Preparation method of DRP1 engineered mitochondrial derived vesicle and application of DRP1 engineered mitochondrial derived vesicle in cell aging resistance

Abstract

The invention discloses a preparation method of DRP1 engineered mitochondrial derived vesicles and application of the DRP1 engineered mitochondrial derived vesicles in cell aging resistance. The method comprises the following steps: firstly, constructing a lentiviral vector of an overexpressed DRP1 gene, and transfecting cells to obtain a cell strain of the stable overexpressed DRP1 gene; and culturing the cell strain, collecting a culture solution, and separating and extracting the mitochondrial derived vesicles to obtain the DRP1 engineered mitochondrial derived vesicles DRP1-MDVs rich in mtDNA. The DRP1-MDVs is used for treating senescent cells, the DRP1-MDVs is successfully internalized into senescent cells, the ROS level can be effectively reduced, the mitochondrial mtDNA and ATP content can be improved, the mtDNA mutation rate can be reduced, the mitochondrial membrane potential and the mitochondrial network structure can be recovered, and finally the expression of senescence-related proteins P16 and P21 can be reduced. The obvious mitochondrial function repairing potential and the cell aging improving effect are shown.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and more specifically, relates to a method for preparing DRP1 engineered mitochondrial-derived vesicles and their application in anti-cellular aging. Background Technology

[0002] Mitochondrial dysfunction is a common phenomenon in the aging process. With age, the production of reactive oxygen species (ROS) in mitochondria increases, triggering oxidative stress and leading to oxidative damage to mitochondrial DNA (mtDNA), lipids, and proteins. Increased mutation rates in mitochondrial DNA can cause errors in the function of encoded enzyme subunits, thereby impairing mitochondrial oxidative phosphorylation and resulting in insufficient cellular energy supply. Due to the secretion of age-related secretory phenotypes, the PINK1 / Parking ubiquitin pathway-mediated mitophagy function declines, leading to reduced clearance efficiency of damaged mitochondria. The decreased efficiency of mitophagy in senescent cells leads to the accumulation of damaged mitochondria, further exacerbating the cellular senescence process. There is a bidirectional relationship between mitochondrial dysfunction and cellular senescence; mitochondrial damage is not only a consequence of aging but also a driving factor. Therefore, improving mitochondrial dysfunction holds promise for delaying the progression of age-related diseases.

[0003] mtDNA mutations are a key inducing factor of cellular senescence, and the formation of senescent cells further exacerbates mtDNA damage and mutations, creating a vicious cycle. This interaction plays a crucial role in cellular physiology, tissue function decline, and various age-related diseases. On the one hand, the accumulation of mtDNA mutations may lead to mitochondrial respiratory chain dysfunction and increased ROS production. Since senescent cells have a reduced ability to process reactive oxygen species (ROS), mtDNA becomes susceptible to ROS attack, resulting in oxidative damage and triggering oxidative stress. This not only damages the mitochondria themselves but also activates intracellular stress response pathways, such as the p53-p21 pathway, further leading to cell cycle arrest and senescence. On the other hand, mtDNA mutations may also lead to a decrease in mitochondrial membrane potential and insufficient energy metabolism, further affecting normal cellular function. A close link exists between mtDNA mutations and senescent cells; this link not only affects normal cellular function but also plays a significant role in various age-related diseases. Therefore, interventions targeting mtDNA mutations and cellular senescence may provide new strategies for delaying aging and preventing age-related diseases.

[0004] Mitochondrial-derived vesicles (MDVs) are important membrane vesicles formed by the outer membrane or the inner and outer membranes and the matrix contents of mitochondria, which contain mitochondrial homologous components such as mtDNA. MDVs have various biological functions, including maintaining mitochondrial mass, enhancing antioxidant and anti-infection capacity, promoting intercellular communication, regulating cell metabolism and immune response, etc., which play an important role in normal physiological processes, and are also closely related to the occurrence and development of various diseases. Because MDVs and mitochondria have homology and low immunogenicity, etc., compared with other extracellular vesicles, MDVs have natural superiority in targeted treatment of mitochondrial-related diseases, and MDV carrier is a new and effective method for treating related diseases, which is of great significance for understanding mitochondrial biology and developing new disease treatment strategies. If normal mtDNA can be delivered to mtDNA mutant senescent cells, the mitochondrial dysfunction of senescent cells can be improved to achieve anti-aging. However, under physiological conditions, the level of MDVs secreted by cells is low, which makes it difficult to achieve treatment due to insufficient mtDNA content. SUMMARY

[0005] The purpose of the present application is to overcome the problem that the level of MDVs secreted by cells is low, resulting in insufficient mtDNA content, and to provide a preparation method of DRP1 engineered mitochondrial-derived vesicles rich in mtDNA.

[0006] The second purpose of the present application is to provide the DRP1 engineered mitochondrial-derived vesicles rich in mtDNA.

[0007] The third purpose of the present application is to provide the use of the DRP1 engineered mitochondrial-derived vesicles rich in mtDNA in the preparation of a medicine for treating DNA inhibition caused by mitochondrial dysfunction.

[0008] The above purposes of the present application are achieved by the following technical solutions: The present application first provides a preparation method of DRP1 engineered mitochondrial-derived vesicles, which comprises the following steps: constructing a lentiviral vector overexpressing DRP1 the gene, transfecting cells to obtain a cell strain stably overexpressing DRP1 the gene; culturing the cell strain, collecting the culture solution, and separating and extracting mitochondrial-derived vesicles to obtain DRP1 engineered mitochondrial-derived vesicles rich in mitochondrial DNA (i.e., DRP1-MDVs).

[0009] The nucleotide sequence of the DRP1 gene is shown in SEQ ID No. 1, and the encoded amino acid sequence is shown in SEQ ID No. 2.

[0010] Since the formation of MDVs involves the participation of multiple proteins, the present application introduces DRP1 (dynamin-related protein 1). DRP1 is a GTPase mainly located in the cytoplasm, but is recruited to the outer mitochondrial membrane when performing functions. Its basic function is to act as a core regulatory protein for mitochondrial fission: by wrapping and contracting the mitochondrial membrane, it "pinches off" mitochondria under the energy of GTP hydrolysis, thereby maintaining the dynamic balance of the mitochondrial network. DRP1-mediated fission is crucial for mitochondrial function. It provides a basis for mitochondrial autophagy by removing damaged mitochondrial fragments, ensuring the stability of mitochondrial quality and cellular energy metabolism. Disorders in the fission process can lead to mitochondrial dysfunction, affecting ATP production, calcium homeostasis, and increasing the production of reactive oxygen species. DRP1 also plays a key role in the formation of mitochondrial-derived vesicles (MDVs). MDVs are vesicles produced by mitochondria for the transport of specific substances. Studies have shown that there is a DRP1-dependent MDV subpopulation that requires the participation of DRP1 for its formation. DRP1 may provide the necessary membrane dynamics for the "budding" and separation of MDVs by regulating the fission site of the mitochondrial membrane, thereby transporting specific proteins or cargo from mitochondria to other organelles such as peroxisomes or lysosomes. Therefore, DRP1 is an important molecular bridge connecting mitochondrial fission and vesicle transport. The present application shows that stable overexpression of DRP1 in L-O2 cells significantly increases the secretion of MDVs and enriches mtDNA in MDVs. DRP1 The cell strain stably overexpressing DRP1 mainly produces inner membrane type MDVs composed of mitochondrial inner membrane, significantly increases the level of cell secretion of MDVs under normal physiological conditions, and is rich in normal intact mtDNA compared with wild-type cells or empty vector cells.

