Application of umbilical cord mesenchymal stem cells combined with exosome in treatment of neurological function deficiency after brain injury

Abstract

The invention belongs to the technical field of biology, and particularly discloses application of an umbilical cord mesenchymal stem cell combined exosome in treatment of neurological function deficiency after brain injury, a tangential flow filtration method and a precipitation method are combined for extraction of an ultrapure exosome, and the umbilical cord mesenchymal stem cell and the exosome are combined for use, so that the survival rate of brain injury cells is increased, and the neurological function deficiency after brain injury is treated. According to the application, the secretion level of an inflammatory factor TNF-alpha is reduced, and meanwhile, the expression of a neurotrophic factor NGF is promoted, so that the repair and improvement of the neurological function after the brain injury are realized, and the application can be used for treating the neurological function deficiency after the brain injury. Compared with a traditional treatment method, the umbilical cord mesenchymal stem cells and the exosome are combined for use, so that the neurological function deficiency can be more effectively improved, the inflammatory response is relieved, the neural restoration is promoted, and a new thought and a new method are provided for clinical treatment of brain injury patients.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to the application of umbilical cord mesenchymal stem cells combined with exosomes in the treatment of neurological dysfunction after brain injury. Background Art

[0002] Currently, emergency treatment for traumatic brain injury (TBI) in clinical practice primarily involves symptomatic support, reducing cerebral edema, and maintaining cerebral blood perfusion. Once vital signs stabilize, functional rehabilitation training and hyperbaric oxygen therapy are then administered to address neurological deficits. While these treatments can save the lives of some TBI patients, they do not significantly improve neurological deficits. Exploring effective TBI treatment models to reduce mortality, alleviate post-traumatic complications, and reduce the burden on families and society is a top priority. Existing drug intervention strategies and combination therapies still have limitations in repairing brain tissue defects. Summary of the Invention

[0003] The purpose of the present invention is to provide an application of umbilical cord mesenchymal stem cells combined with exosomes in the treatment of neurological dysfunction after brain injury, so as to effectively improve neurological dysfunction after brain injury, reduce inflammatory response, and promote nerve repair.

[0004] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted:

[0005] The present invention provides an application of umbilical cord mesenchymal stem cells combined with exosomes in treating neurological dysfunction after brain injury.

[0006] Preferably, the exosomes are umbilical cord mesenchymal stem cell exosomes.

[0007] Specifically, the dosage of the umbilical cord mesenchymal stem cells is 1×10 4 ~5×10 5 cells; the exosome dosage was 50-400 μg protein.

[0008] Preferably, the dosage of the umbilical cord mesenchymal stem cells is 1×10 5 cells; the exosome dose was 400 μg exosomal protein.

[0009] Preferably, the purity of the exosomes is ≥1.0×10 8 particles / μg protein.

[0010] Preferably, the exosomes are prepared by the following method:

[0011] The primary umbilical cord mesenchymal stem cells were subcultured, and the supernatant of the third generation of umbilical cord mesenchymal stem cells was collected, large particles, impurities and dead cells were removed, and the supernatant was concentrated to 10 times, filtered and sterilized to obtain concentrated exosomes;

[0012] Sterile water for injection was used to adjust the particle concentration of concentrated exosomes to 1.0 × 10 10 The exosomes were precipitated by adding polyethylene glycol solution, and the mixture was centrifuged after standing at low temperature. The supernatant was discarded, and the mixture was air-dried and washed with ultrapure water. The mixture was centrifuged again and finally resuspended with sterile water for injection to obtain a purity of ≥1.0×10 8 Ultrapure exosome solution with 1000 particles / μg protein.

[0013] Preferably, the umbilical cord mesenchymal stem cells combined with exosomes are administered by intravenous injection or local intracerebral injection.

