Breast cancer circulating tumor cell line CTC-3, culture medium, and building method and application of CTC-3

A CTC-3, tumor cell technology, applied in the field of cells, can solve problems such as false negative specificity, and achieve the effects of simple operation, wide universality and low cost

Active Publication Date: 2018-10-02
湖南医药集团精准医疗生物产业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The third purpose of the present invention is to provide a method for establishing the above-mentioned breast cancer circulating tumor cell line CTC-3, so as to alleviate the technical problems existing in the prior art that the enrichment of circulating tumor cells is likely to cause false negatives or poor specificity

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  • Breast cancer circulating tumor cell line CTC-3, culture medium, and building method and application of CTC-3
  • Breast cancer circulating tumor cell line CTC-3, culture medium, and building method and application of CTC-3
  • Breast cancer circulating tumor cell line CTC-3, culture medium, and building method and application of CTC-3

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 Obtaining of Breast Cancer Circulating Tumor Cell Line CTC-3

[0071] Using the method for establishing breast cancer circulating tumor cell line CTC-3 provided by the present invention to obtain breast cancer circulating tumor cell line CTC-3 comprises the following steps:

[0072] (a) Extract 6-8mL of venous peripheral blood from breast cancer patients into 10mL blood collection tubes, centrifuge to obtain serum and whole blood cells, separate and collect whole blood cells by Ficoll-diatrizoate glucosamine density gradient centrifugation, and wash with normal saline After 3 times of low-speed centrifugation, peripheral blood mononuclear cells were obtained.

[0073] (b) The obtained peripheral blood mononuclear cells are resuspended in the medium provided by the present invention for culturing primary cells of breast cancer circulating tumor cell lines, and the cells are planted in a 6-well plate at 37° C., 5% CO 2 Cultivate in an incubator with saturated h...

Embodiment 2

[0075] Example 2 Detection of Breast Cancer Circulating Tumor Cell Line CTC-3

[0076] After continuing to culture the breast cancer circulating tumor cell line CTC-3 obtained in Example 1 of the present invention for one week, after the cells cover about 70% of the bottom of the well, digest with 0.25% trypsin at room temperature for 2-3 minutes and then use it for cell climbing and identification ; soak the coverslip in a biological safety cabinet in advance with 75% alcohol for 48h, and use the method provided by the invention for cultivating breast cancer circulating tumor cell line CTC-3 to digest the P2 generation clone cells that are highly proliferative and differentiated The culture medium of primary cells was resuspended and seeded in 6-well plates pre-covered with coverslips, at 37°C, 5% CO 2 Cultivate in an incubator with saturated humidity. After 2-3 days, the cell slides are stabilized and identified by immunofluorescence staining.

[0077] Immunofluorescence st...

Embodiment 3

[0083] Example 3 Identification of breast cancer circulating tumor cell line CTC-3STR

[0084] In this example, the cell line identification kit was used to amplify using the 10-STR amplification scheme, and the STR loci and the sex gene Amelogenin were detected on an ABI3730XL genetic analyzer.

[0085] The method includes the following steps:

[0086] 1. DNA extraction;

[0087] 2. Multiplex PCR uses the cell line identification kit to amplify the cell line DNA;

[0088] 3. Capillary electrophoresis typing;

[0089] 4 Compare the obtained allele types with DSMZ.

[0090] Test samples include: CTC-3, MCF-7, PBMCs in the peripheral blood of breast cancer patients.

[0091] The results of STR identification are shown in the table below:

[0092]

[0093] The identification results showed that the similarity between CTC-3 and MCF-7 in the standard locus alignment was less than 20%, while the similarity between CTC-3 and PBMCs derived from the peripheral blood of breast c...

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Abstract

The invention provides a breast cancer circulating tumor cell line CTC-3, a culture medium, and a building method and application of the CTC-3, and relates to the technical field of cells. The collection number of the breast cancer circulating tumor cell line CTC-3 provided by the invention is CCTCC NO: C201892. The character of the cell line is stable, and large-scale basic researches and clinical tests can be developed, thereby further mastering features of a circulating tumor cell and explaining a tumor development mechanism, and providing a basis for clinical staging, prognosis and individualized treatment for a cancer patient. The culture medium provided by the invention comprises a basic culture medium and an additive. The culture medium can greatly multiply high-quality and high-purity circulating tumor cells. The building method of the breast cancer circulating tumor cell line CTC-3 is easy to operate, low in cost and wide in universality, and can quickly and effectively realize multiplication culture of the breast cancer circulating tumor cells.

Description

technical field [0001] The invention relates to the field of cell technology, in particular to a breast cancer circulating tumor cell line CTC-3, a culture medium and a method for establishing and using the CTC-3. Background technique [0002] Circulating tumor cells (CTCs) are tumor cells that escape from solid tumors and enter the peripheral blood circulation. They exist in various malignant tumors such as breast cancer, prostate cancer, and colorectal cancer. Circulating tumor cells are an important biological marker to characterize the metastatic potential of malignant tumors. The existence and quantity of circulating tumor cells in the blood can reflect the degree of malignancy of the tumor, the effect of tumor treatment and prognosis. Because CTC detection is minimally invasive and real-time, it is called "liquid biopsy". [0003] At present, the main methods for CTC detection include immunological detection technology relying on the detection of tumor-related markers...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09
CPCC12N5/0693C12N2500/32C12N2506/11
Inventor 邹畅赵盼周文斌王建红
Owner 湖南医药集团精准医疗生物产业有限公司
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