[0011] Further, the lentiviral vector is pCDH-CMV-MCS-EF1-copGFP-Puro. pCDH-CMV-MCS-EF1-CopGFP-Puro is a commonly used lentiviral expression vector designed for efficient expression and stable selection of exogenous genes in mammalian cells.

[0012] Further, the cell is an L-O2 cell. L-O2 cells are derived from normal human liver tissue and have typical characteristics of hepatocytes, such as albumin secretion and urea synthesis functions, and are commonly used to simulate the physiological or pathological processes of normal hepatocytes.

[0013] Further, the separation and extraction is performed by iodixanol gradient density centrifugation.

[0014] The present application also provides a DRP1 engineered mitochondrial-derived vesicle enriched in mitochondrial DNA prepared by any of the above preparation methods.

[0015] The application further applies the engineered mitochondria-derived vesicle DRP1-MDVs to an aging cell model of EtBr-induced mitochondrial dysfunction, detects the antioxidant activity indicators of cell mitochondria and the expressions of aging-related proteins P16 and P21, and the results show that the engineered mitochondria-derived vesicle DRP1-MDVs have a therapeutic effect on the aging model cells, indicating that the application successfully plays a role in cell aging treatment in the form of mitochondria-derived vesicle delivery mtDNA, and provides a new strategy for treating aging-related diseases.

[0016] Specifically, the application adds the engineered mitochondria-derived vesicle DRP1-MDVs to an aging cell model of EtBr-induced mitochondrial dysfunction, evaluates the effects of the engineered mitochondria-derived vesicle DRP1-MDVs on the ATP content, mitochondrial membrane potential, ROS level and mtDNA content in the model cells, detects the expression levels of mitochondrial signaling pathway proteins, and the research results show that the constructed engineered mitochondria-derived vesicle DRP1-MDVs are successfully internalized into aging cells, can effectively reduce the ROS level, increase the mitochondrial mtDNA and ATP content, reduce the mtDNA mutation rate, have an effect of promoting the recovery of mitochondrial function, regulate the biological effects of various metabolic enzymes of mitochondria, and enhance the antioxidant capacity, and have application value in treating cell aging caused by mitochondrial dysfunction or treating cell aging-related diseases.

[0017] Therefore, the application first provides the use of the above mtDNA-rich DRP1 engineered mitochondria-derived vesicle in the preparation of a medicine for improving cell aging or treating cell aging-related diseases.

[0018] Further, the cell aging is caused by mitochondrial dysfunction caused by mtDNA mutation.

[0019] Further, the medicine internalizes the MDVs rich in normal mtDNA into aging cells, promotes the oxidative phosphorylation of mitochondria of cells of a subject, regulates the activities of mitochondrial metabolic enzymes and antioxidant enzymes, removes ROS, increases the ATP content, enhances the generation of mitochondrial networks and recovers the mitochondrial function, and reduces the expressions of aging-related proteins P16 and P21, so as to achieve the improvement of cell aging or the treatment of cell aging-related diseases.

[0020] Further, the subject is selected from mammals.

[0021] Further, the mammal is selected from a mouse, a cat, a dog, a pig, a cow, a horse, a sheep, a monkey and a human, etc.

[0022] The application also provides an anti-cell aging medicine, and the medicine contains the above-mentioned any mtDNA-rich DRP1 engineered mitochondria-derived vesicle.

[0023] Furthermore, the drug also includes other pharmaceutically acceptable excipients.

[0024] Compared with the prior art, the present invention has the following beneficial effects: This invention provides engineered mitochondrial-derived vesicles rich in mtDNA, specifically DRP1, and their application in anti-cellular senescence. This invention involves first constructing an overexpressing... DRP1 Lentiviral vectors of the gene are transfected into cells to obtain stable overexpression. DRP1 A cell line containing the gene was developed; this cell line was then cultured, and the culture medium was collected for the separation and extraction of mitochondrial-derived vesicles, yielding DRP1-MDVs engineered mitochondrial-derived vesicles rich in normal mtDNA. The results of this invention show that these engineered mitochondrial-derived vesicles, DRP1-MDVs, can be internalized into senescent cells, effectively reducing ROS levels, increasing mitochondrial mtDNA and ATP content, reducing mtDNA mutation rate, improving mitochondrial network structure, and reducing the expression of aging-related proteins P16 and P21. They thus promote mitochondrial function recovery and improve and treat cellular senescence. This invention successfully utilizes mitochondrial-derived vesicles to deliver mtDNA in the treatment of cellular senescence, providing a new strategy for treating aging-related diseases. Attached Figure Description

[0025] Figure 1 The result of PCR amplification of the DRP1 gene in Example 1 (M: DNA Marker).

[0026] Figure 2 This is the colony PCR of pDRP1 in Example 1.

[0027] Figure 3 This is an image showing the enzyme digestion identification of the recombinant vector in Example 1.

[0028] Figure 4 The relative mRNA expression level of DRP1 in Example 1 (n=3, *: P<0.05).

[0029] Figure 5 The value represents the protein expression level of DRP1 in Example 1.

[0030] Figure 6 The expression levels of MDV marker proteins TOM20 and TIMM44 in a 20% iodixanol concentration in Example 1.

[0031] Figure 7 The particle size of MDVs was detected by nanoparticle tracking analysis in Example 1.

[0032] Figure 8 In Example 1, RT-qPCR was used to detect the expression levels of 13 protein genes encoded by mtDNA in MDVs.

[0033] Figure 9 The expression levels of P16 and P21 proteins in EtBr-induced 293T cells were detected by ELISA in Example 2 (n=3, **: P<0.01, ***: P<0.001).

[0034] Figure 10 The relative content of mtDNA in M293T cells in Example 2 (n=3, *: P<0.05).

[0035] Figure 11 The mutation levels of the top 5 genes with the highest mtDNA mutation rates in M293T cells after treatment with DRP1-MDVs in Example 2 (n=3, *: P<0.05, **: P<0.01, ***: P<0.001, vs M293T).

[0036] Figure 12 The ATP levels of M293T cells after internalization of MDVs in each group in Example 2 are (n=3, *: P<0.05, **: P<0.01, ***: P<0.001, vs M293T).

[0037] Figure 13 The relative ROS levels of cells in each group in Example 2 are (n=3, ***: P<0.001, ns: no significant vs M293T).

[0038] Figure 14 The relative fluorescence intensity of the red-green ratio of mitochondrial membrane potential was measured using JC-1 after internalizing MDVs in each group in Example 2 (n=3, **: P<0.01, ***: P<0.001, vs M293T).

[0039] Figure 15 This is an observation of the mitochondrial network in cells after internalization of MDVs in Example 2.

