CN107970438B

CN107970438B - Nerve regeneration gel and preparation method and application thereof

Info

Publication number: CN107970438B
Application number: CN201711212065.2A
Authority: CN
Inventors: 胡萍萍; 陈谦; 宗斌; 张志坚
Original assignee: Zhejiang Hospital Of Chinese Traditional And Western Medicine
Current assignee: Zhejiang Hospital Of Chinese Traditional And Western Medicine
Priority date: 2017-11-28
Filing date: 2017-11-28
Publication date: 2021-08-13
Anticipated expiration: 2037-11-28
Also published as: CN107970438A

Abstract

The invention discloses a nerve regeneration gel and a preparation method and application thereof, wherein the nerve regeneration gel comprises a matrix composed of fibrinogen, thrombin, laminin and fibronectin, and contains procyanidine, sonic hedgehog factor, epidermal growth factor, neurotrophin-3 and vascular endothelial growth factor. In vitro and animal experiments prove that the gel can promote the regeneration and protrusion extension of neurons and repair damaged nerve tissues. The matrix in the hydrogel comprises fibrin, laminin and fibronectin, and can reduce cavity formation and promote the adhesion and proliferation of endogenous neural stem cells; procyanidin in the gel has effects of resisting oxidation and inhibiting inflammation; hedgehog factor, epidermal growth factor and neurotrophin-3 can promote stem cell proliferation and differentiation to neuron; vascular endothelial growth factor promotes angiogenesis. The gel can be formed in situ at the injured site, and can be used for treating nervous system injury.

Description

Nerve regeneration gel and preparation method and application thereof

Technical Field

The invention relates to a regenerated gel, in particular to a nerve regenerated gel and a preparation method and application thereof, belonging to the field of biological materials.

Background

Central nerve injuries, especially spinal cord injuries, are often caused by trauma, and striking causes massive cell death, while myelin sheaths begin to disintegrate and softening of the spinal cord occurs at the injury site. A large amount of cells and myelin sheath fragments are attacked by high oxygen partial pressure under the condition of bleeding, lipid peroxidation reaction occurs, and a large amount of free radicals are generated in an acute stage, so that severe oxidative damage is caused to the damaged part. Therefore, studies have proposed antioxidant supplementation for patients with spinal cord injury. On the other hand, the blood spinal cord barrier is present in the spinal cord and is destroyed after trauma, causing spinal cord antigens to come into contact with inflammatory cells, thereby causing a long-term inflammatory response, playing an important role in secondary injuries to the spinal cord. Spinal cord inflammation is performed mainly by neutrophils, monocytes and microglia from the blood and spinal cord itself, advantageously enabling the clearance of cellular debris and myelin debris at the site of injury; however, the inflammatory reaction is not limited to this, and inevitably causes apoptosis that survives the primary injury, and thus aggravates the tissue injury. Meanwhile, the oxidative reaction and the inflammatory reaction interact with each other to jointly cause the deterioration of the microenvironment of the injured part and prevent the migration, proliferation and differentiation of endogenous neural stem cells. Although large doses of glucocorticoids are clinically used to suppress inflammation and immune responses in the early stages of spinal cord injury, the effect is not significant, and experts have recently gradually denied this option. The applicant has analysed this solution for drawbacks in two points: firstly, glucocorticoid effect is too extensive, and serious complications such as infection, aseptic femoral head necrosis and the like easily occur to patients due to large dose; secondly, systemic administration of glucocorticoids can amplify their side effects.

