Method for constructing transmitochondrial human umbilical vein endothelial cell model
After deletion of mitochondrial DNA of human umbilical vein endothelial cells treated with rhodamine 6G, it fuses with donor mitochondria carrying pathogenic mutations or normal controls, a model of transmitochondria of human umbilical vein endothelial cells was constructed, which solved the difficulty in the prior art The problem of constructing a specific mitochondrial DNA mutant cell model has been realized to construct a cell model suitable for studying diseases related to vascular endothelial injury.
Patent Information
- Application Number
- CN202411670375.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-21
- Publication Date
- 2025-02-18
AI Technical Summary
The prior art is difficult to effectively construct cellular models of specific mitochondrial DNA mutations, especially when studying maternal hereditary diseases associated with vascular endothelial injury, and there is a lack of appropriate in vitro cellular models.
A transmimochondrial human umbilical vein endothelial cell model was prepared by deletion of mitochondria from rhodamine 6G-treated human umbilical vein endothelial cells and fused with donor mitochondria carrying pathogenic mutations or normal controls.
The constructed transmitochondrial human umbilical vein endothelial cell model has the basic characteristics of vascular endothelial cells and is suitable for studying maternal hereditary diseases related to vascular endothelial injury, providing a platform for clear pathogenic mechanism research and drug target discovery.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedicine and relates to a method for constructing a mitochondrial transgenic human umbilical vein endothelial cell model, which is a method for preparing a mitochondrial transgenic human umbilical vein endothelial cell model by fusing mitochondria of donor cells with human umbilical vein endothelial cells that have been treated with rhodamine 6G and lack mitochondrial DNA. Background Art
[0002] Mitochondrial dysfunction caused by mitochondrial gene mutations can cause a variety of diseases, involving multiple tissues and organs. Some mitochondrial DNA mutations can cause systemic symptoms, such as mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes syndrome (MELAS), myoclonic epilepsy with fragmented red fiber syndrome (MERRF), Kearns-Sayre syndrome, etc. Other mitochondrial gene mutations only occur in specific tissues, such as Leber's hereditary optic neuropathy, diabetes and myocardial hypertrophy. For tissue-specific mitochondrial DNA pathogenic mutations, clarifying their effects on diseased tissues or cells can reveal the mechanism of disease occurrence in depth. Vascular endothelial damage plays an important role in the pathological mechanism of various mitochondrial DNA mutation-related diseases such as maternally inherited hypertension, maternally inherited diabetes, and maternally inherited coronary heart disease. Therefore, clarifying the effects of mitochondrial gene mutations and the mitochondrial dysfunction caused by them on vascular endothelial cell function is of great significance for revealing the role of mutations in the pathological process of maternally inherited diseases related to vascular endothelial damage. However, based on ethics and operability, it is not feasible to directly obtain vascular endothelial tissue from the clinic for experimental research, and there are still technical bottlenecks in constructing animal models of specific mitochondrial DNA mutations. Therefore, using human vascular endothelial cells from in vitro sources to construct mitochondrial gene mutation models has become the best choice. When conducting research related to vascular endothelial cells, the cell model usually selected is human umbilical vein endothelial cells. The present invention inhibits its mitochondrial DNA replication by culturing human umbilical vein endothelial cells using an inhibitory culture medium containing rhodamine 6G. Then, human umbilical vein endothelial cells with mitochondrial DNA deletion are fused with donor mitochondria (immortalized lymphocyte source) carrying pathogenic mutations or normal controls to obtain a transmitochondrial human umbilical vein endothelial cell model carrying mitochondrial DNA mutations. The establishment of such cell models has important research and application value in the study of the pathological mechanism of mitochondrial diseases involving vascular endothelial damage such as maternal hereditary hypertension, maternal hereditary diabetes and maternal hereditary coronary heart disease, exploration of treatment methods, and discovery of drug targets. Summary of the invention
[0003] The purpose of the present invention is to provide a method for constructing a mitochondrial transfected human umbilical vein endothelial cell model, which is suitable for constructing mitochondrial transfected human umbilical vein endothelial cells carrying pathogenic mitochondrial DNA mutations and normal controls without pathogenic mutations.