[0014] Compared with the prior art, the beneficial effects of the present invention include at least:

[0015] 1. By combining tangential flow filtration and precipitation to extract exosomes, the yield and high purity of exosomes were achieved;

[0016] 2. The combined use of umbilical cord mesenchymal stem cells and exosomes can improve the survival rate of brain-damaged cells, reduce the secretion level of the inflammatory factor TNF-α, and promote the expression of the neurotrophic factor NGF, thereby repairing and improving neurological function after brain injury. It can be used to treat neurological deficits after brain injury. Compared with traditional treatment methods, the combined use of umbilical cord mesenchymal stem cells and exosomes can more effectively improve neurological deficits, reduce inflammatory responses, and promote neural repair. It also has the advantages of relatively easy access to raw materials, simple methods of use, and good biosafety, which is conducive to its promotion and application in clinical practice, providing new ideas and methods for the treatment of patients with brain injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the specific implementation of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the specific implementation or the description of the prior art.

[0018] Figure 1 Bright field image of human umbilical cord mesenchymal stem cells (hUC-MSCs).

[0019] Figure 2 This is a diagram showing the identification of hUC-MSCs surface markers.

[0020] Figure 3 This is an Alizarin red staining image of UC-MSCs after 14 days of osteogenic differentiation.

[0021] Figure 4 This is the Oil Red O staining image of UC-MSCs after 14 days of adipogenic differentiation.

[0022] Figure 5 This is the Alcian blue staining image of UC-MSCs after 21 days of chondrogenic differentiation.

[0023] Figure 6 This is the STR analysis chart of UC-MSCs.

[0024] Figure 7 This is a characterization diagram of exosomes (Exos) using nanoparticle tracking analysis (NTA) technology.

[0025] Figure 8 This is a standard curve for determining the concentration of Exos proteins.

[0026] Figure 9 This is a graph showing the effect of UC-MSCs dosage on SH-SY5Y cell viability.

[0027] Figure 10 This is a diagram evaluating the immunomodulatory effect of UC-MSCs administration on SH-SY5Y.

[0028] Figure 11 This is a graph showing the effect of Exos administration dose on SH-SY5Y cell viability.

[0029] Figure 12 This is a diagram evaluating the immunomodulatory effect of Exos administration on SH-SY5Y.

[0030] Figure 13 This is a diagram evaluating the neuromodulatory effect of Exos administration on SH-SY5Y.

[0031] Figure 14 This is a graph showing the effect of combined administration of different ratios of UC-MSCs and EXOs on the cell viability of SH-SY5Y.

[0032] Figure 15 This is a diagram evaluating the immunomodulatory effect of SH-SY5Y on the combined administration of UC-MSCs and EXOs at different ratios.

[0033] Figure 16 This is a diagram evaluating the neuromodulatory effect of combined administration of UC-MSCs and EXOs at different ratios on SH-SY5Y. DETAILED DESCRIPTION

[0034] 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 scope of protection of the present invention.

[0035] It should be noted that, unless otherwise specified, the technical 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.

[0036] Example 1: Extraction and identification of umbilical cord mesenchymal stem cells (UC-MSCs)

[0037] 1. Extraction of primary UC-MSCs: The umbilical cord was removed from the biosafety cabinet and placed in a sterile culture dish. The residual blood was washed with 20 mL of saline containing double-antibody until there was no obvious blood on the surface of the umbilical cord sample. The umbilical cord was cut open along the umbilical vein, and the arteriovenous vessels were removed. The Wharton's jelly was torn and cut into 1 mm pieces. 3 The size of the tissue block is cleaned with physiological saline containing double antibodies, and the tissue block is inoculated into a 15 cm diameter sterile culture dish after adhesion, with the tissue blocks separated by about 2 to 3 mm. After the tissue block is adhered to the wall, the culture dish is inverted and placed in an incubator for 4 hours. The culture dish is removed and complete culture medium containing double antibodies is added to continue culturing. The amount of culture medium added should be enough to submerge the tissue block. The liquid addition action should be gentle to avoid lifting the tissue block. After the tissue block is adhered to the wall, physiological saline without double antibodies is added to the centrifuge tube containing the tissue block for sample quality inspection.

