CN113832109B - Bone marrow mesenchymal stem cell exosome and preparation method and application thereof - Google Patents

Abstract

The application relates to the technical field of exosome biological preparations, in particular to a bone marrow mesenchymal stem cell exosome, a preparation method and application thereof. The method comprises the following steps: s1: preparing bone marrow mesenchymal stem cells which overexpress the Scleraxis protein to obtain BMMSC-SCX cells; s2: stimulating the BMMSC-SCX cell to secrete exosomes and extracting to obtain the exosomes. The application discloses that exosomes produced by bone marrow mesenchymal stem cells over-expressing the Scleraxis protein have the effect of promoting healing of tendinous bones. The exosome of the scheme can be used as a biological agent to be applied to the preparation of related medicaments for healing tendinous bones, especially to the practical operation of front fork reconstruction and rotator cuff reconstruction, and plays a role in promoting the front fork reconstruction process with higher difficulty.

Description

Bone marrow mesenchymal stem cell exosome and preparation method and application thereof

Technical Field

The application relates to the technical field of exosome biological preparations, in particular to a bone marrow mesenchymal stem cell exosome, a preparation method and application thereof.

Background

Exosomes (exosomes) are microvesicles secreted by most cells in the body, approximately 30-150nm in diameter, with a lipid bilayer membrane structure, produced by the physiological process of "endocytosis-fusion-efflux". From the first discovery of exosomes, which were originally considered "waste" by cells, to date, for more than 30 years, has been considered a means by which cells excrete waste. However, through a great deal of research, the exosomes are obviously different from microvesicles and apoptotic bodies, and have distinct characteristics and potential values, so that the exosomes gradually become scientific research hot spots. The exosomes are used as natural intercellular information carriers, have smaller molecular structures and good biocompatibility, have no ethical problem compared with stem cell treatment, and have the potential of developing the exosomes into biological agents or medicines with therapeutic effects.

The tendon-bone junction is located between tendon and bone and is a key structure for mechanical transition, but the tendon-bone junction is difficult to recover to normal tissue structure and normal function after injury due to complex tissue structure. In particular, in the surgical treatment after injury of the anterior and posterior fork ligaments and rotator cuff injury, tendons are often used to replace ligaments and to connect tendons to bones by constructing bone marrow ducts, but healing between tendons and bone marrow ducts is difficult. Therefore, how to promote early tendon bone healing strength is a critical issue to be resolved urgently, and there is no drug with the above functions and good biocompatibility and bioavailability.

Disclosure of Invention

The application aims to provide a preparation method of bone marrow mesenchymal stem cell exosomes, which aims to solve the technical problem of lack of medicines with the function of promoting tendon bone healing and good biocompatibility.

In order to achieve the above purpose, the application adopts the following technical scheme:

the preparation method of the bone marrow mesenchymal stem cell exosome comprises the following steps in sequence:

s1: preparation of bone marrow fragments over-expressing the Scleraxis proteinMesenchymal stem cells, obtaining BMMSCs ^SCX A cell;

s2: stimulating the BMMSC ^SCX The cells secrete exosomes.

The proposal also provides a bone marrow mesenchymal stem cell exosome which is secreted by bone marrow mesenchymal stem cells over-expressing the Scleraxis protein.

The proposal also provides the application of the bone marrow mesenchymal stem cell exosome in preparing the medicine for promoting the healing of the tendinous bone.

The principle of the technical scheme and the beneficial effects are adopted:

the scheme uses genetic engineering technology to modify mesenchymal stem cells, overexpresses scleroxis protein in the cells, and induces the genetically modified mesenchymal stem cells to secrete a large amount of exosomes, wherein the exosomes have the effect of promoting healing of tendinous bones. The exosome can be used as a biological agent for promoting healing of tendinous bones in medical practice. Scleraxis (Scx) is a tendon-specific marker gene specifically expressed in tendon tissues and cells. In the technical scheme, the inventor discovers through a large number of experimental researches that the Scleraxis protein is overexpressed in the bone marrow mesenchymal stem cells, and the Scleraxis protein can change the types and the contents of microRNAs in exosomes of the bone marrow mesenchymal stem cells, so that the microRNAs favorable for healing of tendon bones are enriched, and the effect of enhancing the curative effect is realized.

The proposal discovers that the Scleraxis protein has the effect of inducing the large-scale expression of microRNA related to the healing of tendinous bones for the first time, and utilizes the phenomenon to prepare a large number of exosomes with therapeutic efficacy. The exosome medicine has remarkable advantages over the traditional medicine, and is characterized in that: the exosomes are used as natural intercellular information carriers, and have small molecular structures and good biocompatibility; compared with the common medicine, the exosome has high bioavailability and good targeting property. The exosome biological preparation for promoting the healing of the tendinous bone, which is developed by the scheme, has more ideal application prospect compared with the treatment by directly using the Scleraxis protein.

Further, in S1, the gene sequence of the Scleraxis protein is shown in SEQ ID NO. 1.

The sclearxis protein is a transcription factor that plays an important role in tendinogenesis, differentiation and regeneration. According to the scheme, the exosome is prepared by using cells which overexpress the protein, and the content of microRNA (ribonucleic acid) related to tendon bone healing in the obtained exosome is remarkably improved.

Further, in S1, bone marrow mesenchymal stem cells overexpressing the sleraxis protein were constructed using a lentiviral infection method.

The method for preparing the transgenic cells by lentivirus infection is a conventional method for preparing a cell line which over-expresses a target protein in the prior art, has clear principle, mature technology and easy operation, and can be used for preparing a stable cell line of mesenchymal stem cells expressing the Scleraxis protein.

Further, the procedure of the method for lentiviral infection is as follows: infecting bone marrow mesenchymal stem cells with SCX virus suspension; the preparation method of the SCX virus suspension comprises the following steps: inoculating 293T cells, and culturing the 293T cells to a cell density of 70% -80% by using a DMEM medium; adding a transfection system into a DMEM culture medium, wherein the transfection process lasts for 10-12 hours; the transfection system comprises a plasmid with an integrated SCX gene. The above process is a conventional method for virus suspension and lentivirus infection, and has simple operation and reliable result.