[0004] The present invention realizes the construction of the mitochondrial translocation human umbilical vein endothelial cell model by the following steps:
[0005] 1. Preparation of mitochondrial DNA-deficient human umbilical vein endothelial cells
[0006] (1) Prepare an inhibitory culture medium and a cell suspension containing rhodamine 6G, the formula of which is as follows: Inhibition medium: each ml of the medium contains 15 ng of recombinant human R3 insulin-like growth factor-1, 10 ng of fibroblast growth factor-2, 5 ng of epidermal growth factor, 1 μg of hydrocortisone succinate, 90 μg of heparin, 50 μg of vitamin C, 100 μg of sodium pyruvate, 50 ug of uridine nucleoside, 3 μg of rhodamine 6G and 5% volume percentage of fetal bovine serum, and the remainder is DMEM / F12 medium, wherein the DMEM / F12 medium is a mixture of DMEM high glucose medium and F12 medium in a volume ratio of 1:1; Cell suspension: Each ml of cell suspension contains 15 ng of recombinant human R3 insulin-like growth factor-1, 10 ng of fibroblast growth factor-2, 5 ng of epidermal growth factor, 1 μg of hydrocortisone succinate, 90 μg of heparin, 50 μg of vitamin C and 5% volume percentage of fetal bovine serum, and the remainder is DMEM / F12 medium, which is a mixture of DMEM high glucose medium and F12 medium in a volume ratio of 1:1.
[0007] (2) Human umbilical vein endothelial cells were cultured in an inhibitory medium in a constant temperature incubator at 37°C, 5% CO2, and saturated humidity for 7 days, and the inhibitory medium was replaced every day to inhibit the replication of mitochondrial DNA.
[0008] (3) Human umbilical vein endothelial cells lacking mitochondrial DNA were digested with trypsin, washed with PBS, centrifuged at 1000 rpm for 3 min, the supernatant was removed, and 1×10 6 Resuspend the cells in a cell suspension solution at a concentration of 10 cells / ml, take 2 ml of cells and add them to a centrifuge tube and place it in a constant temperature incubator at 37°C, 5% CO2, and saturated humidity for later use.
[0009] 2. Removal of immortalized lymphocyte nuclei to obtain donor mitochondria
[0010] (1) Preparation of lymphocyte culture medium, centrifugation medium and cytoplasmic fluid Lymphocyte culture medium: Each ml of culture medium contains 100 μg sodium pyruvate, 50 μg uridine nucleoside, 10% volume percentage of fetal bovine serum, and the remainder is RPMI 1640 culture medium; Centrifuge: Each ml of centrifuge contains 12.5% by mass of Ficoll and 20 μg of cytochalasin B, and the remainder is DMEM / F12 culture medium; Cytoplasmic fluid: Each ml of cytoplasmic fluid contains 5% volume percentage of fetal bovine serum and 50ug of uridine nucleoside, and the rest is DMEM / F12 (without Ca 2+ ) culture medium.
[0011] (2) Preparation of Ficoll density gradient a. Prepare 50% (w / w) Ficoll stock solution: Dissolve Ficoll in an equal weight of ultrapure water, sterilize and slowly vent for 20 minutes, and stir overnight at 4°C to dissolve; b. Prepare the denuclearization gradient master solution according to Table 1 below:
[0012] Table 1 c. Slowly add each gradient along the tube wall into a sterilized nitrocellulose centrifuge tube (adapted to the SW41 rotor of Beckman ultracentrifuge L-80) according to the volume in Table 2 below. Clear boundaries can be observed between the 25%, 17%, 16%, 15% and 12.5% gradients. The gradient is prepared before use and can be stored in a constant temperature incubator at 37°C, 5% CO2, and saturated humidity for <12 hours;
[0013] Table 2 Ficoll solution concentration (%) 25 17 16 15 12.5 Volume(ml) 2 2 0.5 0.5 2
[0014] (3) Cultivate the mitochondrial donor immortalized lymphocytes in lymphocyte culture medium until the logarithmic growth phase, and collect the cells (1.5×10 7 ) and resuspend it fully by repeatedly pipetting with 5 ml of centrifuge buffer preheated to 37°C.