[0038] After the cells have grown to full size, they are subcultured. When the cells reach P3, the morphology of the cells is observed using a bright field microscope. Under the microscope, the cells are fibrous, arranged relatively tightly, and are long spindle-shaped, interwoven into a network or vortex distribution. Figure 1 As shown in the figure, these cell morphological characteristics are consistent with the typical manifestations of umbilical cord mesenchymal stem cells.

[0039] 2. Surface marker identification: Follow the instructions of the BD Stemflow hMSC Analysis Kit to detect the expression of CD90, CD105, CD73, CD34, CD45, CD11b, CD19, and HLA·DR proteins on the cell surface. The results are as follows: Figure 2 As shown: the expression levels of CD90+, CD105+, and CD73+ in positive cells were 99.9%, 99.1%, and 100.0%, respectively, and the positive rates were ≥95%; the expression levels of CD34+, CD45+, CD11b+, CD19+, and HLA-DR+ in negative cells were 0.48%, and the negative rates were ≤2%, which met the standards for human mesenchymal stem cell surface markers.

[0040] 3. Induction differentiation function test: All UC-MSCs were tested for osteoblastic, adipogenic and chondrogenic differentiation functions, and were stained with Alizarin Red S, Oil Red O and Alcian Blue respectively. The results showed that after 14 days of culture in the relevant differentiation induction medium, UC-MSCs were positive for Alizarin Red S staining. Figure 3 As shown in the figure, the arrow points to the mineralized matrix, indicating that the cells have the ability to differentiate into osteoblasts; after culturing C-MSCs in the relevant differentiation induction medium for 14 days, they were positive for Oil Red O staining, as shown in the figure. Figure 4As shown in the figure, the arrows point to lipid droplets, indicating that the cells have the ability to differentiate into adipocytes. After 21 days of culture in the relevant differentiation induction medium, UC-MSCs were treated with the relevant differentiation induction medium. UC-MSCs first aggregated into cell spheres and then differentiated into cartilage tissue. The chondrocytes were positive by alcian blue staining, as shown in the figure. Figure 5 As shown, the cells have the ability to differentiate into chondrocytes.

[0041] 4. STR karyotype analysis: DNA samples were extracted using the Biospin cell genomic DNA extraction kit. A reaction system was prepared in a PCR tube according to the components and amounts shown in Table 1 for amplification reaction. Amplification reaction was performed according to the reaction procedure shown in Table 2. After amplification, electrophoresis samples were prepared according to the components and amounts shown in Table 3. After denaturation at 95°C for 3 minutes and rapid ice cooling for 3 minutes, the samples were transferred to 8-tube strips, the strips were sealed, and data were collected on the machine. The results showed that the genotype test results of this UC-MSCs were good after comparison with the STR database. No multiple alleles were found, and there was no possibility of cross-contamination with other cells. Figure 6 shown.

[0042] Table 1 Composition and content of reaction system

[0043]

[0044] Table 2 Reaction procedures for amplification reaction

[0045]

[0046] Table 3 Composition and content of electrophoresis samples

[0047]

[0048] Example 2: Extraction and purification of exosomes (Exos)

[0049] 1. Exosome concentration: 10L of supernatant from the third generation of UC-MSCs culture was collected and firstly used a 10-inch Bursa filter to remove large particles, impurities, and dead cells. Then, a hollow fiber column was used to concentrate the supernatant to 10 times, and finally, a 2.5-inch Bursa filter was used for sterilization.

[0050] Nanoparticle tracking technology (NTA) was used to detect the number of exosome particles after concentration. The specific operation was as follows: first, the sample pool was washed with deionized water, and then the instrument was calibrated with 100nm polystyrene microspheres to ensure the accuracy of particle size detection; the sample pool was washed with 1X PBS buffer, and the concentrated exosomes were diluted with 1X PBS buffer. The particle size and distribution range of the exosomes were observed by NTA technology on the instrument. The results are as follows Figure 7(A) shows that the exosome particle size is mainly distributed in the range of 50-150 nm, and the concentration (measured in the number of small extracellular vesicles) is 3.60×10 10 particles / mL.