Further, the SCX gene was integrated on PGMLV-CMV-MCS-ZsGreen 1-T2A-Blastidin to obtain a plasmid into which the SCX gene was integrated. PGMLV-CMV-MCS-ZsGreen 1-T2A-Blastidin is a conventional empty plasmid in the prior art, and is easy to obtain and operate.

Further, in S2, BMMSC-SCX cells were cultured to a cell density of 80% using CORNING DMEM/F12 medium; the culture medium of Corning DMEM/F12 is replaced by serum culture medium without exosomes, the culture is continued until the cell density reaches 90-100%, and then the cell supernatant is collected. The technology for stimulating the secretion of exosomes by cells is mature, and a large number of exosomes can be stably obtained by adopting the method.

Further, in S2, exosomes are extracted from the cell supernatant by means of gradient centrifugation. The gradient centrifugation method is a conventional method for obtaining exosomes, and can stably obtain exosomes and is simple to operate.

Further, the bone marrow mesenchymal stem cell exosome contains microRNA with a sequence shown as SEQ ID NO. 2.

Through microRNA expression profile analysis, the species and the expression quantity of microRNA in exosomes prepared according to the scheme are found to be greatly changed. However, the function of mir-6924-5p is not reported in the prior art, but is found to be enriched in a large amount in exosomes generated under the condition of overexpression of scleroxis protein in the study, and experiments prove that the microRNA has the efficacy of remarkably promoting healing between tendon tissues and bone tissues.

Drawings

Fig. 1 is a micrograph of a P0 generation BMMSC of example 1.

Fig. 2 is a micrograph of a P3 generation BMMSC of example 1.

Fig. 3 is a graph of the results of flow cytometry detection of BMMSCs of example 1.

FIG. 4 is a graph showing the result of immunofluorescence detection of BMMSC of example 1.

FIG. 5 is a graph showing the result of the SCX gene expression in WB assay of example 1.

FIG. 6 shows the results of cell confocal laser assay after transfection in example 1.

FIG. 7 is a fluorescence image of the transfected cells of example 1.

FIG. 8 shows the results of experiments for identifying the protein expression levels of transfected cells in WB of example 1.

FIG. 9 shows the results of three cell osteogenic differentiation experiments of example 1.

FIG. 10 shows the results of three cell tendinous differentiation experiments of example 1.

FIG. 11 shows the results of the differentiation experiments of the chondrogenic balls of the three cells of example 1.

FIG. 12 shows the results of three cell adipogenic differentiation experiments of example 1.

FIG. 13 shows the results of three apoptosis flow assays of example 1.

FIG. 14 shows the results of three cell proliferation BRDU flow assays of example 1.

FIG. 15 is a graph showing particle size identification of three exosomes of example 2.

FIG. 16 is an electron microscope identification of three exosomes of example 2.

FIG. 17 is a WB identification pattern of three exosomes of example 2.

FIG. 18 is a BMMSC of example 3 ^SCX Volcanic plot of the mirRNA expression profile of exos.

FIG. 19 is a BMMSC of example 3 ^SCX Percentage graph of mirRNA for exos.

FIG. 20 is a BMMSC of example 3 ^SCX Differential expression of exos mirrRNA pie charts (showing mirrRNA up-regulated, down-regulated, and unchanged expression levels).

FIG. 21 is a BMMSC of example 3 ^SCX GO enriched expression profile of exos.

FIG. 22 is a BMMSC of example 3 ^SCX KEGG path analysis results of exos.

Fig. 23 is a schematic diagram and procedure of a mouse tendon-bone healing model according to example 4 of the present application.

FIG. 24 shows the experimental results of in vivo cell therapy according to example 4 of the present application.

FIG. 25 shows the experimental results of in vivo exosome treatment according to example 4 of the present application.

FIG. 26 shows the expression of mir-6924-5p according to example 5 of the present application (RT-PCR result).

FIG. 27 is an experimental result of the dual luciferase reporter system of example 5 of the application verifying the target gene binding site of miR-6924-5p.

FIG. 28 is a graph showing experimental results of mir-6924-5p pathway analysis in example 5 of the present application.

FIG. 29 is an image of TRAP staining and safranin fast green staining for the treatment of agomir-6924-5p according to example 6 of the present application.

FIG. 30 is a statistical plot of the results of biomechanical experiments for the treatment of agomir-6924-5p of example 6 of the present application.

FIG. 31 is a graph showing the results of a study of the effect of conditioned medium of three BMMSCs of example 7 of the present application on osteoclasts.

FIG. 32 shows a BMMSC of example 7 of the present application ^Scx -CM processingResults of studies on mRNA expression of group osteoclast biomarkers.

FIG. 33 is a graph showing the results of examining the effects of the soluble factors and exosomes in example 7 of the present application.

FIG. 34 is a graph showing the results of an in vitro induction of osteoclast differentiation by three BMMSCs-derived exosomes of example 7 of the present application.

FIG. 35 shows the results of an enrichment of mir-6924-5p in example 8 of the present application.

FIG. 36 is a study of the inhibition of human osteoclasts by mir-6924-5p of example 8 of the present application.

FIG. 37 shows mir-6924-5p in BMMSC of example 8 of the present application ^Scx Results of studies of the role in exos-mediated osteoclast inhibition.

Detailed Description

The present application will be described in further detail with reference to examples, but embodiments of the present application are not limited thereto. The technical means used in the following examples are conventional means well known to those skilled in the art unless otherwise indicated; the experimental methods used are all conventional methods; the materials, reagents, and the like used are all commercially available.

Example 1: preparation of overexpressed Bone Marrow Mesenchymal Stem Cells (BMMSC) of the sceraxis protein

1. Extraction of primary BMMSC

BMMSCs were isolated and cultured using prior art whole bone marrow adhesion methods, see prior art documents Isolation of mouse mesenchymal stem cells on the basis of expression of Sca-1 and PDGFR-a, diarmaid D Houlihan, nature Protocols,2012,7 (12): 2103-2111 (DOI: 10.1038/nprot.2012.125). Specific PROCEDUREs are described in detail in "process" on pages 2105-2109 of this document, and are not described here in detail since this PROCEDURE is conventional in the art. The P1 generation cell image is shown in fig. 1, and the P3 generation cell image is shown in fig. 2. Flow cytometry detection showed that the isolated cells highly expressed the two positive stem cell markers CD44 and CD90, the results of which are shown in figure 3. Immunofluorescence detection of BMMSC surface markers expression, the results are shown in figure 4.

