Eye drops containing multi-element trace elements and stem cell extract as well as preparation method and application of eye drops

Abstract

The invention relates to the technical field of biological medicines, in particular to eye drops containing multi-element trace elements and stem cell extracts as well as a preparation method and application of the eye drops. The eye drops are prepared from the following components in parts by weight: 0.5 to 1.5 parts of mesenchymal stem cell supernatant, 0.05 to 0.15 part of mesenchymal stem cell extract and 5 to 15 parts of sodium hyaluronate solution, the mesenchymal stem cell supernatant and the mesenchymal stem cell extract contain multiple trace elements. The invention has the advantages of high tear secretion amount, high tear film stability and good corneal injury repairing effect.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly relates to an eye drop containing multiple trace elements and stem cell extracts, a preparation method thereof, and an application thereof. Background Art

[0002] Common symptoms of dry eye include dryness, redness, pain, foreign body sensation, blurred vision, photophobia and tearing in the eyes. Among various eye diseases, dry eye, as a common disease, seriously affects the quality of life of patients. At present, traditional eye drops applicable to dry eye often contain various antibiotics and various chemical drug components, or are simply aqueous solutions of a single medium that only play a moisturizing role. Their effects in repairing corneal and conjunctival injuries of the eyes and restoring the stability of the tear film are not obvious. Therefore, there is an urgent need for a new type of eye drop to solve these problems.

[0003] Trace elements play an important role in eye development. For example, Zn, Se, Cu, etc. play an important role in retinal health. They are involved in the formation and maintenance of photoreceptor cells in the retina, and help protect the retina from oxidative damage and light damage. Ga, Mg, etc. are involved in nerve conduction and play an important role in visual conduction and visual information processing. They help maintain the normal function of nerve conduction in the eyes. Cu, Zn, Fe, etc. play an important role in the health of eye blood vessels, help maintain the elasticity and stability of eye blood vessels, and help prevent the occurrence of eye blood vessel diseases.

[0004] Sodium hyaluronate is a mucopolysaccharide widely present in the interstitial substance of animal tissue cells. It is currently the best medium in ophthalmic preparations. It can not only increase the bioavailability of drugs, but also reduce the irritation of drugs to the eyes, reduce the adverse reactions of preservatives in the preparations, promote the healing of corneal injuries, and quickly relieve eye discomfort symptoms.

[0005] The prior art (Huang Shouqiang. Efficacy study on the treatment of Sjogren's syndrome-related dry eye in mice by human umbilical cord mesenchymal stem cells [D]. Anhui Medical University, 2016.) discloses three transplantation treatment methods, namely tail vein injection, lacrimal gland injection and eye drop, to transplant human umbilical cord mesenchymal stem cells into dry eye mice.

[0006] Chinese Patent Publication No. CN111658607A discloses a sustained-release eye drop of umbilical cord mesenchymal stem cells, which includes separately packaged powder A and separately packaged solvent B. Each portion of the sustained-release eye drop of umbilical cord mesenchymal stem cells includes 0.1 mg - 0.5 mg of powder A and 10 g of solvent B; powder A is the dried powder of the culture supernatant of umbilical cord mesenchymal stem cells; by weight, solvent B includes 0.2 - 0.6 parts of sodium hyaluronate, 0.15 - 0.5 parts of taurine, 0.1 - 0.3 parts of sodium alginate and 98.6 - 99.55 parts of sterile water.

[0007] However, the above-mentioned eye drops containing umbilical cord mesenchymal stem cells or the supernatant of umbilical cord mesenchymal stem cells and sodium hyaluronate can only supplement the amount of tears in the short term and temporarily relieve the damage of corneal epithelium, and cannot repair the eyes and improve the stability of the tear film.

[0008] Therefore, it is very necessary to develop an eye drop containing multiple trace elements and stem cell extracts, as well as its preparation method and application, which can solve the above technical problems. Summary of the Invention

[0009] The object of the present invention is to overcome the deficiencies of the prior art and provide an eye drop containing multiple trace elements and stem cell extracts, as well as its preparation method and application, which have a high tear secretion volume, a high tear film stability, and a good effect of repairing corneal damage.

[0010] The present invention is achieved by the following technical solutions:

[0011] In the first aspect of the present invention, an eye drop containing multiple trace elements and stem cell extracts is provided. By weight, it includes the following components: 0.5 - 1.5 parts of mesenchymal stem cell supernatant, 0.05 - 0.15 parts of mesenchymal stem cell extract, and 5 - 15 parts of sodium hyaluronate solution;

[0012] The mesenchymal stem cell supernatant and the mesenchymal stem cell extract contain multiple trace elements.

[0013] As an embodiment of the present invention, the mesenchymal stem cells include one or more of umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, placental mesenchymal stem cells, amniotic mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, bone marrow mesenchymal stem cells, skin mesenchymal stem cells, dental pulp mesenchymal stem cells, urine-derived mesenchymal stem cells, and mesenchymal stem cells derived from induced pluripotent stem cells.

