CN117487737B - Fe-containing material 3 O 4 Enucleated cells of nanoparticles, preparation thereof and application thereof in anti-aging - Google Patents
Fe-containing material 3 O 4 Enucleated cells of nanoparticles, preparation thereof and application thereof in anti-aging Download PDFInfo
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Abstract
The invention relates to a Fe-containing alloy 3 O 4 Preparation of enucleated cells of nanoparticles and application of enucleated cells in anti-aging, and Fe-containing nanoparticles 3 O 4 The size of the enucleated cells of the nano particles is 10 mu m-15 mu m, and the enucleated cells comprise Fe 3 O 4 Nanoparticle, mitochondria, do not contain nuclei. The enucleated cells have large diameters and can wrap and transmit more mitochondria. The enucleated cells and target cells are incubated in VSV-G concentrate, vesicular stomatitis virus proteins are inserted on cell membranes, the distance between membranes is pulled in under the action of magnetic force, membrane tension is changed, and fusion occurs between cells under the action of citric acid buffer. The invention improves the cell fusion efficiency through magnetic force and higher fusion temperature, and the transplanted mitochondria maintain normal reticular structure and membrane potential, improves the biological activity of the mitochondria, can effectively enter target cells, can solve the problems of mitochondrial mutation and dysfunction, is used for transferring biological macromolecules and organelles, and can also be used for resisting aging.
Description
Technical Field
The invention belongs to the technology of synthetic biology, in particular to a Fe-containing material 3 O 4 The enucleated cells of the nano-particles, the preparation thereof and the application thereof in anti-aging.
Background
Mitochondria belong to semi-autonomous organelles, and most of the energy required for cellular biochemical reactions is supplied by mitochondria. Mitochondria are also involved in the fundamental activities of many cells. Mitochondrial DNA (mtDNA) is prone to mutations that may occur in mendelian-type inheritance, or maternal inheritance, sporadic, or outbreak at some stage of development, resulting in mitochondrial dysfunction, which plays an important role in aging, metabolic diseases, neurodegenerative diseases, neuromuscular diseases, cancer, and the like. Thus, mitochondria are now becoming the focus of research and the target of biomedical company attention.
Mitochondrial grafting can occur naturally in vivo and can also be transferred through a structure called tunnel nanotubes during cell culture in vitro. In addition, cells can also release vesicles containing mitochondria and transfer to surrounding cells. Such mitochondrial transplantation has been reported to result in reduced free Radical (ROS) and apoptosis, however, the efficiency of these pathways to transplant mitochondria is below grade, limiting their biomedical value.
There are many studies currently reporting on purification of mitochondrial transplantation, which is said to be effective against mitochondrial co-dysfunction in various research models and even in clinical studies. McCully and colleagues reported that mitochondrial grafting can reduce myocardial infarction area, reduce cardiomyocyte loss, and improve post-ischemic myocardial function. Zhang et al reported that mitochondrial grafting could reduce cerebral infarct volume and reverse neurological deficit in a stroke rat model. However, the biological mechanisms studied above have not been elucidated, resulting in the questionable use of purified mitochondria.
Enucleated cells are well known biological carriers that can be used to deliver normal mitochondria or patient mitochondria containing mutant DNA to cells that lack mitochondria (Rho 0 cells). In the above case, mitochondria are coated with plasma membrane, and severe environmental injury can be avoided to maintain the intact state. A small enucleated cell, i.e., plasma Membrane Vesicle (PMV), obtained by mechanically squeezing cells, can be used to transplant functional mitochondria. Rho0 cells and acetaminophen-damaged HepG2 liver cells were well protected from death and proliferation activity after mitochondrial transplantation. However, this approach relies heavily on cell membrane fusion, which is notoriously difficult and often leads to cell death.