2. Preparation of SCX Virus suspension

The SCX virus suspension of this section was commissioned to be prepared by qualified biotechnology companies, and the specific procedures were as follows:

1) Construction of a plasmid integrating the SCX gene:

the SCX gene sequence is shown as SEQ ID NO.1, and the transcription number is NM_198885.3.

The SCX gene was integrated into PGMLV-CMV-MCS-ZsGreen 1-T2A-Blastidin (empty vector) according to the conventional means in the prior art to obtain the target gene overexpression vector. The method comprises the following steps of: cutting out the target fragment through enzyme cutting sites (XhoI-ctcgag/BamHI-ggatcc) contained at two ends of the target fragment, and connecting the target fragment to an over-expression vector PGMLV-CMV-MCS-ZsGreen1-T2A-Blasticidin which is subjected to enzyme cutting; transferring the connection product into the prepared bacterial competent cells, sequencing the grown monoclonal colony, and comparing the correct clone to obtain the target gene overexpression vector which is successfully constructed. The expression of the target gene is detected by a Western Blot method, the experimental result is shown in figure 5 (ECL kit color development and X-ray development), S1 is a blank control, S2 is a negative control (PGMLV-CMV-MCS-eGFP-T2A-Blastidin), S3 is an experimental group (PGMLV-CMV-SCX-eGFP-T2A-Blastidin) for transfecting the target gene overexpression vector, and the experimental result shows that the target gene overexpression vector is successfully constructed and can be used for expressing a large amount of target protein.

2) Virus package

Extracting high-purity endotoxin-free lentiviral vector and auxiliary packaging original vector plasmids thereof by adopting a conventional method in the prior art, co-transfecting the constructed lentiviral vector and auxiliary packaging original vector plasmids thereof into 293T cells by using HG transgene reagent, adding an Enhancing buffer after 10-12h of transfection, replacing fresh culture medium after 8h of continuous culture, collecting cell supernatant rich in lentiviral particles, and concentrating the cell supernatant to obtain the high-titer lentiviral concentrated solution. The type of packaging plasmid is gag/pol; rev; VSV-G (pHelper 1.0 incorporating gag/pol and pHelper 2.0 incorporating VSVG/pHelper 3.0 incorporating rev). The packaging cells for lentiviruses were 293T, anchorage dependent epithelioid cells, and the growth medium DMEM (10% FBS). The adherent cells are grown by culture to form monolayer cells.

The day before transfection, cells that have grown are passaged in appropriate proportions into 10cm dishes and when cells grow to 70% -80% are ready for transfection. Cells to be transfected were replaced with fresh medium 1-2h prior to transfection, 12ml/10cm dish. A sterile 1.5ml EP tube or 15ml centrifuge tube was used to prepare a transfection system (DMEM 1ml, plasmid 10. Mu.g, lenti-HG Mix 10. Mu.l (10. Mu.g), HG transgene reagent. Mu.l). Mixing, standing at room temperature for 15-20 min, dripping into culture dish with liquid replaced in advance, and placing in CO ₂ Culturing in an incubator. After 10-12h of transfection, 100 XEnhancing buffer was added dropwise to promote transfection, 120. Mu.l/dish. After 18-20h of transfection, the cell culture broth was carefully aspirated and discarded in a waste liquid cup containing the disinfectant, and then culture was continued with 15ml of fresh cell culture medium. After 48h of liquid exchange, sucking the cell supernatant in a 50ml centrifuge tube, centrifuging at 4 ℃ for 5min, filtering the supernatant by a 0.45 mu m filter, transferring the filtrate into a new centrifuge tube, transferring the filtrate into a concentrating device in batches, centrifuging at 4 ℃ for 10min, discarding the liquid in the lower layer in a waste liquid cup containing disinfectant, centrifuging at 4 ℃ for 20min at the last time, wherein the liquid in the upper layer of the visible filter is the virus concentrate. The titer of the SCX virus suspension of this protocol was 1.15X109 TU/ml. The virus was packaged and stored at-80 ℃.

3. Lentiviral infection

BMMSC cells are routinely cultured until about 70% of the BMMSC cells are passaged, 6 pore plates are paved in a counting mode, 15-20 ten thousand holes are reserved, and the BMMSC cells are cultured for 24 hours; adding SCX virus suspension according to the proportion of 5 viruses in each cell, and infecting the cells for 48 hours; the cells can be observed under the mirror to see green fluorescence; digesting the cells, and culturing the cells in a complete medium containing 2. Mu.g/ml blasticidin (blasticidin); changing the complete medium containing 2 μg/ml blasticidin every three days, digesting the cells after cell expansion, counting, plating 96-well plates at 1/well, and culturing for about 2 weeks with the complete medium containing 2 μg/ml blasticidin; wells with only one colony were picked under a microscope, cells were digested, transferred to 6-well plates and plated with complete medium containing 2. Mu.g/ml blasticidin (blasticidin)Culturing (taking 3); the equal cells grow to about 70%, and then transferred to a culture flask, and are routinely cultured, passaged and frozen by a complete culture medium containing 2 mug/ml blasticidin (blasticidin); meanwhile, collecting protein for WB detection, and identifying strong positive cell strains; using the infected cells for subsequent cell experiments; infected cells, i.e., BMMSC cells that overexpress the scrrerxis protein (referred to as BMMSC ^SCX Cells). Empty transfected BMMSC cells (called BMMSC) were prepared in the same manner ^Ad Cells).

For BMMSC cells, BMMSC ^Ad Cells and BMMSCs ^SCX The cell is identified, the result of cell laser confocal identification after transfection is shown in fig. 6, the fluorescent image after cell transfection is shown in fig. 7, and the result of WB identification after transfection is shown in fig. 8.