[0014] As an embodiment of the present invention, the multiple trace elements include Al, Mg, Ba, Mn, Ca, Mo, Cd, Ni, Co, Se, Cr, Sr, Cu, Ti, Fe, V, and Zn.

[0015] Preferably, the specific composition of the multiple trace elements is as follows: the Al content is 500 - 5000 mg / L, the Mg content is 500 - 5000 mg / L, the Ba content is 0.01 - 0.1 mg / L, the Mn content is 50 - 500 mg / L, the Ca content is 50 - 500 mg / L, the Mo content is 1 - 10 mg / L, the Cd content is 0.001 - 0.01 mg / L, the Ni content is 1 - 10 mg / L, the Co content is 0.5 - 5 mg / L, the Se content is 10 - 100 mg / L, the Cr content is 1 - 10 mg / L, the Sr content is 0.5 - 5 mg / L, the Cu content is 0.5 - 5 mg / L, the Ti content is 50 - 500 mg / L, the Fe content is 500 - 5000 mg / L, the V content is 1 - 10 mg / L, and the Zn content is 100 - 1000 mg / L.

[0016] As an embodiment of the present invention, the mesenchymal stem cell supernatant is: the mesenchymal stem cell supernatant obtained by centrifugation and filtration after adding multiple trace elements to the mesenchymal stem cell culture system.

[0017] As an embodiment of the present invention, the mesenchymal stem cell extract is: the mesenchymal stem cells obtained after adding multiple trace elements to the mesenchymal stem cell culture system, and the mesenchymal stem cell extract obtained by ultrasonic lysis extraction method.

[0018] In the present invention, adding a certain amount of multiple trace elements to the mesenchymal stem cell culture system can promote cell proliferation, maintain the young cell morphology and enhance cell adhesion ability. After being ingested by cells, the multiple trace elements can exist in the cell matrix and various cell organelles, and at the same time, they can be encapsulated and excreted into the cell culture medium during the formation of intracellular vesicles, forming extracellular vesicles loaded with multiple trace elements, and these extracellular vesicles can transport the multiple trace elements they carry across the membrane into the target cells to play a role.

[0019] The mesenchymal stem cell supernatant is rich in a variety of cytokines secreted by it. The cell extract obtained after cell lysis also contains various components such as cytokines, active proteases, and mitochondria. All of the above components play an active role in repairing eye tissue damage and promoting optic nerve development.

[0020] Preferably, the ultrasonic lysis extraction method specifically includes the following steps: washing the obtained mesenchymal stem cells by centrifugation with physiological saline, resuspending with the mesenchymal stem cell supernatant, ultrasonically lysing, and filtering to obtain.

[0021] The present invention preferably uses the mesenchymal stem cell supernatant to resuspend mesenchymal stem cells. As an alternative, physiological saline, 1‰ sodium hyaluronate solution, or injection water can also be used for resuspension.

[0022] More preferably, the density of resuspension with the supernatant of the mesenchymal stem cells is 1×10 6 -1×10 7 / mL; the time of ultrasonic lysis is 20 - 60 s, wherein each duration is 1 - 3 s and the interval is 3 - 9 s; the filtration is first through a 2000 - mesh sterilized filter screen and then through a 0.22 - μm sterile filter membrane.

[0023] Preferably, the preparation process of the mesenchymal stem cell culture system is as follows: The stem cells are subcultured to the P3 generation using a serum - free stem cell medium, the seed cells are cryopreserved, the P3 - generation seed cells are batch - resuscitated using a serum - free stem cell medium, multiple trace elements are added during the resuscitation process, and the cells are further subcultured to the P5 - P8 generation to obtain the mesenchymal stem cell culture system.

[0024] Preferably, each time multiple trace elements are added, the addition ratio needs to be screened through a cell proliferation experiment. The experimental content is as follows:

[0025] The stock solution of multiple trace elements is diluted with a serum - free stem cell medium at dilution multiples of 1000, 1500, 2000, 2500, and 3000. Umbilical cord stem cells of the 4th generation are resuscitated. When the cell confluence is about 80%, the cells are digested and counted. The cells are evenly divided into 6 parts, centrifuged, and resuspended with the above - mentioned 5 groups of media supplemented with multiple trace elements and the medium without trace elements (control group) respectively. The density is adjusted to 15000 / mL. 200 μL of the above - mentioned 6 groups of resuspended umbilical cord stem cells are added to each column of a 96 - well plate. When the confluence reaches about 80%, the cell proliferation ability is detected by the MTT method. Steps of the MTT method: Under dark conditions in a 96 - well plate, 5 mg / mL MTT solvent (thiazolyl blue) is added, 10 μL is added to each well, and the cells are further cultured at 37°C for 4 h, then the culture medium is discarded. 200 μL of DMSO is added to each well, and the mixture is shaken well. After complete color development (20 min - 30 min), the absorbance OD value at 490 nm is measured with an enzyme - linked immunosorbent assay (ELISA) reader, and the cell proliferation rate is calculated. The cell proliferation (relative activity) rate (%) = OD value of the trace element group / OD value of the control group × 100%. According to the experimental results, the dilution multiple X of multiple trace elements with a cell proliferation (relative activity) rate > 1, especially the optimal cell proliferation rate max (the group with the highest cell proliferation rate) can be selected. Generally, when the cell proliferation (relative activity) rate > 1, the dilution multiple is 2000 - 3000 times.

