Protective agent for in-vitro culture of retinal ganglion cells with mechanical injury as well as preparation method and application of protective agent

Abstract

The invention belongs to the technical field of cell protective agents, and particularly relates to a protective agent for in-vitro culture of mechanically damaged retinal ganglion cells as well as a preparation method and application of the protective agent. The protective agent is composed of etomidate, a protease inactivated substance and calcium chloride, and the ratio of etomidate to the protease inactivated substance to the calcium chloride is 1 mmol: (1-1.5) g: (0.1-0.5 mmol); the protease inactivated substance is a substance obtained by carrying out high-temperature inactivation on water-soluble protease; the temperature of the high-temperature inactivation is 120 DEG C to 140 DEG C. According to the invention, the protease inactivator and the calcium chloride are used for synergistic interaction on the etomidate, so that the protection effect on mechanically damaged retina neuronodule cells cultured in vitro is enhanced.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell protectants, and in particular relates to a protective agent for mechanically damaged in vitro cultured retinal ganglion cells, a preparation method and an application thereof. Background Art

[0002] Retinal ganglion cells (RGCs) are the only neurons that transmit visual information captured by retinal photoreceptors to the central nervous system, and maintaining their normal function is crucial for visual perception. Under the influence of mechanical factors such as eye trauma, increased intraocular pressure in glaucoma leading to compression of the optic nerve, retinal ganglion cells are easily damaged, causing irreversible vision loss. Due to the complex in vivo environment, in vitro culture of retinal ganglion cells for injury research has opened up an important way to deeply understand the mechanism of injury and explore effective protection methods. At present, retinal ganglion cells are mainly isolated and obtained from the retinal tissue of newborn mice or rats. Retinal ganglion cells are cultured in an in vitro culture environment and given external factors to construct a mechanical injury model of retinal ganglion cells. The mechanical injury models of retinal ganglion cells mainly include: scratch injury model, pressure injury model and stretch injury model.

[0003] According to prior art research, protective agents for retinal ganglion cell damage mainly include chemical protective agents and biomaterial protective agents. Among them, common chemical protective agents include neurotrophic factors such as brain-derived neurotrophic factor (BDNF), antioxidants such as vitamin C and vitamin E, and calcium channel blockers such as nimodipine; common biomaterial protective agents include hyaluronic acid-based hydrogels, etc. Hydrogels can also be used as drug sustained-release carriers to slowly release the above-mentioned neurotrophic factors and other drugs to the surrounding retinal tissue, prolonging the drug's action time. In addition, extracellular matrix (ECM) components such as collagen and laminin are also biomaterial protective agents for protecting retinal ganglion cells. However, long-term use of drugs may lead to drug resistance and reduced drug sensitivity, so it is necessary to continue to develop new protective agents for in vitro cultured retinal ganglion cells that act on mechanical damage. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a protective agent for mechanically damaged in vitro cultured retinal ganglion cells, as well as a preparation method and application thereof.

[0005] The present invention aims to provide a protective agent for mechanically damaged in vitro cultured retinal ganglion cells, hereinafter referred to as the protective agent. The protective agent comprises etomidate, a protease inactivator, and calcium chloride, wherein the ratio of etomidate, protease inactivator, and calcium chloride is 1 mmol: 1 g to 1.5 g: 0.1 mmol to 0.5 mmol. The protease inactivator is a water-soluble protease inactivated by high-temperature inactivation, which disrupts the molecular structure network of the protease. The high-temperature inactivation temperature is 120°C to 140°C.

[0006] In the research of the prior art, it was found that etomidate has a protective effect on mechanically damaged in vitro cultured retinal ganglion cells, and the protective effect is enhanced with the increase of etomidate dosage within a certain range. Etomidate is an organic compound with the chemical formula C 14 H 16 N2O2, the prior art also has research on its use as an anesthetic. The present invention uses etomidate as a basic protective agent component, and utilizes a protease inactivated substance and calcium chloride to synergistically enhance the efficacy of etomidate to strengthen the protective effect on mechanically damaged in vitro cultured retinal ganglion cells. In addition, in the present invention, the structure of the protease inactivated substance can load some etomidate and calcium chloride to form a composite material, which on the one hand increases the contact with the retinal ganglion cells, and on the other hand, the etomidate and calcium chloride loaded on the protein inactivated substance can be gradually released, thereby extending the effective action time of the protective agent, achieving an enhanced repair effect on mechanically damaged in vitro cultured retinal ganglion cells, and protecting the retinal ganglion cells.

