CN102716470B - Medicine composite for treating peripheral nerve injury - Google Patents
Medicine composite for treating peripheral nerve injury Download PDFInfo
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- CN102716470B CN102716470B CN201210218575.1A CN201210218575A CN102716470B CN 102716470 B CN102716470 B CN 102716470B CN 201210218575 A CN201210218575 A CN 201210218575A CN 102716470 B CN102716470 B CN 102716470B
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention relates to application of insulin-like growth factor-1 on preparation of medicaments for treating peripheral nerve injury and a medicine composite for treating the peripheral nerve injury. An active ingredient of the medicine composite is the insulin-like growth factor-1. The medicine composite has functions of promoting proliferation and survival of schwann cells, restraining cell death, participating in neuron protection, cynapse rebuilding after injury, restraining denervation sweeny and the like, can directly and effectively improve serious peripheral nerve injury, and is accurate in pesticide effect and high in safety.
Description
Technical field
The present invention relates to the novelty teabag of IGF-1 (IGF-1), be specifically related to IGF-1 for the preparation of the application in treatment peripheral nerve injury medicine, the present invention still further provides a kind of pharmaceutical composition being used for the treatment of peripheral nerve injury.
Background technology
The renewable reservoir of peripheral nerve injury is the problem that numerous No systematic and clinical medicine worker pay close attention to always.Peripheral nerve comprises 31 pairs of spinal nerves and 12 pairs of cranial nerve, and peripheral nerve injury is multiple diseases, and vehicle accident, natural disaster even all perineural damage may occur in the treatment clinical course of other diseases.Show as the peripheral nerve generation pathological change that various factors causes, and the function generation obstacle such as skin, muscle, motion, blood vessel of its domination.Peripheral nerve injury incidence rate is high, repairs difficulty, disables serious, is one of serious challenge of facing of current clinical treatment.Although existing a large amount of basis and clinical research report at present, and achieve some gratifying progress, but the Regeneration and Repair after peripheral nerve injury is a complicated pathophysiological process, the key affecting this repair process how to promote that injured nerve grows fast, and set up contact before distal nerve degeneration.
Perineural ultimate unit is neuron axon, myelin and effect target organ.After peripheral nerve injury, show as the change of pericaryon, nerve fiber degeneration and microenvironment and change.In the transmission of damage signal, relate to damage signal conduct fast, and have the structure component of associated transcription factor, adhesion molecule, growth associated protein and axon regeneration to react.The peripheral nerve of Adult Mammals shows vigorous regeneration capacity, and axonal injury, no matter whether its cell space is at maincenter, all cannot regenerate under normal circumstances.From neurodevelopment, neural axon extends along specific path, arrives and sets up the cell space of the target cell of synaptic contact, dendron or aixs cylinder by with it.A sector structure is there is at nervous process end---growth cone (axonal growth cone), the targeting signal optionally identified by physical ability in born of the same parents' external environment on its surface, cause self stretching, extension and retraction reaction through a series of signal Transduction Mechanism, thus instruct axon growth.When after peripheral nerve injury, there is pericaryon swelling in nerve fiber and connective tissue (being divided into endoneurium, perineurium and epineurium from outside to inside) successively, and Nissl body disappears, and nucleus offsets, and synaptic terminal reduces; There is magnificent Le Shi (Waller) degeneration and disintegrate, regulation of Schwann cell proliferation in far-end Axon and myelin sheath, cell space aixs cylinder produces growth cone, starts the regenerative process under multi factor control.
The treatment of current peripheral nerve injury remains one of clinical problem, and difficulty is that the functional rehabilitation of injured nerve can not be satisfactory more, and its treatment means mainly contains operative treatment, Drug therapy and naturopathy.Drug therapy is the important measures of peripheral nerve injury repairing and treating, and for the peripheral nerve injury do not had after Operation indication or operative treatment, Drug therapy is essential.Current main employing neurotrophy medicine, comprise vitamin(e) B group, vitamin B1, vitamin B6, bendazol, vitamin B12, methycobal (mecobalamin) etc., this kind of medicament categories is a lot, Clinical practice is extensive, but alone curative effect is not good enough and unstable, often must drug combination or share other drug; The exogenous neurotrophic factor such as nerve growth factor half-life is short, and in aqueous solution, activity is easily affected by other factors, and it can not through blood brain barrier and blood-nerve barrier.Therefore, clinical practice is mainly limited to nerve injury topical application, and effect is difficult to long term maintenance.Therefore, how play the effect of NGF around in nerve injury treatment further, also require further study; The basic FGF (bFGF) that current clinical fibroblast growth factor (FGF) is gene recombinaton and Brain Derived Neurotrophic Factor (BDNF), it is obvious to the effect of animal acute spinal cord injury, but has not yet to see report to peripheral nerve injury.For ganglioside, its injection has been applied to clinical at present, commodity are called Cronassial (Cronassial), the peripheral neuropathy such as postoperative and polyneuritis for various nerve injury, neural anastomosis, but the molecular mechanism of its effect is still not quite clear, and at present clinical application cases is few, actual efficacy is still needed large sample case treatment checking.In addition other are also had as hormone medicine, interleukin, the Chinese herbal medicine such as injection such as Radix Angelicae Sinensis, Flos Carthami etc., the medicine methyl meticortelone etc. suppressing local scar to be formed is conducive to neuranagenesis, the drug uses such as the perfusate of proteinase inhibitor, Ca2+ antagonist, simulation aixs cylinder composition are due to the restriction by the different times of nerve injury, inflammation, blood supply and untoward reaction etc., although topical application also can promote peripheral nerve regeneration, but practical clinical is few, and curative effect needs to study confirmation further.Therefore find the factor that a kind of broad spectrum activity urgees nerve growth to be necessary very much.
Insulin-like growth factor-i (insulin-like growth factor-1, IGF-1) belongs to insulin like growth factor family member, is a kind of cytokine relevant to cell differentiation, propagation tissue metabolism.It derives from many cells in body, and neural neuron and neurogliocyte can produce IGFs, and macrophage, fibroblast also can produce IGFs in addition, are played a role by autocrine, paracrine and endocrine mechanism.Research in recent years finds, IGF-1 is the multi-functional neurotrophic factor of a class, has and promotes Schwann Cell Increase and survival, apoptosis inhibit; Axon regeneration and myelinization after promotion nerve injury, participate in neuronic protection and hinder post-synapse rebuilding and suppressing the effects such as denervated muscle atrophy.Prompting IGF-1 may be a kind of effective wide spectrum class repairing of neural injury pharmaceutical preparation, but relevant its promotes that repairing of neural injury research there is not yet report both at home and abroad.
Summary of the invention
The object of the present invention is to provide the application of IGF-1 in the medicine for the preparation for the treatment of peripheral nerve injury, in this medicine, the using dosage of IGF-1 is 10ng/kg-1000ng/kg, and preferably, using dosage is 100ng/kg.
