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A culture method for improving oxidative metabolism of chicken skeletal muscle cells

A technology of skeletal muscle cells and a culture method is applied in the field of culture for improving the oxidative metabolism capacity of chicken skeletal muscle cells, which can solve the problems of strong randomness of muscle fiber types and low oxidative metabolism capacity, so as to improve the oxidative metabolism capacity and inhibit myoblasts. Apoptosis, the effect of promoting myogenic differentiation

Inactive Publication Date: 2016-08-17
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although researchers can obtain myotubes in vitro by using embryonic myoblasts or adult satellite cells, the types of muscle fibers obtained are highly random and their oxidative metabolism capacity is generally low.
Moreover, most of the existing technologies use genetic engineering to interfere or overexpress specific genes that can cause changes in animal skeletal muscle fiber types or oxidative metabolism levels (Semsarian et al., 1999; Lin et al., 2003; Shinichiro et al., 2008), and there is no shortage of methods based on Non-transgenic directional induction platform for skeletal muscle cells. In order to further meet the needs of future applications in the fields of medical treatment and animal husbandry production, the establishment of a non-transgenic directional induction platform for skeletal muscle cells has more practical significance

Method used

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  • A culture method for improving oxidative metabolism of chicken skeletal muscle cells
  • A culture method for improving oxidative metabolism of chicken skeletal muscle cells
  • A culture method for improving oxidative metabolism of chicken skeletal muscle cells

Examples

Experimental program
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Effect test

Embodiment 1

[0038] Embodiment 1 Isolation, purification and proliferation of chicken skeletal muscle myoblasts

[0039] 1. Isolation and purification of chicken skeletal muscle myoblasts

[0040] Fresh eggs were hatched in an incubator at 38°C and 63% humidity for 11 days. After the eggshells are sterilized by 70% ethanol, the eggshells are broken and the chicken embryos are taken out and placed in a petri dish (if the chicken embryos are dead, discard them to prevent contamination). Use tweezers to remove the breast skin of the chicken embryo, separate the pectoralis major muscle, and remove ligaments, connective tissue and blood vessels under a dissecting microscope. Wash the pectoralis tissue 3 times with Hank's solution, shred it thoroughly, add 0.1% collagenase I and incubate at 37°C for 30 minutes, blowing several times during the period. Centrifuge, add Hank's to blow the cells, and filter the cells with a 200-mesh stainless steel filter to obtain a single-cell suspension.

[00...

Embodiment 2

[0044] Embodiment 2 Isolation and cultivation of chicken skeletal muscle myogenic fibroblasts

[0045] Fresh eggs were hatched in an incubator at 38°C and 63% humidity for 11 days. After the eggshells are sterilized by 70% ethanol, the eggshells are broken and the chicken embryos are taken out and placed in a petri dish (if the chicken embryos are dead, discard them to prevent contamination). Use tweezers to remove the breast skin of the chicken embryo, separate the pectoralis major muscle, and remove ligaments, connective tissue and blood vessels under a dissecting microscope. Wash the pectoralis tissue 3 times with Hank's solution, shred it thoroughly, add 0.1% collagenase I and incubate at 37°C for 30 minutes, blowing several times during the period. Centrifuge, add Hank's to blow the cells, and filter the cells with a 200-mesh stainless steel filter to obtain a single-cell suspension. The cells were seeded on a culture dish, and after culturing at 37°C for 2 hours, the a...

Embodiment 3

[0047] The differentiation culture of embodiment 3 chicken skeletal muscle myoblasts

[0048] In order to avoid physical contact between myoblasts and fibroblasts during the culture process, and at the same time exert the myogenic effect of myogenic fibroblasts, this example uses a Transwell cell culture system with a 1um pore size PET membrane. The operation steps of myoblast differentiation culture include: using 2000 cells / cm 2 The density of myogenic fibroblasts (Example 2) through 3-5 passages was inoculated in a Transwell culture dish, and the medium was high-sugar DMEM containing 10% FBS, 100U / ml penicillin, and 100ug / ml streptomycin After culturing at 37°C for 1 day, remove the above-mentioned medium and wash 3 times with PBS; transfer the Transwell Insert (step 2 of embodiment 1) inoculated with chicken skeletal muscle myoblasts into the above-mentioned Transwell culture dish, and along the inner wall of the culture dish Add slowly containing 60umol / L H 2 o 2 Myoge...

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Abstract

The invention discloses a cultivation method for improving the oxidative metabolism ability of chicken skeletal muscle cells, which belongs to the field of biotechnology. The method provided by the present invention is to inoculate the separated and purified chicken skeletal muscle myoblasts and myogenic fibroblasts in the upper chamber and the lower chamber of the Transwell culture dish respectively, and the two are separated by a PET membrane with a pore size of 1 μm but shared. There is H 2 o 2 myogenic differentiation medium; Myotubes with voluntary contraction ability were obtained after myogenic differentiation induction culture for 7-10 days, during which low-temperature shock stimulation was performed every 8-12 hours. The method provided by the invention can significantly increase the mitochondrial content of chicken skeletal muscle cells, the transmembrane potential and the activity of aerobic metabolism enzymes, while reducing the activity of ATPase. This method has the characteristics of short cultivation period and stable effect, and it has laid a foundation for the study of chicken skeletal muscle energy metabolism, fiber type formation mechanism, etc., and has good research and application value.

Description

technical field [0001] The invention relates to a cultivation method for improving the oxidative metabolism ability of chicken skeletal muscle cells, belonging to the field of biotechnology. Background technique [0002] The formation of skeletal muscle is a continuous physiological process. During the embryonic period, skeletal muscle myoblasts proliferate, differentiate, fuse into primary myotubes and gradually mature; after birth, although the number of muscle fibers does not change, when the muscle tissue is damaged, the resting satellite cells are activated and then divide , fusion to form new myotubes, and skeletal muscle to regenerate. Relying on modern cell separation and culture techniques, researchers can use embryonic myoblasts or adult satellite cells to obtain primary myotubes in vitro. [0003] Various movements of animals depend on the contraction of skeletal muscle, and the energy necessary for muscle contraction comes from ATP, skeletal muscle myosin Ca 2...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/073
Inventor 张宇李海刘忠华连正兴
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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