Omerological mucosa mesenchymal stem cell exosome preparation as well as preparation method and application thereof

Abstract

The invention relates to an olfactory mucosa mesenchymal stem cell exosome preparation as well as a preparation method and application thereof, and relates to the technical field of biological medicines and pharmaceutic adjuvants, the olfactory mucosa mesenchymal stem cell exosome preparation comprises the following raw materials: olfactory mucosa mesenchymal stem cell exosome, a freeze-drying protection composition and a phosphate buffer; the freeze-drying protection composition is obtained by carrying out ultrasonic-pulsed electric field coupling treatment on trehalose and mannitol. According to the preparation method, trehalose and mannitol are subjected to synergistic modification treatment by adopting a composite field formed by an ultrasonic-pulse electric field under proper condition parameters, so that the freeze-drying protection effect on the exosome of the olfactory mucosa mesenchymal stem cells is enhanced, and convenience is provided for preservation and transportation of an exosome preparation; meanwhile, the invention also finds that the olfactory mucosa mesenchymal stem cell exosome has an obvious improvement effect on mouse model diseases with osteosclerosis, so that not only is the clinical application field of the exosome widened, but also a new means is provided for the treatment of osteosclerosis.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine and pharmaceutical excipients, and in particular to an olfactory mucosa mesenchymal stem cell exosome preparation, and a preparation method and application thereof. Background Art

[0002] In the vast world of biomedical research, exosomes are becoming a bright new star in the field of disease diagnosis, innovative treatment and tissue regeneration as key carriers of efficient information exchange between cells. These tiny and powerful vesicle structures carry a rich library of bioactive molecules including proteins, nucleic acids, lipids, etc., providing unlimited possibilities for exploring the mysteries of life and treating diseases. In particular, olfactory mucosa mesenchymal stem cells (OM-MSCs), as a newly discovered and highly promising subpopulation of mesenchymal stem cells, are widely distributed in the nasal mucosa of the upper, middle and lower nasal turbinate of the nasal septum and lateral wall. They not only inherit the basic characteristics of mesenchymal stem cells' self-renewal and multidirectional differentiation, but also attract much attention for their unique source stability, easy accessibility, strong regeneration ability and rich in more diverse bioactive factors. However, although OM-MSCs and their exosomes theoretically provide broad prospects for the treatment of a variety of diseases, the current research on their exosomes in clinical applications is still relatively scarce, especially the therapeutic potential for specific diseases needs to be further explored.

[0003] On the other hand, osteopetrosis, a rare systemic bone disease, brings great pain and challenges to patients due to its abnormal bone hardening, increased brittleness and easy fractures. The root cause of this disease lies in osteoclast dysfunction, which leads to the imbalance between bone resorption and bone formation and abnormal increase in bone mass. At present, although hematopoietic stem cell transplantation is regarded as a potentially effective treatment, the scarcity of donors has severely limited its widespread application. In this context, existing treatment options mostly rely on combined drug therapy to relieve symptoms, but the treatment effect is often unsatisfactory and is often accompanied by side effects such as stress ulcers and steroid-induced hyperglycemia, which further aggravates the treatment burden on patients.

[0004] It is worth noting that although the exosomes of olfactory mucosa mesenchymal stem cells have shown significant potential in promoting tissue repair and regulating immune response, there have been no relevant research reports on their application in the treatment of osteosclerosis. Summary of the invention

[0005] In order to solve the above problems, the present invention provides an olfactory mucosa mesenchymal stem cell exosome preparation and a preparation method and application thereof.

[0006] In a first aspect, the present invention provides an olfactory mucosa mesenchymal stem cell exosome preparation, wherein the olfactory mucosa mesenchymal stem cell exosome preparation comprises the following raw materials: olfactory mucosa mesenchymal stem cell exosomes, a lyophilized protective composition, and a phosphate buffer;

[0007] The freeze-dried protective composition is obtained by treating trehalose and mannitol by ultrasound-pulse electric field coupling;

[0008] The working condition parameters of the ultrasound-pulse electric field coupling treatment include: the ultrasonic power is 550-700W, and the pulse power supply constant voltage is 3-7V.

