CN114504595A - CD24 positive expression cell in urine and preparation method and application thereof - Google Patents

Abstract

The invention relates to the field of biological medicine, in particular to a CD24 positive expression cell in urine and a preparation method and application thereof; the application discovers and screens cells with CD24 positive expression in cells derived from urine from a muscle injury patient, wherein the cells with CD24 positive expression in the urine cells can be subjected to myoblast differentiation under in-vitro induction conditions and are fused with muscle cells to form fused muscle tubules; through verification, after CD24 positive expression cells in urine cells are transplanted into an animal body, inflammatory reaction symptoms can be relieved and muscle regeneration and repair can be realized at muscle damage parts, and the cells can be used for preparing related medicines for repairing muscle damage treatment.

Description

CD24 positive expression cell in urine and preparation method and application thereof

[ technical field ] A

The invention relates to the field of biomedicine, in particular to a CD24 positive expression cell in urine and a preparation method and application thereof.

[ background of the invention ]

Muscle damage and muscle loss caused by external force trauma, aging and other factors can cause symptoms such as stress urinary incontinence, skeletal muscle defect and the like, and finally, the patient can have difficulty in moving and inconvenient actions, thereby causing great troubles to the daily life and social activities of the patient. Currently, clinical treatment for such muscle damage is mainly limited to patient rehabilitation physiotherapy and partial chemotherapy. In addition to physical rehabilitation and chemotherapy which are widely used, the transplantation therapy and cell therapy of engineering materials are also applied to large-volume muscle defects in a small amount at present, but the treatment effect is not ideal at present due to the fact that the technology is not mature enough, and related specific drugs are not developed.

Muscle damage caused by trauma or aging, such as stress urinary incontinence, large skeletal muscle defects, and the like, cannot be quickly repaired by physical rehabilitation therapy, that is, indirect external force therapy, and thus, the damaged tissue is damaged too long, the muscle growth time is delayed, and finally, the muscle cannot be cured. Due to the lack of direct intervention on the injured part of the physical therapy, the injured muscle tissue is difficult to directly regenerate and repair, and can only achieve partial relief.

At present, most of chemical medicaments adopted in chemical medicament treatment are traditional Chinese medicine preparations for diminishing inflammation, relieving pain, promoting blood circulation and removing blood stasis, and the medicaments can only relieve the symptoms of patients and still cannot promote the regeneration of new muscle tissues so as to realize the specific repair of the muscle tissues. In recent years, with the development of stem cells and tissue engineering methods, a new method is provided for treating muscle injury, the implantation of tissue engineering materials can assist the treatment of large-volume muscle defects, but the implanted materials can generate larger rejection reaction of an organism due to the technical barriers of bioactivity and biocompatibility existing between the implanted materials and patients, so that the application of muscle tissue regeneration is greatly limited; treatment of stem cells is still in need of further investigation for the therapeutic effects of muscle injury repair due to differences in cell source/subpopulation classification and myogenic differentiation function.

Therefore, in order to perform precise intervention on the muscle injury part, improve the precise treatment effect on muscle injury repair, reduce the pain of patients, and reduce the rejection reaction of organisms to medicines and external materials, the research of the application is necessary to obtain medicines and treatment means which are better and effective in repairing muscle injury.

[ summary of the invention ]

In view of the above, in order to improve the accurate treatment effect of repairing muscle injury, reduce the pain of patients, reduce the use of physical rehabilitation treatment means, perform accurate intervention on muscle injury parts, treat the patient with self-specificity, apply medicine accurately, and quickly recover muscle injury, it is necessary to perform research of the present application.

In order to achieve the purpose, the following technical scheme is adopted in the application:

one embodiment of the invention is the application of CD24 positive expression cells in urine in preparing a muscle repair treatment drug.

Another embodiment of the invention is the use of CD24 positive expression cells in urine for the preparation of a medicament for increasing muscle electrophysiological activity.

The application of the CD24 positive expression cells in the urine described in the above examples in the preparation of medicaments uses the urine as autologous urine of patients.

The use of cells expressing CD24 positively in urine as described in the above embodiment in the preparation of a medicament, wherein the medicament is an injection.

In the injection preparation described in the above embodiment, the effective number of cells is 1X 10⁵-1×10⁸Individual cells/ml.

