Human colorectal adenocarcinoma tumor cell line as well as preparation method and application thereof

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 restrictions on the in-depth study of tumor stem cells, and achieve high in vivo tumorigenic ability and strong drug resistance.

Active Publication Date: 2013-08-14
INST OF ZOOLOGY CHINESE ACAD OF SCI
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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

Method used

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  • Human colorectal adenocarcinoma tumor cell line as well as preparation method and application thereof
  • Human colorectal adenocarcinoma tumor cell line as well as preparation method and application thereof
  • Human colorectal adenocarcinoma tumor cell line as well as preparation method and application thereof

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[0041] Example 1 Isolation and identification of primary colon cancer stem cells

[0042] a) Isolation of primary colon cancer stem cells: fresh colon cancer tissue from colon cancer patients (taken from Beijing Cancer Hospital), in a phosphate buffer solution 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) and 1 mg / mL hyaluronidase (purchased from Sigma) for digestion Digest the solution at 37°C for 30 minutes (min); centrifuge to remove the supernatant, resuspend in PBS and filter with 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) and mouse anti-human CD44FITC monoclonal antibody (1:50, purchased from BD) at 4°C for 15 minutes; then Sterile flow cytometry (FACS) was used to isolate EpCAM+C...

Example Embodiment

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

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

[0047] b) Oct3 / 4 promoter activation indicates the establishment of cell lines: 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. One day before transfection of the cell line P6C, the cell line P6C ​​was digested with 0.25% trypsin from the clo...

Example Embodiment

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

[0049] a) The adherent cultured cell line P6C ​​was digested with 0.2% trypsin at 37°C into a single cell suspension, counted, and pH 7.2 phosphate buffered saline (PBS) was added 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 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 ...

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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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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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