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CBH culture medium for inducing chicken preadipocyte differentiation and differentiation method thereof

A preadipocyte and culture medium technology, which is applied to the CBH medium for inducing chicken preadipocyte differentiation and its differentiation field, can solve the problems of inability to promote expression levels, preadipocyte function defects, and inability to promote lipid droplet deposition, etc., to achieve The effect of simple operation and low cost

Inactive Publication Date: 2016-12-07
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The study found that adding the PPARβ / δ agonist GW501516 to the hormone mixture could not promote the expression level of aP2 in chicken preadipocytes, but adding the PPARβ / δ agonist GW501516 and the PPARγ agonist troglitazone to the hormone mixture at the same time could promote the aP2 expression level in chicken preadipocytes. The expression level of aP2 and the activity of glycerol-3-phosphate dehydrogenase (GPDH) in preadipocytes could not promote the deposition of lipid droplets. Therefore, the differentiation of preadipocytes induced by this method is functionally still flawed

Method used

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  • CBH culture medium for inducing chicken preadipocyte differentiation and differentiation method thereof
  • CBH culture medium for inducing chicken preadipocyte differentiation and differentiation method thereof
  • CBH culture medium for inducing chicken preadipocyte differentiation and differentiation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Isolation and Culture of Chicken Primary Preadipocytes

[0020] Take 10-day-old commercial AA broilers, aseptically collect abdominal adipose tissue, put it into a plate filled with PBS (containing 100 units / mL penicillin and 100 μg / mL streptomycin), rinse repeatedly, and remove fascia and blood vessels as much as possible. Then the tissue was cut with ophthalmic scissors, and then digested in 2 mg / mL type Ⅰ collagenase at 37°C for 65 min (shaking once every 5 min). After the digestion is complete, add growth medium (DMEM / F12 with 10% fetal bovine serum by volume, 100 units / mL penicillin and 100 μg / mL streptomycin) to stop the digestion, pipette pipetting, and pass through 100-mesh and 600-mesh stainless steel sieves respectively net filter. Put the filtrate into 15mL centrifuge tubes, centrifuge at 2000r / m for 10min, remove the culture medium, resuspend the cells with red blood cell lysate to make a cell suspension, incubate at room temperature for 10min, centrifuge a...

Embodiment 2

[0022] Induced Differentiation of Chicken Primary Preadipocytes

[0023] When the chicken preadipocytes reached 50% confluency in the growth medium, the cells were divided into 2 groups (control group and induction group), the control group still used the growth medium, while the induction group replaced the growth medium with CBH medium . The control group and induction group were replaced with fresh growth medium and CBH medium once a day for a total of 6 days of induction. The existing growth medium formula is: DMEM / F12 with volume fraction of 10% fetal bovine serum, 100units / mL penicillin and 100μg / mL streptomycin

[0024] Induction medium contains hormone mixture: 20 μg / mL bovine insulin, 1 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine and 5 μM rosiglitazone.

Embodiment 3

[0026] Comparison of lipid droplet deposition effects

[0027] Discard the culture medium described in Example 2, wash 3 times with PBS, fix with 10% formalin for 30 minutes, wash 3 times with PBS, wash 3 times with distilled water, dry at 32°C, and stain with 1% Oil Red O staining solution for 40 minutes , washed 3 times with PBS, 100% isopropanol was used to dissolve Oil Red O in the cells for 15 min, and the light absorption value was recorded at 510 nm with a microplate reader, and the result was corrected by the number of cells.

[0028] After 6 days of induction, use an inverted microscope to observe the cell morphology of the control group and the induction group, as shown in figure 1 As shown, it can be seen that a large number of lipid droplets were deposited in the cells of the induction group, while there were almost no lipid droplets in the cells of the control group. Such as figure 2 As shown, the oil red O extraction colorimetric results showed that the deposi...

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Abstract

The invention discloses a CBH culture medium for inducing the chicken preadipocyte differentiation and a differentiation method thereof. The CBH culture medium is prepared by adding the following components into a conventional culture medium: bovine insulin (20[mu]g / mL), dexamethasone (1[mu]M), 3-isobutyl-1-methyl xanthine (0.5mM), and rosiglitazone (5[mu]M). The differentiation method comprises the following steps: separating and culturing chicken primary preadipocyte; when cells reach 50% confluence in a growth culture medium, dividing cells into a control group and an induction group; culturing the control group in the growth culture medium, while culturing the induction group in a CBH culture medium, respectively changing the cultures every day, and carrying out induction for six days. The result shows that the lipid droplets of the cells in the induction group deposit, GPDH activity and expression levels of aP2, G0S2, and PPAR[gamma]mRNA of the induction group are prominently higher than those of the control group, and the result show that CBH culture medium can induce chicken preadipocyte differentiation.

Description

technical field [0001] The invention belongs to the field of animal cell biology, in particular to a CBH medium for inducing differentiation of chicken preadipocytes and a differentiation method thereof. Background technique [0002] Excessive fat deposition is one of the main problems faced by the modern broiler industry, and the increase in fat content comes from the increase in the number and volume of fat cells. The preadipocyte orientation of pluripotent stem cells and the proliferation of preadipocytes can lead to an increase in the number of adipocytes, and the differentiation of preadipocytes can lead to an increase in the volume of adipocytes. The biological process of preadipocyte differentiation has been extensively studied in the mammalian preadipocyte cell lines 3T3-L1 and 3T3-F442A. Many transcription factors are involved in mammalian preadipocyte differentiation, the most important of which is peroxisome proliferator-activated receptor (PPAR)γ. PPARγ is a li...

Claims

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Application Information

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IPC IPC(8): C12N5/077
CPCC12N5/0653C12N2500/40C12N2501/30C12N2501/33C12N2501/385
Inventor 李辉程博涵
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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