An umbilical cord mesenchymal stem cell exosome overexpressing anxa1 and a preparation method and application thereof

Abstract

The application relates to a mesenchymal stem cell exosome overexpressing Anxal and a preparation method and application thereof, and belongs to the technical field of inflammatory disease medicine preparation. The umbilical cord mesenchymal stem cell exosome overexpressing Anxal provided by the application is obtained by the following steps: cell electrotransfection of a modified Anxal mRNA sequence which is not easy to degrade into umbilical cord mesenchymal stem cells, culture, collection of cell supernatant, and then super-high-speed centrifugation or tangential flow. The umbilical cord mesenchymal stem cell exosome overexpressing Anxal protein can efficiently improve nerve tissue inflammation caused by trauma, improve nerve function, reduce chronic inflammation of local tissues of limbs caused by diabetes, burns and the like, and accelerate tissue healing.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of inflammatory disease drug preparation, and particularly relates to a mesenchymal stem cell exosome overexpressing Anxa1 and a preparation method and application thereof. BACKGROUND

[0002] Inflammation is accompanied by many diseases, and inflammation itself can manifest as redness, swelling, heat, pain and dysfunction. The factors causing inflammation can be pathogenic microbial infection, physical factors such as impact, tissue necrosis, allergy, etc. The above-mentioned typical diseases causing inflammation include soft tissue infection, neural inflammation after craniocerebral trauma, limb tissue necrotic inflammatory reaction caused by diabetes, rheumatism or rheumatoid arthritis. The incidence of rheumatism and rheumatoid arthritis is about 1% worldwide, and there is currently no effective treatment drug. Inflammatory response also greatly affects the course of the disease, and anti-inflammatory treatment often has a positive effect on the improvement or cure of the symptoms of such diseases. However, there are few anti-inflammatory drugs in clinical practice, and hormone drugs have large side effects. Therefore, we urgently need a highly effective and safe anti-inflammatory biological agent.

[0003] Stem cells have great prospects in the field of regenerative medicine, and they have been proven to participate in the repair of various tissues and organs, such as heart, liver, lung, kidney, and nerve. In recent years, umbilical cord mesenchymal stem cells (MSCs) have been widely studied, which are a kind of multifunctional stem cells present in the umbilical cord of newborns. Exosomes are a kind of extracellular vesicles (EVs, double-layer lipid structure vesicles) secreted by cells. Umbilical cord mesenchymal stem cells can exert various biological effects by secreting exosomes, and exosomes can carry various functional molecules secreted by umbilical cord mesenchymal stem cells. Exosomes are an important way of intercellular communication, and the diameter of exosomes is about 40-200 nm, and the main components include proteins and various types of nucleic acids. Although umbilical cord mesenchymal stem cell exosomes can secrete certain tissue nutrients, their anti-inflammatory effect is still weak, and how to enhance the anti-inflammatory capacity of the exosomes is an important direction for the preparation of exosome drugs.

[0004] Annexins are a superfamily of calcium-dependent phospholipid-binding proteins, and studies have found that they are widely present in animals, plants and fungi in various organisms, and are expressed in almost all organs. ANXA1 is the first member of the family to be discovered, and is involved in anti-inflammatory response, cell differentiation and proliferation, cell death signal regulation, and phagocytosis of apoptotic cells, etc. ANXA1 mainly exerts anti-inflammatory effect by inhibiting the migration of granulocytes, which has been verified in various established acute, chronic and even systemic inflammatory response models.

[0005] The content ANXA1 carried in the umbilical cord mesenchymal stem cell exosome is less, so it is difficult to have a good therapeutic effect on inflammatory diseases. Therefore, increasing the content of Anxa1 in the umbilical cord mesenchymal stem cell exosome is an important way to improve its clinical application value. There is no preparation of umbilical cord mesenchymal stem cell exosome overexpressing Anxa1 at present. SUMMARY

[0006] In order to solve the above technical problems, the first aspect of the present application provides a preparation method of umbilical cord mesenchymal stem cell exosome overexpressing Anxa1, comprising the following steps:

[0007] The artificially synthesized Anxa1 messenger ribonucleic acid (Anxa1 mRNA) is subjected to cap modification, tail modification and pseudouridine modification to obtain modified Anxa1 mRNA (modified Anxa1 mRNA);

