Method for detecting expression level of annexin A3

An annexin, expression level technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, measurement devices, etc.

Active Publication Date: 2010-11-24
PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Based on the correlation between annexin A3 and platinum-based chemotherapeutic drug resistance, the present invention intends to clinically evaluate the sensitivity of tumor plat

Method used

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  • Method for detecting expression level of annexin A3
  • Method for detecting expression level of annexin A3
  • Method for detecting expression level of annexin A3

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: In vitro study on the mechanism of annexin A3 regulating the drug resistance of ovarian cancer epithelial cells to platinum-based chemotherapy drugs

[0056] 1. Experimental method

[0057] 1) Cell lines and their culture conditions

[0058] Human epithelial ovarian cancer cell SKOV3 cell line was purchased from the Basic Medical Cell Center of the Chinese Academy of Medical Sciences. SKOV3 / Cis is the SKOV3 cisplatin-resistant cell subline induced by the inventor (refer to Yan Xuedong, Zhang Mingwei, Pan Lingya. Basic Medicine and Clinical, 2006, 26(7):739-744). This experiment also used A2780 and its cisplatin-resistant cell line A2780 / Cis (refer to LuanYZ, Li L. et al. Zhonghua Fu Chan Ke Za Zhi, 2004, 39(6): 403-407.). These cells were all transfected with an empty pcDNA3.1(+) plasmid. SKOV3 / Ann and A2780 / Ann cell lines are stable transfection clones obtained after SKOV3 and A2780 were transfected with positive-sense Annexin A3 plasmids, respectively, a...

Embodiment 2

[0083] Example 2: Identification of Exocrine Annexin A3 in Epithelial Ovarian Cancer Cells

[0084] 1. Experimental method

[0085] 1) Cell lines and cell culture

[0086] With embodiment 1.

[0087] 2) Collection and concentration of epithelial ovarian cancer cell line culture supernatant

[0088] Take 2×10 5 Cells were seeded at 25cm 2 Cell culture flasks adhered to the wall and replaced serum-free DMEM when the growth reached 70%, about every 1×10 6 The cells were given 5ml of culture medium, and placed in a cell incubator to continue culturing for 48 hours. Then, the serum-free DMEM was collected in a 10ml centrifuge tube, and centrifuged at 1000g for 10 minutes to remove the formed components in DMEM. After centrifugation, the supernatant was carefully collected and placed Centrifuge horizontally at 3000g for 15 minutes in an Amicon Ultra-15 ultrafiltration tube (Millipore, USA), collect the concentrated liquid in an Ep tube, and store at -70°C.

[0089] 3) Semi-qua...

Embodiment 3

[0104] Example 3: The Exocrine Pathway of Annexin A3 in Ovarian Cancer Cells

[0105] 1. Experimental method

[0106] 1) Cell lines and culture

[0107] The cells used in this experiment and their culture conditions are completely consistent with those in "Example 2", and will not be repeated here.

[0108] 2) Immunofluorescence and its laser confocal imaging

[0109] Take 2×10 4 Cells were planted in a 24-well cell culture plate with glass slides, at 37°C, 5% CO2 After culturing overnight in a saturated humidity incubator, discard the medium, wash 3 times with PBS, fix with 4% paraformaldehyde for 20 minutes, wash 3 times with PBS, permeabilize the membrane with 0.5% TritonX-100 for 20 minutes, wash 3 times with PBS, 2% Blocked with BSA for 30 minutes, incubated with 1:50 rabbit anti-human annexin A3 polyclonal antibody (primary antibody), overnight at 4°C. Wash 3 times with PBS, incubate with goat anti-rabbit FITC secondary antibody for 1 hour, wash 3 times with PBS, inc...

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Abstract

The invention generally relates to the field of medicament resistance of chemotherapeutic medicaments, in particular to a method for detecting the expression level of annexin A3 in the supernate of the culture medium of platinum-based chemotherapeutic medicament resisting tumor cells, a method for adjusting the excretion amount of tumor cell annexin A3, a method for evaluating the medicament resistance of patients with tumor to the platinum-based chemotherapeutic medicament, a method for adjusting the platinum releasing of the platinum-based chemotherapeutic medicament resisting tumor cells, a method for combining deoxyribonucleic acid (DNA) and the platinum in the platinum-based chemotherapeutic medicament resisting tumor cells and a method for adjusting the quantity of vesicles in the platinum-based chemotherapeutic medicament resisting tumor cells.

Description

technical field [0001] The present invention generally relates to the field of chemotherapeutic drug resistance, and specifically relates to the following aspects: a method for detecting the expression level of annexin A3 (Annexin A3) in the culture medium supernatant of platinum-based chemotherapeutic drug resistant tumor cells, a A method for regulating the amount of tumor cell annexin A3 exocytosis, a method for assessing the resistance of tumor patients to platinum-based chemotherapy drugs, a method for regulating the release of platinum from platinum-based chemotherapy drug-resistant tumor cells, a method for regulating platinum-based chemotherapy Methods for DNA binding to platinum in drug-resistant tumor cells, and a method for modulating the number of vesicles in platinum-based chemotherapeutic drug-resistant tumor cells. Background technique [0002] Ovarian cancer is the first fatal disease of gynecological tumors. The 5-year survival rate of advanced patients has ...

Claims

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Application Information

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IPC IPC(8): G01N33/574C12N15/12C12N15/113C12N15/85C07K14/47
Inventor 潘凌亚尹婕闫雪冬
Owner PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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