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Establishment method and application of SMA model mouse bone marrow mesenchymal stem cells

A technology of bone marrow mesenchyme and model mice, applied in the field of biomedicine, can solve the problems of not extensive, difficult to collect, complicated to induce, etc., and achieve the effect of promoting proliferation, low degree of differentiation, and strong proliferation ability

Pending Publication Date: 2022-03-01
NANTONG UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The research on SMN2 function and the screening of therapeutic drugs mainly rely on human cell lines, such as HEK293 and hela cell lines, but SMN1 in these cells has normal function and no SMA-related phenotype, so it is not a good research carrier, and some studies have collected SMA patients Cells derived from the body, and undergo IPS induction transformation, but it is not widely used due to ethical, difficult collection, complicated and expensive induction and other issues; there are more SMA mice with SMN2 transgene, but the mice are born It can only survive for about 10 days, and the dosage method is not easy to control, and the development cost is extremely high

Method used

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  • Establishment method and application of SMA model mouse bone marrow mesenchymal stem cells
  • Establishment method and application of SMA model mouse bone marrow mesenchymal stem cells
  • Establishment method and application of SMA model mouse bone marrow mesenchymal stem cells

Examples

Experimental program
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Effect test

Embodiment 1

[0027] BMMSC extraction method and cell culture identification of SMA neonatal mice

[0028] The tail tips of newborn SMA model mice (P4) were taken for rapid genotype identification (Beyotime, D7283M, Shanghai, China), and the genotype was selected as SMA (smn- / -SMN 20 / 2tg ) mouse BMMSC extraction and separation, anesthetized on ice, soaked in 75% ethanol, took its limbs, stripped the muscles, took femur and humerus bone marrow, and used DME / F12 containing 10%-20% FBS (Cytiva, Hyclone, USA) Culture in complete medium, and change the medium the next day. Observe the cell morphology with an inverted microscope, and digest it with 0.25% trypsin after it is overgrown (70%-80%), and then continue to culture. F3 generation cells were used for subsequent experiments ( figure 1 ).

[0029] in 1x10 6 cells / mL were inoculated in a six-well plate containing small round glass slides, added 1 mL DME / F12 complete medium, mixed well, and cultured overnight in a 37°C, 5% CO2 cell incubat...

Embodiment 2

[0034] RT-PCR and Western blot analysis

[0035] SMA BMMSC cell transfection and total RNA and total protein extraction

[0036] Seed the cells in a six-well plate, wait until the SMA model mouse BMMSC grows to 70%-80%, discard the complete culture, wash with PBS (1x) three times, discard the PBS, add 2mL to each well to complete the culture, and set a blank Control group and experimental group, blank control group was added transfection reagent (Lipofectamine™ 3000 Reagent, Inviteogen, Thermo Fisher Scientific, USA), and experimental group was added transfection reagent + ASO 10-29 (abbreviated as ASO), 37°C, 5 %CO 2 Cultivate in the incubator for 48h. Take the cells transfected for 48 hours, discard the complete medium, and wash 3 times with PBS; add 1mL Trizol (Vazyme, R401-01-AA, Nanjing, China) to each well, collect the total RNA, and extract RNA according to the operation instructions of the Trizol kit , centrifuged, and the concentration of cellular RNA was detected ...

Embodiment 3

[0043] Immunofluorescence detection of SMN protein expression

[0044] BMMSC cells were inoculated in a 12-well plate containing small round glass slides, and immunofluorescence experiments were performed 48 hours after transfection. Take out the 12-well plate from the incubator, discard the complete medium, and wash 2-3 times with PBS (1x); add 500 μL of 4% paraformaldehyde solution to each well, and fix it at room temperature for 40 min; discard the paraformaldehyde solution, and use Wash with PBS 3 times, each time for 10 min; add 500 μL of blocking solution to each well, and keep at room temperature for 2 hours; ; α-TubuLin Antibodyx, Cell Signaling Technology, #3873, 1:100, USA), at room temperature for 2 hours, then overnight at 4°C; the next day, take out the small round slides from the 4°C refrigerator and rewarm for 30 minutes; discard the primary antibody , washed 3 times with PBS, 10 min each time; add secondary antibodies (FITC-labeled goat anti-mouse, beyotime, A...

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Abstract

The invention belongs to the technical field of biomedical treatment, and discloses an SMA model mouse bone marrow mesenchymal stem cell establishment method, which comprises: carrying out genotype identification on an SMA model mouse, selecting an SMA mouse BMMSC with a genotype, carrying out extraction separation, culturing with a culture medium, and carrying out liquid change next day; an inverted microscope is used for observing the morphology of the cells, and after the cells are overgrown, pancreatin is used for digestion for continuous passage culture; f3 generation cells are used for subsequent experiments. The invention further discloses a construction and identification kit of the SMA model mouse bone marrow mesenchymal stem cells, and further discloses application of the SMA model mouse bone marrow mesenchymal stem cells constructed by the invention, the primary culture SMA mouse bone marrow mesenchymal stem cells are utilized for the first time, the primary cells are fusiform and only have two copied SMN2 genes, and the SMN2 genes are copied by the primary cells. No interference of the SMN1 gene exists, and due to the fact that the SMN1 gene comes from the SMA mouse, the SMN1 gene is the most real simulation of the spinal muscular atrophy mouse in vitro.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for establishing bone marrow mesenchymal stem cells of an SMA model mouse and an application thereof. Background technique [0002] Spinal muscular atrophy (SMA) is a rare autosomal recessive neurodegenerative disorder due to degeneration of alpha motor neurons in the anterior horn of the spinal cord, resulting in degeneration of motor neurons and muscle atrophy sick. It is currently one of the most common genetic neurological diseases with infant mortality, with a prevalence of 1 in 6000 newborns. The causative gene of SMA is that after the SMN1 gene mutation loses its function, it cannot produce normal SMN protein, which eventually leads to the occurrence of the disease. In humans, exon 7 of the SMN2 gene homologous to SMN1 is mostly cleaved, leaving only a small amount of SMN full-length gene, and a small amount of SMN has functional protein. There...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775C12Q1/02G01N33/68
CPCC12N5/0663G01N33/6872G01N33/5073C12N2509/00C12N2503/02G01N2333/7055G01N2333/70585G01N2333/70589G01N2333/70596G01N2500/10
Inventor 吴刘成华益民朱顺星刘春王旭邵义祥
Owner NANTONG UNIVERSITY
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