[0008] The Wharton's jelly in the human umbilical cord is separated and digested with mixed enzymes, and the digested tissue fragments and suspended cells are cultured to the third generation to obtain umbilical cord mesenchymal stem cells;

[0009] The modified Anxa1 mRNA is introduced into the umbilical cord mesenchymal stem cells by cell electroporation to obtain cell electroporation umbilical cord mesenchymal stem cells, and the cell electroporation umbilical cord mesenchymal stem cells are cultured and expanded in a 3D environment to obtain Anxa1 overexpression umbilical cord mesenchymal stem cell culture supernatant;

[0010] The Anxa1 overexpression umbilical cord mesenchymal stem cell culture supernatant is centrifuged to remove living cells and debris, and the exosomes in the supernatant are separated and purified by ultrahigh speed centrifugation or tangential flow method to obtain umbilical cord mesenchymal stem cell exosomes overexpressing Anxa1.

[0011] The preparation method of the alternative technical solution is that the sequence of the messenger ribonucleic acid (Anxa1 mRNA) of Anxa1 is shown in SEQ ID NO. 1.

[0012] The preparation method of the alternative technical solution is that the cap modification method is:

[0013] 7-methyl guanine is connected to the 5'-end of the artificially synthesized Anxa1 messenger ribonucleic acid (Anxa1 mRNA) through a 5'-5' triphosphate bond.

[0014] The preparation method of the alternative technical solution is that the tail modification method is:

[0015] Polyadenylic acid polyA is connected to the 3'-end of the artificially synthesized Anxa1 messenger ribonucleic acid (Anxa1 mRNA).

[0016] The preparation method of the alternative technical solution is the method for modifying the pseudouracil:

[0017] The messenger ribonucleic acid (AnxalmRNA) of artificially synthesized Anxal is modified by N1-methyl-pseudouridine.

[0018] The preparation method of the alternative technical solution is that the components of the mixed enzyme for digesting Wharton's jelly include:

[0019] 0.2% (w / w) collagenase type II, 0.2% (w / w) collagenase type IV, 0.1% (w / w) hyaluronidase, 0.1% (w / w) trypsin, 15 U / ml DNase.

[0020] The preparation method of the alternative technical solution is the method for preparing umbilical cord mesenchymal stem cells:

[0021] The umbilical cord of an ex vivo full-term newborn is taken, and Wharton's jelly is separated therefrom, cut into 1 mm 3 size on ice, and digested by the mixed enzyme for 2-16 hours. The tissue fragments and suspended cells after digestion are cultured, and the cells are subcultured at a ratio of 1:2-1:4 when they reach 70-80% confluence. The cells in the logarithmic growth phase are obtained by subculturing to the third generation, and umbilical cord mesenchymal stem cells are obtained when the cells grow to 70%-80% confluence.

[0022] The preparation method of the alternative technical solution is that the culture medium for preparing umbilical cord mesenchymal stem cells includes: umbilical cord mesenchymal stem cell culture medium without phenol red and serum; preferably, the umbilical cord mesenchymal stem cell culture medium without phenol red and serum does not contain any animal-derived components.

[0023] The preparation method of the alternative technical solution is that the conditions for cell electroporation include:

[0024] The pulse voltage is 150-250 V, the driving voltage is 10-30 V, the pulse time is 1-30 ms, and the cycle number is 1-30.

[0025] The preparation method of the alternative technical solution is that the method for culturing and expanding the umbilical cord mesenchymal stem cells after cell electroporation in a 3D environment includes:

[0026] The suspension of the umbilical cord mesenchymal stem cells after cell electroporation is dropped into 3D microcarriers;

[0027] The 3D microcarriers containing umbilical cord mesenchymal stem cells are placed into a 3D stirring bioreactor.

