High-load miR-125a-5p human umbilical cord mesenchymal stem cell source small extracellular vesicle as well as preparation method and application of high-load miR-125a-5p human umbilical cord mesenchymal stem cell source small extracellular vesicle

Abstract

The invention provides a high-load miR-125a-5p human umbilical cord mesenchymal stem cell source small extracellular vesicle as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The human umbilical cord mesenchymal stem cell source small extracellular vesicle with high miR-125a-5p loading capacity, which is provided by the invention, is loaded with GGAG modified miR-125a-5p of which the nucleotide sequence is shown as SEQ ID NO. 1. According to the invention, the miR-125a-5p is modified by the exosome motif GGAG, so that the miR-125a-5p can be promoted to enter the hucMSC-sEV through active sorting, and the sEV loading capacity of the miR-125a-5p is specifically increased. The human umbilical cord mesenchymal stem cell source small extracellular vesicle with high miR-125a-5p loading capacity, provided by the invention, has a remarkable treatment effect on acute kidney injury caused by sepsis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a human umbilical cord mesenchymal stem cell-derived small extracellular vesicle with high miR-125a-5p loading, its preparation method, and its application. Background Technology

[0002] Sepsis is a life-threatening syndrome of organ dysfunction caused by infection, with extremely high morbidity and mortality. Currently, apart from traditional symptomatic and supportive therapies such as anti-infection treatment, fluid resuscitation, and vasoactive drugs, there is still a lack of specific treatments. Among the organ dysfunctions caused by sepsis, the kidneys are one of the most commonly affected organs, and sepsis patients have a high probability of developing severe kidney injury (S-AKI). Due to the lack of effective treatments, the mortality rate of S-AKI patients is significantly higher than that of patients without kidney injury. Moreover, the mortality rate of sepsis-related kidney injury is also significantly higher than that of kidney injury from other causes. Even if survivors do not survive, their risk of progressing to chronic kidney disease is significantly increased. Therefore, developing new and effective S-AKI prevention and treatment strategies is of great practical significance.

[0003] Although human umbilical cord mesenchymal stem cell-derived small extracellular vesicles (hucMSC-sEV) based miRNA therapy has shown great potential, the actual content of specific miRNAs within small extracellular vesicles (sEVs) is extremely low. Most individual exosomes do not carry biologically significant miRNA molecules, often requiring high intervention doses to achieve the desired therapeutic effect. However, excessive doses can easily lead to dose toxicity, severely hindering its translational application. Summary of the Invention

[0004] In view of this, one of the objectives of the present invention is to provide a human umbilical cord mesenchymal stem cell-derived small extracellular vesicle (miR-125a-5p) highly loaded with miR-125a-5p. GGAG -sEV), using the exosome motif GGAG to promote the sorting of miR-125a-5p into hucMSC-sEV, significantly increasing the miR-125a-5p loading in sEV.

[0005] A second objective of this invention is to provide the application of the highly loaded miR-125a-5p human umbilical cord mesenchymal stem cell-derived small extracellular vesicles in the preparation of products for treating acute kidney injury in sepsis.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: The present invention provides a human umbilical cord mesenchymal stem cell-derived small extracellular vesicle loaded with miR-125a-5p, wherein the human umbilical cord mesenchymal stem cell-derived small extracellular vesicle is loaded with GGAG-modified miR-125a-5p, and the nucleotide sequence of the GGAG-modified miR-125a-5p is shown in SEQ ID NO.1.

[0007] The present invention also provides a method for preparing the above-mentioned human umbilical cord mesenchymal stem cell-derived small extracellular vesicles with high miR-125a-5p loading, comprising the following steps: transfecting human umbilical cord mesenchymal stem cells with GGAG-modified miR-125a-5p, culturing and collecting the culture supernatant, performing differential ultracentrifugation to obtain a precipitate, resuspending the precipitate in PBS buffer and filtering it through a size exclusion chromatography column, and taking the filtrate to obtain human umbilical cord mesenchymal stem cell-derived small extracellular vesicles with high miR-125a-5p loading.

