CN204840394U - Preparation and application of biological activity support with chemotactic function - Google Patents
Preparation and application of biological activity support with chemotactic function Download PDFInfo
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- CN204840394U CN204840394U CN201520319632.4U CN201520319632U CN204840394U CN 204840394 U CN204840394 U CN 204840394U CN 201520319632 U CN201520319632 U CN 201520319632U CN 204840394 U CN204840394 U CN 204840394U
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Abstract
The utility model provides the utility model discloses a that restores the spinal cord injury has chemotactic endogenous cell migration, promotes the endogenous cell to survive and break up to and the novel biological activity support of utensil back neuroprotection. Utilizing the natural silk albumen that draws as the media in the silk cocoon, utilizing freeze drying technique and ethanol cross -linking agent to make neurotrophic factor -3 (NT -3), vitamin A acid (RA) and silk -fibroin crystalline form change, the crosslinked is fixed in three -dimensional gelatin sponge support. The utility model provides a biological activity support that has the chemotactic function can attract endogenous cell migration to damage / transplanting district by long period release chemokine after being transplanted to the spinal cord, release has the nutrition factor of neuroprotection, improves the microenvironment, reaches the purpose that attracts the endogenous cell to execute real self -regeneration spinal cord injury.
Description
Art
This utility model relates to a kind of timbering material for repairing spinal cord injury, endogenous cell especially can be attracted to move, promote the bioactive bracket of endogenous cell survival and differentiation.
Background technology
At present, the known support for repairing spinal cord injury is made up of support and exogenous cells.Mainly be transplanted to impaired injury to reach the effect of replacing and losing neuron and treatment spinal cord injury by exogenous cells, and support is only as a kind of carrier supporting cell attachment.But in process of clinical application, the transplanting of exogenous cells is faced with the numerous difficulties such as source, ethics and immunologic rejection, and this makes the exogenous cells transplantation treatment of spinal cord injury be greatly limited.Therefore, find a kind of effective mode can evade the cell derived sex chromosome mosaicism of cell transplantation, spinal cord injury/graft area can be met again and move, survive, break up and provide the demand of the nutritional activities factor to be crucial to endogenous cell.
The targeting migration of cell and mechanism thereof are the focuses that scientists is paid close attention to always.Current research mainly concentrates in exogenous cells body the directional migration after transplanting.Can irritation cell migration chemotactic factor be expelled to diseased region, can by the exogenous cells specificity of transplanting attract migration come, and can present treatment and replace effect.Conventional chemotactic factor comprises the [TakeuchiH such as monocyte chemoattractant protein-1 (MCP-1) and medium selection (SDF-1), etal.Intravenouslytransplantedhumanneuralstemcellsmigrat etotheinjuredspinalcordinadultmiceinanSDF-1-andHGF-depen dentmanner.NeurosciLett, 2007,426 (2): 69-74].In recent years; utilize the trophic factors with neuroprotective as chemotactic factor; also good chemotactic effect is shown; these factors also have trophocyte and improve the effect of spinal cord injury microenvironment simultaneously; as [ChenYF such as neurenergen 3s (NT-3); etal.Neurotrophin-3stimulatesmigrationofmesenchymalstemc ellsoverexpressingTrkC.CurrMedChem, 2013,20 (24): 3022-3033].
Retinoic acid (RA) is a kind of metabolite of vitamin A (Vit.A).Animal self can not synthesize Vit.A, must absorb from plant with the form of carotene with in animal with forms such as tretinoins.RA plays dominant role in neurodevelopment.Research shows, RA can induce different types of special cells for transplantation treatment.This seminar early-stage Study finds, in vitro, RA inducing bone mesenchymal stem cell (MSCs) neurad histiocyte direction is utilized to break up, improve associated receptor mrna expression level [ZhangW, etal.Combinationofadenoviralvector-mediatedneurotrophin-3genetransferandretinoicacidpromotesadultbonemarrowcells todifferentiateintoneuronalphenotypes.NeurosciLett, 2006,408 (2): 98].In vivo, under synergism in NT-3 and electro-acupuncture on Du meridian of the MSCs of RA induction, there is the ability [ZhangK of neurad unit direction differentiation, etal.Electro-acupuncturepromotesthesurvivalanddifferenti ationoftransplantedbonemarrowmesenchymalstemcellspre-ind ucedwithneurotrophin-3andretinoicacidingelatinspongescaf foldafterratspinalcordtransection.StemCellRevRep, 2014,10 (4): 612].Therefore, RA has critical effect in the Induction and differentiation of neural tissue cell.