Membrane fusion is a fundamental process of biological cells involved in membrane biogenesis, intracellular trafficking, hormone secretion and viral infection. Membrane fusion is subjected to a well-defined series of intermediate steps, the so-called semi-fusion model. These steps are governed by energy barriers, and various fusogenic agents, such as proteins, polypeptides, fats and ions, have been reported to overcome the energy barriers of the fusion process. The energy barrier can also be released by mechanical or chemical techniques such as PEG induction and electrical stimulation, but such methods are inefficient and result in severe cytotoxicity.
Disclosure of Invention
The object of the present invention is to provide a Fe-containing alloy 3 O 4 The enucleated cells of the nano particles, the preparation and the application thereof in anti-aging promote cell fusion and mitochondrial transplantation mediated by the fusion vesicular stomatitis virus glycoprotein VSV-G under the action of magnetic force, and can realize the efficient transfer of functional mitochondria so as to solve the problems in the prior art.
Fe-containing material 3 O 4 A method for preparing enucleated cells of nanoparticles, comprising the steps of:
(1) Plating BMSC cells in well plates; preferably 24-well plates, the cell density is about 50-70% (about 1x 10) 3 -1x10 6 Individually), the volume of the culture medium is 500 mu L;
(2) Adding 0.5-2mg/mL Fe 3 O 4 Nano particles, placing the pore plate on a magnet for incubation for 12-24 hours, absorbing and discarding supernatant, and cleaning;
(3) Pouring into a centrifuge tube, and adding centrifugate; placing the centrifuge tube in a preheating centrifuge, and centrifuging at 30-35deg.C and 5000-10000rpm for 40-60min;
(4) Adding cell culture solution, and placing into cell culture box for recovering for 30-60min to obtain Fe-containing material 3 O 4 Enucleated cells of the nanoparticles.
Further, the centrifugate contained 10% sucrose, 1mg/mL cytochalasin B, 100. Mu.M calcium chloride, 500mM NAC antioxidant, and 10mg/mL colchicine.
The Fe-containing material prepared by the preparation method 3 O 4 Enucleated cells of the nanoparticles.
Fe-containing material 3 O 4 Removing nuclear cells of the nano particles, wherein the size of the nuclear cells is 10 mu m-15 mu m; comprises Fe 3 O 4 Nanoparticle, mitochondria, do not contain nuclei. Fe (Fe) 3 O 4 The nanoparticles are loaded on bone marrow mesenchymal stem cells BMSCs; the enucleated cells comprise a stem macromolecule of a stem cell; the enucleated cell surface integrates a fusogenic protein VSV-G.
Nanoscale particles can encapsulate proteins and some small molecule drugs easily enter cells, but the larger the nanoparticle size, the less efficient the delivery and the nanoparticles cannot encapsulate larger substances, such as mitochondria with a diameter on the order of microns. The Fe-containing alloy of the present invention 3 O 4 The enucleated cells of the nano particles are about 10 mu m-15 mu m in size, and due to the magnetic force effect and the high fusion temperature, the fusion efficiency between the enucleated cells and the target cells is greatly improved, so that the mitochondrial transfer efficiency is remarkably improved.
The ferric oxide nano-particles are used as a novel nano-material, and have good targeting property, biocompatibility, biodegradability and biosafety; it exhibits excellent stability in terms of physicochemical properties such as biodistribution, metabolic rate, thermal stability, etc. Under the action of an externally applied magnetic field, the membrane tension between cells is changed, and intracellular calcium ion transient and cytoskeleton formation are increased, so that the membrane fusion efficiency is effectively promoted.
BMSC bone marrow mesenchymal stem cells are selected, and the cells contain Fe 3 O 4 Nanoparticles, enucleated by high-speed centrifugation, the obtained enucleated cells contained a large number of mitochondria. Mitochondria are in a reticular structure, and the membrane potential is normal.
Containing Fe 3 O 4 The enucleated cells of the nanoparticle fuse with the target cells under magnetic force and high temperature, transferring a large number of functional mitochondria to the mitochondria-defective cells. The transplanted mitochondria maintain normal reticular structure and membrane potential and exert on target cellsFunction and restore normal function to the cells.
