Mfn1 engineered mitochondrial vesicle and application thereof in preparation of anti-cell aging drugs

Abstract

This invention discloses an MFN1-engineered mitochondrial vesicle and its application in the preparation of anti-cellular aging drugs. The MFN1-engineered mitochondrial vesicle is first constructed by... MFN1 Gene overexpression vectors are used to transfect cells and obtain overexpression. MFN1 Cell lines containing the gene were cultured, culture medium was collected, and mitochondrial vesicles were isolated and extracted to obtain MFN1-MDVs, engineered mitochondrial vesicles rich in mtDNA. MFN1-MDVs were used to treat senescent cells caused by mitochondrial dysfunction. Results showed that MFN1-MDVs could be internalized into senescent cells, effectively increasing mitochondrial mtDNA and ATP content, enhancing oxidative phosphorylation levels, increasing antioxidant enzyme activity and reducing ROS, restoring mitochondrial membrane potential, and ultimately improving mitochondrial network structure, demonstrating significant potential for mitochondrial function repair and improvement of cell senescence.

Description

An MFN1 engineered mitochondrial vesicle and its application in the preparation of anti-cellular aging drugs Technical Field

[0001] This invention belongs to the field of biotechnology, and more specifically, relates to the application of MFN1 engineered mitochondrial vesicles in anti-cellular aging drugs. Background Technology

[0002] Mitochondria contain a small amount of DNA called mitochondrial DNA (mtDNA). mtDNA differs in structure from nuclear DNA (nDNA). It is a closed circular double-stranded DNA composed of 16,569 base pairs, encoding 37 genes (including 13 proteins and some RNA). Because mtDNA lacks the protection of histones, it is more susceptible to damage and mutation.

[0003] mtDNA mutations are a key factor in inducing 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 the decline of cellular physiological function, tissue dysfunction, and the development of various age-related diseases. On the one hand, the accumulation of mtDNA mutations can lead to mitochondrial respiratory chain dysfunction, increasing the production of reactive oxygen species (ROS). Simultaneously, the ROS scavenging capacity of senescent cell mitochondria is significantly reduced, making mtDNA more susceptible to ROS attack and oxidative damage, thereby triggering oxidative stress. This not only damages the structure and function of mitochondria but also activates intracellular stress response pathways (such as the p53-p21 pathway), further inducing cell cycle arrest and accelerating the senescence process. On the other hand, mtDNA mutations may also lead to a decrease in mitochondrial membrane potential and energy metabolism imbalance, ultimately affecting normal cellular physiological function. Therefore, targeted interventions against mtDNA mutations and cellular senescence hold promise for providing new strategies to delay aging and prevent age-related diseases. In recent years, studies have reported that mitochondrial transplantation can increase the number of mitochondria in damaged cells and deliver normal mtDNA to damaged cells, thereby reducing the heterogeneity of damaged cells and improving their mitochondrial function. However, mitochondrial transplantation has problems such as the short duration of mitochondrial activity after in vitro isolation and the possibility of triggering an immune response.

[0004] Mitochondrial-derived vesicles (MDVs) are vesicles directly formed and secreted by mitochondria, containing mitochondrial homologous components such as mtDNA. As intercellular communication mediators, MDVs participate in regulating oxidative stress and mitochondrial function. Delivering MDVs from normal cells to senescent cells may improve mitochondrial dysfunction in senescent cells. Due to their homology with mitochondria and their natural advantages such as low immunogenicity, MDVs exhibit unique natural superiority over other extracellular vesicles in targeted therapy for mitochondrial-related diseases. If normal mtDNA could be delivered to mtDNA-mutated senescent cells, it might improve mitochondrial dysfunction in senescent cells, thereby achieving anti-aging. However, under normal physiological conditions, MDVs form at a low level to maintain mitochondrial homeostasis, making it difficult to deliver normal mtDNA for treatment. Summary of the Invention

[0005] To address the technical problem that mitochondrial vesicles (MDVs) form at low levels under normal physiological conditions, maintaining mitochondrial homeostasis and making it difficult to deliver normal mtDNA for therapeutic purposes, this invention first provides mtDNA-rich MFN1-engineered mitochondrial vesicles (MFN1-MDVs). These mtDNA-rich MFN1-engineered mitochondrial vesicles are obtained by first constructing an MFN1 gene overexpression vector, transfecting cells to obtain a cell line overexpressing the MFN1 gene, culturing the cells, collecting the culture medium, and isolating and extracting the mitochondrial vesicles to obtain MFN1-MDVs rich in normal mtDNA.