[0008] Preferably, the differential ultracentrifugation includes the following steps: first centrifuging at 4°C and 2000×g for 20 minutes, then taking the supernatant and centrifuging at 4°C and 15000×g for 30 minutes, and finally taking the supernatant and ultracentrifuging at 4°C and 100000×g for 120 minutes.

[0009] Preferably, the culture medium for culturing human umbilical cord mesenchymal stem cells is MSCNutriStem® XF Basal Medium containing 5% human platelet lysate.

[0010] Preferably, the transfection reagent includes a liposome transfection reagent, which includes Lipofectamine. TM 3000, the transfection time is 6 hours.

[0011] Preferably, when transfecting human umbilical cord mesenchymal stem cells, the degree of fusion of human umbilical cord mesenchymal stem cells is 70%-80%, and the human umbilical cord mesenchymal stem cells are human umbilical cord mesenchymal stem cells cultured to the 6th generation.

[0012] This invention also provides the application of the above-mentioned high-load miR-125a-5p human umbilical cord mesenchymal stem cell-derived small extracellular vesicles or the above-mentioned preparation method in the preparation of products for treating acute kidney injury in sepsis.

[0013] Preferably, the administration method of the high-load miR-125a-5p human umbilical cord mesenchymal stem cell-derived small extracellular vesicles includes intravenous injection, wherein the intravenous injection volume is 2 × 10⁻⁶. 9 particles and above.

[0014] The present invention also provides a medicament for treating acute kidney injury in sepsis, wherein the active ingredient of the medicament comprises the above-mentioned human umbilical cord mesenchymal stem cell-derived small extracellular vesicles with high miR-125a-5p loading.

[0015] Preferably, the type of drug includes injectable formulations.

[0016] The beneficial effects of this invention are: This invention modifies miR-125a-5p with the exosome motif GGAG, which can promote the active sorting of miR-125a-5p into hucMSC-sEV, thereby specifically increasing the sEV loading of miR-125a-5p.

[0017] The hucMSC-sEV with high miR-125a-5p loading provided by this invention improves kidney injury and survival by enhancing inflammation suppression and reducing serum creatinine and urea nitrogen levels. It can also effectively improve the therapeutic effect of hucMSC-sEV on acute kidney injury in sepsis while reducing the dosage. Attached Figure Description

[0018] Figure 1 For NC-sEV and miR-125a-5p GGAG The characterization results of -sEV are shown in Figure A, where A is the nanoparticle size distribution detected by NTA, B is the morphology observed by TEM, and C is the surface marker protein result detected by Western blot.

[0019] Figure 2 The results of the evaluation of the promoting effect of exosome motif GGAG on miR-125a-5p loading into hucMSC-sEVs are shown. A represents the content of miR-125a-5p in human umbilical cord mesenchymal stem cells (hucMSCs) and hucMSC-sEVs under NC, WT, and GGAG conditions, respectively, as detected by qRT-PCR. B represents the ratio of miR-125a-5p content in hucMSC-sEVs to that in hucMSCs under NC, WT, and GGAG conditions, as detected by qRT-PCR.

[0020] Figure 3 Design diagram for animal experimental protocols.

[0021] Figure 4 For NC-sEV and miR-125a-5p GGAG Results of the effect of -sEV on the survival rate of S-AKI mice.

[0022] Figure 5 NC-sEV and miR-125a-5p were injected into the tail vein. GGAGResults of the effects of -sEV on the kidneys of S-AKI mice, where A represents serum creatinine (Scr) levels in each group of mice; B represents serum blood urea nitrogen (BUN) levels in each group of mice; C represents NGAL mRNA expression levels in kidney tissues in each group of mice; D represents TNF-α mRNA expression levels in kidney tissues in each group of mice; E represents CCL2 mRNA expression levels in kidney tissues in each group of mice; and F represents representative images of HE staining and NGAL immunohistochemical staining of kidney tissues in each group of mice. This indicates that p < 0.05. This indicates that p < 0.01. ns indicates p < 0.001, and ns indicates p ≥ 0.05. Detailed Implementation

[0023] This invention provides a human umbilical cord mesenchymal stem cell-derived small extracellular vesicle loaded with miR-125a-5p, wherein the human umbilical cord mesenchymal stem cell-derived small extracellular vesicle carries GGAG-modified miR-125a-5p, and the nucleotide sequence of the GGAG-modified miR-125a-5p is 5'-UCCCUGAGACCCUUUAACCUGUGAGGAG-3', as shown in SEQ ID NO.1.