At present at home, outward, a kind of self have biological activity and can have not yet to see report by the chemotactic endogenous cell timbering material that moves to spinal cord injury/transplanting place.For this reason, we imagine a kind of bioactive bracket with chemotactic endogenous cell shift function of structure.Plan thisly there is chemotactic activity structural transplantation to enter spinal cord injury place, transfer endogenous cell and move to damage/transplanting place enforcement self-regeneration spinal cord injury.The object of the invention is the deficiency wanting to overcome on the techniques and methods of existing clinical treatment spinal cord injury, apply we build the bioactive bracket with chemotactic performance for endogenous cell repair spinal cord injury brand-new thinking and countermeasure is provided.
Summary of the invention
In order to the scheme overcoming existing bioactive bracket transplantation treatment spinal cord injury is not enough, this utility model provides one to have chemotactic activity support, this support can not only attract endogenous relevant cell to move to internal stent survival and differentiation, and can be improved the microenvironment of spinal cord injury/graft area by slow release neurotrophic factor and inducible factor.
This utility model solves the technical scheme that its technical problem adopts:
Utilize the native silk protein extracted in Bombyx bombycis as medium, Freeze Drying Technique is utilized to make neurenergen 3 (NT-3), retinoic acid (RA) and fibroin mixed liquor anhydrate with degerming, ethanol promotes the crystal phase transition of chemotactic activity factor mixed liquor, chemotactic factor and inducible factor is cross-linked and is fixed in three-dimensional rack.After transplanting, the chemotactic activity factor on support can be released slowly in the microenvironment in spinal cord injury district, a kind of chemotactic factor enrichment region is formed centered by structural transplantation position, promote that relevant endogenous cell is to this zone migration, reach and attract endogenous cell move to internal stent survival and break up the object of repairing spinal cord injury.
The beneficial effects of the utility model are:
This have after functional bioactive timbering material is transplanted to spinal cord injury place, chemotactic factor (NT-3) can be discharged the long period, continue to attract endogenous relevant cell to move in support, under the synergism of inducible factor (RA), these endogenous relevant cells can be survived better, and the differentiation of neurad histiocyte direction; The NT-3 simultaneously discharged has neuroprotective, the microenvironment of continuative improvement spinal cord injury/graft area.
Accompanying drawing explanation
Below in conjunction with drawings and Examples, this utility model is further illustrated.
Fig. 1 is that (1 shows: chemotactic factor, inducible factor and fibroin complex for the schematic diagram of the bioactive bracket with Chemotaxis Function; 2 show: three-dimensional isinglass sponge; 3 show: the irregular multi-pore structure of internal stent; 4 show: PLGA shell).
Fig. 2 is the swelling ratio of the bioactive bracket with Chemotaxis Function.
Fig. 3 is the water absorption rate of the bioactive bracket with Chemotaxis Function.
Fig. 4 is after the bioactive bracket with Chemotaxis Function is transplanted to the complete cross-section place of rat spinal cord, can be observed nerve fiber growth and astrocyte migration enters damage/graft area.
Fig. 5 is that after the bioactive bracket with Chemotaxis Function is transplanted to the complete cross-section place of rat spinal cord, the nerve fiber that can be observed to surround sheath structure grows into damage/graft area.
Fig. 6 is after the bioactive bracket with Chemotaxis Function is transplanted to the complete cross-section place of rat spinal cord, and can be observed damage/graft area has the cell migration of expressing TrkC to enter.
Fig. 7, after the bioactive bracket with Chemotaxis Function is transplanted to the cross-section place of dog spinal cord half, can be observed angioid extensibility of structure and enters damage/graft area.
