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Culture method for improving oxidative metabolic capability of chicken skeletal muscle cells

A technology for skeletal muscle cells and metabolic capacity, which is applied in the field of cultivating the oxidative metabolic capacity of chicken skeletal muscle cells. It can solve the problems of strong randomness of muscle fiber types and low oxidative metabolic capacity, so as to improve oxidative metabolic capacity and inhibit myoblasts. Effects of apoptosis and promotion of myogenic differentiation

Inactive Publication Date: 2014-09-24
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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  • Culture method for improving oxidative metabolic capability of chicken skeletal muscle cells
  • Culture method for improving oxidative metabolic capability of chicken skeletal muscle cells
  • Culture method for improving oxidative metabolic capability 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 culture method for improving oxidative metabolic capability of chicken skeletal muscle cells, belonging to the technical field of biology. The method provided by the invention comprises the following steps: respectively inoculating separated and purified chicken skeletal muscle sarcoblasts and muscle-derived fibroblasts into an upper chamber and a lower chamber of a Transwell culture dish, wherein the chicken skeletal muscle sarcoblasts and the muscle-derived fibroblasts are separated by a PET membrane having a pore size of 1 mu m but share a myogenic differentiation culture medium with added H2O2; and performing myogenic differentiation induced culture for 7-10 days to obtain myotubes having an independent retraction capability, and performing low-temperature impact stimulation once every 8-12 hours in the culture period. According to the method provided by the invention, the mitochondrion content and transmembrane potential of the chicken skeletal muscle cells as well as the activity of aerobic metabolic enzymes can be obviously improved; and meanwhile, the activity of ATPase can be reduced. The method has the characteristics of short culture period and stable effect, and lays a foundation for researching a chicken skeletal muscle energy metabolism law, a fiber type formation mechanism and the like, thus having favorable 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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IPC IPC(8): C12N5/073
Inventor 张宇李海刘忠华连正兴
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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