Preparation method of drp1 engineered mitochondria-derived vesicles and application thereof in anti-cellular senescence

Abstract

This invention discloses a method for preparing DRP1-engineered mitochondrial-derived vesicles 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 mtDNA. The DRP1-MDVs were used for cell senescence therapy, showing successful internalization into senescent cells. This effectively reduced ROS levels, increased mitochondrial mtDNA and ATP content, decreased mtDNA mutation rate, restored mitochondrial membrane potential and mitochondrial network structure, and ultimately reduced the expression of senescence-related proteins P16 and P21. This demonstrates significant potential for mitochondrial function repair and improvement of cell senescence.

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 intracellular membrane vesicles formed by the outer mitochondrial membrane, or the contents of both the inner and outer membranes and the matrix, containing mitochondrial homologous components such as mtDNA. They possess a variety of biological functions, including maintaining mitochondrial mass, enhancing antioxidant and anti-infection capabilities, promoting intercellular communication, and regulating cellular metabolism and immune responses. These functions enable them to play a crucial role in normal physiological processes and are also closely related to the occurrence and development of various diseases. Due to their homology with mitochondria and low immunogenicity, MDVs have a natural advantage over other extracellular vesicles in targeted therapy for mitochondrial-related diseases. MDV vectors represent a novel and effective method for treating these diseases, and are significant for understanding mitochondrial biology and developing new disease treatment strategies. Delivering normal mtDNA to mtDNA-mutated senescent cells could potentially improve mitochondrial dysfunction in senescent cells, thereby achieving anti-aging effects. However, under physiological conditions, the low level of MDV secretion by cells leads to insufficient mtDNA content, hindering therapeutic efficacy. Summary of the Invention

[0005] The purpose of this invention is to overcome the problem of insufficient mtDNA content caused by the low level of MDVs secreted by cells, and to provide a method for preparing DRP1-engineered mitochondrial-derived vesicles rich in mtDNA.

[0006] A second objective of the present invention is to provide the DRP1-engineered mitochondrial-derived vesicles rich in mtDNA.

[0007] A third object of the present invention is to provide the use of the mtDNA-rich DRP1-engineered mitochondrial-derived vesicles in the preparation of medicaments for treating DNA inhibition caused by mitochondrial dysfunction.

[0008] The above-mentioned objective of this invention is achieved through the following technical solution:

[0009] This invention first provides a method for preparing DRP1-engineered mitochondrial-derived vesicles, starting by constructing an 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 (i.e., DRP1-MDVs).

[0010] The 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.

[0011] Since the formation of MDVs involves multiple proteins, this invention introduces DRP1 (Dynamics-associated protein 1). DRP1 is a GTPase primarily located in the cytoplasm, but it is recruited to the outer mitochondrial membrane to perform its function. Its fundamental function is as a core regulatory protein for mitochondrial division: by encircling and contracting the mitochondrial membrane, it "severes" mitochondria under the energy supply of GTP hydrolysis, thereby maintaining the dynamic homeostasis of the mitochondrial network. DRP1-mediated division is crucial for mitochondrial function. It provides the basis for mitophagy by clearing damaged mitochondrial fragments, ensuring the stability of mitochondrial quality and cellular energy metabolism. Dysregulation of the division process leads to mitochondrial dysfunction, affecting ATP production, calcium homeostasis, and increasing reactive oxygen species production. 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 subset whose formation requires DRP1 participation. DRP1, by regulating the mitochondrial membrane splitting sites, may provide the necessary membrane dynamics basis for mitochondrial budding and separation, 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 division and vesicle transport. This invention shows that stable overexpression... DRP1 The gene-derived cell line mainly produces inner membrane MDVs composed of the inner mitochondrial membrane, significantly increasing the level of MDV secretion by cells under normal physiological conditions, and is rich in normal and intact mtDNA compared to wild cells or empty vector cells.

[0012] Furthermore, 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 screening of exogenous genes in mammalian cells.

[0013] Furthermore, the cells are L-O2 cells. L-O2 cells are derived from normal human liver tissue and possess typical characteristics of hepatocytes, such as albumin secretion and urea synthesis. They are often used to simulate the physiological or pathological processes of normal hepatocytes.

[0014] Furthermore, the separation and extraction are performed using iodixanol gradient density centrifugation.

[0015] The present invention also provides DRP1-engineered mitochondrial-derived vesicles rich in mitochondrial DNA prepared by any of the above-described preparation methods.

[0016] This invention further applies the engineered mitochondrial-derived vesicles DRP1-MDVs to an EtBr-induced senescent cell model of mitochondrial dysfunction, and detects the antioxidant activity indicators of mitochondria and the expression of senescence-related proteins P16 and P21. The results show that the engineered mitochondrial-derived vesicles DRP1-MDVs have a therapeutic effect on senescent model cells, indicating that this invention successfully plays a role in the treatment of cellular senescence by delivering mtDNA through mitochondrial-derived vesicles, providing a new strategy for the treatment of senescence-related diseases.

[0017] Specifically, this invention incorporates the engineered mitochondrial-derived vesicles DRP1-MDVs into an EtBr-induced senescent cell model of mitochondrial dysfunction, evaluating their effects on ATP content, mitochondrial membrane potential, ROS level, and mtDNA content in the model cells. The expression levels of mitochondrial signaling pathway proteins were also detected. The results show that the engineered mitochondrial-derived vesicles DRP1-MDVs were successfully internalized into senescent cells, effectively reducing ROS levels, increasing mitochondrial mtDNA and ATP content, and decreasing the mtDNA mutation rate. This demonstrates their ability to promote mitochondrial function recovery, regulate the biological effects of various mitochondrial metabolic enzymes, and enhance their antioxidant capacity. Therefore, this invention has application value in treating cellular senescence caused by mitochondrial dysfunction or in treating cellular senescence-related diseases.