[0026] In the preparation process of the mesenchymal stem cell culture system of the present invention, there is no special requirement for the serum - free stem cell medium, and commercially available serum - free mesenchymal stem cell media can all meet the requirements of the present invention.

[0027] More preferably, the last generation is cultured in an in vitro 3D culture system that simulates the in - vivo microenvironment.

[0028] According to different production scales and cell usage amounts, the cells need to be passaged to P5 or P6 or P7 or P8 generations. Select a suitable passage generation according to the production scale and cell usage amount. Within this range, the passage generation has basically no effect on the effect of the eye drops. Only in the last generation, an in vitro 3D culture system that simulates the in vivo microenvironment is used for culture to harvest cells and supernatants to the maximum extent.

[0029] In the present invention, the multiple trace elements are added in each generation after resuscitation.

[0030] More preferably, the stem cells are extracted from tissues such as neonatal umbilical cord, placenta, amniotic membrane, or healthy adult subcutaneous fat, skin, dental pulp, etc.

[0031] More preferably, during the resuscitation process, the addition ratio of the multiple trace elements in the serum-free stem cell culture medium is X max :1 (referring to the dilution multiple of the multiple trace elements, that is, 1 mL of multiple trace elements is contained in X max mL of serum-free stem cell culture medium).

[0032] As an embodiment of the present invention, the concentration of the sodium hyaluronate solution is 0.05 - 0.5 wt%.

[0033] Preferably, the preparation process of the sodium hyaluronate solution is: weigh 0.5 - 5 g of sodium hyaluronate, measure 1000 ml of physiological saline to completely dissolve the sodium hyaluronate, and pass through a 0.22 μm sterile filter membrane to obtain it.

[0034] As an embodiment of the present invention, the eye drops further include at least one of a pH regulator and an osmotic pressure regulator.

[0035] Preferably, the dosage of the pH regulator is 0.01 - 0.5 parts, and the dosage of the osmotic pressure regulator is 0.01 - 0.5 parts.

[0036] Preferably, the pH regulator includes one or more of sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, boric acid, borax, acetic acid, sodium acetate, citric acid, sodium citrate, tartaric acid, sodium tartrate, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, hydrochloric acid, and phosphoric acid.

[0037] Preferably, the osmotic pressure regulator includes one or more of sodium chloride, potassium chloride, boric acid, borax, sodium sulfate, potassium sulfate, sodium nitrate, potassium nitrate, sodium acetate, mannitol, glycerol, propylene glycol, and glucose.

[0038] As an embodiment of the present invention, the eye drops do not contain preservatives.

[0039] The second aspect of the present invention provides a preparation method of the above eye drops, comprising the following steps: uniformly mixing the mesenchymal stem cell supernatant, the mesenchymal stem cell extract, and the sodium hyaluronate solution, and adding a pH regulator and an osmotic pressure regulator as needed to obtain the eye drops.

[0040] Preferably, the eye drops are stored in an environment of 2-8 °C.

[0041] The third aspect of the present invention provides an application of the above eye drops or the eye drops prepared by the above preparation method in the preparation of a drug for treating dry eye.

[0042] The beneficial effects of the present invention are as follows:

[0043] (1) By adding a certain proportion of multiple trace elements, the present invention can promote the proliferation of mesenchymal stem cells, thereby harvesting more stem cells.

[0044] (2) In the eye drops of the present invention, there are various anti-inflammatory factors and growth factors secreted by mesenchymal stem cells, which can relieve the ocular inflammatory reaction and promote the repair of ocular tissue damage. In addition, among the various trace elements contained in the eye drops, a part exists in a free state and can directly act on various tissues of the ocular surface, and another part exists in the cell matrix components and is encapsulated in exosomes, and is delivered to deep ocular tissue sites to promote local metabolism and maintain normal biological functions, such as promoting the proliferation of lacrimal gland cells and promoting tear secretion.

[0045] (3) The present invention simultaneously adds the mesenchymal stem cell supernatant containing multiple trace elements and the mesenchymal stem cell extract containing multiple trace elements. Compared with adding only any one of them, the two have a significant synergistic effect in increasing the tear secretion volume, improving the tear film stability, and improving the corneal injury repair effect.