[0007] In addition, the activity state of the protease has a crucial impact on the microenvironment and physiological function of the cell. If the protease is not inactivated, it will act on the peptide chains of specific sequences around the retinal ganglion cells, gradually degrading them, which will affect the activity of the retinal ganglion cells. The present invention uses a protease inactivated substance. The protein after high-temperature inactivation loses its enzymatic activity, which can prevent the degradation of the peptide chains around the retinal ganglion cells, thereby effectively protecting the normal morphology of the retinal ganglion cells and surrounding cells, and also protecting the peptide chain structure and activity involved in many key functions such as signal conduction and nutrient transport, providing a guarantee for the stable survival and normal function of the retinal ganglion cells. This can not only be widely used in in vitro experiments, but also has application prospects in in vivo experiments.

[0008] More specifically, the present invention mixes calcium chloride with etomidate and a protease inactivator to repair retinal ganglion cells, improve cell survival rate, repair the protrusion connection relationship of retinal ganglion cells, inhibit the apoptosis of mechanically damaged in vitro cultured retinal ganglion cells, and reduce the cell apoptosis rate.

[0009] Preferably, in the protective agent for mechanically damaged in vitro cultured retinal ganglion cells, the protease is a water-soluble protease containing a disulfide bond.

[0010] Preferably, in the protective agent for mechanically damaged in vitro cultured retinal ganglion cells, the water-soluble protease containing a disulfide bond is at least one of trypsin, chymotrypsin, and papain. These water-soluble proteases containing a disulfide bond undergo structural reconstruction after high-temperature treatment, such as a transformation of the secondary structure of the protein, such as a transformation of α-helix and β-sheet, a breakage and reconstruction of some hydrogen bonds, and a breakage of some disulfide bonds, thereby forming a carrier for loading etomidate and calcium chloride.

[0011] Preferably, the protease-inactivated material is prepared as a protective agent for mechanically damaged cultured retinal ganglion cells in vitro by the following method: dissolving protease in water, treating at a high temperature of 120°C to 140°C for 20 to 30 minutes, and removing water to obtain the protease-inactivated material. For example, water may be removed by rotary evaporation at 40°C to 60°C (e.g., 40°C, 50°C, or 60°C), or by freeze-drying at -10°C.

[0012] The invention provides a method for preparing a protective agent for mechanically damaged in vitro cultured retinal ganglion cells. The method comprises the following steps: preparing etomidate, a protease inactivator and calcium chloride in a ratio of 1 mmol: 1 g to 1.5 g: 0.1 mmol to 0.5 mmol, and uniformly mixing the mixture to obtain the protective agent for mechanically damaged in vitro cultured retinal ganglion cells.

[0013] The present invention also provides a use of a protective agent for mechanically damaged in vitro cultured retinal ganglion cells, wherein the use comprises at least one of the following:

[0014] (1) Improve the survival rate of mechanically damaged retinal ganglion cells in vitro;

[0015] (2) Repairing mechanically damaged neurite connections in cultured retinal ganglion cells;

[0016] (3) Inhibit the apoptosis of mechanically damaged retinal ganglion cells in vitro.

[0017] Preferably, the application of the protective agent for mechanically damaged in vitro cultured retinal ganglion cells is carried out by:

[0018] The protective agent for the mechanically damaged in vitro cultured retinal ganglion cells is added to the in vitro cultured retinal ganglion cell mechanical damage model.

[0019] Preferably, the amount of etomidate added per liter of the in vitro cultured retinal ganglion cell mechanical injury model liquid is: 1 μmol / L to 10 μmol / L, calculated as etomidate.

[0020] Preferably, the protective agent acts on the mechanically damaged in vitro cultured retinal ganglion cells for at least 4 days, preferably 4 to 8 days, more specifically 4, 5, 6, 7, or 8 days.