Proanthocyanidin (PAC) extracted from herbaceous plants is a highly effective antioxidant and has excellent safety. PAC is able to combat fluoride-induced oxidative stress in fetal liver by modulating iron metabolism; can resist oxidative stress caused by arsenic; the protective effect on oxidative stress and apoptosis of the rat hippocampus with diabetes caused by streptozotocin is obviously better than that of vitamin E. A nutritional study directed to the college of military showed that the daily administration of PAC at 200mg could significantly reduce the levels of low density lipoproteins, and also showed that PAC has a function of regulating lipid metabolism. On the other hand, PAC is able to inhibit lipopolysaccharide induced macrophage synthesis and release of multiple inflammatory mediators. PAC has also been found to inhibit the synthesis of inflammatory factors by a variety of mechanisms in asthma models. PAC also alleviates ischemia reperfusion injury in multiple organs by different mechanisms. More importantly, PAC can significantly improve motor function following spinal cord injury, probably by inhibiting oxidative damage due to glutamate. Thus, PAC is able to resist oxidative stress, inhibit inflammatory reactions, and protect damaged tissues. However, systemic administration of the drugs in the whole body has low drug concentration at the injured part and limited effect.

Epidermal growth factor and neurotrophin-3 can promote proliferation of neural stem cells; the sonic hedgehog factor can promote the differentiation and protrusion extension of the neural stem cells to neurons; the vascular endothelial factor can promote the regeneration of blood vessels. However, systemic administration of these cytokines is not only expensive, but also has low concentration at the site of injury and limited efficacy.

Since central nervous system injury, especially spinal cord injury, is often surgically removed of fracture fragments and edema and fixed internally, it is considered that PAC and 4 cytokines are loaded on extracellular matrix and dropped on the injured site.

Fibrin is gel formed by catalyzing fibrinogen by prothrombin and blood coagulation XIII factor, and has the characteristics of wide source, easy preparation and excellent histocompatibility. Fibrin promotes myelination in vivo following spinal cord injury. Laminin and fibronectin are important components in extracellular matrix, and can promote cell adhesion and proliferation.

At present, fibrin is used as a carrier to transfer various nerve factors, for example, in chinese patent publication nos. CN103127494A and CN103230589A, tens of factors are mixed in a fibrinogen solution, and a nerve regeneration biogel is formed through a thrombin procoagulant process for nerve tissue repair. However, such techniques have certain limitations, firstly, the main function of neurotrophic factors is to promote neuronal regeneration and synaptic extension, and there is no significant relief from secondary injury after spinal cord injury, including lipid peroxidation injury due to degenerated myelin, inflammatory cell infiltration after vascular injury, and local immune-enhancing response. It is this secondary injury that significantly interferes with the effects of neurotrophic factors. As a natural strong antioxidant, PAC has the functions of inhibiting inflammation and immunoreaction, can resist the deterioration of microenvironment caused by secondary injury, and improves the efficacy of neurotrophic factors. On the other hand, fibrin alone is not stable enough as a matrix, and our in vitro and in vivo experiments show that fibrin gel with a final concentration of 50mg/ml is maintained at the spinal cord injury site for less than one week, while secondary injury often peaks in the second week after injury. At the same time, fibrin has significantly less capacity to promote cell proliferation than both fibronectin and laminin. However, fibronectin and laminin are difficult to form gel, and are easy to run off when being injected to the injured part. Combining fibrin and fibronectin/laminin can achieve complementary advantages.

Disclosure of Invention

The technical problem to be solved is as follows: aiming at the defects in the prior art, the invention provides a nerve regeneration gel, a preparation method and application thereof, which are used for treating central nerve injury, and promoting nerve repair and protrusion extension while resisting oxidation, resisting inflammation, inhibiting immune response.

The technical scheme is as follows: the invention provides a nerve regeneration gel, wherein the solvent of the gel is phosphate buffer solution, and the solute comprises fibrinogen, laminin, fibronectin, thrombin, procyanidine and 4 cytokines. The concentration of the fibrinogen is 10-100mg/ml, the concentration of the thrombin is 150-300U/ml, the concentrations of the laminin and the fibronectin are both 0.1-0.2 mg/ml, the concentration of the procyanidine is 10-30mg/ml, the 4 kinds of cell factors are sonic hedgehog factor, epidermal growth factor, neurotrophin-3 and vascular endothelial growth factor respectively, and the concentrations are 50-200 ng/ml.