[0040] Figure 16 The expression concentrations of senescence-related proteins P16 and P21 in cells after internalization of MDVs in each group in Example 2 (n=3, **: P<0.01, ns: no significant vs M293T). Detailed Implementation

[0041] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0042] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0043] 1. Experimental Materials Table 1. Cell and plasmid sources

[0044] 2. Experimental Methods 2.1 Cell resuscitation Remove the cryovials from the liquid nitrogen container and quickly place them in a 37°C water bath to thaw completely. Then centrifuge at 800 rpm for 5 minutes, discard the supernatant, and gently resuspend the cells in 1 mL of DMEM complete cell culture medium by pipetting. Add the cell suspension to a culture dish containing room-temperature complete culture medium. The complete culture medium consists of DMEM, fetal bovine serum (FBS), and penicillin-antibody in a ratio of 100:10:1. Incubate the culture dishes in a 37°C, 5% CO2 cell culture incubator.

[0045] 2.2 Cell passage and cryopreservation (1) Cell passage: Observe the cell state. When the cell density reaches 80%, discard the culture medium, rinse once with sterile PBS at room temperature, add an appropriate amount of trypsin, shake the culture dish to ensure that the trypsin is in full contact with the cells, and then place it in a 37℃ incubator for 1-2 min for digestion. Add an appropriate amount of culture medium containing 10% FBS to stop digestion, transfer the cell suspension to a 4mL centrifuge tube, centrifuge at 800rpm for 5 min at room temperature, discard the supernatant, resuspend the cells with fresh culture medium, and transfer them to a new culture dish at a ratio of 1:2. Place the newly seeded cells in a 37℃, 5% CO2 incubator for culture.

[0046] (2) Cell cryopreservation: Digest cells in good growth condition and transfer them to centrifuge tubes for centrifugation. Discard the supernatant and add an appropriate amount of cell cryopreservation solution. Mix gently with a pipette tip. Record the date, cell name and passage number and aliquot into 1.5 mL cell cryopreservation tubes. First, place them in a programmed cooling box and perform programmed cooling in an ultra-low temperature freezer at -80 ℃. After 24 h, transfer them to a liquid nitrogen tank for cryopreservation.

[0047] I. Experimental Methods 1. Construction of the DRP1 lentiviral vector 1.1 DNA Extraction According to the mammalian genomic DNA extraction kit (purchased from Beijing Zhuangmeng International Biotechnology): after completely digesting L-O2 cells with a growth confluence of 80-90% with trypsin, centrifuge, add 1 mL PBS, resuspend by pipetting, centrifuge at 10000 rpm for 1 min, and discard the supernatant. Add 250 μL of buffer A to the precipitate and vortex until completely suspended. Add 10 μL of proteinase K, mix well, then add 250 μL of buffer B, vortex to mix, and place in a 70°C oven for 10 min until the solution becomes clear. Add 250 μL of anhydrous ethanol to the solution and gently vortex for 15 s. At this point, a small amount of suspended flocculent precipitate can be observed in the solution. Transfer this precipitate to the adsorption column and place the adsorption column in a collection tube. Centrifuge at 12000 rpm for 30 s, discard the waste liquid in the collection tube, and return the adsorption column to the collection tube. Add 500 μL of buffer C to the adsorption column, centrifuge at 12000 rpm for 30 s, discard the waste liquid, add 700 μL of wash buffer W2 (wash buffer W2 needs to be diluted with anhydrous ethanol beforehand), centrifuge, and repeat this step once. Discard the waste liquid and centrifuge at 12000 rpm for 2 min. Let the adsorption column stand at room temperature for several minutes to completely dry any remaining wash buffer. Finally, the adsorption column was transferred to a clean 1.5 mL centrifuge tube, and 150 μL of elution buffer TE was added dropwise to the middle of the adsorption membrane. The adsorption column was allowed to stand at room temperature for several minutes to allow the elution buffer to fully wet the adsorption column precipitate. The column was then centrifuged at 12,000 rpm for 2 minutes. The solution obtained after centrifugation was the genomic DNA extract. After the concentration was detected by an ultra-micro UV spectrophotometer, the extract was stored at -20 °C for later use.

[0048] 1.2 PCR amplification According to NCBI DRP1 The transcript NM_001278463.2 of (Gene ID: 10059) was used for amplification, and primers were designed based on PrimerBlast. The primers were synthesized by Qingke Biotechnology, and the primer sequences are shown in Table 2. BamHI and XhoI restriction sites were introduced at both ends of the DRP1 gene. Using whole DNA from L-O2 cells as a template, PCR was performed according to the systems and reaction conditions described in Tables 3 and 4.

[0049] Table 2 PCR amplification primers

[0050] Table 3 Amplification System

[0051] Table 4 Amplification Procedure

[0052] The PCR product was subjected to 1% agarose nucleic acid electrophoresis and the gel was cut and recovered.

[0053] 1.3 Agarose gel electrophoresis (1) Electrophoresis: 0.5 g of agarose was dissolved in 50 mL of electrophoresis buffer in a microwave oven or a heated magnetic stirrer until the agarose was completely dissolved; the dissolved agarose solution was cooled to 60-70 °C, 5 μL of SYBR Safe was added and mixed; the agarose solution was poured into a gel mold, a comb was inserted to ensure that the comb was flat and free of air bubbles; it was left to stand for 10-15 minutes until the gel was completely solidified. During this period, 1x TAE electrophoresis buffer was prepared and poured into the electrophoresis tank, the gel holes of the gel plate were opposite to the negative electrode of the electrophoresis tank, and the gel plate was placed into the tank. The DRP1 gene amplification sample was mixed with 10x Loading Buffer, the DNA Marker and the mixed samples were loaded and electrophoresed at 140 V for 30 min. After electrophoresis, the gel was placed in a gel imaging system and photographed.

[0054] (2) Fragment recovery: the target gene was cut and transferred to a centrifuge tube under ultraviolet light, an equal volume of Binding Buffer was added after weighing, and the solution was treated in a 56 °C water bath for 10 min to accelerate the melting into a DNA-agarose solution. The solution was transferred to an adsorption column, left to stand for 1 min, then placed in a collection tube, centrifuged at 12000 rpm for 1 min, and the lower waste liquid was discarded. Repeat this step. Add the rinse solution WB diluted with anhydrous ethanol twice and centrifuge and discard the supernatant. Repeat this step once to remove possible residual ethanol solution. The adsorption column was fitted on the centrifuge tube, 50 μL of EB eluent was added, and the DNA was collected by centrifugation at 12000 rpm for 1 min. The DNA solution was detected using the Nucleic acid function of the ultraviolet-visible spectrophotometer. When the OD 260 / OD 280 =1.8-2.0, it indicated that the DNA purity was suitable, and the DNA concentration data was obtained, with a unit of ng / μL, which was stored at -20 °C for later use.

[0055] 1.4 Recombinant vector (1) Vector linearization: pCDH-CMV-MCS-EF1-copGFP-Puro vector (referred to as pCDH) was digested with BstX I / Not I or Nhe I / BamH I, respectively, and the reaction system is shown in Table 5. QuickCut BamH I and QuickCut Xho I were used to construct pCDH-DRP1 recombinant vector. Agarose electrophoresis and recovery were performed.

[0056] Table 5 pCDH digestion system

[0057] Gently mix the reaction system, place it in a 37 °C water bath for 1 h, and then move it to a 70 °C water bath for 10 min.

[0058] (2) Ligation: Construct pCDH-DRP1 (referred to as pDRP1) according to the instructions of TreliefTM SoSoo Cloning Kit (purchased from Genecopoeia). The ligation reaction is shown in Table 6, and incubation is performed at 50 °C for 25 min. Transformation and bacterial liquid coating experiments are performed.