[0028] The preparation method of the alternative technical solution is that the method for separating and purifying exosomes in the supernatant by using ultracentrifugation includes:

[0029] The supernatant is first filtered by using a sterile 0.45-micron filter screen, and the filtered supernatant is centrifuged at high speed at 4 DEG C three times to remove cell debris, and the supernatant is collected after each centrifugation, and the centrifugal force is 3000g, 4000g and 5000g, respectively, and the time is 30 minutes each time; after the end, the supernatant is collected and subjected to ultrahigh-speed centrifugation, the first time is 10000g, 70 minutes, the supernatant after the first ultrahigh-speed centrifugation is collected and used for the second centrifugation, and the second centrifugation parameters are 120000g, 70 minutes, and the supernatant after the second ultrahigh-speed centrifugation is removed, and the bottom of the tube is repeatedly washed with sterile PBS for 3 minutes to collect the exosomes.

[0030] The preparation method of the alternative technical solution is that the exosomes in the supernatant are separated and purified by using a tangential flow method.

[0031] The supernatant is circulated in a tangential flow device, and the exosomes are filtered and collected by using the tangential force of the filter screen and liquid flow.

[0032] The second aspect embodiment of the present application provides a human umbilical cord mesenchymal stem cell exosome overexpressing Anxa1.

[0033] The third aspect embodiment of the present application provides an application of the human umbilical cord mesenchymal stem cell exosome overexpressing Anxa1 in the preparation of a drug for treating inflammatory diseases; preferably, the inflammatory diseases include inflammatory reactions of various organs and tissues caused by various traumas, inflammatory diseases of tissues and organs caused by chronic injuries, any one of the liver, the lung, the kidney, the stomach and the muscle, systemic inflammatory diseases or infections caused by diabetes, and autoimmune inflammatory diseases.

[0034] The present application has the advantages and positive effects that the present application prepares a human umbilical cord mesenchymal stem cell exosome overexpressing Anxa1, the mRNA of Anxa1 is greatly reduced in immunogenicity after modification, and is avoided from being rapidly degraded in cells or in vivo. The human umbilical cord mesenchymal stem cell exosome overexpressing Anxa1 can be effectively injected in vivo and reduce inflammatory reactions of various inflammatory diseases, and improve the symptoms and treatment effects of various diseases. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 Optical microscope image of human umbilical cord mesenchymal stem cells (10x);

[0036] Figure 2 Positive rate of flow cytometry identification of surface markers of human umbilical cord mesenchymal stem cells;

[0037] Figure 3 Transmission electron microscope image of human umbilical cord mesenchymal stem cell exosomes overexpressing Anxa1;

[0038] Figure 4Exosome particle size analysis of human umbilical cord mesenchymal stem cells overexpressing Anxal

[0039] Figure 5 Western blot analysis of exosome markers of human umbilical cord mesenchymal stem cells overexpressing Anxal

[0040] Figure 6 Comparison of mNSS scores of rats

[0041] Figure 7 Comparison of IL-1β expression levels of exosomes in two groups

[0042] Figure 8 Comparison of IL10 expression levels of exosomes in two groups

[0043] Figure 9 Comparison of wound healing rates of rats

[0044] Figure 10 Comparison of IL-14 expression levels in wound tissues of rats in two groups

[0045] Figure 11 Comparison of TNFα expression levels in wound tissues of rats in two groups DETAILED DESCRIPTION

[0046] The following examples further illustrate the present application but should not be construed as limiting the application. Modifications or variations of the method, steps or conditions of the application can be made by those skilled in the art without departing from the spirit and scope of the application.

[0047] TERMS

[0048] Annexin A1 (ANXA1) is the first discovered member of the annexin family, which is involved in anti-inflammatory response, cell differentiation and proliferation, cell death signal regulation, and clearance of apoptotic cells, etc.

[0049] Mesenchymal stem cells (MSCs) are a kind of multipotent stem cells existing in the umbilical cord of newborns.

[0050] MSC-Exos is a kind of extracellular vesicles (EVs, double-layer lipid structure vesicles) secreted by MSCs.

[0051] Cell electroporation (electrotransfection), also known as cell electroporation, is to treat cells with a short high-field electric pulse, along the voltage difference of the cell membrane, the electric current will reversibly break through the cell membrane to form a transient water channel or small hole on the membrane, so that exogenous macromolecular substances DNA, RNA, protein, some small molecules, etc. Enter the inside of the cell membrane.

[0052] 3D environment is to place microcarriers containing cells into a 3D stirred bioreactor culture environment.

