Composition for proliferation, differentiation promotion, or senescence inhibition of stem cells, containing JAK1 inhibitor as active ingredient
A technology of active ingredients and cell proliferation, applied to medical preparations containing active ingredients, organic active ingredients, animal cells, etc., can solve the problems of inhibiting aging restrictions, stem cell survival rate, and rising costs
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Embodiment 1
[0085] Confirmation of resistance to inflammatory aging following treatment with tofacitinib as a JAK1 inhibitor
[0086] Confirmation that aging of stem cells was reduced with tofacitinib treatment
[0087] In order to confirm whether there is an effect of reducing aging of stem cells by tofacitinib treatment, the present inventors confirmed using SA-b-gal staining, which is a representative technique for measuring cell aging.
[0088] More specifically, stem cells were plated in 6-well plates (2×10 4 cell / well) and treated with poly IC (1ug / ml) 1 day later. Tofacitinib (1ug / ml) was pretreated 2 hours before poly IC treatment. After 2 days of poly IC treatment, the degree of aging was measured using a senescence cell shistochemical staining kit (CS0030, Sigma). For this purpose, after washing twice with phosphate buffered saline (PBS), fixation buffer (fixation buffer; 2% formaldehyde, 0.2% glutaraldehyde, 7.04mM Na 2 HPO 4 , 1.47mM KH 2 PO 4 , 0.137M NaCl, and 2.6...
Embodiment 2
[0118] Inflammatory aging resistance confirmed in JAK1 knockout stem cells
[0119] Confirmation of reduction in aging of stem cells following JAK1 knockdown
[0120] The present inventors cloned the JAK1 gide RNA sequence in the plentiCRISPRv2 vector in order to create JAK1 knockout cells. Thereafter, for packaging as lentivirus, the plentiCRISPR-JAK1, pMD2.G and psPAX2 plasmids were transfected into 293T cells. After 3 days of transfection, stem cells were treated with virus-containing medium to infect plentiCRISPR-JAK1 lentivirus. Puromycin selection was performed for 2 days using the puromycin resistance gene present in the plentiCRISPRv2 vector, and then only surviving cells were cultured. Knockout of the JAK1 gene was confirmed by Western blot, sequencing (Western blot - see Figure 3C).
[0121] In order to confirm whether the JAK1-knockout cells produced as described above have an effect of reducing stem cell aging, it was confirmed by SA-b-gal staining, which i...
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