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Cell strain subjected to long-term passage after gamma ray irradiation of human skin fibroblasts and construction method of cell strain

A technology of fibroblasts and gamma rays, applied in the field of biomedicine, can solve the problems of not being able to better reveal the reasons for changes in the pathological mechanism of cell irradiation, the method of cell model construction has not been studied in depth, and the value of cells is limited. The effect of high scientific research and production application value

Inactive Publication Date: 2020-01-10
SHANGHAI CHANGZHENG HOSPITAL +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditionally used gamma ray irradiation model mostly uses directly irradiated cells, and the cells are not passaged after irradiation. In this way, the irradiated cells are directly detected and intervened, and only the physiological and biochemical changes of the cells after acute radiation damage are obtained. As a result, this is not consistent with the reality of chronic wound non-healing in clinical practice, so it has limited value in guiding the research on the mechanism of long-term cell damage after irradiation, and cannot better reveal the reasons for the changes in the pathological mechanism of cell irradiation, while long-term can The cell model irradiated by gamma rays has not been reported yet, and the method of constructing the cell model has not been studied in depth.

Method used

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  • Cell strain subjected to long-term passage after gamma ray irradiation of human skin fibroblasts and construction method of cell strain
  • Cell strain subjected to long-term passage after gamma ray irradiation of human skin fibroblasts and construction method of cell strain
  • Cell strain subjected to long-term passage after gamma ray irradiation of human skin fibroblasts and construction method of cell strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Establishment and verification of stable long-term passage human skin fibroblast (HFF-1) cells after irradiation

[0026] Human skin fibroblast (HFF-1) cells were recovered and cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin dual antibiotic solution at 37°C. , 5% CO 2 Cultivate in an incubator; when the degree of cell mixing reaches 90%, use 1ml of trypsin containing EDTA (the mass concentration of EDTA is 0.25%) to digest, after digesting for 5min, use 2ml of culture medium to stop the digestion of cells in the culture flask, and put the culture flask After the 3ml liquid in the medium was repeatedly pipetted evenly, it was transferred into a 15ml centrifuge tube and placed at a Co60γ-ray source for irradiation, with a total irradiation dose of 10Gy. After the irradiated cells were taken back to the cell room, they were laid flat in the culture flask and continued to be cultured. After the cells reached more th...

Embodiment 2

[0027] Example 2: Morphological observation of long-term passage human skin fibroblast (HFF-1) cells after irradiation

[0028] Inverted phase-contrast microscope was used to observe the morphology of living cells: human skin fibroblasts (HFF-1) control cells in the logarithmic growth phase and human skin fibroblasts (HFF-1) irradiated cells were taken respectively, washed with normal saline and replaced in the The living cell morphology of the first and third passage cells was observed under an inverted microscope (×200 times). The cell morphology of the first generation of cells did not change significantly after gamma ray irradiation. The density gradually decreased, the boundary was blurred, irregular vesicles appeared in the cytoplasm of some cells, and the cell membrane became transparent and bright, showing a pre-apoptotic shape ( figure 2 ).

Embodiment 3

[0029] Embodiment 3: Determination of the proliferation curve curve of long-term passage human skin fibroblast (HFF-1) cells

[0030] Normal control cells and irradiated mouse embryonic fibroblasts were digested with trypsin and then added a suitable new medium to resuspend the cells, according to 5×10 3 The concentration of cells / ml was inoculated into a 96-well plate, and each of the control group, the first generation and the third generation of the irradiation group had 5 controls to ensure that the cells were evenly dispersed in each culture well, and placed in the incubator overnight , to be detected after it adheres to the wall. The method is to add 10 μl of CCK-8 solution to each well, incubate in a 37°C incubator for 1 hour, detect the absorbance OD value at a wavelength of 476nm, and measure continuously for 5-7 days according to the actual growth of the cells. SPSS 19.0 software system was used for statistical analysis, and P﹤0.05 was considered statistically signi...

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Abstract

The invention relates to the technical field of biomedicine, in particular to a cell strain subjected to long-term passage after gamma ray irradiation of human skin fibroblasts (HFF-1) and a construction method of the cell strain. According to the invention, human skin fibroblasts (HFF-1) are used as induction objects, after the induction objects are subjected to a specific dose (10Gy) of gamma rays for irradiation and mouse embryo fibroblasts irradiation, the cell proliferation is slowed down, apoptosis is increased, the migration capacity is weakened, and the change of the characteristics ofthe cells is not changed along with the increase of cell passage number and can be inherited to the next generation along with the passage of the cells. The cell model is closer to clinical practiceof long-term non-healing of wounds after skin squamous cell carcinoma radiotherapy in practical application, and has important application prospects in the aspects of research of pathological mechanisms of non-healing of wounds after radiotherapy, research and development of new drugs and the like.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a human skin fibroblast (HFF-1) gamma ray-irradiated cell strain capable of long-term passage and a construction method thereof. Background technique [0002] Radiotherapy refers to the treatment method that uses the effect of radiation ionizing radiation (Ionizing Radiation, IR) to kill tumor cells, and plays an important role in the treatment of malignant tumors of the head and neck. According to literature statistics, there are about 5,492,200 new cases of head and neck cancer in the world every year, and more than 50% of the patients need to receive radiotherapy during treatment. Clinically, in addition to the therapeutic effect of radiotherapy, the complications often caused by radiotherapy are acute skin damage (mainly manifested as skin erythema, dry desquamation, hair loss, ulcers, etc.) and skin wound healing disorders after surgery. The latter mainly manifests as d...

Claims

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Application Information

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IPC IPC(8): C12N5/077C12N13/00G01N33/50
CPCC12N5/0656C12N13/00C12N2509/00G01N33/5005
Inventor 孙鹏张亮王国栋赵云富何文王晓莉
Owner SHANGHAI CHANGZHENG HOSPITAL
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