[0024] This invention utilizes the exosomal motif GGAG to modify miR-125a-5p, promoting its active loading and enrichment within hucMSC-sEVs, thereby specifically increasing the sEV loading of miR-125a-5p. In this invention, hucMSC-sEVs with increased miR-125a-5p loading exhibit stronger therapeutic effects than native hucMSC-sEVs (here, native hucMSC-sEVs refer to hucMSC-sEVs without miR-125a-5p loading) after intervention in CLP-induced S-AKI mice, manifested in a more significant increase in the survival rate of septic mice, improved renal function, and reduced inflammatory response. Compared to human umbilical cord mesenchymal stem cells (hucMSCs), hucMSC-sEVs offer advantages such as higher safety, easier acquisition, and convenient storage and transportation.

[0025] The present invention also provides a method for preparing the above-mentioned human umbilical cord mesenchymal stem cell-derived small extracellular vesicles with high miR-125a-5p loading, comprising the following steps: transfecting human umbilical cord mesenchymal stem cells with GGAG-modified miR-125a-5p, culturing and collecting the culture supernatant, performing differential ultracentrifugation to obtain a precipitate, resuspending the precipitate in PBS buffer and filtering it through a size exclusion chromatography column, and taking the filtrate to obtain human umbilical cord mesenchymal stem cell-derived small extracellular vesicles with high miR-125a-5p loading.

[0026] In this invention, it is preferred to culture human umbilical cord mesenchymal stem cells to passage 6 before transfection; more preferably, transfection is performed when human umbilical cord mesenchymal stem cells are cultured to passage 6 and the confluence of human umbilical cord mesenchymal stem cells is 70%-80%. In this invention, the culture medium for culturing human umbilical cord mesenchymal stem cells is preferably MSCNutriStem® XF Basal Medium containing 5% human platelet lysate. This invention does not have specific limitations on the specific sources of human platelet lysate and MSC NutriStem® XF Basal Medium; commercially available products commonly used in the art can be used. In this invention, 48 hours before collecting the culture supernatant, it is preferred to replace the MSC NutriStem® XF Basal Medium containing 5% human platelet lysate with serum-free DMEM / F12 containing no human platelet lysate. In this invention, the 5% refers to a volume percentage. In this invention, the transfection reagent preferably includes a liposome transfection reagent, and the liposome transfection reagent preferably includes Lipofectamine. TM 3000, the transfection time is preferably 6 hours. This invention does not specifically limit the method for modifying miR-125a-5p using GGAG; conventional sequence synthesis methods in the art are sufficient. After transfection, culturing is performed, preferably for 48 hours.

[0027] In this invention, the differential ultracentrifugation preferably includes the following steps: first, centrifuging at 2000×g for 20 minutes at 4°C; then, centrifuging the supernatant at 15000×g for 30 minutes at 4°C; finally, ultracentrifuging the supernatant at 100000×g for 120 minutes at 4°C. In this invention, the reagent used for resuspending the precipitate is preferably sterilized PBS buffer, and the sterilization method is preferably filtration sterilization, with a preferred pore size of 0.1 μm. This invention does not specifically limit the source of the size exclusion chromatography column; any conventional size exclusion chromatography column for the preparation of small extracellular vesicles in the art can be used. In some embodiments of this invention, the size exclusion chromatography column is purchased from Beijing Enzekangtai Biotechnology Co., Ltd., product model ES933.

[0028] This invention also provides the application of the above-mentioned high-load miR-125a-5p human umbilical cord mesenchymal stem cell-derived small extracellular vesicles or the above-mentioned preparation method in the preparation of products for treating acute kidney injury in sepsis.