[0009] Furthermore, the preparation method of the freeze-dried protective composition comprises the following process:

[0010] Stir and mix trehalose, mannitol and deionized water in a weight ratio of (1-2):(1-2):(15-20) to obtain a first mixed liquid;

[0011] The ultrasonic probe of the ultrasonic device and the electrodes of the pulse device are placed in the first mixed liquid, and the distance between the electrodes is set to 15-20 mm, the constant voltage of the pulse power supply is 3-7 V, the pulse period is 5-10 ms, and the ultrasonic power is 550-700 W; the ultrasonic device and the pulse device are turned on at the same time to perform ultrasonic-pulse electric field coupling treatment for 180-360 seconds to obtain a second mixed liquid;

[0012] The second mixed liquid is concentrated, dried, crushed and sieved to obtain the freeze-dried protection composition.

[0013] Furthermore, the weight ratio of the trehalose, the mannitol and the deionized water is 1.5:1:18, the ultrasonic power is 615W, and the constant voltage of the pulse power supply is 5V.

[0014] Furthermore, the concentration of the olfactory mucosa mesenchymal stem cell exosomes in the olfactory mucosa mesenchymal stem cell exosome preparation is 100 to 1000 μg / mL.

[0015] Furthermore, the concentration of the freeze-dried protective composition in the olfactory mucosa mesenchymal stem cell exosome preparation is 5-10% w / v.

[0016] Furthermore, the physicochemical property parameters of the phosphate buffer include: a concentration of 10 to 30 mmol / L and a pH of 6.5 to 7.5.

[0017] In a second aspect, the present invention provides a method for preparing the olfactory mucosa mesenchymal stem cell exosome preparation according to any one of the first aspects, comprising the following steps:

[0018] Mixing the olfactory mucosa mesenchymal stem cell exosomes, the freeze-dried protective composition and the phosphate buffer to obtain a freeze-dried solution;

[0019] The lyophilized liquid is freeze-dried to obtain the olfactory mucosa mesenchymal stem cell exosome preparation.

[0020] Further, the freeze-drying steps sequentially include a first freezing, a second freezing, a first drying and a second drying;

[0021] The working condition parameters of the first freezing include: temperature of -25 to -15°C and time of 1 to 2 hours;

[0022] The working condition parameters of the second freezing include: temperature of -55 to -50°C and time of 2 to 4 hours;

[0023] The working condition parameters of the first drying include: vacuuming to 0.1-0.2 mbar, temperature of -30--20°C, and time of 12-16 hours;

[0024] The working condition parameters of the second drying include: vacuuming to 0.1-0.2 mbar, temperature of 20-30° C., and time of 2-5 hours.

[0025] In a third aspect, the present invention provides the use of the olfactory mucosa mesenchymal stem cell exosome preparation described in any one of the first aspect and / or the olfactory mucosa mesenchymal stem cell exosome preparation prepared by the preparation method described in any one of the second aspect in the preparation of a drug for treating osteosclerosis.

[0026] The above technical solution provided by the embodiment of the present invention has at least the following advantages compared with the prior art:

[0027] The embodiment of the present invention provides an olfactory mucosal mesenchymal stem cell exosome preparation and its preparation method and application. The present invention uses a composite field formed by an ultrasound-pulsed electric field under appropriate conditions and parameters to synergistically modify trehalose and mannitol, thereby enhancing the freeze-drying protection effect on the olfactory mucosal mesenchymal stem cell exosomes, and providing convenience for the storage and transportation of the exosome preparation; at the same time, the present invention also found that the olfactory mucosal mesenchymal stem cell exosomes have a significant improvement effect on the symptoms of a mouse model with osteosclerosis, which not only broadens the clinical application field of exosomes, but also provides a new means for the treatment of osteosclerosis. Specifically:

[0028] 1) The present invention utilizes the composite field formed by ultrasound-pulsed electric field to synergistically modify trehalose and mannitol, which can destroy the aggregation state of the protective agent molecules and change their surface properties, enhance their interaction with exosomes, and facilitate the formation of a stable protective layer, thereby making the freeze-dried olfactory mucosa mesenchymal stem cell exosomes more distributed in a smaller particle size range, better preventing the aggregation of olfactory mucosa mesenchymal stem cell exosomes during the freeze-drying process, enhancing the stability of the olfactory mucosa mesenchymal stem cell exosome preparation system, and providing convenience for the storage and transportation of the exosome preparation.