The invention provides a preparation method of nerve regeneration gel, which comprises the following preparation steps:

(1) preparing a phosphate buffer solution with the pH value of 7.4;

(2) preparing 10-100mg/ml fibrinogen solution with phosphate buffer solution, and sequentially adding fibronectin, laminin, procyanidine, sonic hedgehog, epidermal growth factor, neurotrophin-3 and vascular endothelial growth factor into the fibrinogen solution to obtain mixed solution A; the final concentrations of the laminin and the fibronectin are both 0.1-0.2 mg/ml, the final concentration of the procyanidine is 10-30mg/ml, and the final concentrations of the sonic hedgehog factor, the epidermal growth factor, the neurotrophin-3 and the vascular endothelial growth factor are all 50-200 ng/ml;

(3) preparing 150-300U/ml thrombin solution by using phosphate buffer solution to obtain mixed solution B; and (3) uniformly mixing the mixed solution A and the mixed solution B according to the volume ratio of A: B =4:1 to obtain the nerve regeneration gel.

The nerve regeneration gel provided by the invention is applied to the preparation of medicines for treating central nervous system injury.

The nerve regeneration gel provided by the invention is applied to preparation of preparations for resisting oxidation, inhibiting inflammatory reaction and protecting nerves.

Has the advantages that: compared with the prior art, the invention has the advantages that: (1) the invention takes the extracellular matrix as a carrier by utilizing the pleiotropic effect of the procyanidine, transplants the procyanidine to the spinal cord injury part, and obviously inhibits the oxidative damage, inflammatory reaction and immune response of the nerve injury part; in vitro cell experiments show that the nerve regeneration gel provided by the invention enhances the oxidation resistance of the neural stem cells by 2-4 times; meanwhile, spinal cord injury animal experiments show that the level of oxidative injury products at spinal cord injury parts can be remarkably reduced by adopting the nerve regeneration gel, the injury parts are connected by the gel rich in a large number of capillary vessels, and a large number of nerve fibers are arranged in the gel and communicated with the proximal end and the distal end of an injury.

(2) The invention considers the defects of cell adhesion promotion and proliferation capacity of fibrin, combines fibronectin and laminin on the basis of the fibrin, and enhances the adhesion and proliferation of residual neural stem cells.

(3) The nerve regeneration gel prepared by the invention can be formed in situ and is convenient for use in operation.

Drawings

FIG. 1 is a diagram of the in vitro cell assay of example 1 showing the protrusion of neurospheres of each group on a nerve regeneration gel;

FIG. 2 is a graph of the cell activity of groups of neurospheres after hypoxia in vitro cells of example 1;

FIG. 3 shows BBB scores of groups of spinal cord injury animal experiments in example 1;

FIG. 4 is a graph showing the degree of oxidative stress at the injury site in each group of animals with spinal cord injury in example 1;

FIG. 5 is a graph showing the results of testing the groups of animals with spinal cord injury in example 1 for tissue repair at the injury site.

Detailed Description

The technical solution of the present invention is further described with reference to the following specific examples, but the scope of the present invention is not limited to the following examples, but is defined by the description of the present invention and the claims.

Example 1

Preparation of nerve regeneration gel

Phosphate Buffer Solution (PBS) with pH of 7.4 is prepared by deionized water, and is used for standby after high-temperature sterilization. Preparing 250U/ml thrombin solution (component B) by PBS; preparing 25mg/ml sterile fibrinogen solution by PBS, and then sequentially adding fibronectin and laminin (final concentration is 0.125mg/ml), procyanidine (concentration is 12.5 mg/ml), sonic hedgehog, epidermal growth factor, neurotrophin-3 and vascular endothelial growth factor (concentration is 125 ng/ml) into the fibrinogen solution, wherein the component A is the component A. And (3) uniformly mixing the component A with the component B by 4 times of the volume of the gel, thus obtaining the transparent gel, namely the nerve regeneration gel.

Second, in vitro cell assay

1. Nerve regeneration gel prepared as described in example 1

2. Experiment grouping

According to culture conditions, the culture medium is divided into four groups:

group A: neuronal medium (Neurobasal medium, 10% FBS, supplemented with B27, EGF, bFGF) without nerve regeneration gel.