[0006] The formation of mitochondrial vesicles (MDVs) involves multiple proteins. The mitochondrial fusion protein 1 (MFN1) introduced in this invention is a GTPase located on the outer mitochondrial membrane and is one of the core proteins regulating mitochondrial homeostasis. Its basic function is to mediate mitochondrial membrane fusion: through interaction with other MFN1 or MFN2 proteins on other mitochondria, it promotes the fusion of two independent mitochondria into a larger network structure, which is crucial for maintaining the morphology, distribution, and functional integrity of mitochondria. For mitochondrial function, the MFN1-mediated fusion process directly affects its energy metabolism and stability. A complete mitochondrial network helps balance membrane potential, optimize oxidative phosphorylation efficiency, and promote stable ATP production. When MFN1 function is abnormal, it leads to mitochondrial fragmentation, resulting in loss of membrane potential, decreased energy production, and accumulation of reactive oxygen species (ROS), thereby inducing apoptosis. MFN1 plays a key regulatory role in the generation of mitochondrial-derived vesicles (MDVs). MDVs are small vesicles formed by mitochondria under stress conditions to clear damaged components or to perform specific signal transduction. This study shows that cell lines that stably overexpress the MFN1 gene mainly produce inner membrane MDVs composed of the mitochondrial inner membrane, which can significantly increase the level of MDV secretion by cells under normal physiological conditions, and are richer in normal and intact mtDNA compared to wild cells or empty vector cells.

[0007] Specifically, the amino acid composition of MFN1 encoded by the MFN1 gene is shown in SEQ ID No. 2.

[0008] Preferably, the nucleotide sequence of the MFN1 gene is shown in SEQ ID No. 1.

[0009] Preferably, the base vector of the overexpression vector is pCDH-CMV-MCS-EF1-copGFP-Puro (abbreviated as pCDH). The overexpression vector of the MFN1 gene is constructed by introducing the MFN1 gene into the multiple cloning site of the pCDH vector.

[0010] Preferably, the cells are L-O2 cells, which are derived from normal human liver tissue and have typical characteristics of hepatocytes, such as secreting albumin and synthesizing urea. They are often used to simulate the physiological or pathological processes of normal hepatocytes.

[0011] As a preferred embodiment, the present invention constructs pCDH-MFN1, then infects L-O2 cells, screens MFN1-L-O2, and then cultures them. The culture medium is collected, and MDVs are centrifuged with iodixanol at a gradient density to obtain MFN1 engineered mitochondrial vesicles rich in normal mtDNA.

[0012] This invention applies engineered mitochondrial vesicles MFN1-MDVs, rich in normal mtDNA, to an EtBr-induced senescent cell model of mitochondrial dysfunction. The antioxidant activity indicators and mitochondrial signaling pathway protein expression in the cells were detected. The results showed that the engineered mitochondrial vesicles MFN1-MDVs have a therapeutic effect on senescent model cells. This invention successfully uses mitochondrial-derived vesicles as mtDNA delivery vectors to exogenously deliver normal mtDNA into the mitochondria of senescent cells, achieving therapeutic effects.

