Method for isolating and identifying human lung adenocarcinoma stem cell

A lung adenocarcinoma cell and stem cell technology, applied in the field of cell biology, can solve problems such as unclear origin of cells

Inactive Publication Date: 2009-06-24
SHANGHAI INST OF ONCOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But the cellular origin of these...

Method used

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  • Method for isolating and identifying human lung adenocarcinoma stem cell
  • Method for isolating and identifying human lung adenocarcinoma stem cell
  • Method for isolating and identifying human lung adenocarcinoma stem cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0138] Example 1 Expression analysis of stem cell markers in lung adenocarcinoma cells

[0139] CD133 and ABCG2 are expressed on the surface of cancer stem cells of various solid tumors, and are a broad spectrum of stem cell markers. Therefore, the inventors detected the expression of CD133 and ABCG2 in A549 and SPC-A1 lung adenocarcinoma cells by RT-PCR and flow cytometry.

[0140] figure 1 A The results showed that both A549 and SPC-A1 cells expressed ABCG2-specific mRNA, but not CD133. Further detection of its protein expression by flow cytometry confirmed that ABCG2 in A549 and SPC-A1 cell populations + The proportion of cells was 6% and 10%, respectively, and no positive cells were detected by CD133 antibody staining, see figure 1 b.

Embodiment 2

[0141] Example 2 Relationship between the expression of ABCG2 and related markers and SP

[0142] ABCG2 in lung + The cells belong to the terminal airways (Distal airways) and alveolar epithelial cells. The former do not have SP characteristics, while the latter are lung stem cells with SP characteristics (Summer R et al., Sidepopulation cells and Bcrpl expression in lung[J].Am J Physiol Lung Cell Mol Physiol, 2003, 285(1): L97-L104). To identify ABCG2 in lung adenocarcinoma cells + The type of the cells, the inventors performed Hoechst staining and ABCG2 antibody labeling on the SPC-A1 cells.

[0143] figure 2 The results showed that SP cells in SPC-A1 ranged from 3.2 to 7.4%, with an average of 5%, and ABCG2 cells in SP and non-SP (non-SP) fractions + The cell ratios were 3.1% and 2.2%, respectively, that is, ABCG2 was mainly expressed on the surface of non-SP cells.

[0144] It is known that the expression of ABCG2 on the surface of cancer cells is regulated by growth...

Embodiment 3

[0146] Example 3 CD24 + IGF-1R + Stem cell gene expression analysis of cell subsets

[0147] In order to search for LASC in lung adenocarcinoma cells, the inventors used MACS technology to divide A549 cells into ABCG2 + , EGFR + , CD24 + and IGF-1R + Cell subpopulations, and then RT-PCR was used to detect the expression of a panel of stem cell genes involved in self-renewal (OCT4 and Bmi-1), differentiation regulation (TTF-1 and GRM), and BASC markers (CCSP and SP- C).

[0148] image 3 The results showed that unsorted A549 parental cells (Parent) expressed SP-C and Bmi-1, while all marker positive cells expressed Bmi-1, SP-C and Gremlin (GRM). In addition, CD24 + Cells also express OCT4, IGF-1R + Cells also express CCSP and TTF-1.

[0149] further from CD24 + Isolation of IGF-1R in Cell Subpopulations + cells, the resulting CD24 + IGF-1R + The expression of all stem cell genes, especially the simultaneous expression of CCSP and SP-C, suggested that these cells w...

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Abstract

The invention belongs to the field of cytobiology, and discloses a method for separating or identifying lung adenocarcinoma stem cells (group) from lung adenocarcinoma cells (group); the invention also discloses a reagent and a kit for separating or identifying lung adenocarcinoma stem cells (group). The invention shows that the lung adenocarcinoma stem cells have special mark CD24 and insulin-like growth factor-1 receptor (IGF-1R) for the first time, and provides efficient path for separating or identifying lung adenocarcinoma stem cells (group).

Description

technical field [0001] The invention belongs to the field of cell biology, and more specifically, the invention relates to a method for isolating and identifying lung adenocarcinoma stem cells (groups). Background technique [0002] Lung adenocarcinoma is one of the most common tissue types of lung cancer, and its incidence rate accounts for about 30-40% of the total number of lung cancers. Lung adenocarcinoma occurs frequently in the peripheral airways, especially the bronchioles and alveoli, and its precancerous lesions are called atypical adenomatous hyperplasia (AAH). Columnar (Clara) cells or type II alveolar (AT2) cells. However, the cellular origin of such early-stage adenocarcinomas remains unclear. [0003] American researcher Kim et al. have revealed for the first time that lung adenocarcinoma is a stem cell disease, and its originating cells are the bronchiole-alveolar junctions (BADJ) at the end of the airway in the mouse lung. Stem cells (Branchio aveolar Ste...

Claims

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Application Information

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IPC IPC(8): C12N5/08C07K14/705C07K14/71C07K16/28C12Q1/68G01N33/574C12N5/095
Inventor 董强刚
Owner SHANGHAI INST OF ONCOLOGY
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