[0029] In this invention, the product preferably includes a drug. In this invention, the preferred method of administration of the highly loaded miR-125a-5p human umbilical cord mesenchymal stem cell-derived small extracellular vesicles is intravenous injection, and the preferred amount injected intravenously is 2 × 10⁻⁶. 9The above particles. In some embodiments of the present invention, a human umbilical cord mesenchymal stem cell-derived small extracellular vesicle group loaded with high miR-125a-5p (referring to Example 1 group) and a human umbilical cord mesenchymal stem cell-derived small extracellular vesicle group loaded with unmodified miR-125a-5p (referring to Comparative Example 1 group) were simultaneously set up to evaluate the therapeutic effect in a mouse model of septic acute kidney injury. The results showed that Example 1 group 2 × 10 9 The therapeutic effect of the particle dosage was better than that of the control group 1 (1×10⁻⁶). 10 The therapeutic effect of the dosage of particles indicates that the human umbilical cord mesenchymal stem cell-derived small extracellular vesicles with high miR-125a-5p loading of the present invention can achieve better therapeutic effects while effectively reducing the dosage.

[0030] The present invention also provides a medicament for treating acute kidney injury in sepsis, wherein the active ingredient of the medicament comprises the above-mentioned human umbilical cord mesenchymal stem cell-derived small extracellular vesicles with high miR-125a-5p loading.

[0031] In the medicament described in this invention, the type of medicament preferably includes injectable formulations. This invention does not specifically limit the types of other excipients in the medicament.

[0032] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0033] Unless otherwise specified, the following embodiments are all conventional methods.

[0034] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0035] The MSC NutriStem® XF Basal Medium used in the following examples was purchased from Sartorius, and the human platelet lysate was purchased from Mill Creek Life Science.

[0036] Example 1 Highly loaded human umbilical cord mesenchymal stem cell-derived small extracellular vesicles (miR-125a-5p) GGAG Preparation of -sEV): (1) First, miR-125a-5p was artificially synthesized. GGAGmimic is specifically prepared by inserting the exosome motif GGAG into the 3' end of the miR-125a-5p nucleotide sequence (5'-UCCCUGAGACCCUUUAACCUGUGA-3' (SEQ ID NO.2)). The sequence after inserting the exosome motif GGAG is: 5'-UCCCUGAGACCCUUUAACCUGUGAGGAG-3' (SEQ ID NO.1) GGAG For mimic powder, the above process was outsourced to Shanghai Jima Pharmaceutical Technology Co., Ltd.

[0037] Human umbilical cord mesenchymal stem cells (hucMSCs) were cultured normally using NutriStem® XF Basal Medium containing 5% human platelet lysate. When the hucMSCs reached passage 6 and cell confluence reached 70-80%, miR-125a-5p was added to sterile DEPC water. GGAG Dissolve mimic powder to a concentration of 1 μg / μL, then mix with Lipofectamine™ 3000 solution and react for 15 minutes. Then add to the culture medium of hucMSCs, mix thoroughly, discard the culture medium after 6 hours, wash the cells twice with sterile PBS, and replace with serum-free and platelet lysate-free DMEM / F12 and continue culturing for 48 hours. Aseptically collect the culture supernatant.

[0038] (2) Centrifuge the culture supernatant at 4°C and 2000×g for 20 minutes to remove dead cells and large cell debris; transfer the supernatant to a new sterile centrifuge tube and centrifuge at 4°C and 15000×g for 30 minutes to remove large vesicles (apoptotic bodies); then transfer the supernatant to a clean, sterile ultracentrifuge tube and centrifuge at 4°C and 100000×g for 120 minutes, discard the supernatant and retain the precipitate, resuspend the precipitate in sterile pre-cooled PBS buffer filtered through a 0.1μm filter membrane; finally, filter the resuspended solution through a size exclusion chromatography column (Beijing Enzekangtai Biotechnology Co., Ltd., ES933), and collect the filtrate of the fraction with the highest exosome purity according to the instructions, which is pure miR-125a-5p. GGAG Store in sEV form at -80℃.