[0015] (4) Take 4 ml of the resuspended cells in the centrifuge solution and add them to the Ficoll gradient described in step c to form the upper layer.
[0016] (5) Place the centrifuge tube into the centrifuge adapter tube and balance it with centrifuge fluid.
[0017] (6) Place the centrifuge adapter tube into the SW41 rotor, and place the rotor into a Beckman L-80 ultracentrifuge. Set the temperature to 37°C, vacuum, 25,000 rpm, and centrifuge for 1 hour.
[0018] (7) After centrifugation, the cytoplasm layer can be seen at the junction of 15% and 12.5% Ficoll, and the cell nucleus is located at the junction of 25% and 17% Ficoll. Use a 2 ml syringe needle to insert horizontally under the cytoplasm layer to extract the white cytoplasm.
[0019] (8) Use DMEM / F12 (without Ca 2+ ) The extracted cytoplasm was diluted 10-fold and centrifuged to remove residual Ficoll and cytochalasin B.
[0020] (9) Resuspend the cells with 2 ml of cytoplasmic solution and incubate in a constant temperature incubator at 37°C, 5% CO2, and saturated humidity for 30 minutes to allow the cytoplasm to restore its spherical shape. During this period, take a drop of resuspended cells and observe under a 400x phase contrast microscope to calculate the proportion of cells with enucleation.
[0021] 3. Fusion of mitochondrial DNA-deficient human umbilical vein endothelial cells and immortalized lymphocyte-derived donor mitochondria
[0022] (1) Prepare a mild culture medium: each ml of the culture medium contains 15 ng of recombinant human R3 insulin-like growth factor-1, 10 ng of fibroblast growth factor-2, 5 ng of epidermal growth factor, 1 μg of hydrocortisone succinate, 90 μg of heparin, 50 μg of vitamin C, 100 μg of sodium pyruvate, 50 ug of uridine and 5% by volume of fetal bovine serum, and the remainder is DMEM / F12 culture medium, wherein the DMEM / F12 culture medium is a mixture of DMEM high glucose culture medium and F12 culture medium in a volume ratio of 1:1.
[0023] (2) Gently mix 2 ml of the cytoplasmic suspension in step 2 and 1 ml of the mitochondrial DNA-deficient human umbilical vein endothelial cells resuspended in step 1 in a centrifuge tube, remove the supernatant after centrifugation, and replace with DMEM / F12 (without Ca 2+ )Wash the cells.
[0024] (3) Add 1 ml of PEG 1500 preheated to 37°C and mix gently at room temperature for 1 minute.
[0025] (4) Add 10 ml DMEM / F12 (without Ca 2+ ), mix thoroughly, and centrifuge to remove PEG in the solution.
[0026] (5) Resuspend the cells in the culture medium and seed the cells in a 96-well plate at a concentration gradient of 1 cell / well, 10 cells / well, and 100 cells / well. The fused cells are cultured for 2 days.
[0027] 4. Screening, Culture and Identification of Transmitochondrial Human Umbilical Vein Endothelial Cells
[0028] (1) Preparation of screening culture medium: each ml of culture medium contains 15 ng of recombinant human R3 insulin-like growth factor-1, 10 ng of fibroblast growth factor-2, 5 ng of epidermal growth factor, 1 μg of hydrocortisone succinate, 90 μg of heparin, 50 μg of vitamin C and 5% volume percentage of fetal bovine serum, and the remainder is DMEM / F12 culture medium, wherein the DMEM / F12 culture medium is a mixture of DMEM high glucose culture medium and F12 culture medium in a volume ratio of 1:1.
[0029] (2) Observe the adhesion status of the fused cells. After culturing the cells in the gentle medium for 2-3 days, replace them with the screening medium and change the medium every three days.
[0030] (3) After 10-14 days of culture in the screening medium, the emergence of single clones of cells can be observed. Single clones are picked and expanded for culture.
[0031] (4) DNA of the transmitochondrial human umbilical vein endothelial cell monoclonal strain was extracted and Sanger sequencing was used to identify whether the cell strain was a transmitochondrial cell (carrying the donor mitochondrial DNA mutation).
