Allergen blocking agent loaded with umbilical cord mesenchymal stem cell culture medium extract and preparation method therefor

Abstract

A nasal allergen blocking agent. Specifically, the present invention relates to a preparation method for an allergen blocking agent loaded with an umbilical cord mesenchymal stem cell culture medium extract. The provided allergen blocking agent is prepared from the following raw materials: lyophilized powders of the umbilical cord mesenchymal stem cell culture medium extract, hydroxypropyl methylcellulose, a honeysuckle extract, and borneol. By means of adding the honeysuckle extract and the umbilical cord mesenchymal stem cell culture medium extract to the allergen blocking agent, under the interaction of various raw materials, capillary permeability can be reduced by means of dilating capillaries, thereby reducing tissue fluid exudation, helping reduce swelling, inhibiting smooth muscle contraction, alleviating the spasticity in the respiratory tract and other parts, and improving related discomfort symptoms. The dry powder mixture is loaded into a nasal spray reagent bottle for use. The anti-allergen blocking agent can not only physically isolate allergens, but also load a variety of stem cell factors contained in the umbilical cord mesenchymal stem cell culture medium extract, thereby eliminating chronic nasal mucosa inflammation, repairing nasal mucosa damage without any toxic and side effects, and effectively resisting the onset of allergic rhinitis.

Description

An allergen barrier loaded with umbilical cord mesenchymal stem cell culture solution extract and a preparation method thereof TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and relates to a nasal allergen barrier, in particular to a preparation method of an allergen barrier loaded with umbilical cord mesenchymal stem cell culture solution extract. BACKGROUND

[0002] Allergic rhinitis, also known as allergic rhinitis, is a multifactorial disease caused by genetic and environmental factors. The characteristic of this disease is that when the body's immune system overreacts to environmental allergens such as pollen, dust mites, etc., it causes chronic inflammation of the mucosa, and the main symptoms are paroxysmal sneezing, rhinorrhea and nasal congestion. Genetic factors and environmental allergens such as fungal spores, dust mites, animal excrement, and some foods can all be triggers for allergic rhinitis. Studies have shown that the incidence of the disease worldwide has been increasing annually, affecting about 500 million people, especially in developed regions such as Western Europe, Northern Europe and North America. Common symptoms of allergic rhinitis include nasal itching, paroxysmal sneezing, clear watery nasal discharge and nasal congestion, and symptoms can be seasonal or irregular throughout the year. In some cases, such as after exposure to allergens, patients may also experience ocular symptoms such as ocular itching, tearing, redness and burning of the eyes, as well as respiratory symptoms such as throat itching, chest tightness, coughing and asthma attacks.

[0003] Current treatments for allergic rhinitis mainly include avoiding contact with allergens, appropriate use of antihistamines and glucocorticoids. For patients with severe allergic rhinitis, monoclonal antibodies or specific immunotherapy targeting IgE can also be considered.

[0004] Drug therapy is the main treatment for allergic rhinitis, including antihistamines, hormone drugs, and antispasmodics. Antihistamines can relieve symptoms such as nasal itching and rhinorrhea; hormone drugs are mainly used to relieve nasal mucosal swelling; and antispasmodics can relieve nasal congestion. The disadvantages are: antiallergic drugs such as levocabastine hydrochloride nasal spray and azelastine hydrochloride nasal spray can improve symptoms such as nasal congestion and rhinorrhea caused by allergic rhinitis. The antihistamine component contained in the anti-allergic drug may cause damage to the central nervous system and a decrease in the body's immunity if used for a long time, resulting in drowsiness, dry mouth, and nasal bleeding. Frequent use of hormone nasal sprays can cause nasal mucosa to be in a state of hyperemia, which can cause damage or erosion of the nasal mucosa, leading to symptoms of nasal bleeding, and can cause irritation of the oral mucosa, resulting in dry mouth and other adverse reactions.

[0005] Immunotherapy: Immunotherapy is a treatment method for allergens, which gradually increases the dose of allergens to make patients tolerant to allergens. However, immunotherapy needs to be carried out under the guidance of a doctor, and the treatment cycle is long, but it is difficult to achieve for some patients. SUMMARY

[0006] In view of the problems in the prior art, the present application provides an allergen blocking agent loaded with umbilical cord mesenchymal stem cell culture solution extract, which can block and reduce allergen contact, effectively reduce the stimulation and damage of allergens to the human body, and help people prevent and relieve allergic reactions.