[0059] Table 6 Ligation reaction system

[0060] 1.5 Extraction and identification of plasmids (1) Transformation: Take 10 μL of the ligation product to 200 μL of DH5a competent bacteria, and incubate in an ice bath for 30 min. Then transfer to a water bath at 42 °C, place for 90 s, and then place on ice for 3 min. Take 1 mL of sterile and antibiotic-free LB medium, mix with the bacterial liquid, and continue to incubate at 250 rpm and 37 °C for 1 h. After the incubation is completed, take 100 μL of bacterial liquid, centrifuge at 3000 rpm for 2 min, and inoculate the precipitated bacteria into LB culture plates containing ampicillin. Continue to culture for 24 h, and after the culture is completed, pick single colonies and inoculate into Amp-containing LB liquid medium, and incubate at 37 °C for 12 h.

[0061] (2) Colony PCR: Perform bacterial liquid PCR detection using pCDH-FP and pCDH-RP primers on pCDH (Table 7) (reaction liquid system and program are shown in Tables 8 and 9). Perform agarose nucleic acid electrophoresis detection on the PCR product, select positive colonies, and incubate at 37 °C on a shaking bed overnight.

[0062] (3) Extraction of recombinant plasmid: DNA plasmid extraction kit (DP103) (purchased from Tiangen Biochemical Technology) was used, 30 mL of fresh bacterial liquid cultured for 12-16 h was centrifuged at 4°C, 12000 rpm for 10 min; first, column equilibration operation was performed, 500 μL of equilibration solution BL was added to the adsorption column, and centrifuged at 12000 rpm for 1 min. After centrifugation of the bacterial liquid, the supernatant was discarded, and the precipitate was left. The precipitate was bacterial cells. 500 μL of P1 solution was added to the bacterial cell precipitate and mixed well; then 500 μL of P2 solution was added, and the bacterial cells were gently inverted to lyse the bacterial cells, and the bacterial liquid became clear; then 700 μL of P3 solution was added, and the bacterial cells were gently inverted until white flocculent precipitate appeared, centrifuged at 12000 rpm for 10 min, and the supernatant was collected. The supernatant was transferred to the CP3 adsorption column, and centrifuged at 12000 rpm for 1 min; then 600 μL of rinse solution PW was added to the adsorption column CP3, and centrifuged at 12000 rpm for 1 min, the waste liquid in the collection tube was discarded, and the operation was repeated once; centrifuged at 12000 rpm for 2 min, the residual rinse solution in the adsorption column was removed, and the adsorption column CP3 was placed at room temperature for several minutes to completely dry the rinse solution. Finally, an appropriate amount of elution solution EB was added to the adsorption membrane, and placed for 2 min, centrifuged at 12000 rpm for 2 min, and the solution after centrifugation was the solution containing the recombinant plasmid. After extracting the plasmid, it was sent for sequencing, and the correct sequencing result indicated that the construction was successful.

[0063] Table 7 Bacterial liquid PCR primer

[0064] Table 8 Bacterial liquid PCR reaction system

[0065] Table 9 Bacterial liquid PCR reaction program

[0066] 2, Construction of L-O2 cell strain stably overexpressing DRP1 2.1 Virus liquid preparation and collection The 293T cells were seeded in 6-well plates at a density of 4×10 6The DRP1 overexpression lentivirus vector (pCDH-DRP1) and 2 μg PAPX2 and 2 μg MD2.G plasmids were taken out, 250 μL DMEM was added, mixed, and incubated at room temperature for 5 min; 8 μL of Lipo 8000 transfection reagent was added and incubated at room temperature for another 5 min; the cell culture medium was aspirated, washed once with PBS, and then fresh complete DMEM medium was added, and the transfection mixed solution was added dropwise, and the cells were incubated in a 37°C, 5% CO2 incubator for 4 h, and then 2 mL of fresh medium was added and cultured for another 48 h; the cell supernatant was collected, filtered through a 0.22 μm filter membrane, and the residual cell debris was removed, thereby obtaining the virus solution, which was stored in a -80°C refrigerator.

[0067] 2.2 Lentivirus infection 5×10 5 L-O2 cells were seeded in a 6-well plate, and the cell density was about 70% after culture, the original culture medium was aspirated, the L-O2 cells were washed with sterile PBS for 3 times, 1 mL of virus solution was added for infection, and the final concentration of polybrene was 8 μg / mL, the whole virus was infected for 6 h, then 1 mL of fresh DMEM medium was added, and the infection was continued for 48 h, and the cell state was observed, and then the secondary infection was carried out according to the above operation.

[0068] 2.3 Screening of stable cell lines 5×10 5 The infected cells were seeded in a 6-well plate and cultured for 24 h, the next day the original culture medium was aspirated and washed with sterile PBS for 3 times, 2 mL of fresh DMEM medium containing 2 μg / mL of puromycin was added, and the screening was cultured for 14 days. Then the RNA of the stable cell line was extracted for RT-qPCR detection of overexpression efficiency or the protein of the stable cell line was extracted for Western blot detection of overexpression efficiency.

[0069] 3. qPCR detection 3.1 Extraction of total cell RNA When the cell density reaches 80%–90%, the cell culture medium is aspirated and the cells are washed three times with PBS. 1 mL of TRNsol is added evenly to the cell culture dish and repeatedly pipetted. The lysate is then transferred to an EP tube, 0.2 mL of chloroform is added, and the mixture is vigorously shaken for 15 seconds. After incubating on ice for 5 minutes, the mixture is centrifuged at 12,000 rpm for 10 minutes at room temperature. The upper aqueous phase is transferred to a new EP tube, and an equal volume of isopropanol is added. The mixture is vortexed and incubated at room temperature for 10 minutes, followed by centrifugation at 12,000 rpm for 10 minutes at 4°C. The supernatant is discarded, and the RNA is washed with 75% ethanol at 10,000 rpm for 5 minutes at 4°C. This step is repeated three times. After thorough washing, the ethanol is carefully aspirated, and an appropriate amount of DEPC water is added to dissolve the RNA. The RNA concentration is then measured for reverse transcription and stored at -80°C.

[0070] 3.2 Reverse transcription of cellular RNA RNA was processed using a reverse transcription kit (PrimeScript). TM II. Prepare reaction solution 1 and reaction solution 2 (see Tables 10 and 11) using the 1st StrandcDNA Synthesis Kit (purchased from Takara) and perform reverse transcription.

[0071] Table 10 RNA Reverse Transcription Reaction Solution 1

[0072] After the above solution was treated at 65 °C for 5 min, it was immediately transferred to ice for cooling. Then, reaction solution 2 was prepared according to Table 11.

[0073] Table 11 RNA Reverse Transcription Reaction Solution 2

[0074] After slow mixing, reverse transcription is performed by placing the mixed sample in a PCR instrument and setting the temperature to 30℃ for 10 min, 42℃ for 30 min, 95℃ for 5 min, and cooling on ice to complete the reverse transcription and obtain cDNA.