Fe-containing material 3 O 4 Nanoparticles and integrated with enucleated cells of VSV-G.
Fe-containing material 3 O 4 A method of fusing enucleated cells of nanoparticles with mitochondrial deficient cells comprising the steps of:
(1) Will contain Fe 3 O 4 Digesting the enucleated cells of the nano-particles with digestive juice and centrifugally collecting;
(2) Collected Fe-containing material 3 O 4 The enucleated cells of the nanoparticles were resuspended using VSV-G concentrate and added to the plated mitochondrial defective cells;
(3) Placing the pore plate on a magnet and incubating in a cell incubator for 2-4 hours;
(4) Absorbing supernatant, and adding citric acid buffer solution at 37-55deg.C for 1min;
(5) The supernatant was aspirated off, DMEM cell culture medium containing 10% serum was added and placed in a cell culture incubator for incubation.
The Fe-containing alloy of the present invention 3 O 4 The enucleated cells of the nano particles and target cells are incubated in a concentrated solution containing VSV-G, vesicular stomatitis virus glycoprotein VSV-G is attached to cell membranes, and cell fusion is induced at 45 ℃ under the action of magnetic force, so that mitochondrial transplantation can be effectively promoted.
The invention preferably selects Ackutase TM The digestive juice Ackutsete (TM) digestive juice contains proteolytic enzyme and collagenase activity, can gently and effectively digest cells, does not destroy cell surface antigens, realizes separation of adherent cells within a few minutes, increases cell yield and survival rate, and enhances cell adherence efficiency.
The VSV-G concentrate was obtained by collecting a VSV-G conditioned medium, and then concentrating by ultracentrifugation. VSV-G conditioned medium Ad293 cells were harvested 48 hours after transfection of the VSV-G expression plasmid.
Further, the preparation of the VSV-G conditioned medium includes the following steps:
(1) Adding 0.8 mu g of plasmid for expressing vesicular stomatitis virus protein into a centrifuge tube, adding 50 mu L of serum-free culture solution, and uniformly mixing;
(2) Add 3. Mu.L Polyjet transfection reagent and 50. Mu.L serum-free medium to another centrifuge tube and gently shake; then adding the mixture into the solution obtained in the step (1); obtaining transfection mixed solution, and standing at room temperature for 15min;
(3) After 15min, the culture solution is sucked off, 1mL of fresh serum-free culture solution is added, the transfection mixture is immediately added into Ad293 cells, and the mixture is gently shaken;
(4) After 12 hours, the culture broth was replaced with new one.
Further, the preparation method of the VSV-G concentrate comprises the following steps:
(1) Transient transfection of Ad293 cells with plasmid expressing 0.8 μg vesicular stomatitis virus protein;
(2) After 48 hours the supernatant will be collected and the cells will be collected after digestion with pancreatin;
(3) After the collected cells are resuspended by cell culture liquid, repeatedly freezing and thawing for 5 times, and centrifuging to collect supernatant;
(4) Collecting the supernatants of (2) and (3), centrifuging at 300g,10min,2000g, 10min and 10000rpm for 30min, and collecting the supernatant;
(5) Placing the obtained product (4) into a 6mL ultracentrifuge tube, performing ultracentrifugation at 47000rpm and 4 ℃ for 2h, and collecting 1mL of sediment per tube, namely the concentrated VSV-G protein conditioned medium.
Further, the mitochondria-deficient cells include Rho0 cells and senescent cells.
Further, the Fe-containing alloy 3 O 4 The number of enucleated cells of the nanoparticle is: 1x10 3 -1x10 6 The concentration of the VSV-G concentrate is 0.5-5mg/mL, and the incubation volume is 10-100L.
Further, the concentration of the citric acid is 0.8-1mM, and the pH is 4.5-6.0. And adding the citric acid solution into the cell culture solution and accounting for 1/10 of the total volume, namely the citric acid buffer solution. The magnetic force of the magnet is 1500Gs-5000Gs.