Fig. 8, after the bioactive bracket with Chemotaxis Function is transplanted to the cross-section place of dog spinal cord half, can be observed a large amount of cell migration and enters damage/graft area.
Detailed description of the invention
Detailed description done by key instrument, chemotactic activity support and the reagent used to this utility model below by specific embodiment:
1. key instrument
Superclean bench (Suzhou purification Electronic Equipment Factory); Generic centrifuge (Kubo field Japan); Constant water bath box (Beijing Medical Equipment Plant); 5%CO
2incubator (the Queue U.S.); Inverted phase contrast microscope (Olympus Japan); Fluorescence microscope (Leica Germany); Scanning electron microscope (Philips Holland); Transmission electron microscope (Philips Holland); Laser confocal imaging system (CarlZeiss Germany); Low temperature baking box (Shanghai leap medical apparatus and instruments factory); High temperature roaster (Shanghai leap medical apparatus and instruments factory); Autoclave sterilizer (Jiangyin Binjiang Medical Equipment Plant); Cryostat microtome (Shandon Britain); Ultra-pure water instrument (Molsheim France); Enzyme-linked immunosorbent assay instrument (the Bio-Rad U.S.); Electrophresis apparatus power supply (the Bio-Rad U.S.); Vertical slab electrophoresis groove (the Bio-Rad U.S.); Electroporation (the Bio-Rad U.S.); Ultrahigh speed refrigerated centrifuge (the Beckman U.S.);-80 DEG C of ultra cold storage freezers (the RevcoTech U.S.); JY92-2D ultrasonic cell disruptor (NingBo XinZhi Biology Science Co., Ltd).
2. chemotactic activity support
The concrete dynamic modulus gelatin cylinder bracket material preparing support is purchased from the product in Nanjing Jinting Pharmaceutical Co., Ltd.---medical gelatin sponge.Surrounding PLGA (50: the 50) thin film forming shell thin-walled around concrete dynamic modulus gelatin cylinder bracket is purchased from the product in Jinan Dai Gang biotechnology company.Bombyx bombycis is given by Silk Worm Inst Zhejiang Prov. Agriculture Science Academy.The equal available from Sigma of NT-3 and RA.
3. main agents
DMEM-LG (Gibico), top grade hyclone (TBD), poly-D-lysine (Sigma), D-Hank's balance liquid (autogamy), trypsin Sigma), EDTA (Sangon), 0.01mol/LPBS (Zhong Shan Golden Bridge), MTT (Ameresco company), dimethyl sulfoxide (DMSO, Sangon), Hoechst33342 (Sigma), DAPI (Sigma), lowlenthal serum (Zhong Shan Golden Bridge), little mouse-anti BrdU monoclonal antibody (Sigma), Cy3 labelling goat anti-mouse igg (JacksonImmunoResearch), calcein-AM/EthD-IIILive/Deadkit (Biotium), the anti-NT-3 polyclonal antibody (SantaCruz) of rabbit, mouse-anti-human T rkC monoclonal antibody (RD), rabbit Chinese People's Anti-Japanese Military and Political College Mus NF monoclonal antibody (Sigma), little anti-rat NF monoclonal antibody (Sigma), rabbit Chinese People's Anti-Japanese Military and Political College Mus GFAP polyclonal antibody (Sigma), little anti-rat MBP monoclonal antibody (Millipore), goat anti-mouse FITC (JacksonImmunologicalResearch), the goat anti-mouse igg (JacksonImmunoResearch) of Cy3 labelling, Cy3 labelling goat anti-rabbit igg (JacksonImmunoResearch), the goat anti-mouse igg (JacksonImmunoResearch) of Dylight405 labelling, AMCA labelling goat anti-rabbit igg (JacksonImmunoResearch), goat antirabbit HRP (JacksonImmunoResearch), protein quantification detection kit (ancient cooking vessel state), cell pyrolysis liquid (Boster), protease inhibitor cocktail (Sigma), ECL luminous substrate detection kit (health is century), Epon-812 (TedPella), Coomassie brilliant blue (Bio-rad), 30% polyacrylamide solution (health is century), X photographic film (Kodak).
