Preparation method of high-mutation urine stem cells in mitochondria mt.3243A > G mutation crowds

Abstract

The invention provides a preparation method of high-mutation urine stem cells in mitochondria mt.3243A > G mutation crowds. The method comprises the following steps: collecting urine cells of mitochondria mt.3243A > G mutant population; obtaining a sediment red tube by adopting a mode of centrifuging and adding a PBS to blow, beat and mix uniformly, obtaining P1-generation high-mutation urine stemcells are obtained, and obtaining the mitochondria mt.3243A > G mutation urine stem cells with the high mutation rate of 95% or above through digestion passage and mutation rate detection. The methodhas the advantages that the method can be obtained in a non-invasive mode, and the method can be obtained regardless of the gender, age and health condition of the patient. As a primary cell, the cell line maintains the basic properties of the original cell. The invention provides a cell application platform for researching the pathogenesis of mt.3243A > G mutant cells in the future and screeningnew drugs aiming at mt.3243A > G.

Description

technical field

[0001] The invention belongs to the field of biochemistry, and relates to mitochondrial mt.3243A>G population, specifically a method for preparing highly mutated urine stem cells in mitochondrial mt.3243A>G mutation population. Background technique

[0002] Stem cells are not fully differentiated and immature cells, which have the potential to regenerate various tissues and organs and the human body, and are called "universal cells" in the medical field. Mesenchymal stem cells are a kind of pluripotent stem cells, which have all the common characteristics of stem cells, that is, self-renewal and multidirectional differentiation capabilities. Mesenchymal stem cells have been clinically applied to solve a variety of blood system diseases, cardiovascular diseases, liver cirrhosis, nervous system diseases, partial knee meniscectomy injury repair, autoimmune diseases and other aspects have made major breakthroughs.

[0003] Urine-derived stem cells are a ne...

Examples

Embodiment 1

[0034] Isolating and culturing stem cells from the urine of mutation patients and monoclonal isolation and culture, including the following steps:

[0035] 1) Add 5ml of a mixed solution of penicillin, streptomycin and amphotericin B (the content of penicillin is 10kU / ml, the content of streptomycin is 10mg / ml, and the content of amphotericin B is 25μg / ml) into the T75 bottle. Spray disinfectant around;

[0036] 2) Select the urine of the mitochondrial mt.3243A>G mutant population, and use the T75 bottle in step 1) for urine collection;

[0037] 3) Open the T75 bottle, add 50ml of urine sample to each of the four 50ml centrifuge red tubes, and centrifuge;

[0038] 4) Use a pipette to suck up the supernatant to less than 5ml in each red tube, add 15ml~20ml PBS to the first red tube, pipette and mix well, then transfer the resuspension to the next red tube, pipette again Mix well, and so on, until finally all the sediments of the tubes are collected in one tube, and centrifuge...

Embodiment 2

[0054] Example 2 Membrane Potential Detection

[0055] The amount of JC-1 staining working solution is 1ml, take an appropriate amount of JC-1 (200X), and dilute JC-1 by adding 8ml of ultrapure water to every 50μl of JC-1 (200X). Vigorously dissolve Vortex and mix JC-1 thoroughly. Then add 2ml of JC-1 staining buffer (5X), and mix well to get the JC-1 staining working solution.

[0056] a. For one well of the six-well plate, suck off the culture medium, wash the cells once with PBS or other appropriate solution if necessary according to the specific experiment, and add 1ml of mesenchymal stem cell culture medium. Cell culture medium may contain serum and phenol red.

[0057] b. Add 1ml JC-1 staining working solution and mix well. Incubate at 37°C for 20 minutes in a cell culture incubator.

[0058] c. During the incubation period, prepare an appropriate amount of JC-1 staining buffer (1X) by adding 4ml of distilled water to every 1ml of JC-1 staining buffer (5X), and place...

Embodiment 3

[0062] Example 3 ROS detection

[0063] Loading probes after collecting urine mesenchymal stem cells: Dilute DCFH-DA with serum-free medium according to 1:1000, so that the final concentration is 10umol / L.