[0075] 3.3 qPCR reaction According to NCBI DRP1 Primers for RT-qPCR were designed from the transcripts (see Table 12), and the primers were synthesized by Qingke Biotechnology. 20 μL of sterile water was added to the cDNA for dilution, and a 20 μL qPCR reaction system was constructed in eight-tube sets according to the qPCR reagent instructions (see Table 13). Each sample was divided into three replicates, and qPCR was performed according to the conditions in Table 14. The resulting CT values ​​were expressed as 2... -△△Ct The analytical calculation formula is: 2-（[实验组目的基因 CT 值-实验组内参基因 CT 值]-[ 对照组目的基因 CT 值-对照组内参基因 CT 值]） Table 12 PCR primers

[0076] Table 13 qPCR reaction system

[0077] Table 14 qPCR reaction procedure

[0078] 4. Western Blot (1) After one cell passage, Western Blot detection was performed, and part of the protein lysate was extracted from the cells for Western Blot detection. First, the cells were washed with pre-cooled PBS at 4°C, each time for 1 minute, a total of 3 times, to ensure complete removal of residual liquid. Then, the lysate was prepared according to the ratio of 1 mL RIPA plus 10 μL PMSF (100 mM), and shaken and placed on ice. Then, 400 μL of lysate was added to each bottle of cells, and lysed on ice for 30 minutes, with intermittent shaking of the culture bottle to facilitate the complete reaction of the cells. After lysis, the cells were quickly scraped to one side of the culture bottle using a cell scraper, and the cell debris and lysate were transferred to a 1.5 mL EP tube using a pipette gun. Then, the EP tubes were centrifuged at 4°C at 8000 g for 10 minutes. After centrifugation, the supernatant was taken and stored at -20°C for subsequent detection.

[0079] (2) Then, the BCA protein concentration was determined. First, BSA was dissolved in PBS to prepare a series of standard samples with concentrations of 5, 2.5, 1, 0.5, 0.25, 0.125, 0.05 and 0.025 mg / mL. Then, 20 μL of each concentration of standard and total protein sample was taken and added to a 96-well plate, with two replicate wells for each standard and sample. Then, the A and B liquids of the BCA kit were mixed at a volume ratio of 50:1 to form a working solution, 200 μL of working solution was added to each well, and the 96-well plate was placed in a 37°C incubator for 30 minutes. Finally, the OD value at 562 nm was read using a microplate reader, and a standard curve was plotted according to the OD value and concentration of the standard, thereby obtaining the protein concentration of the total protein sample.

[0080] (3) Then, SDS-PAGE gel was prepared, including 15% separation gel and 5% concentration gel. First, the separation gel was injected into the gap between the glass plates to 1.5 cm from the upper edge, and an appropriate amount of 75% ethanol was added to the upper layer. After the separation gel was solidified, the ethanol in the upper layer was poured off, and the concentration gel was injected, followed by inserting the comb and naturally air-drying.

[0081] (4) Before electrophoresis, the total protein sample was heated in a 95°C water bath for 5 minutes and mixed with the protein loading buffer. Then, the electrophoresis buffer was poured into the electrophoresis tank, and 10 μL of protein Maker was added to the lanes on both sides, and 15 μL of sample was added to each lane. During electrophoresis, first use 90V voltage for 30 minutes in the concentration gel stage, and then use 160V voltage for electrophoresis in the separation gel stage until the bromophenol blue runs to the bottom of the gel.

[0082] (5) After electrophoresis, membrane transfer was performed. First, filter paper and 0.22 μm PVDF membrane of appropriate size were cut, and the PVDF membrane was activated with methanol for 1 minute. Then, the membrane transfer clamp was assembled in the order of "sponge-filter paper-gel-PVDF membrane-filter paper-sponge", and no air bubbles were ensured. After assembly, the membrane transfer clamp was inserted into the membrane transfer slot, and the membrane transfer solution was poured. Membrane transfer was performed at 200 mA constant current for 60 minutes in an ice bath.

[0083] (6) After membrane transfer, antibody incubation was performed. First, the membrane was washed with TBST solution for 5 minutes, and 5% skim milk powder (prepared with PBS solution) was used for 1 hour of blocking at room temperature. Then, the membrane was washed with TBST solution for 3 times, 5 minutes each time. Then, the primary antibody diluted with the primary antibody diluent was added, and incubated at 4°C overnight. The next day, the membrane was washed with TBST solution for 3 times, 5 minutes each time, then the secondary antibody diluted with the secondary antibody diluent was added, and incubated on a shaker at room temperature for 1 hour. Finally, the membrane was washed with TBST solution for 3 times, 5 minutes each time.

[0084] (7) Finally, luminescence detection was performed. After adding ECL luminescence solution and incubating for 3 minutes, exposure imaging was performed. Finally, the gray values of the internal reference gene and the target gene were measured using ImageJ software, and the entire Western Blot detection process was completed.

[0085] 6. Iodixanol gradient density method for extracting MDVs 6.1 Ultracentrifugation method for extracting extracellular vesicles (1) Collecting culture medium: cells were seeded into 75 cm 2Culture flask, when the cell growth density is 80-90%, collect the culture medium. After the collected culture medium is centrifuged at 300 g for 20 min at 4 ℃, the supernatant is reserved, and then the supernatant is centrifuged at 2000 g for 20 min at 4 ℃, and the supernatant is reserved and the precipitate such as dead cells is discarded. Then, the supernatant is centrifuged at 10000 g for 20 min at 4 ℃, and the supernatant obtained after this time of centrifugation can be stored in a -80 ℃ refrigerator after being sealed and labeled for subsequent large-scale extraction of extracellular vesicles.

[0086] (2) Ultra-high-speed centrifugation: The stored supernatant is thawed at 4 ℃, and then the supernatant is transferred to a centrifuge tube, and the centrifuge samples are weighed and balanced using an analytical balance, with a balance accuracy of 0.01 g. The balanced centrifuge tube is transferred to a SW70Ti rotor and placed in an adapter, covered with a lid, and the rotor is placed in a centrifuge chamber. After checking that the rotor is safely placed, the door is closed, the parameters are set, and the centrifuge is run at 100000 g for 70 min. After centrifugation, the supernatant is discarded, and the precipitate in the centrifuge tube is resuspended with PBS, and then the resuspended liquid is transferred to a 13 mL ultraclean centrifuge tube. The centrifuge tube is balanced, and then carefully transferred to a SW41Ti rotor, centrifuged at 10000 g for 70 min at 4 ℃, and the precipitate is resuspended with pre-cooled PBS and then aliquoted into sterile EP tubes, which are stored in a -80 ℃ refrigerator for subsequent experiments. Avoid repeated freezing and thawing when taking.

[0087] 6.2 Iodixanol gradient density separation of MDVs (1) Preparation of iodixanol gradient solution: Prepare the density gradient solution of iodixanol according to Table 15. The commercial OptiPrep solution is an aqueous solution containing 60% (wt / vol) iodixanol, which needs to be pre-equilibrated in a Tris-HCl buffer system before use. Dissolve 0.17 g sucrose in 2 mL 60 mM Tris-HCl pH 7.4 (final sucrose concentration = 0.25 M) to prepare solution A. Add 1 mL of solution A to 5 mL of OptiPrep to obtain 6 mL of OptiPrep working solution (OWS, equivalent to 50% iodixanol in 10 mM Tris-HCl pH 7.4). Dissolve 1.28 g sucrose in 15 mL Tris-HCl 10 mM pH 7.4 to prepare solution B (final sucrose concentration = 0.25 M).

