Application of miR-27 or analog thereof to in vitro restraining of differentiation of mesenchymal stem cells to hypertrophic chondrocyte
A bone marrow mesenchymal and mir-27 technology, applied in the field of cartilage damage drugs, can solve problems such as prone to ossification and affecting the effect of regenerated cartilage repair
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Embodiment 1
[0053] Primary isolation and purification of human bone marrow mesenchymal stem cells:
[0054] Add 5ml of bone marrow fluid from subjects diagnosed as having no blood disease provided by the Hematology Department of Jilin University First Hospital into a centrifuge tube, add 20ml of PBS buffer solution (pH value 7.4) into the centrifuge tube, centrifuge at 1500rpm for 5min, and centrifuge at room temperature 2 times. Aspirate the supernatant and remove the anticoagulant. After centrifugation, add 10ml of culture medium (DME / F12 containing 10% FBS, 10ng / ml bFGF), and then put it into a 10mm culture dish for adherent cells. 2 , 37 ° C incubator 48h after replacement of culture medium. After changing the medium twice, wash away the red blood cells with PBS buffer, and observe the bone marrow mesenchymal stem cells under a microscope (such as figure 1 -A). When the cells were confluent to 90%, they were subcultured, and when they were subcultured to P4, 2×10 5 Each well was ...
Embodiment 2
[0056] Chondrocyte induction and qRT-PCR identification
[0057] The 4th generation bone marrow mesenchymal stem cells prepared in Example 1 were mixed with 5×10 5 cells / ml density suspended in 0.5ml chondrocyte induction medium (high sugar DMEM medium, 1% ITS, 10ng / ml TGF-β3, 100nM dexamethasone, 40mg / ml proline, 25mg / ml ascorbic acid) and transferred to 15ml centrifuge Centrifuge the cells at 1500rpm for high-density cell pellet induction, replace the induction solution every 2 days, fix the cell mass with 4% paraformaldehyde at 3 weeks, and perform HE, Alcian blue staining and safranin O after embedding in paraffin dyeing. The formed cartilage balls were collected on the 7th day, 14th day, and 21d day of cartilage induction.
[0058] For the qRT-PCR identification of miR-27b-3P, the cartilage balls were collected on the 7th day and 14th day of cartilage induction, and the total RNA was extracted to identify the expression of miR-27b-3P.
[0059]The total RNA was extracte...
Embodiment 3
[0069] Bone marrow mesenchymal stem cells transfected with lentivirus carrying miR-27b-3P and identified by qRT-PCR
[0070] Divide the above by 2×10 5 Bone marrow mesenchymal stem cells seeded per well in a six-well plate were cultured for 24 hours, and when the confluence reached about 70%, they were transfected with a lentivirus carrying miR-27b-3P (Jikai Gene, Shanghai). Add the lentivirus (MOI index is 50) into the normal culture medium, divide into miR-27b-3P group, Scramble group and NC group, mix them and place them at 37°C, 5% CO 2 Incubate overnight in the incubator. After 24 hours, the culture medium containing lentivirus was replaced with normal culture medium. After 3 days of transfection, the fluorescent expression was observed under an inverted fluorescent microscope, and detected by fluorescent quantitative PCR (Gene Copoeia, China).
[0071] qRT-PCR detection kit for miRNAs (Gene Copoeia, USA) was used to detect target genes.
[0072] reaction system
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