[0029] 2) The present invention shows through animal experiments that the exosomes of olfactory mucosa mesenchymal stem cells can effectively upregulate the expression of type II carbonic anhydrase in osteoclasts of osteosclerosis mouse model, indicating that the exosomes of olfactory mucosa mesenchymal stem cells can activate the activity of type II carbonic anhydrase, improve osteosclerosis caused by poor osteoclast absorption, and have a better improvement effect on the symptoms of osteosclerosis. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0032] In a first aspect, the present invention provides an olfactory mucosa mesenchymal stem cell exosome preparation, wherein the olfactory mucosa mesenchymal stem cell exosome preparation comprises the following raw materials: olfactory mucosa mesenchymal stem cell exosomes, a lyophilized protective composition, and a phosphate buffer;

[0033] The freeze-dried protective composition is obtained by treating trehalose and mannitol by ultrasound-pulse electric field coupling;

[0034] The working condition parameters of the ultrasound-pulse electric field coupling treatment include: the ultrasonic power is 550-700W, and the pulse power supply constant voltage is 3-7V.

[0035] The embodiment of the present invention provides a method for preparing an olfactory mucosal mesenchymal stem cell exosome preparation. The present invention uses a composite field formed by an ultrasound-pulsed electric field under appropriate conditions and parameters to synergistically modify trehalose and mannitol, thereby enhancing the freeze-drying protection effect on the olfactory mucosal mesenchymal stem cell exosomes, and providing convenience for the storage and transportation of the exosome preparation.

[0036] In some specific embodiments, the olfactory mucosa mesenchymal stem cell exosomes described in the present invention can be directly commercially available products, or can be prepared by referring to the exosome preparation methods disclosed in the prior art such as US20170296627A1, CN116064389A, CN111808804A, CN108004206A, etc., and can be specifically prepared by the preparation method disclosed in Example 2 in CN108004206A.

[0037] In some specific embodiments, the method for preparing the lyophilized protective composition comprises the following process:

[0038] Stir and mix trehalose, mannitol and deionized water in a weight ratio of (1-2):(1-2):(15-20) to obtain a first mixed liquid;

[0039] The ultrasonic probe of the ultrasonic device and the electrodes of the pulse device are placed in the first mixed liquid, and the distance between the electrodes is set to 15-20 mm, the constant voltage of the pulse power supply is 3-7 V, the pulse period is 5-10 ms, and the ultrasonic power is 550-700 W; the ultrasonic device and the pulse device are turned on at the same time to perform ultrasonic-pulse electric field coupling treatment for 180-360 seconds to obtain a second mixed liquid;

[0040] The second mixed liquid is concentrated, dried, crushed and sieved to obtain the freeze-dried protection composition.

[0041] In some specific embodiments, the weight ratio of the trehalose, the mannitol and the deionized water is 1.5:1:18, the ultrasonic power is 615W, and the pulse power constant voltage is 5V.

[0042] In some specific embodiments, the concentration of the olfactory mucosa mesenchymal stem cell exosomes in the olfactory mucosa mesenchymal stem cell exosome preparation is 100-1000 μg / mL, preferably 400 μg / mL.

[0043] In some specific embodiments, the concentration of the lyophilized protective composition in the olfactory mucosa mesenchymal stem cell exosome preparation is 5-10% w / v, preferably 8% w / v.

[0044] In some specific embodiments, the physicochemical property parameters of the phosphate buffer include: a concentration of 10 to 30 mmol / L, preferably 20 mmol / L; a pH of 6.5 to 7.5, preferably 7.4.