[0046] (4) When multiple trace elements are added during the stem cell culture process in the present invention, compared with directly adding multiple trace elements, some trace elements that are not easily absorbed by the human body can exist in the cell matrix components and be encapsulated in exosomes, which is more conducive to absorption and can increase the tear secretion volume, tear film stability, and corneal injury repair effect.

[0047] (5) The present invention simultaneously adds the mesenchymal stem cell supernatant, the mesenchymal stem cell extract, and multiple trace elements. Compared with adding only the mesenchymal stem cell supernatant and the mesenchymal stem cell extract, or only adding multiple trace elements, the three have a significant synergistic effect in increasing the tear secretion volume, improving the tear film stability, and improving the corneal injury repair effect. Description of the Drawings

[0048] Figure 1Growth state diagram of umbilical cord mesenchymal stem cells in Example 1 of the present invention; in the figure, (a) represents the seed cell diagram of passage P3, and (b) represents the stem cell diagram cultured in a 3D system of passage P6;

[0049] Figure 2 It is a diagram showing the effect of multi - element trace elements with different addition ratios on the proliferation rate of umbilical cord mesenchymal stem cells in Example 1 of the present invention. Detailed implementation manners

[0050] The present invention will be further described below in conjunction with specific embodiments. The advantages and features of the present invention will become clearer as the description progresses. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that without departing from the spirit and scope of the present invention, modifications or substitutions can be made to the details and forms of the technical solutions of the present invention, but these modifications and substitutions all fall within the protection scope of the present invention. All kinds of reagents used in the embodiments of the present invention are obtained through conventional commercial channels unless otherwise specified.

[0051] The specific composition of the multi - element trace elements adopted in each embodiment of the present invention is as follows: the content of Al is 1460 mg / L, the content of Mg is 1790 mg / L, the content of Ba is 0.032 mg / L, the content of Mn is 154 mg / L, the content of Ca is 85.2 mg / L, the content of Mo is 3.9 mg / L, the content of Cd is 0.0074 mg / L, the content of Ni is 4.25 mg / L, the content of Co is 1.39 mg / L, the content of Se is 21.1 mg / L, the content of Cr is 6.87 mg / L, the content of Sr is 0.89 mg / L, the content of Cu is 0.82 mg / L, the content of Ti is 104 mg / L, the content of Fe is 1920 mg / L, the content of V is 6.28 mg / L, and the content of Zn is 606 mg / L.

[0052] Example 1

[0053] An eye drop containing multi - element trace elements and stem cell extract, comprising the following components by weight: 1 part of umbilical cord stem cell supernatant containing multi - element trace elements, 0.1 part of umbilical cord stem cell extract containing multi - element trace elements, and 10 parts of sodium hyaluronate solution.

[0054] The preparation method of the umbilical cord stem cell supernatant containing multi - element trace elements is as follows: Stem cells are extracted from neonatal umbilical cord tissue and passaged and cultured using a serum - free stem cell medium (prepared by mixing MEM Alpha medium of Gibco company and EliteGro serum substitute of EliteCell company according to a volume ratio of 20:1). The cell seeding density is 10000 cell / cm 2, Cultured to passage P3, cryopreserved the seed cells, batch-resuscitated the P3-generation seed cells and passaged them to P6. When resuscitating the P3-generation stem cells, multi-trace elements were added. The addition ratio of this multi-trace element in the culture medium was 2500:1 (dilution factor of the multi-trace element). The supernatant and umbilical cord stem cells containing multi-trace elements were harvested. The supernatant was centrifuged and filtered through a 0.45 μm filter membrane to obtain the supernatant of umbilical cord stem cells containing multi-trace elements.

[0055] The preparation method of the extract of umbilical cord stem cells containing multi-trace elements was that the umbilical cord stem cells were centrifuged and washed 3 times with normal saline, counted, and resuspended with the harvested supernatant of umbilical cord stem cells containing multi-trace elements at a density of 5×10 6 / mL, ultrasonically lysed for 40 s (2 s each time, with an interval of 6 s), passed through a multi-layer sterilized filter screen with 2000 meshes, and then passed through a 0.22 μm sterile filter membrane to obtain the extract of umbilical cord stem cells containing multi-trace elements.

[0056] The growth state diagram of the umbilical cord mesenchymal stem cells in this example is as Figure 1 shown. This example also studied the influence of different addition ratios of multi-trace elements on the proliferation rate of umbilical cord mesenchymal stem cells, as Figure 2 shown. Among them, the "*control" group indicated that no trace elements were added.

[0057] The preparation method of the sodium hyaluronate solution was as follows: Weigh 1 g of sodium hyaluronate, measure 1000 ml of normal saline to completely dissolve the sodium hyaluronate, the solution density was uniform and there were no lumps, and then passed through a 0.22 μm sterile filter membrane.

