CN114432347B - Application of nervonic acid-producing microalgae in preparation of product for treating spinal cord injury - Google Patents
Application of nervonic acid-producing microalgae in preparation of product for treating spinal cord injury Download PDFInfo
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Abstract
The invention relates to application of nervonic acid-producing microalgae in preparation of a product for treating spinal cord injury, belonging to the technical field of biological medicines. Microalgae is tiny in individual and has fluidity, and is not easy to fix at a damaged spinal cord tissue, the microalgae is encapsulated by fibrin glue, the fibrin glue has good biocompatibility and safety proved by clinical practice, in a prepared microalgae-biogel system, the microalgae is used as a natural carrier of oxygen and nervonic acid, the fibrin hydrogel three-dimensionally carries the microalgae to supplement the extracellular matrix missing at the damaged part, and the comprehensive regulation of a spinal cord injury microenvironment is realized from the three aspects of oxygen supply, provision of neurotrophic factors and supplement of the missing extracellular matrix, so that the repair of the central nervous tissue of the spinal cord and the reconstruction of the nerve function are promoted. The invention explores the application potential of the microalgae in the aspect of biomedicine and provides a brand new thought for treating spinal cord injury.
Description
Technical Field
The invention relates to application of nervonic acid-producing microalgae in preparation of a product for treating spinal cord injury, belonging to the technical field of biological medicines.
Background
Spinal Cord Injury (SCI) is a serious disease of the central nervous system with a high disability rate; about 90% of SCI cases are caused by trauma, often occur in young and middle-aged people, and are mainly manifested by limb somatosensory, motor function and autonomic nerve dysfunction, which seriously affects the normal life of patients.
At present, effective means for treating SCI clinically are lacked, and the treatment is only limited to acute-stage operation spinal cord stabilization, hormone drug impact therapy, hyperbaric oxygen therapy and other means for controlling complications and later-stage rehabilitation, but still effective nerve function recovery cannot be realized. The complex inhibitory pathological microenvironment is a major factor impeding neural repair, and the overall regulation of the microenvironment is critical in the treatment of SCI. After the spinal cord injury occurs, a large amount of bleeding and ischemia occur to the affected part, and the affected part is in a microenvironment imbalance state. The microenvironment of ischemia and hypoxia further causes secondary injury, massive diffusion of oxidative stress and inflammatory factors leading to neuronal death, with loss of extracellular matrix at the site of injury as the condition progresses, forming a void surrounded by a glial scar barrier. Therefore, repair of damaged spinal cord tissue should primarily involve the following mechanisms of action: (1) supplementing biocompatible extracellular matrix, protecting spinal cord, and supporting damaged tissue growth; (2) improving the hypoxic microenvironment of the affected part and relieving the secondary injury of spinal cord tissues; (3) provides neurotrophic factors for damaged tissues, promotes development and growth of nerve fibers and promotes recovery of nerve functions.
The microalgae can be degraded in physiological environment and has no obvious toxic action on normal cells. The Zhou Min team of Zhejiang university takes spirulina as a chemotherapeutic drug adriamycin carrier, and the targeted delivery of drugs to breast cancer lung metastasis tumors shows higher drug loading efficiency, ideal lung targeted accumulation, pH-responsive drug release and other performances; 5363 the subject group Zhou Min further uses the oxygen release property of microalgae to improve the tumor hypoxia microenvironment, and applies a magnetic field to target the magnetized spirulina cells on the tumor site, and the autofluorescence property of chlorophyll of microalgae can realize the noninvasive tracking of microalgae in vivo, and the chlorophyll can be used as photosensitizer to realize photodynamic therapy to kill tumor cells; the group also creatively coats the chlorella with a red blood cell membrane, uses the modified chlorella cells for killing cancer cells, and shows certain physiological activity. The research proves the biocompatibility and safety of the microalgae, and simultaneously shows the potential of the microalgae as a natural oxygen carrier in the microenvironment of organisms.