[0045] In a second aspect, based on the same inventive concept, the present invention provides a method for preparing the olfactory mucosa mesenchymal stem cell exosome preparation according to any one of the first aspects, comprising the following steps:

[0046] Mixing the olfactory mucosa mesenchymal stem cell exosomes, the freeze-dried protective composition and the phosphate buffer to obtain a freeze-dried solution;

[0047] The lyophilized liquid is freeze-dried to obtain the olfactory mucosa mesenchymal stem cell exosome preparation.

[0048] The preparation method of the olfactory mucosa mesenchymal stem cell exosome preparation provided by the present invention is simple to operate, does not require additional specific equipment, and is suitable for industrial mass production; at the same time, the preparation method is based on the olfactory mucosa mesenchymal stem cell exosome preparation described in any one of the first aspects, and therefore has at least the beneficial effects of the olfactory mucosa mesenchymal stem cell exosome preparation described in any one of the first aspects, which will not be repeated here.

[0049] In some specific embodiments, the freeze-drying step includes first freezing, second freezing, first drying, and second drying in sequence;

[0050] The working condition parameters of the first freezing include: temperature of -25 to -15°C and time of 1 to 2 hours;

[0051] The working condition parameters of the second freezing include: temperature of -55 to -50°C and time of 2 to 4 hours;

[0052] The working condition parameters of the first drying include: vacuuming to 0.1-0.2 mbar, temperature of -30--20°C, and time of 12-16 hours;

[0053] The working condition parameters of the second drying include: vacuuming to 0.1-0.2 mbar, temperature of 20-30° C., and time of 2-5 hours.

[0054] In the third aspect, based on the same inventive concept, the present invention provides the use of the olfactory mucosa mesenchymal stem cell exosome preparation described in any one of the first aspect and / or the olfactory mucosa mesenchymal stem cell exosome preparation prepared by the preparation method described in any one of the second aspect in the preparation of a drug for treating osteosclerosis.

[0055] The present invention discovered for the first time that exosomes from olfactory mucosa mesenchymal stem cells have a significant improvement effect on the symptoms of a mouse model with osteosclerosis, which not only broadens the clinical application field of exosomes, but also provides a new means for the treatment of osteosclerosis.

[0056] It should be noted that the components and raw materials involved in the olfactory mucosal mesenchymal stem cell exosome preparation, preparation method and application thereof provided in the embodiments of the present invention, unless otherwise specified or specified, can be directly commercially available products or homemade using existing public preparation methods; at the same time, the steps and parameters involved, unless otherwise specified or specified, can be carried out according to existing processing techniques or directly using existing equipment, and the present invention document will not repeat them one by one.

[0057] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually measured according to national standards. If there is no corresponding national standard, then the conditions recommended by the manufacturer are followed.

[0058] Example 1

[0059] This example provides an olfactory mucosa mesenchymal stem cell exosome preparation, which is composed of the following raw materials: olfactory mucosa mesenchymal stem cell exosomes at a concentration of 400 μg / mL, a freeze-dried protective composition at a concentration of 8% w / v, and the remainder is a 20 mmol / L phosphate buffer at a pH of 7.4;

[0060] The freeze-dried protective composition is obtained by treating trehalose and mannitol by ultrasound-pulse electric field coupling. The preparation method of the freeze-dried protective composition includes the following steps:

[0061] Stir and mix trehalose, mannitol and deionized water in a weight ratio of 1.5:1:18 to obtain a first mixed liquid;

[0062] The ultrasonic probe of the ultrasonic device and the electrodes of the pulse device are placed in the first mixed liquid, and the distance between the electrodes is set to 18 mm, the constant voltage of the pulse power supply is 5 V, the pulse period is 8 ms, and the ultrasonic power is 615 W; the ultrasonic device and the pulse device are turned on at the same time to perform ultrasonic-pulse electric field coupling treatment for 240 seconds to obtain a second mixed liquid;

[0063] The second mixed liquid is concentrated and dried to a constant weight, and the dried solid is crushed and passed through a 150-mesh sieve to obtain the freeze-dried protection composition.

