Application of mTORC1 in medicines for regulating and controlling functions and conversion of pancreatic beta cells
A technology of β cells and pancreatic islets, applied in the field of diabetes
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Embodiment 1
[0031] Example 1, Construction of GFP-tracked β cell-specific Raptor knockout mice and GFP sorting and purification of β cells tower
[0032] In order to exclude the interference of other endocrine cells in the islets, this example constructed β cell-specific Raptor knockout GFP tracer mice. The β cell-specific Raptor knockout GFP tracer mice were constructed by the CRE-LoxP system and the Rosa26-LoxP-STOP-LoxP-EGFP system, and the purified β cells were obtained by flow sorting technology to exclude the interference of other endocrine cells in the islets.
[0033] Specifically: the currently widely used ROSA26-EGFP is adopted f mouse system. The ROSA26 gene is located on chromosome 6 of the mouse and is an antisense long-chain non-coding RNA gene, which has been shown to be expressed in most tissues and cells. A -LoxP-STOP-LoxP-EGFP-sequence is specifically inserted in this region, usually after the -STOP-sequence is transcribed, RNA synthesis will stop. The ROSA26 mice...
Embodiment 2
[0036] Example 2. Effects of β cell-specific knockout of Raptor on glucose homeostasis and β cell quality
[0037] 2.1 Raptor knockout mice had significantly elevated blood glucose at four weeks
[0038] From 2 weeks after the birth of the mice, the body weight and random blood glucose of the male mice were continuously monitored every 2 weeks, and the 6-hour fasting blood glucose of the mice was monitored from the 4th week until the mice were 12 weeks old. In this experiment, male WT mice (cre Raptor w / w g / g) as control, male knockout mouse βRapKO GFP (ere Raptor lox / lox g / g) is the experimental group. There was no difference in body weight between the two groups of mice during the observation period. At 2 weeks, βRapKO GFP The random blood glucose levels of the two groups of mice were similar to those of the WT group, and there were differences from the 4th week (12.53±0.48vs 8.72±0.17mmol / l, PGFP The blood glucose of mice in the control group continued to rise, whi...
Embodiment 3
[0046] Example 3. Changes in β-cell identity after β-cell-specific Raptor knockout
[0047] for research in βRapKO GFP Whether there is a phenomenon of transdifferentiation in mice, this example explored the 8-week-old mice.
[0048] 3.1 Changes in the properties of β cells after β cell-specific Raptor knockout
[0049] ROSA26-EGFP was used to specifically trace the mice, and the GFP-positive cells in the islets were all derived from β cells. In order to study whether β cells are transdifferentiated into α cells, this example carried out co-staining of GFP, glucagon and insulin, and found that some of the GFP-labeled β cells knocked out of Raptor expressed glucagon. We counted and found that there was no difference in the proportion of GFP- and glucagon-positive cells in GFP-positive cells at 2 weeks (WT: 0.17±0.02% vs βRapKO GFP : 0.17±0.01%). At 5 weeks, the GFP and glucagon double-positive cells in the knockout group mice were significantly increased, while the presen...
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