Composition containing neural stem cells and application of composition in preparation of medicine for treating retina degenerative diseases

Abstract

The invention provides a composition containing neural stem cells, which comprises the following components in parts by mass: 15-20 parts of neural stem cells which are subjected to hypoxia pretreatment and chemical protective agent treatment; 30 to 40 parts of a collagen-sodium alginate composite carrier; 5-8 parts of compound active factors, wherein the compound active factors comprise endostatin, recombinant human epidermal growth factors and superoxide dismutase; and 100 parts of cell protection liquid, wherein the cell protection liquid comprises thioredoxin and nicotinamide. The composition is used for preparing a medicine for treating retina degenerative diseases, and the medicine is an intravitreal injection preparation. The composition synchronously overcomes the three technical bottlenecks of low cell transplantation survival rate, out-of-control differentiation direction and neural network reconstruction disorder through neural stem cell hypoxia pretreatment, active factor compound network, collagen-sodium alginate carrier mechanical adaptation and multi-channel synergistic intervention of a redox protection system.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a neural stem cell-containing composition and application thereof in preparation of a drug for treating retinal degenerative diseases. BACKGROUND

[0002] The most critical problem of retinal degenerative diseases is that retinal neurons continuously die and cannot be regenerated. Taking glaucoma as an example, all current treatment methods can only slow down the progression of the disease, and ultimately about 30% of patients will lose their vision. Neural stem cell transplantation can solve this problem to some extent, but in actual operation, there are three major difficulties.

[0003] The first difficulty is that the cells cannot survive. The retinal vitreous cavity is both oxygen-deficient and severely inflamed, with a tumor necrosis factor concentration of more than 50 picograms per milliliter. Ordinary stem cells transplanted into it will die more than 80% within three days. Some people have tried to use sodium alginate microcapsules to encapsulate the cells, although this can prevent immune attacks, but the lack of oxygen inside the microcapsules will cause the cells to eat themselves, and the density of living cells will drop to less than 2,000 per cubic millimeter after two days, far below the effective standard.

[0004] The second difficulty is that the cells grow randomly. Stem cells in a damaged environment tend to grow into useless glial cells. Some people use BMP-4 to induce differentiation, but the conversion rate of retinal ganglion cells is less than 18%, and in one-third of the cases, glial scars appear. More dangerously, about 5% of the transplanted cells grow into tumors in animal experiments, causing permanent vision loss.

[0005] The third difficulty is that the transplanted cells become a decoration. Even if the cells survive, they are difficult to establish effective connections with the original neural network. A medical institution in the United States found that the proportion of transplanted cells expressing the key synaptic protein PSD95 is less than 10%, while at least 60% is required to repair nerves. Some people add a layer of laminin on the surface of the cells to help adhesion, but the coating hardness is more than 5,000 pascals, which is much harder than the 0.5 to 1,000 pascals of the retina's own soft tissue, and thus inhibits the growth of nerve fibers.

[0006] Recent improvements mainly fall into three categories: carrier materials such as gelatin hydrogel can delay drug release, but six out of ten active ingredients are lost within the first 12 hours; gene editing can remove certain pathogenic genes, but it cannot stop inflammation-induced mitochondrial damage; a combination of nutritional factors such as BDNF and NT-3 can increase the survival rate to 35%, but it accelerates the desensitization of the recipient. The common weakness of these solutions is that they only solve a single problem and ignore the fact that the retinal microenvironment is actually a dynamic network: hypoxia triggers oxidative stress, oxidative stress exacerbates inflammation, and inflammation hinders neural reconstruction. Therefore, a neural stem cell-containing composition and its application in the preparation of a drug for treating retinal degenerative diseases are needed. SUMMARY

[0007] In order to overcome the defects in the prior art, a neural stem cell-containing composition and its application in the preparation of a medicament for treating retinal degenerative diseases are provided.

[0008] In order to achieve the above-mentioned purpose, the present application provides the following technical solutions: A neural stem cell-containing composition, which comprises the following components in mass parts: Neural stem cells 15-20 parts: pretreated by hypoxia and treated by a chemical protective agent; Collagen-sodium alginate composite carrier 30-40 parts; Compound active factors 5-8 parts: containing endostatin, recombinant human epidermal growth factor, superoxide dismutase; Cell protection solution 100 parts: containing thioredoxin and nicotinamide.