The method uses Mychonastes afer as an experimental algae species, and M.afer as a fresh water microalgae, the cell diameter is 2-8 mu m, the microalgae is a Nervonic acid (Nervonic acid) producing microalgae, the Nervonic acid content can reach 6.5% of the content of neutral fatty acid in cells, the oil content can reach 53.9% of the dry weight of the cells after environmental induction stress, and the microalgae is a good Nervonic acid source species. Nervonic acid was first found in nerve tissue of mammals and was named nervonic acid. Foreign research shows that nervonic acid is a core natural component of brain nerve cells and nerve tissues, is a specific substance which is discovered in the world so far and can promote the repair and regeneration of damaged nerve tissues, is an essential 'higher nutrient' for the growth, regeneration and maintenance of nerve cells, particularly brain cells, optic nerve cells and peripheral nerve cells, and has great effects on improving the activity degree of the brain nerves and preventing the aging of the brain nerves. However, microalgae are small in size, fluid, and not easy to fix, and are not suitable for application in damaged tissues.
Disclosure of Invention
The invention aims to provide application of nervonic acid-producing microalgae in preparing products for treating spinal cord injury. According to the invention, microalgae with a certain amount of nervonic acid is mixed with the biogel, the biogel provides a fixed support for the microalgae on the basis of not influencing the biological activity of the microalgae, the growth and oxygen production of the microalgae are promoted to a certain extent, the microalgae-biogel system is embedded in a spinal cord injury part, extracellular matrix is supplemented, oxygen release of the microalgae is promoted by additional red light irradiation, and meanwhile, nervonic acid generated by the microalgae is used for providing a neurotrophic factor for repairing nervous tissue, so that the aims of improving the microenvironment of the affected part and promoting the repair of the nervous tissue of the affected part are fulfilled.
The technical scheme of the invention is as follows:
in a first aspect, the present invention provides a microalgae-gel system consisting essentially of nervonic acid-producing microalgae and a biogel.
Preferably, the nervonic acid-producing microalgae is Mychonastes afer with the preservation number of CGMCC NO.4654.
Preferably, the density of the microalgae cells in the microalgae-gel system is 1 × 10 8 ～1×10 9 Per mL; more preferably 5X 10 8 one/mL.
Preferably, the biogel is fibrin glue.
In a second aspect, the present invention provides a method for preparing the above microalgae-gel system, comprising the steps of:
(1) Dispersing microalgae for generating nervonic acid in a thrombin solution to be used as solution A; marking the fibrinogen solution as solution B;
(2) Mixing the solution A and the solution B, and forming gel in situ to obtain the microalgae-gel system.
Preferably, the method for culturing microalgae in step (1) comprises the following steps:
culturing microalgae producing nervonic acid at 23-25 deg.C for 6-8 days with light intensity of 40-60 μmol photons.m -2 ·s -1 The photoperiod is 15-17 h of illumination and 7-9 h of darkness, the culture medium is BG-11 liquid culture medium, and the pH value of the culture medium is 7.0-7.5. Ensure that the culture is not contaminated by other substances during the culture.
Preferably, the concentration of the thrombin in the solution A in the step (1) is 4-10 mg/mL; the concentration of fibrinogen in the solution B is 50-100 mg/mL.
Preferably, the volume ratio of the solution A to the solution B in the step (2) is 1:1-2, and the density of the microalgae in the microalgae-gel system is 1 × 10 8 ～1×10 9 one/mL.
In a third aspect, the present invention provides the use of nervonic acid-producing microalgae for the preparation of a product for the treatment of spinal cord injury.
Preferably, the nervonic acid-producing microalgae is Mychonastes afer with the preservation number of CGMCC NO.4654.
In the above applications, in order to ensure the application effect of the nervonic acid-producing microalgae in preparing products for treating spinal cord injury, other physiologically/pharmacologically acceptable carriers or excipients, such as biogel, can be added as required.
In a fourth aspect, the invention provides the use of the above microalgae-gel system for the preparation of a product for the treatment of spinal cord injury.