The concrete operations technology that this utility model is detailed is described as follows:
1. the structure of chemotactic activity support
The preparation of 1.1 rack shells
Shell thin-walled is around cylindrical PLGA (polyD, L-lactic-co-glycolicacid, PLGA), it is made up of degradable high molecular synthetic material PLGA (polylactic acid and polyglycolic acid ratio are 50: 50, and molecular weight is 100000).Getting a certain amount of PLGA is dissolved in dichloromethane, be made into 5% solution, after PLGA dissolves completely, in the polytetrafluoro mould of cast and level-off, room temperature (control temperature is at 20 DEG C), volatilizees 24 hours, within the 2nd day, carefully takes thin film off, to be inverted in mould 24 hours, to cut out kept dry after suitable size.Thin film is surrounded a circle on rustless steel cylinder grinding tool that diameter is 3mm, edge acetone is pasted, and forms the cylindric PLGA shell that diameter is 3mm.During use, PLGA shell is cut into 2mm length, alcohol-pickled 15 minutes, embathes 3 times with aseptic D-Hank's balance liquid subsequently, each 10 minutes.
The structure of 1.2 three-dimensional materials
Sterile gelatin sponge, purchased from Nanjing Pharma Inc., is cut into diameter 3mm in superclean bench, thickness 2mm size (about 1.0mg), and its pattern is cylinder, pore diameter wherein about 200 ~ 600 μm.Aseptic kept dry is stand-by.
The preparation of 1.3 chemotactic activity differential bulbecs
By 20gNa
2cO
3be dissolved in 4 premium on currency, be heated to 100 DEG C, put into 75g silkworm Bombyx bombycis (being given by Silk Worm Inst Zhejiang Prov. Agriculture Science Academy), keep that solution is micro-boils, and constantly stir.After 1 hour, remove solution.Repeat said process again 1 time.By the Bombyx bombycis natural cooling boiled, clean with deionized water rinsing, to be placed in baking oven 24 hours, 50 DEG C dry after for subsequent use.Get CaCl
244.40g, ethanol 46.00ml, deionized water 57.60ml makes mixed solution, puts into the Bombyx bombycis 15.00g of oven dry in this solution.After making the abundant submergence Bombyx bombycis of solution, 80 DEG C of heating in water bath, stirring and dissolving.After 1 hour, Bombyx bombycis is all dissolved as silk protein solution.Stop heating and stir, natural cooling silk protein solution is to room temperature.The salt ion in silk protein solution is removed with bag filter (purchased from Guangzhou Qi Yun Bioisystech Co., Ltd) dialysis.Within first 2 days, soak the bag filter containing silk protein solution with tap water, within latter 1 day, use deionized water instead and soak, dialyse 3 days altogether.Between dialysis period, changed 1 tap water or deionized water every 3 hours.Silk protein solution after dialysis is poured in graduated cylinder, leaves standstill 4 hours, the solid impurity in removing solution.The silk protein solution after leaving standstill is collected by conical flask.By 0.5 μ g recombined human NT-3 and 10
-6mmol/LRA mixes with 3% fibroin, prepares mixed liquor.
The assembling of 1.4 supports
The natural gelatin sponge absorption chemotactic activity differential bulbec of having reduced, to saturated, puts into lyophilizing bottle ,-80 DEG C of stable crystalline forms.In freezer dryer, freezing evacuation 12 hours, to remove the gas in moisture and material.After balance, 70% ethanol cross-linked material, D-Hanks washes 3 times, and material is put into PLGA sleeve pipe, is prepared into the bioactive bracket of 2mm.
2. the performance of vitro detection support
2.1 swelling ratio
In certain hour, the change in volume of support in PBS solution is expressed as its swelling ratio, and its swelling ratio amplitude of variation of stable biomaterial is less.Detection method is as follows: by each for support Duplicate Samples 5, surveys its diameter R respectively
0with length h
0(being accurate to 0.01mm); Steeped in PBS solution by material material Seepage, test sample is at the change in size R of 1h, 2h, 6h, 12h and 24h
tand h
t.
