A method for absolute quantification of the number of tumor cell resistance pro-apoptotic protein molecules

Abstract

The invention provides a measurement method for absolute quantification of the number of pro-apoptotic protein molecules tolerated by tumor cells. According to the invention, EGFP is used for marking pro-apoptotic proteins; flow cytometry is employed for measuring the value of EGFP when tumor cells can still tolerate pro-apoptotic protein molecules; and absolute quantification measurement of the number of pro-apoptotic protein molecules tolerated by tumor cells can be realized through conversion between fluorescent microspheres labeled by FITC and the excitation fluorescence intensities of FITC and EGFP. The method provided by the invention overcomes the problem that traditional research can only measure what multiplication ratio, instead of what absolute value, of the increment of protein content will lead to cell apoptosis, avoids problems caused by the activity of BH3 small peptides in methods of analysis with BH3, prevents problems in difference between BH3 small peptides and BH3 proteins, and realizes absolute quantification testing of pro-apoptotic protein molecules tolerated by tumor cells.

Description

technical field [0001] The invention belongs to the field of analysis and detection, and in particular relates to a method for absolutely quantifying the number of tumor cell tolerance and pro-apoptosis protein molecules. Background technique [0002] Tumors seriously endanger human health. In recent years, a large number of studies have shown that the occurrence, development and treatment of tumors are closely related to apoptosis. Apoptotic signaling pathways mainly include death receptor pathway and mitochondrial pathway. In the death receptor pathway, when death receptors (such as DR4, DR5, etc.) bind to their ligands (such as TRAIL), they can promote cell apoptosis, so these death receptors are all pro-apoptotic proteins. The mitochondrial apoptotic pathway is mainly controlled by the BCL2 family. BCL2 protein family members are divided into anti-apoptotic proteins and pro-apoptotic proteins, and pro-apoptotic proteins include pro-apoptotic proteins containing only B...

AI Technical Summary

Problems solved by technology

The BH3 analysis method developed in recent years (Deng J, et al. Cancer Cell, 2007, 12: 171-185; Vo TT, et al. Cell, 2012, 151: 344-355), however, this method has its limitations : (1) The BH3 small peptide corresponding to the BH3 protein does not fully reflect the BH3 protein. Studies have shown that the activity of the BH3 small peptide is different from that of the BH3 protein alone (DaiH, et al. The Journal of Biological Chemistry, 2014 , 289:89-99); (2) Because this method is done using isolated mitochondria, the method using BH3 small peptide detection is only applicable to the mitochondrial apoptotic pathway, and cannot be tested on other pro-apoptotic proteins use

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