The preparation method of the CD24 positive expression cells in urine in the embodiment of the invention is as follows:

(1) collecting urine of a patient, centrifuging, separating, and collecting cell precipitate;

(2) resuspending the cell precipitate obtained in step (1) with KSFM culture fluid, and continuing primary culture;

(3) when urine cells appear in the primary culture in the step (2), continuously culturing by changing the liquid and carrying out subculture amplification to obtain subcultured cells;

(4) and (4) collecting the cells subcultured in the step (3), carrying out flow sorting by using a CD24 antibody to obtain a CD24 positive cell population, and collecting the cell population to obtain the cell.

The invention has the following beneficial effects:

1. the application finds and screens a cell with CD24 positive expression from urine cells in the own urine of a muscle injury patient, and finds that the cell with CD24 positive expression in the urine cells can be myogenic to differentiate and fused with muscle cells to form a fused muscle tubule under the condition of in vitro induction.

2. Through animal experiments, the following results are found: CD24 positive expression cell transplants into internal back in the urine cell can alleviate the inflammatory reaction symptom and realize muscle regeneration and repair at muscle injury position, because the cell that this application treatment used derives from patient autologous, possesses the characteristic of the accurate treatment of individuation, can realize patient autologous cell treatment fast, avoids the immune rejection who transplants.

[ description of the drawings ]

FIG. 1 shows the proportion of CD 24-positive expressing cells in urine, on the abscissa, the fluorescence intensity of CD24 positivity, and on the ordinate, SSC (lateral angle); p1 in the figure represents the population of cells falling within a preset CD24 positive threshold range;

FIG. 2 is a schematic representation of a cell flow chart;

FIG. 3 is a diagram of the cell morphology of sub-cultured CD24 positive expression cell subset;

FIG. 4 is a graph showing the expression of CD24 by subpopulation of urine cell-derived CD 24-positive expressing cells, wherein A is the expression level of CD 24-negative cell population, non-grouped cells, and CD 24-positive cell population for CD24 gene; b is the expression level of CD24 negative cell population, non-grouped cells, CD24 positive cell population for CD24 protein;

FIG. 5 is a CD24 cell distribution map of in vivo injected subpopulations of urine cell-derived CD 24-positively expressing cells after muscle injury treatment;

FIG. 6 is a graph of the effect of in vivo injection of a subpopulation of urine cell-derived CD 24-positively expressing cells on inflammatory repair after muscle injury treatment;

FIG. 7 is a graph showing the effect of tissue injury regeneration and repair after muscle injury treatment by in vivo injection of a subpopulation of urine cell-derived CD 24-positive expressing cells;

FIG. 8 is a graph showing the electrophysiological activity of muscle tissues after the rat muscle injury model is 7d without injection of CD 24-positively expressing cells;

FIG. 9 is a graph showing the electrophysiological activity of muscle tissues after injection of 7d CD 24-positive expression cells in a rat muscle injury model.

[ detailed description ] embodiments

The invention will be further described with reference to the following drawings, examples and experiments, and the embodiments of the present application will further explain the technical solutions of the present invention, but should not be construed as limiting the present invention:

example 1:

this example is the separation of CD24 positive expression cells in urine and its amplification culture method:

(1) the source of urine: collecting 100-300ml of urine from a patient to a sterile container, subpackaging the urine into a 50ml centrifuge tube, centrifuging at a speed of 400g for 5 minutes, and collecting cell precipitates;

(2) resuspending the cell pellet obtained in step (1) in 5ml of KSFM culture medium, transferring to a T25 culture flask, and performing primary culture at 37 ℃;

(3) changing the liquid once every 2 days for urine cells which appear after the primary culture for 3-4 days in the step (2), performing subculture amplification on the cells by using a KSFM culture solution when the cell fusion degree reaches 90%, and performing cryopreservation or use after the cells are amplified to the required number to obtain subculture cells;

(4) collecting the cells subcultured in the step (3), carrying out flow sorting on the CD24 antibody by using a flow sorting magnetic bead kit of Meitianni company to obtain a CD24 positive expression cell population, and preserving or continuously culturing the cells;

(5) inducing differentiation culture solution containing horse serum and dexamethasone to perform myogenic induced differentiation to obtain well-differentiated myocytes; the induced differentiation culture solution comprises the following components in percentage by weight: 2% of horse serum, 1% of dexamethasone and the balance of KSFM culture solution; the induction culture conditions are as follows: changing the liquid 1 time every 2 days, and inducing for 14 days; discarding the culture solution, washing with PBS buffer solution for 2 times, fixing with 4% paraformaldehyde for 15 min, and washing with PBS buffer solution for 3 times; collecting the washed cells to obtain CD24 positive expression cells;

(6) centrifuging the CD24 positive expression cells collected in the step (5), collecting precipitates, and then resuspending the precipitates by using physiological saline for injection to reach the cell amount of 10⁵-10⁸And selecting proper cell concentration for injection of the muscle tissue at the damaged part for repair treatment.

