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Cell line based on low-dose nano-TiO2 induction, and preparation method and application thereof

A technology of inducing cells and low doses, applied in the field of cell biology, can solve the problems of uncertain correlation, limited value, large investment, etc., and achieve the effect of eliminating species differences, strengthening characterization research, and long time period

Inactive Publication Date: 2018-12-14
SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The safety research of nanomaterials mainly faces the following problems: (1) The correlation between animal experiments and the human body is not sure enough, and animal experiments are time-consuming and expensive, which is not conducive to high-throughput detection of the toxicity of unknown chemicals; (2) It is difficult to obtain A comprehensive grasp of the physical and chemical properties of itself, many current studies only focus on the health effects of nanomaterials under specific conditions of specific sizes, ignoring their physical and chemical properties such as size, surface area, surface composition, charge, shape, etc.; (3) Exposure experiments of high-concentration nanomaterials The value of the obtained data in toxicological health assessment is limited, and the artificial high-concentration nano environment is not the real exposure level; (4) most of the existing evaluation methods are based on short-term exposure studies, which is not conducive to judging the long-term health effects of nanomaterials on individuals
[0004] Traditional safety assessment techniques cannot be fully applied to nanomaterials. The physical and chemical properties of nanomaterials are very different from those in the macroscopic state, which means that their toxicokinetic and toxicological data cannot be fully used as a reference in the macroscopic state.

Method used

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  • Cell line based on low-dose nano-TiO2 induction, and preparation method and application thereof
  • Cell line based on low-dose nano-TiO2 induction, and preparation method and application thereof
  • Cell line based on low-dose nano-TiO2 induction, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Construction of cell lines

[0026] Select normal human bronchial epithelial cells Beas2B as the source cells to induce and screen new cell lines. The specific steps are as follows: take out the Beas2B cells in liquid nitrogen, quickly place them in a water bath at 37°C and shake them continuously until the Beas2B cells are rapidly thawed; Beas2B cells were transferred into a culture dish, and RPMI-1640 medium including 10% fetal bovine serum, 1% penicillin and streptomycin was added, and the volume ratio of air and carbon dioxide was 19:1 (v / v), and the temperature was 37°C. Cultivate overnight under the same conditions, change the medium after the Beas2B cells adhere to the wall, and continue to culture. When the Beas2B cells grow to a density of 90%, they are passaged; then select Beas2B cells that are growing vigorously and in good condition, washed twice with PBS, and digested with trypsin until Beas2B The cells became round, and the reaction was termin...

Embodiment 2

[0027] Example 2 Cell Growth Curve Determination

[0028] The cells in good culture state were digested with trypsin and counted. According to the standard of 4000 cells in 200 μL cell suspension per well, the cells were evenly seeded in a 96-well plate, and no cells were planted in the microwells on the four sides of the 96-well plate to culture Liquid seal. Place in an incubator at 37°C, 5% CO 2 Cultivate, add 10uLCCK-8 reagent after 6h, incubate under cell culture conditions for 3-4h, measure the absorbance value at 450nm, and add CCK-8 reagent at the same time for 4 consecutive days, measure the absorbance value at 450nm at the same time, and The absorbance value on the first day was normalized as 100%, and the following days were normalized according to the absorbance value of different cells on the first day, and the growth curve was drawn, see appendix figure 1 , for different low-dose nano-TiO 2 The Beas2B cell proliferation changes after long-term treatment of Be...

Embodiment 3

[0031] Example 3 Cell Migration Test

[0032]Add 500uL of medium to the 24-well plate, put the transwell chamber into it, and incubate in a 37°C incubator for 30min, trypsinize and count the cells in good culture state, and use serum-free medium at a rate of 70,000 per 200uL suspension. A standard of 1 cell was added to the transwell chamber, and placed in an incubator at 37°C, 5% CO 2 Cultured, stained with eosin or 0.5% crystal violet for 10 min after 18 h, and stained with ddH 2 O Rinse, wipe off the upper layer of cells with a cotton swab, and take pictures after drying, as attached Figure 4 As shown in A, nano-TiO 2 The longitudinal migration ability of the cells treated for a long time is reduced; through the scratch test, the cell monolayer cultured in the 6-well plate is scratched with a line through the sterile wall, and the floating cells are washed away, and observed under the microscope at 24 hours and 48 hours , the result is attached Figure 4 As shown in ...

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Abstract

The invention discloses a cell line based on low-dose nano-TiO2 induction, and a preparation method and an application thereof. The cell line is screened after long-term stimulation of human-derived normal cells by low-concentration nano-TiO2; and the cell line can be used as a cell model to evaluate the potential toxicity and the pathogenic effects of the of nano-TiO2 or screen nutritional intervention components or drugs for reducing the toxicity of the nano-TiO2, and can be used for evaluating the safety of food additives.

Description

technical field [0001] The invention relates to the field of cell biology, in particular to a 2 Long-term low-dose induced cell lines for evaluating the safety of food additives. Background technique [0002] At present, the safety and toxicology research of traditional food is mainly evaluated through animal experiments. However, with the advocacy and implementation of the 3R principle of using experimental animals and the transformation of biomedical research models, overall animal experiments are facing severe challenges. In vitro model research, especially the human cell model based on in vitro culture, has become an important development direction. [0003] The unique nano-effects (size effect, surface effect, quantum effect, self-assembly, etc.) of nanomaterials make their toxicity characteristics significantly different from those of corresponding micron-scale materials. For example, nanomaterials can penetrate biological barriers and enter through the pores of biolo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12Q1/02
CPCC12N5/0625G01N33/5008
Inventor 王慧巴乾贾旭东
Owner SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE
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