The present invention also provides a kind of pharmaceutical composition being used for the treatment of peripheral nerve injury, and active component is IGF-1.
Preferably, this pharmaceutical composition also comprises chitosan further.
Preferably, in this pharmaceutical composition, the using dosage of IGF-1 is 10ng/g-1000ng/kg.
More preferably, in described pharmaceutical composition, the using dosage of IGF-1 is 100ng/g.
Beneficial effect of the present invention: the invention provides the new opplication of IGF-1 in the medicine for the preparation for the treatment of peripheral nerve injury, and further provide a kind of pharmaceutical composition being used for the treatment of peripheral nerve injury, it comprises IGF-1 and chitosan, this pharmaceutical composition has promotion Schwann Cell Increase and survival, suppresses it dead; Axon regeneration and myelinization after promotion nerve injury, participate in neuro-protective and hinder post-synapse rebuilding and suppressing the effects such as denervated muscle atrophy, directly effectively can improve serious peripheral nerve injury, clearly, safety is high for drug effect.
Accompanying drawing explanation
Figure 1A and Figure 1B is plane and the three-dimensional structure diagram of IGF-1 respectively.
Fig. 2 is nerve injury and sews up schematic diagram.
Fig. 3 A to Fig. 3 I is IGF-1 various dose treatment group gross specimen figure after peripheral nerve injury.Fig. 3 A-C uses silica gel tube, and treat the situation after 4 weeks, Fig. 3 A: Calf muscle atrophy, toes are stiff; Fig. 3 B: neural from disconnected; Fig. 3 C: without the tissue blood vessel of regeneration around silica gel tube; Fig. 3 D-F uses silica gel tube+chitosan to treat the situation after 4 weeks; Fig. 3 D: Calf muscle is atrophy a little, toes can micro-flexing; Fig. 3 E: neural disconnected section has filament sample regenerating tissues; Fig. 3 F: have blood vessel sample tissue encapsulation around silica gel tube; Fig. 3 G-I uses silica gel tube+chitosan+IGF-1 (100ng/kg) to treat the situation after 4 weeks; Fig. 3 G: Calf muscle atrophy is not obvious, toes can stretch simultaneously, flexing; Fig. 3 H: the neural broken ends of fractured bone has macroscopic toughness fiber tissue regeneration; Fig. 3 I: silica gel tube surrounding tissue parcel is more regular, and broken ends of fractured bone regenerating tissues is similar to basic stitch.
Fig. 4 A to Fig. 4 E is that after peripheral nerve injury, various dose IGF-1 treats histological observation (H.E dyeing) after 4 weeks continuously.Fig. 4 A is that matched group is neural; Fig. 4 B is the situation using chitosan to treat 4 weeks; Fig. 4 C is the situation using chitosan+IGF-1 (10ng/kg) to treat 4 weeks; Fig. 4 D is the situation using chitosan+IGF-1 (100ng/kg) to treat 4 weeks; Fig. 4 E is the situation using chitosan+IGF-1 (1000ng/kg) to treat 4 weeks.
Fig. 5 A to Fig. 5 E is that after peripheral nerve injury, various dose IGF-1 treats histological observation after 4 weeks (Holmes silver dye) continuously.Fig. 5 A is that matched group is neural; Fig. 5 B is the situation using chitosan to treat 4 weeks; Fig. 5 C is the situation using chitosan+IGF-1 (10ng/kg) to treat 4 weeks; Fig. 5 D is the situation using chitosan+IGF-1 (100ng/kg) to treat 4 weeks; Fig. 5 E is the situation using chitosan+IGF-1 (1000ng/kg) to treat 4 weeks.
Fig. 6 A to Fig. 6 D is gastrocnemius motor end plate dyeing in 4 weeks after different group peripheral nerve injury.Fig. 6 A is that matched group is neural; Fig. 6 B is the situation using chitosan to treat 4 weeks; Fig. 6 C is the situation using chitosan+IGF-1 (10ng/kg) to treat 4 weeks; Fig. 6 D is the situation using chitosan+IGF-1 (100ng/kg) to treat 4 weeks; Fig. 6 E is the situation using chitosan+IGF-1 (1000ng/kg) to treat 4 weeks.
Fig. 7 is control rats sensory cortex Evoked ptential.
Fig. 8 is that IGF-1 (10ng) treats 4 weeks sensory cortex Evoked ptential.
Fig. 9 is that IGF-1 (100ng) treats 4 weeks sensory cortex Evoked ptential.
Figure 10 is matched group motor cortex Evoked ptential.
Figure 11 is that IGF-1 (10ng) treats 4 weeks motor cortex Evoked ptential.
Figure 12 is that IGF-1 (100ng) treats 4 weeks motor cortex Evoked ptential.
Figure 13 A to Figure 13 C is that endotheliocyte is differentiated.Figure 13 A is DAPI labelling endotheliocyte core; Figure 13 B is VIII factor marker endotheliocyte; Figure 13 C is DAPI, VIII agents labelling.
Figure 14 is the impact (1 × 100) of IGF-1 on cell injury.Experimental group culture medium is containing 40ng/ml IGF-l, and matched group adds conventional medium.A1, B1: the instant situation after damage; A2, B2: the situation of 24h after damage, during 24h, experimental group endotheliocyte is own starts to the growth of intermediate score place, and matched group is without obvious change; C1, C2: the situation of 48h after damage, after 48h, the quantity of experimental group cell migration is obviously many than matched group cell migration number, the gap width of experimental group cut also comparatively matched group obviously narrow, have oneself dead cells float many in matched group in culture medium.
Figure 15 is the impact of IGF-1 on cell death.DAPI nucleus blue dyeing shown in arrow b, d, the red nuclear staining of dead cell that to be PI shown in arrow a, c be.
Detailed description of the invention
In order to make the object of the invention, technical scheme and advantage clearly understand, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Experiment material:
Animal: SD rat, male, body weight 210 ± 26g, Third Military Medical University of Chongqing City the 3rd Affiliated Hospital's Experimental Animal Center provides, regular grade animal, licence: SCXK (yu) 2007017.