[0007] The present application also provides a preparation method of the above-mentioned allergen blocking agent loaded with umbilical cord mesenchymal stem cell culture solution extract.

[0008] The technical scheme adopted by the present application to achieve the above-mentioned purposes is:

[0009] The present application provides an allergen blocking agent loaded with umbilical cord mesenchymal stem cell culture solution extract, which is prepared from the following raw materials: umbilical cord mesenchymal stem cell culture solution extract freeze-dried powder, hydroxypropyl methyl cellulose, honeysuckle extract, and borneol.

[0010] Further, the above-mentioned allergen blocking agent is composed of the following mass percentage raw materials: umbilical cord mesenchymal stem cell culture solution extract freeze-dried powder 0.2%, hydroxypropyl methyl cellulose 98%, honeysuckle extract 1%, and borneol 0.8%.

[0011] The present application also provides a preparation method of the above-mentioned allergen blocking agent, characterized in that it comprises the following steps:

[0012] (1) Culturing umbilical cord mesenchymal stem cells and collecting the culture solution of umbilical cord mesenchymal stem cells; extracting the culture solution of umbilical cord mesenchymal stem cells, concentrating and freeze-drying to obtain umbilical cord mesenchymal stem cell culture solution extract freeze-dried powder;

[0013] (3) Mixing the umbilical cord mesenchymal stem cell culture solution extract freeze-dried powder, hydroxypropyl methyl cellulose, honeysuckle extract, and borneol uniformly to obtain the allergen blocking agent.

[0014] Further, in step (1), the specific culture and extraction process is as follows: the human umbilical cord mesenchymal stem cells of p4 generation are cultured in saturated humidity, and the mesenchymal stem cell culture medium without serum is used for culture; when the mesenchymal stem cells grow to a certain confluence degree, the culture supernatant is collected; the cells are digested with 0.125% trypsin solution, subcultured, and the fresh mesenchymal stem cell culture medium is used for further expansion culture of the mesenchymal stem cells, and the process is repeated until the cells are subcultured to P6 generation; the cell culture supernatant in each generation is collected, concentrated, dialyzed, then the freeze-drying protective agent is added, and after freeze-drying, the mesenchymal stem cell culture supernatant freeze-dried powder is obtained.

[0015] Further, the saturated humidity culture is carried out in a 37℃, 5% carbon dioxide incubator; and the confluence degree is 70-90%.

[0016] Further, the freeze-drying protective agent contains 2% trehalose and 3% mannitol in terms of volume percentage.

[0017] Further, the parameters of freeze-drying are as follows:

[0018] a. Pre-freezing stage: temperature -10℃ to 20℃, time 2-5 hours; vacuum degree 0 mbar;

[0019] b. Primary drying: temperature -10℃ to -45℃, time 10-20 hours; vacuum degree 0.3-0.5 mbar.

[0020] c. Secondary drying: temperature 15℃ to 35℃, time 4-8 hours; vacuum degree 0.3-0.5 mbar.

[0021] The hydroxybenzyl cellulose in the allergen barrier provided by the application has high hydrophilicity, can absorb and lock water, form a protective film on the skin, effectively prevent the allergens such as pollen and dust mites in the external environment from entering the body, and also can adsorb pollutants in the air, play a purifying role, thereby improving the dryness of the nasal mucosa, reducing the itching, and relieving symptoms such as nasal congestion, runny nose, sneezing, etc.

[0022] The extract of honeysuckle and the extract of umbilical cord mesenchymal stem cell culture medium are added in the allergen barrier, and under the interaction of various raw materials, the capillary vessels can be expanded, the capillary permeability can be reduced, and the tissue fluid exudation can be reduced, which is helpful for swelling. The smooth muscle contraction can be inhibited, the spasm state of the respiratory tract and other parts can be relieved, and the related discomfort symptoms can be improved. The dry powder mixture is filled into a nasal spray reagent bottle for use. The anti-allergen barrier can not only physically isolate the allergen, but also load various stem cell factors contained in the extract of umbilical cord mesenchymal stem cell culture medium, eliminate the chronic inflammatory response of the nasal mucosa, repair the damage of the nasal mucosa, and has no any toxic and side effects, and can effectively resist the onset of allergic rhinitis.

[0023] The umbilical cord mesenchymal stem cell culture solution extract allergen barrier agent can not only anti-inflammatory and bacteriostatic repair nasal mucosa damage, but also form a physical isolation layer on the nasal mucosa, block allergen substances and inhale into the respiratory tract, so as to avoid the occurrence of allergic reaction.