[0032] (5) Use real-time quantitative PCR to determine the mitochondrial DNA copy number of monoclonal cell lines, and select cell lines with similar DNA copy numbers for other related experimental studies.
[0033] (6) Detect whether the monoclonal cell line of mitochondrial transfected human umbilical vein endothelial cells still possesses the basic characteristics of vascular endothelial cells, such as immunofluorescence detection of cell surface expression of vascular endothelial cell markers such as CD31, VE-cadherin, Flk1 and vWF; cell scratch assay to detect the migration ability of the monoclonal cell line; in vitro angiogenesis assay to detect the angiogenesis ability of the constructed mitochondrial transfected human umbilical vein endothelial cells.
[0034] The reagents described in the present invention can all be commercially available.
[0035] Another object of the present invention is to provide an application of the method in constructing a transmitochondrial human umbilical vein endothelial cell model carrying a pathogenic mutation.
[0036] Compared with the prior art, the present invention has the following advantages: Mitochondrial disease is a type of heritable disease caused by nuclear gene defects or mitochondrial gene defects, with an incidence rate of about 1:5000 and a complex molecular pathogenic mechanism. Mitochondrial diseases caused by some mitochondrial DNA mutations only affect a single or specific tissue organ, that is, mitochondrial gene mutations are tissue-specific. Only by clarifying the effects of pathogenic mutations of such diseases on diseased tissues or cells can we deeply reveal the physiological and pathological mechanisms of the disease. For a variety of mitochondrial diseases such as maternally inherited hypertension, maternally inherited diabetes, and maternally inherited coronary heart disease, vascular endothelial damage plays a key role in the occurrence and development of the disease. However, based on ethics and operability, it is not feasible to directly obtain vascular endothelial tissue from the clinic for experimental research, and there are still technical bottlenecks in constructing animal models of specific mitochondrial DNA mutations. Therefore, the use of human vascular endothelial cells derived in vitro to construct a mitochondrial gene mutation model has become the best choice. Compared with vascular endothelial cells induced by patient-derived iPSCs, the transmitochondrial vascular endothelial cells constructed by the method of the present invention have a consistent nuclear gene background, which excludes the modification effect of nuclear genes on the pathogenicity of mitochondrial gene mutations. Studies using such models can more clearly reveal the pathogenic mechanism of mitochondrial DNA mutations. With 143B.TK - Compared with the transmitochondrial cell model constructed by using ρ0 cells from the source as the cell nucleus donor, the transmitochondrial cell model constructed by the method of the present invention is a vascular endothelial cell, which is more suitable for the study of diseases related to vascular endothelial injury and helps to reveal the tissue specificity of mitochondrial DNA mutation-induced pathogenicity. The method of the present invention is highly operable and has high batch-to-batch repeatability, which is convenient for analyzing disease manifestations. The transmitochondrial human umbilical vein endothelial cells carrying pathogenic mutations constructed by the method of the present invention provide a good model for studying maternal genetic diseases related to vascular endothelial injury. BRIEF DESCRIPTION OF THE DRAWINGS
[0037] Figure 1 It shows the morphological changes of human umbilical vein endothelial cells cultured in an inhibitory medium containing rhodamine 6G from 0 to 7 days. Under the action of rhodamine 6G, the replication of mitochondrial DNA of human umbilical vein endothelial cells is inhibited, some cells die, and the morphology of the remaining cells also changes.
[0038] Figure 2 This is a schematic diagram of preparing Ficoll gradient in a nitrocellulose centrifuge tube. The volumes of 25%, 17%, 16%, 15%, 12.5% Ficoll and centrifuge fluid are 2ml, 2ml, 0.5ml, 0.5ml, 2ml and 4ml respectively. After centrifugation, it can be seen that the cytoplasm layer is located at the junction of 15% and 12.5% Ficoll, and the cell nucleus is located at the junction of 25% and 17% Ficoll. Use a 2ml syringe needle to insert horizontally under the cytoplasm layer to extract the white cytoplasm.
[0039] Figure 3 It is the Sanger sequencing result near the mitochondrial 5512 site of human umbilical vein endothelial cells and transmitochondrial human umbilical vein endothelial cells, where the 5512 site of human umbilical vein endothelial cells is A, while the 5512 site of transmitochondrial human umbilical vein endothelial cells is G.