[0039] Comparative Example 1 Preparation of human umbilical cord mesenchymal stem cell-derived small extracellular vesicles (WT-sEVs, also labeled WT) loaded with unmodified miR-125a-5p: (1) Human umbilical cord mesenchymal stem cells (hucMSCs) were cultured normally using MSC NutriStem® XF Basal Medium containing 5% human platelet lysate. When the hucMSCs reached the 6th generation and the cell fusion reached 70-80%, miR-125a-5p powder (SEQ ID NO.2) was dissolved in sterile DEPC water to a concentration of 1 μg / μL. Then, it was mixed with Lipofectamine™ 3000 solution and reacted for 15 minutes. Then, it was added to the hucMSCs culture medium and mixed thoroughly. After 6 hours, the culture medium was discarded, and the cells were washed twice with sterile PBS. The culture medium was then replaced with DMEM / F12 without exosomes and cultured for another 48 hours. The culture supernatant was collected aseptically.

[0040] Step (2) is the same as step (2) in Example 1, to obtain WT-sEV, which is then marked as WT.

[0041] Comparative Example 2 Preparation of natural human umbilical cord mesenchymal stem cell-derived small extracellular vesicles (NC-sEVs): (1) Use MSC NutriStem Xeno & serum-free BasalMedium containing 5% human platelet lysate to culture human umbilical cord mesenchymal stem cells (hucMSCs) normally. When the hucMSCs reach the 6th generation and the cell fusion reaches 70-80%, discard the culture medium, wash the cells twice with sterile PBS, and replace with DMEM / F12 without exosomes for 48 hours. Collect the culture supernatant aseptically.

[0042] Step (2) is the same as step (2) in Example 1, to obtain NC-sEV, which is then labeled as NC.

[0043] Example 2 The miR-125a-5p prepared in Example 1 and Comparative Example 2 GGAG -sEV and NC-sEV are tested as follows: NTA detection: After calibrating the Zetaview instrument (Particle Metrix, Germany) using NTA standards, the miR-125a-5p samples obtained in Example 1 and Comparative Example 2 were analyzed using ultrapure water. GGAG -sEV and NC-sEV were diluted separately, and 1 mL was injected into the instrument. miR-125a-5p was measured in EV 488C mode. GGAG -sEV and NC-sEV particle size distributions, the results are as follows Figure 1 As shown in A in the diagram. miR-125a-5p GGAGThe average diameter of -sEV is 157.6 nm, and the average diameter of NC-sEV is 142.2 nm.

[0044] Transmission electron microscopy observation of miR-125a-5p GGAG The basic morphology of -sEV and NC-sEV: 20 μL of each of the samples prepared in Example 1 and Comparative Example 2 were sent to Beijing Zhongke Baice Testing Technology Co., Ltd. for testing. The samples were observed and photographed under a transmission electron microscope. The results are as follows: Figure 1 As shown in B in the image. miR-125a-5p GGAG -sEV and NC-sEV have a double-layered cup-shaped structure.

[0045] Western blot detection of miR-125a-5p GGAG Surface marker proteins of -sEV and NC-sEV (human umbilical cord mesenchymal stem cells (hucMSCs) were used as a control group): Total protein was extracted with RIPA lysis buffer containing 1% protease inhibitor, and protein concentration was determined by BCA method. Proteins were separated by 4%–20% SDS-PAGE and transferred to 0.22 μm PVDF membranes. The membranes were blocked with 5% skim milk at room temperature for 1 hour, followed by overnight incubation at 4°C with primary antibodies against Calnexin, TSG101, CD9, and CD81, respectively. The next day, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody at room temperature for 1 hour, and washed three times with TBST solution for 5 minutes each time. Protein signals were detected using a chemiluminescent gel imaging system. Results are as follows: Figure 1 As shown in C, miR-125a-5p GGAG TSG101, CD9, and CD81 were positively expressed in -sEV and NC-sEV, while Calnexin was negatively expressed.

