Application of lycium ruthenicum anthocyanin to preparing medicine for inducing apoptosis of two types of cancer cells

Abstract

The invention discloses application of lycium ruthenicum anthocyanin to preparing a medicine for inducing the apoptosis of two types of cancer cells. A method of the application includes the followingsteps of firstly, culturing four types of cells MCF-7, HepG2, Mac-T and RAW264.7; secondly, continuing to culture the four types of cells with an anthocyanin culture medium and a normal culture medium for 30 hours; thirdly, conducting a cell flow apoptosis experiment and EDU cell proliferation and using a GeminiSEM 300 Zeiss field emission scanning electron microscope and a fluorescent quantitative PCR for the cells in the second step so that it can be proved that the lycium ruthenicum anthocyanin promotes the apoptosis of two types of cancer cells and has no influences on the other two typesof normal cells. The effect of the lycium ruthenicum anthocyanin of regulating and controlling the apoptosis of the liver cancer cells and breast cancer cells is disclosed for the first time. Compared with the prior art, it is proved that the lycium ruthenicum anthocyanin can induce the apoptosis of the human breast cancer cells MCF-7 and the liver cancer cells HepG2 but has no remarkable toxic effect on the cattle mammary gland epithelial cells Mac-T and mouse macrophages RAW264.7, so the lycium ruthenicum anthocyanin can be effectively applied to the research and development of anti-cancerpreparations.

Description

technical field

[0001] The invention relates to the application of anthocyanin from Lycium barbarum in the preparation of medicines for inducing apoptosis of two cancer cells, and belongs to the field of molecular biology. Background technique

[0002] Lycium barbarum anthocyanin is a purple-red pigment extracted from the fruit of Lycium barbarum. It has bright and natural color and no special smell. It is a rare and natural edible anthocyanin pigment, which belongs to bioflavonoids. A water-soluble pigment widely found in plants. Anthocyanins are biologically active compounds present in plant foods with strong antioxidant and anticancer properties. The anti-tumor mechanism of anthocyanins is mainly through anti-mutation, anti-oxidation, anti-inflammation, induction of transformation, and regulation of signal transduction pathways to inhibit tumor cell proliferation, induce cell cycle arrest, promote tumor cell apoptosis, induce autophagy, and resist Tumor invasion and met...

Examples

Embodiment 1

[0031] Embodiment 1: In this embodiment, the anthocyanin concentration and processing time of Lycium barbarum are explored.

[0032] (1) Cell culture: MCF-7 uses DMEM / F12 medium, the proportion of complete medium is 90% medium and 10% fetal bovine serum, cultured at 37°C, CO 2 The concentration is 5%.

[0033] (2) Cell treatment: MCF-7 cells were divided into 5 groups, the first group was the control group, and the 2-5 groups were the treatment groups. Cells were cultured in a six-hole plate. When the cells were cultured to 50%, the treatment groups 2 and 3 were replaced with anthocyanin medium with an anthocyanin concentration of 20ug / mL, and the culture was continued for 20h and 30h respectively; 5. Change to the anthocyanin medium with the anthocyanin concentration of Lycium barbarum 2mg / mL, and continue to culture for 20h and 30h respectively. HepG2 cells were divided into three groups, the first group was the control group, and the second and third groups were the treat...

Embodiment 2

[0035] Example 2: In this example, four kinds of cells were detected by flow cytometry.

[0036] (1) Cell culture: MCF-7 and Mac-T use DMEM / F12 medium, HepG2 uses MEM medium, RAW264.7 uses DMEM medium, and the proportion of complete medium is 90% medium and 10% fetal bovine serum , cultured at 37°C, CO 2 The concentration is 5%. , when the cells were cultured to 50%, the experimental group was replaced with an anthocyanin medium with an anthocyanin concentration of 20ug / mL and continued to cultivate for 30 hours.

[0037] (2) Sample preparation: After 30 hours, the culture medium was discarded, washed twice with PBS buffer, digested with trypsin and centrifuged at low speed to collect the cells. Adjust the cell concentration 1x10 with buffer 6 / 100uL / test; add 5uL Annexin V and 5uL PI, keep away from light for 20min at room temperature; add 500uL buffer to test on the machine.

[0038] (3) Image analysis: Annexin V-FITC and PI are the most commonly used reagents for apoptosi...

Embodiment 3

[0039] Example 3: In this example, the cells were treated with the EDU kit, and the cell proliferation was observed with an immunofluorescence microscope.

[0040] (1) Cell culture: same as Example 2(1).