For BMMSC cells, BMMSC ^Ad Cells and BMMSCs ^SCX The cells were subjected to a four-line differentiation experiment, and the results of the cell osteogenic differentiation experiment are shown in FIG. 9 (E: osteogenic staining image, BMMSC cells, BMMSC were sequentially arranged from left to right) ^Ad Cells and BMMSCs ^SCX A cell; f: an osteogenic related marker mRNA expression quantity statistical chart, each gene test group comprises BMMSC cells and BMMSCs from left to right ^Ad Cells and BMMSCs ^SCX A cell); the results of the cell tendinous differentiation experiment are shown in FIG. 10 (G: tendinous staining image; H: tendinous related marker mRNA expression level statistical chart); the results of the cell chondroblastomere differentiation experiment are shown in FIG. 11 (I: chondroblastomere staining image; H: chondroblastomere-related marker mRNA expression level statistical chart); the results of the cell adipogenic differentiation experiment are shown in FIG. 12 (C: adipogenic staining image; D: adipogenic related marker mRNA expression amount statistical chart). For BMMSC cells, BMMSC ^Ad Cells and BMMSCs ^SCX The cells were subjected to flow cytometry analysis, the apoptosis flow analysis results are shown in FIG. 13, and the proliferation BRDU flow analysis results are shown in FIG. 14.

Example 2: preparation of exosomes

1. Collecting cell supernatant

BMMSC cells (cells not subjected to lentivirus infection), BMMSC were resuscitated using CORNING DMEM/F12 medium (50/50, 1×, meditech 10-092-CVR) ^Ad Cell, BMMSC ^SCX A cell; changing the liquid the next day, and carrying out passage when the cell density reaches 80% -100%; observing the state of the passaged cells, and changing into a culture solution containing Exosome-reduced FBS (namely, an Exosome-free serum culture medium, which is a commercial product purchased directly, EXO-FBS-50A-1, SBI) when the cell density reaches more than 80%; after culturing for 24-48h, observing the cell state, and collecting cell supernatant when the cell density reaches 90% -100%, wherein the cell supernatant is used as a sample for extracting exosomes; the sample preservation adopts a gradient freezing method, and is preserved for 2 hours in a refrigerator at the temperature of minus 4 ℃, then preserved for 2 hours in a refrigerator at the temperature of minus 20 ℃ and finally preserved in a refrigerator at the temperature of minus 80 ℃.

2. Extraction and preparation of exosomes

Taking out the sample, thawing in water bath at 25 ℃, and placing on ice; centrifuging at 4deg.C for 10min at 2,000Xg, and collecting supernatant; centrifuging at 4deg.C for 30min at 10,000Xg, and collecting supernatant; transferring the sample into a super-high speed centrifuge tube, centrifuging at 4 ℃ and 110,000Xg for 75min, and discarding the supernatant; the pellet was resuspended in 1mL of 1 XPBS, and each of the resuspended pellets was diluted with 1 XPBS and filtered through a 0.22 μm membrane; transferring the sample to a super-high speed centrifuge tube, centrifuging at 4 ℃ for 75min at 110,000Xg, and discarding the supernatant; the pellet was resuspended in the corresponding 1 XPBS, sub-packaged and stored at-80 ℃. Obtaining BMMSC cells (without lentiviral infection), BMMSC-NC cells and exosomes of BMMSC-SCX cells (BMMSC-exos, BMMSC, respectively) ^Ad Exos and BMMSC ^SCX Exos). The particle size identification chart of the three exosomes is shown in fig. 15; the electron microscope identification chart is shown in fig. 16; the WB identification chart is shown in fig. 17.

Example 3:

the exosomes prepared in example 2 were subjected to mirrRNA expression profiling, part of which was delegated to qualified biotechnology companies. BMMSC ^SCX The volcanic diagram of exos is shown in FIG. 18, in which the arrow indicates Mir-6924-5P, and it can be seen that the expression level of Mir-6924-5P is in BMMSC ^SCX Maximum up-regulation amplitude in exos (relative to BMMSC ^Ad Exos). For more detail, the expression profile of mirRNA is shown in FIG. 19, and the content of mir-6924-5P is 8.33% of all mirRNAs. Wherein mir6924-5p has the sequence 3'-UGAAGCGGUUUAGGGGUAGGAGA-5' (SEQ ID NO. 2).

BMMSC is also compared to ^SCX Exos and BMMSC ^Ad Between exosAs a result of the differential expression of mirRNA, 1765 mirRNAs, 38 upregulated mirRNAs, and 171 downregulated mirRNAs were found in BMMSC-SCX-exos, as shown in FIG. 20. GO analysis was performed on BMMSC-SCX-exos, and the GO enrichment expression profile is shown in FIG. 21, in which the mirRNA that up-regulates expression of BMMSC-SCX-exos contained mirRNA involved in bone marrow cell differentiation. The results of the KEGG Pathway analysis are shown in FIG. 22, in which BMMSC-SCX-exos contains mirRNA associated with the osteoclast differentiation Pathway.

Example 4: in vivo experimental study

1. Construction of tendon bone healing model

Constructing a bone marrow duct: 0.5% sodium pentobarbital anesthetized mice, and then the skin in front of the achilles tendon of the right hind limb was cut, the achilles tendon was revealed and dissociated (see left 1 of fig. 23), and the removed achilles tendon was kept in a bacterial culture dish placed on an ice box with PBS. A longitudinal incision was then made in the anterior aspect of the knee of the right hind limb, and a bone marrow tunnel was constructed by drilling from the anterior medial aspect of the proximal end of the tibial stem to the posterior lateral aspect with a 1mL syringe needle perpendicular to the tibial axis (left 2 of fig. 23).