[0009] The specific steps of pretreating the neural stem cells by hypoxia and treating the neural stem cells by a chemical protective agent are as follows: placing the neural stem cells in a culture box containing 5% oxygen for 24 hours, and then transferring the neural stem cells into a culture medium containing nicotinamide for 48 hours of immersion.

[0010] The concentration of the nicotinamide in the culture medium is 12-18 parts of nicotinamide per hundred parts of the culture medium.

[0011] The mass ratio of endostatin, recombinant human epidermal growth factor and superoxide dismutase in the compound active factors is 1: (1-3): (3-10).

[0012] The preparation method of the collagen-sodium alginate composite carrier is as follows: dissolving sodium alginate in physiological saline, adding collagen to adjust the pH value to 6.8, adding a calcium chloride solution for crosslinking for 30 minutes, freezing at -20°C, and then gradiently increasing the temperature for drying.

[0013] The concentration of the calcium chloride solution is 0.095 M-0.105 M.

[0014] The mass ratio of the sodium alginate to the collagen is 1.5-3:1, and the gradient temperature increase is as follows: maintaining at 22-26°C for 1-3 hours, maintaining at 28-32°C for 3-6 hours, and maintaining at 36-38°C for 4-8 hours.

[0015] The cell protection solution further comprises buffered saline.

[0016] The mass ratio of the buffered saline to thioredoxin and nicotinamide is 98: (0.04-0.06): (1-2).

[0017] The application of the neural stem cell-containing composition, which is used for the preparation of a medicament for treating retinal degenerative diseases, the medicament being a glass body cavity injection preparation.

[0018] Compared with the prior art, the application has the advantages and beneficial effects that: 1. The composition of the application has significant advantages in solving the treatment problem of retinal degenerative diseases. The hypoxic pretreatment of neural stem cells combined with the chemical protective agent treatment process effectively improves the survival of transplanted cells. The process promotes the establishment of endogenous defense mechanisms by cells and enhances their tolerance to low-oxygen microenvironments. The simultaneous implementation of nicotinamide reinforcement treatment maintains the energy metabolism homeostasis of cells. Compared with the traditional physical isolation type carrier scheme, the present method avoids cell damage caused by the accumulation of metabolic waste.

[0019] 2. The specific component ratio of the compounded active factors realizes precise regulation of cell differentiation. Among them, endostatin inhibits abnormal vascular signaling pathways, recombinant human epidermal growth factor promotes neuronal synapse development, and superoxide dismutase maintains local oxidation-reduction balance. This group eliminates abnormal differentiation inducers from the molecular signal level and guides the directional differentiation of neural stem cells into target cell types.

[0020] 3. The preparation process of the collagen-sodium alginate composite carrier creates an ideal physical microenvironment. The carrier structure formed by specific cross-linking parameters has pore size characteristics that promote the exchange of nutrients while blocking the invasion of inflammatory factors. The mechanical properties of the carrier are highly compatible with the retina tissue, providing a suitable interface for neural synapse extension and overcoming the inhibition of neural growth by traditional carrier materials.

[0021] 4. The synergistic effect of the cell protection solution system maintains the homeostasis of the microenvironment in the transplanted area. Thioredoxin effectively removes active oxygen free radicals, and the nicotinamide component continuously supplies coenzyme precursors required for cell metabolism. The optimized ratio of the solution to buffered saline ensures stable physiological osmotic pressure and systematically blocks the cascade amplification of oxidative stress and inflammatory response.

[0022] 5. The components of the present application form a step-by-step reinforced protection mechanism: pretreated neural stem cells have enhanced stress resistance, collagen-sodium alginate composite carriers create a suitable three-dimensional growth space, compounded active factors precisely regulate the development process, and cell protection solution continuously optimizes the local microenvironment. This multi-dimensional synergistic effect breaks through the limitations of existing single-link improvement schemes and significantly improves the efficiency of retinal neural network reconstruction. DETAILED DESCRIPTION

[0023] The technical solutions in the embodiments of the application will be described below in a clear and complete manner. Obviously, the described embodiments are only a part of the embodiments of the application, not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of the present application.

