MiR-181c-based diabetic fracture repair method and application

Abstract

The invention provides a miR-181c-based diabetic fracture repair method and application, and the miR-181c-based diabetic fracture repair method realizes targeted delivery of miR-181c in a fracture microenvironment by using an exosome as a miR-181c delivery carrier, and comprises the following steps: carrying out miR-181c gene overexpression treatment on bone marrow mesenchymal stem cells (BMSCs), extracting the exosome rich in miR-181c, and carrying out miR-181c gene overexpression treatment on the BMSCs to obtain the miR-181c-based diabetic fracture repair method. The miR-181c can be accurately injected to a diabetic fracture area under the guidance of CT, proliferation and differentiation of osteoblasts and angiogenesis are promoted by activating an ERK signal channel at the fracture part, fracture repair is effectively accelerated, healing quality is improved, meanwhile, the fracture microenvironment is optimized by combining with anti-inflammatory factors, and the treatment effect of the miR-181c on the fracture part is improved. The common chronic inflammatory response in the diabetic environment is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of fracture repair, and in particular to a diabetic fracture repair method based on miR-181c and application thereof. Background Art

[0002] Diabetic patients face significant difficulties in the fracture healing process. Due to osteoblast dysfunction and local inflammatory response caused by the hyperglycemic environment, the patients' fractures heal slowly and with poor quality. In addition, the bone tissue regeneration capacity of diabetic patients is limited, and angiogenesis is inhibited, leading to delayed fracture healing and increasing the risk of infection, malformation and non-healing. Current treatment methods mainly alleviate the problem through fracture fixation, drug support and blood sugar control, but have failed to significantly improve the speed and quality of fracture healing. Traditional treatment methods have limited effects in the special environment of diabetic fractures, and long-term drug support may bring side effects.

[0003] Studies have found that microRNA plays an important regulatory role in bone tissue repair and regeneration. In particular, miR-181c shows potential in promoting osteogenic differentiation and regulating the activity of bone marrow mesenchymal stem cells (BMSCs). However, due to chronic inflammation and unfavorable cellular microenvironment in the diabetic environment, miR-181c is difficult to play an effective role in damaged tissues. Therefore, a diabetic fracture repair method based on miR-181c and its application are proposed. Summary of the invention

[0004] In view of this, the present invention provides a diabetic fracture repair method and application based on miR-181c to solve or alleviate the technical problems existing in the prior art and at least provide a beneficial option.

[0005] The technical solution of the present invention is implemented as follows: a diabetic fracture repair method based on miR-181c, comprising the following steps:

[0006] S1. Overexpression of the miR-181c gene in bone marrow mesenchymal stem cells (BMSCs) to achieve high levels of miR-181c expression in secreted exosomes; extract the exosomes by ultracentrifugation and ensure that the exosomes are structurally intact and contain functionally active miR-181c molecules to support the osteogenic differentiation process;

[0007] S2. Precisely inject exosomes containing miR-181c into the fracture site of diabetic patients, so that the miR-181c molecules in the exosomes can directly act on osteoblasts and local bone marrow mesenchymal stem cells, thereby increasing the concentration and biological activity of miR-181c at the fracture repair site through targeted action;

[0008] S3. After miR-181c delivered by exosomes enters the fracture microenvironment, it activates the local ERK signaling pathway, specifically regulates the differentiation and proliferation of osteoblasts, and promotes the formation of new blood vessels to achieve tissue repair and regeneration in the diabetic fracture area.

[0009] S4. Regularly assess bone density, angiogenesis, and callus formation in the fracture area through imaging tests to monitor and ensure the sustainability and effectiveness of miR-181c exosome therapy.

[0010] Further preferably, in step S1, bone marrow mesenchymal stem cells BMSCs are treated with miR-181c gene overexpression transfection using a lentiviral vector to stably enhance the sustained expression of miR-181c by BMSCs, so that BMSCs can secrete exosomes containing high levels of miR-181c; the lentiviral vector inserts miR-181c into the vector sequence and introduces it into BMSCs cells through a high-efficiency transfection medium, and cultures the cells in a culture medium at 37°C and 5% CO2 for 24 to 48 hours, so that the exosomes are enriched with a stable concentration of miR-181c.

