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Method for establishing tree shrew immortalized skin fibroblasts

A fibroblast and establishment method technology, applied in the field of cell immortalization construction, can solve problems such as research obstacles, slow cell proliferation, and impact on experimental efficiency, and achieve the goals of reducing changes and impacts, strong cell proliferation ability, and saving culture costs Effect

Pending Publication Date: 2021-11-12
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The team previously isolated primary tree shrew skin fibroblasts. However, the primary cells often only grow steadily for a few specific generations. Proliferation slows down, and it is easy to miss the optimal growth period of cells during the experiment, which is a big obstacle for the construction of cell models related to skin diseases and the research of related mechanisms
At the same time, the primary cells isolated from each batch will inevitably be different, and it is almost impossible to repeat the experiment, and it takes a long time, which will greatly affect the efficiency of the experiment, and the immortalized cells can effectively make up for the above-mentioned shortcomings of the primary cells.

Method used

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  • Method for establishing tree shrew immortalized skin fibroblasts
  • Method for establishing tree shrew immortalized skin fibroblasts
  • Method for establishing tree shrew immortalized skin fibroblasts

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Separation and purification of tree shrew primary skin fibroblasts

[0024] Take a tree shrew about 1 year old, inject 0.5 mL of 3% pentobarbital sodium into its intraperitoneal anesthesia, and then kill it. Disinfect the skin with 75% alcohol, scrape off the hair on the inner thigh of the tree shrew, and cut off the skin of about 2.5 cm × 1.5 cm For the tissue block, scrape and wash 3 times repeatedly in PBS containing 2% penicillin-streptomycin, then cut the tissue block into 1mm pieces 3 Put the left and right blocks into a 50mL centrifuge tube; add 0.1% type Ⅰ collagenase (prepared in PBS), digest in a 37°C incubator for 45 minutes, and shake once every 15 minutes to fully contact the tissue block with type Ⅰ collagenase; digestion is complete Finally, filter the cell suspension with a 100-mesh steel mesh sieve, then centrifuge at 1000rpm for 10min, discard the supernatant and add complete medium to resuspend the cells (complete medium formula: MEM basal ...

Embodiment 2

[0025] Example 2: Establishment of Tree Shrew Immortalized Skin Fibroblasts

[0026] 1. Lentivirus transfection of tree shrew primary skin fibroblasts

[0027]Primary skin fibroblasts in good growth state were treated with 1×10 5 cells / mL inoculated in 6-well plate, 2mL / well, placed in 37℃, 5%CO 2 Cultivate in an incubator. When the confluence of the cells reaches more than 80%, wash them twice with PBS buffer, add 1 mL of culture solution containing polybrene (4 μg / mL), and inoculate HBLV-SV40T-3xflag-PURO at MOI=30 Lentivirus (blank wells are not inoculated), placed at 37°C, 5% CO 2 Culture in an incubator, add 1 mL of culture solution containing polybrene (4 μg / mL) after 4 hours, replace with complete medium without polybrene after 24 hours of cultivation, and place at 37°C, 5% CO 2 Continue culturing in the incubator; after 24 hours, replace the culture medium with a complete medium containing 4 μg / mL puromycin, continue culturing, and replace the medium every 2 days un...

Embodiment 3

[0030] Example 3: Characteristic identification of tree shrew immortalized skin fibroblasts---identification of fibroblast-specific Vimentin protein and immortalized gene SV40T

[0031] Take the 52nd passage cells and dilute to 1×10 5 cells / mL, inoculated in 24-well plate, 500μ / well, and used tree shrew primary brain endothelial cells as a control. Fix the cells for 20 min, wash with PBS 3 times, 5 min each time, permeabilize with 0.5% Triton X-100 (diluted in PBS) at room temperature for 20 min, wash 3 times with PBS, 5 min each time, 20% diluted goat serum blocking solution (use 0.01 mmol / L diluted in PBS) for blocking for 30min, washed with PBS for 3 times, 5min each time, adding 1:500 mouse anti-vimentin monoclonal antibody and 1:50 rabbit anti-SV40T monoclonal antibody (primary antibody dilution, each Antibody in 2 duplicate wells), incubate overnight at 4°C, wash with PBS 3 times, 5min each time, add 1:500 goat anti-mouse secondary antibody and donkey anti-rabbit secon...

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Abstract

The invention discloses a method for establishing immortalized tree shrew skin fibroblasts, which comprises the following steps of: separating and purifying tree shrew skin tissues by using an enzyme digestion method to obtain primary tree shrew skin fibroblasts, transfecting the primary cells by using lentiviruses carrying SV40T, and performing passage for more than 50 generations to obtain the immortalized tree shrew skin fibroblasts,in the implementation process of the method, a complete culture medium used for cell culture is an MEM culture medium containing 10% by volume of fetal calf serum, 1% by volume of Penicillin-Streptomycin, 1-3% by volume of NaHCO3 and 1-3% by volume of non-essential amino acid; the cells obtained by the method can be stably passed to more than 50 generations, the stable growth state and multiplication capacity of the tree shrew skin fibroblasts are effectively maintained, the cells can be taken once being used, and the skin fibroblasts capable of being stably passed are provided for research on human skin related diseases.

Description

technical field [0001] The invention relates to a method for establishing tree shrew immortalized skin fibroblasts, and belongs to the technical field of cell immortalization construction. Background technique [0002] Skin fibroblasts are a relatively common type of cells in the reticular dermis, which can secrete and deposit a large amount of integrins and extracellular matrix (ECM), including collagen, elastic fibers, binding proteins, etc. . Due to the ability to secrete a series of ECM, dermal fibroblasts play a vital role in various skin repair processes such as skin aging, photoaging, inflammation, skin burn or mechanical damage repair, skin scarring, tumors and other skin-related diseases. important role. [0003] At present, mouse skin fibroblasts, rat skin fibroblasts, human skin fibroblasts, naked mole rat skin fibroblasts and donkey skin fibroblasts have been isolated, and based on skin fibroblasts of different species Research on skin diseases in various dire...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/62C12N5/071C12N5/10
CPCC12N15/86C07K14/005C12N5/0656C12N2740/15043C12N2800/107C12N2510/04C12N2509/00C12N2509/10C12N2710/22022C07K2319/43
Inventor 陆彩霞奎秀莹代解杰罕园园王文广李娜仝品芬孙晓梅
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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