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Mouse embryo fibroblast malignant transformant and application thereof

A technology of fibroblasts and mouse embryos, applied in the fields of biology and oncology, can solve the problems of lack of dynamic interaction and inaccurate reflection of tumor growth, and achieve good application prospects

Pending Publication Date: 2022-02-01
ZHEJIANG MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that the tumor microenvironment in vivo plays an important role in the proliferation, differentiation, metastasis, and drug resistance of tumor cells, while the two-dimensional culture system lacks the dynamic interaction between cells and cells and between cells and extracellular matrix in the tumor microenvironment in vivo. function, and cannot accurately reflect the real growth of tumors in the body
[0007] The three-dimensional cell culture (3DCC) technology developed in recent years can make up for the above shortcomings, but there is no report on the application of three-dimensional cell culture technology to the malignant transformation of chrysotile-induced cells

Method used

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  • Mouse embryo fibroblast malignant transformant and application thereof
  • Mouse embryo fibroblast malignant transformant and application thereof
  • Mouse embryo fibroblast malignant transformant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038]Fiber preparation: Chrysotile was obtained from the Japan Mineral Fiber Association. When in use, weigh 5 mg of chrysotile fiber and suspend it in 1 ml of PBS solution to make a 5 mg / ml mother solution, and ultrasonically treat it in ice water to form a uniform suspension. Ultrasonic conditions: 180W, 10s on, 5s off, a total of 30 cycles. Autoclave after sonication.

[0039] Cell culture: Mouse embryonic fibroblasts (NIH / 3T3) were cultured in DMEM containing 10% fetal bovine serum at 37°C, 5% CO 2 Cultured in a saturated humidity environment, the cells were subcultured once every 3-4 days, and inoculated at a ratio of 1:3. The cells were divided into a transformation group and a negative control group, and 3 wells were set up in each group as a parallel control.

[0040] Asbestos-induced cell transformation under 3D conditions: 1 day before the experiment, put Matrigel in a 4°C refrigerator overnight. On the day of the experiment, NIH / 3T3 cells were digested with try...

Embodiment 2

[0042] CCK8 assay measures cell viability.

[0043] Discard the medium for the cells, add 110 μL of a mixture of CCK-8 reagent and culture medium (volume ratio: 1:10) to each well, and store at 37°C, 5% CO 2 After continuing to incubate in the incubator for 3 h, the OD value of each well was detected by a microplate reader at a wavelength of 450 nm, and the cell viability was calculated according to the OD value. The formula is [(ODAs-ODAb) / (ODAc-ODAb)]×100%. Among them, ODAs, ODAc, and ODAb represent the OD values ​​of the experimental group, control group, and blank group, respectively. see results figure 1 , the activity of 3D-Asb cells was higher than that of the control group, but not statistically significant.

Embodiment 3

[0045] Soft agar colony formation assay was used to measure the malignant cell biological characteristics of transformed cells.

[0046] The removed cells were digested with 0.25% trypsin and gently pipetted to make them single cells, and the cells were counted, and the cell density was adjusted to 1000 cells / mL with DMEM medium containing 20% ​​fetal bovine serum. Two concentrations of low-melting point agarose solutions of 1.2% and 0.6% were prepared respectively with distilled water, and maintained at 40° C. after autoclaving. After mixing 1.2% agarose and 2×DMEM medium (containing 2×antibiotics and 20% calf serum) at a ratio of 1:1, pour 1.5mL of the mixture into a 6-well plate, cool and solidify, and use it as the bottom layer Agar with CO 2 Reserve in the incubator. Mix 0.6% agarose and 2×DMEM medium in a sterile test tube at a ratio of 1:1, then add 0.2mL of cell suspension to the tube, mix well, and pour into the bottom layer of 1.2% agarose In the 6-well plate (tha...

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Abstract

The invention discloses a chrysotile-induced mouse embryo fibroblast malignant transformant based on a 3D culture condition and an application thereof, and belongs to the technical field of biology and oncology. The malignant transformant of the mouse embryo fibroblast is classified and named as a mouse embryo fibroblast line NIH3T3 / 3As-T, and the preservation number of the malignant transformant of the mouse embryo fibroblast is CCTCC NO: C2021138. The mouse embryo fibroblast malignant transformant provided by the invention is obtained by carrying out malignant transformation on mouse embryo fibroblasts subjected to chrysotile multi-stage multi-time contamination treatment under a 3D culture condition. The 3DCC simulates a microenvironment for in-vivo tumor cell growth, and the constructed mouse embryo fibroblast malignant transformant has morphological and cytobiological characteristics similar to in-vivo tumor cells, and has good application prospects in a plurality of aspects such as a cell model for studying a carcinogenic mechanism of carcinogens.

Description

technical field [0001] The invention relates to the technical fields of biology and oncology, in particular to a mouse embryonic fibroblast malignant transformation strain induced by chrysotile based on 3D culture conditions and its application. Background technique [0002] Carcinogenicity is a toxic effect with serious consequences. Therefore, the evaluation of carcinogenicity of chemicals is an extremely important, prudent and complicated task. There are currently methods for evaluating the carcinogenicity of chemicals to humans, such as population epidemiological investigations, mammalian There are three types of long-term carcinogenicity experiments and in vitro experiments. Before long-term animal experiments, mutagenesis experiments, cell malignant transformation experiments and short-term mammalian carcinogenicity experiments can be carried out to conduct preliminary speculation and screening on the carcinogenicity of chemicals, among which cell malignant transformat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077C12Q1/02C12R1/91
CPCC12N5/0656G01N33/5017G01N33/5044C12N2513/00C12N2503/02C12N2500/05G01N2500/10
Inventor 张芳芳朱丽瑾苑修源张敏肖芸鞠莉余珉应士波高雅楠
Owner ZHEJIANG MEDICAL COLLEGE
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