[0021] Preferably, the mechanical damage model refers to a scratch damage model, a pressure damage model or a tensile damage model.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention uses etomidate as a protective agent component. In a ratio of 1 mmol of etomidate, 1 g to 1.5 g of protease inactivated substance, and 0.1 mmol to 0.5 mmol of calcium chloride, the protease inactivated substance and calcium chloride synergistically enhance the protective effect of etomidate on mechanically damaged in vitro cultured retinal ganglion cells. Furthermore, the structure of the protease inactivated substance can load etomidate and calcium chloride to form a composite material with a sustained release effect, which helps to enhance the repair effect of mechanically damaged in vitro cultured retinal ganglion cells. Experimental results of the present invention show that when calcium chloride is mixed with etomidate and the protease inactivated substance, it can be used to increase the survival rate of retinal ganglion cells and inhibit retinal ganglion cell apoptosis. DETAILED DESCRIPTION

[0024] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below with reference to specific embodiments.

[0025] In the description of the present invention, unless otherwise specified, all reagents used are commercially available and all methods used are conventional techniques in the art.

[0026] Example 1

[0027] A protective agent for mechanically damaged in vitro cultured retinal ganglion cells, comprising etomidate, a protease inactivator, and calcium chloride, wherein the ratio of etomidate, protease inactivator, and calcium chloride is 1 mmol:1 g:0.1 mmol. The protease inactivator is prepared by the following method: dissolving the protease in water, treating it at a high temperature of 120°C for 30 minutes, and then rotary evaporating the water at 40°C. The solid matter collected is the protease inactivator. The water-soluble protease is papain (Wuhan Lanabai Pharmaceutical Chemical Co., Ltd., with a specific activity ≥10 units / mg protein).

[0028] Example 2

[0029] A protective agent for mechanically damaged in vitro cultured retinal ganglion cells comprises etomidate, a protease inactivator, and calcium chloride, wherein the ratio of etomidate, protease inactivator, and calcium chloride is 1 mmol:1.5 g:0.1 mmol. The protease inactivator is prepared by dissolving a protease in water, treating the protease at 120°C for 30 minutes, and then rotary evaporating the water at 40°C. The solid material collected is the protease inactivator. The water-soluble protease is papain (the source of the papain is the same as in Example 1).

[0030] Example 3

[0031] A protective agent for mechanically damaged in vitro cultured retinal ganglion cells comprises etomidate, a protease inactivator, and calcium chloride, wherein the ratio of etomidate, protease inactivator, and calcium chloride is 1 mmol:1.0 g:0.1 mmol. The protease inactivator is prepared by dissolving a protease in water, treating the solution at 120°C for 20 minutes, and then rotary evaporating the water at 40°C. The solid matter collected is the protease inactivator. The water-soluble protease is papain (the source of papain is the same as in Example 1).

[0032] Example 4

[0033] A protective agent for mechanically damaged in vitro cultured retinal ganglion cells comprises etomidate, a protease inactivator, and calcium chloride, wherein the ratio of etomidate, protease inactivator, and calcium chloride is 1 mmol:1.0 g:0.1 mmol. The protease inactivator is prepared by dissolving a protease in water, treating the protease at 140°C for 30 minutes, and then rotary evaporating the water at 40°C. The solid material collected is the protease inactivator. The water-soluble protease is papain (the source of the papain is the same as in Example 1).

[0034] Example 5

[0035] A protective agent for mechanically damaged in vitro cultured retinal ganglion cells, comprising etomidate, a protease inactivator, and calcium chloride, wherein the ratio of etomidate, protease inactivator, and calcium chloride is 1 mmol:1.0 g:0.1 mmol. The protease inactivator is prepared by dissolving the protease in water, treating it at 120°C for 30 minutes, and then rotary evaporating the water at 40°C. The solid material collected is the protease inactivator. The water-soluble protease is trypsin (Mingguang Guangjulong Laboratory Equipment Business Department, trypsin 1:250, enzyme activity ≥50,000 U / g).