[0053] w / w refers to the mass percentage.

[0054] Example 1 Preparation of umbilical cord mesenchymal stem cell exosomes overexpressing Anxal

[0055] 1. Isolation and culture of umbilical cord mesenchymal stem cells

[0056] 1.1 Primary cells

[0057] Fresh, full-term healthy fetal umbilical cord specimens were obtained, immersed in PBS containing 1% penicillin and streptomycin, and placed on ice. The umbilical cord sample was voluntarily donated by the mother and her family, and the sample was applied in accordance with the ethical committee's specification for human specimen experiments. In a clean bench, the umbilical cord was cut into tissue blocks of 3 cm in size with an ophthalmic scissors, longitudinally cut, and washed with sterile PBS until no bloody liquid flowed out. The umbilical cord artery and vein were separated, and the outer membrane of the umbilical cord was peeled off, leaving the remaining tissue as Wharton's jelly. The Wharton's jelly tissue was cut into 1 mm 3 small pieces, transferred to mixed enzymes (0.2% type II collagenase, 0.2% type IV collagenase, 0.1% hyaluronidase, 0.1% trypsin, 15 U / ml DNase, which is prepared by mixing the components (enzymes) together to meet the above-mentioned concentration of each component (enzyme) in the Wharton's jelly tissue pieces), and digested at 37°C for 6h, with gentle shaking of the digestion solution every 30 minutes to allow mesenchymal stem cells to detach from the tissue pieces. The tissue digestion residue was filtered with a 70um filter, and the cell-containing filtrate was collected and centrifuged at 1500 rpm / min at 4°C for 5 minutes. The supernatant was discarded, and the cells at the bottom of the tube were umbilical cord mesenchymal stem cells (primary cells).

[0058] 1.2 Subculture cells

[0059] The umbilical cord mesenchymal stem cells were cultured in a phenol red-free serum-free medium (composition: DMEM medium, supplemented with 5 ml / L transferrin, 10 mg / L insulin, 10 ug / L bFGF, 10 ug / L bFGF, 10 ug / L EGF, 10 ug / L LIF, 20 ug / L SCF, 20 ug / L G--C-CSF, 25 mM L-glutamine), and the primary cells were inoculated in a T75 culture flask. The primary cells were left to stand for 7 days, and then the medium was changed for the first time. When the primary cells were close to 75%, they were digested with 0.25% trypsin and subcultured at a ratio of 1:3. When the subculture reached the third generation, the umbilical cord mesenchymal stem cells obtained were used for subsequent experiments. The umbilical cord mesenchymal stem cells were observed and photographed under a light microscope. Figure 1

[0060] 1.3 Flow cytometry detection

[0061] The third generation of umbilical cord mesenchymal stem cells in good growth state were selected, and six cell surface molecules, i.e., CD29, CD34, CD44, CD45, CD90, and CD105, were detected by flow cytometry. The required amount of cells for each test group was 1.0 x 10 5 The CD29, CD44, CD90, and CD105 are marker proteins of umbilical cord mesenchymal stem cells, and the CD45 and CD34 are non-marker proteins of umbilical cord mesenchymal stem cells. The positive rates of the surface molecule markers were calculated. The results are shown in Table 1. The positive rates of CD29, CD44, CD90, and CD105 were all above 98.5%, and the positive cell rates of CD45 and CD34 were both less than 1%. Figure 2

[0062] 2. Synthesis, modification of Anxa1 mRNA, cell electroporation of umbilical cord mesenchymal stem cells, and culture and expansion in a 3D environment

[0063] 2.1 Synthesis of Anxa1 mRNA

[0064] The sequence of Anxa1 mRNA is shown in SEQ ID NO. 1. The complete sequence of Anxa1 mRNA was synthesized by gene synthesis method, and the synthesized Anxa1 mRNA sequence was verified by sequencing.

[0065] 2.2 Capping modification of Anxa1 mRNA using capping enzyme

[0066] ​​Vaccinia Capping Enzyme (VCE) was used. Vaccinia virus can encode a 2'-O-methyltransferase enzyme that uses S-Adenosylmethionine (SAM) as a methyl donor to add a methyl group to the 2'-O site of the first nucleotide immediately adjacent to the cap structure (Cap0) at the 5' end of the RNA, forming mRNA with Cap1 structure.

