Method for calcification of cancer cells

A technology of cancer cells and seeding density, applied in the field of calcified cancer cells, can solve the problems of affecting mammalian cell activity and lack of cell wall protection, and achieve the effect of inhibiting cell activity and improving biological safety.

Active Publication Date: 2016-06-15
ZHEJIANG UNIV
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  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

In our previous research, we used chemical modification to achieve calcification of yeast cell walls. Calcification would cause yeast cells to enter a dormant period and stop dividing. This is due to the good stress resistance of yeast cells. Compared with mammalian cells, it was found that The cell membrane of mammalian cells is relatively fragile and has no cell wall protection, and some studies have shown that after the siliconization of the cell surface forms a layer of SiO2, the cell's time does not exceed 12h, indicating that the formation of a layer of minerals on the outer layer of the cell membrane will seriously affect the activity of mammalian cells

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  • Method for calcification of cancer cells
  • Method for calcification of cancer cells
  • Method for calcification of cancer cells

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Experimental program
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Embodiment 1

[0034] 1. Preparation of targeting calcified cancer cells—A

[0035] Spread HeLa cells in a 24-well plate, the number of cells per well is about 2×10 5, when the cells grow to about 70% of the bottom area of ​​the orifice plate, the calcification process is started. First pour off the culture medium of the cells, wash with PBS 1-2 times, then add DMEM medium containing 300 μg / mLFA to the well plate, place the cells in the incubator, and adjust the CO 2 The concentration is 5%, the air is 95%, the temperature is 37°C, after incubation for 15min, add 11.8mM Ca 2+ (including newly added 10mMCaCl 2 ) DMEM medium calcification solution, place the orifice plate in a cell culture incubator for calcification treatment for 30 minutes, absorb the calcification solution and add fresh calcification solution to continue the treatment for 60 minutes. The FA and calcification solution were treated alone as a control, and the solution was sucked off after the treatment to obtain calcified ...

Embodiment 2

[0047] Laser confocal microscopy, life-and-death staining and MTT assay were used to detect the killing effect of calcification treatment on cancer cells. The specific operation is as follows:

[0048] 1. Changes after cell calcification

[0049] Before calcification treatment, the cell membrane and nucleus of cancer cells were fluorescently stained with living cell dyes PKH26 and Hoechst33342, respectively. After the calcification treatment, the calcium-specific fluorescent dye calcein was used to fluorescently stain the mineral layer. After the staining was completed, the changes in cell morphology over time were observed under a laser confocal microscope. For the results, see Figure 5 .

[0050] 2. Cell death staining

[0051] (1) HEK293, MCF7 and HeLa cells were treated with 1×10 4 Cells / well were inoculated in 96-well plates respectively, and after 24 hours of culture, the cells were subjected to calcification treatment, and the calcification treatment method was the...

Embodiment 3

[0059] A HeLa tumor animal model was constructed to test the effect of targeted calcification in vivo. The operation and detection process is as follows:

[0060] (1) HeLa cell line with 5×10 6 The amount of one / only was inoculated into nude mice (20 nude mice, female) subcutaneously as a mouse drug resistance model, and after 10 days, the tumor volume grew to 80mm 3 , start the test.

[0061] (2) Inject 200 μL, 1 mg / mL folic acid intraperitoneally, wait for about 15 minutes, wait for the folic acid to accumulate in the tumor site, inject high calcium DMEM medium (50 mMCa 2+ ), injected once a day, treated continuously for 15 days, and observed the effect of tumor calcification. Injection of calcification solution and folic acid alone and blank group were used as controls.

[0062] (3) Use micro-CT technology to scan the tumor, and perform three-dimensional reconstruction after scanning to determine the result of tumor calcification; slice the calcified tumor, perform fluo...

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Abstract

The invention discloses a method for calcification of cancer cells. The method comprises the following steps: (1) cancer cells are inoculated to a folic acid containing medium, incubation is carried out, and cancer cells with folic acid enriched on the surface are obtained; (2) the cancer cells with folic acid enriched on the surface are placed in calcification liquid for incubation, and calcified cancer cells are prepared. The invention also discloses an application of folic acid to preparation of a cancer cell calcification inducer. The method is based on the characteristic that cancer cells highly express folic acid receptors and folic acid carboxyl provides a nucleation site for calcification, calcium phosphate calcified layer is formed on the surface of the cancer cells, so that cell activity is inhibited, cell membrane structures are broken, and final cancer cell death is leaded to; animal experiments show that the calcification treatment does not influence blood cells and does not have hepatotoxicity, the calcification treatment almost does not damage organs and has good biosecurity, so that the technical scheme provides a feasible scheme for tumor treatment.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for calcifying cancer cells. Background technique [0002] Cancer is one of the diseases with the highest mortality rate in the world, and maintains a high incidence rate. At present, the methods of cancer treatment recognized by the international medical community are still traditional chemotherapy and radiotherapy. However, while radiotherapy and chemotherapy drugs kill cancer cells, they will cause serious damage to the body, such as kidney failure, hair loss, weight loss, etc. In addition, tumor metastasis is an important cause of death in cancer patients, and a high proportion of patients die from organ failure caused by tumor metastasis, while chemoradiotherapy drugs cannot effectively inhibit tumor metastasis. According to statistics, the annual cost of cancer treatment is very high, and it is increasing year by year, causing serious economic losses and psychological...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09
CPCC12N5/0693C12N2500/38
Inventor 赵瑞波唐睿康
Owner ZHEJIANG UNIV
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