[0024] In the present application, the sources of various raw materials are briefly described as follows: Sodium alginate: Shandong Fentai Biotechnology Co., Ltd., CAS No. 9005-38-3, Model No. FT-203 Collagen: Jiangsu Jiangshan Juyuan Biological Engineering Co., Ltd., CAS No. 9007-34-5, Model No. JS-I Calcium chloride: National Pharmaceutical Group Chemical Reagent Co., Ltd., CAS No. 10043-52-4, Model No. AR grade anhydrous particles Endostatin: Wuhan Huamei Biological Engineering Co., Ltd., CAS No. 183903-27-9, Model No. HM-Human Endostatin Recombinant human epidermal growth factor: Beijing Yiqiao Godz Sci-Tech Co., Ltd., CAS No. 62253-63-8, Model No. rec-hu EGF Superoxide dismutase: Shanghai Yuan Ye Biological Technology Co., Ltd., CAS No. 9054-89-1, Model No. SOD-W30 Thioredoxin: Shanghai Baiolai Biological Technology Co., Ltd., CAS No. 52500-60-4, Model No. Thioredoxin-1 Human Nicotinamide: Tianjin Xinsisheng Biological Technology Co., Ltd., CAS No. 98-92-0, Model No. V802536 The technical solution of the present application is: a composition containing neural stem cells, which comprises the following components in mass parts: Neural stem cells 15-20 parts: pretreated by hypoxia and treated by chemical protectant; Collagen-sodium alginate composite carrier 30-40 parts; Compound active factor 5-8 parts: containing endostatin, recombinant human epidermal growth factor, superoxide dismutase; Cell protection solution 100 parts: containing thioredoxin and nicotinamide.

[0025] The specific steps of pretreating the neural stem cells by hypoxia and treating by chemical protectant are: placing the neural stem cells in a culture box containing 5% oxygen for 24 hours, and then transferring them into a culture medium containing nicotinamide for 48 hours.

[0026] The concentration of nicotinamide in the culture medium is 12-18 parts of nicotinamide per hundred parts of culture medium.

[0027] The mass ratio of endostatin, recombinant human epidermal growth factor and superoxide dismutase in the compound active factor is 1: (1-3): (3-10).

[0028] The preparation method of the collagen-sodium alginate composite carrier is as follows: sodium alginate is dissolved in physiological saline, collagen is added to adjust the pH value to 6.8, calcium chloride solution is added for crosslinking for 30 minutes, and then the mixture is frozen at-20℃ and dried by gradient temperature rising.

[0029] The concentration of the calcium chloride solution is 0.095 M-0.105 M.

[0030] The mass ratio of the sodium alginate to the collagen is 1.5-3:1, and the gradient temperature rising is as follows: 22-26℃ for 1-3 hours, 28-32℃ for 3-6 hours, and 36-38℃ for 4-8 hours.

[0031] The cell protection solution further comprises buffered saline.

[0032] The mass ratio of the buffered saline to the thioredoxin to the nicotinamide is 98:(0.04-0.06):(1-2).

[0033] The application of a composition containing neural stem cells for preparing a medicine for treating retinal degenerative diseases, wherein the medicine is a preparation for intravitreal injection.

[0034] The technical solutions of the present application are further illustrated by the following examples and comparative examples, but the protection scope of the present application is not limited thereto.

[0035] All the examples and comparative examples use neural stem cells of the same source (HUM-iPS001 from the Cell Bank of Chinese Academy of Sciences), and the chemical reagents are purchased from the manufacturers specified in the foregoing.

[0036] Example 1 Preparation of the neural stem cell component: 20 parts of neural stem cells are placed in a 5% oxygen incubator for 24 hours, and then transferred into a culture medium containing 18 parts of nicotinamide (per hundred parts of culture medium) for 48 hours of immersion.

[0037] Preparation of the collagen-sodium alginate composite carrier: 3 parts of sodium alginate are dissolved in physiological saline, 1 part of collagen (mass ratio 3:1) is added, and the pH value is adjusted to 6.8. 0.105 M calcium chloride solution is added for crosslinking for 30 minutes, and then the mixture is frozen at-20℃ and dried by gradient temperature rising: 22℃ for 1 hour→28℃ for 3 hours→36℃ for 4 hours.