[0064] Centrifuge the cells at 600g for 4min (or 3000rpm / 5min), remove the supernatant, suspend the cells in diluted DCFH-DA (1ml), the cell concentration is 10^6-2*10^7 / ml, and culture the cells at 37°C Incubate in the box for 20 minutes. Mix by inverting every 3-5 minutes to make the probe fully contact with the cells. The cells were centrifuged at 600 g for 4 min, and the cells were washed (centrifuged, broken, and blown) three times with serum-free cell culture medium to fully remove DCFH-DA that did not enter the cells. Add about 500ul (depending on the cell density) of culture medium, blow and mix well, smear 10ul sample onto the glass slide, and observe under the fluorescence microscope. Each sample has three fields of view, and each field of view is first shot wi...

technical field

[0001] The invention belongs to the field of biochemistry, and relates to mitochondrial mt.3243A>G population, specifically a method for preparing highly mutated urine stem cells in mitochondrial mt.3243A>G mutation population. Background technique

[0002] Stem cells are not fully differentiated and immature cells, which have the potential to regenerate various tissues and organs and the human body, and are called "universal cells" in the medical field. Mesenchymal stem cells are a kind of pluripotent stem cells, which have all the common characteristics of stem cells, that is, self-renewal and multidirectional differentiation capabilities. Mesenchymal stem cells have been clinically applied to solve a variety of blood system diseases, cardiovascular diseases, liver cirrhosis, nervous system diseases, partial knee meniscectomy injury repair, autoimmune diseases and other aspects have made major breakthroughs.

[0003] Urine-derived stem cells are a ne...

Examples

Embodiment 1

[0034] Isolating and culturing stem cells from the urine of mutation patients and monoclonal isolation and culture, including the following steps:

[0035] 1) Add 5ml of a mixed solution of penicillin, streptomycin and amphotericin B (the content of penicillin is 10kU / ml, the content of streptomycin is 10mg / ml, and the content of amphotericin B is 25μg / ml) into the T75 bottle. Spray disinfectant around;

[0036] 2) Select the urine of the mitochondrial mt.3243A>G mutant population, and use the T75 bottle in step 1) for urine collection;

[0037] 3) Open the T75 bottle, add 50ml of urine sample to each of the four 50ml centrifuge red tubes, and centrifuge;

[0038] 4) Use a pipette to suck up the supernatant to less than 5ml in each red tube, add 15ml~20ml PBS to the first red tube, pipette and mix well, then transfer the resuspension to the next red tube, pipette again Mix well, and so on, until finally all the sediments of the tubes are collected in one tube, and centrifuge...

Embodiment 2

[0054] Example 2 Membrane Potential Detection

[0055] The amount of JC-1 staining working solution is 1ml, take an appropriate amount of JC-1 (200X), and dilute JC-1 by adding 8ml of ultrapure water to every 50μl of JC-1 (200X). Vigorously dissolve Vortex and mix JC-1 thoroughly. Then add 2ml of JC-1 staining buffer (5X), and mix well to get the JC-1 staining working solution.

[0056] a. For one well of the six-well plate, suck off the culture medium, wash the cells once with PBS or other appropriate solution if necessary according to the specific experiment, and add 1ml of mesenchymal stem cell culture medium. Cell culture medium may contain serum and phenol red.

[0057] b. Add 1ml JC-1 staining working solution and mix well. Incubate at 37°C for 20 minutes in a cell culture incubator.

[0058] c. During the incubation period, prepare an appropriate amount of JC-1 staining buffer (1X) by adding 4ml of distilled water to every 1ml of JC-1 staining buffer (5X), and place...

Embodiment 3

[0062] Example 3 ROS detection

[0063] Loading probes after collecting urine mesenchymal stem cells: Dilute DCFH-DA with serum-free medium according to 1:1000, so that the final concentration is 10umol / L.

[0064] Centrifuge the cells at 600g for 4min (or 3000rpm / 5min), remove the supernatant, suspend the cells in diluted DCFH-DA (1ml), the cell concentration is 10^6-2*10^7 / ml, and culture the cells at 37°C Incubate in the box for 20 minutes. Mix by inverting every 3-5 minutes to make the probe fully contact with the cells. The cells were centrifuged at 600 g for 4 min, and the cells were washed (centrifuged, broken, and blown) three times with serum-free cell culture medium to fully remove DCFH-DA that did not enter the cells. Add about 500ul (depending on the cell density) of culture medium, blow and mix well, smear 10ul sample onto the glass slide, and observe under the fluorescence microscope. Each sample has three fields of view, and each field of view is first shot wi...

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