The sources and manufacturers of the solutions of this example are as follows:

each solution and related compositional factors in this example were purchased from Life, Inc. under the Gibco brand.

The detection results of flow cytometry are shown in fig. 1-3:

as can be seen, 80% of the isolated primary cells were CD24 positive expressing cells (see fig. 1), and flow sorting further enriched for CD24 positive expressing cells by measuring the proportion of CD24 positive expressing cells as high as 95% or more (see fig. 2); the cell morphology of the sub-population of CD 24-positive expressing cells obtained after flow sorting was shown in FIG. 3. it can be seen from the figure that the CD 24-positive expressing cells were spherical and spindle-shaped.

Example 2:

in this example, expression identification is performed on CD24 positive expression cells isolated in example 1, and the specific identification method is as follows:

(1) respectively collecting each group of cells, extracting cell RNA samples, and detecting the expression of each group of cell populations on CD24 genes through PCR reaction;

(2) and collecting each group of cells, extracting cell protein samples, and detecting the expression of the CD24 protein of each group of cells through a western blot reaction and a CD24 antibody.

The final results are shown in fig. 4, and fig. 4A shows the expression levels of CD24 gene in CD 24-negative cell population, non-divided cell population and CD 24-positive cell population; fig. 4B is a graph showing the results of western blot reactions of CD 24-negative cell populations, non-divided cell populations, and CD 24-positive cell populations on the expression level of CD24 protein, and it can be seen from the graph that the expression level of the CD 24-positive cell population is the highest among the cell populations obtained in example 1, and it is also apparent from the western blot reaction that: the CD24 positive expression band is obviously brighter and more obvious in color development than the non-clustered cells and the CD24 negative expression cells.

Example 3:

the embodiment is an application of the CD24 positive expression cell obtained in the embodiment 1, and is mainly applied to muscle regeneration and repair, and the specific experimental method comprises the following steps:

(1) establishing a rat muscle injury model:

a rat muscle injury model was created by injecting 0.2mg/ml CTX (cardiotoxin) into the mid-tibial anterior muscle of rat.

(2) Rats of the rat muscle injury model were divided on average into two groups, one of which was the experimental group and the other was the control group:

the experimental group adopts CD24 positive expression cell injection for treatment, and the specific method comprises the following steps: CD 24-positive expression cells separated in example 1 were resuspended in physiological saline for injection to prepare an injection solution of CD 24-positive expression cells at 1X 10⁵-1×10⁸The injected amount of each cell/ml is used for in situ repair treatment of muscle injury sites of rats.

The control group was not treated with the injection solution of CD 24-positive expressing cells, but the same volume of physiological saline for injection was used instead of the injection solution of CD 24-positive expressing cells of the experimental group.

The test groups and the control group are subjected to detection: collecting tibialis anterior muscles of each group of animal models, taking out muscle tissues of the injured parts, carrying out tissue frozen section after fixing with 4% paraformaldehyde, and detecting the expression of an antibody CD206 related to the immune inflammatory reaction and an antibody Desmin related to muscle tissue repair and regeneration through immunohistochemical staining; meanwhile, the electromyography detector is used for carrying out the electrophysiological detection of muscle tissues.

The results obtained are shown in FIGS. 5 to 9.

Fig. 5-7 show CD24 cell distribution (fig. 5), inflammatory repair effect (CD206) (fig. 6) and expression of antibodies related to muscle tissue repair and regeneration (Desmin) (fig. 7) after muscle injury is treated by in vivo injection of subpopulation of urine cell-derived CD24, respectively, and it can be seen that, after injection of CD24 positive expression cell solution, CD24 positive expression cells can undergo myogenic differentiation under in vitro and in vivo induction conditions, express muscle cell-related marker proteins, and form fused multinuclear myotubular structures, and can be directly fused with muscle cells, form multinuclear myotubular structures, and reduce inflammation.

FIGS. 8 to 9 are graphs showing the electrophysiological activity and muscle regeneration repair effect of muscle after injection of CD 24-positive expression cell solution after muscle injury in experimental and control groups, and it can be seen that: the muscle electrophysiological activity of the control group (figure 8) which is not injected with the CD24 positive expression cell solution is obviously inferior to that of the experimental group (figure 9), so that the CD24 positive expression cell solution has the advantages of obviously reducing inflammatory reaction after muscle injury, effectively promoting muscle regeneration and repair, and playing a good role in repairing muscle injury.