The above-mentioned Fe-containing material 3 O 4 Enucleated cells of nanoparticles in delivery of biomacromolecules and organellesUse, said use being for diagnosis and treatment of non-diseases.
Containing Fe 3 O 4 The enucleated cells of the nanoparticle may be used for mitochondrial grafting to target cells.
The target cell may be a mitochondrial deficient cell Rho0.Rho0 cells were obtained after 7 days of continuous treatment of C2C12 cells with dideoxycytosine (10. Mu.M) and ethidium bromide (5. Mu.M).
The target cell may also be a senescent cell. The senescent cells may be induced by C2C12 cells. The induction conditions are as follows: obtained after one week in 2% FBS serum medium containing D-gal (40G/L) and then 1 day of incubation in medium containing R6G (5. Mu.g/mL).
The above-mentioned Fe-containing material 3 O 4 Use of enucleated cells of nanoparticles for anti-aging.
The present invention introduces a magnetic force to bring enucleated bone marrow mesenchymal stem cells (BMSCs) into close contact with target cells. The pH-induced membrane fusion is carried out at 45 ℃, and the higher temperature is favorable for breaking the liquid crystal state of the cell membrane. Fusion results in engrafted target cells, such as Rho0 cells and senescent cells, of intact mitochondria of enucleated BMSCs. Similarly, mitochondria were also successfully transplanted into gastrocnemius cells.
Compared with the prior art, the invention greatly improves the cell fusion efficiency through magnetic force and higher fusion temperature, and the fusion leads to mitochondrial transplantation, so that the transplanted mitochondria maintain normal reticular structure and membrane potential, thereby improving the biological activity of the mitochondria. The Fe-containing alloy of the present invention 3 O 4 The nanoparticle enucleated cells have a larger diameter and can encapsulate and deliver more mitochondria. The enucleated cells and target cells are incubated together in VSV-G concentrated solution, vesicular stomatitis virus proteins are inserted on cell membranes, the distance between the membranes is pulled in under the action of magnetic force, membrane tension is changed, and fusion occurs between the cells under the action of 45 ℃ citric acid buffer solution. The mitochondria in the enucleated cells effectively enter the target cells, can solve the problems of mitochondrial mutation and dysfunction, and can be used for resisting aging.
Drawings
FIG. 1 shows the Fe-containing alloy of the present invention 3 O 4 Nanoparticle enucleated cellsIs a schematic of the preparation and fusion of (a);
FIG. 2 shows the concentration of Fe 3 O 4 Schematic after nanoparticle loading of cells; loading 293 cells with nano particles with different concentrations, incubating for 24 hours under the condition of magnetic force, photographing and detecting CCK 8;
in fig. 3, (a) is a cell fusion map under different concentrations of the fusion membrane protein, and 1x represents the concentration: 0.5mg/mL, wherein a group of 293 cells are loaded with nanoparticles and labeled green and target cells 293 are labeled red; the rightmost column of the graph is a cell split graph, and the other three columns are sequentially cell DiI fluorescence, bright field, calcein and cell nucleus from left to right; (B) is a statistical plot of diameter after cell fusion; (C) is a statistical plot of cell fusion rates;
in FIG. 4 (A) the Fe concentration is different 3 O 4 An effect map of nanoparticles on cell fusion; wherein a group of 293 cells are loaded with nanoparticles at different concentrations and marked green, and target cells 293 are marked red; the rightmost column of the graph is a cell split graph, and the other three columns are sequentially cell DiI fluorescence, bright field, calcein and cell nucleus from left to right; (B) is a statistical plot of diameter after cell fusion; (C) is a statistical plot of cell fusion rates;