[0067] 2.3 Tailing modification of Anxa1 mRNA using polyA

[0068] PolyA was linked to the 3'-end of the artificially synthesized Anxa1 messenger ribonucleic acid (Anxa1 mRNA).

[0069] 2.4 Pseudouridine modification of Anxa1 mRNA using N1-methyl-pseudouridine

[0070] The artificially synthesized Anxa1 messenger ribonucleic acid (Anxa1 mRNA) was modified with N1-methyl-pseudouridine.

[0071] 2.5 Cell electroporation of umbilical cord mesenchymal stem cells

[0072] Take 200 μl of umbilical cord mesenchymal stem cell suspension (cell amount is 4 x 10 5 6, add 20 μg of modified Anxa1 mRNA, and place the umbilical cord mesenchymal stem cell suspension with modified Anxa1 mRNA into an electroporation cup. Turn on the power supply and connect the instrument. The parameters are set as follows: voltage 250 V, driving voltage 10-30 V, interval 125 ms, pulse time 1-30 ms, and cycle number 1-10.

[0073] 2.6 Culture and expansion in 3D environment

[0074] After electroporation, the cell suspension was dropped into 3D microcarriers (preparation method see Chinese patent CN113249311A), and then placed into a Tyiloong2.0 3D bioreactor produced by Shanghai Tang Yihuike Biological Engineering Equipment Co., Ltd. for mass expansion.

[0075] 3. Purification of umbilical cord mesenchymal stem cell exosomes overexpressing Anxa1

[0076] 3.1 After umbilical cord mesenchymal stem cells have been cultured in a 3D bioreactor for 7 days, flow cytometry is used to detect the positive rate of CD29, CD44, CD90, and CD105 antibodies on the cell surface. When the cell positive rate exceeds 98%, the culture medium supernatant can be collected. Collect the cell and culture medium suspension, centrifuge at 1500rpm / min for 10 minutes, collect the supernatant after centrifugation, and discard the umbilical cord mesenchymal stem cells. Then filter the supernatant with a sterile 0.45μm filter. Centrifuge the filtered supernatant three times at 4°C to remove cell debris. Collect the supernatant after each centrifugation. The centrifugal force is 3000g, 4000g, and 5000g, respectively, and the time is 30min each time. The supernatant after high-speed centrifugation was then subjected to two ultrahigh-speed centrifugations, the first at 10,000 g for 70 min. The supernatant after the first ultrahigh-speed centrifugation was collected and used for a second centrifugation, with the second centrifugation parameters being 120,000 g for 70 min. The supernatant after the second ultrahigh-speed centrifugation was removed, and the bottom of the tube was repeatedly rinsed with sterile PBS for 3 min to collect exosomes, thereby obtaining umbilical cord mesenchymal stem cell exosomes overexpressing Anxa1.

[0077] In other embodiments, the tangential flow method can be used to separate and purify exosomes in the supernatant, that is, the supernatant after centrifugation at 1500 rpm / min is circulated in a tangential flow device, and the exosomes are filtered and collected by using the tangential force of the filter and the liquid flow. The yield of exosome collection is 1.6x 10 12 / L supernatant.

[0078] 3.2 Pipette 10 μL of the extracted exosome sample and carefully drop it onto the electron microscope copper grid. After 2 minutes of precipitation, use filter paper to gently absorb the liquid at the edge of the copper grid. Add 10 μL of uranyl acetate to the copper grid and stain for 2 minutes. Drain the excess floating liquid at the edge of the copper grid and let it air dry for several minutes at room temperature. Observe the exosomes under a transmission electron microscope to obtain the exosome morphology results as shown below. Figure 3 As shown. Use the nanometer to analyze the exosome particle size. Take 10μL of the extracted exosome sample and perform gradient dilution of the sample according to the operating procedures. After the automatic detection is completed, the exosome particle size results can be obtained as shown. Figure 4 As shown in Figure 2, the average particle size of exosomes is 168.3 nm. The surface protein markers of the extracted exosomes were identified by Western blot, and the identification markers were CD9, CD81, as shown in Figure 2. Figure 5 As shown, the expression levels of CD9 and CD81 were 1.1 times that of β-actin.