[0036] Example 6

[0037] A protective agent for mechanically damaged in vitro cultured retinal ganglion cells, comprising etomidate, a protease inactivator, and calcium chloride, with the ratio of etomidate, protease inactivator, and calcium chloride being 1 mmol:1.0 g:0.1 mmol. The protease inactivator is prepared by dissolving the protease in water, treating it at 120°C for 30 minutes, and then rotary evaporating the water at 40°C. The solid material collected is the protease inactivator. The water-soluble protease is chymotrypsin (Beijing Wokai Biotechnology Co., Ltd., USP grade 1500 U / mg).

[0038] Example 7

[0039] A protective agent for mechanically damaged in vitro cultured retinal ganglion cells comprises etomidate, a protease inactivator, and calcium chloride, wherein the ratio of etomidate, protease inactivator, and calcium chloride is 1 mmol:1.0 g:0.3 mmol. The protease inactivator is prepared by dissolving a protease in water, treating the protease at 120°C for 30 minutes, and then rotary evaporating the water at 40°C. The solid material collected is the protease inactivator. The water-soluble protease is papain (the source of the papain is the same as in Example 1).

[0040] Example 8

[0041] A protective agent for mechanically damaged in vitro cultured retinal ganglion cells comprises etomidate, a protease inactivator, and calcium chloride, wherein the ratio of etomidate, protease inactivator, and calcium chloride is 1 mmol:1.0 g:0.5 mmol. The protease inactivator is prepared by dissolving a protease in water, treating the protease at 120°C for 30 minutes, and then rotary evaporating the water at 40°C. The solid matter collected is the protease inactivator. The water-soluble protease is papain (the source of the papain is the same as in Example 1).

[0042] The differences in the key points of operation in the above-mentioned Examples 1 to 8 are shown in Table 1.

[0043] Table 1 Differences in key operating points in Examples 1 to 8

[0044]

[0045]

[0046] In order to demonstrate the effect of the present invention, we conducted the following experiments:

[0047] 1. Culture of primary retinal ganglion cells

[0048] The study was conducted with reference to the literature “Zhao Xuan, Bai Jue, You Siwei, Cui Yuanyuan, Wu Mingmei, Protective effect of etomidate on mechanically damaged cultured retinal ganglion cells in vitro [J], Chinese Journal of Fundus Diseases, 2023, 39(6): 489-493”, as follows:

[0049] The present invention selected 20 healthy newborn SD rats, all of which were between 0 and 3 days old. Before the experiment, the SD rats were adaptively raised and cared for to ensure the health of the experimental animals.

[0050] In order to obtain the retinal tissue required for the experiment of the present invention, the SD rats must first be anesthetized and then killed. The eyeballs of the rats are then removed to avoid unnecessary damage to the eyeballs and surrounding tissues. After the eyeballs are removed, they are immediately transferred to a sterile operating environment, the retinal tissue is separated, and it is cut into pieces to obtain retinal tissue fragments. It should be noted that this experiment is only an exemplary introduction to the method of obtaining retinal tissue fragments, and those skilled in the art can also replace it with other methods of obtaining retinal tissue.

[0051] The retinal tissue fragments were digested with a 0.25g / 100mL trypsin solution. Trypsin can specifically act on the junction proteins between cells, allowing the cells to gradually dissociate from the tissue blocks. When the retinal tissue fragments were observed to become loose and most of the cells had been freed, Dulbecco's modified Eagle's medium (Beijing Noble Technology Co., Ltd.) containing 10% fetal bovine serum by volume was added to terminate the digestion reaction. Use a sterile pipette to repeatedly blow the tissue digestion solution after digestion, and through this physical method, the free cells are further dispersed to form a cell suspension. In order to remove incompletely digested tissue fragments and other impurities, the cell suspension was filtered through a sterile filter and then transferred to a centrifuge tube and centrifuged at 1000rpm and 4°C. After the centrifugation is completed, the supernatant is discarded. At this time, the precipitate at the bottom of the centrifuge tube is the relatively pure retinal cells. Next, add Neurobasal-A neuron culture medium containing 2% B27 and 2 mmol / L glutamine to the centrifuge tube. Use a sterile pipette to pipette the pellet at the bottom of the centrifuge tube again, resuspending the cells to create a single-cell suspension. B27 is a complex additive that provides comprehensive and essential nutrients to neurons, promoting cell growth and survival. Glutamine, as an important substrate for cellular energy metabolism, plays a key role in maintaining normal cellular physiological functions.

