CN107367455B - Method for measuring absolute quantification of number of tumor cell tolerance pro-apoptosis protein molecules - Google Patents
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Abstract
The invention provides an absolute quantitative measuring method for the number of tumor cell tolerance apoptosis-promoting protein molecules. According to the method, the apoptosis-promoting protein is marked by the EGFP, the EGFP numerical value when the tumor cells can tolerate the apoptosis-promoting protein molecules is measured by flow cytometry, and the absolute quantitative measurement of the number of the apoptosis-promoting protein molecules tolerated by the tumor cells is realized by the FITC marked fluorescent microspheres and the conversion of the excitation fluorescence intensity of the FITC and the EGFP. The method not only solves the problem that cell apoptosis is caused by how many times of protein content is increased in the traditional research and the absolute numerical value is lacked, but also avoids the activity problem of BH3 small peptide and the difference problem between BH3 small peptide and BH3 protein in a BH3 analysis method, and realizes the absolute quantitative test of tumor living cell tolerance apoptosis-promoting protein molecules.
Description
Technical Field
The invention belongs to the field of analysis and detection, and particularly relates to an absolute quantitative measuring method for the number of tumor cell tolerance pro-apoptosis protein molecules.
Background
Tumors seriously harm human health. In recent years, a great deal of research shows that the occurrence, development and treatment of tumors are closely related to apoptosis. Apoptotic signaling pathways include mainly death receptor pathways and mitochondrial pathways. The death receptors (e.g., DR4, DR5, etc.) in the death receptor pathway are pro-apoptotic proteins because they promote apoptosis when bound to their ligands (e.g., TRAIL). The mitochondrial apoptotic pathway is controlled primarily by the BCL2 family. The BCL2 protein family members are classified into anti-apoptotic proteins and pro-apoptotic proteins, which in turn include pro-apoptotic proteins (BIM, PUMA, NOXA, BMF, BAD, HRK, etc.) containing only the BH3 domain and multi-domain pro-apoptotic proteins BAX and BAK. BCL2 family regulates the permeability of mitochondrial membrane through protein interaction, controls the release of a series of apoptosis-promoting proteins such as cytochrome c in the mitochondrial membrane, and activates caspase, thereby causing a series of apoptosis reactions. Many other intracellular proteins are also pro-apoptotic proteins (e.g., P53, ERK), which promote apoptosis by promoting the transcriptional expression of downstream pro-apoptotic proteins or by increasing the intracellular stability of downstream pro-apoptotic proteins.
Apoptosis is essential for tumor research. In one aspect, anti-apoptotic BCL2 family proteins are highly expressed in a variety of tumors. On the other hand, many anti-tumor drugs, including DNA damaging drugs, kinase inhibitors, microtubule aggregation inhibitors, etc., also kill tumor cells by inducing mitochondrial apoptosis pathway. Furthermore, many analogs of pro-apoptotic proteins have become anti-tumor drugs or are undergoing clinical trials. For example, the analogue of BH3 of the pro-apoptotic protein BAD, navitoclax, specifically inhibits BCL2, BCLxLAnd BCLw, thereby causing apoptosis of tumor cells, are currently undergoing clinical trials in a variety of tumors. While the more specific BCL2 inhibitor venetoclax has been approved for the treatment of relapsed chronic lymphocytic leukemia. Therefore, the measurement of the absolute molecular number of the tumor cell apoptosis tolerance pro-apoptosis protein is not only helpful for predicting the treatment effect of the anti-tumor drug on a certain tumor, but also helpful for judging which protein molecules are effective drug target molecules.
Earlier studies have generally found out which pro-apoptotic proteins are expressed in cells by immunoblotting methods and then confirmed that these proteins indeed have pro-apoptotic effects by overexpression methods, which has not allowed quantitative analysis (Puthalakath H, et al. Cell death and differential,2002, 9 (5): 505-512; Li R, et al. Cell death and differential, 2005, 12 (3): 292-303). The method of BH3 analysis developed successively in recent years (Deng J, et al. Cancer Cell, 2007, 12: 171-: (1) the corresponding BH3 small peptide of The BH3 protein does not completely react with The BH3 protein, and studies have shown that The activity of BH3 small peptide differs from that of BH3 protein alone (DaiH, et al. The Journal of Biological Chemistry, 2014,289: 89-99); (2) since this method is performed using isolated mitochondria, the method of detection using the BH3 small peptide is only applicable to the mitochondrial apoptotic pathway and cannot be applied to other pro-apoptotic proteins.