[0079] 3.3 The expression of Anxa1 protein in the exosomes of overexpressing Anxa1 umbilical cord mesenchymal stem cells and conventional umbilical cord mesenchymal stem cells was detected by ELISA method. According to the ELISA kit instructions, the standard was gradiently diluted for standard curve. The sample to be tested was gradiently diluted, mixed with reaction reagent and color developing reagent, and then the absorbance value was detected on the enzyme label instrument. The Anxa1 protein concentration of the sample to be tested was calculated according to the standard curve. The Anxa1 protein concentration in the exosomes of overexpressing Anxa1 umbilical cord mesenchymal stem cells was 26.4 mg / L, while the Anxa1 protein concentration in the exosomes of conventional umbilical cord mesenchymal stem cells was 3.2 mg / L.

[0080] Example 2 Therapeutic effect of intravenous transplantation of exosomes of overexpressing Anxa1 umbilical cord mesenchymal stem cells on acute craniocerebral trauma

[0081] 1. Preparation of acute craniocerebral trauma model of rats

[0082] Twenty-four female adult SD rats (2 months old) were selected, and anesthetized by intraperitoneal injection of 40 mg / kg sodium pentobarbital. The scalp was incised along the midline, and the subcutaneous soft tissue was separated to expose the skull surface. A micro drill was used to drill a hole with a diameter of 5 mm at 1 mm behind the right coronal suture and 2 mm lateral to the midline. A 40 g hammer was placed at a height of 25 cm to prepare the injury model by free fall. The depth of the hammer was 5 mm. Then the scalp was sutured and the wound was bandaged.

[0083] 2. Intravenous injection of exosomes of overexpressing Anxa1 umbilical cord mesenchymal stem cells and evaluation of therapeutic effect

[0084] 2.1 The successfully modeled rats were placed in the tail vein injection instrument, and the tail vein was developed. Twenty-four rats were divided into Anxa1 exosome group and conventional exosome group, with 12 rats in each group. The rats in the Anxa1 exosome group were injected with 100 μl of exosomes of overexpressing Anxa1 umbilical cord mesenchymal stem cells through the tail vein, while the rats in the conventional exosome group were injected with 100 μl of umbilical cord mesenchymal stem cell exosomes through the tail vein. After injection of the exosomes, the rats were placed in cages for normal feeding for 7 days.

[0085] 2.2 The neurological impairment scores of the rats in the two groups were evaluated at 7 days after the operation. The motor, sensory and reflex functions of the rats at 1 day, 3 days, 7 days, 14 days and 21 days after the injury were evaluated using the modified neurological severity score (mNSS) scale. The differences in the scores of the rats in the two groups were compared. Figure 6 It is shown that the mNSS scores of the rats in the two groups were significantly decreased with the extension of the treatment time, but the mNSS scores of the rats in the Anxa1 exosome group were significantly lower than those of the rats in the conventional exosome group at 3 days, 7 days, 14 days and 21 days after the injury. It indicates that the Anxa1 exosomes can more effectively improve the functional recovery of the rats after craniocerebral trauma than the conventional exosomes.

[0086] 2.3 At 7 days after operation, after the rat nerve function was evaluated, the brain tissue was taken after the rats were sacrificed, the whole brain tissue protein was extracted, and the BCA kit was used for quantification. The expression levels of inflammatory proteins IL-1β and IL10 in the brain tissues of the two groups of rats were detected and compared by using an ELISA kit, and the results are shown in Figures 7-8 It is shown that the IL-1β in the brain tissue of the Anxa1 exosome group is significantly lower than that in the conventional exosome group, and the expression level of IL10 is significantly higher than that in the conventional exosome group.

[0087] The umbilical cord mesenchymal stem cell exosome overexpressing Anxa1 injected intravenously can more effectively improve the nerve function of rats after craniocerebral trauma, and also greatly reduces the inflammatory reaction in the brain tissue after trauma.

