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Isolated culture and induction method of nibea albiflora precursor adipose cells

A technique for separating and culturing adipocytes, which is applied in the field of cell culture and can solve problems such as low cell survival rate and small single cell volume

Active Publication Date: 2019-01-11
MARINE FISHERIES RES INST OF ZHEJIANG
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0004] However, when the team of the present invention carried out the adipocyte separation experiment on yellow croaker using the above method, it was found that the amount of single cells in the adipose tissue of croaker croaker obtained by the collagen enzymatic hydrolysis method was very small, and if the concentration of collagenase was increased, the cell survival rate was low
Therefore, the above-mentioned traditional collagen enzymatic hydrolysis method is not suitable for yellow croaker
In addition, the team of the present invention also found through experiments that the traditional induction methods of other fish could not successfully induce the differentiation and maturation of the preadipocytes of croaker

Method used

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  • Isolated culture and induction method of nibea albiflora precursor adipose cells

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preparation example Construction

[0039] The preparation method of the type II collagenase digestion solution is: adding type II collagenase, fetal bovine serum, penicillin and streptomycin to Hanks balanced salt solution containing calcium and magnesium ions; wherein the quality of type II collagenase The fraction is 0.08-0.12%, the volume fraction of fetal bovine serum is 1.2-2.2%, the concentration of penicillin is 90-110IU / mL, and the concentration of streptomycin is 90-110μg / mL.

[0040] 2) After collagenase digestion, use 100-mesh nylon sieve to filter the adipose tissue block of croaker twice, centrifuge the obtained single cell suspension at 700-900rpm for 8-12min, discard the supernatant and supplement with fresh precursor fat Cell culture medium, repeated 3 times; adjust cell concentration to 0.95-1.05×10 6 cells / mL, draw 1mL of single cell suspension and inoculate it in a cell culture flask, shake gently to disperse the cells evenly, let stand for 3-4h, add preadipocyte culture medium to 5mL, and pl...

Embodiment 1

[0047] Isolation and culture method of preadipocytes of yellow croaker.

[0048] Proceed as follows:

[0049] (1) Preparation of cell culture reagents, the steps are as follows:

[0050] ①Metallic croaker preadipocyte culture medium:

[0051] 10% fetal bovine serum, 1mmol / L sodium pyruvate, 2mmol / L L-alanyl-L-glutamine, 0.1g / L N-acetylglucosamine, 100IU / mL penicillin and 100μg / mL chain Mycin DMEM / F12 medium. Adjust the pH to 7.2-7.4 with autoclaved 1M sodium hydroxide solution.

[0052] ② Type II collagenase solution:

[0053] The mass of 0.1g type II collagenase (Thermo Fisher Scientific, USA) was dissolved in 97mL of Hanks balanced salt solution containing calcium and magnesium ions, and 2mL of fetal bovine serum and 1mL of 100× double antibody solution (10000IU / mL penicillin, 10mg / mL streptomycin).

[0054] (2) Pre-coating treatment of cell culture flasks, specific steps: take blood from the tail vein of yellow croaker, get serum by high-speed centrifugation, inactiva...

Embodiment 2

[0076] Induction of croaker preadipocytes (on the basis of Example 1)

[0077] (1) Preparation of croaker preadipocyte induction medium, the steps are as follows:

[0078] ①Preparation of liver lipid extract of yellow croaker:

[0079] Take 10g croaker liver homogenate, 10mL distilled water and 0.1g vitamin E acetate into a 50mL Erlenmeyer flask, seal it with a stopper, place it in a constant temperature oscillator and heat it with water for 1 hour. Wash with hot saturated sodium chloride solution and a small amount of glacial acetic acid for 3 times, and finally wash with distilled water for 1 time, separate the upper layer of grease, blow dry and dry at low temperature.

[0080] Weigh 1 g of fat and add enough 1M sodium hydroxide solution, and heat it with water to make it completely esterified.

[0081] Add 0.2 g of vitamin E acetate and 2.5 g of Tween 20 to the oil treated above, and dilute the volume of DMEM / F12 to 1 L to prepare 100× yellow croaker liver lipid extract....

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Abstract

The invention relates to the technical field of cell culture and discloses isolated culture and an induction method of nibea albiflora precursor adipose cells. The isolated culture comprises the following steps: 1) firstly, digesting nibea albiflora adipose tissue blocks by utilizing a pancreatin solution; then treating the nibea albiflora adipose tissue blocks by utilizing a type II collagenase digestion solution; and 2) inoculating dissociated nibea albiflora precursor adipose cells into a cell culture bottle, and culturing by adopting a precursor adipose cell culture medium to obtain a cellsingle layer. The induction method comprises the following steps: after carrying out digestion treatment on the obtained nibea albiflora precursor adipose cells through the pancreatin solution, inoculating into a 6-pore plate and culturing; and then transferring into a precursor cell induction culture medium and continually culturing. According to the isolated culture and the induction method, the nibea albiflora precursor adipose cells can be successfully obtained and are further induced to obtain mature adipose cells; the isolated culture and the induction method have the advantages of simple and feasible method, good repeatability, low cost and rapid cell growth; and an effective in vitro cell model can be provided for molecular cell biology researches of nutrition metabolism of nibeaalbiflora.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a method for isolating and culturing preadipocytes of croaker carp and inducing them. Background technique [0002] Adipose tissue is an important organ that stores fat in the form of triglyceride. In recent years, with the development of aquatic biology, the understanding of adipose tissue in aquatic organisms has been further deepened. As an important farmed fish in the East China Sea, yellow croaker has been extensively studied on its physiological metabolism, but there are few studies on adipose tissue. [0003] At present, fish fat cell culture is mainly carried out by collagen enzymatic hydrolysis. For example, the Chinese patent application number 201110030738.9 discloses a method for in vitro culture of large yellow croaker fat cells, which includes the following steps in sequence: 1) Take the fat tissue from the abdominal wall of the large yellow croaker; 2) The ad...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077
CPCC12N5/0653C12N2500/30C12N2500/34C12N2500/40C12N2500/80C12N2501/33C12N2501/855C12N2501/999C12N2509/00
Inventor 谭朋楼宝王立改詹炜刘峰谢庆平陈睿毅徐冬冬
Owner MARINE FISHERIES RES INST OF ZHEJIANG
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