[0064] The preparation method of the olfactory mucosa mesenchymal stem cell exosome preparation comprises the following steps:

[0065] Mixing the olfactory mucosa mesenchymal stem cell exosomes, the freeze-dried protective composition and the phosphate buffer to obtain a freeze-dried solution;

[0066] The lyophilized liquid is sequentially subjected to a first freezing, a second freezing, a first drying, and a second drying to obtain the olfactory mucosa mesenchymal stem cell exosome preparation;

[0067] Among them, the working condition parameters of the first freezing include: temperature is -20℃, time is 1.5 hours; the working condition parameters of the second freezing include: temperature is -52℃, time is 3 hours; the working condition parameters of the first drying include: vacuum to 0.1mbar, temperature is -25℃, time is 14 hours; the working condition parameters of the second drying include: vacuum to 0.1mbar, temperature is 25℃, time is 3 hours.

[0068] Example 2

[0069] This example provides an olfactory mucosa mesenchymal stem cell exosome preparation, which is composed of the following raw materials: olfactory mucosa mesenchymal stem cell exosomes at a concentration of 400 μg / mL, a freeze-dried protective composition at a concentration of 8% w / v, and the remainder is a 20 mmol / L phosphate buffer at a pH of 7.4;

[0070] The freeze-dried protective composition is obtained by treating trehalose and mannitol by ultrasound-pulse electric field coupling. The preparation method of the freeze-dried protective composition includes the following steps:

[0071] Stir and mix trehalose, mannitol and deionized water in a weight ratio of 1:1:15 to obtain a first mixed liquid;

[0072] The ultrasonic probe of the ultrasonic device and the electrodes of the pulse device are placed in the first mixed liquid, and the distance between the electrodes is set to 15 mm, the constant voltage of the pulse power supply is 3 V, the pulse period is 5 ms, and the ultrasonic power is 700 W; the ultrasonic device and the pulse device are turned on at the same time to perform ultrasonic-pulse electric field coupling treatment for 360 seconds to obtain a second mixed liquid;

[0073] The second mixed liquid is concentrated and dried to a constant weight, and the dried solid is crushed and passed through a 150-mesh sieve to obtain the freeze-dried protection composition.

[0074] The preparation method of the olfactory mucosa mesenchymal stem cell exosome preparation comprises the following steps:

[0075] Mixing the olfactory mucosa mesenchymal stem cell exosomes, the freeze-dried protective composition and the phosphate buffer to obtain a freeze-dried solution;

[0076] The lyophilized liquid is sequentially subjected to a first freezing, a second freezing, a first drying, and a second drying to obtain the olfactory mucosa mesenchymal stem cell exosome preparation;

[0077] Among them, the working condition parameters of the first freezing include: temperature is -20℃, time is 1.5 hours; the working condition parameters of the second freezing include: temperature is -52℃, time is 3 hours; the working condition parameters of the first drying include: vacuum to 0.1mbar, temperature is -25℃, time is 14 hours; the working condition parameters of the second drying include: vacuum to 0.1mbar, temperature is 25℃, time is 3 hours.

[0078] Example 3

[0079] This example provides an olfactory mucosa mesenchymal stem cell exosome preparation, which is composed of the following raw materials: olfactory mucosa mesenchymal stem cell exosomes at a concentration of 400 μg / mL, a freeze-dried protective composition at a concentration of 8% w / v, and the remainder is a 20 mmol / L phosphate buffer at a pH of 7.4;

[0080] The freeze-dried protective composition is obtained by treating trehalose and mannitol by ultrasound-pulse electric field coupling. The preparation method of the freeze-dried protective composition includes the following steps:

[0081] Stir and mix trehalose, mannitol and deionized water in a weight ratio of 2:1:20 to obtain a first mixed liquid;

[0082] The ultrasonic probe of the ultrasonic device and the electrodes of the pulse device are placed in the first mixed liquid, and the distance between the electrodes is set to 20 mm, the constant voltage of the pulse power supply is 7 V, the pulse period is 10 ms, and the ultrasonic power is 550 W; the ultrasonic device and the pulse device are turned on at the same time to perform ultrasonic-pulse electric field coupling treatment for 180 seconds to obtain a second mixed liquid;

[0083] The second mixed liquid is concentrated and dried to a constant weight, and the dried solid is crushed and passed through a 150-mesh sieve to obtain the freeze-dried protection composition.