Group B: preparing 25mg/ml sterile fibrinogen solution and 250U/ml thrombin solution with PBS, mixing at a ratio of 4:1 to form fibrin gel without other matrixes, procyanidins and cytokines, and culturing cells with neuron culture solution.

Group C: the neuron culture solution is added with procyanidin (the final concentration is 10 mg/ml) and cytokine (the final concentration is 100 ng/ml) without gel.

Group D: neuron culture solution and the nerve regeneration gel.

3. In vitro culture of neural stem cells

Pregnant mice were sacrificed by cervical dislocation and sterilized in iodophors. The uterus was dissected and transferred to a dish containing D-Hanks solution. Separating fetal rat, taking brain, stripping meninges, and washing with pre-cooled D-Hanks solution and culture solution for several times. The brain tissue suspension was transferred into a centrifuge tube, digested with 1.25% papain for 15 minutes, then centrifuged at 1000 rpm for 3 min, and the supernatant was discarded. Digestion was stopped with Neurobasal broth containing 10% FBS. DNAse was added to the tissue suspension at a final concentration of 0.5 mg/L and incubated for 3 min (37 ℃), the suspension was gently blown down and centrifuged at 1000 rpm for 3 min, and the supernatant was discarded. Subsequently, serum-free neurosphere medium was added for resuspension.

And (3) passage of neurospheres, namely collecting the cell balls formed by primary culture in a centrifuge tube, centrifuging for 5 min at 1000 rpm, removing supernatant, and adding the cell balls containing accutase for digestion for 10 min. Washing with D-Hanks solution for 3 times, re-suspending cells, centrifuging at 1000 rpm for 5 min, discarding supernatant, adding neurosphere culture solution, re-suspending cells, and culturing neurospheres in bottles. Cultured neurospheres were characterized by Nestin and CD133 staining following the general procedure of immunofluorescence staining. The identification result shows that the obtained neurospheres are positive for Nestin and CD 133.

4. Neurospheres were cultured for 3 weeks under the four conditions described above, and expression of the cellular neuron marker NF-200 was identified by immunofluorescence staining, while nuclei were counterstained. Representative results are shown in figure 1, the neurospheres in group A are poorly attached, and the partially attached neurospheres are mostly differentiated into polygonal glial cells and only a small amount of the partially attached neurospheres are differentiated into neuron-like cells with elongated processes; group B only grows on common fibrin gel, after 2 weeks, the gel is degraded into a silk strip by cells, and the number of positive cells is small; the group C can see NF-200 cells with strong positive expression, but the positive cells are less, and the neurosphere adherence effect is poorer; the neurospheres of group D grow on the nerve regeneration gel, the neurospheres adherence is obviously superior to other groups, the cells are in a filament shape and are interwoven into a net shape, and simultaneously, the stability of the gel is obviously higher than that of group B due to the cross-linking effect of procyanidine.

5. Neurospheres were cultured for 1 day under the above four conditions to acclimate the neurospheres. Subsequently, the cells were placed in an anoxic cassette, purged with nitrogen, and subjected to hypoxia for 4 hours, followed by culture for 3 days, and the cell activity was measured using CCK8 kit. The result is shown in figure 2, the group A is taken as 1, the hypoxia tolerance of the group B is not obviously improved, the hypoxia tolerance of the group C is about 2 times of that of the group A, and the hypoxia tolerance of the group D is about 4 times of that of the group A, which shows that the nerve regeneration gel can obviously enhance the antioxidant capacity of the neural stem cells.

Spinal cord injury animal test

1. Culturing neural stem cells

Neurospheres were suspension cultured and identified as described in example 2.

2. Rat spinal cord transection model making

Animals were anesthetized with 10% chloral hydrate, fixed for skin preparation, sterilized, dorsal muscles were isolated, spinous and vertebral plates of segment T9-T11 were exposed, T10 vertebral plates were excised, spinal cords were exposed, spinal cords were cut, and different treatments were given according to the following groups. After hemostasis, suture layer by layer, injection of antibiotics to prevent infection, and squeezing urine after operation.

