A biological preparation for treating rheumatoid arthritis and its preparation method

Abstract

The present invention discloses a biological preparation for treating rheumatoid arthritis and a preparation method thereof, wherein the biological preparation is prepared from the following raw materials: human umbilical cord mesenchymal stem cell exosomes, phycocyanin, honeysuckle extract, sodium alginate and normal saline. The present invention adopts natural sodium alginate as a polymer matrix, so that the prepared biological preparation has good biocompatibility and is easily degraded in vivo, and can respond to calcium ions at the arthritis site to form a hydrogel, so as to achieve the slow release of exosomes, phycocyanin and honeysuckle extract, and achieve the effect of lasting treatment; in addition, through the combined action of exosomes, phycocyanin and honeysuckle extract, the level of local inflammatory cytokines can be effectively improved, blood circulation in the joints and repair of bone and joint injuries can be promoted, and the purpose of treating rheumatoid arthritis can be achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of rheumatoid arthritis treatment, and in particular to a biological preparation for treating rheumatoid arthritis and a preparation method thereof. Background Art

[0002] Rheumatoid arthritis (RA) is a chronic, systemic disease characterized by inflammatory synovitis, which is characterized by polyarticular, symmetrical, invasive joint inflammation of the small joints of the hands and feet, often accompanied by extra-articular organ involvement and positive serum rheumatoid factor, which can lead to joint deformity and loss of function.

[0003] The treatments for rheumatoid arthritis mainly include drugs and surgery. Among them, drugs used to treat rheumatoid arthritis include ibuprofen, non-steroidal anti-inflammatory drugs, methotrexate, prednisone and other drugs. Although the above drugs have certain anti-inflammatory, analgesic and joint function improvement effects, they will inevitably cause some side effects to the body. For example, non-steroidal anti-inflammatory drugs can cause gastrointestinal discomfort, and hormones can cause osteoporosis and other problems.

[0004] Exosomes are disc-shaped vesicles (30-150nm) secreted by cells into the extracellular fluid. Many cells can secrete exosomes under normal and pathological conditions. They are mainly derived from multivesicular bodies formed by the invagination of lysosomal microparticles in cells, and are released into the extracellular matrix after the fusion of the multivesicular outer membrane with the cell membrane. Studies have shown that exosomes are involved in a variety of biological activities, such as regulating immune responses, transporting proteins and RNA, etc.

[0005] Therefore, it is particularly important to provide a biological agent that is effective in treating rheumatoid arthritis without side effects. Summary of the invention

[0006] The purpose of the present invention is to provide a biological preparation for treating rheumatoid arthritis and a preparation method thereof. The biological preparation has a significant therapeutic effect on rheumatoid arthritis and is prepared by combining exosomes with natural plant components and has no toxic side effects on the human body.

[0007] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0008] The first aspect of the present invention provides a biological preparation for treating rheumatoid arthritis, wherein the biological preparation is prepared from the following raw materials:

[0009] Human umbilical cord mesenchymal stem cell exosomes, phycocyanin, honeysuckle extract, sodium alginate and normal saline.

[0010] Phycocyanin in the present invention is a dark blue pigment extracted from blue-green algae. It is one of the rare pigment proteins in nature. It is not only brightly colored, but also a nutritious protein with a complete amino acid composition and a high content of essential amino acids. It is both a protein and an excellent natural edible pigment, and also has antioxidant, anti-tumor, immune system regulation and anti-inflammatory properties.

[0011] The present invention adopts natural sodium alginate as a polymer matrix, so that the prepared biological preparation has good biocompatibility and is easily degradable in the body. At the same time, it can respond to calcium ions in the arthritis to form a hydrogel, realize the slow release of exosomes, phycocyanin, and honeysuckle extract, and achieve a lasting therapeutic effect; in addition, through the combined action of exosomes, phycocyanin and honeysuckle extract, it can effectively improve the level of local inflammatory cytokines, promote blood circulation in the joints and repair of bone and joint injuries, and achieve the purpose of treating rheumatoid arthritis.