[0041] (2) Sample preparation: 1) Dilute the EdU solution (reagent A) with the cell culture medium at a ratio of 1000:1 to prepare an appropriate amount of 50 μM EdU medium; add 100 μL of 50 μM EdU medium to each well and incubate for 2 hours, discard the medium; PBS Wash the cells 1-2 times, 5 minutes each time. 2) Add 50 μL cell fixative solution (i.e. PBS containing 4% paraformaldehyde) to each well and incubate at room temperature for 30 minutes, discard the fixative solution; add 50 μL 2 mg / mL glycine to each well, incubate on a decolorizing shaker for 5 minutes, discard the glycine solution; Add 100 μL PBS to each well, wash on a decolorizing shaker for 5 minutes, discard PBS; add 100 μL osmotic agent (0.5% TritonX-100 in PBS) to each well and incubate on a decolorizi...

technical field

[0001] The invention relates to the application of anthocyanin from Lycium barbarum in the preparation of medicines for inducing apoptosis of two cancer cells, and belongs to the field of molecular biology. Background technique

[0002] Lycium barbarum anthocyanin is a purple-red pigment extracted from the fruit of Lycium barbarum. It has bright and natural color and no special smell. It is a rare and natural edible anthocyanin pigment, which belongs to bioflavonoids. A water-soluble pigment widely found in plants. Anthocyanins are biologically active compounds present in plant foods with strong antioxidant and anticancer properties. The anti-tumor mechanism of anthocyanins is mainly through anti-mutation, anti-oxidation, anti-inflammation, induction of transformation, and regulation of signal transduction pathways to inhibit tumor cell proliferation, induce cell cycle arrest, promote tumor cell apoptosis, induce autophagy, and resist Tumor invasion and met...

Examples

Embodiment 1

[0031] Embodiment 1: In this embodiment, the anthocyanin concentration and processing time of Lycium barbarum are explored.

[0032] (1) Cell culture: MCF-7 uses DMEM / F12 medium, the proportion of complete medium is 90% medium and 10% fetal bovine serum, cultured at 37°C, CO 2 The concentration is 5%.

[0033] (2) Cell treatment: MCF-7 cells were divided into 5 groups, the first group was the control group, and the 2-5 groups were the treatment groups. Cells were cultured in a six-hole plate. When the cells were cultured to 50%, the treatment groups 2 and 3 were replaced with anthocyanin medium with an anthocyanin concentration of 20ug / mL, and the culture was continued for 20h and 30h respectively; 5. Change to the anthocyanin medium with the anthocyanin concentration of Lycium barbarum 2mg / mL, and continue to culture for 20h and 30h respectively. HepG2 cells were divided into three groups, the first group was the control group, and the second and third groups were the treat...

Embodiment 2

[0035] Example 2: In this example, four kinds of cells were detected by flow cytometry.

[0036] (1) Cell culture: MCF-7 and Mac-T use DMEM / F12 medium, HepG2 uses MEM medium, RAW264.7 uses DMEM medium, and the proportion of complete medium is 90% medium and 10% fetal bovine serum , cultured at 37°C, CO 2 The concentration is 5%. , when the cells were cultured to 50%, the experimental group was replaced with an anthocyanin medium with an anthocyanin concentration of 20ug / mL and continued to cultivate for 30 hours.

[0037] (2) Sample preparation: After 30 hours, the culture medium was discarded, washed twice with PBS buffer, digested with trypsin and centrifuged at low speed to collect the cells. Adjust the cell concentration 1x10 with buffer 6 / 100uL / test; add 5uL Annexin V and 5uL PI, keep away from light for 20min at room temperature; add 500uL buffer to test on the machine.

[0038] (3) Image analysis: Annexin V-FITC and PI are the most commonly used reagents for apoptosi...

Embodiment 3

[0039] Example 3: In this example, the cells were treated with the EDU kit, and the cell proliferation was observed with an immunofluorescence microscope.

[0040] (1) Cell culture: same as Example 2(1).

[0041] (2) Sample preparation: 1) Dilute the EdU solution (reagent A) with the cell culture medium at a ratio of 1000:1 to prepare an appropriate amount of 50 μM EdU medium; add 100 μL of 50 μM EdU medium to each well and incubate for 2 hours, discard the medium; PBS Wash the cells 1-2 times, 5 minutes each time. 2) Add 50 μL cell fixative solution (i.e. PBS containing 4% paraformaldehyde) to each well and incubate at room temperature for 30 minutes, discard the fixative solution; add 50 μL 2 mg / mL glycine to each well, incubate on a decolorizing shaker for 5 minutes, discard the glycine solution; Add 100 μL PBS to each well, wash on a decolorizing shaker for 5 minutes, discard PBS; add 100 μL osmotic agent (0.5% TritonX-100 in PBS) to each well and incubate on a decolorizi...

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