Reagent reagent:
IGF-1 article No.: MSDS I8779, Sigma Pharmaceuticals Ltd of the U.S. produces;
Medical chitose, lot number 070674-01, Qisheng Biopreparations Co., Ltd., Shanghai produces;
Medical silicone tube, long 15mm, its external diameter 2.0mm, internal diameter 1.5mm, Suzhou rubber plant
Pentobarbital sodium, Denmark's import subpackage;
Paraformaldehyde analytical pure, lot number 20100501, Chengdu Ke Long chemical reagent factory produces;
Dehydrated alcohol, lot number 20100509, Chongqing Mao Ye chemical reagent company limited produces;
Sodium dihydrogen phosphate, lot number 20091105, Chongqing Chuan Dong Chemical Group company limited produces;
Sodium hydrogen phosphate, lot number 90519, Chongqing game chemical reagent company limited produces;
Sucrose analytical pure, lot number 20081113, Chengdu Ke Long chemical reagent factory produces;
Dimethylbenzene, lot number 20090911, Chongqing Chuan Dong Chemical Group company limited chemical reagent factory is produced;
Sodium acetate lot number 20070920 Chongqing Chuan Dong Chemical Group company limited chemical reagent factory is produced;
Acetic acid lot number 20080310 Chongqing Chuan Dong Chemical Group company limited chemical reagent factory is produced;
Sodium citrate lot number 20090910 Chongqing Chuan Dong Chemical Group company limited chemical reagent factory is produced;
Copper sulfate lot number 2010718 Chongqing Chuan Dong Chemical Group company limited chemical reagent factory is produced;
Potassium ferricyanide lot number 20090908 Chengdu Ke Long chemical reagent factory produces;
Acetylcholine article No.: Sigma Pharmaceuticals Ltd of the 60-31-1 U.S. produces
Human umbilical vein endothelial (being provided by Medical University Of Chongqing's contagious department)
Dimethyl sulfoxide article No.: SH30021.01 (Hyclone company of the U.S.)
DMEM/F12 culture medium article No.: SH30023.01B (Hyclone company of the U.S.)
10% hyclone article No.: SH30070.01 (Hyclone company of the U.S.)
0.25% trypsin article No.: SH30042.01B (Hyclone company of the U.S.)
Tetramethyl azo azoles salt article No.: 708569 (Sigma Co., USA)
0.3%Tritonx-100 article No.: 9002-93-1 (Sigma company)
4', 6-diamidino-2-phenylindone article No.: 11032 (DAPl) antibody (Sigma company)
Propidium iodide (Pl) article No.: P4170 (sigma company)
Rabbit resists VIII factor antibody article No.: F3520 (Sigma company)
Goat anti-rabbit igg-FITC article No.: ZPR5211 (Beijing Zhong Shan company)
Normal closed lowlenthal serum article No.: C-0005 (Beijing Zhong Shan company)
VEGF-ELISA test kit article No.: EK0540 (Wuhan doctor's moral company)
Experimental apparatus:
Physiology leads instrument (Australian ADI company) more, freezing microtome (CM1900 U.S. Leica), laser confocal scanning system (TCSSP2, Germany Leica), block sections machine (SM2000R, German Leica), desk-top refrigerated centrifuge (U.S. Sigma-16k), Ix-50 inverted phase contrast microscope (Olympus company), MD type microplate reader (Thermo company), 80-3 centrifuge (Shanghai Surgical Operation Equipment Factory)
Embodiment 1: the preparation of IGF-1
IGF-1, is also referred to as " somatomedin " (i.e. somatomedin C), be a kind of on molecular structure with polypeptide protein material like insulin type.IGF-1 of the present invention is a kind of activated protein peptide material, and being one has 70 amino acid whose strand basic proteins, is formed, molecular weight 7649Da by 3 cystine linkage interconnections, heat-resisting; The 26S Proteasome Structure and Function about 50% former to human insulin is similar.Described IGF-1 is that Sigma biotech firm of the U.S. buys (article No.: MSDS I8779), also gene recombination method can be adopted to synthesize, specifically see: (oriole a species of orchid, Zhu abundant ancient cooking vessel CN98106111.7 insulin-like growth factor-i engineering bacteria builds and prepares the method for IGF-1; Zhu CN200410053399.6 becomes steel, a kind of loyal method extracting insulin like growth factor (IGF-1) from fresh Cornu Cervi Pantotrichum of Wang Li; The graceful CN200810084902.2 of Zhang Xinyu, Liu Yue, Liu Yang, Yuan Jing mono-kind extracts the method for insulin like growth factor (IGF-1) from Cornu Cervi Pantotrichum) to obtain the required finished product of experiment.Plane and the three-dimensional structure diagram of IGF-1 please refer to Figure 1A and Figure 1B.
Embodiment 2: the preparation of pharmaceutical composition
Medical chitose gel (Qisheng Biopreparations Co., Ltd., Shanghai) adds IGF-1 group: after injecting medical chitose gel 10 μ l in pipe, again with microsyringe respectively by 5 μ l IGF-1 (2ng/ μ l, 20ng/ μ, 200ng/ μ l Sigma Co., USA) inject in sterilization medical silicone tube.Except adopting said method can ensure effective ingredient lastingly and slowly local action, conventional syringe local injection, semar technique etc. also can be adopted to act on injured nerve local, and distinct methods all has the drug effect improving peripheral nerve injury.
Beneficial effect of the present invention is proved below by way of concrete pharmacodynamic experiment.
Embodiment 3: IGF-1 is to the observation of the general state of peripheral nerve injury in rats
SD rat 210, male and female are regardless of, body weight 210 ± 26g, adds that silica gel tube adds chitosan group 20, chitosan adds IGF-1 (10ng/kg) 40, chitosan adds IGF-1 (100ng/kg) 40, chitosan adds IGF-1 (1000ng/kg) 40 after experiment is grouped into sham operated rats (NE) 20, neural cutting; Often organize and be divided into: 1,2,3, phase point when 4 weeks.
Method: 1% pentobarbital sodium presses 30mg/kg intraperitoneal injection anesthesia.Lateral position, after shaving hair, sterilization, get angular cut after stock, expose right sciatic nerves, free sciatic nerve under 10 times of operating microscopes, and at 5mm place, piriformis outlet below, sciatic nerve denervation is about 8mm, let alone retraction, cause 10mm defect, by the silica gel tube bridge joint defect section disinfected, sew up with nerve is near, the far away broken ends of fractured bone of 9-0 not damaged micro suture line and bridge pipe and fix 2 pins.Sewing method adopts two fixed point cover connections: namely respectively sew up 1 pin at nerve trunk all 180 ° two, footpaths relative position respectively, the regenerating nerve broken ends of fractured bone is inserted in bridge pipe, and is about 2.5mm(Fig. 2 apart from pipe).Add that silica gel tube adds chitosan group, chitosan adds IGF-1 (10ng/kg), chitosan adds IGF-1 (100ng/kg), chitosan adds IGF-1 (1000ng/kg) after dividing 5 groups to be respectively sham operated rats (NE), neural cutting at random animal according to the medicine difference added in pipe; Often organize and be divided into: 1,2,3, phase point when 4 weeks, only often organizing 5-10.It is inject medical chitose gel 20 μ l (Qisheng Biopreparations Co., Ltd., Shanghai) in silica gel tube respectively that chitosan adds silica gel tube group; Chitosan adds IGF-1 group: after injecting medical chitose gel 10 μ l in pipe, again with microsyringe respectively by 5 μ l IGF-1 (2ng/ μ 1,20ng/ μ, 200ng/ μ 1 Sigma Co., USA) in ascending pipe, with 9-0 silk thread, bridge pipe stitching is fixed on contiguous soft tissue, layer-by-layer suture sarolemma and skin incision, sub-cage rearing under unified standard condition.