[0052] In order to accurately grasp the number of cells in the prepared cell suspension so as to perform precise inoculation according to the experimental design, the cells were counted using trypan blue staining.

[0053] Primary retinal ganglion cells were identified by immunofluorescence. Specifically, when the retinal ganglion cells were cultured for 7 days, the coverslip covered with cells was carefully removed from the culture vessel. Then, the coverslip was placed in a 4% paraformaldehyde solution and fixed for 4 hours. Paraformaldehyde can effectively fix the cell morphology and various components in the cell to prevent changes during subsequent operations. After fixation, the cells on the coverslip were blocked with 3% (v / v) bovine serum albumin-0.3% (v / v) Triton-X100 solution for 30 minutes. Subsequently, the coverslip was incubated with MAP2 (1:1000) and Thy1.1 (1:100) primary antibodies at 4°C overnight. The next day, the cells were incubated with AlexaFluor594 / 488-labeled secondary antibodies (1:800) for 2 hours. After incubation, 50% glycerol was used for sealing. Finally, the cells were observed under a fluorescence microscope to determine whether they were retinal ganglion cells by observing whether corresponding fluorescent signals appeared.

[0054] 2. Establishment of the Retinal Ganglion Cell Mechanical Injury Model and Experimental Group Setup

[0055] During the cell experiment, in order to further explore the effects of relevant factors on retinal ganglion cells, the present invention constructed a corresponding mechanical injury model.

[0056] Retinal ganglion cells were counted at a total of 5 × 10 5 The cells were inoculated at a density of 100 cells / well in a 24-well culture plate. The inoculated culture plate was placed in an incubator at 37°C and 5% CO2 for incubation for 7 days. When the cell fusion reached 80%, the cells were reasonably grouped. The following experimental groups were included: control group, scratch group, low-dose group in Example 1, medium-dose group in Example 1, high-dose group in Example 1, medium-dose group in Example 2, medium-dose group in Example 3, medium-dose group in Example 4, medium-dose group in Example 5, medium-dose group in Example 6, medium-dose group in Example 7, medium-dose group in Example 8, medium-dose group in control 1, and medium-dose group in control 2. The specific treatment methods of each group are as follows:

[0057] Control group: The cells were routinely cultured in neuronal culture medium without any additional treatment.

[0058] The scratch injury model uses a specific experimental method to simulate mechanical injury. Specifically, an iris knife is used to scratch the cultured retinal ganglion cells at intervals of 5 mm, thus establishing a mechanical injury model.

[0059] Example 1 low-dose group, Example 1 medium-dose group, and Example 1 high-dose group: First, refer to the treatment method of the scratch group to construct a mechanical injury model, and then immediately add different final concentrations of the protective agent of Example 1 to each well. Among them, the final concentration of Example 1 added to the low-dose group of Example 1 was 1 μmol / L (i.e., the amount added per liter of the in vitro cultured retinal ganglion cell mechanical injury model liquid was: calculated as etomidate, the concentration was 1 μmol / L), the final concentration of Example 1 added to the medium-dose group of Example 1 was 5 μmol / L (i.e., the amount added per liter of the in vitro cultured retinal ganglion cell mechanical injury model liquid was: calculated as etomidate, the concentration was 5 μmol / L), and the final concentration of Example 1 added to the high-dose group of Example 1 was 10 μmol / L (i.e., the amount added per liter of the in vitro cultured retinal ganglion cell mechanical injury model liquid was: calculated as etomidate, the concentration was 10 μmol / L).

[0060] In the dose group of Example 2, a mechanical injury model was first established by referring to the treatment method of the scratch group, and then the protective agent of Example 2 was immediately added to each well at a final concentration of 5 μmol / L (i.e., the amount of the protective agent added per liter of the in vitro cultured retinal ganglion cell mechanical injury model liquid was: calculated as etomidate, the concentration was 5 μmol / L).