[0058] The preparation method of the eye drops included the following steps: Mix the supernatant of umbilical cord stem cells containing multi-trace elements, the extract of umbilical cord stem cells containing multi-trace elements, and the sodium hyaluronate solution evenly to obtain the eye drops.

[0059] Example 2

[0060] The difference between Example 2 and Example 1 was the different composition ratios. In this example, it included the following composition components, by weight: 0.5 parts of the supernatant of umbilical cord stem cells containing multi-trace elements, 0.05 parts of the extract of umbilical cord stem cells containing multi-trace elements, and 10 parts of the sodium hyaluronate solution. The rest was the same as in Example 1.

[0061] Example 3

[0062] The difference between Example 3 and Example 1 was the different composition ratios. In this example, it included the following composition components, by weight: 1.5 parts of the supernatant of umbilical cord stem cells containing multi-trace elements, 0.15 parts of the extract of umbilical cord stem cells containing multi-trace elements, and 10 parts of the sodium hyaluronate solution. The rest was the same as in Example 1.

[0063] Example 4

[0064] An eye drop containing multiple trace elements and stem cell extract, comprising the following components by weight: 1 part of adipose stem cell supernatant containing multiple trace elements, 0.1 part of adipose stem cell extract containing multiple trace elements, and 10 parts of sodium hyaluronate solution.

[0065] The preparation method of adipose stem cell supernatant containing multiple trace elements is as follows: Stem cells are extracted from the abdominal adipose tissue of healthy adults and passaged and cultured using a serum-free stem cell medium (prepared by mixing MEM Alpha medium from Gibco and EliteGro serum substitute from EliteCell in a volume ratio of 20:1). The cell seeding density is 10000 cells / cm 2 , cultured to passage 3 (P3), the seed cells are cryopreserved, and a batch of P3 seed cells are resuscitated and passaged to P6. Multiple trace elements are added when the P3 stem cells are resuscitated. The addition ratio of this multiple trace element in the medium is 2500:1 (dilution factor of multiple trace elements). The supernatant and adipose stem cells containing multiple trace elements are harvested. The supernatant is centrifuged and filtered through a 0.45 μm filter membrane to obtain adipose stem cell supernatant containing multiple trace elements.

[0066] The preparation method of adipose stem cell extract containing multiple trace elements is as follows: The adipose stem cells are centrifuged and washed 3 times with physiological saline, counted, and resuspended with the harvested adipose stem cell supernatant containing multiple trace elements at a density of 5×10 6 / mL, ultrasonically lysed for 40 s (2 s each time, with an interval of 6 s), passed through a multi-layer sterilized filter screen with 2000 meshes, and then through a 0.22 μm sterile filter membrane to obtain adipose stem cell extract containing multiple trace elements.

[0067] The preparation method of sodium hyaluronate solution is as follows: Weigh 1 g of sodium hyaluronate, measure 1000 ml of physiological saline, completely dissolve the sodium hyaluronate, and the solution has a uniform density and no lumps, and is filtered through a 0.22 μm sterile filter membrane.

[0068] The preparation method of the eye drop includes the following steps: Mix the adipose stem cell supernatant containing multiple trace elements, the adipose stem cell extract containing multiple trace elements, and the sodium hyaluronate solution evenly to obtain the eye drop.

[0069] Example 5

[0070] The difference between Example 5 and Example 4 lies in the different component ratios. In this example, it includes the following components by weight: 0.5 part of adipose stem cell supernatant containing multiple trace elements, 0.05 part of adipose stem cell extract containing multiple trace elements, and 10 parts of sodium hyaluronate solution. The rest is the same as Example 4.

[0071] Example 6

[0072] Example 6 is different from Example 4 in that the composition ratio is different. In this example, it includes the following components, by weight: 1.5 parts of adipose-derived stem cell supernatant containing multiple trace elements, 0.15 part of adipose-derived stem cell extract containing multiple trace elements, and 10 parts of sodium hyaluronate solution. The rest is the same as in Example 4.

[0073] Example 7

[0074] Example 7 is different from Example 1 in that the component contents of the added trace elements are different. Among them, the content of Al is 500 mg / L, the content of Mg is 500 mg / L, the content of Ba is 0.01 mg / L, the content of Mn is 50 mg / L, the content of Ca is 50 mg / L, the content of Mo is 1 mg / L, the content of Cd is 0.001 mg / L, the content of Ni is 1 mg / L, the content of Co is 0.5 mg / L, the content of Se is 10 mg / L, the content of Cr is 1 mg / L, the content of Sr is 0.5 mg / L, the content of Cu is 0.5 mg / L, the content of Ti is 50 mg / L, the content of Fe is 500 mg / L, the content of V is 1 mg / L, and the content of Zn is 100 mg / L.