[0038] Recompounding of active factors: the total amount of 5 parts is weighed according to the mass ratio of endostatin: recombinant human epidermal growth factor: superoxide dismutase = 1:1:3.

[0039] Preparation of the cell protection solution: 100 parts are prepared according to the mass ratio of buffered saline: thioredoxin: nicotinamide = 98:0.04:1.

[0040] Final composition: the above neural stem cells are mixed with the composite carrier, and after loading the active factors, they are suspended in the protective solution.

[0041] Example 2 In this example, the same as in Example 1 will not be repeated, and the differences are as follows: Neural stem cell component: 15 parts of stem cells are treated under 5% oxygen for 24 hours, and then transferred to a culture medium containing 12 parts of nicotinamide for 48 hours of immersion.

[0042] Composite carrier: sodium alginate 1.5 parts and collagen 1 part (mass ratio 1.5:1) are mixed, cross-linked with 0.095 M calcium chloride for 30 minutes, and gradient temperature: 26°C for 3 hours→32°C for 6 hours→38°C for 8 hours.

[0043] Compound active factor: endostatin: epidermal growth factor: superoxide dismutase = 1:3:10 (mass ratio), total amount 8 parts.

[0044] Protective solution: buffer saline: thioredoxin: nicotinamide = 98:0.06:2 (mass ratio).

[0045] Example 3 In this example, the same as in Example 1 will not be repeated, and the differences are as follows: Neural stem cell component: 15 parts of stem cells are treated under 5% oxygen for 24 hours, and then transferred to a culture medium containing 12 parts of nicotinamide for 48 hours of immersion.

[0046] Composite carrier: sodium alginate 2 parts + collagen 1 part (mass ratio 2:1), cross-linked with 0.1 M calcium chloride, gradient temperature: 24°C for 2 hours→30°C for 4.5 hours→37°C for 6 hours.

[0047] Compound active factor: the mass ratio of the three factors is 1:2:6, and the total amount is 6 parts.

[0048] Protective solution: buffer saline: thioredoxin: nicotinamide = 98:0.05:1.5 (mass ratio).

[0049] Comparative Example 1

[0050] In this comparative example, the same as in Example 1 will not be repeated, and the differences are as follows: Cancel the hypoxic pretreatment (use normal oxygen culture).

[0051] Comparative Example 2

[0052] In this comparative example, the same as in Example 2 will not be repeated, and the differences are as follows: The ratio of endostatin: recombinant human epidermal growth factor: superoxide dismutase in the compound active factor is changed to 1:0.5:15.

[0053] Comparative Example 3

[0054] In this comparative example, the same as Example 3 is not repeated, and the differences are described as follows: The composite carrier only uses sodium alginate, and the collagen is cancelled.

[0055] Comparative Example 4

[0056] In this comparative example, the same as Example 1 is not repeated, and the differences are described as follows: The cell protection solution lacks thioredoxin.

[0057] Comparative Example 5

[0058] In this comparative example, the same as Example 1 is not repeated, and the differences are described as follows: Directly use the traditional scheme, the neural stem cell suspension is used with the gelatin hydrogel carrier (Adv. Mater 2021 method).

[0059] Performance test results and analysis

[0060] According to the parameters of the examples and comparative examples, the compositions are obtained, and each composition is injected into the vitreous cavity of the glaucoma model rabbit (n=10 / group), and the test results are shown in Table 1.

[0061] As can be seen from Table 1, the survival rate of the example group is 65-69%, which is significantly higher than that of all comparative examples, proving that hypoxic preconditioning and nicotinamide synergistically improve cell resistance. Comparative Example 1 lacks hypoxic preconditioning, and the cell cannot activate the HSP70 pathway, so the survival rate drops to 32.6%; Comparative Example 4 lacks thioredoxin, resulting in a ROS clearance rate of only 36.2%, proving that oxidative damage is the main cause of death.

[0062] The neuron differentiation rate of the three example groups is 36-41%, and the PSD95 positive rate is 52-59%, which meets the demand for nerve repair. Comparative Example 2 lacks epidermal growth factor and has excessive superoxide dismutase, which destroys the signal balance, resulting in a differentiation rate of only 15.7%, proving the necessity of the proportion limit in the present application. The pure sodium alginate carrier of Comparative Example 3 has a hardness exceeding the standard, which inhibits synaptic extension, resulting in a PSD95 positive rate of only 32.1%. The gelatin hydrogel of Comparative Example 5 has a burst effect, releasing 68% of the active factors within 12 hours, resulting in insufficient long-acting performance.