Drug treatment: slowly injecting the medicinal solution into bone marrow canal, taking care to avoid leakage of the solution, waiting for 2-3min, and completely absorbing the solution by surrounding bone. In this example, the drug solution used a blank (specifically PBS), BMMSC cell suspension (cell density 10) ⁶ Preparing suspension of cells with PBS (phosphate buffer solution) at a volume of 1ml per ml, culturing cells in conventional culture cell, performing exosome induction), and BMMSC ^Ad Cell suspension (10) ⁶ Individual/ml, 1 ml), BMMSC ^SCX Cell suspension (10) ⁶ Individual/ml, 1 ml), BMMSC-exos suspension (concentration 10 ⁸ Particles/mL,60ul in volume, formulated with PBS), BMMSC ^Ad Exos suspension (10) ⁸ Particles/mL,60 ul) and BMMSC ^SCX Exos suspension (10) ⁸ Particles/mL，60ul)。

Constructing a tendon bone healing model: one end of the achilles tendon stored in PBS was marked with a suture, and the achilles tendon was passed through the medullary canal by pulling the suture, and both ends of the achilles tendon were fixed to the surrounding periosteum, respectively (right 1 of fig. 23).

1. Biomechanical test experiments

The animal specimen was tested 21 days after molding using the existing biomechanical tester protocol. Tendon-bone recovery strength was tested by following the biomechanical tester instructions. The specific operation is as follows: fresh tibial specimens were taken out and immersed in PBS in an ice box cell culture dish, and sutures at both ends of the achilles tendon were cut off to free the achilles tendon attachment portion. One end of the achilles tendon was then fixed to a biomechanical testing machine and the achilles tendon was pulled continuously at a rate of 0.05mm/s in the direction of the longitudinal axis of the medullary canal. The extraction force is the maximum force when the achilles tendon is completely extracted or the force when the achilles tendon breaks. The tendon bone healing strength was then calculated: extraction force (N)/bone marrow tract length (mm).

Experimental results of the healing of tendinous bone are shown in fig. 24C and 25G. In fig. 24C, the data statistics columns are, in order from left to right: blank control, BMMSC cell suspension, BMMSC ^Ad Cell suspension and BMMSC ^SCX A cell suspension; in fig. 25G, the data statistics pillars are in order from left to right: blank, BMMSC-exos suspension, BMMSC ^Ad Exos suspension and BMMSC ^SCX Exos suspension. Experimental results show the use of BMMSCs ^SCX After exos treatment, the tendon bone healing strength was enhanced, indicating BMMSC ^SCX Exos can promote the healing process of the tendinous bone. And use BMMSC ^SCX Effect ratio of exos using BMMSCs ^SCX The effect of the cells is better.

2. Tissue staining experiments

14 days after molding, tibial samples were collected, fixed with 4% paraformaldehyde for 48 hours, then decalcified with EDTA for 10 days, and paraffin sections were made.

TRAP staining: the effect of exosome treatment on osteoclast activation in bone surrounding the bone marrow tract was observed by TRAP staining, sections were dewaxed with xylene and rehydrated in gradient alcohol, PBS washed off alcohol. The tissue samples on the sections were covered with 0.5mL TRAP staining solution and incubated for 30 minutes at room temperature. Excess dye was washed with PBS and then the sections were soaked in 0.1M AMPD-HCl buffer for 10 min.

Safranine solid green staining: the area of the bone trabecula is observed through safranine solid-green staining, soaked for 3min with 0.1% safranine solution, then soaked for 10 seconds in 0.1% solid-green solution, then separated by 1% acetic acid solution, and finally sealed for observation after washing off the redundant solution.

The results of TRAP staining and safranin fast green staining experiments are shown in fig. 24A and 25E, and statistics of TRAP positive area versus total bone trabecular area are shown in fig. 24B and 25F. Fig. 24A shows the experimental results of cell therapy, and histochemical analysis shows that osteolysis is prevented after treatment with bone marrow mesenchymal stem cells (fig. 24A, safranin fast green staining). Fig. 25E shows the experimental results of exosome treatment, and histochemical analysis showed that osteolysis was prevented after treatment with exosomes (fig. 25E, safranin fast green staining). Since osteoclasts are responsible for osteolysis during tendon-bone healing, we examined the state of osteoclasts in vivo. TRAP staining shows (FIGS. 24A and B), BMMSC ^SCX Osteoclast osteolysis was reduced and osteoclastogenesis was significantly inhibited compared to the other groups. TRAP staining shows (FIGS. 25E and F), BMMSC ^SCX Exos has reduced osteolysis of osteoclasts compared to the other groups, and osteoclastogenesis is significantly inhibited. These results indicate that BMMSC ^SCX Exos and BMMSC ^SCX Tendon-bone healing may be promoted by inhibiting the production of osteoclasts. In fig. 24B, the statistics columns are, in order from left to right: blank control, BMMSC cell suspension, BMMSC ^Ad Cell suspension and BMMSC ^SCX A cell suspension; in fig. 25F, the statistics bars are, in order from left to right: blank, BMMSC-exos suspension, BMMSC ^Ad Exos suspension and BMMSC ^SCX Exos suspension.

Example 5:

research on action target of mir-6924-5p

The sequence of mir-6924-5p used in the scheme is 3'-UGAAGCGGUUUAGGGGUAGGAGA-5' (SEQ ID NO. 2), and after transfection, the expression level of mir-6924-5p in the primary osteoclast precursor cells of the mice is up-regulated, and the gene expression condition in the osteoclast precursor cells of the mice is detected. The primary osteoclast precursor cells of mice were extracted by rinsing bone marrow from the bone marrow cavity of the mice and plating in 24-well plates. Cells were cultured in complete medium (10% FBS,1% diabody,. Alpha. -Minimal Essential Medium (MEM) medium) and stimulated with M-CSF (macrophage colony stimulating factor) for 24h. The suspension cells were then transferred to a new cell culture plate and stimulated with M-CSF (50 ng/mL) and RANKL (50 ng/mL). The culture medium is replaced every two days, and the primary osteoclast precursor cells of the mice are obtained through culture.