[0013] Specifically, this invention incorporates the engineered mitochondrial vesicles MFN1-MDVs into an EtBr-induced senescent cell model of mitochondrial dysfunction, evaluating their effects on ATP content, mitochondrial membrane potential, ROS level, cellular oxygen consumption rate (OCR) 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 vesicles MFN1-MDVs rich in normal mtDNA constructed in this invention can be internalized into senescent cells, effectively reducing ROS levels, increasing mitochondrial mtDNA and ATP content, reducing mtDNA mutation rate, significantly promoting mitochondrial oxidative phosphorylation, promoting the expression of antioxidant enzymes in animal cells, and improving mitochondrial network structure. This has the effect of promoting mitochondrial functional recovery, regulating the biological effects of various mitochondrial metabolic enzymes, and enhancing their antioxidant capacity, thus having application value in the treatment of cell senescence caused by mtDNA mutations.

[0014] Therefore, the present invention also provides the use of the above-mentioned MFN1 engineered mitochondrial vesicles rich in normal mtDNA in the preparation of drugs to improve cell senescence or treat cell senescence-related diseases.

[0015] Specifically, the cellular senescence is caused by mtDNA mutations.

[0016] Preferably, the drug promotes the activity of mitochondrial antioxidant enzymes in senescent cells to clear ROS, increases ATP and mtDNA content, reduces the mtDNA mutation rate in senescent cells, enhances the generation of mitochondrial networks and restores mitochondrial function in senescent cells, and reduces the expression of senescence-related proteins P16 and P21, thereby improving cell senescence or treating cell senescence-related diseases.

[0017] The present invention also provides an anti-cellular aging drug, wherein the drug contains any of the above-described MFN1 engineered mitochondrial vesicles rich in normal mtDNA.

[0018] Preferably, the drug further includes other pharmaceutically acceptable excipients.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] This invention first constructs an overexpression vector for the MFN1 gene, transfects cells to obtain a cell line overexpressing the MFN1 gene, cultures the cells, collects the culture medium, and isolates and extracts mitochondrial vesicles to obtain engineered MFN1-MDVs rich in normal mtDNA, overcoming the problem of insufficient MDV secretion under normal physiological conditions. Furthermore, these engineered mitochondrial vesicles MFN1-MDVs are used to treat cell senescence caused by mtDNA mutations. Results show that MFN1-MDVs can be internalized into senescent cells, effectively reducing ROS levels, increasing mitochondrial mtDNA and ATP content, reducing the mtDNA mutation rate, significantly promoting mitochondrial oxidative phosphorylation in senescent cells, promoting the expression of cellular antioxidant enzymes, and improving mitochondrial network structure, thus promoting mitochondrial function recovery and improving and treating cell senescence. This invention successfully utilizes mitochondrial-derived vesicles as an mtDNA delivery vector to exogenously deliver normal mtDNA into the mitochondria of senescent cells, reducing the proportion of mutant mtDNA in senescent cells, thereby achieving cell anti-aging. Attached Figure Description

[0021] Figure 1 shows the PCR amplification results of the MFN1 gene in Example 1 (M: DNA Marker).

[0022] Figure 2 shows the colony PCR of pMFN1 in Example 1.

[0023] Figure 3 shows the enzyme digestion identification diagram of the recombinant vector in Example 1.

[0024] Figure 4 shows the relative mRNA expression level of MFN1 in Example 1 (n=3, **: p<0.01).

[0025] Figure 5 shows the protein expression level of MFN1 in Example 1.

[0026] Figure 6 shows the expression levels of MDV marker proteins TOM20 and TIMM44 at a 20% iodixanol concentration in Example 1. (n=3, ns: p>0.05, *: p<0.05, ****: p<0.0001).

[0027] Figure 7 shows the particle size of MDVs detected by nanoparticle tracking analysis in Example 1.

[0028] Figure 8 shows the expression levels of 13 genes encoded by mtDNA in MDVs detected by RT-PCR in Example 1.

[0029] Figure 9 shows the Western blot analysis of the expression levels of P16 and P21 proteins in 293T cells induced by EtBr in Example 2.

[0030] Figure 10 shows the results of M293T cells taking up MDVs at 24, 48, 72 and 96 h in Example 2.

[0031] Figure 11 shows the relative level of mtDNA in M293T cells in Example 2 (n=3, *: p<0.05).

