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A kind of human colorectal cancer tumor cell line and its preparation method and application

A cancer stem cell line and colorectal cancer technology, applied in the field of cell lines, can solve the problems of large sample differences, low tumor stem cell viability, and scarce number of tumor stem cells, and achieve high in vivo tumorigenic ability and strong drug resistance.

Active Publication Date: 2014-10-01
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the main difficulty restricting the research of tumor stem cells is that the number of tumor stem cells is scarce, and it is easy to differentiate into common cancer cells in vitro.
[0006] At present, there are no reports of tumor stem cell lines at home and abroad. Cancer stem cells are mainly derived from primary cancer cells marked with specific surface proteins (such as CD44, CD133, CD24, etc.) from clinical samples using flow cytometry technology, which leads to Solved the problem of low viability of tumor stem cells and large differences between samples
Because primary cancer stem cells are easily differentiated into normal cancer cells under ordinary in vitro culture conditions, the in-depth study of cancer stem cells is greatly restricted.

Method used

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  • A kind of human colorectal cancer tumor cell line and its preparation method and application
  • A kind of human colorectal cancer tumor cell line and its preparation method and application
  • A kind of human colorectal cancer tumor cell line and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Isolation and Identification of Primary Colon Cancer Stem Cells

[0042] a) Isolation of primary colon cancer stem cells: take fresh colon cancer tissues from patients with colon cancer (taken from Beijing Cancer Hospital), and in phosphate buffer containing 300 units / mL (U / mL) penicillin and 300 U / mL streptomycin ( Rinse 3 times in PBS); cut it into pieces with sterile ophthalmic scissors; add 1 mg / ml (mg / mL) collagenase (purchased from Sigma Company) and 1 mg / mL hyaluronidase (purchased from Sigma Company) to digest solution, digested at 37°C for 30 minutes (min); centrifuged to remove the supernatant, resuspended in PBS, and filtered through a 40-micron cell sieve to obtain a single-cell suspension. The cells were incubated with mouse anti-human EpCAM Alexa Fluor647 monoclonal antibody (1:50, purchased from Cell Signaling Company) and mouse anti-human CD44FITC monoclonal antibody (1:50, purchased from BD Company) at 4°C for 15 minutes; EpCAM+CD44+ primar...

Embodiment 2

[0045] Example 2 Establishment of a cell line with characteristics of tumor stem cells

[0046] a) Establishment of a tumor stem cell line expressing CD44 membrane protein: the colon cancer cells isolated and cultured by the above method have the characteristics of tumor stem cells, have extremely high tumor formation ability and clone formation ability, and are named P6C. P6C can be stably passaged in vitro, and has been passed down to the 120th passage. P6C forms suspended cloning balls under low adhesion culture conditions, and can adhere to the wall to form stem cell-like clones (holoclone) under adhesion conditions.

[0047] b) The activation of the Oct3 / 4 promoter indicates the establishment of a cell line: when the Oct3 / 4 promoter is regulated and activated, the expression of EGFP emits green fluorescence, which indicates the activation state of the stem cell factor Oct3 / 4. The day before the transfection of the cell line P6C, the cell line P6C ​​was digested with 0....

Embodiment 3

[0048] Example 3 Detection of differentiation characteristics of tumor stem cell line P6C

[0049] a) Digest the adherent cultured cell line P6C ​​with 0.2% trypsin at 37°C to form a single cell suspension, count, add pH7.2 phosphate buffer saline (PBS) to a final concentration of 10 6 cells / ml.

[0050] b) Add fluorescently labeled mouse anti-human monoclonal antibody anti-CD24 (1:50, purchased from Biolegend); anti-CD44 (1:50, purchased from BD pharmingen); anti-CD133 (1:50, purchased from from Miltenyi); anti-CD45-FITC (1:50, purchased from BD pharmingen); anti-CD31 (1:50, purchased from BD pharmingen); anti-CK1 (1:50, purchased from Santa Cruz); anti- CK20 (1:50, purchased from Santa Cruz); anti-CDX2 (1:100, purchased from DAKO).

[0051] c) Incubate at 4°C for 20 minutes, and then use flow cytometry to detect the expression of the corresponding antigen; FITC-labeled goat anti-mouse IgG is incubated with the P6C ​​cell line as a negative control.

[0052] Such as Fi...

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Abstract

The invention provides a human colorectal adenocarcinoma tumor cell line as well as a preparation method and an application thereof. The cell line P6C of human colorectal adenocarcinoma tumor stem cell line has the preservation number of CGMCC No.5558. The preparation method comprises the following steps of: 1) separating original colon cancer stem cells; 2) culturing the primary colon cancer stem cells in vitro; 3) culturing and identifying the colon cancer stem cells; and 4) separating and culturing the obtained colon cancer stem cells according to the same method in the steps 1), 2) and 3) to screen and express the tumor stem cell of CD44 membrane protein. The cell line can be applied in the preparation of the medicine for inducing tumour formation, a medicine for tumor metastasis and medicines for screening antituvmorigenesis, anti-tumor growth and anti-tumor metastasis.

Description

technical field [0001] The present invention relates to a cell line, in particular to a human colorectal cancer tumor cell line, which expresses factors related to normal stem cell self-renewal, has high clone formation ability and in vivo tumorigenic ability, has the characteristics of tumor stem cells, and can Applied to the research of tumor stem cells, drug development and related fields. Background technique [0002] Cancer is a disease that seriously threatens human survival and health. In recent decades, with the development of science, people's understanding of cancer has improved significantly. However, traditional treatment methods cannot completely cure cancer, and recurrence and metastasis are often the cause of death of cancer patients. Studies have found that both primary tumors and metastatic cancers contain cancer cells with different characteristics and functions: highly differentiated cancer cells have weak proliferation ability; while cancer cells with p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/095C12N5/10C12Q1/02A61K35/38C12R1/91A61K35/13
Inventor 陈佺杜蕾王珺金海京王晓慧
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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