FIG. 5 (A) is a photograph showing cell fusion under different magnetic forces; one group of 293 cells was loaded with 2mg/mL of nanoparticles and labeled green, and the target cells 293 were labeled red; membrane fusion was performed at a concentration of 1 XVSV-G at a concentration of 0.5 mg/mL; the rightmost column of the graph is a cell split graph, and the other three columns are sequentially cell DiI fluorescence, bright field, calcein and cell nucleus from left to right; (B) is a statistical plot of diameter after cell fusion; (C) is a statistical plot of cell fusion rates;
FIG. 6 (A) is a graph showing the cell fusion efficiency under different temperature conditions of citrate buffer and incubation temperature; (B) Labeling live and dead cells for Calcein and PI staining of fused cells; (C) Making a histogram for counting the cell fusion efficiency under different conditions;
FIG. 7 shows the mitochondrial defective cell C2C12 Rho0 (mitochondrial defective) constructed after Ethidium Bromide (EB), dideoxycytosine (ddC) and uridine (uridine) are added, followed by mitochondrial staining reagent, bright field and split map from left to right;
FIG. 8 shows the identification of mitochondrial morphology fluorescence staining after removal of Ethidium Bromide (EB), zalcitabine (ddC), uridine (uridine), fusion of mitochondrial defective cell C2C12 Rho0 (mitochondrial defective) with nanoparticle-containing BMSC cells after enucleation (mitochondria with red fluorescence), and after 30min of fusion, CFSE, mitochondrial staining reagent, bright field, nuclei and split in sequence from left to right;
FIG. 9 shows the fusion of mitochondrial deficient cells C2C12 Rho0 with nanoparticle-containing BMSC cells after enucleation after removal of Ethidium Bromide (EB), zalcitabine (ddC) and uridine (uridine), wherein (A) shows the morphology of the cells after two to three days of fusion, respectively, (B) shows the statistics of cell numbers, (C) shows the PCR analysis, and (D) shows the quantitative PCR analysis of mitochondrial DNA.
FIG. 10 is an analytical graph of senescent cells C2C12 and senescent cells fused with enucleated BMSC cells for 30 min;
FIG. 11 is a graph showing β -galactosidase staining of cells after 3 days of fusion of normal C2C12 cells, senescent cells C2C12 and senescent cells with enucleated BMSC cells.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings, for the purpose of making the objects, technical solutions and advantages of the present invention more apparent.
Example 1
1. Fe-containing material 3 O 4 A method for preparing enucleated cells of nanoparticles, comprising the steps of:
(1) BMSC cells were plated in 24 wells at a cell density of about 70% (cell number 2X 10) 5 ) The volume of the culture medium is 500 mu L;
(2) Adding Fe to (1) 3 O 4 Nanoparticles (0.5-2 mg/mL) and incubating the well plate on a magnet for 12-24 hours;
(3) Inverted in a 50mL centrifuge tube; the centrifuge tube contained 10mL of a centrifuge containing 10% sucrose, 1mg/mL cytochalasin B, 100. Mu.M calcium chloride, 500mM NAC antioxidant and 10mg/mL colchicine;
(4) Placing the centrifuge tube in a preheating centrifuge, centrifuging at 30-35deg.C and 5000-10000rpm for 40-60min;
(5) Adding cell culture solution, and recovering in cell culture box for 30-60min to obtain Fe-containing material 3 O 4 Enucleated cells of the nanoparticles.
2. Containing Fe 3 O 4 A method of fusing a nanoparticle of enucleated cells and Rho0 cells or senescent cells, comprising the steps of:
(1) Will contain Fe 3 O 4 Ackutase for enucleated cells of nanoparticles TM Digesting and centrifugally collecting digestive juice;
(2) The collected cells were resuspended in VSV-G concentrate (at a concentration of 0.5 mg/ml) and added to Rho0 cells or senescent cells plated the day before (cell density around 20%, i.e., about 5X 10) 4 ）；
(3) Placing the pore plate on a magnet and incubating in a cell incubator for 2-4 hours;
(4) Absorbing supernatant, and adding 45 ℃ citric acid buffer solution for 1min;
(5) The supernatant was aspirated off, DMEM cell culture medium containing 10% serum was added and placed in a cell incubator for incubation, which was the confluent cells.