In the application, other physiologically/pharmacologically acceptable carriers or excipients and other auxiliary materials can be added into the microalgae-gel system according to requirements, so that the application, carrying or storage of the product is facilitated.
In a fifth aspect, the present invention provides the use of the above microalgae-gel system for the treatment of spinal cord injury.
In the application, the microalgae-gel system is applied to treatment of spinal cord injury, the solution A and the solution B are simultaneously injected into a spinal cord injury part to form gel in situ at the spinal cord injury part, and infrared light irradiation treatment is given for a certain time every day subsequently to promote oxygen release of microalgae and repair of spinal cord injury tissues.
In a sixth aspect, the invention provides a microalgae-gel-illumination remediation system, which comprises the above microalgae-gel system and illumination system.
Preferably, the illumination wavelength of the illumination system is 750-800 nm, the illumination intensity is 4000-4500 lux, and the whole illumination treatment or the intermittent illumination treatment is carried out in the repair cycle.
The invention has the beneficial effects that:
1. microalgae is tiny in individual and has fluidity, and is not easy to fix at a damaged spinal cord tissue, the microalgae is encapsulated by fibrin glue, the fibrin glue has good biocompatibility and safety proved by clinical practice, in a prepared microalgae-biogel system, the microalgae is used as a natural carrier of oxygen and nervonic acid, the fibrin hydrogel three-dimensionally carries the microalgae to supplement the extracellular matrix missing at the damaged part, and the comprehensive regulation of a spinal cord injury microenvironment is realized from the three aspects of oxygen supply, provision of neurotrophic factors and supplement of the missing extracellular matrix, so that the repair of the central nervous tissue of the spinal cord and the reconstruction of the nerve function are promoted.
2. The invention mixes microalgae producing nervonic acid with biogel, injects the mixture into the spinal cord injury, forms colloid in situ, and gives a certain time of red light irradiation treatment every day to promote the oxygen release of the microalgae and the generation of nervonic acid, thereby effectively improving the environments of ischemia and anoxia, extracellular matrix deficiency and neurotrophic deficiency of the affected part, and promoting the repair of damaged nervous tissues and the recovery of motor functions.
3. The invention explores the application potential of the microalgae in the aspect of biomedicine and provides a brand new thought for treating spinal cord injury.
Drawings
Fig. 1 is an electron microscope image of a microalgae-gel system consisting of nervonic acid-producing microalgae m.afer and fibrin glue;
FIG. 2 shows the M.afer photosynthetic parameter F in a microalgae-gel system under different treatment conditions 0 A change line graph;
fig. 3 is a plot of the m.afer oxygen release in a microalgae-gel system under different treatment conditions;
FIG. 4 is a line graph of BBB score 28 days after spinal cord injury model rat surgery;
FIG. 5 is a graph of immunohistochemical staining of nerve fibers in spinal cord tissue of rats in a model of spinal cord injury;
FIG. 6 shows the photosynthetic parameter F in the microalgae-gel system of Chlorella of comparative example 2 0 A change line graph;
FIG. 7 is a line graph showing oxygen evolution in the microalgae-gel system of Chlorella of comparative example 2.
Detailed Description
The technical solutions of the present invention are further described below with reference to the following embodiments and the drawings of the specification, but the scope of the present invention is not limited thereto.
The source of the biological material is as follows:
microalgae: mychonases afer, deposited in China center for type microorganism Collection, with the collection number CGMCC No.4654 (see the record of application No. CN201110119480. X);
chlorella: is Chlorella sorokiniana, is generally sold in the market, and has no nervonic acid after being analyzed and determined by algae cell grease.
SD rats at 3 weeks old are purchased from the experimental animal breeding company Limited of Jinmenfeiye, and relevant animal experiments strictly comply with the relevant regulations of animal ethics and national experimental animal management.
And other raw material sources are as follows:
bovine thrombin, available from bevercy technologies ltd, beijing, and bovine fibrinogen from saint next biotechnology (shanghai).