(1) Transfection: a method of up-regulating mir-6924-5p in mouse primary osteoclast precursor cells is to transfect mouse osteoclast precursor cells with agomiR-6924-5p (synthesized by Shanghai Ji Ma company), a nucleic acid conventionally used in the prior art to raise endogenous mir-6924-5p levels in cells. The agoniR-6924-5 p is a complementary double-chain structure, and comprises a sense strand with a sequence shown as SEQ ID NO.3 and an antisense strand with a sequence shown as SEQ ID NO.4, and the specific steps are as follows:

sense strand: 5'-ACACTCCAGCTGGGAGAGGATGGGGATTTGG-3' (SEQ ID NO. 3);

antisense strand: 5'-TGGTGTCGTGGAGTCG-3' (SEQ ID NO. 4).

More specifically, the method comprises the following steps: the mixture of the transfection reagent INVI DNA RNA Transfection reagent with the agomiR-6924-5p (concentration 0.06 OD) or the control (agomiR N.C, a general random control, from commercial sources) was mixed for 15min, and the volume ratio of the agomiR-6924-5p or the control to INVI DNA RNA Transfection reagent was 1:1, to obtain a mixed reagent. The primary osteoclast precursor cells of mice were cultured in vitro in a medium of alpha-Minimal Essential Medium (MEM) in a conventional manner according to the prior art, and then a mixed reagent was added to the cell culture medium in an amount of 15 μl. Incubate in a 37℃cell incubator for 24h. After 24h, the medium was removed, replaced with fresh medium for further culture for 24h, and the cells were taken to obtain transfected cell samples.

(2) RT-PCR detection: the transfected cell samples were collected, treated with TRIzol, total RNA was extracted according to the conventional method of the prior art, reverse transcribed into cDNA, and then the mRNA level of the predicted target gene was detected using GAPDH as an internal reference. And screening potential target genes of miR-6924-5p through a database of three miRNA target gene predictions to obtain a large number of candidate target genes (116). Then, by transfecting agoniR-6924-5 p into the osteoclast precursor cells, PCR was used to examine whether the target genes to be determined exhibited a phenomenon in which the expression level was significantly decreased. As shown in fig. 26 (mean±sd, n=3), it was found from the experimental results that miR-6924-5p was overexpressed in the cells, and the expression levels of OCSTAMP, CXCL12, and PRLR in the mouse osteoclast precursor cells were significantly reduced, suggesting that these three genes could be potential target genes for miR-6924-5p.

OCSTAMP (osteoclast stimulatory trans-membrane protein) is a novel gene found in osteoclasts that is up-regulated during differentiation of monocytes into osteoclasts. The gene expression product of OCSTAMP is conserved at its carboxy terminus and its main role is as a key molecule in the fusion and differentiation process of osteoclasts. Upregulation of ocstmp gene expression promotes osteoclast formation and inhibits tendon bone healing formation. The chemokine CXCL12 (C-X-C motif) ligand 12, also known as stromal cell derived factor-1 (SDF-1), is a small molecule cytokine. CXCL12 overexpression can promote differentiation of osteoclast precursor cells. Prolactin receptor (Prolactin Receptor, PRLR) regulates osteoclast activation in bone metastasis of breast cancer.

2. Dual luciferase reporter system validation

The construction method of the target plasmids m-Cxcl12-3UTR, m-OCSTAMP-3UTR and m-PRLR-3UTR comprises the following steps: the 3' UTR fragment of Cxcl12 (wild type wt and mutant mut), the 3' UTR fragment of OCSTAMP (wild type wt and mutant mut) and the 3' UTR fragment of PRLR (wild type wt and mutant mut) were integrated into the empty plasmid pSI-Check2, respectively, according to the conventional methods in the prior art, to obtain six plasmids of interest.

293T cells were seeded in 96-well plates and transfected until cell densities reached 50% -70%. 10ul of DMEM was thoroughly mixed with 0.16ug of the plasmid of interest and 5pmol of agoniR-6924-5 p (or negative control, agonir N.C) and then left at room temperature (solution A), after which 10ul of DMEM was thoroughly mixed with 0.3ul of transfection reagent (transfection reagent was a Hantao product, at a concentration of 0.8 mg/ml) (solution B), and left at room temperature for 5min. And (3) fully and uniformly mixing the solution A and the solution B, and standing at room temperature for 20min to obtain a transfection mixture. Cells were exchanged for fresh medium prior to transfection, and the transfection mixture of A and B was then added to mix. 37 ℃,5% CO ₂ Culturing. Fresh medium was exchanged after 6h of transfection and cell detection was collected after 48h of transfection.

5X PLB (Passive Lysis Buffer) was diluted to 1 XPLB with distilled water, added in an amount of 100. Mu.l per well of a 96-well plate, scattered by pipetting with a pipetting gun, slowly shaking for 15min on a shaking table at room temperature, and then the cell lysate was sucked into a 1.5ml centrifuge tube, centrifuged at 12000rpm for 10min at 4℃and the supernatant was transferred into a new centrifuge tube. A new 96-well plate was added with 100. Mu.l of working solution of Luciferase Assay Reagent II (LAR II) (Luciferase Assay Reagent, promega) and 20. Mu.l of cell lysate, and the mixture was blown and mixed with a pipette for 2 to 3 times, and the firefly luciferase (Firefly luciferase) value was measured and recorded as an internal reference value. 100. Mu.l of Stop solution (Stop) was added&Reagent, luciferase Assay Reagent, promga), a pipette is used for blowing and mixing for 2-3 times, and the value of Renilla luciferase (Renilla luciferase) is measured and recorded, namely the luminescence value of the reporter gene. As shown in fig. 27 (mean±sd, n=3), four data columns from left to right in each histogram are represented by: using agomir N.C and m-ocstmp-3 UTR (wild type); using agoniR-6924-5 p and m-OCSTAMP-3UTR (wild type); using agoniR-6924-5 p and m-OCSTAMP-3UTR (mutant); using agomir N.C and m-OCSTAMP-3UTR (mutant), it was found from the experimental results that mir-6924-5p could control gene expression via Cxcl12 and the 3' UTR region of the OCSTAMP gene, allowing down-regulation of both genes and thus achieving control of tendinous bone healing.

