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Use of reagent in preparing medicine, interference fragment, method for inhibiting self-renewal of liver cancer tumor stem cells and medicine for treating liver cancer

A tumor stem cell and drug preparation technology, which can be used in anti-tumor drugs, botanical equipment and methods, biochemical equipment and methods, etc., and can solve problems such as limited strategies and unclear regulation mechanism of liver cancer stem cells.

Active Publication Date: 2021-08-17
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the regulatory mechanism of HCC CSCs is still unclear, and strategies targeting HCC CSCs are very limited

Method used

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  • Use of reagent in preparing medicine, interference fragment, method for inhibiting self-renewal of liver cancer tumor stem cells and medicine for treating liver cancer
  • Use of reagent in preparing medicine, interference fragment, method for inhibiting self-renewal of liver cancer tumor stem cells and medicine for treating liver cancer
  • Use of reagent in preparing medicine, interference fragment, method for inhibiting self-renewal of liver cancer tumor stem cells and medicine for treating liver cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1 Expression of MCM7 in human liver cancer tumor stem cell spheres (Tumorsphere)

[0065] In this embodiment, to detect the expression of MCM7 in human Tumorsphere:

[0066] 1. Liver cancer cell tumor stem cell sphere culture

[0067] 1. Cell preparation: After digestion and centrifugation of liver cancer cells, resuspend in serum-free DMEM medium, blow and beat repeatedly to form a single cell suspension, and count for later use;

[0068] 2. Preparation of spheroidizing medium: DMEM medium and DMEM / F-12 medium are mixed according to the volume of 1:1, and B27 (1:100), N2 (1:50), 10ng / ml EGF, 5ng / ml bFGF are added , 4μg / ml heparin, 100U / ml penicillin and 100ng / ml streptomycin, the prepared medium can be stored at 4°C for 7 days.

[0069] 3. Add the spheroidizing medium to a low-adsorption six-well plate, inoculate cells at a density of 3000 cells / well, and place them in a cell culture incubator for static culture.

[0070] 2. Fluorescent quantitative real-tim...

Embodiment 2

[0112] Example 2 Effect of MCM7 knockdown on the sphere formation ability of human liver cancer tumor stem cell spheres (Tumorsphere)

[0113] 1. Construction of pSicoR-shMCM7-GFP vector

[0114] 1. Recovery of pSicoR-GFP vector by enzyme digestion: pSicoR-GFP vector was digested with HpaⅠ / XhoⅠ double enzymes, and the enzyme digestion system is shown in Table 3:

[0115] Table 3 enzyme digestion system

[0116] pSicoR-GFP 1μg HpaⅠ 1μl XhoI 1μl 10×CutSmart Buffer 2μl h 2 o

Make up to 20μl

[0117] Digest at 37°C for 4 hours, and recover the DNA fragments using a gel extraction kit.

[0118] 2. Interference sequence annealing and phosphorylation:

[0119] 2.1 Interference sequence: the nucleotide sequence of the following sense oligo is the nucleotide sequence shown in SEQ ID NO: 1, the nucleotide sequence of the anitsense oligo is the nucleotide sequence shown in SEQ ID NO: 2, and the sequence consists of Synthesized by Shangh...

Embodiment 3

[0158] Example 3 Effect of MCM7 knockdown on the ability of human liver cancer tumor stem cells to initiate tumorigenesis in vivo

[0159] 1. Establishment of subcutaneous tumor-bearing model in NOD / SCID mice

[0160] 1. Take the HCCLM3 liver cancer cells stably transfected with the pSicoR-GFP-Scramble / shMCM7 plasmid in the exponential growth phase, digest and centrifuge, and wash with PBS to eliminate the stimulation of fetal bovine serum to the mice;

[0161] 2. Count the cells using a live cell counter. Take 10 for each group 6 、10 5 cells into 1.5ml EP tubes, and each order of magnitude of cells in each group takes 8 tubes;

[0162] 3. Centrifuge at 1200rpm for 5min, discard the supernatant, and resuspend the cells with MatriGel:serum-free medium = 1:3. The volume of resuspension is about 30-50μl / tube. The steps should be carried out on ice as much as possible, and the tips used should be pre-cooled;

[0163] 4. Randomly divide NOD / SCID mice into 10 6 group and 10 5...

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Abstract

The invention proposes the application of the reagent in the preparation of medicine, the medicine is used for treating liver cancer, and the reagent is used for inhibiting the expression of MCM7 protein. The MCM7 protein is involved in the regulation of the self-renewal of the liver cancer stem cells, and by inhibiting the expression of the MCM7 protein, the self-renewal of the liver cancer stem cells can be inhibited, thereby achieving the purpose of treating liver cancer.

Description

technical field [0001] The invention relates to the field of biomedicine. Specifically, the present invention relates to the use of the reagent in the preparation of medicines, the interference fragment, the method for inhibiting the self-renewal of liver cancer stem cells and the medicine for treating liver cancer. More specifically, the present invention relates to the use of reagents in the preparation of medicines, interference fragments, constructs, methods for inhibiting the self-renewal of liver cancer stem cells, and medicines for treating liver cancer. Background technique [0002] Hepatocellular carcinoma is the fifth most common cancer in the world, and its cancer-related mortality rate ranks third among malignant tumors, which is inseparable from its high metastasis rate of 60% and postoperative recurrence rate. [0003] The common key step of tumor recurrence and metastasis is the reconstruction of tumor tissue. Since surgery, radiotherapy, chemotherapy and ot...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K31/713A61K48/00A61P35/00C12N15/113C12N15/867
CPCA61K31/713A61P35/00C12N15/113C12N15/86C12N2310/141C12N2740/15043
Inventor 周军年裴雪涛岳文王海洋张彪王东星曾泉
Owner ACADEMY OF MILITARY MEDICAL SCI
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