[0012] Preferably, the biological preparation is prepared from the following raw materials in percentage:

[0013] 0.1% to 0.4% human umbilical cord mesenchymal stem cell exosomes, 1% to 5% phycocyanin, 0.5% to 0.8% honeysuckle extract and 2% to 3% sodium alginate, and the balance is normal saline.

[0014] More preferably, the biological preparation is prepared from raw materials having the following percentage contents:

[0015] 0.2% human umbilical cord mesenchymal stem cell exosomes, 3% phycocyanin, 0.6% honeysuckle extract and 2.5% sodium alginate, and the balance is normal saline.

[0016] In the present invention, by limiting the ratio of each raw material, the synergistic effect of each raw material can be better adjusted to improve the overall efficacy of the biological preparation.

[0017] Preferably, the human umbilical cord mesenchymal stem cell exosomes are prepared by the following method:

[0018] The primary umbilical cord mesenchymal stem cells are subcultured, and the third generation culture supernatant of the umbilical cord mesenchymal stem cells is collected; the supernatant is then centrifuged at (3000-4000)×g to remove cells and cell debris;

[0019] Add exosome precipitation reagent to the supernatant, mix thoroughly, let stand, and then centrifuge at (10,000-50,000) × g for 20-40 min to collect the precipitate;

[0020] The precipitate was resuspended in PBS and placed in a purification column, and the purification column was placed in a collection tube. The collection tube was centrifuged at (3000-4000)×g for 8-12 minutes at 4° C., and the solution in the collection tube was freeze-dried to obtain the human umbilical cord mesenchymal stem cell exosomes.

[0021] Preferably, the honeysuckle extract is prepared by the following method:

[0022] The honeysuckle is crushed and placed in water for stirring extraction, and the extraction is continued for 2 to 3 times. The extracts are combined, concentrated, and freeze-dried to obtain the honeysuckle extract.

[0023] Preferably, the mesh size of the honeysuckle after crushing is not less than 50 mesh.

[0024] Preferably, the stirring extraction temperature is 50-60° C., and the extraction time is 3-4 hours.

[0025] The second aspect of the present invention provides a method for preparing the above-mentioned biological agent, the preparation method comprising the following steps:

[0026] (a) placing human umbilical cord mesenchymal stem cell exosomes in physiological saline and mixing to obtain a mixed solution;

[0027] (b) placing the honeysuckle extract and phycocyanin in the mixed solution and stirring to mix well, then adding sodium alginate and shaking to mix, thereby obtaining the biological preparation.

[0028] Compared with the prior art, the beneficial effects of the present invention include at least:

[0029] The present invention adopts natural sodium alginate as a polymer matrix, so that the prepared biological preparation has good biocompatibility and is easily degradable in the body. At the same time, it can respond to calcium ions in the arthritis to form a hydrogel, realize the slow release of exosomes, phycocyanin, and honeysuckle extract, and achieve a lasting therapeutic effect; in addition, through the combined action of exosomes, phycocyanin and honeysuckle extract, it can effectively improve the level of local inflammatory cytokines, promote blood circulation in the joints and repair of bone and joint injuries, and achieve the purpose of treating rheumatoid arthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for the specific embodiments or the description of the prior art. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn according to the actual scale.

[0031] Figure 1 This is a photograph of the toes of mice in the control group on the 30th day in the experimental example of the present invention;

[0032] Figure 2 This is a photograph of the toes of the model mice on the 30th day in the experimental example of the present invention;

[0033] Figure 3 This is a photograph of the toes of mice in the EVs-Lj-Phy-SA group on day 30 in the experimental example of the present invention;

[0034] Figure 4 This is a photograph of the toes of mice in the EVs-Lj-SA group on day 30 in the experimental example of the present invention;

[0035] Figure 5 This is a photograph of the toes of mice in the EVs-Lj-Phy group on day 30 in the experimental example of the present invention;

[0036] Figure 6 This is a photograph of the toes of mice in the Lj-Phy-SA group on the 30th day in the experimental example of the present invention. DETAILED DESCRIPTION

[0037] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.

[0038] It should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application should have the common meanings understood by those skilled in the art to which the present invention belongs.

