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Application of P glycoprotein to influence of INS-1 832/13 cell insulin secretion

A technology of INS-18721 and insulin secretion, which is applied in the field of P-glycoprotein, can solve problems such as unclear influence of insulin

Pending Publication Date: 2018-11-23
李代清
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there have been more and more studies on the relationship between P-glycoprotein and blood glucose homeostasis in recent years, its influence on insulin secretion has not yet been clarified.

Method used

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  • Application of P glycoprotein to influence of INS-1 832/13 cell insulin secretion
  • Application of P glycoprotein to influence of INS-1 832/13 cell insulin secretion
  • Application of P glycoprotein to influence of INS-1 832/13 cell insulin secretion

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Construction of P-glycoprotein overexpressed insulinoma cells INS-1 832 / 13 cells.

[0022] Prepare the full cell culture medium and common equipment for cell culture and place them in the sterile environment of the cell room. Remove the cell culture medium and gently wash the cells twice with sterile PBS. Add 5mL of cell culture medium, and blow the cells with a 3mL pipette until the wall of the bottle is clear, that is, the cells at the bottom of the bottle have been blown off. Move the suspension to a centrifuge tube, add an appropriate amount of medium, and gently blow and mix the suspension with an electric gun. Move the cell suspension to a centrifuge tube, add an appropriate amount of medium, and then blow with a 10ml pipette gun for 10 minutes and mix well, then spread the suspension on a 6-well plate, grow for 12-24 hours, and wait for the cells to be placed in a logarithmic During the growth period, replace with serum-free medium, and then use diff...

Embodiment 2

[0023] Example 2: Cell viability was detected by MTT method to determine the appropriate concentration of adenovirus and the optimal infection time of adenovirus.

[0024] Cells into a cell suspension, add 100 μL of cell suspension to each well of a 96-well plate, adjust the cell density to 5×10 3 After every 3-5 columns, mix the cell suspension in the "8" pattern, and set up corresponding control wells, that is, only the cell culture medium, MTT, and DMSO groups, and the edge wells of the 96-well plate are used Fill with sterile PBS to rule out edge effects. After 24 hours, different concentrations of adenovirus were added, and 6 replicate wells with 5 gradients were set up. After adenovirus infection, observe the state of the cells under a microscope. Add 10 μL of 0.5% MTT solution (5 mg / ml) to each well, continue to incubate for 4 hours in the dark, and then terminate the MTT incubation, and add 150 μL DMSO to each well, shake on a shaker at low speed for 10 minutes in th...

Embodiment 3

[0025] After embodiment 3P-gp is overexpressed, the efflux pump function of P-gp is significantly enhanced

[0026] After culturing the cells in a twelve-well plate for 24 hours, use 50×10 6 The cells were incubated with Ad-Control and Ad-Abcb1b adenoviruses at pfu / mL concentration, and after the infection, the cells were washed twice with preheated serum-free 1640 culture medium. Add 1 mL of serum-free 1640 culture solution containing 1 μl rhodamine stock solution (1 μg / μl) to each well, and keep the incubator away from light for 3 hours. After incubation, the cells were washed twice with serum-free 1640 to extract cell proteins. Add 220 μL of strong RIPA lysate to each well for lysis for 30 minutes, centrifuge at 12000 r / min for 10 minutes, take the supernatant and add it to a white 96-well plate dedicated to the fluorescence intensity in the dark, add 100 μL of the supernatant to each well; then add rhodamine-prepared For the standard product, under the conditions of λex=...

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Abstract

The invention discloses application of P glycoprotein as a protection factor to influence of INS-1 832 / 13 cell insulin secretion. The action mechanism of the P glycoprotein on rat insulinoma cell INS-1 832 / 13 cell insulin secretion is shown by researches. As found by in-vitro test, the P glycoprotein can further activate a PKA (Protein Kinase A) signal transduction pathway by increasing in-cell cAMP (Cyclic Adenosine monophosphate) content in order to improve the PKA and CERB activity. Meanwhile, the existence of interaction between the P glycoprotein and a lipid raft relevant protein, Caveolinl, an L-shaped calcium ion channel protein 1.2 (Cav1.2), a potassium ion channel protein (Kir6.2) and adenylate cyclase is verified by the experiment, so that the secretion of insulin is influenced.A theoretical basis is laid for the research of pancreatic islet protection factors, and improvement of pancreatic islet transplantation clinical outcome.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a P-glycoprotein capable of affecting insulin secretion of INS-1 832 / 13 cells. Background technique [0002] Diabetes mellitus (DM) is a metabolic disorder characterized by chronic hyperglycemia caused by insulin resistance and / or insufficient insulin secretion, which seriously endangers human life and quality of life. Its pathophysiology is extremely complex, it is prevalent worldwide, and its incidence continues to increase. In 2014, the global incidence of T2DM in people over 18 years old was approximately 9%. In the United States, excluding the impact of accidents, diabetes is the fifth leading cause of death in women and the fourth leading cause of death in men. In China, the incidence rate of diabetes is 9.7% (924 million adults), and the incidence rate of prediabetes is 15.5% (1.4 billion adults). Increased incidence of diabetes is associated with rapid economic developmen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/17A61P3/10
CPCA61K38/1709
Inventor 李代清朱楠楠田琳琳
Owner 李代清
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