The contents of the examples, which are not specified in specific conditions, were carried out under conventional conditions; the reagents or instruments used are not indicated by manufacturers, and are all common commercial products.
Example 1:
the culture of the nervonic acid-producing microalgae Mychonastes afer comprises the following steps:
culturing microalgae Mychonastes afer producing nervonic acid at 24 deg.C for 7 days with illumination intensity of 50 μmol photons.m -2 ·s -1 The illumination period is 16h illumination and 8h darkness, the culture medium is BG-11 liquid culture medium, and the pH value of the culture medium is 7.2. The culture process ensures that the culture medium is not contaminated by other substances.
The preparation of the microalgae-gel system comprises the following steps:
(1) Dispersing the cultured microalgae for producing nervonic acid Mychonastes afer into a thrombin solution to serve as an A solution, wherein the concentration of thrombin in the A solution is 8mg/mL; marking the fibrinogen solution as B solution, wherein the concentration of fibrinogen is 80mg/mL;
(2) Mixing the solution A and solution B at 30 μ L, and in-situ gelatinizing to obtain microalgae-gel system with microalgae cell density of 1 × 10 8 ～1×10 9 One per mL.
The electron micrograph of the prepared microalgae-gel system is shown in FIG. 1, and the microalgae cell concentration of the microalgae-gel system in FIG. 1 is 5 × 10 8 one/mL, as can be seen from the figure, the biogel is used as a fixing support to fix the microalgae in the biogel, and the porosity of the biogel is large, so that the stability of the microalgae is improved, and the biological function of the microalgae is not affected.
Example 2: in vitro survival experiments of microalgae-gel systems
The microalgae-gel system prepared according to example 1 (system volume 100. Mu.L, density of microalgae cells in the system 5X 10) 8 one/mL) and microalgae cell concentration of 5 × 10 8 100 mu L of algae solution per mL is placed in an incubator at 37 ℃ and cultured in a simulated in vivo temperature environment, and four treatment conditions are set in total: white light, white light + skin tissue, red light + skin tissue, wherein the red light has a wavelength of 750nm and an illumination intensity of 4500lux, the skin tissue is illuminated all day long, the white light has a wavelength of a normal visible light range and an illumination intensity of 4500lux, and the skin tissue is illuminated all day long; the light treatment is to directly irradiate light on a microalgae-gel system or a microalgae liquid; the illumination + skin tissue treatment is to place the microalgae-gel system or the microalgae solution under the skin tissue and the light directly irradiates the skin tissue. Determination of photosynthetic parameters F of microalgae-gel system or algae liquid by using Imaging-PAM (polyacrylamide) modulation chlorophyll fluorometer 0 Using photosynthetic parameter F of microalgae-gel system or algae liquid 0 As a marker for characterizing whether microalgae cells survived, the results are shown in fig. 2.
The results show that the sets of optical parameters F are measured over a 5 day measurement period 0 The numerical values are all larger than 0.04, which indicates that the microalgae cells are always in a survival state in the experimental period and can play an oxygen release function; the survival time of the microalgae-gel system did not show significant difference compared to microalgae alone, indicating that the biogel had no significant effect on the survival time of microalgae in vitro. At the same time, photosynthetic parameters F of the microalgae-gel system in the red light treatment group and the red light + skin tissue treatment group 0 Photosynthetic parameters F higher than those of individual microalgae 0 It is demonstrated that under red light conditions, the gel has a promoting effect on the growth of microalgae, which is beneficial for the photosynthetic oxygen production of microalgae, but the white light treatment group does not have the phenomenon.