[0011] Further preferably, the exosome extraction in step S1 is separated by ultracentrifugation, comprising the following steps:

[0012] S11, centrifuging the BMSCs culture medium at a low speed to remove cell debris and larger particles;

[0013] S12, subjecting the supernatant after low-speed centrifugation to ultracentrifugation to collect small vesicles and further enrich the exosomes secreted by BMSCs;

[0014] S13. The obtained small vesicles are further purified by density gradient centrifugation to extract exosomes with uniform particle size and complete lipid bilayer membrane structure for efficient delivery in subsequent diabetic fracture repair.

[0015] Further preferably, in step S2, exosomes containing miR-181c are precisely injected into the fracture area of ​​diabetic patients under CT image guidance to ensure that miR-181c in the exosomes is released around osteoblasts and local bone marrow mesenchymal stem cells BMSCs; wherein CT guidance is used to locate and adjust the position and depth of the injection needle in real time to accurately deliver the exosomes so that they are concentrated in the fracture healing site.

[0016] Further preferably, the activation of the ERK signaling pathway in step S3 is to specifically regulate the ERK pathway of osteoblasts through miR-181c delivered by exosomes, wherein miR-181c acts on the nodes of the ERK pathway, and enhances the proliferation, differentiation and anti-apoptosis ability of osteoblasts by upregulating the expression of pro-differentiation and pro-proliferation genes in the ERK signaling pathway;

[0017] The regulatory process includes miR-181c activating the phosphorylation reaction of ERK in osteoblasts and bone marrow mesenchymal stem cells, which is used to promote neovascularization and bone tissue regeneration at the fracture repair site.

[0018] Further preferably, the imaging evaluation in step S4 includes regular monitoring of fracture repair using micro-computed tomography and digital X-ray technology, wherein the micro-CT is used to obtain high-resolution three-dimensional images of the fracture site to accurately evaluate changes in bone density, callus formation, and distribution of new blood vessels; and the digital X-ray is used to dynamically observe the progress of fracture healing, including the disappearance of the fracture line and the degree of bone healing.

[0019] Further preferably, the exosomes containing miR-181c are further co-injected in combination with anti-inflammatory factors to improve the fracture microenvironment of diabetic fracture patients, wherein the anti-inflammatory factors are appropriate amounts of non-steroidal anti-inflammatory drugs or anti-inflammatory cytokines.

[0020] Further preferably, the expression level of miR-181c is detected by qRT-PCR before and after the extraction and injection of the exosomes to ensure that the concentration of miR-181c in the exosomes is stable and effectively delivered to the fracture repair site; before extraction, the expression level of miR-181c is detected in the BMSCs culture medium to confirm the overexpression effect of lentiviral transfection; after injection, the presence and content of miR-181c are detected in the tissue samples of the fracture area to ensure that miR-181c is successfully delivered to the target site and remains active.

[0021] In addition, the present invention also provides an application of a diabetic fracture repair method based on miR-181c in accelerating bone healing, supporting postoperative recovery, and improving chronic bone healing disorders in the treatment of fractures in diabetic patients.

[0022] The embodiment of the present invention has the following advantages due to the adoption of the above technical solution:

[0023] 1. The present invention utilizes exosomes as carriers of miR-181c to achieve targeted delivery of miR-181c in the fracture microenvironment, so that it can play a more efficient role in the fracture healing process of diabetic patients. Exosomes have natural biocompatibility and low immunogenicity, and can safely deliver miR-181c to the fracture site, while avoiding the immune rejection problem that may be caused by traditional carriers. The targeted delivery characteristics of exosomes enable them to concentrate the release of miR-181c in the diabetic fracture area, directly act on osteoblasts and bone marrow mesenchymal stem cells BMSCs, and effectively activate the local osteogenic signaling pathway.

[0024] 2. In the osteogenic microenvironment of the present invention, miR-181c promotes the proliferation, differentiation and angiogenesis of osteoblasts by activating the ERK signaling pathway. miR-181c can upregulate the expression of osteoblast-related genes, enhance the activity of osteoblasts and BMSCs, and further accelerate new bone formation. At the same time, miR-181c effectively inhibits cell apoptosis and chronic inflammatory response caused by the diabetic environment, optimizes the fracture microenvironment, makes the osteogenesis process smoother and more continuous, and improves the quality of fracture repair.

[0025] The above summary is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present invention will be readily apparent by reference to the accompanying drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 It is a flow chart of the steps of the present invention. DETAILED DESCRIPTION

[0028] In the following, only some exemplary embodiments are briefly described. As those skilled in the art will appreciate, the described embodiments may be modified in various ways without departing from the spirit or scope of the present invention. Therefore, the drawings and descriptions are considered to be exemplary and non-restrictive in nature.

