Method for separating and purifying high-purity chicken-precursor intramuscular fat cells and establishing intramuscular-fat-cell-and-muscle-satellite-cell co-culturing system

A technique for intramuscular fat cells and muscle satellite cells, applied in the field of cytology, can solve the problems of uneven distribution of intramuscular fat tissue in chickens, inability to directly collect fat tissue, and limiting molecular regulation mechanism, etc. well-defined effects

Active Publication Date: 2017-04-26
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Unlike domestic animals such as pigs, cattle, and sheep, the adipose tissue in chicken muscle is unevenly distributed and generally invisible, and pure adipose tissue cannot be collected directly; Little difference, only a mixture of cells is obtained
Therefore, it has been unable to effectively isolate high-purity precursor intramuscular adipocytes
This technical problem greatly limits the in vitro research on chicken intramuscular adipocytes, the mutual influence relationship between chicken intramuscular adipocytes and muscle cells and the molecular regulation mechanism, as well as the screening and development of related nutrients or drugs.

Method used

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  • Method for separating and purifying high-purity chicken-precursor intramuscular fat cells and establishing intramuscular-fat-cell-and-muscle-satellite-cell co-culturing system
  • Method for separating and purifying high-purity chicken-precursor intramuscular fat cells and establishing intramuscular-fat-cell-and-muscle-satellite-cell co-culturing system
  • Method for separating and purifying high-purity chicken-precursor intramuscular fat cells and establishing intramuscular-fat-cell-and-muscle-satellite-cell co-culturing system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Isolation and acquisition of high-purity chicken precursor intramuscular fat cells

[0058] 10-21-day-old Beijing oily chickens were bled to death, completely soaked in 75% (V:V) alcohol solution for 5 minutes, and separated cells on a sterile ultra-clean workbench in the cell room.

[0059] Remove the chest skin, subcutaneous fat, and connective tissue using sterile scissors and forceps. The ophthalmology department cut out the pectoralis major muscle, put it into the PBS buffer containing double antibodies (penicillin 100U / ml and streptomycin 100U / ml) and washed it 3 times, then cut the muscle tissue into 1mm 3 After the size of the minced meat, move it into a 10ml centrifuge tube, add PBS solution containing 1% double antibody and let it stand for 1 minute, wait for the muscle tissue to settle, discard the supernatant and floating tissue; then add 9 times the volume of 0.1% Type I collagenase was shaken and digested in a water bath at 37°C for 30 minutes. ...

Embodiment 2

[0062] Identification of chicken precursor intramuscular fat cells obtained in Example 2

[0063] The chicken precursor intramuscular adipocytes obtained in Example 1 were separated, and the chicken abdominal fat preadipocytes obtained by existing conventional methods were used as a control, and were respectively divided into 1 × 10 5 / ml density was seeded in 6-well cell culture plates, when the cells grew to 100% confluence, the cell morphology was observed under a microscope, and the two types of cells were treated with differentiation induction and non-induction. Induced group DMEM / F12 medium (containing 10% FBS, 1% double antibody) added 10 μg / ml INS (insulin) and 1 μmol / L DEX (dexamethasone) and 115ng / ml IBMX (3-isobutyl-1 - methylxanthine) induction differentiation agent, after 96 hours of induction, all cells were tested for lipid content by Oil Red O staining, and each treatment of each cell was repeated in 3 wells. After staining, observe and take pictures under the ...

Embodiment 3

[0066] Proliferation and differentiation ability identification of chicken precursor intramuscular fat cells obtained in Example 3

[0067] The chicken pre-body intramuscular adipocytes obtained by the isolation of Example 1, the chicken abdominal fat pre-adipocytes obtained by the existing conventional method and the abdominal fat pre-adipocytes obtained by the same method as in Example 1 were used as controls, and compared The proliferation and differentiation abilities of the chicken precursor intramuscular adipocytes obtained by this method were analyzed. The three types of cells were seeded in 96-well cell culture plates at a final density of 15% of the cells, and cultured in DMEM / F12 medium (containing 10% FBS, 1% double antibody), and were cultured for 12h, 24h, 48h, 72h and 96h respectively The number of cells was detected by the MTT method, and each treatment was repeated in 8 wells. After 4 hours of MTT staining, DMSO was eluted, and the OD value of the eluate was m...

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Abstract

The invention relates to a method for separating and purifying high-purity chicken-precursor intramuscular fat cells and establishing an intramuscular-fat-cell-and-muscle-satellite-cell co-culture system. The method includes the steps that chicken breast muscle tissue is cut into meat paste, and the meat paste is digested with I-type collagenase and centrifuged; upper-layer mature intramuscular fat cells are sucked, inoculated into a culture bottle and subjected to dedifferentiation treatment, and the chicken-precursor intramuscular fat cells are finally obtained; lower-layer cells obtained after centrifuging are precipitated and resuspended, the cells are purified in a differential-wall-attachment mode, and high-purity muscle satellite cells are obtained; the precursor intramuscular fat cells and the muscle satellite cells are respectively inoculated into a transwell chamber or a matched culture plate, and the co-culturing system is established. By means of the method, the technical bottleneck that the chicken-precursor intramuscular fat cells cannot be independently separated and purified all the time is broken through, and the method can be applied to quantitatively simulating muscular tissue, accurately researching the interactional relationship and the molecular regulation mechanism of the chicken intramuscular fat cells and muscle cells in vitro, and screening, researching and developing related nutrients or medicine.

Description

technical field [0001] The invention relates to the field of cytology, in particular to a method for separating and purifying chicken precursor intramuscular adipocytes and constructing a co-culture system of chicken precursor intramuscular adipocytes and muscle satellite cells. Background technique [0002] Animal fat is mainly distributed around viscera, subcutaneous tissue and muscles. Among them, intramuscular fat (IMF) refers to the lipids deposited inside and outside the endomysium or perimuscular membrane, which can affect muscle quality (tenderness, taste, flavor). Within a certain range, the higher the intramuscular fat content, the better the meat quality; however, excessive accumulation of fat in the belly of chickens will reduce feed utilization efficiency, increase processing costs, and cause certain environmental pollution. Therefore, intramuscular fat and abdominal fat deposition are related to two indicators of meat quality and production efficiency, and the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077
CPCC12N5/0653C12N2502/1335
Inventor 崔焕先文杰赵桂苹刘冉冉郑麦青李庆贺
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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