Result: when clinical follow finds 3 weeks bridge pipe without breach, distortion, thinning, variable color, be shifted and subside, it is surperficial holds for membranaceous tissue.In each group of pipe, medical chitose gel is degradable, and in pipe, regeneration sciatic nerve near-end be that the time silica gel tube that regenerates of bud shape adds chitosan group and occurred regrowth in 4 weeks, and has membranaceous tissue to hold; 12 weeks after operation occurs that disconnected section is coincide regrowth; Chitosan added IGF-1 (10ng/kg) and occurred thread new capillary vessel 1 week time, and increased gradually 2 weeks time, and when 3 weeks, two broken ends of fractured bone bud shape regenerating nerves extend forward; The regeneration pencil thing fitted like a glove is had as seen when 4 weeks; Chitosan starts to occur the membranaceous tissue of comparatively dense when adding IGF-1 (100ng/kg) 1 week, have the regenerating tissues coincide as pencil when 2 weeks as seen, and when 4 weeks, visible regrowth two ends are thick, the thin diameter 1/2 as silica gel tube in centre; Chitosan added IGF-1 (1000ng/kg) 4 weeks time visible 2/5 has regrowth to coincide, and 3/5 does not coincide, and in filament shape.
Conclusion: from ordinary circumstance, IGF-1 (100ng/kg) treatment group significantly can promote peripheral nerve regeneration in 4 weeks.
Embodiment 4: the pathological change that IGF-1 is repaired peripheral nerve injury
Adopt H.E and silver to catch an illness and manage colouring method detection.SD rat, male and female are regardless of, body weight 210 ± 26g; Experiment is grouped into: add after sham operated rats (NE) 20, neural cutting that silica gel tube adds chitosan group 20, chitosan adds IGF-1 (10ng/kg) 40, chitosan adds IGF-1 (100ng/kg) 40, chitosan adds IGF-1 (1000ng/kg) 40; Often organize and be divided into: 1,2,3, phase point when 4 weeks.
Method: 1% pentobarbital sodium presses 30mg/kg intraperitoneal injection anesthesia.Lateral position, after shaving hair, sterilization, get angular cut after stock, expose right sciatic nerves, free sciatic nerve under 10 times of operating microscopes, and at 5mm place, piriformis outlet below, sciatic nerve denervation is about 8mm, let alone retraction, cause 10mm defect, by the silica gel tube bridge joint defect section disinfected, sew up with nerve is near, the far away broken ends of fractured bone of 9-0 not damaged micro suture line and bridge pipe and fix 2 pins.Sewing method adopts two fixed point cover connections: namely respectively sew up 1 pin at nerve trunk all 180 ° two, footpaths relative position respectively, the regenerating nerve broken ends of fractured bone is inserted in bridge pipe, and is about 2.5mm(as Fig. 2 apart from pipe).Add that silica gel tube adds chitosan group, chitosan adds IGF-1 (10ng/kg), chitosan adds IGF-1 (100ng/kg), chitosan adds IGF-1 (1000ng/kg) after dividing 5 groups to be respectively sham operated rats (NE), neural cutting at random animal according to the medicine difference added in pipe; Often organize and be divided into: 1,2,3, phase point when 4 weeks, only often organizing 5-10.It is inject medical chitose gel 20 μ l (Qisheng Biopreparations Co., Ltd., Shanghai) in silica gel tube respectively that chitosan adds silica gel tube group; Chitosan adds IGF-1 group: after injecting medical chitose gel 10 μ l in pipe, again with microsyringe respectively by 5 μ l IGF-1 (2ng/ μ l, 20ng/ μ l, 200ng/ μ l Sigma Co., USA) in ascending pipe, with 9-0 silk thread, bridge pipe stitching is fixed on contiguous soft tissue, layer-by-layer suture sarolemma and skin incision, sub-cage rearing under unified standard condition.Specifically please refer to Fig. 3 A to Fig. 3 I, it is IGF-1 various dose treatment group gross specimen figure after peripheral nerve injury.Fig. 3 A to Fig. 3 I is applying silicon sebific duct after peripheral nerve injury, chitosan, IGF-1 various dose treatment group gross specimen figure.Fig. 3 A-C uses silica gel tube, and treat the situation after 4 weeks, Fig. 3 A: Calf muscle atrophy, toes are stiff; Fig. 3 B: neural from disconnected; Fig. 3 C: without the tissue blood vessel of regeneration around silica gel tube; Fig. 3 D-F uses silica gel tube+chitosan to treat the situation after 4 weeks; Fig. 3 D: Calf muscle is atrophy a little, toes can micro-flexing; Fig. 3 E: neural disconnected section has filament sample regenerating tissues; Fig. 3 F: have blood vessel sample tissue encapsulation around silica gel tube; Fig. 3 G-I uses silica gel tube+chitosan+IGF-1 (100ng/kg) to treat the situation after 4 weeks; Fig. 3 G: Calf muscle atrophy is not obvious, toes can stretch simultaneously, flexing; Fig. 3 H: the neural broken ends of fractured bone has macroscopic toughness fiber tissue regeneration; Fig. 3 I: silica gel tube surrounding tissue parcel is more regular, and broken ends of fractured bone regenerating tissues is similar to basic stitch.Above-mentioned each treated animal is after the test through 1% pentobarbital sodium (40mg/kg) intraperitoneal injection of anesthesia, expose heart, through left ventricular cannulation to ascending aorta, quick filling 0.01M PBS 200ml, pour into 4% paraformaldehyde-PB liquid 200ml (0.1mol/L subsequently, pH:7.4) internal fixtion is carried out, then brain specimen is taken out, the paraformaldehyde putting into 4% liquid-solidly determines 24h, spend the night after abundant washing, row dehydration, transparent, paraffin embedding, often open the thick 5um ~ 10um of brain sheet, row HE dyes, transparent, sealing.Row Holmes silver dye, evaluates the quality of regenerated nervous fibers simultaneously.
Result: H.E dyes and observes finding, and compared with matched group, chitosan group 4 weeks group regenerates " nerve " and is only and dyes more cell spline structure, nerve fiber shape structure, but fascicular texture space is many; The visible a small amount of regenerated fiber of IGF-1 low dose group regenerating nerve, in IGF-1, hemocyte and fibrocyte can interweave by dosage group, fiber fasciculation arrangement and intensive; Chitosan high dose group regenerated nervous fibers has fracture end, coincide bad, and arrangement is comparatively mixed and disorderly, specifically please refer to Fig. 4 A to Fig. 4 E, and Fig. 4 A is that matched group is neural; Fig. 4 B is the situation using chitosan to treat 4 weeks; Fig. 4 C is the situation using chitosan+IGF-1 (10ng/kg) to treat 4 weeks; Fig. 4 D is the situation using chitosan+IGF-1 (100ng/kg) to treat 4 weeks; Fig. 4 E is the situation using chitosan+IGF-1 (1000ng/kg) to treat 4 weeks.