[0088] Table 15 Configuration of iodixanol gradient concentration

[0089] Ultracentrifugation: Add 40% iodixanol solution to the centrifuge tube first, then add iodixanol with decreasing concentration on top of the solution, first 20%, then 15%, 13%, 11%, 9%, 7%. Finally, add the collected extracellular vesicle solution, balance the centrifuge tube, and place it in the SW41Ti rotor. Set the parameters: 4 ℃, 200000 g, centrifuge for 16 h. After centrifugation, the liquid of different concentration layers is divided into EP tubes. The 20% layer solution is diluted to 10 mL with sterile PBS, and then centrifuged at 4 ℃, 100,000 g for 70 min. After centrifugation, the supernatant is discarded, and the MDV precipitate is resuspended with 1 ml of pre-cooled PBS and then divided into sterile EP tubes. Store in the -80 ℃ refrigerator for subsequent experiments, and avoid repeated freezing and thawing when taking.

[0090] 6.3 Western Blot of MDVs marker protein isolated from stable overexpression cells Isolate MDVs of L-O2, Empty-L-O2 (cell strain with pCDH empty vector), and DRP1-L-O2. Group MDVs: MDVs secreted by L-O2 cells are WT-MDVs; MDVs secreted by Empty-L-O2 cells are Empty-MDVs; MDVs secreted by DRP1-L-O2 cells are DRP1-MDVs.

[0091] Determine the concentration of the collected sample by BCA method. Add loading buffer to the protein sample with a proportion (sample volume: 5x loading buffer = 5:1) and place it in a 100 ℃ metal bath for 10 min to prepare the sample. Then, detect the expression levels of MDVs markers TOMM20 and TIMM44 proteins by Western Blot.

[0092] 6.4 Nanoparticle size detection Use a 1 mL syringe to inject the extracted MDVs suspension into the sample cell, ensuring full coverage of the liquid surface. Set the parameters and adjust the focus through the microscope to make the particles clearly visible. Select the sample for particle size observation and calculation in the standard operating procedure.

[0093] 6.5 Detection of mtDNA integrity in MDVs Use PCR to detect the integrity of mtDNA in extracted MDVs. Design primers for amplification according to the sequences of 13 genes encoded by mtDNA. Including ND1, ND2, ND3, ND4, ND4L, ND5, ND6, COI, COII, COIII, ATPase8, ATPase6, CYTB.

[0094] 7. Enzyme-linked immunosorbent assay (ELISA) was used to detect the expression levels of aging proteins P16 and P21 First, the cells were treated with a lysis solution containing PMSF, and the supernatant was centrifuged and diluted. Then, standard working solution, biotinylated antibody working solution, HRP enzyme conjugate working solution and washing solution were prepared. In the enzyme-labeled plate, samples or standards were added in turn, incubated at 37°C, then discarded, biotinylated antibody working solution was added and incubated again. After washing the plate, enzyme conjugate working solution was added, incubated, then washed the plate, TMB substrate was added to develop color in the dark, finally stop solution was added, and OD value was measured at 450 nm, and sample concentration was calculated by standard curve.

[0095] 8. Statistical analysis Statistical analysis of continuous variables was expressed as "mean ± standard deviation". Statistical analysis was performed using GraphPad Prism 10.0. Independent sample t-test or one-way ANOVA was used to analyze the statistics between groups. Differences were statistically significant when p was less than 0.05.

[0096] II. Experimental results 1. Identification of pDRP1 plasmid 1.1 DRP1 PCR amplification of gene fragment DRP1 The gene size is 2178 bp, which is amplified by PCR DRP1 Gene fragment, the amplified fragment was electrophoresed by agarose gel electrophoresis, and the electrophoresis result is shown in Figure 1 The electrophoretic band position is similar to the expected gene size, indicating that the gene amplification is successful. DRP1 The nucleotide sequence of the gene is shown in SEQ ID No. 1, and the encoded amino acid sequence is shown in SEQ ID No. 2.

[0097] 1.2 Colony PCR identification of plasmid After transforming the pDRP1 plasmid into E. coli competent cells, colony lysis solution PCR was performed. Figure 2 The results of colony lysis solution PCR of transfected plasmid pDRP1 showed that pDRP1 existed in the band around 2000-3000 bp, indicating that the plasmid was successfully transformed into E. coli.

[0098] 1.3 Enzymatic identification of plasmid The constructed pDRP1 plasmid was subjected to double enzyme digestion identification. The pDRP1 plasmid was subjected to NheI and XhoI enzyme digestion identification, and the enzyme digestion result is shown in Figure 3 After pDRP1 enzyme digestion, a band appeared below 2000-3000 bp, which was consistent with DRP1 the gene size of 2178 bp. The enzyme digestion result was consistent with the expectation, indicating that the gene was successfully cloned into the plasmid pCDH.

[0099] 2. Identification of stable overexpression DRP1 L-O2 cell lines 2.1 mRNA relative expression level of DRP1 After screening stable overexpression cells, RNA of stable cell lines was extracted to detect overexpression efficiency by RT-qPCR. The results are shown in Figure 4 Compared with the control group, the mRNA level of DRP1 in stable overexpression cells was higher, which preliminarily indicated that the L-O2 cell line overexpressing DRP1 was successfully constructed. DRP1

[0100] 2.2 Protein expression level of DRP1 In order to further verify whether the stable overexpression cell line was successfully constructed, the protein of stable cell line was extracted to detect the protein expression level by ELISA. The results are shown in Figure 5 The protein expression level of DRP1 in stable overexpression cells was higher than that in the control group, which further indicated that the L-O2 cell line overexpressing DRP1 (referred to as DRP1-L-O2) was successfully constructed.

[0101] 3. Western Blot of MDVs content and marker proteins TOMM20 and TIMM44 It is known that DRP1 protein is closely related to the production and secretion of MDVs, but it is not clear which type of MDVs is promoted. Therefore, we extracted MDVs of L-O2 and DRP1-L-O2 using iodixanol density gradient centrifugation, and used the 20% iodixanol concentration layer containing the most MDVs for Western Blot analysis of MDVs outer membrane marker protein TOMM20 and mitochondrial inner membrane protein TIMM44. The results of Western Blot analysis are shown in Figure 6 The TOMM20 band of mitochondrial outer membrane of DRP1-MDVs was the lightest, but the TIMM44 band of mitochondrial inner membrane was the deepest, indicating that DRP1-L-O2 mainly produced inner membrane type MDVs composed of mitochondrial inner membrane. We found that compared with the DRP1-MDVs group, the TOMM20 and TIMM44 protein contents of the WT-MDVs group and the Empty-MDVs group were abnormally low, indicating that the MDVs produced by wild type L-O2 cells and Empty-L-O2 were extremely small.

[0102] 5. Analysis of MDVs particle size The Nanosight particle size analyzer was used to detect the diameter of extracted WT-MDVs, Empty-MDVs and DRP1-MDVs. The results are shown in Figure 7 ​As shown, the particle size range is mainly in the range of 50-200 nm, and the peak particle size is about 100 nm, which is consistent with the morphological characteristics of MDVs.