[0075] Example 8

[0076] Example 8 is different from Example 1 in that the component contents of the added trace elements are different. Among them, the content of Al is 5000 mg / L, the content of Mg is 5000 mg / L, the content of Ba is 0.1 mg / L, the content of Mn is 500 mg / L, the content of Ca is 500 mg / L, the content of Mo is 10 mg / L, the content of Cd is 0.01 mg / L, the content of Ni is 10 mg / L, the content of Co is 5 mg / L, the content of Se is 100 mg / L, the content of Cr is 10 mg / L, the content of Sr is 5 mg / L, the content of Cu is 5 mg / L, the content of Ti is 500 mg / L, the content of Fe is 5000 mg / L, the content of V is 10 mg / L, and the content of Zn is 1000 mg / L.

[0077] Comparative Example 1

[0078] On the basis of Example 1, the addition of multiple trace elements was cancelled during the resuscitation and culture of P3-generation stem cells.

[0079] Comparative Example 2

[0080] On the basis of Example 4, the addition of multiple trace elements was cancelled during the resuscitation and culture of P3-generation stem cells.

[0081] Comparative Example 3

[0082] On the basis of Examples 1 and 4, the addition of the stem cell supernatant and the stem cell extract in the eye drop formulation was cancelled, and it was only a sodium hyaluronate solution.

[0083] Comparative Example 4

[0084] On the basis of Example 1, the umbilical cord stem cell supernatant containing multiple trace elements was adjusted to 1.1 parts, and the umbilical cord stem cell extract containing multiple trace elements was not added.

[0085] Comparative Example 5

[0086] On the basis of Example 1, the umbilical cord stem cell supernatant containing multiple trace elements was not added, and the umbilical cord stem cell extract containing multiple trace elements was adjusted to 1.1 parts.

[0087] Comparative Example 6

[0088] On the basis of Example 1, the multiple trace elements were not added during the resuscitation and culture of P3 generation stem cells, but were directly added during the preparation of the eye drops. The specific steps are as follows:

[0089] Mix the mesenchymal stem cell supernatant, the mesenchymal stem cell extract, the sodium hyaluronate solution and the multiple trace elements evenly.

[0090] Comparative Example 7

[0091] On the basis of Examples 1 and 4, the addition of the stem cell supernatant and the stem cell extract in the formulation was cancelled, and the multiple trace elements were directly added to the sodium hyaluronate solution, and the eye drops were obtained. The specific steps are as follows:

[0092] Weigh 1 g of sodium hyaluronate, measure 1000 ml of normal saline to completely dissolve the sodium hyaluronate, and filter it through a 0.22 μm sterile filter membrane to obtain a 1‰ sodium hyaluronate solution. Dilute the multiple trace elements 2500 times with the 1‰ sodium hyaluronate solution to obtain the eye drops.

[0093] Experimental Example

[0094] I. The protein concentration, osmotic pressure and pH of the eye drops prepared in the above Examples 1-8 and Comparative Examples 1-7 were detected respectively, and the specific results are shown in Table 1.

[0095] II. The irritation tests of the eye drops prepared in the above Examples 1-8 and Comparative Examples 1-7 were compared respectively:

[0096] Experimental method: BALB / c mice were selected, and a dry eye model was established by locally instilling benzalkonium chloride solution into the eyes. The animals were randomly divided into a control group and an experimental group. A 2 g / L benzalkonium chloride solution was instilled into both eyes twice a day for 2 consecutive weeks to establish a moderate to severe dry eye model. After successful modeling, treatment began. The control group was not treated. The experimental group was treated with the eye drops of Examples 1-8 and Comparative Examples 1-7. 20 μL was instilled into each eye three times a day for 7 consecutive days. Record the changes before and after drug intervention. For example, observe and record the tear film function-related indicators of the mice 1, 4, and 7 days before and after treatment.

[0097] Tear secretion test (Schirmer I test, SIT): Take a phenol red cotton thread, place one end at the outer 1 / 3 of the outer canthus of the right eye of the mouse, and time for 60 s. Use a vernier caliper to measure the length of the red part of the phenol red cotton thread and calculate the tear secretion volume. When conducting each examination, attention should be paid to controlling variables, and it should be operated by the same person at the same time, place, lighting brightness, humidity, and temperature.

[0098] Tear film break-up time (BUT): Instill 1 μL of 10 g / L sodium fluorescein eye drops into the conjunctival sac of the experimental mouse, and let it blink. Observe and record the time when the first break point appears in the corneal staining area under the cobalt blue light of a slit lamp microscope. When conducting each examination, attention should be paid to controlling variables, and it should be operated by the same person at the same time, place, lighting brightness, humidity, and temperature.

