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Application of stem cell marker CD133 interference nucleic acid to preparation of tumor proliferation restraining preparation

A stem cell marker and nucleic acid interference technology, applied in the field of preparation of adjuvant therapy drugs or reagents for liver cancer, can solve the problems of high and limited mortality of liver cancer, lack of sensitive markers of liver cancer, etc., and achieve the effect of reducing the mortality of liver cancer

Inactive Publication Date: 2019-06-11
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The current clinical treatment methods are relatively limited, mainly relying on surgical resection, chemotherapy and a small number of liver transplantation. Therefore, the reason for the high mortality rate of liver cancer is that there is a lack of sensitive markers for early diagnosis of liver cancer. Surgical resection is not possible; the genetic characteristics and molecular mechanisms of the occurrence and development of liver cancer are not well understood, making chemotherapy resistance inevitable

Method used

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  • Application of stem cell marker CD133 interference nucleic acid to preparation of tumor proliferation restraining preparation
  • Application of stem cell marker CD133 interference nucleic acid to preparation of tumor proliferation restraining preparation
  • Application of stem cell marker CD133 interference nucleic acid to preparation of tumor proliferation restraining preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The magnetic bead sorting method sorted out CD133 in Huh7 and PLC / PRF / 5 liver cancer cells + Liver cancer stem cells, using shCD133-1 and shCD133-2 two shRNAs targeting different sequences of CD133, respectively knocked down the liver cancer cell line Huh7-CD133 + Cells and PLC / PRF / 5-CD133 + The expression of CD133 in the cells was compared with shLacz not targeting any human gene sequence, and the knockdown effect of CD133 and the expression of the cell cycle regulatory protein CyclinD1 were detected by western blot hybridization;

[0029] 1) CD133 was isolated from liver cancer cell line Huh7 and PLC / PRF / 5 magnetic beads + Cells, detection of CD133, Cyclin D1 protein expression after CD133 interference nucleic acid

[0030] 1. CD133 was isolated from liver cancer cell line Huh7 and PLC / PRF / 5 magnetic beads + cell

[0031] In this experiment, human liver cancer cell lines Huh7 and PLC / PRF / 5 were used. The cells were cultured in DMEM medium (Gibco) containing 10% fe...

Embodiment 2

[0049] Liver cancer cell lines Huh7 and PLC / PRF / 5CD133 + After knocking down the expression of CD133 in the cells, the proliferation and self-renewal ability of the cells were detected by cell sphere formation assay

[0050] 2) Liver cancer cell lines Huh7 and PLC / PRF / 5CD133 + Knock down the expression of CD133 in cells to detect the ability of cells to form spheres

[0051] 1. Detection of CD133 knockdown of Huh7 and PLC / PRF / 5CD133 + Experimental process and result analysis of cell spheroidizing ability

[0052] 1) Digest the cells of the control group and the CD133 knockdown group cultured in the above DMEM culture medium with trypsin, and resuspend the cells in DMEM culture medium to a single state; centrifuge at 2000rpm for 5 minutes, and aspirate the supernatant; 2) Add vitamin A-free B27 (Gibco), 100 μg / ml penicillin with 20ng / mL EGF (Chemicon), 20ng / mL FGF-2 (Chemicon), 2μg / mL heparin (Sigma), 1:50 ratio Wash the cells with DMEM / F12 medium (Gibco) of 50 μg / ml strept...

Embodiment 3

[0056] Liver cancer cell lines Huh7 and PLC / PRF / 5CD133 + After knocking down the expression of CD133 in the cells, the tumorigenic ability of the cells was detected by subcutaneous tumorigenesis in nude mice

[0057] 3) Liver cancer cell lines Huh7 and PLC / PRF / 5CD133 + Knock down the expression of CD133 in the cells, and detect the subcutaneous tumorigenesis ability of nude mice

[0058] 1. Detection of CD133 knockdown of Huh7 and PLC / PRF / 5CD133 + Experimental process and result analysis of cell tumor formation in nude mice subcutaneously

[0059] 1) Digest the cells with 0.25% trypsin for 1-2min, and blow the cells with DMEM containing 10% fetal bovine serum

[0060] 2) Centrifuge at 2000rpm for 5min, remove the supernatant, wash the cells 3 times with PBS, centrifuge at 2000rpm for 5min, remove the supernatant;

[0061] 3) resuspend the cells with PBS, and count the number of cells with a cell counter;

[0062] 4) will 10 5 Each cell was diluted to 150 μl with PBS, inj...

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Abstract

The invention belongs to the field of biomedicine, and relates to an application of stem cell marker CD133 interference nucleic acid to preparation of a tumor proliferation restraining preparation, and the use of stem cell marker CD133 interference nucleic acid to preparation of an auxiliary treatment medicine or reagent for liver cancer. By a magnetic bead sorting method, in Huh7 liver cancer cells and PLC / PRF / 5 liver cancer cells, CD133+ cancer liver stem cells are sorted, then two shRNA in accordance with different sequences of CD133, of shCD133-1 and shCD133-2, are used for knocking down expression of CD133 in Huh7-CD133+ cells and PLC / PRF / 5-CD133+ cells, shLacz which is not in accordance with any human gene sequence is used as contrast, effective interference of a shRNA sequence to CD133 protein expression is certificated, and the CD133 protein expression is knocked down. After the situation that CD133 expression is knocked down in CD133+ cells is confirmed through in vitro stem cell balling experiment and tumour formation tumour formation in nude mouse bodies, self replacement and proliferation capacity of cells are significantly restrained. The CD133 interference nucleic acid can be used for preparing the liver cancer proliferation restraining preparation and preparing the auxiliary treatment medicine for liver cancer.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to a new medical application of CD133 interfering nucleic acid, in particular to the application of a stem cell marker CD133 interfering nucleic acid in inhibiting the proliferation of liver cancer, and its use in preparing liver cancer adjuvant therapy drugs or reagents. Background technique [0002] Data show that in my country, liver cancer is one of the most common malignant tumors with a high mortality rate. Clinical studies have shown that liver cancer has no typical symptoms in the early stage. When most patients feel unwell and are diagnosed, they are already in the advanced stage of liver cancer and have missed the best treatment period. The current clinical treatment methods are relatively limited, mainly relying on surgical resection, chemotherapy and a small number of liver transplantation. Therefore, the reason for the high mortality rate of liver cancer is that there is a lack ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/7105A61P35/00A61P1/16
Inventor 魏湲颜江建海刘婵娟李亦楠邢洋杨帆陈晓宁
Owner FUDAN UNIV
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