Holmes silver dye result: regenerating nerve form and matched group in postoperative silica gel tube, chitosan organizes regenerating nerve pluckeds in 4 weeks and nerve has the end that do not coincide, but arrangement is more neat; IGF-1 low dose group regenerated nervous fibers bundle is thicker, but arranges coarse; In IGF-1, the arrangement of dosage group is comparatively neat, even thickness, and has neural waviness curve, and the arrangement of IGF-1 high dose group is in corrugated, but coarse texture, plucked, specifically please refer to Fig. 5 A to Fig. 5 E, and Fig. 5 A is that matched group is neural; Fig. 5 B is the situation using chitosan to treat 4 weeks; Fig. 5 C is the situation using chitosan+IGF-1 (10ng/kg) to treat 4 weeks; Fig. 5 D is the situation using chitosan+IGF-1 (100ng/kg) to treat 4 weeks; Fig. 5 E is the situation using chitosan+IGF-1 (1000ng/kg) to treat 4 weeks.
Conclusion: IGF-1 can effectively reduce perineural cell death, and the neurocyte number of regeneration increases gradually, and fiber alignment is neat.
Embodiment 5: IGF-1 is on the impact of the motor end plate arranged after peripheral nerve injury
SD rat, male and female are regardless of, body weight 210 ± 26g; Experiment is grouped into: add after sham operated rats (NE) 20, neural cutting that silica gel tube adds chitosan group 20, chitosan adds IGF-1 (10ng/kg) 40, chitosan adds IGF-1 (100ng/kg) 40, chitosan adds IGF-1 (1000ng/kg) 40; Often organize and be divided into: 1,2,3, phase point when 4 weeks.
Method: 1% pentobarbital sodium presses 30mg/kg intraperitoneal injection anesthesia.Lateral position, after shaving hair, sterilization, get angular cut after stock, expose right sciatic nerves, free sciatic nerve under 10 times of operating microscopes, and at 5mm place, piriformis outlet below, sciatic nerve denervation is about 8mm, let alone retraction, cause 10mm defect, by the silica gel tube bridge joint defect section disinfected, sew up with nerve is near, the far away broken ends of fractured bone of 9-0 not damaged micro suture line and bridge pipe and fix 2 pins.Sewing method adopts two fixed point cover connections: namely respectively sew up 1 pin at nerve trunk all 180 ° two, footpaths relative position respectively, the regenerating nerve broken ends of fractured bone is inserted in bridge pipe, and is about 2.5mm(Fig. 2 apart from pipe).Add that silica gel tube adds chitosan group, chitosan adds IGF-1 (10ng/kg), chitosan adds IGF-1 (100ng/kg), chitosan adds IGF-1 (1000ng/kg) after dividing 5 groups to be respectively sham operated rats (NE), neural cutting at random animal according to the medicine difference added in pipe; Often organize and be divided into: 1,2,3, phase point when 4 weeks, only often organizing 5-10.It is inject medical chitose gel 20 μ l (Qisheng Biopreparations Co., Ltd., Shanghai) in silica gel tube respectively that chitosan adds silica gel tube group; Chitosan adds IGF-1 group: after injecting medical chitose gel 10 μ l in pipe, again with microsyringe respectively by 5 μ l IGF-1 (2ng/ μ l, 20ng/ μ, 200ng/ μ l Sigma Co., USA) in ascending pipe, with 9-0 silk thread, bridge pipe stitching is fixed on contiguous soft tissue, layer-by-layer suture sarolemma and skin incision, sub-cage rearing under unified standard condition.
The detection of motor end plate: above-mentioned each treated animal is after the test through 1% pentobarbital sodium (40mg/kg) intraperitoneal injection of anesthesia, expose heart, through left ventricular cannulation to ascending aorta, quick filling 0.01M PBS 200ml, pours into 4% paraformaldehyde-PB liquid 200ml (0.1mol/L, pH:7.4) subsequently and carries out internal fixtion, take out the gastrocnemius of damage side, the sucrose solution putting into 30% spends the night, and after tissue sinks to the bottom, carries out frozen section (30-40um).The situation of change of motor end plate is observed with the dyeing of sulfo-acidylate choline.
Colouring method
Get liquid respectively, composition mixed liquor: 0.06N (0.82%) sodium acetate 6.32ml; 0.1N (0.6%) acetic acid 0.20ml; 0.1M (2.94%) sodium citrate 0.48ml; 30mM (0.75%) copper sulfate 1ml; 5mM (0.165%) potassium ferricyanide 1ml; H
2o 0.8ml; Acetylcholine 5mg is dissolved in above-mentioned mixed liquor, regulates PH5.5-5.6, frozen section is dipped in aforesaid liquid, 37 DEG C, 30-120 minute.Motor end plate dye brownish red under light microscopic.
Result: Post operation 4 weeks, the motor end plate of damage group shows as regression, and soleplate dyeing shoals, and the olistherozone of cavity sample appears in edge, and the quantity of soleplate is without significant change; The soleplate number of chitosan group 4 weeks treatment groups has minimizing, edge generation shrinkage, has a small amount of significantly dyeing disappearance district.IGF-1 low dose group invariable number, but there is the shrinkage of soleplate edge, a small amount of blank scarce zone; In IGF-1, some edge of dosage group is fuller, and shrinkage is less; High dose group part soleplate has shrinkage and scarce zone.Specifically please refer to Fig. 6 A to Fig. 6 D, Fig. 6 A is that matched group is neural; Fig. 6 B is the situation using chitosan to treat 4 weeks; Fig. 6 C is the situation using chitosan+IGF-1 (10ng/kg) to treat 4 weeks; Fig. 6 D is the situation using chitosan+IGF-1 (100ng/kg) to treat 4 weeks; Fig. 6 E is the situation using chitosan+IGF-1 (1000ng/kg) to treat 4 weeks.
Conclusion: IGF-1 (100ng/kg) effectively can improve the recovery of peripheral nerve injury after effect device motor end plate.
Embodiment 6: IGF-1 is on the impact of peripheral nerve injury rat sensation, motor function
SD rat, male and female are regardless of, body weight 210 ± 26g; Experiment is grouped into: add after sham operated rats (NE) 20, neural cutting that silica gel tube adds chitosan group 20, chitosan adds IGF-1 (10ng/kg) 40, chitosan adds IGF-1 (100ng/kg) 40, chitosan adds IGF-1 (1000ng/kg) 40; Often organize and be divided into: 1,2,3, phase point when 4 weeks.
