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Construction method of immortalized human sebaceous gland cell line and application thereof

A construction method and sebaceous gland technology, applied in the field of cell biology, can solve problems such as large cost and workload, difficulty in obtaining specimens, and unfavorable research development, achieve the effect of strong vitality and passaging ability, and expand the value of research and application.

Active Publication Date: 2021-04-06
SHANGHAI DERMATOLOGY HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The primary sebocytes isolated from the human body are affected by the division algebra, and it is difficult to expand them on a large scale for related research. If they want to continue to use them, they must be separated from living skin again and again. It is difficult to obtain specimens, and the cost and workload are huge. conducive to research

Method used

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  • Construction method of immortalized human sebaceous gland cell line and application thereof
  • Construction method of immortalized human sebaceous gland cell line and application thereof
  • Construction method of immortalized human sebaceous gland cell line and application thereof

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Experimental program
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Effect test

Embodiment 1

[0054] Construction method of embodiment 1 cell line

[0055] A method for constructing an immortalized human sebocyte cell line, comprising the steps of:

[0056] 1) Treatment of tissue blocks: cut out human facial skin, soak it in a penicillin-streptomycin double antibody solution with a concentration of 2000IU / ml for 30 minutes, disinfect it with 75% alcohol, and use amphotericin B with a concentration of 2ug / ml Rinse 3 times with HBSS, remove subcutaneous fat under aseptic conditions, and cut skin tissue into 1mm 3 The tissue pieces were soaked in 0.025% neutral protease and 0.01% EDTA, and stored overnight at 4 degrees; using microsurgery, the epidermis was peeled off under a stereo microscope to separate the sebaceous glands;

[0057] 2) Primary culture: transplant the tissue block to a bottom area of ​​25cm 2 In a culture flask, keep the temperature at 37°C and put in 5% CO 2 Culture in the cell incubator for 72 hours, and replace half of the proliferation medium eve...

Embodiment 2

[0065] The construction method of embodiment 2 cell lines

[0066] A method for constructing an immortalized human sebocyte cell line, comprising the steps of:

[0067] 1) Treatment of tissue blocks: cut out human facial skin, soak it in a penicillin-streptomycin double antibody solution with a concentration of 200IU / ml for 30 minutes, disinfect it with 50% alcohol, and use amphotericin B with a concentration of 1ug / ml Rinse 3 times with HBSS, remove subcutaneous fat under aseptic conditions, and cut skin tissue into 1mm 3 The tissue pieces were soaked in 0.1% neutral protease and 0.03% EDTA, and stored overnight at 4 degrees; using microsurgery, the epidermis was peeled off under a stereo microscope to separate the sebaceous glands;

[0068] 2) Primary culture: transplant the tissue block to a bottom area of ​​25cm 2 In the culture flask, keep the temperature at 35°C and put in 10% CO 2 Culture in the cell incubator for 72 hours, and replace half of the proliferation mediu...

Embodiment 3

[0075] The construction method of embodiment 3 cell lines

[0076] A method for constructing an immortalized human sebocyte cell line, comprising the steps of:

[0077] 1) Treatment of tissue blocks: Cut out human facial skin, soak it in a penicillin-streptomycin double-antibody solution with a concentration of 1000IU / ml for 30 minutes, disinfect it with 60% alcohol, and use amphotericin B with a concentration of 4.5ug / ml Rinse 3 times with HBSS, remove subcutaneous fat under aseptic conditions, and cut skin tissue into 1mm 3 The tissue pieces were soaked in 0.01% neutral protease and 0.005% EDTA, and stored overnight at 4 degrees; using microsurgery, the epidermis was peeled off under a stereo microscope to separate the sebaceous glands;

[0078] 2) Primary culture: transplant the tissue block to a bottom area of ​​25cm 2 In a culture flask, keep the temperature at 36°C and put in 8% CO 2 Culture in the cell incubator for 72 hours, and replace half of the proliferation med...

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Abstract

The invention relates to a construction method of an immortalized human sebaceous gland cell line and application thereof. The method comprises the following steps: cutting human facial skin, removing epidermis and subcutaneous tissues, performing separating to obtain sebaceous glands, adding a proliferation culture solution, carrying out primary culture, preparing H-tert lentivirus infected cells, and carrying out continuous passage to 50 generations to obtain stable cell strains, and thus successfully constructing the immortalized sebaceous gland cell strain. Aiming at the characteristics of human sebaceous gland tissue cells, neutral protease and EDTA (Ethylene Diamine Tetraacetic Acid) are adopted to digest tissue blocks, the digested tissue blocks are transplanted into a culture bottle, a proliferation culture solution is added for primary culture, and after virus solution infected cell strains are screened, continuous passage is performed to successfully construct a human sebaceous gland cell line. The human sebaceous gland cell line constructed by the method disclosed by the invention has a continuous passage capability; after stable passage, the cells are in a long shuttle shape, and related cell strain identification proves that the cell line is a Chinese sebaceous gland cell line with stable characteristics and uniform components, and is a good material for researching sebaceous gland related diseases.

Description

technical field [0001] The invention relates to the field of cell biology, in particular to a method for constructing an immortalized human sebaceous gland cell line and its application. Background technique [0002] Human skin has important physiological and protective functions. The sebaceous glands, which are skin appendages, play an important role in ensuring the normal function of the skin. The sebaceous gland is located on the side of the hair follicle and consists of multiple lobes. The periphery of the gland is covered by a basement membrane, and there are undifferentiated flat cells on the basement membrane. Secreted and released, sebum flows into the hair follicle through the sebaceous duct, may be decomposed by the microorganisms in the hair follicle, and then flows out through the hair follicle opening, and spreads on the skin surface through an unexplained mechanism, forming an acidic "sebum film" to moisturize the skin and hair, Natural anti-ultraviolet B, in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/54C12N5/10C12N5/071
CPCC12N15/86C12N9/1276C12N5/0633C12Y207/07049C12N2740/10043C12N2800/107C12N2510/04C12N2509/10C12N2509/00C12N2501/11C12N2501/105C12N2513/00C12N2533/54Y02A50/30
Inventor 王秀丽许德田刘佳
Owner SHANGHAI DERMATOLOGY HOSPITAL
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