[0407] Figure 4 It is the relative value of mitochondrial copy number of 10 different monoclonal cell lines of transmitochondrial human umbilical vein endothelial cells relative to that of human umbilical vein endothelial cells. If relevant experimental research is conducted, it is necessary to select monoclonal cell lines with mitochondrial copy number close to that of human umbilical vein endothelial cells.
[0041] Figure 5 The results of immunofluorescence staining of endothelial cell-specific marker proteins indicate that mitochondrial-transfected human umbilical vein endothelial cells still possess the basic characteristics of endothelial cells and express endothelial cell-specific marker proteins CD31, VE-Cadherin, VEGFR-2 and vWF.
[0042] Figure 6 The results of the cell scratch experiment show that mitochondrial transferred human umbilical vein endothelial cells still have the migration and movement ability of vascular endothelial cells.
[0043] Figure 7 This is the result of an in vitro angiogenesis experiment, which shows that mitochondrial-transfected human umbilical vein endothelial cells still have the basic characteristics of vascular endothelial cells, namely, in vitro angiogenesis. DETAILED DESCRIPTION
[0044] The present invention is further described below with reference to the accompanying drawings and embodiments, but is not intended to limit the present invention.
[0045] Example 1: Carrying tRNA Trp Construction of a transgenic human umbilical vein endothelial cell model with 5512A>G mutation
[0046] Mitochondrial tRNA Trp The 5512A>G mutation is a pathogenic mutation associated with essential hypertension. This mutation affects tRNA TrpPost-transcriptional processing and normal function of the 5' end lead to mitochondrial dysfunction, which in turn promotes the occurrence of essential hypertension. Vascular endothelial damage plays an important role in the pathological mechanism of essential hypertension. Clarifying the effects of the mitochondrial 5512A>G mutation and the mitochondrial dysfunction it causes on vascular endothelial cells is of great significance for revealing its role in the pathological process of essential hypertension. Using patient-derived immortalized lymphocytes as mitochondrial donors, transmitochondrial human umbilical vein endothelial cells carrying the 5512A>G mutation were constructed, providing the best cell model for exploring the role of this mutation in the pathological process of essential hypertension at the molecular and cellular levels. The specific steps for constructing the cell model are as follows:
[0047] (1) Human umbilical vein endothelial cells were cultured in an incubator at 37°C, 5% CO2, and saturated humidity for 7 days using an inhibitory medium, with the inhibitory medium replaced daily to inhibit mitochondrial DNA replication (see Figure 1 );
[0048] (2) Human umbilical vein endothelial cells lacking mitochondrial DNA were digested with trypsin and washed with PBS. 6 Resuspend the cells in a cell suspension solution at a concentration of 10 cells / ml, take 2 ml of cells and add them to a centrifuge tube and place it in a constant temperature incubator at 37°C, 5% CO2, and saturated humidity for later use;
[0049] (3) Using lymphocyte culture medium to culture cells carrying tRNA Trp Immortalized lymphocytes with 5512A>G mutation were grown to the logarithmic growth phase and the cells (1.5×10 7 ) and resuspend it fully by repeatedly pipetting with 5 ml of centrifuge solution preheated to 37°C;
[0050] (4) Take 4 ml of the resuspended cells in the centrifuge solution and add them to a nitrocellulose centrifuge tube (a 25%, 17%, 16%, 15% and 12.5% Ficoll gradient has been prepared) to form the upper layer (see Figure 2 );
[0051] (5) Use centrifuge liquid to balance the centrifuge tube, put the centrifuge adapter tube with the centrifuge tube into the SW41 rotor, put the rotor into the Beckman L-80 ultracentrifuge, 37°C, vacuum, 25,000 rpm, and centrifuge for 1 hour;
[0052] (6) Use a 2 ml syringe needle to horizontally insert the needle at the junction of 15% and 12.5% Ficoll to extract the white cytoplasm (see Figure 2 ), DMEM / F12 (without Ca 2+) The extracted cytoplasm was diluted 10 times, centrifuged to remove residual Ficoll and cytochalasin B, and the cells were resuspended in 2 ml of cytoplasmic solution and incubated in a constant temperature incubator at 37°C, 5% CO2, and saturated humidity for 30 minutes to allow the cytoplasm to restore its spherical shape;
[0053] (7) Gently mix 2 ml of the cytoplasmic suspension in step (6) and 1 ml of the mitochondrial DNA-deficient human umbilical vein endothelial cells resuspended in step (2) in a centrifuge tube, remove the supernatant after centrifugation, and replace with DMEM / F12 (without Ca 2+ )Wash the cells.