[0039] The embodiment of the present invention provides a biological preparation for treating rheumatoid arthritis, wherein the biological preparation is prepared from the following raw materials:

[0040] Human umbilical cord mesenchymal stem cell exosomes, phycocyanin, honeysuckle extract, sodium alginate and normal saline.

[0041] The present invention adopts natural sodium alginate as a polymer matrix, so that the prepared biological preparation has good biocompatibility and is easily degradable in the body. At the same time, it can respond to calcium ions in the arthritis to form a hydrogel, realize the slow release of exosomes, phycocyanin, and honeysuckle extract, and achieve a lasting therapeutic effect; in addition, through the combined action of exosomes, phycocyanin and honeysuckle extract, it can effectively improve the level of local inflammatory cytokines, promote blood circulation in the joints and repair of bone and joint injuries, and achieve the purpose of treating rheumatoid arthritis.

[0042] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0043] Example 1

[0044] This embodiment is a method for preparing human umbilical cord mesenchymal stem cell exosomes, which comprises the following steps:

[0045] 1. Instruments and consumables, D-MEM / F12 culture medium, fetal bovine serum, inverted microscope, clean bench, CO2 incubator, centrifuge, cytokines, ITS and PBs, etc.;

[0046] 2. Reagent configuration

[0047] 1. D-MEM / F12 medium configuration: D-MEM / F12 medium containing 10% fetal bovine serum, one cytokine, and 5ml ITS;

[0048] 2. Cytokines: containing 20ng / ml EGF, 5ng / ml bFGF;

[0049] 3. Preparation before the experiment

[0050] 5 pairs of scissors, 5 pairs of hemostats, 3 pairs of tissue forceps, 3 pairs of ordinary forceps, 2 kidney-shaped dishes, 3 50ml beakers, 2 medicine spoons (about 20cm), 500ml PBS autoclaved at 121℃ for 30 minutes;

[0051] 4. Experimental operation

[0052] (1) Expose the clean bench to ultraviolet light for 30 minutes and wipe the surface;

[0053] (2) Place the sterilized laboratory equipment on the workbench, light the alcohol lamp, take out a kidney-shaped dish, pour half of 75% alcohol into it, place the umbilical cord into the kidney-shaped dish, and sterilize for 30 seconds;

[0054] (3) Take a 50 ml beaker, pour half of the sterilized PBS into it, put the umbilical cord into the beaker, and wash it three times;

[0055] (4) Take out another kidney-shaped dish, put the umbilical cord in it, pour in an appropriate amount of PBS, take scissors in the right hand and ordinary tweezers in the left hand, and cut the umbilical cord into small pieces of about 2 cm;

[0056] (5) Use the hemostatic forceps in your left hand and the tissue forceps in your right hand to slowly remove the outer skin and blood vessels of the umbilical cord;

[0057] (6) Place the remaining tissue in a clean beaker and cut it into pieces of about 3 mm using scissors;

[0058] (7) Use a medicine spoon to spread the tissue blocks in the T75 bottle, with about 100 tissue blocks in each bottle, until all the tissue blocks are spread;

[0059] (8) Slowly add 15 ml of culture medium to one side of the T75 bottle and place in a CO2 incubator for incubation;

[0060] (9) Change the medium every 48 hours. After one week, observe the crawling of stem cells. If the crawling is good, discard the tissue block and add culture medium.

[0061] (10) After 48 h, the cells were digested by adding 1 ml of trypsin for digestion, and the cells were collected and centrifuged (1200 rpm, 5 min). The supernatant was discarded, and the cells were resuspended in culture medium. The cells were divided into 4 groups according to the number of cells (1×10 6 ) cells are inoculated, which is the P1 generation;

[0062] (11) After 48 hours, digest and centrifuge, count, and passage to obtain the P2 generation;

[0063] (12) Two days later, digest and centrifuge the supernatant (to test for sterility, mycoplasma, and endotoxin), and count the cells at 4.5 × 10 6 Cryopreservation;

[0064] (13) Resuscitate P2 cells according to production needs, transfer to P3 cells and collect supernatant;

[0065] (14) Centrifuge the supernatant at 4000 × g to remove cells and cell debris;

[0066] (15) Add exosome precipitation reagent to the supernatant, mix thoroughly, let stand, and then centrifuge at 10,000 × g for 30 min to collect the precipitate;

[0067] (16) The precipitate was resuspended in PBS and placed in a purification column, which was then placed in a collection tube. The collection tube was centrifuged at 4000 × g for 10 min at 4 °C, and the solution in the collection tube was freeze-dried to obtain human umbilical cord mesenchymal stem cell exosomes.