[0061] Dose group in Example 3: First, refer to the treatment method of the scratch group to construct a mechanical injury model, and then immediately add the protective agent of Example 3 to each well at a final concentration of 5 μmol / L (i.e., the amount added per liter of in vitro cultured retinal ganglion cell mechanical injury model liquid is: calculated as etomidate, the concentration is 5 μmol / L).

[0062] Dose group in Example 4: First, refer to the treatment method of the scratch group to construct a mechanical injury model, and then immediately add the protective agent of Example 4 to each well at a final concentration of 5 μmol / L (i.e., the amount added per liter of in vitro cultured retinal ganglion cell mechanical injury model liquid is: calculated as etomidate, the concentration is 5 μmol / L).

[0063] Dose group in Example 5: First, refer to the treatment method of the scratch group to construct a mechanical injury model, and then immediately add the protective agent of Example 5 to each well at a final concentration of 5 μmol / L (i.e., the amount added per liter of in vitro cultured retinal ganglion cell mechanical injury model liquid is: calculated as etomidate, the concentration is 5 μmol / L).

[0064] In the dose group of Example 6, a mechanical injury model was first established by referring to the treatment method of the scratch group, and then the protective agent of Example 6 was immediately added to each well at a final concentration of 5 μmol / L (i.e., the amount of the protective agent added per liter of the in vitro cultured retinal ganglion cell mechanical injury model liquid was: calculated as etomidate, the concentration was 5 μmol / L).

[0065] Dose group in Example 7: First, refer to the treatment method of the scratch group to construct a mechanical injury model, and then immediately add the protective agent of Example 7 to each well at a final concentration of 5 μmol / L (i.e., the amount added per liter of in vitro cultured retinal ganglion cell mechanical injury model liquid is: calculated as etomidate, the concentration is 5 μmol / L).

[0066] Dose group in Example 8: First, refer to the treatment method of the scratch group to construct a mechanical injury model, and then immediately add the protective agent of Example 8 to each well at a final concentration of 5 μmol / L (i.e., the amount added per liter of in vitro cultured retinal ganglion cell mechanical injury model liquid is: calculated as etomidate, the concentration is 5 μmol / L).

[0067] Medium-dose etomidate group: First, refer to the treatment method of the scratch group to construct a mechanical injury model, and then immediately add etomidate to each well at a final concentration of 5 μmol / L (i.e., the amount of etomidate added per liter of mechanical injury model liquid of retinal ganglion cells cultured in vitro is: calculated as etomidate, the concentration is 5 μmol / L).

[0068] Control 1 medium-dose group: First, refer to the treatment method of the scratch group to construct a mechanical injury model, and then immediately add a protective agent of the control 1 medium-dose group to each well at a final concentration of 5 μmol / L (that is, the amount of liquid added per liter of in vitro cultured retinal ganglion cell mechanical injury model is: calculated as etomidate, the concentration is 5 μmol / L). The protective agent of the control 1 medium-dose group is composed of etomidate and protease inactivator, and the ratio of etomidate to protease inactivator is 1 mmol: 1 g. The protease inactivator is prepared according to the following method: the protease is placed at a high temperature of 120°C for 30 minutes, and the water is evaporated by rotary evaporation at 40°C. The collected solid substance is the protease inactivator. The water-soluble protease is papain (source is the same as in Example 1).

[0069] Control 2 Medium-Dose Group: First, a mechanical injury model was established using the same treatment as the scratch group. Immediately, a final concentration of 5 μmol / L (i.e., the amount of etomidate added per liter of in vitro cultured retinal ganglion cell mechanical injury model fluid) of the Control 2 Medium-Dose Group protective agent was added to each well. The protective agent for the Control 2 Medium-Dose Group consisted of etomidate and calcium chloride, with the ratio of etomidate to calcium chloride being 1 mmol:0.1 mmol.

[0070] 3. CCK-8 proliferation assay to detect the survival rate of retinal ganglion cells in each group

[0071] On day 7 after modeling, add 50 μl of CCK-8 reagent to each well. After adding the CCK-8 solution, place the culture plate in an incubator and incubate for 4 hours to allow the cells to fully react with the CCK-8. After the incubation period, transfer 200 μl of culture medium from each well to a 96-well plate. The 96-well plate is then placed on a microplate reader and the absorbance (A) of the cells is measured at a wavelength of 450 nm.