Example 3: determination of oxygen release amount and nervonic acid content of microalgae-gel system
Setting five treatment conditions of red light, red light plus skin tissue, white light plus skin tissue and darkness in a 37 ℃ incubator, wherein the wavelength of the red light is 750nm, the illumination intensity is 4500lux, and the whole day illumination is given; the wavelength of the white light is within the normal visible light range, the illumination intensity is 4500lux, and the illumination is given all day long; culturing the microalgae-gel system under five treatment conditions, measuring the oxygen content of the microalgae-gel system once every 24 hours by using a probe type sensor of a fluorescent optical fiber oxygen measuring instrument, wherein the measuring period is 5 days, and comparing the oxygen release capacity of the microalgae under different illumination conditions, the result is shown in figure 3.
The results show that compared with the white light treatment group, the oxygen content of the white light + skin tissue treatment group is reduced, which indicates that the white light can not completely penetrate through the skin tissue to irradiate into the microalgae-gel system, the shielding of the skin tissue blocks the penetration of the white light, and the light energy oxygen release activity of microalgae cells is blocked; compared with the red light treatment group, the oxygen content of the red light + skin tissue treatment group is not changed obviously, and the oxygen content is higher, which indicates that the red light can penetrate through the skin tissue and act on the light energy oxygen release activity of the microalgae-gel system.
After the oxygen release measurement period is finished, taking out the microalgae-gel system, placing the microalgae-gel system in a glass tube, adding 1.5mL of chloroform-methanol mixed solution (2, 1 v/v) and 3.5mL of methanol sulfate solution (the content of sulfuric acid is 2 wt%), shaking and mixing uniformly, screwing a bottle cap, placing the bottle cap in an oven at 85 ℃ for methyl esterification reaction for 2 hours, taking out the microalgae-gel system, and placing the microalgae-gel system on ice to cool to room temperature. Adding 2mL of n-hexane solution into the glass tube, and fully shaking; adding 700 μ L KCl solution (concentration of 0.9 wt%), shaking thoroughly for several times, standing or centrifuging for 3-5min, and collecting the upper organic phase in a centrifuge tube for gas chromatography. The composition of fatty acid methyl ester in the sample was analyzed by Agilent 7890A gas chromatography using HP-5 (30 m.times.320. Mu.m.times.0.25 μm) as a column. Gas chromatography analysis procedure: the injection inlet temperature is 250 ℃, the injection volume is 1 mu L, and high-purity nitrogen is used as carrier gas; setting a temperature rise program: the temperature of 120 ℃ is maintained for 5min, then the temperature is increased to 240 ℃ at the heating rate of 3.5 ℃/min and the temperature is maintained for 10min, and the experimental results are shown in the following table.
TABLE 1 Peak time of nervonic acid and relative nervonic acid content in microalga-gel systems of different treatment groups
The result shows that the content of nervonic acid in the system is the largest in the red light treatment group, and is 4.057%; and compared with a white light treatment group, the red light has a small promotion effect on the nervonic acid content in the system.
Example 4: construction of rat spinal cord injury model
SD rats at 3 weeks of age were weighed, and 24 SD rats weighing 200-230g were randomly assigned to 5 groups, SCI group: no plants are buried after spinal cord injury surgery; gel group: embedding fibrin glue after spinal cord injury surgery, wherein the fibrin glue is prepared by mixing 8mg/mL thrombin solution and 80mg/mL fibrinogen solution according to the volume ratio of 1:1; gel-M.afer-H group: a microalgae-gel system prepared according to example 1 was implanted after spinal cord injury surgery, the concentration of microalgae cells in the system was 5X 10 8 Per mL; gel-M.afer-L group post-spinal cord injury surgery Implantation of the microalgae-Gel system prepared according to example 1, with a concentration of microalgae cells of 1X 10 8 Per mL; sham group, only skin tissue was cut, and spinal cord was not damaged.