[0068] Example 2

[0069] This embodiment is a method for preparing a honeysuckle extract, which comprises the following steps:

[0070] The honeysuckle was crushed and passed through an 80-mesh sieve, the sieve material was collected, and the sieve material was placed in 55°C water for stirring extraction for 4 hours, and the extraction was repeated 3 times. The extracts were combined, concentrated, and freeze-dried to obtain the above honeysuckle extract.

[0071] Example 3

[0072] This embodiment is a biological preparation for treating rheumatoid arthritis, and the biological preparation is prepared from raw materials with the following percentage contents:

[0073] 0.2% human umbilical cord mesenchymal stem cell exosomes of Example 1, 3% phycocyanin, 0.6% honeysuckle extract of Example 2, and 2.5% sodium alginate, and the balance is normal saline.

[0074] The above-mentioned biological preparation for treating rheumatoid arthritis is prepared by the following method:

[0075] (a) placing human umbilical cord mesenchymal stem cell exosomes in physiological saline and mixing to obtain a mixed solution;

[0076] (b) The honeysuckle extract and phycocyanin are placed in the mixed solution and stirred to mix well, and then sodium alginate is added and shaken to mix, thereby obtaining the above-mentioned biological preparation (denoted as EVs-Lj-Phy-SA).

[0077] Comparative Example 1

[0078] This comparative example is a biological preparation for treating rheumatoid arthritis, which is basically the same as the biological preparation in Example 3, except that phycocyanin is not added.

[0079] The above-mentioned biological preparation for treating rheumatoid arthritis is prepared by the following method:

[0080] (a) placing human umbilical cord mesenchymal stem cell exosomes in physiological saline and mixing to obtain a mixed solution;

[0081] (b) The honeysuckle extract is placed in the mixed solution and stirred to mix well, and then sodium alginate is added and shaken to mix, thereby obtaining the above-mentioned biological preparation (denoted as EVs-Lj-SA).

[0082] Comparative Example 2

[0083] This comparative example is a biological preparation for treating rheumatoid arthritis, which is basically the same as the biological preparation in Example 3, except that sodium alginate is not added.

[0084] The above-mentioned biological preparation for treating rheumatoid arthritis is prepared by the following method:

[0085] (a) placing human umbilical cord mesenchymal stem cell exosomes in physiological saline and mixing to obtain a mixed solution;

[0086] (b) The honeysuckle extract and phycocyanin are placed in the mixed solution and stirred to obtain the above biological preparation (denoted as EVs-Lj-Phy).

[0087] Comparative Example 3

[0088] This comparative example is a biological preparation for treating rheumatoid arthritis, which is substantially the same as the biological preparation in Example 3, except that no exocrine human umbilical cord mesenchymal stem cells are added.

[0089] The above-mentioned biological preparation for treating rheumatoid arthritis is prepared by the following method:

[0090] The honeysuckle extract and phycocyanin were placed in physiological saline and stirred to mix well, and then sodium alginate was added and shaken to mix, thereby obtaining the above biological preparation (referred to as Lj-Phy-SA).

[0091] Experimental example

[0092] This experimental example is a study on the effects of different biological agents on rheumatoid arthritis:

[0093] Experimental groups: Thirty-six Wistar male rats were obtained and randomly divided into 6 groups (6 rats in each group), including control group, model group, EVs-Lj-Phy-SA group, EVs-Lj-SA group, EVs-Lj-Phy group and Lj-Phy-SA group;

[0094] Animal modeling: The rats in the model group, EVs-Lj-Phy-SA group, EVs-Lj-SA group, EVs-Lj-Phy group, and Lj-Phy-SA group were modeled as follows:

[0095] 0.1 ml of complete Freund's adjuvant was injected intradermally into the plantar of the right hind paw of each rat, and after 12 days of normal feeding, the rats developed rheumatoid arthritis lesions.