Example 4:

this example is an example of the use of the CD24 positive expressing cells of example 1 for the preparation of a medicament:

the CD24 positive expression cells prepared and isolated in example 1 were used to prepare therapeutic drugs, and the corresponding drugs were applied to the muscle injury model rats prepared in example 3:

the prepared medicine is as follows: the injection preparation comprises in-situ injection and intravenous injection, and has effective CD24 positive cell content of 1 × 10⁵-1×10⁸Individual cells/ml.

Muscle model rats of muscle injury of the above formulation were divided into 4 groups of equal number:

group 1: injecting a liquid injection containing CD24 positive cells into a rat vein (the solution in the injection is normal saline);

group 2: injecting a liquid injection containing CD24 positive cells into a rat muscle injury in situ (the solution in the injection is normal saline);

positive control: rats were injected intravenously with diclofenac sodium solution according to the instructions;

negative control: rats were injected intravenously with an equal amount of physiological saline.

In this experiment, the effective CD24 positive cell content in the formulations of groups 1-2 was 1X 10⁶Individual cells/ml.

Observing the speed of the rat mobility recovery capability of the groups 1-2, the positive control and the negative control, and finally finding that the speed of the rat mobility recovery capability is as follows:

group 2 recovered faster than group 1, group 1 recovered faster than the positive control, and the positive control recovered faster than the negative control.

At the time 7d, the electrophysiological activity of the muscle of each group was measured on the tibialis anterior of each animal model group by the electrophysiological muscle tissue measurement instrument, and finally, it was found that:

the muscle electrophysiological activity of the group 2 is superior to that of the group 1, and the muscle electrophysiological activity of the group 1 is superior to that of the positive control group; the muscle electrophysiological activity of the positive control group was superior to that of the negative control group.

Therefore, the administration effect is better by in situ injection than intravenous injection, and after the CD24 positive expression cells in the urine of the example 1 of the application are injected, the mice with muscle injury can be quickly recovered.

Therefore, the CD24 positive expression cells prepared in example 1 of the present application can be induced in vitro to differentiate into myoblasts and fuse with myocytes to form fused myotubules, which can effectively repair damaged muscle parts, and is a novel drug capable of performing targeted therapy and repair on damaged muscles, which has a good therapeutic effect on muscle damage, and at the same time, since CD24 positive cells are derived from urine of patients, the cells are autologous cells of patients, and basically do not generate rejection reaction with patients.

In summary, the method for realizing regeneration and repair of damaged muscle cells by using autologous cells of a patient has the advantages of wide source of the obtained CD24 positive expression cells, easily available raw materials and low cost, and meanwhile, the cells for treatment are derived from the autologous cells of the patient and have the characteristic of individualized and accurate treatment, so that autologous cell treatment of the patient can be quickly realized, and immune rejection reaction of transplantation is avoided; the injection preparation prepared by the CD24 positive expression cell also has good repairing and treating effects on muscle injury.

The above description is intended to describe in detail the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the claims of the present invention, and all equivalent changes and modifications made within the technical spirit of the present invention should fall within the scope of the claims of the present invention.

Claims (6)

1. Application of CD24 positive expression cells in urine in preparing muscle repair treatment medicines.

2. Application of CD24 positive expression cells in urine in preparing medicine for improving muscle electrophysiological activity.

3. Use of CD 24-positive expression cells in urine according to claim 1 or 2 for the preparation of a medicament, wherein the urine is autologous urine of a patient.

4. Use of cells positively expressing CD24 in urine according to claim 1 or 2 for the preparation of a medicament for injection.

5. Use of the urine-containing cells positively expressing CD24 according to claim 4, wherein the effective number of the cells in the preparation for injection is 1X 10⁵-1×10⁸Individual cells/ml.

6. The method for preparing CD 24-positive expression cells in urine according to claim 1, wherein the method comprises:

(1) collecting urine of a patient, centrifuging, separating, and collecting cell precipitate;

(2) resuspending the cell precipitate obtained in step (1) with KSFM culture fluid, and continuing primary culture;

(3) when urine cells appear in the primary culture in the step (2), continuously culturing by changing the liquid and carrying out subculture amplification to obtain subcultured cells;

(4) and (4) collecting the cells subcultured in the step (3), carrying out flow sorting by using a CD24 antibody to obtain a CD24 positive cell population, and collecting the cell population to obtain the cell.

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