Method: 1% pentobarbital sodium presses 30mg/kg intraperitoneal injection anesthesia.Lateral position, after shaving hair, sterilization, get angular cut after stock, expose right sciatic nerves, free sciatic nerve under 10 times of operating microscopes, and at 5mm place, piriformis outlet below, sciatic nerve denervation is about 8mm, let alone retraction, cause 10mm defect, by the silica gel tube bridge joint defect section disinfected, sew up with nerve is near, the far away broken ends of fractured bone of 9-0 not damaged micro suture line and bridge pipe and fix 2 pins.Sewing method adopts two fixed point cover connections: namely respectively sew up 1 pin at nerve trunk all 180 ° two, footpaths relative position respectively, the regenerating nerve broken ends of fractured bone is inserted in bridge pipe, and is about 2.5mm(as Fig. 2 apart from pipe).Add that silica gel tube adds chitosan group, chitosan adds IGF-1 (10ng/kg), chitosan adds IGF-1 (100ng/kg), chitosan adds IGF-1 (1000ng/kg) after dividing 5 groups to be respectively sham operated rats (NE), neural cutting at random animal according to the medicine difference added in pipe; Often organize and be divided into: 1,2,3, phase point when 4 weeks, only often organizing 5-10.It is inject medical chitose gel 20 μ l (Qisheng Biopreparations Co., Ltd., Shanghai) in silica gel tube respectively that chitosan adds silica gel tube group; Chitosan adds IGF-1 group: after injecting medical chitose gel 10 μ l in pipe, again with microsyringe respectively by 5 μ l IGF-1 (2ng/ μ l, 20ng/ μ, 200ng/ μ l Sigma Co., USA) in ascending pipe, with 9-0 silk thread, bridge pipe stitching is fixed on contiguous soft tissue, layer-by-layer suture sarolemma and skin incision, sub-cage rearing under unified standard condition.Sensory evoked potential detects: postoperative 1 week, 2 weeks, 3 weeks, 4 weeks random selecting, 3 rats, and the Powerlab polygraph produced with Australian ADI company surveys cortex sensory evoked potential (CSEP).Adopt intensity of electric stimulus 3mA, time delay 2ms, cycle 200ms, superpose 128 repetitive stimulations, with Scope software records and analysis.
Result: the regenerating nerve that disconnected section is coincide appears in IGF-1 (10ng/kg) group in the 4th week silica gel tube, and sensory evoked potential P1 and N1 ripple compared with normal incubation period group obviously extend, and the variation of P ripple is comparatively large, and stimulus intensity increases to 8v; There is the regrowth coincide in IGF-1 (100ng/kg) group, P1 and N1 ripple compared with normal incubation period group is short, and waveform is irregular the 2nd week time; When the 3rd week, P1 and N1 ripple shortens incubation period further, and different wave shape is large; When the 4th week, P1 ripple incubation period is close with normal group, but N1 ripple compared with normal incubation period group extends, and when stimulus intensity is 5v, waveform is tending towards normal.Specifically please refer to shown in Fig. 7-Fig. 9 and table 1-table 4, Fig. 7 is control rats sensory cortex Evoked ptential; Fig. 8 is IGF-1 (10ng) 4 weeks sensory cortex Evoked ptential; Fig. 9 is that IGF-1 (100ng) treats 4 weeks sensory cortex Evoked ptential; Figure 10 is matched group motor cortex Evoked ptential; Figure 11 is that IGF-1 (10ng) treats 4 weeks motor cortex Evoked ptential; Figure 12 is that IGF-1 (100ng) treats 4 weeks motor cortex Evoked ptential.
The latency change of P1 ripple in the different group sensory evoked potential of table 1.
Group | 1 week | 2 weeks | 3 weeks | 4 weeks |
Normal control | 6.20±0.41 | 6.20±0.41 | 6..20±0.41 | 6..20±0.41 |
Damage untreated fish group | 0 | 0 | 0 | 0 |
Silica gel tube chitosan | 0 | 0 | 0 | 0 |
IGF-1 10ng | 0 | 0 | 0 | 10.52±2.70** |
IGF-1 100ng | 0 | Cannot measure | 5.85±1.09** | 7.13±1.91** |
IGF-1 1000ng | 0 | Cannot measure | Cannot measure | Cannot measure |
Compare with corresponding Normal group: * P<0.05, * * P<0.01
The latency change of N1 ripple in the different group sensory evoked potential of table 2.
Group | 1 week | 2 weeks | 3 weeks | 4 weeks |
Normal control | 16.03±0..87 | 16.03±0..87 | 16.03±0..87 | 16.03±0..87 |
Damage untreated fish group | 0 | 0 | 0 | 0 |
Silica gel tube chitosan | 0 | 0 | 0 | 0 |
IGF-1 10ng | 0 | 0 | 0 | 27.69±3..65** |
IGF-1 100ng | 0 | 17.71±4.14* | 10.55±0.63** | 19.57±3..87** |
IGF-1 1000ng | 0 | Cannot measure | Cannot measure | Cannot measure |
Compare with corresponding Normal group: * P<0.05, * * P<0.01
The latency change of N1 ripple in the different group Motion Evoked Potential of table 3.
Group | 1 week | 2 weeks | 3 weeks | 4 weeks |
Normal control | 6.00±0.84 | 6.00±0.84 | 6.00±0.84 | 6.00±0.84 |
Damage untreated fish group | 0 | 0 | 0 | 0 |
Silica gel tube chitosan | 0 | 0 | 0 | 0 |
IGF-1 10ng | 0 | 0 | 0 | 9.63±0.63** |
IGF-1 100ng | 0 | Cannot measure | 16.15±5.44* | 8.60±1.02** |
IGF-1 1000ng | 0 | Cannot measure | Cannot measure | Cannot measure |
Compare with corresponding Normal group: * P<0.05, * * P<0.01
The latency change of N2 ripple in the different group Motion Evoked Potential of table 4.
Group | 1 week | 2 weeks | 3 weeks | 4 weeks |
Normal control | 24.35±1.01 | 24.35±1.01 | 24.35±1.01 | 24.35±1.01 |
Damage untreated fish group | 0 | 0 | 0 | 0 |
Silica gel tube chitosan | 0 | 0 | 0 | 0 |
IGF-1 10ng | 0 | 0 | 0 | 29.70±1.04* |
IGF-1 100ng | 0 | Cannot measure | 41.60±6.99** | 29.85±3.86* |
IGF-1 1000ng | 0 | Cannot measure | Cannot measure | Cannot measure |
Compare with corresponding Normal group: * P<0.05, * * P<0.01
Conclusion: IGF-1 (100ng/kg) can have the sensation of efficient recovery injured peripheral nerves and motion to pass to function after 4 weeks.
Embodiment 7: IGF-1 is on the impact of peripheral nerve injury rat conduction velocity
SD rat, male and female are regardless of, body weight 210 ± 26g; Experiment is grouped into: add after sham operated rats (NE) 20, neural cutting that silica gel tube adds chitosan group 20, chitosan adds IGF-1 (10ng/kg) 40, chitosan adds IGF-1 (100ng/kg) 40, chitosan adds IGF-1 (1000ng/kg) 40; Often organize and be divided into: 1,2,3, phase point when 4 weeks.