[0096] Dosage:

[0097] After modeling, the medication of each group is as follows: the control group and the model group were injected with normal saline, the EVs-Lj-Phy-SA group, the EVs-Lj-SA group, the EVs-Lj-Phy group and the Lj-Phy-SA group were injected with EVs-Lj-Phy-SA, EVs-Lj-SA, EVs-Lj-Phy and Lj-Phy-SA, respectively. The injection volume was 0.02 ml / mouse, and the injection method was intra-articular injection; the injection was once every two weeks; the medication cycle was 45 days, and the plantar thickness of the right hind foot of the rats was measured on the 1st, 15th and 30th days of medication, and the average value was calculated. The typical mouse toe pictures in each group of mice on the 30th day are shown in the following figure. Figures 1 to 6 As shown, Figure 1 As the control group, Figure 2 For the model group, Figure 3 For the EVs-Lj-Phy-SA group, Figure 4 For the EVs-Lj-SA group, Figure 5 For the EVs-Lj-Phy group, Figure 6 It is the Lj-Phy-SA group.

[0098] Results: The average values ​​of plantar thickness in different groups are shown in Table 1:

[0099] Table 1

[0100]

[0101] From the data in Table 1, we can see that:

[0102] Compared with the control group, the plantar thickness of the model group and the experimental group on the first day was higher, proving that the modeling was successful; compared with the experimental group and the model group, the plantar thickness of the plantar was effectively reduced, and the swelling caused by inflammation was effectively relieved; compared with the EVs-Lj-Phy-SA group, the plantar thickness of the EVs-Lj-SA group, the EVs-Lj-Phy group and the Lj-Phy-SA group was higher, proving that the EVs-Lj-Phy-SA of the present application has the best inhibitory effect on rheumatoid arthritis, however, at 15 days, the inhibitory effect of the EVs-Lj-Phy group was better than that of the EVs-Lj-Phy-SA group, but its long-term 9 (30th) arthritis inhibition effect was worse than that of the EVs-Lj-Phy-SA group, which shows that by adding sodium alginate, the various components can be better slowly released to achieve a lasting inhibitory effect on inflammation.

[0103] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. These modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and specification of the present invention.

Claims

1. A biological preparation for treating rheumatoid arthritis, characterized in that: The biological preparation is prepared from the following raw materials in percentage: 0.2% human umbilical cord mesenchymal stem cell exosomes, 3% phycocyanin, 0.6% honeysuckle extract and 2.5% sodium alginate, the balance is normal saline; The human umbilical cord mesenchymal stem cell exosomes are prepared by the following method: The primary umbilical cord mesenchymal stem cells are subcultured, and the third generation culture supernatant of the umbilical cord mesenchymal stem cells is collected; the supernatant is then centrifuged at (3000-4000)×g to remove cells and cell debris; Add exosome precipitation reagent to the supernatant, mix thoroughly, let stand, and then centrifuge at (10,000-50,000) × g for 20-40 min to collect the precipitate; Resuspending the precipitate with PBS and placing it in a purification column, placing the purification column in a collection tube, centrifuging the collection tube at (3000-4000)×g for 8-12 minutes at 4° C., freeze-drying the solution in the collection tube to obtain the human umbilical cord mesenchymal stem cell exosomes; The honeysuckle extract is prepared by the following method: The honeysuckle is crushed and placed in water for stirring extraction, and the extraction is performed continuously for 2 to 3 times, and the extracts are combined, and then concentrated and freeze-dried to obtain the honeysuckle extract; The mesh size of the honeysuckle after crushing is not less than 50 mesh; The stirring extraction temperature is 50-60°C and the extraction time is 3-4h; The preparation method of the biological agent comprises the following steps: (a) placing human umbilical cord mesenchymal stem cell exosomes in physiological saline and mixing to obtain a mixed solution; (b) placing the honeysuckle extract and phycocyanin in the mixed solution and stirring to mix well, then adding sodium alginate and shaking to mix, thereby obtaining the biological preparation.

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