3. Animal grouping and handling

48 SD female rats (with the body weight of 200-250 g) were cut at the T10 segment into 4 groups, and group A was the injured group, and injected with PBS; b group is transplanted neural stem cell group after injury, and neural stem cell suspension is injected at the injury part; the group C is a group of transplanted nerve regeneration gel after injury, and the components A, B of nerve regeneration gel prepared in the example 1 are dripped into the injured part according to the proportion of 4:1 and are mixed uniformly to form gel; and the group D is a group of nerve regeneration gel and nerve stem cells transplanted after the injury, the nerve stem cells are mixed in the group A, and the group D and the group B are dripped into the injury part according to the ratio of 4: 1.

4. Detecting the index

Closely observing the condition of the rats after operation, performing BBB exercise score (Basso, Beattie & Bresnahan sports scoring scale) every week, and killing the rats in batches at 2, 8, 10 and 12 weeks after operation, wherein the spinal cord specimen at 2 weeks detects TBARS level to measure the tissue oxidation level; other spinal cord specimens were stained with NF-200 and observed for neurite outgrowth under a fluorescent microscope.

5. Test results

The BBB movement score result shows that B, C, D groups of rats have better limb movement recovery than A group, wherein D group has the best recovery condition, and the average score can reach more than 10 points at 12 weeks; whereas group B had poor recovery, which could be associated with loss of transplanted neural stem cells or death in harsh environments (fig. 3). Post-operative spinal cord specimens showed that the nerve regeneration gel significantly reduced the level of oxidation at the spinal cord injury site 2 weeks after injury, regardless of the presence of neural stem cells (fig. 4). Immunofluorescence staining showed a large number of cavities in the group a lesion sites; group B had very little fiber; group C has few cavities, the damaged broken ends are connected by nerve regeneration gel, and a large amount of new blood vessels and a small amount of nerve fibers are arranged in the cavity; group D lesions were not only connected by a gel rich in capillaries, but also had a large number of nerve fibers within them, connecting the proximal and distal ends of the lesion (fig. 5).

6. Conclusion

The nerve regeneration gel containing the procyanidine, the multiple cytokines, the laminin and the fibronectin can relieve the oxidative damage of the damaged part, creates a good microenvironment for nerve regeneration, is convenient for transplanting the nerve stem cells, promotes the proliferation of the nerve stem cells, stretches out the protrusion, connects the two damaged ends and promotes the functional recovery of experimental animals.

Claims

1. A nerve regeneration gel is characterized in that a solvent of the gel is phosphate buffer solution, a solute comprises fibrinogen, laminin, fibronectin, thrombin, procyanidine and 4 cytokines, wherein the 4 cytokines are sonic hedgehog factor, epidermal growth factor, neurotrophin-3 and vascular endothelial growth factor respectively; the preparation method of the nerve regeneration gel comprises the following preparation steps:

(1) preparing a phosphate buffer solution with the pH value of 7.4;

(2) preparing 10-100mg/ml fibrinogen solution with phosphate buffer solution, and sequentially adding fibronectin, laminin, procyanidine, sonic hedgehog, epidermal growth factor, neurotrophin-3 and vascular endothelial growth factor into the fibrinogen solution to obtain mixed solution A; the final concentrations of the laminin and the fibronectin are both 0.1-0.2 mg/ml, the final concentration of the procyanidine is 10-30mg/ml, and the final concentrations of the sonic hedgehog factor, the epidermal growth factor, the neurotrophin 3 and the vascular endothelial growth factor are all 50-200 ng/ml;

2. The nerve regeneration gel as claimed in claim 1, wherein the concentration of fibrinogen is 10-100mg/ml, the concentration of thrombin is 150-300U/ml, the concentrations of laminin and fibronectin are 0.1-0.2 mg/ml, the concentration of procyanidin is 10-30mg/ml, and the concentrations of 4 cytokines are 50-200 ng/ml.

3. Use of the nerve regeneration gel of claim 1 in the manufacture of a medicament for treating central nervous system injury.

4. Use of the nerve regeneration gel of claim 1 for the preparation of an antioxidant and neuroprotective formulation.