[0103] 6. Detection of mtDNA content in MDVs Studies have shown that MDVs carry certain mitochondrial contents including proteins and mtDNA, and mtDNA encodes the genes of 13 proteins related to oxidative phosphorylation. In order to explore whether the MDVs extracted in the present application contain mtDNA, RT-PCR detection of gene expression of the 13 protein genes encoded by mtDNA was performed. The results are shown in Figure 8 As shown, the nucleic acid electrophoretogram of mtDNA of the DRP1-MDVs group showed obvious bands in each lane, indicating that the inner membrane type MDVs produced by DRP1-L-O2 contained complete mtDNA. However, the content of MDVs produced by L-O2 and Empty-L-O2 cells was extremely low, and therefore the content of mtDNA in the MDVs was also extremely low and could not be detected.

[0104] Example 2. Effect of MDVs on improving the EtBr-induced mtDNA mutation to construct a cell aging model in vitro I. Experimental method 1. Cell model establishment 293T cells were inoculated in a 6-well plate containing DMEM medium containing 10% FBS and cultured at 37°C and 5% CO2. When the confluence reached 90%, the cells were washed with PBS, trypsin was added to digest the cells, and the cells were transferred to a 6-well plate containing fresh culture medium for further culture. DMEM complete medium containing 25 ng / mL EtBr was prepared. 293T cells were inoculated in a 6-well plate containing 25 ng / mL EtBr in DMEM complete medium, and cultured for 3 days to induce M293T cells. After the induction was completed, the cells were inoculated in DMEM complete medium without EtBr for amplification culture. Fresh medium was changed every 2 days, and after 5 days, subsequent experiments were performed. The principle of induction is that EtBr (ethidium bromide) selectively inhibits mitochondrial DNA replication and transcription by embedding in mtDNA double strands, leading to mitochondrial dysfunction and ultimately triggering cell aging.

[0105] The cell experiment was divided into 5 groups, namely: 293T group (Normal), 293T group after EtBr induction (M293T), wild type 293T group (WT-MDVs), empty plasmid transfection group (Empty-MDVs), and M293T group with internalized DRP1-MDVs (DRP1-MDVs). MDVs extract solution 100 ul / well was added to each group.

[0106] 2、Western blot of P16 and P21 expression levels The P16 and P21 expression levels of the cells in the Normal group and the M293T group were detected.

[0107] 3、Detection of mtDNA content in cells The cells were digested with trypsin, centrifuged at 4°C and 12000 rpm for 2 min, and the cell precipitate was collected. 250 μL of ice-cold A solution was added and blown apart. 250 μL of room-temperature B solution was added and mixed well. It was placed on ice for 6 min. 350 μL of ice-cold C solution was added and shaken until white precipitate was produced. It was placed on ice for 25 min. The supernatant was transferred to an adsorption column after centrifugation at 12000 rpm for 10 min. After standing for 5 min, the waste liquid was discarded after centrifugation at 12000 rpm for 1 min. 500 μL of column washing solution was added and centrifuged at 12000 rpm for 1 min. The residual liquid was removed by repeating the centrifugation once to obtain the mtDNA extract.

[0108] Genomic DNA was extracted according to the animal DNA extraction kit (purchased from Beijing Zhuangmeng International Biology). mtDNA was quantified by qPCR. The primers were designed according to NCBI and synthesized by Genescript. The ND1 subunit gene of NADH dehydrogenase was used to quantify mtDNA, and the β-actin gene was used to quantify nDNA. SYBR green Master Mix and the corresponding primer configuration system were added to each group for qPCR, with β-actin as the internal reference gene. The corresponding primer sequences and PCR reaction conditions are shown in Tables 16 and 17, and three independent biological repeats were performed. According to 2 -△△Ct The relative expression content of the gene was calculated.

[0109] Table 16 PCR amplification primers

[0110] Table 17 PCR reaction conditions

[0111] 4、Detection of mtDNA mutation rate in cells The mtDNA products of the M293T group and the DRP1-MDVs group were extracted, 100 μL per group, divided into 3 parallel samples, and sent to Shanghai Shenguo for sequencing. The obtained sequencing report was analyzed by gene alignment.

[0112] 5、ATP level detection Add 200 μL lysis solution to each well of a 6-well plate, and after repeated pipetting, centrifuge at 12000 g at 4°C for 5 min, take the supernatant, and use it for subsequent determination. Dissolve the reagents to be used, dilute the ATP standard solution to 0.01, 0.03, 0.1, 0.3, 1, 3 and 10 μM. Add 100 μL ATP detection working solution to a 96-well plate, and stand at room temperature for 3-5 min to eliminate background interference. Add 20 μL of the test solution or standard solution to each well, mix quickly, and then detect the chemiluminescence value with an enzyme-labeled instrument, draw a standard curve, and calculate the ATP content of each group of cells according to the luminescence value.

[0113] 6. ROS level determination Dilute DCFH-DA to 10 μM with serum-free medium. Add 100 μL of the DCFH-DA dilution to the cells to be tested after washing with PBS, and incubate at 37°C, 5% CO2 for 1 h. Wash the cells with serum-free medium 3 times, add fresh medium and continue to incubate for 1 h. Collect the cells and observe the fluorescence intensity of the cells in real time with a laser confocal microscope (EX 488 nm, EM 525 nm).

[0114] 7. Mitochondrial membrane potential detection Dilute JC-1 with ultrapure water at a ratio of 1:160, add 2 mL of JC-1 staining buffer (5x), mix well to obtain JC-1 staining working solution, and store at -20°C. Discard the medium, add the JC-1 staining working solution and incubate in the incubator for 20 min, then discard the supernatant, wash twice with pre-cooled JC-1 staining buffer (1x), add fresh culture medium, and observe and take pictures under an inverted fluorescence microscope by setting 490 nm and 525 nm excitation light, respectively. Quantitatively analyze the red-green fluorescence intensity ratio of the cells by Image J software.

[0115] 8. Fluorescent detection of mitochondrial network structure Use anhydrous DMSO to configure the Mito-Tracker Green solution into a 1 mM stock working solution, and store it at -20°C in the dark; transfer cells in good growth condition to a laser confocal culture dish at a density of 1×10 5 After 24 h of culture, remove the culture medium, add preheated Mito-Tracker Green staining working solution to 37°C, incubate for 2 h; remove the staining solution, add fresh culture medium, and observe the mitochondrial network structure using a laser confocal microscope.

[0116] 9. ELISA detection of the expression level of senescence-related proteins First, the cells were treated with a lysis solution containing PMSF, the supernatant was centrifuged and diluted. Then, the standard working solution, biotinylated antibody working solution, HRP enzyme conjugate working solution and washing solution were prepared. The sample or standard was added to the enzyme-labeled plate in turn, incubated at 37°C, then discarded, biotinylated antibody working solution was added and incubated again. After washing the plate, the enzyme conjugate working solution was added, incubated, then washed the plate, TMB substrate was added to develop color in the dark, finally the stop solution was added, and the OD value was measured at 450 nm. The sample concentration was calculated by the standard curve.

[0117] 10. Statistical analysis Statistical analysis of continuous variables was expressed as "mean ± standard deviation". Statistical analysis was performed using GraphPad Prism 10.0. Independent sample t test or one-way ANOVA was used to analyze the statistics between groups. Differences were statistically significant when p was less than 0.05.