[0099] Corneal FL score: Instill 1 μL of 10 g / L sodium fluorescein eye drops into the conjunctival sac of the experimental mouse, and let it blink. Observe the part and scope of corneal epithelial defects of each group of mice under a slit lamp microscope. Scoring criteria: The cornea is divided into 4 quadrants, with 0-3 points for each quadrant. The scoring criteria for each quadrant are as follows: (1) 0 points: no staining; (2) 1 point: mild staining < 5 points; (3) 2 points: moderate staining ≥ 5 points; (4) 3 points: severe staining ≥ 5 points, with filamentous staining or ulceration. The total score limit is 12 points.

[0100] The specific results are shown in Tables 2, 3, and 4.

[0101] Experimental results:

[0102] Table 1 Comparison results of physical and chemical indexes

[0103]

[0104]

[0105] Table 2 Results of tear secretion test (SIT)

[0106]

[0107] Note: Compared with the control group in each group,* P < 0.05, ** P < 0.01, *** P < 0.001.

[0108] Compared with Example 1, Comparative Examples 1, 3 - 7 # P < 0.05, ## P < 0.01, ### P < 0.001.

[0109] Compared with Example 4, Comparative Examples 2, 3, 7 △ P < 0.05, △△ P < 0.01, △△△ P < 0.001.

[0110] The SIT value can indirectly reflect the basal tear secretion volume of mice. Compared with each comparative example, the tear secretion volume of mice in each example was significantly higher than that of mice in the comparative examples.

[0111] Table 3 Tear film break-up time (BUT)

[0112]

[0113]

[0114] Note: Compared with the control group in each group, * P < 0.05, ** P < 0.01, *** P < 0.001.

[0115] Compared with Example 1, Comparative Examples 1, 3 - 7 # P < 0.05, ## P < 0.01, ### P < 0.001.

[0116] Compared with Example 4, Comparative Examples 2, 3, 7 △ P < 0.05, △△ P < 0.01, △△△ P < 0.001.

[0117] The tear film break-up time is a direct and relatively sensitive detection method for reflecting the stability of the tear film. Compared with each comparative example, the tear film break-up time of mice in each example was longer than that of mice in the comparative examples.

[0118] Table 4 Corneal FL score

[0119]

[0120]

[0121] Note: Compared with the control group in each group, * P < 0.05, ** P < 0.01, *** P < 0.001.

[0122] Compared with Example 1, Comparative Example 1, Comparative Examples 3 - 7 # P < 0.05, ## P < 0.01, ### P < 0.001.

[0123] Compared with Example 4, Comparative Examples 2, 3, 7 △ P < 0.05, △△ P < 0.01, △△△ P < 0.001.

[0124] The corneal FL score is used to evaluate the health status of corneal epithelial tissue through corneal fluorescein staining examination. The higher the score value, the more severe the damage to the corneal epithelial tissue. Compared with each comparative example, the FL scores of the mice in each example are significantly lower than those of the mice in the comparative examples.

[0125] It can be seen from the data in Table 2 - Table 4 that in Example 1 of the present invention, the mesenchymal stem cell supernatant containing multiple trace elements and the mesenchymal stem cell extract containing multiple trace elements are added simultaneously. Compared with Comparative Example 4 or Comparative Example 5 that only add any one of them, the two have a significant synergistic effect in increasing tear secretion, improving tear film stability, and improving the corneal injury repair effect.

[0126] In Example 1 of the present invention, multiple trace elements are added during the stem cell culture process. Compared with Comparative Example 6 that directly adds multiple trace elements during the preparation of eye drops, some trace elements that are not easily absorbed by the human body can exist in the cell matrix components and be encapsulated in exosomes, which is more conducive to absorption, and can increase tear secretion, tear film stability, and corneal injury repair effect.

[0127] In Example 1 and Example 4 of the present invention, the mesenchymal stem cell supernatant, the mesenchymal stem cell extract, and multiple trace elements are added simultaneously. Compared with Comparative Example 1 or Comparative Example 2 that only add the mesenchymal stem cell supernatant and the mesenchymal stem cell extract, or Comparative Example 7 that only adds multiple trace elements, the three have a significant synergistic effect in increasing tear secretion, improving tear film stability, and improving the corneal injury repair effect.

[0128] When the addition ratio of multiple trace elements in Example 1 is 1000:1, compared with the addition ratio of 2500:1, the SIT, BUT, and FL score effects of the eye drops are equivalent, and there is no significant difference.

[0129] The above detailed description is a specific description of one of the feasible embodiments of the present invention. This embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification made without departing from the present invention shall be included within the scope of the technical solution of the present invention.

Claims

1. An eye drop containing multiple trace elements and stem cell extract, characterized in that, By weight, it includes the following components: 0.5 - 1.5 parts of mesenchymal stem cell supernatant, 0.05 - 0.15 parts of mesenchymal stem cell extract, and 5 - 15 parts of sodium hyaluronate solution; The mesenchymal stem cell supernatant and the mesenchymal stem cell extract contain multiple trace elements.