2.2 water absorption rate
Support is placed on the change of quality in PBS solution, W
0represent blank quality, W
tbiao Shi Seepage steeps the quality after the PBS solution t time.
3. the usefulness of Bracket for Inspection in body
3.1 are transplanted to the usefulness in rat body
The complete cross-section model preparation of rat spinal cord: select adult female rats, body weight is about 220g, only often organize each 3-5 of rat, in 3 groups of rat abdominal cavities, inject pentobarbital sodium (30mg/kg) anaesthetize, cut skin in sterile conditions, separating muscle, excision T9 and T10 spinal lamina, and the complete cross-section spinal cord of T9 vertebral plate meta (being positioned at T9 and T10 segments of spinal cord boundary herein), and excise 2mm myeloid tissue block thereafter, remove the nerve fiber that damage intracavity is residual.Be implanted into NT-3/ fibroin albumen gelfoam support and silk fibroin bracket respectively at spinal cord transection place, gelfoam size is 2 × 2 × 2mm
3.After abundant hemostasis, layer-by-layer suture muscle and skin.Postoperative every animal intraperitoneal injection penicillin 50,000 units/d, injects 3d continuously, gives if desired and fluid infusion.Every day manually urinates, routinely raising rat.
Transplant after there is the rat feeding 30d of the bioactive bracket of Chemotaxis Function, use 4% paraformaldehyde to fix.Every rat is got the spinal cord that before and after damage/graft area, 1cm is long altogether and carries out longitudinal section, cuts into slices to get 1 every 5.Detect the situation of the bioactive bracket internal migration cell transplanted.
3.2 are transplanted to the usefulness in dog body
The cross-section model preparation of dog spinal cord half: select beasle dog childhood, often organize each 5, intraperitoneal injection pentobarbital sodium (30mg/kg) is anaesthetized, cut skin in sterile conditions, separating muscle, excision T9 and T10 spinal lamina, and the cross-section spinal cord of T9 vertebral plate meta half (being positioned at T9 and T10 segments of spinal cord boundary herein), and excise right half side 2mm myeloid tissue block, remove the nerve fiber that damage intracavity is residual.The bioactive bracket with Chemotaxis Function is implanted at spinal cord transection place.After abundant hemostasis, layer-by-layer suture muscle and skin.Postoperative every animal intraperitoneal injection penicillin 50,000 units/d, injects 3d continuously, gives if desired and fluid infusion.Every day manually urinates.
Transplant after there is the dog breeders 30d of the bioactive bracket of Chemotaxis Function, use 4% paraformaldehyde to fix.Every dog is got the spinal cord that before and after damage/graft area, 1cm is long altogether and carries out longitudinal section, cuts into slices to get 1 every 5.Detect the situation of the bioactive bracket internal migration cell transplanted.
Claims (2)
1., for repairing a bioactive bracket with Chemotaxis Function for spinal cord injury, it is characterized in that the three-dimensional isinglass sponge being filled with absorption described chemotactic factor, inducible factor and fibroin in PLGA conduit;
2. the bioactive bracket with Chemotaxis Function for repairing spinal cord injury according to claim 1, it is characterized in that being transplanted in spinal cord injury animal body, those expression can be attracted to move in support with the endogenous cell of chemotactic factor associated receptor and survive and break up.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104800885A (en) * | 2015-05-13 | 2015-07-29 | 中山大学 | Preparation method and application of bioactive bracket with chemotactic function |
| CN115671028A (en) * | 2022-11-11 | 2023-02-03 | 西北工业大学 | PLGA-based local ophthalmic sustained-release composite preparation and preparation method and application thereof |
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2015
- 2015-05-13 CN CN201520319632.4U patent/CN204840394U/en active Active
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104800885A (en) * | 2015-05-13 | 2015-07-29 | 中山大学 | Preparation method and application of bioactive bracket with chemotactic function |
| CN115671028A (en) * | 2022-11-11 | 2023-02-03 | 西北工业大学 | PLGA-based local ophthalmic sustained-release composite preparation and preparation method and application thereof |
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