[0051] The absorbance (OD value) of the sample solution (concentrated exosomes were diluted 5 times with 1X PBS buffer) at 562 nm was measured using a BCA kit, and the standard curve ( Figure 8 The protein concentration of the concentrated exosomes was calculated (as shown in Table 4). The results are shown in Table 4: the OD value of the concentrated exosomes was 1.1649, and the calculated protein concentration was 5.19 μg / uL.

[0052] The ratio of the number of small extracellular vesicles to the amount of protein, T, was used as the purity of exosomes. According to the formula T = X / P (where T is the ratio of the number of small extracellular vesicles to the amount of protein, X is the number of small extracellular vesicles, and P is the amount of protein in small extracellular vesicles), the purity of concentrated exosomes, T, was calculated to be 6.94×10 6 particles / μg protein.

[0053] 2. Exosome purification: Ultrapure exosomes were extracted using polyethylene glycol (PEG) precipitation method. The specific experimental steps were as follows: Sterile water for injection was used to adjust the particle concentration of concentrated exosomes to 1.0×10 10 The tube was centrifuged at 10000 rpm for 12-16 h, and then 250 μL of 5X PEG6000 solution was added. The tube was allowed to stand at 4°C for 12-16 h, and then centrifuged at 10,000 rpm. At this time, white flocculent precipitates were visible at the bottom of the centrifuge tube. The supernatant was discarded, and the centrifuge tube was inverted on absorbent paper and air-dried for 5-10 min. The tube was then washed twice with ultrapure water, and then centrifuged at 15,000 rpm. The ultrapure Exos solution was then obtained by resuspending the tube in 1 mL of sterile water for injection.

[0054] Table 4 Protein concentration determination of concentrated exosomes and ultrapure exosomes

[0055]

[0056]

[0057] The particle concentration of ultrapure exosomes detected by NTA was 1.3×10 11 Particles / mL, e.g. Figure 7 (B); According to the absorbance value of ultrapure exosomes (diluted 5 times) and the standard curve of Exos protein concentration determination, the protein concentration of ultrapure exosomes was calculated to be 0.393 μg / uL, as shown in Table 4. According to the formula T=X / P, the purity of ultrapure exosomes was calculated to be 3.3×10 8 particles / μg protein, in line with group standards>1.0×10 8 particles / μg protein.

[0058] Example 3: Screening of the ratio of combined administration of UC-MSCs and EXOs

[0059] 1. Construction of a cellular brain injury model: Human neuroblastoma (SH-SY5Y) cells were seeded in a 12-well plate, with 10,000 cells per well. After culturing to about 70% confluence, oxygen glucose deprivation (OGD) treatment (1% O2, sugar-free medium, 12 hours) was performed.

[0060] 2. Single-dose screening of umbilical cord mesenchymal stem cells (UC-MSCs): After 12 hours of oxygen and glucose deprivation, the supernatant was discarded and 1×10 4 , 5×10 4 , 1×10 5 , 5×10 5 The SH-SY5Y cells were cultured in 1×10 cells / mL of UC-MSCs complete medium / well and the cell viability and TNF-α secretion level of SH-SY5Y cells were observed after 48 hours of culture. The experimental results showed that the cell viability of SH-SY5Y cells gradually increased with the increase of the number of UC-MSCs, but when the number of UC-MSCs reached 1×10 5 After 10 cells enter the plateau phase, Figure 9 As shown; the secretion level of TNF-α gradually decreased, and 1×10 5 cells and 5×10 5 There was no significant difference in the therapeutic effect of individual cells. Figure 10 shown.

[0061] Based on the above results, the optimal dosage of UC-MSCs is 1×10 5 cells.