2. The eye drops according to claim 1, characterized in that, The mesenchymal stem cells include one or more of umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, placental mesenchymal stem cells, amniotic mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, bone marrow mesenchymal stem cells, skin mesenchymal stem cells, dental pulp mesenchymal stem cells, urine-derived mesenchymal stem cells, and induced pluripotent stem cell-derived mesenchymal stem cells; and / or the multiple trace elements include Al, Mg, Ba, Mn, Ca, Mo, Cd, Ni, Co, Se, Cr, Sr, Cu, Ti, Fe, V, and Zn.

3. The eye drops according to claim 2, characterized in that, The specific composition of the multiple trace elements is as follows: the content of Al is 500 - 5000 mg / L, the content of Mg is 500 - 5000 mg / L, the content of Ba is 0.01 - 0.1 mg / L, the content of Mn is 50 - 500 mg / L, the content of Ca is 50 - 500 mg / L, the content of Mo is 1 - 10 mg / L, the content of Cd is 0.001 - 0.01 mg / L, the content of Ni is 1 - 10 mg / L, the content of Co is 0.5 - 5 mg / L, the content of Se is 10 - 100 mg / L, the content of Cr is 1 - 10 mg / L, the content of Sr is 0.5 - 5 mg / L, the content of Cu is 0.5 - 5 mg / L, the content of Ti is 50 - 500 mg / L, the content of Fe is 500 - 5000 mg / L, the content of V is 1 - 10 mg / L, and the content of Zn is 100 - 1000 mg / L.

4. The eye drops according to claim 1, wherein The mesenchymal stem cell supernatant is: the mesenchymal stem cell supernatant obtained by centrifugation and filtration after adding multiple trace elements to the mesenchymal stem cell culture system; and / or the mesenchymal stem cell extract is: the mesenchymal stem cells obtained after adding multiple trace elements to the mesenchymal stem cell culture system, and the mesenchymal stem cell extract obtained by ultrasonic lysis extraction method.

5. The eye drops according to claim 1, characterized in that, The concentration of the sodium hyaluronate solution is 0.05 - 0.5 wt%.

6. The eye drops according to claim 1, characterized in that, The eye drops also include at least one of a pH regulator and an osmotic pressure regulator.

7. The eye drops according to claim 6, characterized in that, By weight, the dosage of the pH regulator is 0.01 - 0.5 parts, and the dosage of the osmotic pressure regulator is 0.01 - 0.5 parts; the pH regulator includes one or more of sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, boric acid, borax, acetic acid, sodium acetate, citric acid, sodium citrate, tartaric acid, sodium tartrate, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, hydrochloric acid, and phosphoric acid; the osmotic pressure regulator includes one or more of sodium chloride, potassium chloride, boric acid, borax, sodium sulfate, potassium sulfate, sodium nitrate, potassium nitrate, sodium acetate, mannitol, glycerol, propylene glycol, and glucose.

8. The eye drops according to claim 4, characterized in that, The preparation process of the mesenchymal stem cell culture system is as follows: The stem cells are subcultured to the P3 generation using a serum-free stem cell culture medium, the seed cells are cryopreserved, the P3 generation of seed cells are batch-resuscitated using a serum-free stem cell culture medium, and multiple trace elements are added during the resuscitation process, and then continue to be subcultured to the P5-P8 generation to obtain it.

9. The eye drops according to claim 8, characterized in that, During the resuscitation process, the dilution ratio of multiple trace elements in the serum-free stem cell culture medium is 2000-3000:

1.

10. The eye drops according to claim 4, characterized in that, The ultrasonic lysis extraction method specifically includes the following steps: The obtained mesenchymal stem cells are centrifuged and washed with physiological saline, resuspended, ultrasonically lysed, and filtered to obtain; the solution used for resuspension is at least one of the mesenchymal stem cell supernatant, physiological saline, 1‰ sodium hyaluronate solution, and injection water.

11. The eye drops according to claim 10, characterized in that, Resuspended with the supernatant of the mesenchymal stem cells at a density of 1×10 6 -1×10 7 / mL; the time of ultrasonic lysis is 20 - 60 s, wherein each time lasts for 1 - 3 s and the interval is 3 - 9 s; the filtration is first carried out by filtering with a 2000-mesh sterilized filter screen and then with a 0.22-μm sterile filter membrane.

12. A method for preparing the eye drops according to any one of claims 1-11, characterized in that, It includes the following steps: Mix the mesenchymal stem cell supernatant, mesenchymal stem cell extract, and sodium hyaluronate solution evenly to obtain the eye drops.

13. Use of the eye drops according to any one of claims 1-11 or the eye drops prepared by the preparation method according to claim 12 in the preparation of a drug for treating dry eye.

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