[0088] Example 3 Efficacy of umbilical cord mesenchymal stem cell exosome overexpressing Anxa1 intravenous injection in the treatment of diabetic foot ulcers

[0089] 1. Preparation of rat diabetic foot ulcer model

[0090] Take 24 one-month-old male SD rats, after adaptive feeding for 3 days, fast for 12 hours, measure the fasting blood glucose by tail vein blood sampling. The preparation of diabetic model adopts 1% streptozotocin intraperitoneal injection. After intraperitoneal injection for 3 days, the fasting blood glucose is detected, and when the blood glucose is greater than 16.7mmol / L, the diabetic modeling is successful. After the rats are anesthetized with 30mg / kg sodium pentobarbital, a 3*3mm mold is used to manufacture a rat foot skin defect model, which is sterilized with 75% alcohol, and a diabetic foot skin ulcer rat model is manufactured

[0091] 2. Intravenous injection of umbilical cord mesenchymal stem cell exosome overexpressing Anxa1 and evaluation of efficacy

[0092] The 24 successfully modeled rats are divided into two groups, Anxa1 exosome group and conventional exosome group, each group has 12 rats. The Anxa1 exosome group is injected with 150μl umbilical cord mesenchymal stem cell exosome overexpressing Anxa1 through the tail vein, while the rats in the conventional exosome group are injected with 150μl umbilical cord mesenchymal stem cell exosome through the tail vein. Continuous injection for 7 days. The wound healing of rats in each group is recorded every day, and the final healing time is recorded. Image analysis is used to record the wound healing rate. Wound healing rate=(original total wound area-unhealed wound area) / original total wound area*100%. The results are shown in Figure 9As shown, the wound healing rates of the Anxa1 exosome group rats were higher than those of the conventional exosome group rats after 4 days of treatment, and the wound healing rate was as high as 79% at 7 days of treatment. The expression levels of inflammatory proteins TNFα and IL-14 in the wound tissues of the two groups of rats were detected by ELISA kit at 7 days and 14 days of treatment, and the results are shown in Table 2. Figures 10-11 As shown, the pro-inflammatory proteins of the Anxa1 exosome group gradually decreased with the extension of the treatment time, and were significantly lower than those of the conventional exosome group, while the anti-inflammatory proteins gradually decreased with the extension of the treatment time, and were significantly higher than those of the conventional exosome group.

[0093] The above results show that the umbilical cord mesenchymal stem cell exosomes overexpressing Anxa1 can improve the efficacy of diabetic foot ulcers in rats by inhibiting the levels of inflammatory factors. The exosomes have strong anti-inflammatory effects, and can replace the amount of hormones to a certain extent in clinical applications, and reduce the side effects of drugs in clinical treatment.

[0094] Although the present application has been described in detail above with general description, specific embodiments and experiments, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of the present application.

Claims

1. A method for preparing exosomes of umbilical cord mesenchymal stem cells overexpressing Anxa1, characterized in that: The following steps are involved: Isolation and culture of umbilical cord mesenchymal stem cells; The artificially synthesized Anxa1 messenger RNA (Anxa1mRNA) is capped, tailed, and pseudouridylated to obtain a modified Anxa1mRNA, wherein the capping method comprises linking 7-methylguanine to the 5'-end of the artificially synthesized Anxa1 messenger RNA (Anxa1mRNA) via a 5'-5' triphosphate bond, the tailing method comprises linking polyadenylic acid (polyA) to the 3'-end of the artificially synthesized Anxa1 messenger RNA (Anxa1mRNA), and the pseudouridylation method comprises modifying the artificially synthesized Anxa1 messenger RNA (Anxa1mRNA) with N1-methyl-pseudouridine; The modified Anxa1 mRNA is introduced into umbilical cord mesenchymal stem cells by cell electrofection to obtain electrofected umbilical cord mesenchymal stem cells, and the electrofected umbilical cord mesenchymal stem cells are then cultured and expanded in a 3D environment to obtain a culture supernatant of umbilical cord mesenchymal stem cells overexpressing Anxa1; The supernatant of the culture of umbilical cord mesenchymal stem cells overexpressing Anxa1 is centrifuged to remove living cells and debris, and the exosomes in the supernatant are separated and purified using ultracentrifugation or tangential flow to obtain exosomes of umbilical cord mesenchymal stem cells overexpressing Anxa1; The sequence of the Anxa1 messenger RNA (Anxa1 mRNA) is shown in SEQ ID NO.