[0062] 3. Screening of exosome (EXOs) single-drug administration concentration: After 12 hours of oxygen and glucose deprivation, the supernatant was discarded and 50, 100, 200, and 400 μg protein / mL exosome solutions were added, and the cell viability, inflammatory factor TNF-α secretion level, and neurotrophic factor NGF release level of SH-SY5Y cells were observed after 48 hours of culture. The experimental results showed that as the amount of exosome protein increased, the cell viability of SH-SY5Y cells gradually increased, but there was no significant difference in cell viability between 200 μg protein and 400 μg protein. Figure 11 As shown in Figure 2, the secretion level of TNF-α gradually decreased, and there was no significant difference between 200 μg protein and 400 μg protein. Figure 12 As shown, it is consistent with the results of cell viability; the release level of neurotrophic factor NGF gradually increased, and the NGF release level of 400μg protein was the highest, as shown in Figure 13 shown.

[0063] Based on the above results, the optimal dosage of exosomes is 200 μg protein.

[0064] 3. Screening of the ratio of combined administration: Based on the optimal concentration of single drug administration, the combined administration ratio of UC-MSCs and EXOs was designed to be 1:1 (1×10 5 cells + 200 μg protein), 1:2 (1×10 5 cells + 400 μg protein), 2:1 (2×10 5 The orthogonal experiment (100 cells + 200μg protein) was conducted. After 48 hours of intervention, the cell viability of SH-SY5Y cells, the secretion level of inflammatory factor TNF-α and the release level of neurotrophic factor NGF were detected to determine whether the combined treatment had a synergistic effect. The results showed that the cell viability of SH-SY5Y cells was the highest when UC-MSCs and EXOs were co-administered at a ratio of 1:2, and the cell viability of the combined administration group was higher than that of the single-dose group. Figure 14 As shown in the figure, when the ratio of UC-MSCs to EXOs was 1:2, the TNF-α secretion level was the lowest, and the TNF-α secretion level of the combined administration group was lower than that of the single administration group. Figure 15 As shown in the figure, when the ratio of UC-MSCs to EXOs was 1:2, the release level of neurotrophic factor NGF was the highest, and the NGF release level of the combined administration group was higher than that of the single administration group. Figure 16 shown.

[0065] Based on the above results, the optimal ratio of UC-MSCs and EXOs co-administration is 1:2, that is, the UC-MSCs dose is 1×10 5 cells, and the EXOs dose was 400 μg protein.

[0066] 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 above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or make equivalent replacements for some or all of the technical features therein. These modifications or replacements do not deviate the essence of the corresponding technical solutions 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 description of the present invention.

Claims

1. Application of umbilical cord mesenchymal stem cells combined with exosomes in the treatment of neurological deficits after brain injury.

2. The use according to claim 1, characterized in that: The exosomes are umbilical cord mesenchymal stem cell exosomes.

3. The use according to claim 2, characterized in that: The dosage of the umbilical cord mesenchymal stem cells is 1×10 4 ~5×10 5 cells; the exosome dosage was 50-400 μg exosomal protein.

4. The use according to claim 3, characterized in that: The dosage of the umbilical cord mesenchymal stem cells is 1×10 5 cells; the exosome dose was 400 μg exosomal protein.

5. The use according to claim 3 or 4, characterized in that: The purity of exosomes is ≥1.0×10 8 particles / μg protein.

6. The use according to claim 5, characterized in that The exosomes are prepared by the following method: The primary umbilical cord mesenchymal stem cells were subcultured, and the supernatant of the third generation umbilical cord mesenchymal stem cell culture was collected. Large particles, impurities, and dead cells were first filtered to remove them, and then the supernatant was concentrated to 10 times using tangential flow filtration. Finally, the bacteria were removed by filtration to obtain concentrated exosomes. Sterile water for injection was used to adjust the particle concentration of concentrated exosomes to 1.0 × 10 10 After the mixture was dried in air, it was washed with ultrapure water and centrifuged again. Finally, sterile water for injection was added to resuspend the mixture to obtain a purity of ≥1.0×10 8 Ultrapure exosome solution with 1000 particles / μg protein.

7. The use according to any one of claims 1 to 6, characterized in that The umbilical cord mesenchymal stem cells combined with exosomes are administered by intravenous injection or local intracerebral injection.

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