Constructing a model: the method comprises the steps of shaving off hairs around T10 spinous processes on the back of an anesthetized SD rat, smearing skin with iodophor, disinfecting the skin by taking the T10 spinous processes as the center, cutting the skin along the midline of the back, separating fascia bluntly, separating muscle tissues around T9-T11, exposing vertebral plates, opening the T10 vertebral plates by using rongeurs, exposing the spinal cords, cutting the spinal cords at the T9-T10 position by using microdissection scissors, manufacturing a notch with the length of about 4mm, and constructing a model rat with the spinal cord injury after hemostasis. Then the corresponding implants of different treatment groups are implanted into the gap of the spinal cord, and after the implants of each group are implanted, hemostasis is realized and muscle tissues and skin are sutured. Penicillin is given within seven days after operation to prevent infection, and the bladder urination function is recovered by massaging urination every day. During the treatment period, red light irradiation treatment is given to the affected part of each model, the wavelength of red light is 750nm, the irradiation intensity is 4500lux, and the irradiation is given for 0.5 hour in the morning and afternoon every day.
Example 5: rat hind limb motor function score
SD rats of example 4 were scored weekly for Baso Beattie Bresnahan (BBB) of hindlimb motor function within 4 weeks of treatment. The rat is placed on a wide plane to freely crawl, two observers who cannot know the grouping condition monitor the motion condition of the rat, the motion amplitude and the motion posture of three joints of hind limbs of the rat and the coordination between the hind limbs and the forelimbs are observed, each observer independently observes for 4min, and the hind limb motion function of the rat is scored according to a BBB scoring method. The results are shown in FIG. 4, the BBB score of the Sham group reached full 21 points, and the hind limb motor function of the SD rats was normal; compared with the SCI group, hind limb motor functions of other groups of rats subjected to the implant treatment of the implant plant are recovered to a certain degree, wherein the recovery condition of the Gel-M.afer-H group is the best, and the recovery conditions of the Gel-M.afer-L group and the Gel group are equivalent, which indicates that the low-density microalgae in a microalgae-Gel system has an unobvious repair effect on spinal cord injury, but has a certain repair effect, and the high-density microalgae and the biogel can repair the spinal cord injury together.
Example 6: tissue collection and immunohistochemical staining
The rats of example 4 were anesthetized 4 weeks after treatment, the right atrial appendages of the rats were excised, and the rats were perfused with saline via the left ventricle until the livers of the rats turned yellow. A rat spinal column is taken and fixed by 4% paraformaldehyde for 8-24h, and then a spinal cord with the total length of 1.5cm and the injury part as the center is taken and soaked in 30% sucrose for dehydration until the spinal cord sinks. After embedding and quick freezing with a tissue embedding medium, 20 μm coronal sections were cut out in a cryomicrotome and immunofluorescent staining was performed with a neurofibrillary primary antibody and a fluorescent secondary antibody. The effects of different implants on the recovery of spinal cord injury were compared. As shown in FIG. 5, the rat nerve fibers of the Gel-M.afer-H group were well repaired and regenerated, and the density of microalgae cells was 5X 10, compared to the SCI group and the Gel group 8 The highest BBB score is obtained in the Gel-M.afer-H group of each/mL, which shows that the microalgae-Gel system is beneficial to the repair of spinal cord injured nervous tissues and has obvious effect on the recovery of motor functions.
Comparative example 1
The nervonic acid-producing microalgae Mychonastes afer is mixed with common medical sodium hyaluronate gel on the market to prepare a microalgae-gel system, and the microalgae-gel system can not keep complete shape and flow after the next day of preparation of the microalgae-gel system, so that the microalgae cells can not be fixed at the spinal cord injury.
When the fibrin glue is used, microalgae cells can be fixed in the gel, and gel tissues can keep complete shapes for a long time, so that the fibrin glue can directionally play a role at the affected part of spinal cord injury.
The fibrin glue mainly comprises preparations such as thrombin and fibrinogen, etc., through simulating the final stage of blood coagulation, under the combined action of thrombin and calcium ions, fibrinogen molecules are cracked into fibrin peptides A and B to form fibrin monomer protein, and meanwhile, under the action of thrombin activated factor XX, fibrin is cross-linked to form stable gel. And the fibrin glue as a natural product has no tissue toxicity and can be absorbed by tissues.