[0029] The embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0030] Example

[0031] like Figure 1 As shown, this embodiment discloses a diabetic fracture repair method based on miR-181c, comprising the following steps:

[0032] Bone marrow mesenchymal stem cells (BMSCs) were transfected with miR-181c gene overexpression. The gene vector carrying miR-181c was introduced into BMSCs cells using a lentiviral vector system and incubated in a culture medium at 37°C and 5% The cells were cultured continuously for 24 to 48 hours in a CO2 environment to ensure that BMSCs continuously expressed and secreted high concentrations of miR-181c exosomes. Then, the exosomes were separated and purified by multi-step ultracentrifugation, including low-speed centrifugation to remove cell debris, ultracentrifugation to collect small vesicles, and density gradient centrifugation to purify exosomes, in order to extract exosomes with a complete lipid bilayer membrane structure and rich in miR-181c, ensuring their stable biological activity and structural integrity. Then, the exosomes containing miR-181c were precisely injected into the fracture site of diabetic patients under the guidance of CT images. CT guidance was used to locate and adjust the position and depth of the injection needle in real time to ensure that the exosomes were precisely distributed in the fracture area and acted directly on osteoblasts and local BMSCs. Targeted injection could increase the local concentration of miR-181c at the fracture site, enhance its biological activity in fracture repair, and reduce drug distribution and potential side effects in non-target sites, further optimizing the efficacy.

[0033] miR-181c is delivered in the fracture microenvironment through exosomes, specifically activating the local ERK signaling pathway and regulating the proliferation and differentiation of osteoblasts. The role of miR-181c in the ERK pathway includes upregulating the expression and phosphorylation level of related genes, enhancing the proliferation and differentiation of osteoblasts and BMSCs, and inhibiting apoptosis, further promoting the formation of new blood vessels, so as to improve the bone tissue regeneration rate and quality of patients with diabetic fractures. During the fracture healing process after injection, the repair of the fracture site is regularly monitored by imaging evaluation, and micro-CT is used to obtain high-resolution three-dimensional images of bone tissue to accurately evaluate bone density, callus formation and new blood vessel distribution. At the same time, digital X-rays are used to dynamically observe the disappearance of fracture lines and the progress of healing to ensure the durability and effectiveness of miR-181c exosome therapy in the repair of diabetic fractures.

[0034] In order to ensure the delivery effect of miR-181c in exosomes, qRT-PCR was used to detect the expression level of miR-181c before and after exosome extraction and injection. Before exosome extraction, the miR-181c content in the BMSCs culture medium was detected to confirm the overexpression effect of lentiviral transfection; after injection, tissue samples from the fracture site were tested to ensure that miR-181c was stably delivered to the fracture area and remained active, so as to exert a sustained repair effect.

[0035] This method also includes a combined injection step of miR-181c exosomes and anti-inflammatory factors. By adding an appropriate amount of non-steroidal anti-inflammatory drugs or anti-inflammatory cytokines, the chronic inflammatory response caused by diabetes can be alleviated, the microenvironment of the fracture area can be improved, and the inflammatory response during osteogenesis can be effectively reduced, thereby supporting the effects of osteogenesis and angiogenesis, and further enhancing the overall repair effect of miR-181c exosome therapy.

[0036] The diabetic fracture repair method of the present invention is based on miR-181c. It utilizes the gene overexpression and exosome delivery of miR-181c, combines CT-guided precision injection, activation of the ERK signaling pathway and real-time imaging monitoring, and cooperates with the combined treatment of anti-inflammatory factors to form a complete and efficient diabetic fracture repair method, which not only improves the quality of fracture healing, but also accelerates the healing speed.

[0037] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of various changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A diabetic fracture repair method based on miR-181c, characterized in that: The following steps are involved: S1. Overexpression of the miR-181c gene in bone marrow mesenchymal stem cells (BMSCs) to achieve high levels of miR-181c expression in secreted exosomes; extract the exosomes by ultracentrifugation and ensure that the exosomes are structurally intact and contain functionally active miR-181c molecules to support the osteogenic differentiation process; S2. Precisely inject exosomes containing miR-181c into the fracture site of diabetic patients, so that the miR-181c molecules in the exosomes can directly act on osteoblasts and local bone marrow mesenchymal stem cells, thereby increasing the concentration and biological activity of miR-181c at the fracture repair site through targeted action; S3. After miR-181c delivered by exosomes enters the fracture microenvironment, it activates the local ERK signaling pathway, specifically regulates the differentiation and proliferation of osteoblasts, and promotes the formation of new blood vessels to achieve tissue repair and regeneration in the diabetic fracture area. S4. Regularly assess bone density, angiogenesis, and callus formation in the fracture area through imaging tests to monitor and ensure the sustainability and effectiveness of miR-181c exosome therapy.