Method: 1% pentobarbital sodium presses 30mg/kg intraperitoneal injection anesthesia.Lateral position, after shaving hair, sterilization, get angular cut after stock, expose right sciatic nerves, free sciatic nerve under 10 times of operating microscopes, and at 5mm place, piriformis outlet below, sciatic nerve denervation is about 8mm, let alone retraction, cause 10mm defect, by the silica gel tube bridge joint defect section disinfected, sew up with nerve is near, the far away broken ends of fractured bone of 9-0 not damaged micro suture line and bridge pipe and fix 2 pins.Sewing method adopts two fixed point cover connections: namely respectively sew up 1 pin at nerve trunk all 180 ° two, footpaths relative position respectively, the regenerating nerve broken ends of fractured bone is inserted in bridge pipe, and is about 2.5mm(Fig. 2 apart from pipe).Add that silica gel tube adds chitosan group, chitosan adds IGF-1 (10ng/kg), chitosan adds IGF-1 (100ng/kg), chitosan adds IGF-1 (1000ng/kg) after dividing 5 groups to be respectively sham operated rats (NE), neural cutting at random animal according to the medicine difference added in pipe; Often organize and be divided into: 1,2,3, phase point when 4 weeks, only often organizing 5-10.It is inject medical chitose gel 20 μ l (Qisheng Biopreparations Co., Ltd., Shanghai) in silica gel tube respectively that chitosan adds silica gel tube group; Chitosan adds IGF-1 group: after injecting medical chitose gel 10 μ l in pipe, again with microsyringe respectively by 5 μ l IGF-1 (2ng/ μ l, 20ng/ μ, 200ng/ μ l Sigma Co., USA) in ascending pipe, with 9-0 silk thread, bridge pipe stitching is fixed on contiguous soft tissue, layer-by-layer suture sarolemma and skin incision, sub-cage rearing under unified standard condition.
Nerve conduction velocity detects: expose bilateral sciatic nerve, and peripheral nerve is done and experimental side bridge joint regenerating tissues.Stimulating electrode is 2 hook-type shield electrodes (die opening 2mm); be placed in the nearly heart of nerve trunk, distal end that bridge pipe near-end distance piriformis lower edge is about 0.3cm; recording electrode adopts metal electrode; above ankle joint, 10mm place inserts in the middle part of gastrocnemius belly of muscle; the two poles of the earth orientation is vertical with gastrocnemius machine direction; the two poles of the earth are most advanced and sophisticated at a distance of 2mm, and the degree of depth is 5mm.Earth lead clamp Mus tail ground connection.Stimulus intensity 3mA, interstimulus interval 2ms, every laboratory animal measures three times, gets wave amplitude person placed in the middle and adds up, and take nerve conduction velocity as stimulating electrode range difference/time difference.
Result: postoperative detection each group of nerve conduction velocity (see table 5), normal group Sciatic Nerve Conduction Velocity is 20.60 ± 2.10m/s, it is 11.76 ± 2.10m/s that IGF-1 (10ng/kg) organizes nerve conduction velocity, and waveform is low flat, short and small.IGF-1 (100ng/kg) is organized conduction velocity and is extended in time and speed gradually, and slightly faster than normal group, action potential waveform is wide flat.IGF-1 (1000ng/kg) cannot detect because neuranagenesis is bad.
The conduction velocity change in 4 weeks of the different group regenerating nerve of table 5.
Group | Conduction velocity |
Normal control | 20.60±2.10 |
Damage untreated fish group | 0 |
Silica gel tube chitosan | 0 |
IGF-1 10ng | 11.76±2.10** |
IGF-1 100ng | 24.41±5.22* |
IGF-1 1000ng | Cannot measure |
Compare with corresponding Normal group: * P<0.05, * * P<0.01
Conclusion: IGF-1 (100ng/kg) is treated peripheral ner ve conduction velocity after 4 weeks and is improved effect.
Embodiment 8: IGF-1 is on the impact of damaged blood vessels Endothelial Cell Survival and propagation
Endotheliocyte is inoculated in the DMEM/F12 culture medium containing 10% hyclone, in 37 DEG C, 5%CO
2incubator is cultivated; Every 3d changes a subculture, and every 7d trypsinization is gone down to posterity (1:3), and trophophase cell of taking the logarithm is for experiment.
(l) by endotheliocyte with 1 × 10
6the density of individual/ml is inoculated in oneself and coats poly-D-lysine sterile cover slips, cultivates 3d, takes out slide carry out immunofluorescence dyeing when cell is paved with slide about 90% with tweezers;
(2) discard culture medium, 0.0lmmol/LpH7.4PBS cleans 3min × 3 time;
(3) fix 20min with 4% paraformaldehyde, 0.01lmmol/LpH7.4PBS cleans 5min × 3 time;
(4) with 0.3%TritonX-100 punch 10min, 0.01mmol/LpH7.4PBS clean 5min × 3 time;
(5) close 20min with Normal Goat Serum, discard serum, do not clean;
(6) anti-VIII factor (1:200 dilution) of rabbit is added, 4 DEG C of overnight incubation;
(7) suck antibody, 0.01mmol/LpBS cleans smin × 3 time;
(8) add two anti-FITC (1:50 dilution), hatch 1h for 37 DEG C;
(9) suck antibody, 0.01mmol/LpH7.4PBS cleans 3min × 3 time;
(10) DAPI is added, incubated at room 3min;
(11) suck liquid, 0.0lmmol/LPBS cleans 5min × 3 time;
(12) use glycerol mounting, observe under laser confocal microscope.
Adopt mtt assay to measure the impact of insulin-like growth factor-l endothelial cell proliferation, concrete operations are as follows:
L endotheliocyte suspension cell density is adjusted to l × 10 by culture medium by ()
7/ mI, is inoculated on 96 orifice plates, every hole 100 μ l.
(2) divide into groups: experiment component is A, B, C, D, E5 subgroup: add the insulin-like growth factor-l Human Umbilical Vein Endothelial Cells that concentration is respectively 10ng/ml, 20ng/ml, 40ng/ml, 80ng/ml, 160ng/ml in the medium respectively and cultivate.Matched group: endotheliocyte suspension is added in the culture medium without IGF-1 and cultivate; Blank group: only have media alone, acellular, blank group is used for zeroing.Test every hole and 6 multiple holes are set.
(3) at 37 DEG C, 5%CO
2after incubator cultivates 48h, every hole adds 5mg/ml MTT 20 μ l, hatches 4h for 37 DEG C.
(4) add 100 μ l DMSO, be placed in shaking table low-speed oscillation 5min, fully after colour developing, measure each hole A value by microplate reader at wavelength 570nm place.