[0032] Figure 12 shows the top 5 gene mutation levels of mtDNA mutation rate in M293T cells after treatment with MFN1-MDVs in Example 2 (n=3, *: p<0.05, **: p<0.01, vs M293T).

[0033] Figure 13 shows the ATP levels of cells after internalizing MDVs in each group in Example 2 (n=3, *: p<0.05, **: p<0.01, ***: p<0.001, vs M293T).

[0034] Figure 14 shows the relative ROS levels of cells in each group in Example 2 (n=3, ***: p<0.001, ns: no significant vs M293T).

[0035] Figure 15 shows the relative mitochondrial membrane potential detected in JC-1 cells after internalization of MDVs in each group in Example 2 (n=3, *: p<0.05, **: p<0.01, ***: p<0.001, ns: no significant vs M293T).

[0036] Figure 16 shows the mitochondrial network observation in cells after internalizing MDVs in each group in Example 2.

[0037] Figure 17 shows the expression concentrations of aging-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

[0038] 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.

[0039] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0040] 1. Experimental Materials

[0041] Table 1. Cell and plasmid sources

[0042]

[0043] 2. Experimental Methods

[0044] 2.1 Cell resuscitation

[0045] 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.

[0046] 2.2 Cell passage and cryopreservation

[0047] (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.

[0048] (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.

[0049] Example 1: Preparation of mtDNA-rich MFN1-engineered mitochondrial vesicles and isolation and characterization of MDVs

[0050] I. Experimental Methods

[0051] 1. Construction of the MFN1 lentiviral vector

[0052] 1.1 DNA Extraction

[0053] 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.

[0054] 1.2 PCR amplification

[0055] Primers for amplification were designed based on the MFN1 gene (Gene ID: 55669, transcript: XM_005247596.5) from NCBI and PrimerBlast. The primers were synthesized by Qingke Biotechnology, and their sequences are shown in Table 2. NheⅠ and XhoⅠ restriction enzyme sites were introduced at both ends of the MFN1 gene. PCR was performed using whole DNA from L-O2 cells as a template, following the systems and reaction conditions described in Tables 3 and 4.

[0056] Table 2 PCR amplification primers

[0057]

[0058] Table 3 Amplification System

[0059]

[0060] Table 4 Amplification Procedure

[0061]

[0062] The PCR products were recovered by gel extraction after 1% agarose gel electrophoresis.

[0063] 1.3 Agarose gel electrophoresis

[0064] (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, and ensure that the comb is flat and free of air bubbles. 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 MFN1 gene amplification sample with 10×Loading Buffer. 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 pictures for recording.

[0065] (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.

[0066] 1.4 Recombinant Vector

[0067] (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-MFN1 recombinant vector. Agarose gel electrophoresis and recovery were performed.

[0068] Table 5 pCDH digestion system

[0069]

[0070] 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.

[0071] (2) Ligation: pCDH-MFN1 (pMFN1 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.

[0072] Table 6 Connection Reaction System

[0073]

[0074] 1.5 Plasmid Extraction and Identification

[0075] (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.

[0076] (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.

[0077] (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. Let the CP3 column air dry at room temperature for several minutes to completely dry the wash buffer. Finally, add an appropriate amount of eluent EB to the adsorption membrane, let stand for 2 min, and centrifuge at 12000 rpm for 2 min. The solution after centrifugation is the solution containing the recombinant plasmid.

[0078] Table 7. Primers for bacterial culture PCR

[0079]

[0080] Table 8. Bacterial PCR Reaction System

[0081]

[0082] Table 9. PCR reaction procedure for bacterial culture

[0083]

[0084] 2. Construction of L-O2 cell lines stably overexpressing MFN1

[0085] 2.1 Preparation and collection of viral fluid

[0086] 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 overexpressing lentiviral vector, 2 μg of PAPX2, and 2 μg of MD2.G plasmid were placed in sterile EP tubes, and 250 μL of DMEM was added and mixed thoroughly. 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 and filtered through a 0.22 μm filter to remove residual cell debris, yielding the viral solution, which was then stored at -80°C.

[0087] 2.2 Lentiviral infection

[0088] 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.

