Method for constructing human dermal fibroblast in-vitro thermal damage model and application of method

A fibroblast, thermal damage technology, applied in the field of cell culture, can solve the problems of weak cell proliferation, low cell yield, and other types of cell contamination.

Pending Publication Date: 2020-08-11
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the long-term in vitro 2D culture of the cell line, the morphology and function of the cells are far from those of the fibroblasts in vivo, resulting in unreliable experimental results
However, the existing primary dermal fibroblast isolation and culture methods have disadvantages such as low cell yield, often containing other types of cell contamination, and weak cell proliferation ability, which greatly limits its application.

Method used

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  • Method for constructing human dermal fibroblast in-vitro thermal damage model and application of method
  • Method for constructing human dermal fibroblast in-vitro thermal damage model and application of method
  • Method for constructing human dermal fibroblast in-vitro thermal damage model and application of method

Examples

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Effect test

Embodiment 1

[0068] Example 1: Isolation and culture method of primary human dermal fibroblasts

[0069] Under the premise of obtaining the verbal consent of the child's family, the discarded skin tissue of the child's circumcision was obtained from the hospital. After carefully removing the fat, blood vessels and mesangium in the skin tissue, wash the skin with PBS. 0.25% Dispase neutral protease, digest overnight in refrigerator at 4°C, rinse with PBS to terminate digestion. The epidermis and dermis were separated with ophthalmic forceps, and then the dermal tissue was completely cut up with ophthalmic scissors. The shredded dermal tissue was attached to the six-well plate, and after being inverted for 1 hour in a 37°C incubator, 1 mL of DFL cell culture medium was added to each well. After 3-5 days, the cells crawled out, and when the confluence of the cells reached 80%, they were digested and passaged with trypsin / EDTA.

[0070] The specific operating procedures are as follows:

[...

Embodiment 2

[0112] Example 2: Construction of In Vitro Thermal Injury Model of Dermal Fibroblasts

[0113] In order to construct the in vitro heat loss model of dermal fibroblasts, we first digested the well-growing P3 amniotic stem cells and pressed 1.5×10 5 Inoculate six-well plates at a density of cells / well at 37°C in 5% CO 2 cultured in an incubator. The next day, after the cells were completely adhered to the wall, the medium was changed to remove unattached cells and dead cells. The temperature of the water bath was adjusted to 41°C, 43°C and 45°C respectively in advance for the experiment. Completely seal the six-well plates containing cells and place them in water baths at different temperatures for heat treatment for 50 minutes. After the heat treatment, the six-well plate was taken out of the water bath, and the culture dish was opened in the ultra-clean bench to change the medium of the cells. After heat treatment for 24h and 48h, the cell growth and death were observed re...

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Abstract

The invention relates to the field of cell culture, and particularly discloses a method for constructing a human dermal fibroblast in-vitro thermal damage model and application of the method. The method mainly comprises the following steps: 1) separating and culturing human dermal fibroblasts; (2) purifying the human dermal fibroblasts: digesting primary culture cells for 1-2 minutes by using 0.05% pancreatin / EDTA according to the difference of the sensitivities of the dermal fibroblasts and the epidermal keratinocytes to pancreatin, wherein dermal fibroblasts are digested; the cells are transferred to a third generation by using the method, so that the dermal fibroblasts with the purity of 100% can be obtained; and 3) constructing the dermal fibroblast in-vitro thermal damage model: placing a culture plate inoculated with the dermal fibroblasts in a 37 DEG C water bath kettle, and carrying out heat treatment for 50 minutes to obtain the dermal fibroblast thermal damage model.

Description

technical field [0001] The invention relates to the field of cell culture, in particular to a method for constructing an in vitro thermal injury model of human dermal fibroblasts and its application. Background technique [0002] As the largest organ of the human body, the skin is the first natural barrier between the human body and the outside world. It plays a role in regulating the body temperature, providing sensation, maintaining a stable internal environment, preventing the entry of microorganisms and chemicals, and preventing dehydration. Normal skin tissue is divided into epidermis, dermis and subcutaneous tissue layers. The dermis is a connective tissue composed of fibroblasts, extracellular matrix, vascular endothelial cells, and skin appendages (hair follicles, sweat glands). Dermal fibroblasts (DFL) secrete collagen and elastin molecules, which provide mechanical strength and elasticity to the skin. [0003] Burns mainly refer to the damage of skin, mucous memb...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12Q1/02A61K35/36A61P17/02
CPCC12N5/0625G01N33/5044A61K35/36A61P17/02C12N2509/00C12N2509/10C12N2503/02G01N2500/10
Inventor 柳全文李婧嫄辛洪波
Owner NANCHANG UNIV
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