[0063] The ROS clearance rate of the three examples is 79-85%, which is in sharp contrast to Comparative Example 4. It is proved that thioredoxin directly protects mitochondria by reducing peroxynitrite, and nicotinamide maintains the NAD+ pool capacity, both of which are indispensable.

[0064] Table 1 Analysis test results

[0065] The composition of the present application effectively solves the core problem of retinal transplantation through a systematic multi-level synergistic mechanism: the endogenous defense system is established by hypoxic preconditioning and nicotinamide intensive treatment of neural stem cells, which significantly improves the tolerance of low oxygen microenvironment; the specific ratio of the compound active factor network precisely regulates the differentiation path, inhibits abnormal proliferation and promotes the development of nerve synapse; the collagen-sodium alginate composite carrier forms a biomimetic microporous structure through controllable crosslinking, which provides an ideal interface for synapse reconstruction due to its mechanical adaptability; the redox protection system composed of thioredoxin and nicotinamide continuously maintains the microenvironment homeostasis and completely blocks the hypoxia-oxidation-inflammation cascade reaction. The functions of each component are complementary and synergistic, breaking through the limitations of traditional single-link intervention, and realizing the synchronous improvement of cell survival rate, differentiation accuracy and neural network function reconstruction efficiency without introducing exogenous gene modification.

[0066] The above is the preferred embodiment of the present application. It should be noted that for those skilled in the art, without departing from the principles of the present application, several improvements and refinements can be made, which should also be considered within the scope of protection of the present application.

Claims

1. A composition containing neural stem cells, characterized in that, The composition comprises the following components in parts by weight: 15-20 neural stem cells: pretreated with hypoxia and chemical protectants; 30-40 parts of collagen-sodium alginate complex carrier; 5-8 parts of compound active factors: including endostatin, recombinant human epidermal growth factor, and superoxide dismutase; 100 portions of cell protection solution: containing thioredoxin and nicotinamide.

2. The composition containing neural stem cells according to claim 1, characterized in that, The specific steps for the hypoxia pretreatment and chemical protection treatment of the neural stem cells are as follows: the neural stem cells are placed in a culture incubator containing 5% oxygen for 24 hours, and then transferred to a culture medium containing nicotinamide for 48 hours.

3. The composition containing neural stem cells according to claim 2, characterized in that, The concentration of nicotinamide in the culture medium is 12-18 parts nicotinamide per 100 parts culture medium.

4. The composition containing neural stem cells according to claim 1, characterized in that, The mass ratio of endostatin, recombinant human epidermal growth factor and superoxide dismutase in the compound active factor is 1:(1-3):(3-10).

5. The composition containing neural stem cells according to claim 1, characterized in that, The preparation method of the collagen-sodium alginate composite carrier is as follows: Sodium alginate is dissolved in physiological saline, collagen is added to adjust the pH value to 6.8, calcium chloride solution is added for cross-linking for 30 minutes, and the mixture is frozen at -20℃ and then dried by gradient temperature increase.

6. The composition containing neural stem cells according to claim 5, characterized in that, The concentration of the calcium chloride solution is 0.095 M-0.105 M.

7. The composition containing neural stem cells according to claim 5, characterized in that, The mass ratio of sodium alginate to collagen is 1.5-3:1, and the gradient temperature is specifically as follows: 22-26℃ for 1-3 hours, 28-32℃ for 3-6 hours, and 36-38℃ for 4-8 hours.

8. The composition containing neural stem cells according to claim 1, characterized in that, The cell protection solution also includes buffered saline.

9. A composition containing neural stem cells according to claim 8, characterized in that, The mass ratio of the buffered saline to thioredoxin and nicotinamide is 98:(0.04-0.06):(1-2).

10. The use of a composition containing neural stem cells as described in any one of claims 1-9, characterized in that, The composition is used to prepare a medicament for treating retinal degenerative diseases, wherein the medicament is a vitrectomy formulation.

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