Wherein, the preparation of the VSV-G conditioned medium comprises the following steps:
(1) Adding 0.8 mu g of plasmid for expressing vesicular stomatitis virus protein into a centrifuge tube, adding 50 mu L of serum-free culture solution, and uniformly mixing;
(2) Add 3. Mu.L Polyjet transfection reagent and 50. Mu.L serum-free medium to another centrifuge tube and gently shake; then adding the mixture into the solution obtained in the step (1); obtaining transfection mixed solution, and standing at room temperature for 15min;
(3) After 15min, the culture solution is sucked off, 1mL of fresh serum-free culture solution is added, the transfection mixture is immediately added into Ad293 cells, and the mixture is gently shaken;
(4) After 12 hours, the culture broth was replaced with new one.
The preparation method of the VSV-G concentrated solution comprises the following steps:
(1) Transient transfection of Ad293 cells with plasmid expressing 0.8 μg vesicular stomatitis virus protein;
(2) After 48 hours the supernatant was collected and the cells were subjected to Acceutase TM Digestion and collection of digestive juice;
(3) After the collected cells are resuspended by cell culture liquid, repeatedly freezing and thawing for 5 times, and centrifuging to collect supernatant;
(4) Collecting the supernatants of (2) and (3), centrifuging at 300g,10min,2000g, 10min and 10000rpm for 30min, and collecting the supernatant;
(5) Placing the obtained mixture (4) into a 6mL ultracentrifuge tube, performing ultracentrifugation at 47000rpm and 4 ℃ for 2h, and collecting 1mL of sediment per tube, namely concentrated VSV-G protein conditioned medium, wherein the concentration is as follows: 0.5-5mg/mL.
Example 2
Monitoring of nanoparticle-loaded cells
For Ad293 cells with a fusion of about 50% (number approximately 1X 10) 5 ) Loading Fe with different concentrations 3 O 4 Nanoparticles were placed on a magnet and incubated for 24 hours in cell culture, after which the cells were stained for nuclei and digested for observation under a fluorescent microscope. FIG. 2 shows the results with Fe 3 O 4 The concentration of the nano particles continuously increases Fe in cells 3 O 4 The loading of nanoparticles also gradually increased; display of Fe by three-dimensional photographing of cells 3 O 4 The nanoparticles confirm localization inside the cells. In addition, CCK8 detection is carried out on the cells, and the result shows that the cells are loaded with Fe 3 O 4 The nanoparticles did not have an effect on cell viability and proliferation afterwards.
Example 3
Fe-containing material 3 O 4 Fusion between nanoparticle Ad293 cells and normal Ad293 cells
Ad293 cells were plated in 24-well plates with a cell fusion of about 50% (number about 1)x10 5 ) Adding 0.01-2mg/mL of nanoparticles with different concentrations according to experimental conditions, placing on a magnet, incubating for 12-24 hours, then labeling cells by Calcein staining, digesting, centrifugally collecting, re-suspending according to experimental design by using different VSV-G (concentration range of 0.5-5 mg/mL) conditioned culture solution, and adding DiI-labeled target cells 293 (cell density about 30% and number about 5×10) 4 ) Placing on a magnet with different magnetic forces of 1500Gs-5000Gs, incubating for 30min in an incubator, acidifying for 1min by using 0.8-1mM citric acid buffer solution with different temperatures of 37-55 ℃ and concentrations according to experimental design, adding cell culture solution, incubating for 30min, and staining cell nuclei.
Under the action of external magnetic force and VSV-G, the cells marked by the two different dyes are finally subjected to membrane fusion by regulating the pH value to 5.5, and the fused cells have fluorescence of two different colors. The analysis was performed by counting the diameter size and the cell area of the cells, and the cells having a diameter of 20 μm or less were not fused cells, and the cells having a diameter of 20 μm or more were fused cells.