[0054] (8) Add 1 ml of PEG 1500 preheated to 37°C and mix gently at room temperature for 1 minute.
[0055] (9) Add 10 ml DMEM / F12 (without Ca 2+ ), mix thoroughly, and centrifuge to remove PEG in the solution.
[0056] (10) Resuspend the cells in a mild culture medium and seed the cells in a 96-well plate at a concentration gradient of 1 cell / well, 10 cells / well, and 100 cells / well. The fused cells were cultured for 2 days.
[0057] (11) Observe the adhesion status of the fused cells. After culturing the cells in the gentle medium for 2-3 days, replace them with the screening medium. Change the medium every three days.
[0058] (12) After 10-14 days of culture in the screening medium, the emergence of single clones of cells can be observed. Single clones are picked and expanded for culture.
[0059] Example 2: Identification of transmitochondrial human umbilical vein endothelial cells
[0060] (1) Extract DNA from monoclonal human umbilical vein endothelial cell lines transfected with mitochondria and use Sanger sequencing to identify whether the monoclonal cell lines carry mitochondrial tRNA Trp 5512A>G mutation, results are shown in Figure 3 ;
[0061] (2) DNA was extracted from different monoclonal cell lines, and the mitochondrial DNA copy number of the monoclonal cell lines was determined by qPCR. The results are shown in Figure 4 ;
[0062] (3) Immunofluorescence staining was used to detect the expression of endothelial cell-specific marker proteins CD31, VE-Cadherin, VEGFR-2, and vWF in transmitochondrial human umbilical vein endothelial cells. The results are shown in the figure. Figure 5 ;
[0063] (4) Cell scratch assay was used to detect the migration ability of transmitochondrial human umbilical vein endothelial cells. The results are shown in Figure 6 ;
[0064] (5) In vitro angiogenesis assay was used to detect the angiogenesis ability of human umbilical vein endothelial cells transfected with mitochondria. The results are shown in Figure 7 .
[0065] It can be seen from the above examples that the method provided by the present invention can obtain a transmitochondrial human umbilical vein endothelial cell model carrying a pathogenic mitochondrial DNA mutation, and the constructed cell model has the basic characteristics of vascular endothelial cells, such as expressing vascular endothelial cell-specific marker proteins CD31, VE-Cadherin, VEGFR-2 and vWF; and has the migration ability and in vitro angiogenesis ability of vascular endothelial cells. The method is highly operable, has high batch-to-batch repeatability, and is convenient for analyzing disease manifestations. The transmitochondrial human umbilical vein endothelial cells carrying pathogenic mutations constructed by the method of the present invention provide a good model for studying maternal inherited disease mitochondria associated with vascular endothelial damage.
Claims
1. A method for constructing a transmitochondrial human umbilical vein endothelial cell model, characterized in that: This is accomplished by following these steps: (1) Preparation of human umbilical vein endothelial cells lacking mitochondrial DNA; (2) removing the nuclei of immortalized lymphocytes to obtain donor mitochondria; (3) Fusion of donor mitochondria derived from mitochondrial DNA-deficient human umbilical vein endothelial cells and immortalized lymphocytes; (4) Screening, culture and identification of mitochondrial transfected human umbilical vein endothelial cells.
