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Establishment method of tonalid-induced human normal hepatocyte malignant transformation model

A technology of spitting musk and establishing a method, which is applied in the field of cell biology, can solve the problems that the carcinogenic effect of spitting musk has not been effectively verified, and achieves the effect of strong cell migration ability, simple operation and low cost

Inactive Publication Date: 2020-01-17
XIAMEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The embodiment of the present invention provides a method for establishing a malignant transformation model of normal human liver cells induced by spitting musk, aiming to solve the problem in the prior art that there is no method that can effectively verify the carcinogenic effect of spitting musk on the human body

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  • Establishment method of tonalid-induced human normal hepatocyte malignant transformation model
  • Establishment method of tonalid-induced human normal hepatocyte malignant transformation model
  • Establishment method of tonalid-induced human normal hepatocyte malignant transformation model

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Embodiment 1

[0037]The embodiment of the present invention provides a method for establishing a musk-induced malignant transformation model of human normal liver cells. 10 μg / L, 100 μg / L, 1000 μg / L; (2) Culture L02 normal human liver cells to the logarithmic phase for later use; (3) Divide the L02 normal human liver cells in the logarithmic phase into four groups, respectively group of cells and three groups of transformed cells, among which the cells of the three groups of transformed groups were treated with the prepared 10μg / L, 100μg / L, 1000μg / L musk working solution for 24h, and at the same time, they were treated with DMEM culture solution containing an equal volume of DMSO Treat the control group cells for 24h; the transformed group cells treated by the treated control group cells and the musk working solution of 10 μg / L, 100 μg / L, and 1000 μg / L in step (3) are normally cultured to the 20th generation; ( 4) Get the 10th, 15th, and 20th generations of control cells and transformation ...

Embodiment 2

[0040] In an embodiment of the invention, the above step (1) specifically includes: weighing Tuna musk powder and dissolving it in dimethyl sulfoxide solution (DMSO), preparing a stock solution with a concentration of 1 mg / mL, and suction-filtering the stock solution to obtain Concentration is 1mg / mL Tuna musk mother liquor. The musk musk mother solution with a concentration of 1 mg / mL was diluted with DMEM culture solution into musk musk working solutions with concentrations of 10 μg / L, 100 μg / L, and 1000 μg / L, respectively, and stored at 4°C in the dark for future use.

Embodiment 3

[0042] In the embodiment of the present invention, the above step (3) specifically includes: digesting and centrifuging the L02 normal human liver cells in the logarithmic phase with 0.25% trypsin, collecting the cells, resuspending the normal DMEM culture medium, and dissolving the cells Adjust the density to about 2×10 6 cells per milliliter;

[0043] The cells with adjusted density were divided into 4 groups, one group of control group and three groups of transformed groups were inoculated in 6-well plates, and each group was set up with 3 parallel cells, placed in a 37°C incubator, cultured for 24 hours, discarded Remove the old culture medium;

[0044] To the three groups of transformation group cells that discarded the old culture medium, add the Tuna musk working solution prepared in the step (1) at a concentration of 10 μg / L, 100 μg / L, and 1000 μg / L, and discard the old culture medium. Add DMEM medium containing an equal volume of DMSO to the cells of the control gro...

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Abstract

The invention belongs to the field of cell biology, and provides an establishment method of a tonalid-induced human normal hepatocyte malignant transformation model. The establishment method comprisesthe steps: a tonalid working solution is prepared; L02 human body normal hepatocyte is cultured to the logarithmic phase and then divided into a transformation group and a control group; and cells inthe transformation group are treated through tonalid; the treated cells in the transformation group and the treated cells in the control group are cultured to 20 generations, the anchoring independent growth capacity of the cells is detected through a soft agarose cloning experiment, and then the healing and migration capacity of the cells is detected through a scratch experiment. According to the establishment method provided by the invention, clone cell clusters with the large size and the orderly edge can be observed in the transformation group through the soft agarose cloning experiment,through the scratch experiment, it can be observed that scratches of the clone cells in the transformation group is healed more rapidly than scratches of the cells in the control group, the cell migration capacity is higher, it is effectively verified that the tonalid has a carcinogenic effect on human bodies, and the reference basis is further provided for research of the toxic effect of the tonalid; and the method is easy to operate, convenient and low in cost.

Description

technical field [0001] The invention belongs to the field of cell biology, and in particular relates to a method for establishing a human normal liver cell malignant transformation model induced by musk musk. Background technique [0002] Artificial synthetic musk is a kind of synthetic semi-volatile organic compound. Because of its unique fragrance, good aroma enhancing effect and low price, it has gradually replaced natural musk. It is widely used in perfumes and soaps as flavor and fragrance additives. , shower gel and other daily chemical industries. These compounds have low polarity and strong lipophilicity, so they are difficult to degrade in the environment and easy to bioaccumulate. As it is widely used, a large amount of artificial musk remains in the environment, which has caused environmental pollution to a certain extent, and has caused potential harm to the ecological environment and human health, which has aroused widespread concern from governments and scient...

Claims

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Application Information

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IPC IPC(8): C12N5/09C12N5/071
CPCC12N5/067C12N5/0693C12N2501/999
Inventor 章幼玉陆楠程舜李文柳清伙
Owner XIAMEN UNIV
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