2. The diabetic fracture repair method based on miR-181c according to claim 1, characterized in that: In step S1, bone marrow mesenchymal stem cells (BMSCs) are subjected to miR-181c gene overexpression transfection treatment using a lentiviral vector to stably enhance the sustained expression of miR-181c by BMSCs, so that BMSCs can secrete exosomes containing high levels of miR-181c; the lentiviral vector inserts miR-181c into the vector sequence and introduces it into BMSCs cells through a high-efficiency transfection medium, and cultures the cells in a culture medium at 37°C and 5% CO2 for 24 to 48 hours, so that the exosomes are enriched with a stable concentration of miR-181c.

3. The diabetic fracture repair method based on miR-181c according to claim 1, characterized in that: The exosome extraction in step S1 is separated by ultracentrifugation, comprising the following steps: S11, centrifuging the BMSCs culture medium at a low speed to remove cell debris and larger particles; S12, subjecting the supernatant after low-speed centrifugation to ultracentrifugation to collect small vesicles and further enrich the exosomes secreted by BMSCs; S13. The obtained small vesicles are further purified by density gradient centrifugation to extract exosomes with uniform particle size and complete lipid bilayer membrane structure for efficient delivery in subsequent diabetic fracture repair.

4. The diabetic fracture repair method based on miR-181c according to claim 1, characterized in that: In step S2, exosomes containing miR-181c are precisely injected into the fracture area of ​​diabetic patients under CT image guidance to ensure that miR-181c in the exosomes is released around osteoblasts and local bone marrow mesenchymal stem cells BMSCs; wherein CT guidance is used to locate and adjust the position and depth of the injection needle in real time to accurately deliver the exosomes so that they are concentrated in the fracture healing site.

5. The diabetic fracture repair method based on miR-181c according to claim 1, characterized in that: The activation of the ERK signaling pathway in step S3 is to specifically regulate the ERK pathway of osteoblasts through miR-181c delivered by exosomes, wherein miR-181c acts on the nodes of the ERK pathway, and enhances the proliferation, differentiation and anti-apoptosis ability of osteoblasts by upregulating the expression of differentiation-promoting and proliferation-promoting genes in the ERK signaling pathway; The regulatory process includes miR-181c activating the phosphorylation reaction of ERK in osteoblasts and bone marrow mesenchymal stem cells, which is used to promote neovascularization and bone tissue regeneration at the fracture repair site.

6. The diabetic fracture repair method based on miR-181c according to claim 1, characterized in that: The imaging assessment in step S4 includes regular monitoring of fracture repair using micro-computed tomography and digital X-ray technology, wherein the micro-CT is used to obtain high-resolution three-dimensional images of the fracture site to accurately assess changes in bone density, callus formation, and distribution of new blood vessels; and the digital X-ray is used to dynamically observe the progress of fracture healing, including the disappearance of the fracture line and the degree of bone healing.

7. The diabetic fracture repair method based on miR-181c according to claim 1, characterized in that: The exosomes containing miR-181c are further co-injected with anti-inflammatory factors to improve the fracture microenvironment of diabetic fracture patients, wherein the anti-inflammatory factors are appropriate amounts of non-steroidal anti-inflammatory drugs or anti-inflammatory cytokines.

8. The diabetic fracture repair method based on miR-181c according to claim 1, characterized in that: The expression level of miR-181c was detected by qRT-PCR before and after the extraction and injection of the exosomes to ensure that the concentration of miR-181c in the exosomes was stable and effectively delivered to the fracture repair site; before extraction, the expression level of miR-181c was detected in the BMSCs culture medium to confirm the overexpression effect of lentiviral transfection; after injection, the presence and content of miR-181c were detected in the tissue samples of the fracture area to ensure that miR-181c was successfully delivered to the target site and remained active.

9. Application of a diabetic fracture repair method based on miR-181c according to any one of claims 1 to 8 in accelerating bone healing, supporting postoperative recovery and improving chronic bone healing disorders in fracture treatment in diabetic patients.

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