2. The method according to claim 1, characterized in that The step (1) of preparing human umbilical vein endothelial cells lacking mitochondrial DNA is achieved by the following steps: (1) Human umbilical vein endothelial cells were cultured in an inhibitory medium in a constant temperature incubator at 37° C., 5% CO 2 , and saturated humidity for 7 days, and the inhibitory medium was replaced every day to inhibit mitochondrial DNA replication; (2) Human umbilical vein endothelial cells lacking mitochondrial DNA were trypsinized, washed with PBS, centrifuged at 1000 rpm for 3 min, and the supernatant was removed; (3) According to 1×10 6 Resuspend the cells in a cell suspension at a concentration of 10 cells / ml, take 2 ml of cells and add them to a centrifuge tube and place it in a constant temperature incubator at 37°C, 5% CO2, and saturated humidity for later use.
3. The method according to claim 2, characterized in that The inhibition culture medium comprises: each ml of culture medium contains 15 ng of recombinant human R3 insulin-like growth factor-1, 10 ng of fibroblast growth factor-2, 5 ng of epidermal growth factor, 1 μg of hydrocortisone succinate, 90 μg of heparin, 50 μg of vitamin C, 100 μg of sodium pyruvate, 50 ug of uridine nucleoside, 3 μg of rhodamine 6G and 5% volume percentage of fetal bovine serum, and the remainder is DMEM / F12 culture medium, and the DMEM / F12 culture medium is a mixture of DMEM high glucose culture medium and F12 culture medium in a volume ratio of 1:
1.
4. The method according to claim 2, characterized in that: The cell suspension is as follows: each ml of the cell suspension contains 15 ng of recombinant human R3 insulin-like growth factor-1, 10 ng of fibroblast growth factor-2, 5 ng of epidermal growth factor, 1 μg of hydrocortisone succinate, 90 μg of heparin, 50 μg of vitamin C and 5% volume percentage of fetal bovine serum, and the remainder is DMEM / F12 culture medium, and the DMEM / F12 culture medium is a mixture of DMEM high-glucose culture medium and F12 culture medium in a volume ratio of 1:
1.
5. The method according to claim 1, characterized in that The step (2) of removing the cell nucleus of the immortalized lymphocyte to obtain the donor mitochondria is achieved by the following steps: (1) Cultivate mitochondrial donor immortalized lymphocytes in lymphocyte culture medium until the logarithmic growth phase and collect cells (1.5×10 7 ) and resuspend it fully by repeatedly pipetting with 5 ml of centrifuge solution preheated to 37°C; (2) Take 4 ml of the resuspended cells in the centrifuge solution and add them to the Ficoll gradient in the nitrocellulose centrifuge tube to form the upper layer; (3) Place the centrifuge tube into the centrifuge adapter tube and balance it with centrifuge fluid; (4) Place the centrifuge adapter tube into the SW41 rotor, place the rotor into a Beckman L-80 ultracentrifuge, set the temperature to 37°C, vacuum, 25,000 rpm, and centrifuge for 1 hour; (5) After centrifugation, the cytoplasm layer is located at the junction of 15% and 12.5% Ficoll, and the cell nucleus is located at the junction of 25% and 17% Ficoll. Use a 2 ml syringe needle to insert horizontally under the cytoplasm layer to extract the white cytoplasm; (6) Use Ca-free 2+ The extracted cytoplasm was diluted 10-fold with DMEM / F12 and centrifuged to remove residual Ficoll and cytochalasin B; (7) Resuspend the cells in 2 ml of cytoplasmic solution and incubate in a constant temperature incubator at 37°C, 5% CO2, and saturated humidity for 30 minutes to allow the cytoplasm to restore its spherical shape.
6. The method according to claim 5, characterized in that The lymphocyte culture medium is as follows: each ml of the culture medium contains 100 μg of sodium pyruvate, 50 μg of uridine nucleoside, 10% volume percentage of fetal bovine serum, and the remainder is RPMI 1640 culture medium.
7. The method according to claim 5, characterized in that The centrifuge solution is as follows: each ml of the centrifuge solution contains 12.5% by mass of Ficoll and 20 μg of cytochalasin B, and the remainder is DMEM / F12 culture medium.
8. The method according to claim 5, characterized in that The cytoplasmic fluid is as follows: each ml of cytoplasmic fluid contains 5% by volume of fetal bovine serum and 50ug of uridine nucleoside, and the remainder is Ca-free. 2+ DMEM / F12 medium.