From fig. 3 to 6, it can be seen that after the cells are loaded with nanoparticles, the fusion efficiency is increased under the incubation of the target cells with VSV-G conditioned medium, and the higher the VSV-G concentration, the higher the fusion efficiency; when the temperature of the citric acid buffer solution and the incubation temperature are increased, the cell fusion efficiency is increased to a certain extent; according to the experimental results, after loading 2mg/mL of nanoparticles on cells, the cells were incubated for 30min in a 1 XVSV-G (0.5 mg/mL concentration) conditioned medium, and after acidification for 1min with 45℃citrate buffer, the cells were incubated in an incubator for optimal fusion.
Selecting optimal experimental conditions to prepare enucleated nano cells, wherein the size of the prepared enucleated cells is 10 mu m-15 mu m, a large number of mitochondria are contained in the enucleated nano cells, and under the condition of externally applied magnetic force and VSV-G, membrane fusion is induced by adjusting the pH value to 5.5, so that substances in the enucleated cells are released to target cells. The biomacromolecule and organelle of interest are delivered into biomacromolecule and organelle deficient cells.
Example 4
Verification that BMSC cells transfer mitochondria to Rho0 cells
Rho0 cells were obtained after treatment of C2C12 cells with Ethidium Bromide (EB), dideoxycytosine (Zalcitabine, ddC) and uridine (uridine). Normal cells and Rho0 cells were stained with 1 μm MitoTracker (mitochondrial green staining reagent) and observed under confocal microscopy; in addition, to detect the cell membrane potential, staining was performed with 500nM TMRE (mitochondrial membrane potential staining reagent), and analysis under confocal microscopy as shown in FIG. 7 revealed that the mitochondrial morphology of the obtained Rho0 cells was significantly impaired.
The BMSC cells loaded with the nanoparticles are enucleated and fused with Rho0 cells; briefly, after enucleation of cells, rho0 cells added with the marker CSFE were resuspended with VSV-G and placed on a magnet and incubated in an incubator for 2 hours, then acidified with 45℃citrate buffer for 1min, then added with cell culture medium and placed in the incubator for 30min, followed by mitochondrial fluorescent staining and nuclear staining of the fused cells and Rho0 cells. From fig. 8, it can be seen that Rho0 cells contain a large number of normal forms of mitochondria, and the above results demonstrate that the BMSC cells of the present invention can efficiently deliver mitochondria into Rho0 cells.
Example 5
BMSC cells transmit mitochondria to Rho0 cell function verification.
The BMSC cells loaded with the nanoparticles are fused with Rho0 cells after enucleation; the fused cells obtained by the fusion method described in example 4 were examined under a microscope three-day later. In addition, the characteristics are also carried out by the techniques of real-time PCR, gel electrophoresis and the like through a cell counting means. From FIG. 9, it can be seen that the cell morphology of Rho0 cells became significantly normal after 3 days of fusion, while cells not fused were not full in cell morphology with or without uracil addition, and wiredrawing and death occurred. In fig. 9 (B), it can be seen that the number of fused cells was significantly higher on the second day of cell fusion than in the other two groups, while the cells in the fused group were about twice more than in the other groups after the third day, and the above results further demonstrate that the cells recovered to growth after cell fusion; the quantitative PCR detection in the result in FIG. 9 (C) shows that the copy number of mitochondrial DNA of Rho0 cells is significantly increased after transplantation with the nuclear beta-2M as a standard; further, PCR electrophoresis band analysis shows that the mitochondrial content in the fused cell is obviously increased. The above results demonstrate that enucleated BMSCs can effectively deliver mitochondria into Rho0 cells and perform biological functions, allowing them to resume Rho0 cell growth proliferation in normal cell culture without uracil addition.
Example 6
Monitoring and validation of BMSC cell transfer mitochondria to senescent C2C12 cells.