Mir-6924-5p action pathway study

Next, we examined whether overexpression of OCSTAMP or CXCL12 could reverse the inhibitory effect of mir-6924-5p on osteoclast formation. We first demonstrated that plasmid transfection with OCSTAMP (OCSTAMP plasmid) or CXCL12 (CXCL 12 plasmid) genes is effective in upregulating the mRNA levels of both genes (FIG. 28A). After transfection of the OCTAMP plasmid or CXCL12 plasmid, agomir-6924-5p was transfected into osteoclast precursor cells (OCPs). TRAP activity assays showed that agomir6924-5p significantly down-regulated TRAP protein expression in the OCSTAMP plasmid-treated group and CXCL12 plasmid group (FIG. 28B). Consistently, TRAP staining showed that overexpression of these two genes reversed the effect of mir-6924-5p on osteoclast formation (FIGS. 28C and 28D). Real-time PCR analysis showed that osteoclast biomarker expression was down-regulated after transfection of agomir-6924-5p in the OCSTAMP plasmid-treated group and CXCL12 plasmid group (FIGS. 28E and 28F). These data indicate that mir-6924-5p inhibits osteoclast formation by targeting OCSTAMP and CXCL 12.

Example 6: mir-6924-5p in vivo Experimental study

In vivo experiments were performed with reference to the experimental method of Experimental example 4 to verify the effect of mir-6924-5p. The experimental grouping included: blank, agomir-6924-5p and agomir N.C. The treatment method comprises the following steps: an agomir-6924-5p solution (20. Mu.M, 50. Mu.l) or agomir N.C or PBS was slowly injected into the bone marrow tract, taking care to avoid leakage of the solution, waiting for 2-3min, until the solution was completely absorbed by surrounding bone. As shown in the results of the tissue staining experiments in FIG. 29, TRAP staining shows that the area of osteoclasts around the bone marrow tract is obviously reduced compared with that of the control group after the injection of agomir-6924-5p, and the osteolysis is relieved; safranin solid green staining shows bone trabecula area, and shows that agomir-6924-5p promotes mir-6924-5p expression level to be up-regulated, so that bone trabecula can be reserved, and bone dissolution can be inhibited.

The experimental results of the tendon-bone healing strength are shown in fig. 30 (mean±sd, n=4, significant difference exists between the group of agomir-6924-5p and the group of agomir N.C, p is less than 0.5), and after the mir-6924-5p level is up-regulated, the mechanical strength of the tendon-bone junction can be obtained to be beneficial to the tendon-bone healing process.

Example 7: BMMSC ^SCX And BMMSC ^SCX In vitro experimental study of the effect of exos on osteoclasts

To investigate BMMSC ^Scx Effect on OCPs we collected Conditioned Medium (CM) from three groups of BMMSCs, acting on OCPs in the presence of RANKL and M-CSF (for inducing osteoclast differentiation maturation), respectively. TRAP staining showed that only BMMSC was found ^Scx The collected CM can inhibit the formation of osteoclasts (fig. 31). BMMSC ^Scx The number of osteoclasts in the CM treated group was about 50% of that in the other two control groups (fig. 31, left). The results showed that BMMSC ^Scx The TRAP protein concentration was significantly lower in the group than in the other 3 groups (bottom right in fig. 31). The Real-time PCR analysis showed that,compared with the other two groups, BMMSC ^Scx mRNA expression of the classical osteoclast biomarkers MMP2, MMP9, pu1, OCSTAMP, NFATc1 and Cathepsin K was all significantly down-regulated in the CM treated group (fig. 32). These findings indicate that BMMSCs can prevent osteoclastogenesis only after Scx overexpression. Furthermore, from BMMSC ^Scx Is a key regulator of this inhibition. In FIGS. 31 and 32, the columns of each statistical chart are DMEM medium, BMMSC-CM, BMMSC, respectively, from left to right ^AD -CM、BMMSC ^Scx -CM. In the above experiments, the specific method for preparing the conditioned medium (collected cell supernatant) was: BMMSC cells (cells not subjected to lentivirus infection), BMMSC were resuscitated using CORNING DMEM/F12 medium (50/50, 1×, meditech 10-092-CVR) ^Ad Cell, BMMSC ^SCX And (3) cells. The next day of liquid exchange, when the cell density reaches 80% -100%, the passage cell state is observed, when the cell density reaches more than 80%, the cell is exchanged into a culture solution containing exoome-reduced FBS, after culturing for 24-48h, the cell state is observed, when the cell density reaches 90% -100%, the cell supernatant is collected, and the part of cell supernatant is the conditioned medium.

To search for BMMSC ^Scx Which components of CM inhibit the formation of osteoclasts, we isolated Soluble Factors (SF) and exosomes (exosomes). Then we compare BMMSCs respectively ^Scx -SF and BMMSC ^Scx Influence of exos on osteoclast formation. TRAP staining showed the formation of osteoclasts. Exosomes were more effective at preventing osteoclast formation compared to the effect of SF (fig. 33E, F). The TRAP concentration was significantly reduced in the exosome-treated group compared to the control group and SF-treated group (fig. 33G). mRNA expression was significantly down-regulated in exosome groups pu.1, OCSTAMP, NFATc1 and CSTK compared to SF group (fig. 33H). These results indicate that BMMSCScx-derived exosomes are mainly involved in the inhibition of osteoclasts. In the above experiments, the Soluble Factor (SF) was prepared by the following method: 1. melting the sample at 37 ℃ at medium speed; 2. the sample is moved into a new centrifuge tube, 2000 Xg, 4 ℃ and centrifuged for 30 min; 3. carefully transfer the supernatant to a new centrifuge tube (50 ml for subsequent steps, redundant designated CM, cryopreserved for sample return), 10,000×g, centrifuging again at 4 ℃ for 60min, and collecting supernatant for carrying out the operation of the step 4; the pellet was resuspended in 15ml PBS and continued for 10,000Xg, centrifuged again at 4℃for 60min to obtain a pellet, which was resuspended in 50ul PBS and designated IEV (frozen stock); 4. filtering the supernatant obtained in the last step by a 0.22 mu m filter membrane, and collecting filtrate; 5. transferring the filtrate into a new centrifuge tube, selecting an overspeed rotor, centrifuging at 4deg.C, 100,000Xg for 80min, collecting supernatant, concentrating at 14000g for 10min with 10K column (Amicon Ultra-0.5 filter), collecting concentrate, named SF, and storing at-80deg.C; after the pellet was resuspended in 15mL of pre-chilled 1 XPBS, the overspeed rotor was selected and ultracentrifuged for 70min at 4℃again at 100,000 Xg; 7. removing supernatant, re-suspending with 50 μl of pre-chilled 1×PBS, designated sEV, and storing at-80deg.C; 8. finally, sample CM, IEV, SF, sEV is returned.