[0089] 2.3 Screening for stable cell lines

[0090] 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.

[0091] 3. qPCR detection

[0092] 3.1 Total RNA extraction from cells

[0093] 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.

[0094] 3.2 Reverse transcription of cellular RNA

[0095] RNA was processed using a reverse transcription kit (PrimeScript). TM II. Prepare reaction solution 1 and reaction solution 2 (see Tables 10 and 11) according to the instructions of the 1st Strand cDNA Synthesis Kit (purchased from Takara) and perform reverse transcription.

[0096] Table 10 RNA Reverse Transcription Reaction Solution 1

[0097]

[0098] 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.

[0099] Table 11 RNA Reverse Transcription Reaction Solution 2

[0100]

[0101] 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.

[0102] 3.3 qPCR reaction

[0103] Primers for RT-PCR were designed based on the MFN1 sequence from NCBI (see Table 12), and 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 bundles 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]）

[0104] Table 12 qPCR Primers

[0105]

[0106] Table 13 qPCR reaction system

[0107]

[0108] Table 14 qPCR reaction procedure

[0109] 4. Western Blot

[0110] (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.

[0111] (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.

[0112] (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.

[0113] (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.

[0114] (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.

[0115] (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.

[0116] (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.

[0117] 6. Extraction of MDVs using the iodixanol gradient density method

[0118] 6.1 Extraction of extracellular vesicles by ultracentrifugation

[0119] (1) Collection of culture medium: Seed cells into 75 cm2 culture flasks. When the cell growth density is 80-90%, collect the culture medium. Centrifuge the collected culture medium at 300 g for 20 min at 4 ℃, retain the supernatant, and then centrifuge at 2000 g for 20 min at 4 ℃, retain the supernatant, and discard dead cells and other precipitates. Then, centrifuge the supernatant at 10000 g for 20 min at 4 ℃. The supernatant obtained after this centrifugation can be sealed and labeled and stored in a -80 ℃ freezer for subsequent large-scale extraction of extracellular vesicles.

[0120] (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.

[0121] 6.2 Iodixanol gradient density separation of MDVs

[0122] (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).

[0123] Table 15 Preparation of Iodixanol Gradient Concentrations

[0124]

[0125] 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.

[0126] 6.3 Western Blot analysis of MDV marker proteins isolated from cells stably overexpressing MDVs

[0127] MDVs were isolated from L-O2, Empty-L-O2, and MFN1-L-O2 cells. 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 MFN1-L-O2 cells were MFN1-MDVs.

[0128] 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.

[0129] 6.4 Detection of mtDNA Integrity in MDVs

[0130] We used PCR to test the integrity of the mtDNA extracted from MDVs, and designed primers for amplification based on the 13 gene sequences encoded by the mtDNA. These include ND1, ND2, ND3, ND4, ND4L, ND5, ND6, COⅠ, COⅡ, COⅢ, ATPase8, ATPase6, and CYTB.

[0131] 6.5 nm particle size detection

[0132] 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.

[0133] II. Experimental Results

[0134] 1. Amplification of the MFN1 target gene

[0135] Figure 1 shows the PCR electrophoresis results of the MFN1 gene, which exhibit an extended band at 2000-3000 bp, consistent with the theoretical size of the MFN1 gene (2226 bp), indicating successful gene amplification. The nucleotide sequence of the MFN1 gene is shown in SEQ ID No. 1, and the encoded amino acid sequence is shown in SEQ ID No. 2.

[0136] 2. Colony PCR identification of plasmids

[0137] After transforming the pMFN1 plasmid into competent E. coli cells, PCR was performed on the colony lysate. Figure 2 shows the PCR results of the colony lysate transfected with the pMFN1 plasmid. A band of pMFN1 was observed at approximately 2000-3000 bp, indicating that the plasmid was successfully transformed into E. coli.