9. The method according to claim 5, characterized in that The step (2) of nitrocellulose centrifuge tube Ficoll gradient is achieved by the following steps: (1) Prepare 50% by weight Ficoll stock solution: dissolve Ficoll in an equal weight of ultrapure water, sterilize and slowly release air for 20 minutes, and stir overnight at 4°C to dissolve; (2) Prepare the denuclearization gradient master solution according to the following table: (3) Slowly add each gradient along the wall of the sterilized nitrocellulose centrifuge tube according to the volume in the table below. Clear boundaries can be observed between the 25%, 17%, 16%, 15% and 12.5% gradients. The gradients are prepared before use and can be stored in a constant temperature incubator at 37°C, 5% CO2, and saturated humidity for <12 hours; 。 10. The method according to claim 1, characterized in that The step (3) of fusing the mitochondrial DNA-deficient human umbilical vein endothelial cells and the donor mitochondria derived from immortalized lymphocytes is achieved by the following steps: (1) Gently mix 2 ml of cytoplasmic suspension and 1 ml of resuspended mitochondrial DNA-deficient human umbilical vein endothelial cells in a centrifuge tube. After centrifugation, remove the supernatant and remove the Ca-free 2+ Wash the cells with DMEM / F12; (2) Add 1 ml of PEG 1500 preheated to 37°C and mix gently for 1 minute at room temperature; (3) Adding Ca-free 2+ 10 ml DMEM / F12, mix thoroughly, and centrifuge to remove PEG in the solution; (4) Resuspend the cells in a mild culture medium and seed the cells in a 96-well plate at a concentration gradient of 1 cell / well, 10 cells / well, and 100 cells / well. The fused cells are cultured for 2 days.
11. The method according to claim 10, characterized in that The mild culture medium is as follows: each ml of the culture medium contains 15 ng of recombinant human R3 insulin-like growth factor-1, 10 ng of fibroblast growth factor-2, 5 ng of epidermal growth factor, 1 μg of hydrocortisone succinate, 90 μg of heparin, 50 μg of vitamin C, 100 μg of sodium pyruvate, 50 ug of uridine and 5% volume percentage of fetal bovine serum, and the remainder is DMEM / F12 culture medium, and the DMEM / F12 culture medium is a mixture of DMEM high glucose culture medium and F12 culture medium in a volume ratio of 1:
1.
12. The method according to claim 1, characterized in that The step (4) of screening, culturing and identifying mitochondrial transfected human umbilical vein endothelial cells is achieved by the following steps: (1) Observe the adhesion status of fused cells, culture the cells in the mitigation medium for 2-3 days, then replace them with the screening medium, and change the medium every three days; (2) After 10-14 days of culture in the screening medium, single clones of cells can be observed. Single clones are picked and expanded for culture; (3) DNA of the transmitochondrial human umbilical vein endothelial cell monoclonal strain was extracted and Sanger sequencing was used to identify whether the cell strain was a transmitochondrial cell carrying the donor mitochondrial DNA mutation; (4) Use real-time quantitative PCR to determine the mitochondrial DNA copy number of monoclonal cell lines, and select cell lines with similar DNA copy numbers for other related experimental studies; (5) Detect whether the monoclonal cell line of mitochondrial transfected human umbilical vein endothelial cells still possesses the basic characteristics of vascular endothelial cells, such as immunofluorescence detection of cell surface expression of vascular endothelial cell markers such as CD31, VE-cadherin, Flk1 and vWF; cell scratch assay to detect the migration ability of the monoclonal cell line; in vitro angiogenesis assay to detect the angiogenesis ability of the constructed mitochondrial transfected human umbilical vein endothelial cells.
13. The method according to claim 12, characterized in that The mild culture medium is as follows: each ml of the culture medium contains 15 ng of recombinant human R3 insulin-like growth factor-1, 10 ng of fibroblast growth factor-2, 5 ng of epidermal growth factor, 1 μg of hydrocortisone succinate, 90 μg of heparin, 50 μg of vitamin C and 5% volume percentage of fetal bovine serum, and the remainder is DMEM / F12 culture medium, and the DMEM / F12 culture medium is a mixture of DMEM high glucose culture medium and F12 culture medium in a volume ratio of 1:1.