Long non-coding RNA and application thereof in diagnosis and/ or treatment of T2DM
A non-coding, therapeutic target technology, applied in the field of genetic engineering, can solve problems such as beta cell developmental defects and impaired glucose tolerance in adult mice
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Embodiment 1
[0077] Example 1 detects the expression of Linpint in normal Balb / c mice
[0078] Extraction of primary islet cells, with HBSS solution (KCl 0.4g, KH2PO 40.06g, MgCl2.6H2O 0.1g, MgSO4.7H2O 0.1g, CaCl2 0.1g, NaCl 8g, NaHCO3 0.35g, NaHPO4.7H2O 0.09g deionized water To IL, PH7.4; prepare collagenase (collagenase V prepared with HBSS solution, 1mg / ml); mice fasted for more than 12h, prepare pentobarbital sodium, and anesthetize mice. , ligated with a thread; cut the heart to bleed, wipe off the blood, find the common pancreatic duct, and intubate; push the syringe first slowly and then quickly. Pancreas filling generally takes 2ml. Peel off the pancreas, put it in a sterilized 50ml centrifuge tube, and then Put it on ice; add collagenase V to 5ml centrifuge tube to 5ml. Bath in 37°C water for 25min; take out the centrifuge tube and put it on ice; vortex the centrifuge tube to vortex the tissue into sand. Add HBSS with serum 2 times the volume of the solution, mix well to stop dig...
Embodiment 2
[0098] Example 2 In db / db mouse islets, the expression of Linpint is down-regulated
[0099] The db / db (diabetes) mouse is a mouse model of T2DM. There is a recessive mutation in the db gene of the mouse autosome 4. It is a spontaneous type 2 diabetic mouse caused by leptin receptor deficiency. It is a good way to study T2DM. animal models.
[0100] The db / db mice began to develop hyperglycemia, hyperinsulinemia, and insulin resistance in about 2 weeks, and then became obese. Generally, after 4 weeks, the blood sugar increased significantly, and the islet beta cells were obviously damaged. After eight weeks, the random blood sugar reached Above 20mmol / L, typical symptoms of diabetes appear. 8-week-old and 12-week-old control mice and db / db mice were simultaneously detected for random blood glucose changes (Fig.2.A): Compared with control mice, the random blood glucose of db / db mice was elevated (P <0.05). The islets of control mice and db / db mice were extracted, and the exp...
Embodiment 3
[0102] Example 3 In mouse cell lines, down-regulation of Linpint inhibits the secretion of insulin
[0103] In order to detect the effect of Linpint on the function of islet cells, Invitrogen Company designed and synthesized three interference sequences of mouse Linpint, and synthesized the corresponding scrambled sequences as a control. We transfected the three interference sequences of Linpint in MIN6 and NIT-1 cells, and detected their interference efficiency by real-time quantitative PCR. The results showed (Fig.4A-B): Compared with the control, the interference efficiency of Linpint siRNA#1 Up to 80±3% and 68±3% (P<0.01).
[0104] The MIN6 cells were planted in a 48-well culture plate, and when the cell density reached 60-70%, the medium was discarded and transfected. After 48h, the GSIS experiment was carried out. Prepare glucose-free Krebs-Ringer bicarbonate (Krb) (115mM NaCl, 4.7mM KCl, 1.2mM MgSO4.7H2O, 1.2mM KH2PO4, 20mM NaHCO3, 16mM HEPES, 2.56mM CaCl2, 0.2% BSA) ...
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