[0018] Therefore, the present invention first provides the application of the above-mentioned mtDNA-rich DRP1 engineered mitochondrial-derived vesicles in the preparation of drugs to improve cell senescence or treat cell senescence-related diseases.

[0019] Furthermore, the cellular senescence is caused by mitochondrial dysfunction resulting from mtDNA mutations.

[0020] Furthermore, the drug promotes mitochondrial oxidative phosphorylation in senescent cells by internalizing MDVs rich in normal mtDNA, regulating the activity of mitochondrial metabolic enzymes and antioxidant enzymes, clearing ROS, increasing ATP content, enhancing mitochondrial network generation and restoring mitochondrial function, and reducing the expression of senescence-related proteins P16 and P21, thereby improving cellular senescence or treating cellular senescence-related diseases.

[0021] Furthermore, the subjects were selected from mammals.

[0022] Furthermore, the mammals mentioned are selected from rats, cats, dogs, pigs, cattle, horses, sheep, monkeys, and humans.

[0023] The present invention also provides an anti-cellular aging drug, wherein the drug contains any of the above-described DRP1-engineered mitochondrial-derived vesicles rich in mitochondrial DNA.

[0024] Furthermore, the drug also includes other pharmaceutically acceptable excipients.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 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

[0027] Figure 1 The result of PCR amplification of the DRP1 gene in Example 1 (M: DNA Marker).

[0028] Figure 2 This is the colony PCR of pDRP1 in Example 1.

[0029] Figure 3 This is an image showing the enzyme digestion identification of the recombinant vector in Example 1.

[0030] Figure 4 The relative mRNA expression level of DRP1 in Example 1 (n=3, *: P<0.05).

[0031] Figure 5 The value represents the protein expression level of DRP1 in Example 1.

[0032] Figure 6 The expression levels of MDV marker proteins TOM20 and TIMM44 in a 20% iodixanol concentration in Example 1.

[0033] Figure 7 The particle size of MDVs was detected by nanoparticle tracking analysis in Example 1.

[0034] Figure 8In Example 1, RT-qPCR was used to detect the expression levels of 13 protein genes encoded by mtDNA in MDVs.

[0035] 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).

[0036] Figure 10 The relative content of mtDNA in M293T cells in Example 2 (n=3, *: P<0.05).

[0037] 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).

[0038] 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).

[0039] 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).

[0040] 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).

[0041] Figure 15 This is an observation of the mitochondrial network in cells after internalization of MDVs in Example 2.

[0042] 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

[0043] 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.

[0044] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0045] 1. Experimental materials

[0046] Table 1. Cell and plasmid sources

[0047]

[0048] 2. Experimental Methods

[0049] 2.1 Cell resuscitation

[0050] 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.

[0051] 2.2 Cell passage and cryopreservation

[0052] (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.

[0053] (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.

[0054] I. Experimental Methods

[0055] 1. Construction of the DRP1 lentiviral vector

[0056] 1.1 DNA Extraction

[0057] 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.

[0058] 1.2 PCR amplification

[0059] 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.

[0060] Table 2 PCR amplification primers

[0061]

[0062] Table 3 Amplification System

[0063]

[0064] Table 4 Amplification Procedure

[0065]

[0066] The PCR products were recovered by gel extraction after 1% agarose gel electrophoresis.

[0067] 1.3 Agarose gel electrophoresis

[0068] (1) Electrophoresis: Weigh 0.5 g of agarose and dissolve it in 50 mL of electrophoresis buffer. Heat the solution in a microwave oven or a heated magnetic stirrer until the agarose is completely dissolved. Cool the dissolved agarose solution to 60-70 °C and add 5 μL of SYBRSafe. Pour the agarose solution into a gel mold, insert a comb to ensure it is flat and free of air bubbles, and let it stand for 10-15 minutes until the gel is completely solidified. During this time, prepare 1×TAE electrophoresis buffer and inject it into the electrophoresis tank. Place the gel wells of the gel plate against the negative electrode of the electrophoresis tank. Mix the DRP1 gene amplification sample with 10×Loading Buffer, and load the DNA Marker and the mixed samples. Perform electrophoresis at 140 V for 30 min. After electrophoresis, place the gel in a gel imaging system and take a picture.

[0069] (2) Fragment recovery: The target gene was cut and transferred to a centrifuge tube under UV light. After weighing, an equal volume of Binding Buffer was added, and the mixture was melted in a 56 ℃ water bath for 10 min to accelerate the melting into a DNA-agarose solution. The solution was transferred to an adsorption column, allowed to stand for 1 min, and then placed in a collection tube. The tube was centrifuged at 12000 rpm for 1 min, and the lower layer was discarded. This step was repeated. Wash buffer diluted with anhydrous ethanol was added, and the tube was centrifuged again and the subsoil was discarded. This step was repeated once to remove any possible residual ethanol solution. The adsorption column was placed in a centrifuge tube, 50 μL of EB elution buffer was added, and the tube was centrifuged at 12000 rpm for 1 min to collect the DNA. The DNA solution was detected using the Nucleic acid function of an ultra-micro UV-Vis spectrophotometer. When the OD value was... 260 / OD 280 A concentration between 1.8 and 2.0 indicates suitable DNA purity, yielding DNA concentration data in ng / μL. Store at -20 ℃ for later use.

[0070] 1.4 Recombinant Vector

[0071] (1) Vector linearization: pCDH-CMV-MCS-EF1-copGFP-Puro vector (pCDH for short) was digested with BstXⅠ / NotⅠ or NheⅠ / BamHⅠ, respectively. The reaction systems are shown in Table 5. QuickCut BamHⅠ and QuickCutXhoⅠ were used to construct the pCDH-DRP1 recombinant vector. Agarose gel electrophoresis and recovery were performed.