[0024] The present application has the following advantages:

[0025] (1) The umbilical cord mesenchymal stem cell culture solution extract contains many cytokines, such as EGF, VEGF, TGF-β, HGF, KGF, FGF, IGF, PDGF, etc. These cytokines act as signal molecules between cells and play an important role in restoring the function of damaged tissues and cells in vivo. For example, TGF-β transforming growth factor can resist inflammation and promote wound healing. Through the awakening of dormant cells by cytokines, the damaged tissues and cells are regenerated and the lost functions are restored. It can resist inflammation, promote cell metabolism and repair damage of nasal mucosa.

[0026] (2) The umbilical cord mesenchymal stem cell culture solution extract is first loaded on hydroxybenzyl cellulose in the present application. Under the synergistic effect of each raw material, the extract first absorbs the moisture in the air in the nasal cavity to form a safe and effective mucosa that isolates allergens. The mucosa adheres to the nasal mucosa and blocks the contact between allergens and nasal mucosa from the source through this physical method. The mixed umbilical cord mesenchymal stem cell culture solution extract can be absorbed by the nasal mucosa to eliminate chronic inflammation of the mucosa and repair damage of the nasal mucosa, thereby controlling the symptoms of allergic rhinitis and significantly improving the quality of life.

[0027] (3) The allergen blocking agent provided by the present application can block allergens through physical isolation, and non-drugs will not produce related drug adverse reactions. The physical isolation of allergens can repair damage of the nasal mucosa and eliminate inflammation. DETAILED DESCRIPTION

[0028] The technical solutions of the present application are further explained and described below through specific examples.

[0029] The honeysuckle extract used in the present application is purchased from Shaanxi Bolin Biotechnology Co., Ltd. The main component is chlorogenic acid, and the specification of the honeysuckle extract is 25%. EMBODIMENT

[0030] 1. Umbilical cord mesenchymal stem cell culture, collect the culture solution of umbilical cord mesenchymal stem cells:

[0031] Step one, hUMSC cell recovery: the P2 generation of umbilical cord mesenchymal stem cell seed bank cells, the frozen hUMSC cells were taken out from liquid nitrogen, and the frozen tube was quickly placed in a 37℃ water bath pot for rapid melting. The thawed cell suspension was slowly added to an appropriate amount of culture medium (the volume ratio of cell suspension to culture medium was 1:8; the culture medium was NC0103 mesenchymal stem cell serum-free medium from Youkang Biotechnology), centrifuged at 200g for 5 minutes, and the supernatant was discarded. The mesenchymal stem cell serum-free medium was added to resuspend the cells, the cell density was adjusted to 1*104 cell / ml, 25ml was taken, and evenly distributed in a T175 cell culture bottle. The culture bottle was shaken to evenly distribute the cells, and cultured at 37℃, 5% CO2, and saturated humidity. After 24 hours, the cell state was observed.

[0032] Step two, hUMSC cell subculture: under a microscope, when the cell fusion degree reached 90%, the cells could be subcultured. The culture supernatant was discarded, and DPBS solution was added for washing once. 5mL of 0.125% trypsin digestion solution was added to each T175 cell culture bottle to completely cover the bottom of the culture bottle. Incubate at room temperature for 2 minutes, gently tap the culture bottle, and stop digestion immediately after observing that most cells have fallen off the bottom of the culture bottle under a microscope. Add 2 times the volume of mesenchymal stem cell serum-free medium to terminate digestion, and use a pipette to gently blow the surface of the culture bottle to completely detach the cells and evenly disperse them. Transfer the cell suspension to a centrifuge tube and centrifuge at 200g for 5 minutes. Discard the supernatant, add mesenchymal stem cell serum-free medium, resuspend the cells, and count. Subculture at a ratio of 1:3-1:6, evenly distribute in the culture bottle, and culture in a 37℃, 5% CO2, and saturated humidity incubator for 4 days.

[0033] Step three, continue to subculture and culture the cells to P6 generation cells. Collect the cell culture solution during the cell culture process and freeze for later use.

[0034] 2. Preparation of umbilical cord mesenchymal stem cell culture medium extract freeze-dried powder:

[0035] Step one: collection of cell culture supernatant: collect the cell culture solution, centrifuge at 700g, 4℃ for 10min; carefully aspirate the supernatant, taking care not to aspirate cells or cell fragments, centrifuge at 10000g, 4℃ for 15min, and ensure that the cells or cell fragments are completely removed.