Then, we compared the ability of three types of BMMSCs-derived exosomes to induce osteoclast differentiation in vitro. TRAP staining showed the number of osteoclasts, BMMSC ^Scx The exos treated group was significantly lower than the other 3 groups. Furthermore, BMMSC-exos treatment group and BMMSC ^Ad No significant difference in osteoclast numbers was seen in exos treated groups compared to control groups (figures 34A and B). In comparison with the other three groups, only BMMSC ^Scx TRAP activity was inhibited in the exos-treated group of osteoclast precursors (FIG. 34C). BMMSC ^Scx The mRNA levels of the exos groups MMP2, MMP9, pu.1, NFATc1 and CXCL12 were also significantly lower than those of the other 3 groups. These results indicate that only BMMSCs ^Scx The exosomes of origin can down regulate the production of osteoclasts. In FIG. 34, the statistics bars of each statistical chart are blank, BMMSC-exos, BMMSC, respectively, from left to right ^AD -exos、BMMSC ^Scx -exos。

Example 8: in vitro experimental study of mir-6924-5p inhibiting osteoclast

We examined the levels of mir-6924-5p in three groups of BMMSCs, BMMSCs ^Scx The levels of mir-6924-5p were up-regulated more than 20-fold over the other two groups (FIG. 35F). Furthermore, when osteoclast precursor cells were treated with exosomes from these three BMMSCs, respectively, BMMSCs compared to the other three control groups ^Scx And mir-6924-5p in exosomesRich (fig. 35G). These results indicate that mir-6924-5p is present only in BMMSC ^Scx And BMMSC ^Scx Enrichment in exos. Next we examined the way in which Scx promoted mir-6924-5p over-expression. We used actinomycin D to block transcription and detect mir-6924-5p in BMMSC ^Ad Or BMMSC ^Scx Time-varying expression of (c). The results showed that, in BMMSC ^Ad The abundance of mir-6924-5p decreased 2 hours after actinomycin D stimulation, but in BMMSC ^Scx The expression of mir-6924-5p decreased 4 hours after stimulation with actinomycin D. This indicated an increase in mir-6924-5p stability after overexpression of Scx (fig. 35L).

To investigate whether mir-6924-5p might be a potential therapeutic target for tendon bone healing, we transfected agomir-6924-5p into osteoclast precursor cells (human origin) and then induced osteogenesis (induction using M-CSF and RANKL). TRAP staining showed fewer osteoclasts in the agomir-6924-5p treated group compared to the NC group (FIGS. 36A and B). The mRNA levels of the osteoclast biomarkers ACP5, CALCR, NFATc1 and ITGB3 were significantly down-regulated in the agomir-6924-5 p-treated group compared to the negative control group (FIG. 36C). These results indicate that mir-6924-5p is also effective in preventing human osteoclast formation.

We have discussed mir-6924-5p in BMMSC ^Scx Importance in exos-mediated osteoclast inhibition. The osteoclast precursor was transfected with antagomir-6924-5 p (commercially available) and then treated with exosomes derived from three BMMSCs. Both TRAP staining and TRAP ELISA detection showed BMMSC ^Scx Exos significantly inhibited osteoclast formation and TRAP concentration was significantly down-regulated, but overexpression of antagomir-6924-5 p was reversible (FIGS. 37A-C) our results indicate that mir-6924-5p is involved in BMMSC ^Scx Exos affects key regulatory factors for osteoclast formation.

The foregoing is merely exemplary of the present application, and specific technical solutions and/or features that are well known in the art have not been described in detail herein. It should be noted that, for those skilled in the art, several variations and modifications can be made without departing from the technical solution of the present application, and these should also be regarded as the protection scope of the present application, which does not affect the effect of the implementation of the present application and the practical applicability of the patent. The protection scope of the present application is subject to the content of the claims, and the description of the specific embodiments and the like in the specification can be used for explaining the content of the claims.

SEQUENCE LISTING

<110> first affiliated Hospital of the university of the force army of the Chinese people's liberation army

<120> an exosome of mesenchymal stem cells, and preparation method and application thereof

<130> 2021.10.09

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 624

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atgtccttcg ccatgctgcg ttcagcgccg ccgccgggtc gctacctgta ccctgaggtg 60

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tgccgtgtgc atgctgcgcg ctgtggcctc cagggcgccc ggcggcgggc aggaggacgg 180

agggccgcgg gtagcgggcc aggacccggg gggcggccag gccgcgagcc ccggcagcgg 240

cacacagcga atgcgcgcga gcgggaccgc accaacagcg tgaacacggc cttcactgcg 300

ctgcgcacac tcatccccac cgagccagcg gaccgcaagc tctccaagat tgagacgctg 360

cgcctggcct ccagctacat ttctcacctg ggcaatgtgc tgctggtggg tgaggcctgt 420

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Claims (2)

1. An application of bone marrow mesenchymal stem cell exosome in preparing a preparation for inhibiting osteoclast, which is characterized in that the preparation method of the bone marrow mesenchymal stem cell exosome comprises the following steps in sequence:

s1: preparation of bone marrow mesenchymal Stem cells overexpressing the Scalarxis protein to obtain BMMSC ^SCX A cell;

s2: stimulating the BMMSC ^SCX The cell secretes exosomes;

the bone marrow mesenchymal stem cell exosome is enriched with Mir-6924-5P.

2. A method for enriching Mir-6924-5P, comprising:

s1: preparation of bone marrow mesenchymal Stem cells overexpressing the Scalarxis protein to obtain BMMSC ^SCX A cell;

s2: stimulating the BMMSC ^SCX The cell secretes exosomes; the Mir-6924-5P is enriched in the exosome.