[0072] Table 5 pCDH digestion system

[0073]

[0074] The reaction system was gently mixed and placed in a 37 ℃ water bath for 1 h, then moved to a 70 ℃ water bath for 10 min.

[0075] (2) Ligation: pCDH-DRP1 (pDRP1 for short) was constructed according to the Trelief™ SoSoo Cloning Kit (purchased from Qingke Biotechnology). The ligation reaction is shown in Table 6, and the mixture was incubated at 50°C for 25 min. Transformation and bacterial coating experiments were then performed.

[0076] Table 6 Connection Reaction System

[0077]

[0078] 1.5 Plasmid Extraction and Identification

[0079] (1) Transformation: 10 μL of the ligation product was added to 200 μL of DH5α competent bacterial culture and incubated on ice for 30 min. Then, it was transferred to a water bath at 42℃ and incubated for 90 s, followed by 3 min on ice. 1 mL of sterile, antibiotic-free LB medium was added and mixed with the bacterial culture. The culture was then incubated at 250 rpm and 37℃ with shaking for 1 h. After incubation, 100 μL of the bacterial culture was centrifuged at 3000 rpm for 2 min, and the precipitated bacterial cells were inoculated onto an LB plate containing ampicillin. The culture was continued for 24 h. After incubation, a single colony was picked and inoculated into LB liquid medium containing ampicillin and incubated at 37℃ for 12 h.

[0080] (2) Colony PCR: Colony PCR was performed using pCDH-FP and pCDH-RP primers (Table 7) on pCDH (the reaction system and procedure are shown in Tables 8 and 9). The PCR products were detected by agarose gel electrophoresis, positive colonies were screened, and cultured overnight at 37°C in a shaker.

[0081] (3) Recombinant plasmid extraction: Using a DNA plasmid miniprep kit (DP103) (purchased from Tiangen Biotech), 30 mL of fresh bacterial culture cultured for 12-16 h was centrifuged at 12000 rpm for 10 min at 4℃. First, column equilibration was performed by adding 500 μL of equilibration buffer BL to the adsorption column and centrifuging at 12000 rpm for 1 min. After centrifugation, the supernatant was discarded, leaving the precipitate, which consisted of 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 cells were gently inverted to lyse them, resulting in a clear bacterial solution. Subsequently, 700 μL of P3 solution was added, and the cells were gently inverted until a white flocculent precipitate appeared. The solution was centrifuged at 12000 rpm for 10 min, and the supernatant was collected. Transfer the supernatant to a CP3 adsorption column and centrifuge at 12000 rpm for 1 min. Then add 600 μL of wash buffer PW to the CP3 column, centrifuge at 12000 rpm for 1 min, discard the waste liquid in the collection tube, and repeat the operation once. Centrifuge at 12000 rpm for 2 min to remove the residual wash buffer from the adsorption column, and let the CP3 column air dry at room temperature for several minutes to completely dry the wash buffer. Finally, add an appropriate amount of elution buffer EB to the adsorption membrane, let it stand for 2 min, and centrifuge at 12000 rpm for 2 min. The solution after centrifugation is the solution containing the recombinant plasmid. After plasmid extraction, send it for sequencing. Correct sequencing results indicate successful construction.

[0082] Table 7. Primers for bacterial culture PCR

[0083]

[0084] Table 8. Bacterial PCR Reaction System

[0085]

[0086] Table 9. PCR reaction procedure for bacterial culture

[0087]

[0088] 2. Construction of L-O2 cell line stably overexpressing DRP1

[0089] 2.1 Preparation and collection of viral fluid

[0090] 293T cells were fed at a rate of 4 × 10 6Cells were seeded at a density of 100 cells / plate in 100 mm culture dishes and cultured until the cell density was approximately 80%. 3 μg of the constructed DRP1 overexpression lentiviral vector (pCDH-DRP1), 2 μg of PAPX2, and 2 μg of MD2.G plasmid were placed in sterile EP tubes, 250 μL of DMEM was added, and the mixture was incubated at room temperature for 5 min. 8 μL of Lipo 8000 transfection reagent was added, and the mixture was incubated again at room temperature for 5 min. The cell culture medium was aspirated, the cells were washed once with PBS, and fresh complete DMEM was added. The prepared transfection mixture was then added dropwise, and the cells were incubated at 37°C with 5% CO2 for 4 h. 2 mL of fresh culture medium was added, and the cells were cultured for another 48 h. The cell supernatant was collected, filtered through a 0.22 μm filter to remove residual cell debris, and the viral solution was obtained and stored at -80°C.

[0091] 2.2 Lentiviral infection

[0092] 5×10 5 L-O2 cells were cultured in 6-well plates until the cell density reached approximately 70%. The original culture medium was aspirated, and the L-O2 cells were washed three times with sterile PBS. 1 mL of virus solution was added for infection, and polybrene was added to a final concentration of 8 μg / mL. After 6 h of whole virus infection, 1 mL of freshly prepared DMEM culture medium was added, and the infection continued for 48 h. The cell status was observed, and a second infection was performed 48 h later according to the above procedure.

[0093] 2.3 Screening for stable cell lines

[0094] Inoculate 5×10⁶ cells into 6-well plates 5 Infected cells were cultured for 24 h. The following day, the original culture medium was aspirated, and the cells were washed three times with sterile PBS. 2 mL of fresh DMEM medium containing puromycin at a final concentration of 2 μg / mL was added, and the cells were cultured for 14 days for selection. Subsequently, RNA was extracted from stable cell lines for RT-qPCR to detect overexpression efficiency, or protein was extracted from stable cell lines for Western blot to detect overexpression efficiency.