[0138] 3. Identification by plasmid digestion

[0139] The constructed pMFN1 plasmid was identified by double enzyme digestion. The digestion results of NheI and XhoI on the pMFN1 plasmid are shown in Figure 3. After digestion, bands appeared in the 2000-3000 bp range, consistent with the MFN1 gene size of 2226 bp. The digestion results were as expected, indicating that the gene was successfully cloned into the pCDH plasmid.

[0140] 4. Identification of L-O2 cell lines stably overexpressing MFN1

[0141] 4.1 Relative mRNA expression level of MFN1

[0142] After screening for stable overexpressing cells, RNA was extracted from the stable cell lines for RT-qPCR to detect the overexpression efficiency. The results are shown in Figure 4. Compared with the control group, the MFN1 mRNA level in the stable overexpressing cells was higher, which preliminarily indicates that the L-O2 cell line with stable MFN1 gene overexpression was successfully constructed.

[0143] 4.2 Protein expression level of MFN1

[0144] To further verify whether the stable overexpression cells were successfully constructed, we also extracted proteins from the stable cell line and performed Western blot analysis to detect protein expression levels. The results are shown in Figure 5. The MFN1 protein expression level in the stable overexpression cells was higher than that in the control group, further demonstrating the successful construction of stable MFN1 overexpressing L-O2 cell beads (hereinafter referred to as MFN1-L-O2).

[0145] 5. Western Blot analysis of MDV content and marker proteins TOMM20 and TIMM44

[0146] It is known that MFN1 protein is closely related to the production and secretion of MDVs, but it is unclear which type of MDV it specifically promotes. Therefore, we extracted MDVs from L-O2 and MFN1-L-O2 cells using iodixanol density gradient centrifugation, and performed Western blot analysis on the 20% iodixanol concentration layer (containing the highest concentration of MDVs) for the outer membrane marker protein TOMM20 and the inner mitochondrial membrane protein TIMM44. The Western blot analysis is shown in Figure 6. The TOMM20 band in MFN1-MDVs was the lightest, while the TIMM44 band was the darkest, indicating that MFN1-L-O2 mainly produces inner membrane MDVs composed of the inner mitochondrial membrane. Furthermore, we found that compared to the MFN1-MDVs group, the levels of TOMM20 and TIMM44 proteins in the WT-MDVs and Empty-MDVs groups were abnormally low, indicating that wild-type L-O2 cells and Empty-L-O2 cells produce very few MDVs.

[0147] 6. MDVs particle size analysis

[0148] The diameters of the extracted WT-MDVs, Empty-MDVs, and MFN1-MDVs were detected using a Nanosight particle size analyzer. The results are shown in Figure 7. The particle size range is mainly 50-200 nm, with the peak particle size appearing at around 100 nm, which is consistent with the morphological characteristics of MDVs.

[0149] 7. Detection of mtDNA content in MDVs

[0150] 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 shown in Figure 8. In the MFN1-MDVs group, the mtDNA electrophoresis diagram showed clear bands in each lane, indicating that the inner membrane MDVs produced by MFN1-L-O2 clearly contain complete mtDNA. However, because the MDVs produced by L-O2 and Empty-L-O2 cells are extremely rare, the mtDNA content in their MDVs is also abnormally low and undetectable.

[0151] Example 2: Effects of MDVs on Improving EtBr-Induced Cellular Senescence by mtDNA Mutations

[0152] I. Experimental Methods

[0153] 1. Establishment of cell model

[0154] 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.

[0155] The cell experiments were divided into 5 groups: Normal group (293T), EtBr-induced 293T group (M293T), wild-type 293T group (WT-MDVs), transfected with blank plasmid group (Empty-MDVs), and M293T group internalized with MFN1-MDVs (MFN1-MDVs). 100 μl of MDVs extraction solution was added to each well.

[0156] 2. Western blot analysis of expression levels of aging-related proteins P16 and P21

[0157] The expression levels of senescence-related proteins P16 and P21 were detected in cells from the 293T group and the M293T group.