[0118] II. Experimental results 1. Expression level of senescence marker P16, P21 protein mtDNA mutation leads to cell senescence, and P16 and P21 proteins are up-regulated during cell senescence. In order to further confirm whether the senescent cells caused by mtDNA mutation are successfully constructed, the total protein of the cells was extracted and detected by ELISA. As shown in Figure 9 , the expression levels of P16 and P21 proteins in M293T cells were significantly increased, indicating that the senescent cell model was successfully constructed.

[0119] 2. Detection of mtDNA content Since the mtDNA of MDVs without DRP1 is very little, only the DRP1-MDVs group was detected here. In order to detect the change of mtDNA content in DRP1-MDVs group after MDVs internalization into M293T, the inventors extracted the mtDNA and nDNA of the cells, quantified the mtDNA by ND1 subunit gene, β-actin quantified the nDNA by gene, and detected the relative content of mtDNA in each group of cells. As shown in Figure 10 , the mtDNA content of DRP1-MDVs group cells increased significantly, indicating that DRP1-MDVs successfully carried mtDNA into M293T cells.

[0120] 3. Detection of mtDNA mutation rate Since the mtDNA of MDVs without DRP1 is very little, only the DRP1-MDVs group was detected here. Figure 11The results show that the genes in the top 5 of mutation rate are analyzed, and compared with the M293T group, the percentage of mutant genes in normal genes in the DRP1-MDVs group is significantly reduced, including ND6 (mutation rate 28.5%→10.5%), ATP6 (mutation rate 20.2%→5.7%), COX1 (mutation rate 15.5%→4.7%), ND1 (mutation rate 10.4%→2.4%) and Cytb (mutation rate 5.7%→3.0%), which shows that DRP1-MDVs plays a repair role in mtDNA in senescent cells and can significantly reduce the mutation rate of mtDNA.

[0121] 4. Changes in ATP levels of M293T cells after internalization of MDVs in each group With cell aging, mitochondrial function gradually declines, manifested as a decrease in mitochondrial number, an increase in mtDNA mutation accumulation, and damage to the electron transport chain. These changes result in weakened ATP production capacity. MDVs, as an important way to regulate mitochondrial quality control, can regulate mitochondrial function in cells. The separated MDVs were internalized into M293T senescent cells, and the ATP levels of the cells in each group were detected, and the results are shown in Figure 12 As shown, the ATP production of the WT-MDVs group, the Empty-MDVs group and the DRP1-MDVs group increased significantly, close to that of normal 293T cells.

[0122] 5. Changes in ROS levels of M293T cells after internalization of MDVs in each group Mitochondrial function in senescent cells is weakened, leading to increased ROS production. However, with aging, the antioxidant defense capacity of the cell decreases, resulting in a decrease in ROS clearance capacity. This imbalance makes ROS more likely to accumulate, further exacerbating cell damage. By internalizing MDVs in each group into M293T, the results are shown in Figure 13 As shown, the ROS levels of the WT-MDVs group, the Empty-MDVs group and the DRP1-MDVs group were reduced, with the most obvious reduction in the DRP1-MDVs group.

[0123] 6. Mitochondrial membrane potential detection of M293T cells after internalization of MDVs in each group In senescent cells, due to the decline in oxidative phosphorylation efficiency and the excessive production of ROS, the mitochondrial membrane potential is usually significantly reduced. The present application performed JC-1 staining in M293T cells internalized with MDVs in each group, and observed the red / green relative fluorescence intensity, and the results are shown in Figure 14 As shown, in normal M293T cells, the red / green relative fluorescence intensity is high; in M293T cells, it is significantly reduced; and after treatment with DRP1-MDVs, the red / green relative fluorescence intensity is significantly increased, indicating an increase in membrane potential and a restoration of mitochondrial state.

[0124] 7. The internalization of MDVs in each group improves the mitochondrial network structure of M293T cells The health status of mitochondrial network can be evaluated by its morphology, distribution, connectivity and density. Healthy mitochondrial network usually shows continuous tubular or branched structure, while damaged mitochondria in senescent cells can show fragmentation, swelling or uneven distribution. After Mito-Tracker Green fluorescent staining of cells, the results observed under fluorescence microscopy are shown in Figure 15 As shown in the results, EtBr-induced mitochondrial fragmentation of M293T cells is severe, and the connection between branches is less. After internalization of MDVs, it is found that the improvement of mitochondrial network in DRP1-MDVs group is obvious, and the mitochondrial network is relatively complete and continuous.

[0125] 9. MDVs reduce the expression levels of senescence-related proteins P16 and P21 in cells As shown in Figure 16 , after internalization of MDVs in each group, the expression levels of senescence-related proteins P16 and P21 in DRP1-MDVs group cells decreased significantly; while compared with M293T, there was no significant difference in WT-MDVs group and Empty-MDVs group.

[0126] In summary, it is shown that stable overexpression of DRP1 gene can significantly improve the level of MDVs secretion by cells, mainly producing inner membrane type MDVs, which are rich in complete mtDNA compared with wild type cells or empty vector cells. The use of DRP1-MDVs for cell senescence treatment shows that DRP1-MDVs are successfully internalized into senescent cells, which can effectively reduce the ROS level, increase the mitochondrial mtDNA and ATP content, reduce the mtDNA mutation rate, restore the mitochondrial membrane potential and mitochondrial network structure, and ultimately reduce the expression of senescence-related proteins P16 and P21. It shows obvious mitochondrial function repair potential and anti-cell senescence effect.

Claims

1. A method for preparing DRP1-engineered mitochondrial-derived vesicles, characterized in that, First construct overexpression DRP1 Lentiviral vectors of the gene are transfected into cells to obtain stable overexpression. DRP1 The cell line containing the gene was cultured again, and the culture medium was collected for separation and extraction of mitochondrial-derived vesicles to obtain DRP1-engineered mitochondrial-derived vesicles rich in mitochondrial DNA.

2. The preparation method according to claim 1, characterized in that, The lentiviral vector is pCDH-CMV-MCS-EF1-copGFP-Puro.

3. The preparation method according to claim 1, characterized in that, The cells in question are L-O2 cells.

4. The preparation method according to claim 1, characterized in that, The separation and extraction were performed using iodixanol gradient density centrifugation.

5. DRP1-engineered mitochondrial-derived vesicles rich in mitochondrial DNA prepared by any of the preparation methods described in claims 1 to 4.

6. The use of the DRP1-engineered mitochondrial-derived vesicles rich in mitochondrial DNA as described in claim 5 in the preparation of medicaments for improving cellular senescence or treating cellular senescence-related diseases.

7. The application according to claim 6, characterized in that, The drug improves cell senescence or treats cell senescence-related diseases by internalizing MDVs rich in normal mtDNA into senescent cells, reducing the mtDNA mutation rate of senescent cells, promoting the activity of mitochondrial antioxidant enzymes to clear ROS, increasing ATP and mtDNA content, enhancing mitochondrial network generation and restoring mitochondrial function in senescent cells, and reducing the expression of senescence-related proteins P16 and P21.

8. An anti-cellular aging drug, characterized in that, The drug contains the DRP1 engineered mitochondrial-derived vesicles rich in mitochondrial DNA as described in claim 5.

9. The drug according to claim 8, characterized in that, It also contains pharmaceutically acceptable excipients.

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