[0095] 3. qPCR detection

[0096] 3.1 Total RNA extraction from cells

[0097] 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.

[0098] 3.2 Reverse transcription of cellular RNA

[0099] 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.

[0100] Table 10 RNA Reverse Transcription Reaction Solution 1

[0101]

[0102] 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.

[0103] Table 11 RNA Reverse Transcription Reaction Solution 2

[0104]

[0105] 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.

[0106] 3.3 qPCR reaction

[0107] According to NCBI DRP1Primers 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]）

[0108] Table 12 PCR Primers

[0109]

[0110] Table 13 qPCR reaction system

[0111]

[0112] Table 14 qPCR reaction procedure

[0113]

[0114] 4. Western Blot

[0115] (1) After one cell passage, Western blotting was performed, and a portion of the protein lysis buffer was extracted from the cells for Western blotting. First, the cells were washed with pre-chilled PBS at 4°C for 1 minute each time, for a total of 3 times, to ensure complete removal of residual liquid. Next, lysis buffer was prepared at a ratio of 1 mL RIPA to 10 μL PMSF (100 mM), and after shaking, it was placed on ice. Subsequently, 400 μL of lysis buffer was added to each flask of cells, and the culture flasks were lysed on ice for 30 minutes, with intermittent shaking during the process to promote full cell reaction. After lysis, the cells were quickly scraped to one side of the culture flask on ice using a cell scraper, and the cell debris and lysis buffer were transferred to 1.5 mL EP tubes using a pipette. Then, these EP tubes were centrifuged at 8000 g for 10 minutes at 4°C. After centrifugation, the supernatant was collected and stored at -20°C for subsequent detection.

[0116] (2) Subsequently, the BCA protein concentration was determined. First, BSA was dissolved in PBS to prepare a series of standards at 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 were added to a 96-well plate, with two replicates for each standard and sample. Next, solutions A and B of the BCA kit were mixed at a volume ratio of 50:1 to prepare the working solution. 200 μL of the working solution was added to each well, and the 96-well plate was incubated at 37°C for 30 minutes. Finally, the OD value at 562 nm was read using a microplate reader, and a standard curve was plotted based on the OD values ​​and concentrations of the standards to determine the protein concentration of the total protein sample.

[0117] (3) Next, SDS-PAGE gel was prepared, consisting of 15% separating gel and 5% stacking gel. First, the separating gel was poured into the gap between the glass plates to 1.5 cm from the top edge, and an appropriate amount of 75% ethanol was added to the top layer. After the separating gel solidified, the ethanol on top was poured off, and the stacking gel was poured in. Then, a comb was inserted, and the gel was allowed to air dry naturally.

[0118] (4) Before electrophoresis, heat the total protein sample in a 95°C water bath for 5 minutes and mix it with the protein loading buffer. Then, pour the electrophoresis buffer into the electrophoresis tank and add 10 μL of protein marker to each lane, and add 15 μL of sample to each lane. During electrophoresis, first use 90V for 30 minutes in the stacking gel stage, and then use 160V for the separating gel stage until the bromophenol blue reaches the bottom of the gel.

[0119] (5) After electrophoresis, the membrane was transferred. First, appropriate sizes of filter paper and a 0.22 μm PVDF membrane were cut, and the PVDF membrane was activated with methanol for 1 minute. Then, the transfer clamp was assembled in the order of "sponge-filter paper-gel-PVDF membrane-filter paper-sponge", ensuring that there were no air bubbles. After assembly, the transfer clamp was inserted into the transfer tank and the transfer buffer was poured in. The transfer was performed for 60 minutes under ice bath conditions using a constant current of 200 mA.

[0120] (6) After transfer, antibody incubation was performed. First, the membrane was washed with TBST solution for 5 minutes and then blocked with 5% skim milk powder (prepared with PBS solution) at room temperature for 1 hour. Then, the membrane was washed three times with TBST solution for 5 minutes each time. Next, primary antibody diluted with primary antibody dilution buffer was added, and the membrane was incubated overnight at 4°C. The next day, the membrane was washed three times with TBST solution for 5 minutes each time, then secondary antibody diluted with secondary antibody dilution buffer was added, and the membrane was incubated on a shaker at room temperature for 1 hour. Finally, the membrane was washed three times again with TBST solution for 5 minutes each time.

[0121] (7) Finally, luminescence detection was performed. After adding ECL luminescent 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, thus completing the entire Western Blot detection process.

[0122] 6. Extraction of MDVs using the iodixanol gradient density method

[0123] 6.1 Extraction of extracellular vesicles by ultracentrifugation

[0124] (1) Collecting culture medium: Seed cells to a height of 75 cm 2 When the cell growth density in the culture flask reaches 80-90%, collect the culture medium. Centrifuge the collected medium at 300 g for 20 min at 4 °C, retain the supernatant, and then centrifuge again at 2000 g for 20 min at 4 °C, retaining the supernatant and discarding any dead cells or other precipitates. Next, centrifuge the supernatant at 10000 g for 20 min at 4 °C. The supernatant obtained after this centrifugation can be sealed, labeled, and stored at -80 °C for subsequent large-scale extraction of extracellular vesicles.