Aging C2C12 cells were obtained by treating C2C12 cells with 2% FBS and D-gal for seven days and then further treating the cells with R6G for one day. R6G is a membrane permeable cationic fluorescent dye that, upon entry into a cell, is free to cross the outer mitochondrial membrane and localize to the inner mitochondrial membrane. In addition, it is an effective oxidative phosphorylation inhibitor, which inhibits ATP synthesis by inhibiting ATP synthase and blocking hydrogen ion channels. Cells treated with R6G can eliminate endogenous mitochondrial and mtDNA. Fusing senescent cells and enucleated nanoparticle-containing BMSC cells by the fusion method of example 3; it can be seen in fig. 10 that the endogenous mitochondria substantially disappeared after the cells were treated with R6G; after the senescent cells are fused with the enucleated BMSC cells, mitochondria with normal morphological structures appear in the senescent cells; again, the above performance demonstrates that enucleated BMSC cells are effective in delivering mitochondria into senescent cells under conditions of magnetic force, VSVG, and elevated temperature. After three days of cell fusion, the cells are respectively stained with cell senescence beta-galactosylglycanase with normal cells and senescent cells, and as can be seen from fig. 11, the degree of staining of the fused cells becomes shallow, which proves that after the enucleated cells effectively transfer mitochondria to the senescent cells, the mitochondria can replicate in the senescent cells and exert biological functions.
Claims (6)
1. Fe-containing material 3 O 4 A method for preparing a enucleated cell of a nanoparticle, comprising the steps of:
(1) Plating BMSC cells in well plates;
(2) Adding 0.5-2mg/mL Fe 3 O 4 Nano particles, and placing the pore plate on a magnet for incubation for 12-24 hours; sucking and cleaning supernatant;
(3) Pouring into a centrifuge tube, and adding centrifugate; placing the centrifuge tube in a preheating centrifuge, and centrifuging at 30-35deg.C and 5000-10000rpm for 40-60min; the centrifugate contained 10% sucrose, 1mg/mL cytochalasin B,100 μm calcium chloride, 500mM NAC antioxidant, and 10mg/mL colchicine;
(4) Adding cell culture solution, and placing into cell culture box for recovering for 30-60min to obtain Fe-containing material 3 O 4 Enucleated cells of the nanoparticles.
2. The Fe-containing material prepared by the method of claim 1 3 O 4 Enucleated cells of the nanoparticles.
3. An Fe-containing material as claimed in claim 2 3 O 4 A method for fusing enucleated cells of nanoparticles with mitochondrial deficient cells, wherein the method is not diagnostic and therapeutic of a disease, comprising the steps of:
(1) The Fe-containing alloy according to claim 2 3 O 4 Digesting the enucleated cells of the nano-particles with digestive juice and centrifugally collecting;
(2) Collected Fe-containing material 3 O 4 The enucleated cells of the nanoparticles were resuspended using VSV-G concentrate and added to the plated mitochondrial defective cells;
(3) Placing the pore plate on a magnet and incubating in a cell incubator for 2-4 hours;
(4) Absorbing supernatant, and adding citric acid buffer solution at 37-55deg.C for 1min;
(5) The supernatant is removed, and DMEM cell culture solution containing 10% of serum is added and placed into a cell culture box for incubation;
the mitochondria-deficient cells are selected from Rho0 cells and senescent cells.
4. A method according to claim 3, characterized in that the Fe-containing material 3 O 4 The number of enucleated cells of the nanoparticle is: 1x10 3 -1x10 6 And the concentration of the VSV-G concentrated solution is 0.5-5mg/mL per hole.
5. A method according to claim 3, wherein the citric acid concentration is 0.8-1mm, the ph is 4.5-6.0, and the magnetic force of the magnet is 1500Gs-5000Gs.
6. The Fe-containing material according to claim 2 3 O 4 Use of nanoparticle enucleated cells for delivery of mitochondria into mitochondria-deficient cells, wherein said mitochondria-deficient cells are selected from the group consisting of Rho0 cells and senescent cells, said use being in the diagnosis and treatment of non-disease.
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| Fe3O4磁性纳米药物用于克服肿瘤多药耐药性的研究;唐昭敏 等;《材料导报》;20220530;第第37卷卷(第第15期期);第50-56页 * |
| Strategies for Engineering of Extracellular Vesicles;Danilushkina 等;《International Journal of Molecular Sciences》;20230826;第24卷(第17期);摘要、引言部分 * |
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