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Culturing and identifying method of transit amplifying fibroblast

A technology of precursor cells and identification methods, which is applied in the field of fibroblast precursor cell culture and identification, can solve the problems of lack of wrinkle-removing plastic culture and identification methods, etc., and achieves the effects of natural curative effect, enhanced thickness and less trauma.

Inactive Publication Date: 2019-04-02
北京华宣医疗美容诊所有限公司
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  • Summary
  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no effective culture and identification method for TAF in improving skin defects and wrinkle removal.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0029] 3. Cultivation of TAF

[0030] Take a small amount of human thigh skin samples and cut them into pieces, wash them in PBS containing double antibodies for 3 times, use sterile tweezers and scissors to cut up the tissues and place them evenly in a petri dish, add 0.2ml of DMEM culture medium (DMEM +10% FBS+100U / ml penicillin+100U / ml streptomycin), at 37°C, 5% CO 2 Cultivate in an incubator, change the medium every 2 days, subculture when the cells cover 85%-95% of the culture dish, measure the growth curve of TAF by the MTT method, and select P 5 -P 10 generation of cells. .

[0031] 4. Detection of TAF-specific marker genes

[0032] Take the cells with normal adherent growth, use the instructions of Trizol reagent to extract the medium RNA, and measure the obtained RNA, select the high-purity ones for subsequent experiments, take 5 μg of RNA and invert in a 20 μl reaction system containing 200UM-MuLV reverse transcriptase 20 μl of reverse transcription products wer...

Embodiment 3

[0038] 5. Cultivation of TAF

[0039] Take a small amount of human thigh skin samples and cut them into pieces, wash them in PBS containing double antibodies for 3 times, use sterile tweezers and scissors to cut up the tissues and place them evenly in a petri dish, add 0.2ml of DMEM culture medium (DMEM +10% FBS+100U / ml penicillin+100U / ml streptomycin), at 37°C, 5% CO 2 Cultivate in an incubator, change the medium every 2 days, subculture when the cells cover 85%-95% of the culture dish, measure the growth curve of TAF by the MTT method, and select P 5 -P 10 generation of cells. .

[0040] 6. Detection of TAF-specific marker genes

[0041] Take the cells with normal adherent growth, use the instructions of Trizol reagent to extract the medium RNA, and measure the obtained RNA, select the high-purity ones for subsequent experiments, take 5 μg of RNA and invert in a 20 μl reaction system containing 200UM-MuLV reverse transcriptase 20 μl of reverse transcription products wer...

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PUM

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Abstract

The invention relates to a culturing and identifying method of transit amplifying fibroblast (TAF). The culturing and identifying method comprises the following steps of in order of culturing TAF; extracting RNA and PT-PCR; performing detecting through a flow cytometer; performing immunofluorescence detecting; inducing polarization; and performing cloning and shaping. According to the culturing and identifying method, the TAF cells obtained by separation are subjected to preliminary evaluation, besides, injection is performed on nude mice, and histology inspection results show that the obtained thigh skin transit amplifying fibroblast can generate I type collagen, can promote the thickness of skin lamination, can reset skin elasticity, and can achieve the purpose of taking materials once,and continuing performing amplification, so that the pain of patients can be reduced. The culturing and identifying method has the advantages that the wound is small, and the treatment effect is natural and durable.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a fibroblast precursor cell culture and identification method. Background technique [0002] With the development of our country's economy, people's quality of life has been improved, and cosmetic plastic surgery technology has also developed rapidly. Various tissue engineering biomaterials are continuously applied to facial cosmetic and trauma treatment. The current development of aesthetic medicine tends to be simple and minimally invasive. and safe direction, and the treatment of facial aging is also transformed from the past search-book treatment to biomedical preparation injection treatment. The current main biological injection preparations include collagen, hyaluronidase, embryonic stem cells and botulinum toxin, etc., except Botulinum toxin acts on facial dynamic wrinkles, and others are suitable for treating fine wrinkles caused by skin aging. Collagen and hyaluronidase are...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12Q1/686G01N15/14G01N33/68
CPCG01N15/14G01N33/56966G01N33/68C12N5/0625C12Q1/686C12N2509/00C12Q2521/107C12Q2565/125
Inventor 张磊
Owner 北京华宣医疗美容诊所有限公司
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