[0125] (2) Ultracentrifugation: Thaw the stored supernatant at 4 °C, then transfer the supernatant to centrifuge tubes. Balance the centrifuged samples pairwise using an analytical balance with a balancing accuracy of 0.01 g. Transfer the balanced centrifuge tubes to an SW70Ti rotor and insert the adapter. Cover the tubes and place the rotors in the centrifuge chamber. After confirming that the rotors are safely in place, close the chamber door, set the parameters, and centrifuge at 100,000 g for 70 min. After centrifugation, discard the supernatant, resuspend the precipitate in the centrifuge tubes with PBS, and then transfer the resuspended liquid to a 13 mL ultra-clean centrifuge tube. Balance the centrifuge tubes and carefully transfer them to an SW41Ti rotor. Centrifuge at 4 °C, 10,000 g for 70 min. Discard the supernatant, resuspend the precipitate in pre-cooled PBS, and aliquot into sterile EP tubes. Store in a -80 °C freezer for subsequent experiments. Avoid repeated freeze-thaw cycles when handling the tubes.

[0126] 6.2 Iodixanol gradient density separation of MDVs

[0127] (1) Preparation of Iodixanol gradient solutions: Prepare density gradient solutions of iodixanol according to Table 15. The commercial OptiPrep solution is an aqueous solution containing 60% (wt / vol) iodixanol and needs to be pre-equilibrated in a Tris-HCl buffer system before use. Dissolve 0.17 g of sucrose in 2 mL of 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 of sucrose in 15 mL of 10 mM Tris-HCl pH 7.4 to prepare solution B (final sucrose concentration = 0.25 M).

[0128] Table 15 Preparation of Iodixanol Gradient Concentrations

[0129]

[0130] Ultracentrifugation: First, add 40% iodixanol solution to the centrifuge tube. Then, add iodixanol solutions of decreasing concentrations sequentially on top of the solution: first 20%, then 15%, 13%, 11%, 9%, and 7%. Finally, add the collected extracellular vesicle solution. Balance the centrifuge tube and place it in an SW41Ti rotor. Set the parameters to 4 °C, 200,000 g, and centrifuge for 16 h. After centrifugation, aliquot the different concentration layers into EP tubes. Make up the volume of the 20% layer solution to 10 mL with sterile PBS and centrifuge again at 4 °C, 100,000 g, for 70 min. Discard the supernatant after centrifugation. Resuspend the MDVs precipitate in 1 mL of pre-cooled PBS and aliquot into sterile EP tubes. Store at -80 °C for subsequent experiments. Avoid repeated freeze-thaw cycles during handling.

[0131] 6.3 Western Blot analysis of MDV marker proteins isolated from cells stably overexpressing MDVs

[0132] MDVs were isolated from L-O2, Empty-L-O2 (cell line transfected with pCDH empty vector), and DRP1-L-O2. MDVs were grouped as follows: MDVs secreted by L-O2 cells were WT-MDVs; MDVs secreted by Empty-L-O2 cells were Empty-MDVs; and MDVs secreted by DRP1-L-O2 cells were DRP1-MDVs.

[0133] The concentration of collected samples was determined using the BCA method. Loading buffer was added to the protein sample at the concentration to be determined at a ratio of 5:1 (sample volume: 5 × loading buffer = 5:1) and placed in a 100 °C metal bath for 10 min to complete sample preparation. Western blotting was then used to detect the protein expression levels of the MDV markers TOMM20 and TIMM44.

[0134] 6.4 Nanoparticle Size Detection

[0135] Inject the extracted MDVs suspension into the sample cell using a 1mL syringe, ensuring full coverage of the liquid surface. Set the parameters, adjust the focus of the microscope to make the particles clearly visible, and select to observe and calculate the particle size of the sample in the standard operating procedure.

[0136] 6.5 Detection of mtDNA Integrity in MDVs

[0137] The integrity of the mtDNA extracted from MDVs was detected using PCR. Primers were designed based on the 13 gene sequences encoded by the mtDNA for amplification, including ND1, ND2, ND3, ND4, ND4L, ND5, ND6, COⅠ, COⅡ, COⅢ, ATPase8, ATPase6, and CYTB.

[0138] 7. Enzyme-linked immunosorbent assay (ELISA) was used to detect the expression levels of aging proteins P16 and P21.

[0139] First, cells were treated with lysis buffer containing PMSF, centrifuged, and the supernatant was collected and diluted. Then, standard working solutions, biotinylated antibody working solutions, HRP enzyme conjugate working solutions, and washing buffer were prepared. Samples or standards were added sequentially to the ELISA plate, incubated at 37°C, and the buffer was discarded. Biotinylated antibody working solution was added and incubated again. After washing the plate, enzyme conjugate working solution was added, incubated, washed again, and TMB substrate was added for color development in the dark. Finally, stop solution was added, and the OD value was measured at 450 nm. The sample concentration was calculated using a standard curve.

[0140] 8. Statistical Analysis

[0141] Statistical analysis of continuous variables is expressed as mean ± standard deviation. GraphPad Prism 10.0 was used for statistical analysis. Independent samples t-tests or one-way ANOVA were used to analyze the statistical differences between groups. A p-value less than 0.05 was considered statistically significant.

[0142] II. Experimental Results

[0143] 1. Identification of pDRP1 plasmid

[0144] 1.1 DRP1 PCR amplification of gene fragments

[0145] DRP1 The gene size is 2178 bp, and it was amplified by PCR. DRP1 Gene fragments, amplified fragments were subjected to agarose gel electrophoresis, and the electrophoresis results are as follows: Figure 1 As shown, the position of the electrophoretic bands is close to the expected gene size, indicating that the gene amplification was 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.

[0146] 1.2 Colony PCR Identification of Plasmids

[0147] After transforming the pDRP1 plasmid into competent E. coli cells, PCR was performed on the colony lysate. Figure 2 The PCR results of colony lysates transfected with plasmid pDRP1 show that pDRP1 has a band at around 2000-3000 bp, indicating that the plasmid was successfully transfected into E. coli.

