Nucleotide molecule SR1B2 and application of the same in producing antidiabetic medicament
An anti-diabetic and nucleotide technology, applied in the field of biomedicine, can solve problems such as anti-diabetic gene drugs that have not yet been reported
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Embodiment 1
[0032] Example 1 Effect of siRNA on insulin secretion after interfering with endogenous BRSK2 expression
[0033] (1) Dilute NIU 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%.
[0034] (2) Invitrogen lipofectamine 2000 eukaryotic transfection kit was used for transfection. 5 μl of Nonsilence (negative control for siRNA interference), siRNA (GAUCGUCAACCGUGAGAAGdTdT and dTdTCUUCUCACGGUUGACGAUC)-51 μl, 3 μl and 5 μl of siRNA (20 μM per μl) were transfected, respectively. Four wells were replicated for each transfection.
[0035] (3) 16 hours after transfection, replace with complete medium (DMEM high glucose medium + 15% fetal calf serum) and continue to culture for 24 hours.
[0036] (4) The complete medium (sugar concentration: 25 mM) was replaced again, and the culture supernatant was collected after culturing for 20 minutes.
[0037] (5...
Embodiment 2
[0042] Example 2 Effect of BRSK2 overexpression on insulin secretion
[0043] (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%.
[0044] (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.
[0045] (3) 16 hours after transfection, replace with complete medium (DMEM high glucose medium + 15% fetal calf serum) and continue to culture for 24 hours.
[0046] (4) The complete medium (sugar concentration: 25 mM) was replaced again, and the culture supernatant was collected after culturing for 20 minutes.
[0047] (5) The concentration of insulin in the culture supernatant was measured by radioimmunoassay. The detection kit was pro...
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