Result: endotheliocyte carries out that visible cell after immunofluorescence dyeing is many in fusiformis, triangle, and cluster exists, and karyon is clear, and endochylema enriches, and Growth of Cells enlivens, after the 48h that goes down to posterity " paving stone " aligned growth (Figure 13 A to Figure 13 C).Found by immunocytochemical stain qualification, cultured cell expresses mark VIII factor of endothelial-cell specific.TTC testing result shows, and control group A value is 0.33 soil 0.03, and along with the increase of dosage, the cultivation effect of IGF-1 Human Umbilical Vein Endothelial Cells is more obvious.But the proliferation function of E group insulin-like growth factor-l to cell that IGF-1 concentration is 160ng/ml weakens.In table 6.
Table 6. IGF-1 is on the impact of vascular endothelial cell proliferation
* P<0.05, * * P<0.01 compared with matched group
Conclusion: IGF-1 has the effect promoting hypertrophy to vascular endothelial cell.
Embodiment 9: IGF-1 is on the impact of the endotheliocyte (cell cut) of damage
(l) by endotheliocyte with 1 × 10
6the density of/ml is inoculated in 35mm culture dish, in 37 DEG C, and 5%CO
2incubator is cultivated;
(2), after cultivating 3d, bottom culture dish, " one " stroke trace of wide about 1.0mm is drawn;
(3) 5min × 3 time are cleaned, replaced medium with 0.01mmol/L pH7.4PBS;
(4) experimental group and matched group 2 groups is set: get 40ng/ml insulin-like growth factor-l and add culture medium as experimental group; Contrast adds conventional medium, in 37 DEG C, and 5%CO
2(PLSCONFM) incubator is cultivated, and observes the growing state of each group of cell under inverted microscope.
Part cultured cell at room temperature fixes 20min with 4% paraformaldehyde;
(5) 5min × 3 time are cleaned with 0.01mmol/L pH7.4PBS;
(6) at room temperature 3min is hatched with DAPI;
(7) use glycerol mounting, under laser confocal microscope, observe two groups of cell death situations respectively.Often group gets 10 visuals field at random, records cell death number in every visual field.
Result: when inverted phase contrast microscope is presented at 24h experimental group endotheliocyte oneself start to grow to intermediate score place, and matched group is without obvious change, after 48h, the quantity of experimental group cell migration is obviously many than matched group cell migration number, the gap width of experimental group cut also comparatively matched group obviously narrow, the cells float of oneself death much is had in culture medium in matched group, illustrative experiment group Growth of Cells is very fast, and IGF-1 has the effect (as Figure 14) promoting damaged endothelial cells propagation.Laser confocal scanning is observed dead cell karyon and is taken on a red color painted, and DAPI labelling all cells karyon dead cell number of experimental group after cut 48h is 0.90 scholar 1.52, and cellular control unit death toll is 5.90 scholar 4.93 (as Figure 15).Experimental group cell death number is significantly less than matched group, Variant statistical meaning P<0.05.
Conclusion: IGF-1 is capable of inhibiting cell in cell damage situation death is described and promotes the survival of damaging cells.
Embodiment 10: IGF-1 is to endothelial cell injury secretion of VEGF content detection
Endothelial cell culture method is the same.Grouping is the same.
Method: experimental group and matched group VEGF level adopt ELISA method to measure, and detection method is as follows:
L each for the standard substance of 2000pg/ml, 1000pg/ml, 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.2pg/ml 0.lml adds in hand-hole by () successively, only add sample diluting liquid as zero hole.Cell conditioned medium liquid sample diluting liquid is by adding in hand-hole after 1:20 dilution;
(2) ELISA Plate adds upper cover, 37 DEG C of reaction 90min;
(3) reaction after get rid of liquid in ELISA Plate, then facing to absorbent paper clap several under;
(4) anti-human for biotin vEGF antibody working solution is added successively by every hole 0.lml, 37 DEG C of reaction 60min;
(5) 3 times are washed with 0.01mmol/LpH7.4PBS, each immersion about 1min;
(6) ready ABC working solution is added successively by every hole 0.lml, at 37 DEG C, react 30min;
(7) 3 times are washed with 0.01mmol/LpH7.4PBS, each immersion about 1min;
(8) add at the TMB nitrite ion of 37 DEG C of balance 30min successively by every hole 90 μ l, 37 DEG C of lucifuge reaction 5-20min, now before naked eyes visual standard product, there is obvious blue gradient in 3-4 hole, and rear 3-4 hole difference is not obvious;
(9) TMB stop buffer, TMB is added successively by every hole 0.1ml, now blue turn of yellow;
(10) under wavelength 450nn, A value is measured by microplate reader.
Result: experimental group VEGF average detectable value is the average measured value of 130.44 scholar 9.13pg/ml, matched group VEGF is 47.68 scholar 12.70pg/ml, and two groups of comparing differences have statistical significance (P<0.05).
Conclusion: IGF-1 promotes that injured nerve reparation may be the ability by promoting and repair neural supply vessels endothelial cells secrete VEGF.
In sum: IGF-1 causes ordinary circumstance, the pathology HE of peripheral nerve severe trauma rat to serious sciatic nerve cutting, Nissl and Holmes dyes the pathological change of the sciatic nerve neuron number, Nissl body form and the nerve fiber that show, the recovery of muscular movement soleplate, sensation, the recovery that shows of detection of movement conduction function and raising, show that IGF-1 can be used for the repairing and treating of peripheral nerve injury.Simultaneously application cell damage model observes the effect and mechanism that IGF-1 repairs cell injury.The present invention is that IGF-1 application provides novelty teabag, also for the drug development of the caused damage for the treatment of traumatic nerve injury provides new, available resources widely.
The foregoing is only preferred embodiment of the present invention, be not used for limiting practical range of the present invention; If do not depart from the spirit and scope of the present invention, the present invention is modified or equivalent to replace, in the middle of the protection domain that all should be encompassed in the claims in the present invention.
Claims (4)
1. the regeneration reconstructing device of a peripheral nerve long distance defect, it is characterized in that: comprise one for the long silica gel tube apart from neurologic defect section of bridge joint, 2.5 mm are left at these two ends being used for the silica gel tube of bridge joint long distance neurologic defect section respectively and the nerve broken ends of fractured bone near, far away is sewed up fixing, and the neural broken ends of fractured bone is inserted in; And be the pharmaceutical composition of the treatment that forms of IGF-1 and chitosan long distance neurologic defect peripheral nerve injury by active component in injection silica gel tube.
2. the regeneration reconstructing device of peripheral nerve according to claim 1 long distance defect, it is characterized in that: in described pharmaceutical composition, chitosan is 10 μ 1, active component is IGF-1 is 5 μ l.
3. the regeneration reconstructing device of peripheral nerve according to claim 2 long distance defect, is characterized in that: described active component is the concentration of IGF-1 is 2 ng/ μ 1 or 20 ng/ μ 1 or 200 ng/ μ 1.
4. the regeneration reconstructing device of peripheral nerve according to claim 2 long distance defect, is characterized in that: described chitosan is medical chitose gel.
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CN201210218575.1A CN102716470B (en) | 2012-06-28 | 2012-06-28 | Medicine composite for treating peripheral nerve injury |
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