[0148] 1.3 Identification by plasmid digestion

[0149] The constructed pDRP1 plasmid was identified by double enzyme digestion. The results of NheI and XhoI digestion of the pDRP1 plasmid are shown below. Figure 3 As shown, pDRP1 enzyme digestion resulted in bands below 2000-3000 bp, consistent with... DRP1 The gene size is 2178 bp. The restriction enzyme digestion results are as expected, indicating that the gene has been successfully cloned into plasmid pCDH.

[0150] 2. Identification of L-O2 cell lines stably overexpressing DRP1

[0151] 2.1 Relative mRNA expression level of DRP1

[0152] After screening for stable overexpression cells, RNA was extracted from the stable cell lines and RT-qPCR was used to detect the overexpression efficiency. The results are as follows: Figure 4 As shown, compared with the control group, the mRNA level of DRP1 in cells with stable overexpression was higher, which preliminarily indicates that stable overexpression... DRP1 The L-O2 cell line of the gene was successfully constructed.

[0153] 2.2 DRP1 protein expression level

[0154] To further verify whether the stable overexpression cells were successfully constructed, proteins were extracted from the stable cell line and their expression levels were detected by ELISA. The results are as follows: Figure 5As shown, the protein expression level of DRP1 in the stably overexpressing cells was higher than that in the control group, further demonstrating that the L-O2 cell beads stably overexpressing DRP1 (referred to as DRP1-L-O2) were successfully constructed.

[0155] 3. Western Blot analysis of MDV content and marker proteins TOMM20 and TIMM44

[0156] DRP1 protein is known to be closely related to the production and secretion of MDVs, but it is unclear which specific type of MDV it promotes. Therefore, we extracted MDVs from L-O2 and DRP1-L-O2 layers using iodixanol density gradient centrifugation, and performed Western blotting analysis on the outer membrane marker protein TOMM20 and the inner mitochondrial membrane protein TIMM44 of MDVs using a 20% iodixanol concentration layer, which contained the highest concentration of MDVs. The Western blotting analysis is as follows: Figure 6 As shown, the TOMM20 band in the outer mitochondrial membrane of DRP1-MDVs was the lightest, while the TIMM44 band in the inner mitochondrial membrane was the darkest. This indicates that DRP1-L-O2 mainly produces inner membrane MDVs composed of the inner mitochondrial membrane. Furthermore, we found that compared with the DRP1-MDVs group, the TOMM20 and TIMM44 protein contents in the WT-MDVs group and Empty-MDVs were abnormally low, indicating that the MDV content produced by wild-type L-O2 cells and Empty-L-O2 cells was extremely low.

[0157] 5. MDVs particle size analysis

[0158] The diameters of the extracted WT-MDVs, Empty-MDVs, and DRP1-MDVs were determined using a Nanosight particle size analyzer. The results are as follows: Figure 7 As shown, the particle size range is mainly 50-200nm, with the peak particle size appearing at around 100nm, which is consistent with the morphological characteristics of MDVs.

[0159] 6. Detection of mtDNA content in MDVs

[0160] Studies have shown that MDVs carry certain mitochondrial contents, including proteins and mtDNA. The mtDNA encodes genes for 13 proteins related to oxidative phosphorylation. To investigate whether the MDVs extracted in this invention contain mtDNA, RT-PCR was used to detect the gene expression of the 13 protein genes encoded by mtDNA. The results are as follows: Figure 8As shown, the nucleic acid electrophoresis diagram of mtDNA in the DRP1-MDVs group showed obvious bands in each lane, indicating that the inner membrane type MDVs produced by DRP1-L-O2 clearly contain complete mtDNA. However, since the MDVs produced by L-O2 and Empty-L-O2 cells are extremely low, the mtDNA content in their MDVs is also abnormally low and cannot be detected.

[0161] Example 2: The effect of MDVs on improving in vitro studies of EtBr-induced mtDNA mutations in constructing cell senescence models.

[0162] I. Experimental Methods

[0163] 1. Establishment of cell model

[0164] 293T cells were seeded in 6-well plates containing DMEM medium (containing 10% FBS) and cultured at 37°C and 5% CO2. When confluence reached 90%, cells were washed with PBS, digested with trypsin, and transferred to 6-well plates containing fresh medium for further culture. Complete DMEM medium containing 25 ng / mL EtBr was prepared. 293T cells were seeded in 6-well plates containing 25 ng / mL EtBr and cultured for 3 days to induce M293T cell line formation. After induction, cells were seeded in complete DMEM medium without EtBr for scale-up culture. The medium was changed every 2 days, and subsequent experiments were performed after 5 days. The induction mechanism is as follows: EtBr (ethidium bromide) selectively inhibits mitochondrial DNA replication and transcription by intercalating into the mtDNA double strand, leading to mitochondrial dysfunction and ultimately cellular senescence.

[0165] The cell experiments were divided into 5 groups: 293T group (Normal), 293T group induced by EtBr (M293T), wild-type 293T group (WT-MDVs), group transfected with blank plasmid (Empty-MDVs), and M293T group internalized with DRP1-MDVs (DRP1-MDVs). 100 μL of MDVs extraction buffer was added to each well.

[0166] 2. Western blot analysis of expression levels of aging-related proteins P16 and P21

[0167] The expression levels of senescence-related proteins P16 and P21 were detected in cells from the Normal group and the M293T group.