Disclosure of Invention
The invention provides an absolute quantitative measuring method for the number of tumor cell tolerance apoptosis-promoting protein molecules. The method comprises the steps of marking the apoptosis-promoting protein by EGFP, measuring the value of EGFP when tumor cells can tolerate the apoptosis-promoting protein molecules by flow cytometry, and realizing the absolute quantitative measurement of the number of the apoptosis-promoting protein molecules tolerated by the tumor cells by FITC marked fluorescent microspheres and the conversion of the excitation fluorescence intensity of FITC and EGFP.
The technical scheme adopted by the invention is as follows:
(1) firstly, EGFP end is used for marking apoptosis-promoting molecular DNA and is transferred into living cells by a transduction method;
(2) after a certain time of cell growth (24 hours or 48 hours), these pro-apoptotic molecules are overexpressed;
(3) then adding Annexin V marked by a fluorescent dye APC to mark apoptotic cells, and detecting and analyzing the EGFP fluorescence intensity of EGFP-coupled apoptosis-promoting molecules of the tumor cells by a flow cytometer;
(4) when the flow cytometry is used for detection and analysis, a reference tube with standard FITC fluorescent microspheres is manufactured, and the fluorescent microspheres can obtain related straight lines of the FITC quantity and the fluorescence intensity on each fluorescent microsphere;
(5) then, determining the conversion relation of emission light intensity within a flow cytometer measurement wavelength range between a certain amount of FITC and EGFP by a fluorescence spectrometer, and obtaining the absolute numerical value of EGFP of tumor cell tolerance apoptosis-promoting molecules; since an EGFP is linked to a pro-apoptotic molecule, this value is the absolute value of the tumor cell tolerance pro-apoptotic molecules.
The detection method comprises the following specific working processes: (1) connecting and cloning cDNA of a target apoptosis-promoting molecule to be measured and cDNA of an EGFP molecule in a high expression vector by a molecular cloning method, and placing the cDNA and the cDNA at the N end or the C end of the EGFP according to requirements; (2) transfecting the plasmid into tested tumor cells by methods such as electrotransformation and virus transfection; (3) collecting cells after 24 hours or 48 hours, staining the cells by using APC-labeled Annexin V, and collecting fluorescence intensity information of EGFP/APC in tumor cells by using a flow cytometer, thereby causing the tumor cells to tolerate the fluorescence intensity information of EGFP at the critical point of apoptosis-promoting molecules; (4) when a sample is collected, simultaneously collecting fluorescence intensity information of a certain number of gradient FITC standard fluorescent microspheres under the same working condition by using a flow cytometer, and making a linear relation graph of FITC fluorescence intensity and number; (5) purifying standard EGFP protein in vitro, purchasing FITC, and detecting the conversion relation between EGFP protein with the same number of molecules and FITC excitation light intensity in the same wavelength range as that detected by a flow cytometer by using a fluorescence spectrophotometer; (6) and converting the fluorescence intensity information of the EGFP at the critical point of the tumor cell tolerance apoptosis-promoting molecules into the molecular number of the EGFP, thereby obtaining the absolute numerical value of the required apoptosis-promoting molecules.
Compared with the prior art, the invention has the following differences and advantages:
(1) the number of the apoptosis-promoting protein molecules in the cell measured by the method is different from the number of the apoptosis-promoting molecules tolerated by the living cell in an extracellular experiment; (2) the method measures an absolute number, and the apoptosis can be caused by the relative increase of the protein content which is not more than the multiple of the protein content, so that the concentration of the apoptosis-promoting molecule-like drugs required by the tumor cells can be estimated according to the absolute number; (3) the method measures the number of molecules of the protein with activity causing apoptosis, and can solve the problem of the activity of the small peptide in the small peptide method and the problem of the activity difference between the small peptide and the protein; (4) the method can be used for measuring and calculating the absolute quantity of the BH3 analogue apoptosis-promoting molecules, and can also be used for measuring and calculating the absolute quantity of other protein molecules (such as DR4, ERK, P53 and the like) which are tolerated by tumor cells.
(2) The innovation of the invention is that: the absolute quantification of the tumor living cell tolerance apoptosis-promoting protein molecules is successfully realized by connecting the apoptosis-promoting protein with the EGFP molecules, combining the EGFP-marked crude apoptosis protein with APC-Annexin V to monitor cell apoptosis, quantifying the application of FITC-marked fluorescent microspheres and skillfully converting the FITC and the EGFP. The method not only solves the problem that cell apoptosis is caused by how many times of protein content is increased in the traditional research and the absolute numerical value is lacked, but also avoids the activity problem of BH3 small peptide and the difference problem between BH3 small peptide and BH3 protein in a BH3 analysis method, and realizes the absolute quantitative test of tumor living cell tolerance apoptosis-promoting protein molecules.