[0046] Example 3 Evaluation of the promoting effect of exosome motif GGAG on miR-125a-5p loading into hucMSC-sEV: Real-time quantitative PCR (qRT-PCR) detection: Following the kit instructions, total RNA was obtained from human umbilical cord mesenchymal stem cells (hucMSCs) and human umbilical cord mesenchymal stem cell-derived small extracellular vesicles (hucMSC-sEVs) using an RNA extraction kit (Nanjing Novizan Biotechnology Co., Ltd.), and the concentration was measured using a NanoDrop micro-spectrophotometer. Then, cDNA was obtained by reverse transcription using a miRNA reverse transcription kit (Sangon Biotech (Shanghai) Co., Ltd.), and the expression level of miRNA was measured by qRT-PCR using ChamQ SYBR qPCR Master Mix (Nanjing Novizan Biotechnology Co., Ltd.). U6 was used as an internal control, and 2...−ΔΔCT The relative expression levels of miRNAs were standardized. The groups in this experiment were NC (referring to those obtained in Comparative Example 2), WT (referring to those obtained in Comparative Example 1), and GGAG (referring to those obtained in Example 1).

[0047] The results are as follows Figure 2 As shown, Figure 2 As shown in Figure A, the content of miR-125a-5p carrying GGAG was significantly increased in both hucMSCs and hucMSC-sEVs. Specifically, the loading amount in hucMSC-sEVs was approximately 3 times higher than that in the WT group (without GGAG), and more than 1000 times higher than that in the NC group. Crucially, compared to hucMSCs, GGAG more specifically promoted the active sorting of miR-125a-5p into hucMSC-sEVs (see Figure A). Figure 2 (B in the middle).

[0048] Example 4 miR-125a-5p obtained in Example 1 GGAG Evaluation of the therapeutic effect of sEV on a mouse model of acute kidney injury with sepsis: (1) Model construction: Cecal ligation and puncture (CLP) induced sepsis in small animals can well simulate clinical reality in terms of pathophysiology and symptoms, and is considered the most classic sepsis model. Therefore, this invention uses this method to construct a mouse sepsis model. The procedure is as follows: The mouse is anesthetized by intraperitoneal injection of 1.25% tribromoethanol (0.2mL / 10g), the surgical area is prepared, the abdominal cavity is opened 1cm longitudinally along the linea alba, the cecum is located and exposed outside the abdominal cavity, and a sterile No. 4 silk suture is used to ligate the cecum at the root and about 1 / 2 of the free distal end. Then, a sterile 21G needle is used to puncture the distal center of the ligated cecum, a small amount of intestinal contents are squeezed out and wiped away with a cotton swab, the cecum is carefully returned to the abdominal cavity, the abdominal cavity is sutured layer by layer, and finally 1mL of sterile physiological saline is injected subcutaneously in the chest and back to replenish fluid and fight shock. The resuscitated body is kept on a constant temperature pad to wait for awakening.

[0049] (2) miR-125a-5p GGAG Applications of -sEV: such as Figure 3 As shown, 60 eight-week-old C57BL6 / J mice were randomly divided into four groups: sham-operated group (Sham), model group (CLP+PBS), control group (CLP+NC-sEV (obtained from Comparative Example 2)), and experimental group (CLP+miR-125a-5p). GGAG -sEV). The CLP model was constructed according to step (1). The experimental group mice were injected with 2×10 sEVs via the tail vein at 0, 24 and 48 hours after surgery. 9 miR-125a-5p particles GGAG-sEV, the model group was injected with an equal volume of sterile PBS buffer, while the control group was injected with 2×10 9 The NC-sEVs of particles were monitored for survival daily, and blood and kidney samples were taken after 72 hours for further testing.

[0050] (3) Survival analysis: After CLP surgery, mice in each group experienced varying degrees of mortality. The results are as follows: Figure 4 As shown, at the same intervention dose, miR-125a-5p GGAG -sEV significantly improved mouse survival compared to NC-sEV (80% vs 73.33%).

[0051] (4) Serological assessment of renal function: 72 hours after CLP surgery, mice were sacrificed, blood was collected, and serum was collected after centrifugation at 3000 rpm for 15 minutes. Serum creatinine (Scr) and blood urea nitrogen (BUN) levels were measured using the microplate method to assess renal function in each group of mice. Results are as follows: Figure 5 As shown in A and B, at the same intervention dose, miR-125a-5p GGAG -sEV significantly reduced Scr and BUN levels more than NC-sEV (P<0.05).

