniotic membrane treated with Traut's reagent at room temperature for 2 hours, and then reacted with PBS (pH7 .2, 4mM EDTA) to wash thoroughly, and use 5% glycine to neutralize excess reactive groups, obtain the human amniotic membrane immobilized with the inhibitor of calpain;

[0029] Take human amniotic membrane immobilized with calpain inhibitor and place it in a 6-well plate, add one or mor...

Embodiment 1

[0035] Preparation of human amniotic membrane

[0036] Amniotic membrane material: Human amniotic membranes are from fetal membranes from healthy mothers without virus infection (informed consent has been signed with the donor), namely hepatitis B 5 items, human hepatitis C virus, hepatitis C virus, human immunodeficiency virus, human immunodeficiency virus, Other relevant indicators of infectivity, including syphilis and human papillomavirus, were negative.

[0037] Human amniotic membrane containing antibiotics (gentamicin 4000U / ml, penicillin 500U / ml, streptomycin 500μg / ml) 1×PBS (pH7.2) to wash away the blood clot of the placenta, bluntly strip the amniotic membrane, and make it with The chorion was separated, rinsed with PBS containing antibiotics, soaked for 20min, and spread on the nitrocellulose membrane with the epithelial side up. Trim the above paper sheet with amnion attached to a small piece of 2.5cm×2.5cm. Rinse twice with PBS, soak in DMEM / F12 culture medium (...

Embodiment 2

[0040] Preparation of GDNF fusion protein containing collagen-binding domain CBD

[0041] After designing and synthesizing primers with biological software Primer5.0, taking GDNF as an example, the TKKTLRT gene sequence of CBD was introduced into the upstream primer of GDNF,

[0042] Upstream primer 1: 5'-GGTCTACGGAGACCGGATCCG-3';

[0043] Upstream primer 2: 5′-CATATGACTAAGAAAACCCTGCGTACTGGGGTCTACGGAG-3′;

[0044] Downstream primer: 5′-CTCGAG TCTCTGGAGCCAGAGATCT-3′

[0045] The GDNF mature fragment was amplified from human cDNA by using the upstream primer 1 and the downstream primer. The PCR amplification reaction solution was: 2μL 10×PCR buffer (Dalian TaKaRa Company), 2U-5U Taq enzyme (Dalian TaKaRa Company), 0.2~ 1.0 mM dNTPs (Dalian TaKaRa Company) and 0.2 μmol of upstream primer 1 and downstream primer (synthesized by Invitrogen, USA); put the prepared system into a PCR machine (Boao Company), and the application amplification program is as follows: 94 ° C 5 minutes; ...

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Abstract

The present invention provides a functional tissue engineering material for nerve repair. The functional tissue engineering material comprises human amnion, a neurotrophic factor specifically bound with the collagen, and an inhibitor fixedly bound with calpain. The present invention further provides a preparation method for the functional tissue engineering material, wherein neurotrophic factor fusion protein specifically bound with the collagen binding domain and an inhibitor for cross-linking calpain are constructed to form the functional tissue engineering material for guiding nerve repair, such that the human amnion can be specifically bound with the neurotrophic factor so as to effectively promote nerve repair, the regeneration nerve forms correct synaptic connections, the nerve re-injury effect of calpain can be eliminated, and the material can be used for targeting treatment of central nerve injury diseases and neural function repair.

Description

technical field [0001] The invention relates to a functional tissue engineering material for promoting nerve repair in central nervous injury diseases, preparation of the functional tissue engineering material and its use in targeted treatment of central nervous injury diseases, nerve function restoration and inhibition of nerve re-injury. The functional tissue engineering material involved in the present invention includes human amniotic membrane and neurotrophic factors specifically bound to its collagen, and is immobilized with inhibitors of calpain, which can be used for targeted therapy of central nervous system injury patients, nerve function repair and clinical re-injury of nerves medication. Background technique [0002] Central nervous system injury is one of the diseases with the highest disability rate, among which traumatic brain injury, cerebral hemorrhage, cerebral ischemia and spinal cord injury are currently the highest incidence. After central nervous syste...

Claims

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Application Information

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IPC IPC(8): A61L27/36A61L27/54A61L31/00A61L31/16
Inventor 张剑宁钱阳明田丽丽刘爽
Owner PLA NAVY GENERAL HOSIPTAL
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