Comparative example 2
At a cell density of 5X 10 8 The chlorella per mL is prepared into a microalgae-gel system together with a fibrin glue solution according to the method described in the embodiment 1, and an in-vitro microalgae cell survival experiment and system oxygen content detection are carried out according to the methods described in the embodiments 2 and 3.
Determination of photosynthetic parameters of microalgae-gel System F 0 As a result, as shown in FIG. 6, the photosynthetic parameter F of the chlorella cells was about 3 days in the environment at 37 ℃ 0 Namely, the cell activity is reduced to 0.01, which shows that the cell activity is obviously reduced and is obviously lower than that of a Gel-M. And performing oil extraction and component analysis on the microalgae-gel system, wherein the nervonic acid component is not detected. Further, the oxygen content in the microalgae-gel system 3 days before the culture of the chlorella is detected, and the result is shown in fig. 7, and under the condition of red light irradiation, the oxygen content in the microalgae-gel system of the chlorella is superior to that in other illumination conditions, but still is obviously lower than that in the microalgae-gel system of m.afer. The M.afer has better photosynthetic oxygen release activity and viability than chlorella under the same cell density, illumination condition and environmental condition.
In conclusion, when chlorella is fixed in fibrin glue, the fibrin glue has a significant effect on the cell activity of chlorella, and the chlorella cells are basically inactivated after being cultured for about 3 days, but the fibrin glue has no significant effect on the in vitro survival time of microalgae m.afer, which is detailed in example 2. In addition, under the same cell density and experimental conditions, the photosynthetic oxygen release activity of chlorella is lower than that of the microalgae m.afer, which indicates that the fibrin glue also inhibits the oxygen production of chlorella, but has no significant influence on the oxygen production of the microalgae m.afer.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and the best mode for practicing the invention, and is not intended to limit the invention in any way, so that any simple modification, equivalent change or modification made to the above embodiments according to the technical spirit of the present invention will still fall within the scope of the technical solution of the present invention without departing from the content of the technical solution of the present invention.
Claims (7)
1. A microalgae-gel system, wherein the microalgae-gel system consists essentially of nervonic acid-producing microalgae and a biogel; the nervonic acid-producing microalgae areMychonastesaferThe preservation number is CGMCC NO.4654, and the biological gel is fibrin glue;
the microalgae-gel system is prepared according to the following method:
(1) Dispersing microalgae producing nervonic acid in a thrombin solution to be used as solution A; marking the fibrinogen solution as solution B; the concentration of the thrombin in the solution A is 4 to 10mg/mL; the concentration of fibrinogen in the solution B is 50 to 100 mg/mL;
(2) Mixing the solution A and the solution B, and forming gel in situ to obtain a microalgae-gel system; the volume ratio of the solution A to the solution B is 1: 1~2, and the density of the microalgae in the microalgae-gel system is 1 x 10 8 ~1×10 9 one/mL.
2. The microalgae-gel system of claim 1, wherein the microalgae-gel system has a density of microalgae cells of 5 x 10 8 one/mL.
3. The microalgae-gel system as claimed in claim 1, wherein the cultivation method of microalgae in step (1) comprises the following steps:
culturing microalgae for producing nervonic acid at 23-25 deg.C for 6-8 days with light intensity of 40-60 μmol photons m −2 ·s −1 The photoperiod is 15 to 17 hours of illumination and 7 to 9 hours of darkness, the culture medium is BG-11 liquid culture medium, and the pH value of the culture medium is 7.0 to 7.5.
4. Use of the microalgae-gel system of claim 1 for the preparation of a product for the treatment of spinal cord injury.
5. The use of claim 4, wherein the product for the treatment of spinal cord injury further comprises a physiologically/pharmacologically acceptable carrier or excipient.
6. A microalgae-gel-illumination remediation system comprising the microalgae-gel system of claim 1 and an illumination system.
7. The microalgae-gel-illumination restoration system as claimed in claim 6, wherein the illumination wavelength of the illumination system is 750 to 800nm, and the illumination intensity is 4000 to 4500 lux.
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