[0158] 3. Internalization detection of MDVs

[0159] WT-MDVs, Empty-MDVs, and MFN1-MDVs were extracted using the iodixanol gradient density method. Following the kit instructions, 100 μg of MDVs were labeled with 5 μL of EvLINK505 and gently incubated in the dark at room temperature for 30 min. The purified samples were collected for subsequent experiments to assess cellular uptake of MDVs. Cells were seeded in confocal culture dishes and cultured for 24 h. Subsequently, M293T cells were incubated with EvLINK505-labeled MDVs for 12, 24, 36, and 48 h, and washed with PBS. To visualize the cell membrane, M293T cells were labeled with Cel-ILINK 555 and incubated in the dark at room temperature for 30 min. After incubation, cells were washed with PBS and fixed with 4% formaldehyde for 30 min. Then, they were stained with DAPI for 5 min. Internalization of labeled MDVs was observed using a laser confocal scanning microscope.

[0160] Detection of mtDNA content in cells

[0161] 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.

[0162] Genomic DNA was extracted according to the DNA extraction kit. 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 added to SYBR Green Master Mix and the corresponding primer preparation system for qPCR, with 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.

[0163] Table 16 PCR Amplification Primers

[0164]

[0165] Table 17 PCR Reaction Conditions

[0166]

[0167] 5. Detection of mtDNA mutation rate in cells

[0168] mtDNA products were extracted from cells in the M293T group and the MFN1-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.

[0169] 6. ATP level detection

[0170] 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.

[0171] 7. ROS level measurement

[0172] 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).

[0173] 8. Mitochondrial membrane potential detection

[0174] 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.

[0175] 9. Fluorescence detection of mitochondrial network structure

[0176] 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.

[0177] 10. Enzyme-linked immunosorbent assay (ELISA) to detect the expression levels of aging-related proteins.

[0178] 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.

[0179] 11. Statistical Analysis

[0180] 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.

[0181] Experimental results

[0182] 1. Expression levels of aging markers P16 and P21 proteins

[0183] 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 Western blot. The results are 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.

[0184] 2. Internalization of MDVs

[0185] M293T cells were co-cultured with MFN1-MDVs labeled with EvLINK505 and observed using laser confocal scanning microscopy at time points of 24 h, 48 h, 72 h, and 96 h. Figure 10 shows that after 72 h of co-culture, MFN1-MDVs reached the peak of internalization in the cells.

[0186] 3. Detection of mtDNA content and mutation rate in cells

[0187] Since the mtDNA of MDVs not converted to MFN1 is extremely low, only the MFN1-MDVs group was tested here. To detect the change in mtDNA content after MDVs in the MFN1-MDVs group were internalized into M293T cells, mtDNA and nDNA were extracted from the cells. The mtDNA was quantified using the ND1 subunit gene and the nDNA was quantified using the β-actin gene. The relative content of mtDNA in each group of cells was detected. The results are shown in Figure 11. The mtDNA content in the cells of the MFN1-MDVs group showed a significant increase, indicating that MFN1-MDVs successfully carried mtDNA into M293T cells.

[0188] Since the mtDNA of MDVs not converted to MFN1 is extremely low, only the MFN1-MDVs group was tested here. Figure 12 shows that, compared with the M293T group, the percentage of mutated genes among normal genes was significantly reduced in the MFN1-MDVs group, including ND6 (mutation rate 27.0%→14.7%), ATP6 (mutation rate 22.1%→8.2%), COX1 (mutation rate 15.4%→7.7%), ND1 (mutation rate 14.6%→5.2%), and Cytb (mutation rate 11.2%→4.3%). This indicates that MFN1-MDVs play a repair role in mtDNA in senescent cells and can significantly reduce the mtDNA mutation rate.

[0189] 4. Changes in ATP levels after MDVs internalized M293T cells in each group

[0190] 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 regulate mitochondrial function. Isolated MDVs were internalized into senescent M293T cells, and ATP levels in each group were measured. The results are shown in Figure 13. ATP production was significantly increased in the WT-MDVs group, the Empty-MDVs group, and the MFN1-MDVs group, approaching that of normal 293T cells.

