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Autophagy monitoring method for fat cells

A technology for adipocytes and preadipocytes, applied in botany equipment and methods, biochemical equipment and methods, microbial measurement/testing, etc., can solve problems such as difficult transfection and difficulty in achieving expectations in plasmid transfection

Inactive Publication Date: 2014-06-18
SHANGHAI INST FOR ENDOCRINE & METABOLIC DISEASES
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Problems solved by technology

In addition, due to the characteristics of mature adipose fats that are not easily transfected, ordinary plasmid transfection is difficult to achieve the desired effect

Method used

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  • Autophagy monitoring method for fat cells

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Embodiment

[0015] Culture and subculture of 3T3-L1 preadipocyte cell line

[0016] 3T3-L1 fibroblasts were cultured in normal high-glucose DMEM solution at 37°C, 5% CO 2 In the incubator, observe under the microscope that the cells have adhered to the wall and are spindle-shaped and translucent, and the culture medium is replaced until the cells reach 90% confluence. Aspirate the culture solution from the culture bottle, add 4ml of 0.25% trypsin to digest, and the cells can be seen under the microscope to shrink from irregular polygons or spindles to round shapes, and the process takes about 2 minutes. Add normal culture medium to stop the reaction of trypsin, blow the remaining cells on the bottle wall repeatedly with a pipette to make the cells detach from the culture bottle wall, suck into the centrifuge tube already filled with culture medium, centrifuge at 1000rpm for 3min, and pellet the cells. After centrifugation, discard the supernatant, then add normal culture medium, and blow...

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Abstract

The invention discloses an autophagy monitoring method for fat cells. Due to the adoption of a special structure, mature fat cells are full of plenty of lipid droplets, so that organelle and nucleus are displaced towards the periphery, and a typical autophagosome is hard to be seen under an electron microscope. Proved by a plurality of experiments, the method disclosed by the invention finally and successfully observes autophagy structures in various autophagy stages in mature 3T3-L1 fat cells by continuously improving the experiment condition. Besides, mature fat has a characteristic of being not easy to be transfected, so common plasmid transfection is hard to play an expectant effect. Therefore, the method disclosed by the invention utilizes a GFP-LC3 lentivirus method to infect 3T3-L1 fat cells, and the infection efficiency is as high as 80% or greater by observation under a fluorescence microscope. At nutrition and hunger condition, by the method disclosed by the invention, GFP-LC3 point-like aggregation phenomenon is successfully observed. The establishment of the autophagy monitoring method for mature fat cells lays the foundation of research on the relationship between autophagy and fat metabolism for us.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for monitoring autophagy in adipocytes. Background technique [0002] In 1962, Ashford and Porten discovered the phenomenon of autophagy. Through targeted knockout of genes necessary for autophagy, it was found that autophagy plays an important role in many aspects, such as tumor suppression, neuroprotection, and red blood cell differentiation. It has also been reported that autophagy is involved in the control of growth and development, aging, and tissue homeostasis. Neurodegeneration, tumors, Huntington's disease, Parkinson's disease, myopathy and cardiomyopathy are all involved in the disorder of autophagy regulation, and autophagy also induces type 2 programmed death. [0003] With the "explosive" development of research in the field of autophagy in the past decade, scientists with different professional backgrounds are attracted to enter this field from var...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/02C12N15/867G01N33/68
Inventor 宁光邓玉杰杨颖张志国
Owner SHANGHAI INST FOR ENDOCRINE & METABOLIC DISEASES
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