[0168] 3. Detection of mtDNA content in cells

[0169] Cells were digested with trypsin using a column-based animal mitochondrial DNA extraction kit (purchased from Lianmai Biotechnology). The cells were centrifuged at 12,000 rpm for 2 min at 4°C, and the cell pellet was collected. 250 μL of solution A (ice-bathed) was added, and the pellet was dispersed. Then, 250 μL of solution B (room temperature) was added, and the mixture was stirred. The mixture was incubated on ice for 6 min. 350 μL of solution C (ice-bathed) was added, and the mixture was stirred until a white precipitate formed. The mixture was incubated on ice for 25 min. The pellet was centrifuged at 12,000 rpm for 10 min, and the supernatant was transferred to the adsorption column. After incubation for 5 min, the pellet was centrifuged at 12,000 rpm for 1 min, and the waste liquid was discarded. 500 μL of column wash buffer was added, and the pellet was centrifuged at 12,000 rpm for 1 min. The centrifugation was repeated once, and the residual liquid was removed to obtain the mtDNA extract.

[0170] Genomic DNA was extracted using an animal DNA extraction kit (purchased from Beijing Zhuangmeng International Biotechnology). mtDNA was quantified using qPCR. Primers were designed based on NCBI and synthesized by Qingke Biotechnology. mtDNA was quantified using the ND1 subunit gene of NADH dehydrogenase, and nDNA was quantified using the β-actin gene. Each group was treated with SYBR Green Master Mix and the corresponding primers for qPCR, using β-actin as an internal control gene. The corresponding primer sequences and PCR reaction conditions are shown in Tables 16 and 17. Three independent biological replicates were performed. -△△Ct Calculate the relative gene expression levels.

[0171] Table 16 PCR Amplification Primers

[0172]

[0173] Table 17 PCR Reaction Conditions

[0174]

[0175] 4. Detection of mtDNA mutation rate in cells

[0176] mtDNA products were extracted from cells in the M293T group and the DRP1-MDVs group, 100 μL per group, divided into 3 replicates, and sent to Shanghai Sangon Biotech for sequencing. Gene alignment analysis was performed on the obtained sequencing reports.

[0177] 5. ATP level detection

[0178] Add 200 μL of lysis buffer to each well of a 6-well plate, repeatedly pipet and centrifuge at 12000 g for 5 min at 4°C. Collect the supernatant for subsequent assays. Dissolve the reagents to be used and dilute the ATP standard solution to 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 µM. Add 100 µL of ATP detection working solution to each well of a 96-well plate and let it 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 detect the chemiluminescence value using a microplate reader. Plot a standard curve and calculate the ATP content of cells in each group based on the chemiluminescence value.

[0179] 6. ROS level measurement

[0180] Dilute DCFH-DA to 10 μM using serum-free medium. Add 100 μL of DCFH-DA dilution to the cells washed with PBS and incubate at 37°C and 5% CO2 for 1 h. Wash the cells three times with serum-free medium and incubate for another 1 h with fresh medium. Collect the cells and observe the fluorescence intensity in real time using a laser confocal microscope (EX 488 nm, EM 525 nm).

[0181] 7. Mitochondrial membrane potential detection

[0182] JC-1 was diluted with ultrapure water at a ratio of 1:160, and 2 mL of JC-1 staining buffer (5×) was added. The mixture was then stirred to obtain the JC-1 staining working solution for later use. The culture medium was discarded, and the JC-1 staining working solution was added and incubated in an incubator for 20 min. After incubation, the supernatant was discarded, and the cells were washed twice with pre-cooled JC-1 staining buffer (1×). Fresh culture medium was then added, and the cells were observed and photographed under an inverted fluorescence microscope with excitation light of 490 nm and 525 nm, respectively. The ratio of red to green fluorescence intensity of the cells was quantitatively analyzed using ImageJ software.

[0183] 8. Fluorescence detection of mitochondrial network structure

[0184] Prepare a 1 mM stock working solution of Mito-Tracker Green solution using anhydrous DMSO and store at -20°C protected from light; inoculate well-grown cells at 1×10⁻⁶ mM. 5 The density was transferred to a laser confocal culture dish, and after culturing for 24 h, the culture medium was removed, and Mito-Tracker Green staining working solution preheated to 37°C was added. After incubation for 2 h, the staining solution was removed, fresh culture medium was added, and the mitochondrial network structure was observed using a laser confocal microscope.

[0185] 9. Enzyme-linked immunosorbent assay (ELISA) to detect the expression levels of aging-related proteins.

[0186] First, cells were treated with lysis buffer containing PMSF, centrifuged, and the supernatant was collected and diluted. Then, standard working solutions, biotinylated antibody working solutions, HRP enzyme conjugate working solutions, and washing buffer were prepared. Samples or standards were added sequentially to the ELISA plate, incubated at 37°C, and the buffer was discarded. Biotinylated antibody working solution was added and incubated again. After washing the plate, enzyme conjugate working solution was added, incubated, washed again, and TMB substrate was added for color development in the dark. Finally, stop solution was added, and the OD value was measured at 450 nm. The sample concentration was calculated using a standard curve.

[0187] 10. Statistical Analysis

[0188] Statistical analysis of continuous variables is expressed as mean ± standard deviation. GraphPad Prism 10.0 was used for statistical analysis. Independent samples t-tests or one-way ANOVA were used to analyze the statistical differences between groups. A p-value less than 0.05 was considered statistically significant.

[0189] II. Experimental Results

[0190] 1. Expression levels of aging markers P16 and P21 proteins

[0191] mtDNA mutations lead to cellular senescence, and the expression of P16 and P21 proteins is upregulated during cellular senescence. To further confirm the successful construction of senescent cells induced by mtDNA mutations, total protein was extracted from the cells and analyzed by ELISA. The results are as follows: Figure 9 As shown, the expression levels of P16 and P21 proteins in M293T cells were significantly increased, indicating that the senescent cell model was successfully constructed.

