Method for authenticating activity of bone marrow mesenchymal stem cells
A bone marrow mesenchymal and stem cell technology, applied in the direction of material inspection products, measuring devices, instruments, etc., can solve the problems of reduced cell number, low content of BMSCs, complicated operation process, etc., to achieve strong proliferation ability, reduce damage, and simple process Effect
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Embodiment 1
[0022] Example 1 Activity Identification of Bone Marrow Mesenchymal Stem Cells
[0023] (1) Immunofluorescence localization of the expression distribution of GAPDH in BMSCs
[0024] Bone marrow mesenchymal stem cells were sliced, soaked, fixed, membrane ruptured, and sealed, then incubated overnight with GAPDH primary antibody, then incubated with FITC-labeled fluorescent secondary antibody, and then observed and collected images under a fluorescent microscope. The specific operation is as follows:
[0025] first day:
[0026] ①In the culture plate, soak the slides with bone marrow mesenchymal stem cells on which the bone marrow mesenchymal stem cells have climbed up with PBS for 3 times, each time for 3 minutes;
[0027] ②Fix slides with 4% paraformaldehyde for 15 minutes, soak and wash slides with PBS 3 times, 3 minutes each time;
[0028] ③ 0.5% Triton X-100 (prepared in PBS) permeabilized at room temperature for 20 minutes;
[0029] ④ Soak slides in PBS for 3 times, 3 ...
Embodiment 2
[0036] Example 2 Effect of the expression distribution level of GAPDH protein in BMSCs on the therapeutic effect of pulmonary hypertension
[0037] 1. Establishment of animal model of pulmonary hypertension
[0038] Animal-related operations were performed in accordance with international standards, and 180 g male SD mice were selected to inject monocrotaline (MCT, 60 mg / kg) into the tail vein to establish a pulmonary hypertension model for a total of 30 days. From the fourth day of modeling, the cell therapy group was injected with H-GAPDH and L-GAPDH BMSCs (1×10 6 per 0.5mL PBS), and the blank control group was injected with 0.5mL PBS. Rats were sacrificed 30 days after modeling, and the muscularization of pulmonary artery and right ventricular hypertrophy were detected.
[0039] 2. Detection of right ventricular hypertrophy and pulmonary artery muscularization
[0040] The right ventricle and left ventricle were separated from the septum, weighed and recorded respectivel...
Embodiment 3
[0047] Example 3 The influence of the expression distribution level of GAPDH protein in BMSCs on the proliferation ability of BMSCs
[0048] 1. Detection of proliferation ability of BMSCs
[0049] BMSCs with different expression types were digested and made into single-cell suspensions. After counting, single-cell suspensions with a certain concentration were prepared for cell plating. Each sample was set up with 5 to 6 multiple wells, and sterile water or PBS was added to the edge wells. , 37 degrees, 5% CO 2 cultured in an incubator. After 72 hours, treat with CCK-8, detect with a microplate reader, and analyze according to the statistics of the data.
[0050] 2. Experimental results
[0051] Such as Figure 4 As shown, the normal 24h proliferation rate of cells in the high-expression GAPDH (H-GAPDH) group was 1 (1.005±0.046, n=7) for normalization, and the low-expression GAPDH (L-GAPDH) normal 24h proliferation was H-GAPDH (0.909±0.054, n=8); after H-GAPDH cells were s...
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