[0084] The preparation method of the olfactory mucosa mesenchymal stem cell exosome preparation comprises the following steps:

[0085] Mixing the olfactory mucosa mesenchymal stem cell exosomes, the freeze-dried protective composition and the phosphate buffer to obtain a freeze-dried solution;

[0086] The lyophilized liquid is sequentially subjected to a first freezing, a second freezing, a first drying, and a second drying to obtain the olfactory mucosa mesenchymal stem cell exosome preparation;

[0087] Among them, the working condition parameters of the first freezing include: temperature is -20℃, time is 1.5 hours; the working condition parameters of the second freezing include: temperature is -52℃, time is 3 hours; the working condition parameters of the first drying include: vacuum to 0.1mbar, temperature is -25℃, time is 14 hours; the working condition parameters of the second drying include: vacuum to 0.1mbar, temperature is 25℃, time is 3 hours.

[0088] Comparative Example 1

[0089] This example provides an olfactory mucosal mesenchymal stem cell exosome preparation and a preparation method thereof, which is different from Example 1 only in that the lyophilized protective composition is composed of trehalose and mannitol in a weight ratio of 1.5:1; the remaining steps and parameters are the same.

[0090] Comparative Example 2

[0091] This example provides an olfactory mucosal mesenchymal stem cell exosome preparation and a preparation method thereof, which differs from Example 1 only in that: the freeze-dried protective composition is obtained by ultrasonic treatment of trehalose and mannitol in a weight ratio of 1.5:1, and the specific process is: trehalose, mannitol and deionized water in a weight ratio of 1.5:1:18 are stirred and mixed to obtain a first mixed liquid; an ultrasonic probe of an ultrasonic device is placed in the first mixed liquid, and the ultrasonic power is set to 615W; the ultrasonic device is turned on for ultrasonic treatment for 240 seconds to obtain a second mixed liquid; the second mixed liquid is concentrated and dried to a constant weight and the dried solid is crushed and passed through a 150-mesh sieve to obtain the freeze-dried protective composition; the remaining steps and parameters are the same.

[0092] Comparative Example 3

[0093] This example provides an olfactory mucosal mesenchymal stem cell exosome preparation and a preparation method thereof, which differs from Example 1 only in that: the freeze-dried protective composition is obtained by treating trehalose and mannitol in a weight ratio of 1.5:1 with a pulse electric field, and the specific process is: stirring and mixing trehalose, mannitol and deionized water in a weight ratio of 1.5:1:18 to obtain a first mixed liquid; placing electrodes of a pulse device in the first mixed liquid, setting the distance between the electrodes to 18 mm, the constant voltage of the pulse power supply to 5 V, and the pulse period to 8 ms; turning on the pulse device for pulse electric field treatment for 240 seconds to obtain a second mixed liquid; concentrating and drying the second mixed liquid to a constant weight and crushing the dried solid to pass through a 150-mesh sieve to obtain the freeze-dried protective composition; the remaining steps and parameters are the same.

[0094] Comparative Example 4

[0095] This example provides an olfactory mucosal mesenchymal stem cell exosome preparation and a preparation method thereof, which is different from Example 1 only in that the ultrasonic power in the preparation method of the freeze-dried protective composition is adjusted to 200W and the pulse power supply constant voltage is adjusted to 10V; the remaining steps and parameters are the same.

[0096] Test Example 1

[0097] In this example, the particle size distribution of the olfactory mucosal mesenchymal stem cell exosome preparation samples obtained in the above Examples 1 to 3 and Comparative Examples 1 to 4 was tested according to the test method disclosed in the prior art. The results showed that compared with Comparative Examples 1 to 4, the particle size distribution of the mucosal mesenchymal stem cell exosome preparation samples prepared in Examples 1 to 3 of the present invention was in a smaller particle size range, indicating that the present invention uses the composite field formed by ultrasound-pulsed electric field to synergistically modify trehalose and mannitol, which can better prevent the aggregation of olfactory mucosal mesenchymal stem cell exosomes during the freeze-drying process and enhance the stability of the olfactory mucosal mesenchymal stem cell exosome preparation system. The average particle size of each group of test samples is shown in Table 1.