[0192] 2. Detection of mtDNA content

[0193] Since MDVs not converted to DRP1 contain very little mtDNA, only the DRP1-MDVs group was tested here. To detect changes in mtDNA content after MDVs in the DRP1-MDVs group were internalized to M293T, this invention extracts mtDNA and nDNA from the cells and uses... ND1 Subunit gene quantification of mtDNA, β-actin Gene quantification of nDNA and detection of the relative content of mtDNA in cells of each group are shown in the following results. Figure 10 As shown, the mtDNA content in the DRP1-MDVs group cells increased significantly, indicating that DRP1-MDVs successfully carried mtDNA into M293T cells.

[0194] 3. Detection of mtDNA mutation rate

[0195] Since there is very little mtDNA in MDVs that have not been converted to DRP1, we only tested the DRP1-MDVs group here. Figure 11 The results showed that, compared with the M293T group, the percentage of mutated genes among normal genes in the DRP1-MDVs group was 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%). This indicates that DRP1-MDVs play a repair role in mtDNA in senescent cells and can significantly reduce the mutation rate of mtDNA.

[0196] 4. Changes in ATP levels after MDVs internalized M293T cells in each group

[0197] As cells age, mitochondrial function gradually declines, manifested as a decrease in mitochondrial number, an increase in the accumulation of mtDNA mutations, and damage to the electron transport chain. These changes lead to a weakened ATP production capacity. MDVs, as an important mechanism for regulating mitochondrial quality control, can modulate mitochondrial function. Isolated MDVs were internalized into M293T senescent cells, and ATP levels in each group were measured. The results are as follows: Figure 12 As shown, ATP production in the WT-MDVs group, Empty-MDVs group, and DRP1-MDVs group was significantly increased, approaching that of normal 293T cells.

[0198] 5. Changes in ROS levels after M293T cells were internalized by MDVs in each group

[0199] In senescent cells, weakened mitochondrial function leads to increased ROS production. However, with aging, cells' antioxidant defense capabilities decline, resulting in a weakened ability to scavenge ROS. This imbalance makes ROS more likely to accumulate, further exacerbating cell damage. Internalizing MDVs from each group to M293T yielded the following results: Figure 13 As shown, the ROS levels in the WT-MDVs group, Empty-MDVs group, and DRP1-MDVs group were all reduced, with the DRP1-MDVs group showing the most significant reduction in ROS levels.

[0200] 6. Detection of mitochondrial membrane potential after MDVs internalize M293T cells in each group

[0201] In senescent cells, mitochondrial membrane potential is typically significantly reduced due to decreased efficiency of oxidative phosphorylation and excessive ROS production. In this invention, JC-1 staining was performed on M293T cells with internalized MDVs in each group, and the relative red / green fluorescence intensity was observed. The results are as follows: Figure 14As shown, the relative fluorescence intensity of red / green is high in normal M293T cells, but significantly lower in M293T cells. After treatment with DRP1-MDVs, the relative fluorescence intensity of red / green is significantly enhanced, indicating an increase in membrane potential and suggesting that the mitochondrial state has recovered.

[0202] 7. Internalization of MDVs in each group led to an improvement in the mitochondrial network structure of M293T cells.

[0203] The health of the mitochondrial network can be assessed through its morphology, distribution, connectivity, and density. A healthy mitochondrial network typically exhibits a continuous tubular or branching structure, while damaged mitochondria in senescent cells may appear fragmented, swollen, or unevenly distributed. This invention, after staining cells with Mito-Tracker Green fluorescence, observes the following under fluorescence microscopy: Figure 15 The results showed that EtBr-induced mitochondrial fragmentation in M293T cells was severe, with few connections between branches. After internalizing MDVs, the mitochondrial network in the DRP1-MDVs group was significantly improved, and the mitochondrial network was more complete and continuous.

[0204] 9. MDVs reduce the expression levels of cellular senescence-related proteins P16 and P21.

[0205] like Figure 16 After internalization of MDVs in each group, the expression levels of aging-related proteins P16 and P21 in cells of the DRP1-MDVs group decreased significantly; while the WT-MDVs group and the Empty-MDVs group showed no significant difference compared with M293T.

[0206] In summary, this demonstrates stable overexpression. DRP1 The gene significantly increased the level of MDV secretion in cells, mainly producing inner membrane MDVs, which were rich in intact mtDNA compared to wild-type cells or empty vector cells. Using DRP1-MDVs for cell senescence therapy showed that DRP1-MDVs were successfully internalized into senescent cells, effectively reducing ROS levels, increasing mitochondrial mtDNA and ATP content, reducing mtDNA mutation rate, restoring mitochondrial membrane potential and mitochondrial network structure, and ultimately reducing the expression of senescence-related proteins P16 and P21. This demonstrates significant potential for mitochondrial function repair and anti-cellular senescence effects.

Claims

1. A method for preparing DRP1-engineered mitochondrial-derived vesicles, characterized in that, First, a lentiviral vector overexpressing the DRP1 gene was constructed and transfected into cells to obtain a cell line stably overexpressing the DRP1 gene. Then, the cell line was cultured, 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. The cells were L-O2 cells. The separation and extraction were performed using iodixanol gradient density centrifugation. 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.

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

3. DRP1-engineered mitochondrial-derived vesicles rich in mitochondrial DNA prepared by any of the preparation methods described in claims 1 to 2.

4. The use of the DRP1-engineered mitochondrial-derived vesicles rich in mitochondrial DNA as described in claim 3 in the preparation of a drug for improving cellular senescence, wherein the cells are 293T cells.

5. The application according to claim 4, characterized in that, The drug improves cell senescence 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.

6. An anti-cellular aging drug, characterized in that, The drug contains DRP1 engineered mitochondrial-derived vesicles rich in mitochondrial DNA as described in claim 3, and the cells are 293T cells.

7. The drug according to claim 6, characterized in that, It also contains pharmaceutically acceptable excipients.

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