[0191] 5. Changes in ROS levels after M293T cells were internalized by MDVs in each group

[0192] Mitochondrial function weakens in senescent cells, leading to increased ROS production. However, with aging, the cell's antioxidant defense capacity declines, resulting in a reduced ability to scavenge ROS. This imbalance makes ROS more likely to accumulate, further exacerbating cell damage. By internalizing MDVs from each group to M293T, the results are shown in Figure 14. ROS levels were reduced in the WT-MDVs, Empty-MDVs, and MFN1-MDVs groups, with the most significant reduction observed in the MFN1-MDVs group.

[0193] 6. Detection of mitochondrial membrane potential after MDVs internalize M293T cells in each group

[0194] 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. Fluorescence analysis showed a significant increase in relative red / green fluorescence intensity in the WT-MDVs, Empty-MDVs, and MFN1-MDVs groups, indicating an increase in membrane potential and suggesting recovery of mitochondrial function. The recovery was most pronounced in the MFN1-MDVs group (Figure 15).

[0195] 7. Internalization of MDVs in each group led to an improvement in the mitochondrial network structure of M293T cells.

[0196] 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 branched structure, while damaged mitochondria in senescent cells may appear fragmented, swollen, or unevenly distributed. In this invention, cells were stained with Mito-Tracker Green fluorescence and observed under a fluorescence microscope. The results, as shown in Figure 16, indicate that EtBr-induced M293T cells showed severe mitochondrial fragmentation and few connections between branches. Internalization of MFN1-MDVs revealed a significant improvement in the mitochondrial network in the MFN1-MDVs group, resulting in a more complete and continuous mitochondrial network.

[0197] 8. Internalization of MDVs in each group reduced the expression levels of cellular senescence-related proteins P16 and P21.

[0198] As shown in Figure 17, after internalizing MDVs in each group, the expression levels of aging-related proteins P16 and P21 in the cells of the MFN1-MDVs group decreased significantly.

[0199] In summary, these results indicate that cell lines stably overexpressing the MFN1 gene primarily produce inner-membrane MDVs composed of the mitochondrial inner membrane, significantly increasing the level of MDV secretion under normal physiological conditions. Compared to wild-type cells or empty vector cells, these cells are rich in normal, intact mtDNA. Application of MFN1-MDVs in senescent cell therapy demonstrates successful internalization into senescent cells, effectively reducing ROS levels, increasing mitochondrial mtDNA and ATP content, decreasing 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-senescence effects.

Claims

1. An MFN1 engineered mitochondrial vesicle, characterized in that, First, an overexpression vector for the MFN1 gene was constructed, transfected into cells, and a cell line overexpressing the MFN1 gene was obtained. The cells were cultured, the culture medium was collected, and mitochondrial vesicles were isolated and extracted to obtain MFN1-MDVs, which are MFN1-engineered mitochondrial vesicles rich in mtDNA. The cells were L-O2 cells. The mitochondrial vesicles were isolated and extracted using iodixanol gradient density centrifugation. The nucleotide sequence of the MFN1 gene is shown in SEQ ID No.

1.

2. The MFN1 engineered mitochondrial vesicles according to claim 1, characterized in that, The overexpression vector is pCDH-CMV-MFN1-EF1-copGFP-Puro.

3. The use of the MFN1 engineered mitochondrial vesicles according to claim 1 or 2 in the preparation of a drug for improving cellular senescence, characterized in that, The cells in question are 293T cells.

4. The application according to claim 3, characterized in that, The drug improves cell senescence by internalizing MDVs rich in normal mtDNA into senescent cells, promoting the activity of mitochondrial antioxidant enzymes in senescent cells to clear ROS, increasing ATP and mtDNA content, reducing the mtDNA mutation rate in senescent cells, enhancing mitochondrial network generation and restoring mitochondrial function in senescent cells, and reducing the expression of senescence-related proteins P16 and P21.

5. An anti-cellular aging drug, characterized in that, The drug contains MFN1 engineered mitochondrial vesicles as described in claim 1 or 2, and the cells are 293T cells.

6. The drug according to claim 5, characterized in that, It also contains pharmaceutically acceptable excipients.

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