[0052] (5) qRT-PCR detection of kidney tissue: 72 hours after CLP surgery, mice were sacrificed to obtain kidney tissue and RNA was extracted. The mRNA expression levels of NGAL (an acute kidney injury marker) and inflammatory factors TNF-α and CCL2 were detected by qRT-PCR. The results are as follows: Figure 5 As shown in the CE diagram, at the same intervention dose, miR-125a-5p GGAG -sEV significantly reduced the expression of NGAL, TNF-α and CCL2 more than NC-sEV (P<0.05).

[0053] (6) HE and immunohistochemical staining of kidney tissue: 72 hours after CLP surgery, mice were sacrificed to obtain kidney tissue, which was fixed, embedded in paraffin, and sectioned for HE staining to assess morphological changes. In addition, immunohistochemical staining was performed to detect NGAL expression levels. Results are as follows: Figure 5 As shown in F, at the same intervention dose, miR-125a-5p GGAG -sEV significantly reduced renal tissue damage (tubular cell shedding, interstitial congestion, inflammatory cell infiltration, etc.) and NGAL expression compared to NC-sEV.

[0054] The above results show that the miR-125a-5p described in this invention... GGAG -sEV can effectively prevent and treat sepsis-induced acute kidney injury, and its effect is significantly better than NC-sEV.

[0055] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A high load of miR-125a-5p human umbilical cord mesenchymal stem cell-derived small extracellular vesicles, characterized in that, The human umbilical cord mesenchymal stem cell-derived small extracellular vesicles carry the GGAG-modified miR-125a-5p, and the nucleotide sequence of the GGAG-modified miR-125a-5p is shown as SEQ ID NO.

1.

2. The method of claim 1, wherein the preparation of the high-loading miR-125a-5p human umbilical cord mesenchymal stem cell-derived small extracellular vesicles is characterized by, The method comprises the following steps: transfecting human umbilical cord mesenchymal stem cells with the GGAG-modified miR-125a-5p, collecting the culture supernatant, performing differential ultracentrifugation to obtain a precipitate, resuspending the precipitate in PBS buffer, filtering through a size exclusion chromatography column, and obtaining the high-load miR-125a-5p human umbilical cord mesenchymal stem cell-derived small extracellular vesicles from the filtrate.

3. The production method according to claim 2, characterized by, The differential ultracentrifugation comprises the following steps: first, centrifugation at 2000xg for 20 minutes at 4°C, then centrifugation of the supernatant at 15000xg for 30 minutes at 4°C, and finally ultracentrifugation of the supernatant at 100000xg for 120 minutes at 4°C.

4. The production method according to claim 2, characterized by, The culture medium for culturing the human umbilical cord mesenchymal stem cells is MSC NutriStem® XF Basal Medium containing 5% human platelet lysate.

5. The preparation method according to claim 2, characterized in that, The reagents for the transfection include lipofectamine reagents for transfection, including Lipofectamine TM 3000, the transfection time is 6 hours.

6. The preparation method according to claim 2, characterized in that, When the human umbilical cord mesenchymal stem cells are transfected, the confluence of the human umbilical cord mesenchymal stem cells is 70%-80%, and the human umbilical cord mesenchymal stem cells are human umbilical cord mesenchymal stem cells cultured to the 6th generation.

7. Use of the high-load miR-125a-5p human umbilical cord mesenchymal stem cell-derived small extracellular vesicles of claim 1 or the preparation method of any one of claims 2-6 in the preparation of a product for treating sepsis acute kidney injury.

8. Use according to claim 7, characterized in that, The administration mode of the high-load miR-125a-5p human umbilical cord mesenchymal stem cell-derived small extracellular vesicles includes intravenous injection, and the amount of the intravenous injection is 2 x 10 9 particles or more.

9. A medicament for treating sepsis acute kidney injury, characterized by, The active ingredient of the drug comprises the high-load miR-125a-5p human umbilical cord mesenchymal stem cell-derived small extracellular vesicles of claim 1.

10. The medicament according to claim 9, characterized in that, The type of the drug comprises an injection.

Images