[0072] In order to more intuitively represent the survival of cells, we use a specific formula to calculate the cell survival rate. Cell survival rate (%) = 100% × (A 1干预 -A 空白 ) / (A 0干预 -A 空白 ).

[0073] In the above formula, A 0干预 represents the absorbance value of the control group; A 1干预 represents the absorbance value of the experimental group; A 空白 is the absorbance value of blank (culture medium + CCK-8 reagent).

[0074] Six replicate wells were set up for each experiment to reduce experimental errors.

[0075] 4. Annexin V / PI double staining to detect retinal ganglion cell apoptosis in each group

[0076] To further explore the impact of modeling on retinal ganglion cell apoptosis, we conducted a cell apoptosis rate detection experiment 7 days after modeling.

[0077] The total number is 2×10 6 The cell suspension was inoculated into a 24-well culture plate at a seeding density of 100 cells / well. Finally, the inoculated culture plate was placed in an incubator at 37°C and 5% CO2 for 7 days.

[0078] First, the cells were digested using 0.125g / 100g trypsin (without EDTA). During the digestion process, the cell state must be closely observed. Once the cell protrusions are found to retract, this indicates that the cells have begun to detach from the culture dish surface, and the digestion should be stopped immediately. Premature termination will result in incomplete cell digestion, while too late termination will cause excessive damage to the cells, affecting the results of subsequent experiments. After terminating the digestion, the cells were collected by centrifugation. Next, the cells were resuspended in 100μl binding buffer. Subsequently, 10μl Annexin V / fluorescein isothiocyanate was added, mixed thoroughly with the cells, placed on ice and incubated in the dark for 15 minutes. After that, 400μl binding buffer was added to further dilute the reaction system, and 5μl PI (propidium iodide) was added. After mixing thoroughly with the cells again, the mixture was incubated on ice in the dark for 5 minutes. Finally, the stained cell suspension was thoroughly mixed and detected by flow cytometry. The cell apoptosis rate was calculated. Six replicate wells were set up for each group of experiments, and the average value was taken.

[0079] Apoptosis rate = 100% × (number of early apoptotic cells + number of late apoptotic cells) / (number of live cells + number of early apoptotic cells + number of late apoptotic cells + number of necrotic cells)

[0080] 5. Experimental results

[0081] It should be noted that since the survival rate and apoptosis rate are calculated using different methods, the sum of the two is not exactly equal to 100%.

[0082] The results of retinal ganglion cell survival rate and apoptosis rate are shown in Table 2. The results show that compared with the scratch group, the protective agents of Examples 1 to 8 can protect mechanically damaged in vitro cultured retinal ganglion cells. By comparing the retinal ganglion cell survival rate and retinal ganglion cell apoptosis rate of the medium-dose group of Control 1, the medium-dose group of Control 2, and the medium-dose group of Example 1, it can be seen that the combination of calcium chloride and etomidate alone, or the combination of etomidate and protease inactivator alone, resulted in a lower retinal ganglion cell survival rate and a higher retinal ganglion cell apoptosis rate than the groups in the examples.

[0083] Table 2 Survival rate and apoptosis rate of retinal ganglion cells

[0084]

[0085]

[0086] The protective agent for repairing mechanically damaged in vitro cultured retinal ganglia provided by the present invention has at least the following application prospects:

[0087] (1) It is convenient for experimenters to precisely control experimental conditions during the study of mechanical damage in an in vitro culture environment and observe a series of changes in cells after mechanical damage, including the cell repair process, activation of related signaling pathways, and changes in gene expression, which helps to reveal the intrinsic mechanism of retinal ganglion cell damage and repair.

[0088] (2) The survival and apoptosis of retinal ganglion cells are associated with a variety of ophthalmic diseases, including glaucoma, retinal vascular occlusion, and diabetic retinopathy. The protective agent of the present invention repairs mechanically damaged retinal ganglion cells by studying in vitro culture, providing clues to the pathophysiological mechanisms of these diseases, thereby providing a theoretical basis for the development of new treatments and drug targets.