[0098] Table 1

[0099] Test samples Average particle size (nm) Example 1 48 Example 2 64 Example 3 59 Comparative Example 1 97 Comparative Example 2 85 Comparative Example 3 81 Comparative Example 4 73

[0100] Test Example 2

[0101] In this example, BALB / C mice with osteosclerosis were used as an animal model according to the experimental method disclosed in the prior art to investigate the effect of the olfactory mucosa mesenchymal stem cell exosome preparation (after reconstitution) prepared in Example 1 on the expression of type II carbonic anhydrase in osteoclasts of the osteosclerosis mouse model.

[0102] The experimental method is as follows:

[0103] Experimental animals: 20 BALB / C mice with osteosclerosis and 10 normal BALB / C mice, with the weight and age kept consistent to reduce the influence of individual differences on the experimental results.

[0104] Grouping and administration: 20 BALB / C mice with osteosclerosis were randomly divided into 2 groups: a treatment group and a model control group, with 10 mice in each group; 10 normal BALB / C mice were used as a normal control group. The treatment group was administered with the olfactory mucosa mesenchymal stem cell exosome preparation prepared in Example 1 at a dosage of 30 mg / kg / d, and administered by gavage for 2 months; the model control group and the normal control group were administered with the same volume of physiological saline as the treatment group, and administered by gavage for 2 months.

[0105] Data detection and statistical analysis: After the experiment was terminated, osteoclasts were separated and extracted according to the method disclosed in the prior art to obtain an osteoclast suspension; the osteoclast suspension was blown evenly and then transferred to a 12-well plate for incubation for 24 hours; after protein quantification by the BCA method, the expression of type II carbonic anhydrase was determined using a type II carbonic anhydrase ELISA enzyme-linked immunosorbent assay kit. The test results of type II carbonic anhydrase content in osteoclasts of each group of mice are shown in Table 2.

[0106] Table 2

[0107] Test Group Type II carbonic anhydrase content (mg / L) Normal control group 24.3 Model control group 5.1 Drug group 21.9

[0108] As shown in Table 2, 1) compared with the normal control group, the content of type II carbonic anhydrase in osteoclasts of mice in the model control group was significantly reduced, indicating that the bone resorption capacity of osteoclasts in the mouse model of osteosclerosis was seriously insufficient; 2) compared with the model control group, the content of type II carbonic anhydrase in osteoclasts of mice in the group administered with the olfactory mucosa mesenchymal stem cell exosome preparation provided in the embodiment of the present invention was significantly increased, indicating that the olfactory mucosa mesenchymal stem cell exosomes provided in the embodiment of the present invention can effectively promote the expression of type II carbonic anhydrase, promote osteoclastic bone resorption, and have a better improvement effect on the symptoms of osteosclerosis.

[0109] In addition, the olfactory mucosa mesenchymal stem cell exosome preparation (after reconstitution) prepared in Example 1 of the present invention was dripped into the conjunctival sac of healthy New Zealand rabbits with no damage in eye examination, compressing the nasolacrimal duct and passively closing the eyes, 4 times a day, 1-2 drops each time, for 14 consecutive days. After the experiment, the cornea, iris, and conjunctiva were observed to show no swelling, congestion, turbidity, etc., indicating that it is non-irritating and safe to use.

[0110] In summary, the embodiments of the present invention provide an olfactory mucosa mesenchymal stem cell exosome preparation, a preparation method and application thereof. The present invention uses a composite field formed by an ultrasound-pulsed electric field under appropriate conditions and parameters to synergistically modify trehalose and mannitol, thereby enhancing the freeze-drying protection effect on the olfactory mucosa mesenchymal stem cell exosomes, and providing convenience for the storage and transportation of the exosome preparation; at the same time, the present invention also found that the olfactory mucosa mesenchymal stem cell exosomes have a significant improvement effect on the symptoms of a mouse model with osteosclerosis, which not only broadens the clinical application field of exosomes, but also provides a new means for the treatment of osteosclerosis.