[0036] Step two, ultrafiltration: place the culture supernatant in an ultrafiltration tube for ultrafiltration and concentration, 3000g, 4℃, and the volume of the concentrated solution is 1 / 100; obtain the concentrated solution.

[0037] Step three, dialysis: transfer the umbilical cord mesenchymal stem cell culture supernatant concentrate in the centrifuge tube to a dialysis bag, and bury the dialysis bag in PEG20000 at 4℃ for 4 hours to dehydrate.

[0038] Step four, preparation of freeze-dried powder: the umbilical cord mesenchymal stem cell culture supernatant dialysate is mixed with a freeze-drying protective agent, and then freeze-dried. The freeze-drying protective agent is 2% trehalose and 3% mannitol based on the mass percentage of the umbilical cord mesenchymal stem cell culture supernatant dialysate. The umbilical cord mesenchymal stem cell culture supernatant dialysate is filled into a test tube, in a semi-plugged state, and placed into a vacuum freeze-drying machine. The freeze-drying parameters are set as follows:

[0039] a. Pre-freezing stage: temperature -10℃ to 20℃, time 2-5 hours; vacuum degree 0 (mbar).

[0040] b. Primary drying: the water molecules are directly sublimated from ice state to gas state and removed from the preparation at a temperature of -10℃ to -45℃, time 10-20 hours; vacuum degree 0.3-0.5 (mbar).

[0041] c. Secondary drying: the residual bound water molecules are dried at a low temperature; temperature 15℃ to 35℃, time 4-8 hours; vacuum degree 0.3-0.5 (mbar).

[0042] Step five, sealing and storage: the dried test tube is capped and sealed for storage.

[0043] 3. Preparation of the umbilical cord mesenchymal stem cell culture supernatant extract allergen barrier agent:

[0044] Step one, mixing: accurately weigh the umbilical cord mesenchymal stem cell culture supernatant extract freeze-dried powder, hydroxypropyl methyl cellulose, honeysuckle extract, and borneol, and mix them in a three-dimensional motion mixer for 20 minutes. The mixing frequency of the mixer is 20 times / min. The mixing ratio is: 0.2% umbilical cord mesenchymal stem cell culture supernatant extract freeze-dried powder, 98% hydroxypropyl methyl cellulose, 1% honeysuckle extract, and 0.8% borneol.

[0045] Step two, filling: the mixture is loaded into the feeding port of an automatic powder dispensing machine. The automatic filling and sealing machine is turned on, the dispensing amount is set, and the equipment automatically dispenses the mixture into plastic bottle reagent bottles, adds a long plug, and tightens the bottle cap. The preparation of the umbilical cord mesenchymal stem cell culture supernatant extract allergen barrier agent is completed.

[0046] In use, only need to open the bottle cap, align the nostrils, pinch the bottle body, and spray the mixture into the nasal cavity by the pressure generated by squeezing the bottle body. The moist air in the nasal cavity contacts the hydroxypropyl methyl cellulose to form a thin film gel, forming a natural barrier to effectively physically isolate the allergen from the nasal cavity. The stem cell culture extract can repair normal or damaged cells, regulate cell division function, reduce inflammation, and immune regulation, which can effectively improve the problem of nasal mucosa damage. The honeysuckle extract and borneol can expand capillaries, reduce capillary permeability, inhibit leukocyte migration and release of inflammatory mediators, reduce tissue fluid exudation, help anti-inflammatory and detumescence, improve local blood circulation, accelerate tissue repair, and have certain antibacterial effect.

[0047] Comparative Example 1

[0048] The allergen barrier loaded with umbilical cord mesenchymal stem cell culture extract has the following formula: hydroxypropyl methyl cellulose 98%, honeysuckle extract 1.2%, and borneol 0.8%.

[0049] Comparative Example 2

[0050] The allergen barrier loaded with umbilical cord mesenchymal stem cell culture extract has the following formula: umbilical cord mesenchymal stem cell culture extract freeze-dried powder 0.2%, hydroxypropyl methyl cellulose 99%, and borneol 0.8%. Example

[0051] (I) Construction of mouse allergic rhinitis model:

[0052] Modeling method: systemic and local challenge method

[0053] The model is prepared by injecting ovalbumin (OVA) antigen suspension to induce immune response, and then dropping the antigen solution into the nose to induce AR symptoms; or without in vivo injection, directly dropping the antigen solution into the nose to cause local induction. The OVA suspension is used for initial immunization by intraperitoneal injection, and then the OVA solution is used for nasal instillation for reinforcement sensitization.