[0089] (3) The protective agent of the present invention has a protective effect on retinal ganglion cells and can therefore also be used to develop neuroprotective and regenerative therapies. For example, it can be combined with growth factors, stem cell transplantation, gene therapy, and other methods to promote the activity recovery and regeneration of damaged retinal ganglion cells.

[0090] (4) The research on the protective effect of the protective agent of the present invention on the repair of mechanically damaged retinal ganglion cells will help promote the application of tissue engineering in the field of ophthalmology. It is also possible to attempt to construct functional retinal neural tissue by simulating the in vivo environment and utilizing biomaterials and cell engineering technologies, laying the foundation for the future realization of retinal tissue replacement therapy.

[0091] It should be noted that when numerical ranges are mentioned in the present invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints may be selected. Since the steps and methods used are the same as those in the embodiments, in order to avoid redundancy, the present invention describes preferred embodiments. Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they understand the inventive concept of the basic invention, and such changes and modifications fall within the scope of the present invention.

[0092] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. If these modifications and variations of the present invention fall within the scope of the equivalent technology of the present invention, the present invention is intended to include these modifications and variations.

Claims

1. A protective agent for mechanically damaged in vitro cultured retinal ganglion cells, characterized in that: The invention is composed of etomidate, protease inactivator and calcium chloride, wherein the ratio of etomidate, protease inactivator and calcium chloride is 1mmol:1g-1.5g:0.1mmol-0.5mmol; the protease inactivator refers to a substance obtained by inactivating water-soluble protease at high temperature, wherein the temperature of high temperature inactivation is 120°C-140°C.

2. The protective agent for mechanically damaged in vitro cultured retinal ganglion cells according to claim 1, characterized in that: The protease is a water-soluble protease containing a disulfide bond.

3. The protective agent for mechanically damaged in vitro cultured retinal ganglion cells according to claim 2, characterized in that: The water-soluble protease containing a disulfide bond is at least one of trypsin, chymotrypsin and papain.

4. The protective agent for mechanically damaged in vitro cultured retinal ganglion cells according to claim 1, characterized in that: Protease inactivated material was prepared according to the following method: The protease is dissolved in water, placed under a high temperature of 120° C. to 140° C. for treatment for 20 min to 30 min, and the water is removed to obtain the protease inactivated product.

5. The method for preparing a protective agent for mechanically damaged in vitro cultured retinal ganglion cells according to claim 1, characterized in that: Etomidate, protease inactivator and calcium chloride are prepared in a ratio of 1 mmol: 1 g to 1.5 g: 0.1 mmol to 0.5 mmol, and mixed evenly to obtain a protective agent for mechanically damaged in vitro cultured retinal ganglion cells.

6. Use of the protective agent for mechanically damaged in vitro cultured retinal ganglion cells according to claim 1, characterized in that: The application refers to at least one of the following: (1) Improve the survival rate of mechanically damaged retinal ganglion cells in vitro; (2) Repairing mechanically damaged neurite connections in cultured retinal ganglion cells; (3) Inhibit the apoptosis of mechanically damaged retinal ganglion cells in vitro.

7. Use of the protective agent for mechanically damaged in vitro cultured retinal ganglion cells according to claim 6, characterized in that: The operation method of the application is: The protective agent for the mechanically damaged in vitro cultured retinal ganglion cells is added to the in vitro cultured retinal ganglion cell mechanical damage model.

8. Use of the protective agent for mechanically damaged in vitro cultured retinal ganglion cells according to claim 7, characterized in that: The amount of the protective agent added to the mechanically damaged in vitro cultured retinal ganglion cells is: 1 μmol / L to 10 μmol / L calculated as etomidate.

9. Use of the protective agent for mechanically damaged in vitro cultured retinal ganglion cells according to claim 8, characterized in that: The protective agent for mechanically damaged in vitro cultured retinal ganglion cells has an action time of no less than 4 days.

10. Use of the protective agent for mechanically damaged in vitro cultured retinal ganglion cells according to claim 6, characterized in that: The mechanical damage model refers to a scratch damage model, a pressure damage model or a tensile damage model.

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