1.

2. The preparation method according to claim 1, characterized in that The method for isolating and culturing umbilical cord mesenchymal stem cells is as follows: Wharton's jelly from human umbilical cord was isolated and digested with mixed enzymes. The digested tissue fragments and suspension cells were cultured to the third generation to obtain umbilical cord mesenchymal stem cells.

3. The preparation method according to claim 2, characterized in that The method for preparing the umbilical cord mesenchymal stem cells is: Take the umbilical cord of a full-term newborn from the body, separate the Wharton's jelly from it, and cut it into 1mm pieces on ice. 3 The size of the tissue was determined by mixed enzyme digestion for 2-16 hours. The digested tissue fragments and suspension cells were cultured. When the cells grew to 70-80% confluence, they were passaged at a ratio of 1:2-1:

4. The cells were in the logarithmic growth phase when the third generation was reached. When they grew to 70%-80% confluence, umbilical cord mesenchymal stem cells were obtained.

4. The preparation method according to claim 3, characterized in that The components of the Wharton's glue mixed enzyme include: 0.2% (w / w) collagenase type II, 0.2% (w / w) collagenase type IV, 0.1% (w / w) hyaluronidase, 0.1% (w / w) pancreatin, 15U / ml DNase.

5. The preparation method according to claim 4, characterized in that The culture medium for preparing umbilical cord mesenchymal stem cells is selected from umbilical cord mesenchymal stem cell phenol red-free and serum-free culture medium.

6. The preparation method according to claim 5, characterized in that The phenol red-free and serum-free culture medium for umbilical cord mesenchymal stem cells does not contain any animal-derived components.

7. The preparation method according to claim 6, characterized in that The conditions for cell electrotransfection include: The pulse voltage was 150-250 V, the driving voltage was 10-30 V, the interval was 125 ms, the pulse time was 1-30 ms, and the number of cycles was 1-10.

8. The preparation method according to claim 1, characterized in that The method for culturing and amplifying the electro-transfected umbilical cord mesenchymal stem cells in a 3D environment comprises: Drop the suspension of umbilical cord mesenchymal stem cells after cell electroporation into the 3D microcarrier; The 3D microcarriers containing umbilical cord mesenchymal stem cells were placed into a 3D stirred tank bioreactor.

9. The preparation method according to claim 1, characterized in that The method for separating and purifying exosomes in the supernatant using ultracentrifugation is as follows: The supernatant was first filtered through a sterile 0.45 μm filter; the filtered supernatant was high-speed centrifuged three times at 4°C to remove cell debris, and the supernatant after each centrifugation was collected. The centrifugal forces were 3000g, 4000g, and 5000g, respectively, and the time was 30 minutes each time; the supernatant after high-speed centrifugation was then ultracentrifuged twice, the first time at 10,000g and 70 minutes. The supernatant after the first ultracentrifugation was collected and used for the second centrifugation. The second centrifugation parameters were 120,000g and 70 minutes. The supernatant after the second ultracentrifugation was removed, and the bottom of the tube was repeatedly rinsed with sterile PBS for 3 minutes to collect exosomes to obtain umbilical cord mesenchymal stem cell exosomes overexpressing Anxa1.

10. The preparation method according to claim 1, characterized in that The method for isolating and purifying exosomes in the supernatant using tangential flow is as follows: The supernatant is circulated in a tangential flow device, and the exosomes are filtered and collected using the tangential force of the filter and liquid flow. 11 . An Anxa1-overexpressing umbilical cord mesenchymal stem cell exosomes obtained by the preparation method according to any one of claims 1 to 10.

12. The umbilical cord mesenchymal stem cell exosomes overexpressing Anxa1 according to claim 11, characterized in that The Anxa1 protein concentration in the exosomes of umbilical cord mesenchymal stem cells overexpressing Anxa1 was 26.4 mg / L.

13. Use of the Anxa1-overexpressing umbilical cord mesenchymal stem cell exosomes according to any one of claims 11 to 12 in the preparation of a drug for treating an inflammatory disease, wherein the inflammatory disease is an inflammatory response after craniocerebral trauma or an inflammatory response in a diabetic foot ulcer wound tissue.

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