[0054] Modeling process: select mice, on days 1, 5, 14, and 21, give the mice intraperitoneal injection of OVA 25 μg, aluminum hydroxide gel [Al(OH)3] 1 mg, and 0.5 ml of isotonic saline to perform basic sensitization. After basic sensitization, on day 28, give the mice bilateral nasal instillation of 500 μg OVA and 20 μl isotonic saline, for 5 days. The experiment starts on the sixth day and continues for three days.

[0055] Observe the mice and score according to the score table (Table 1).

[0056] Table 1

[0057]

[0058] The mice were given bilateral intranasal instillation of 500 μg OVA plus 20 μl isotonic saline to induce allergic reaction. The amount of nasal discharge, sneezing and scratching of the nose of the mice within 30 min were observed and scored. The total score was calculated by using the superposition method. This was used as a control.

[0059] The allergen barrier prepared by Example 1, Comparative Example 1 and Comparative Example 2 was sprayed into the nasal cavity, and then the mice were given bilateral intranasal instillation of 500 μg OVA plus 20 μl isotonic saline to induce allergic reaction. The amount of nasal discharge, sneezing and scratching of the nose of the mice within 30 min were observed and scored. The total score was calculated by using the superposition method.

[0060] The experimental results are shown in Table 2.

[0061] Table 2

[0062]

[0063] The allergic reaction induced by OVA instillation in AR mice was significantly lower in the mice treated with the allergen barrier prepared by Example 1 than in the control group in terms of sneezing, scratching and nasal discharge.

[0064]

Claims

1. An allergen blocker loaded with an extract from umbilical cord mesenchymal stem cell culture medium, characterized in that, It is prepared from the following raw materials: lyophilized powder of umbilical cord mesenchymal stem cell culture medium extract, hydroxypropyl methylcellulose, honeysuckle extract, and borneol.

2. The allergen blocker according to claim 1, characterized in that, The ingredients are composed of the following percentages by weight: 0.2% lyophilized powder of umbilical cord mesenchymal stem cell culture medium extract, 98% hydroxypropyl methylcellulose, 1% honeysuckle extract, and 0.8% borneol.

3. A method for preparing an allergen blocker as described in claim 1 or 2, characterized in that, Includes the following steps: (1) Culture umbilical cord mesenchymal stem cells and collect the culture medium of umbilical cord mesenchymal stem cells; extract the culture medium of umbilical cord mesenchymal stem cells, concentrate and freeze-dry to obtain freeze-dried powder of umbilical cord mesenchymal stem cell culture medium extract. (2) Mix the freeze-dried powder of umbilical cord mesenchymal stem cell culture medium extract, hydroxypropyl methylcellulose, honeysuckle extract and borneol evenly to obtain an allergen blocker.

4. The preparation method according to claim 3, characterized in that, In step (1), the specific culture and extraction process is as follows: human umbilical cord mesenchymal stem cells at passage p4 are cultured in saturated humidity and cultured in serum-free mesenchymal stem cell culture medium. When the mesenchymal stem cells grow to a certain degree of confluence, the culture supernatant is collected. The cells are passaged using 0.125% trypsin digestion solution and then cultured in fresh mesenchymal stem cell culture medium to expand the mesenchymal stem cells. This process is repeated until the cells are passaged to passage p6. The cell culture supernatant from each passage is collected, concentrated, dialyzed, and then a freeze-drying protectant is added. After freeze-drying, the freeze-dried powder of mesenchymal stem cell culture supernatant is obtained.

5. The preparation method according to claim 4, characterized in that, The saturated humidity culture was carried out in a 37°C, 5% CO2 incubator; the confluence was 70-90%.

6. The preparation method according to claim 4 or 5, characterized in that, By volume percentage, the freeze-drying protectant contains 2% trehalose and 3% mannitol.

7. The preparation method according to any one of claims 4-6, characterized in that, The parameters for the freeze-drying process are as follows: a. Pre-freezing stage: Temperature -10℃~20℃, time 2~5 hours; Vacuum degree 0 mbar; b. Single drying: temperature -10℃~-45℃, time 10~20 hours; vacuum degree 0.3~0.5 mbar. 8.c. Secondary drying: temperature 15℃~35℃, time 4~8 hours; vacuum degree 0.3~0.5 mbar.

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