[0111] Various embodiments of the present invention may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be understood as a rigid limitation on the scope of the present invention; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, the range description from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

[0112] The foregoing is merely a specific embodiment of the present invention, which enables those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features claimed herein.

Claims

1. An olfactory mucosa mesenchymal stem cell exosome preparation, characterized in that: The olfactory mucosa mesenchymal stem cell exosome preparation comprises the following raw materials: Olfactory mucosa mesenchymal stem cell exosomes, a lyophilized protective composition, and phosphate buffer; The freeze-dried protective composition is obtained by treating trehalose and mannitol by ultrasound-pulse electric field coupling; The working condition parameters of the ultrasound-pulse electric field coupling treatment include: the ultrasonic power is 550-700W, and the pulse power supply constant voltage is 3-7V.

2. The olfactory mucosa mesenchymal stem cell exosome preparation according to claim 1, characterized in that: The preparation method of the freeze-dried protective composition comprises the following steps: Stir and mix trehalose, mannitol and deionized water in a weight ratio of (1-2):(1-2):(15-20) to obtain a first mixed liquid; The ultrasonic probe of the ultrasonic device and the electrodes of the pulse device are placed in the first mixed liquid, and the distance between the electrodes is set to 15-20 mm, the constant voltage of the pulse power supply is 3-7 V, the pulse period is 5-10 ms, and the ultrasonic power is 550-700 W; the ultrasonic device and the pulse device are turned on at the same time to perform ultrasonic-pulse electric field coupling treatment for 180-360 seconds to obtain a second mixed liquid; The second mixed liquid is concentrated, dried, crushed and sieved to obtain the freeze-dried protection composition.

3. The olfactory mucosa mesenchymal stem cell exosome preparation according to claim 2, characterized in that: The weight ratio of the trehalose, the mannitol and the deionized water is 1.5:1:18, the ultrasonic power is 615W, and the constant voltage of the pulse power supply is 5V.

4. The olfactory mucosa mesenchymal stem cell exosome preparation according to claim 1, characterized in that: The concentration of the olfactory mucosa mesenchymal stem cell exosomes in the olfactory mucosa mesenchymal stem cell exosome preparation is 100 to 1000 μg / mL.

5. The olfactory mucosa mesenchymal stem cell exosome preparation according to claim 1, characterized in that: The concentration of the freeze-dried protective composition in the olfactory mucosa mesenchymal stem cell exosome preparation is 5-10% w / v.

6. The olfactory mucosa mesenchymal stem cell exosome preparation according to claim 1, characterized in that: The physicochemical property parameters of the phosphate buffer include: a concentration of 10 to 30 mmol / L and a pH of 6.5 to 7.

5.

7. A method for preparing the olfactory mucosa mesenchymal stem cell exosome preparation according to any one of claims 1 to 6, characterized in that: The preparation method comprises the following steps: Mixing the olfactory mucosa mesenchymal stem cell exosomes, the freeze-dried protective composition and the phosphate buffer to obtain a freeze-dried solution; The lyophilized liquid is freeze-dried to obtain the olfactory mucosa mesenchymal stem cell exosome preparation.

8. The method for preparing the olfactory mucosa mesenchymal stem cell exosome preparation according to claim 7, characterized in that: The freeze-drying steps sequentially include a first freezing, a second freezing, a first drying, and a second drying; The working condition parameters of the first freezing include: temperature of -25 to -15°C and time of 1 to 2 hours; The working condition parameters of the second freezing include: temperature of -55 to -50°C and time of 2 to 4 hours; The working condition parameters of the first drying include: vacuuming to 0.1-0.2 mbar, temperature of -30--20°C, and time of 12-16 hours; The working condition parameters of the second drying include: vacuuming to 0.1-0.2 mbar, temperature of 20-30° C., and time of 2-5 hours.

9. Use of the olfactory mucosa mesenchymal stem cell exosome preparation according to any one of claims 1 to 6 and / or the olfactory mucosa mesenchymal stem cell exosome preparation prepared by the preparation method according to any one of claims 7 to 8 in the preparation of a drug for treating osteosclerosis.