Nucleotide molecule SR2B and application of the same in producing antidiabetic medicament
A nucleic acid molecule and anti-diabetic technology, applied in the field of biomedicine, can solve problems such as anti-diabetic gene drugs that have not been reported yet
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Embodiment 1
[0033] Example 1 Effect of siRNA interference on endogenous BRSK2 expression on insulin secretion
[0034] (1) Dilute NIT cells at 1×10 5 Cells / well were seeded on a 24-well plate, and the cells were placed in CO 2 Cultivate in an incubator at 37° C. for 18-24 hours to make the cell abundance reach 70-80%.
[0035](2) Invitrogen lipofectamine 2000 eukaryotic transfection kit was used for transfection. 5 μl of Nonsilence (negative control for siRNA interference), 1 μl, 3 μl and 5 μl of siRNA (GCUGCACGACGUUUUAUGAAdTdT and dTdT UUCAUAAACGUCGUGCAGC)-5 (concentration of 20 μM per μl) were transfected respectively. Four wells were replicated for each transfection.
[0036] (3) 16 hours after transfection, replace with complete medium (DMEM high glucose medium + 15% fetal calf serum) and continue to culture for 24 hours.
[0037] (4) The complete medium (sugar concentration: 25 mM) was replaced again, and the culture supernatant was collected after culturing for 20 minutes.
[0...
Embodiment 2
[0043] Example 2 Effect of BRSK2 overexpression on insulin secretion
[0044] (1) Dilute NIT cells at 1×10 5 Cells / well were seeded on a 24-well plate, and the cells were placed in CO 2 Cultivate in an incubator at 37° C. for 18-24 hours to make the cell abundance reach 70-80%.
[0045] (2) Invitrogen lipofectamine 2000 eukaryotic transfection kit was used for transfection. Two plasmids, Pcmv-Myc empty vector and Pcmv-Myc-BRSK2, were transfected respectively, and each plasmid was transfected into 4 wells at a concentration of 200ng / well.
[0046] (3) 16 hours after transfection, replace with complete medium (DMEM high glucose medium + 15% fetal calf serum) and continue to culture for 24 hours.
[0047] (4) The complete medium (sugar concentration: 25 mM) was replaced again, and the culture supernatant was collected after culturing for 20 minutes.
[0048] (5) The concentration of insulin in the culture supernatant was measured by radioimmunoassay. The detection kit was pro...
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