Drawings
FIG. 1 is a flow chart of the practice of the method of the present invention;
FIG. 2 is a flow cytometer analysis of Jurkat cell-tolerant pro-apoptotic molecule BIMELPUMA, NOXA, BMF threshold schematic;
FIG. 3 is a diagram illustrating the conversion of fluorescence intensity and the number of FITC molecules by FITC standard fluorescent microspheres;
FIG. 4 is a graph of the fluorescence intensity of emitted light converted from purified EGFP and FITC;
fig. 5 is a graph showing the results of measuring the threshold absolute number of apoptosis-promoting molecules that are resistant to Jurkat cells and SKW6.4 cells obtained by this method.
Detailed Description
The process flow of the invention is shown in FIG. 1. The method comprises the following specific steps of (1) connecting and cloning cDNA of target apoptosis-promoting molecules and cDNA of EGFP molecules to be measured in a high-expression vector by a molecular cloning method, and placing the cDNA and the cDNA in the N end or the C end of the EGFP according to requirements; (2) transfecting the plasmid into tested tumor cells by methods such as electrotransformation and virus transfection; (3) collecting cells after 24 hours or 48 hours, staining the cells by using APC-labeled Annexin V, and collecting fluorescence intensity information of EGFP/APC in tumor cells by using a flow cytometer, thereby collecting fluorescence intensity information of EGFP at a critical point of tumor cell tolerance apoptosis-promoting molecules; (4) when a sample is collected, simultaneously collecting fluorescence intensity information of a certain number of gradient FITC standard fluorescent microspheres under the same working condition by using a flow cytometer, and making a linear relation graph of FITC fluorescence intensity and number; (5) purifying standard EGFP protein in vitro, purchasing FITC, and detecting the conversion relation between EGFP protein with the same number of molecules and FITC excitation light intensity in the same wavelength range as that detected by a flow cytometer by using a fluorescence spectrophotometer; (6) and converting the fluorescence intensity information of the EGFP at the critical point of the tumor cell tolerance apoptosis-promoting molecules into the molecular number of the EGFP, thereby obtaining the absolute numerical value of the tolerance apoptosis-promoting molecules.
In practice, we will BIMELPro-apoptotic proteins such as PUMA, NOXA and BMF were linked to the back of EGFP and constructed into cell expression vectors. We selected two tumor cell lines, Jurkat and SKW6.4, and transduced these plasmids into tumor cells by electrotransformation, respectively. The cells were cultured for 24 hours to over-express the corresponding pro-apoptotic proteins. For observing apoptosis, QVD was added or not added separately for each assay. Then, the cells were collected and reacted with APC-labeled Annexin V to label apoptotic cells. The GFP channel and APC channel were analyzed by flow cytometry, and Annexin V negative cells were selected.
EGFP-BIM in Jurkat tumor cellsELFor example, Annexin V negative cells were selected as non-apoptotic cells, and boundary EGFP fluorescence intensity was determined by analysis software without adding QVD groups (see fig. 2 a). The same procedure was used for the application of EGFP-PUMA, EGFP-NOXA and EGFP-BMF to these pro-apoptotic protein molecules (FIG. 2b, FIG. 2c and FIG. 2 d). A reference tube containing a quantity of FITC fluorescent microspheres was tested simultaneously while analyzing the sample tube with a flow cytometer (fig. 3 a). By the number of molecules of FITC on the standard fluorescent microsphere, a standard curve (FIG. 3 b) of the number of molecules of FITC and the fluorescence intensity of FITC can be obtained, so that the number of FITC corresponding to the fluorescence intensity of EGFP at the boundary can be calculated. By using the relationship between the fluorescence intensity of the purified EGFP protein (FIG. 4 a) and the purchased standard FITC (FIG. 4 b) in the corresponding detection emission wavelength range of the flow cytometer, the FITC per molecule and the FITC per molecule can be obtainedThe number of molecules of EGFP corresponding to the fluorescence intensity of EGFP at the above-described boundary was calculated from the conversion relationship of the fluorescence intensity obtained by EGFP (fig. 4 c). Since one EGFP molecule is linked to one pro-apoptotic protein, the absolute value of the threshold for tumor cell tolerance to pro-apoptotic molecules can be obtained. The absolute values of the thresholds for the resistance of Jurkat cells, as well as SKW6.4 cells to pro-apoptotic molecules determined according to the above method are shown in FIG. 5.
The present description is not set forth in detail in order to not unnecessarily obscure the present invention.
The above description is only a part of the embodiments of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.
Claims (1)
1. A method for measuring the absolute quantification of the number of tumor cell tolerance pro-apoptotic protein molecules is characterized by comprising the following specific operation methods:
(1) connecting and cloning cDNA of a target apoptosis-promoting molecule to be measured and cDNA of an EGFP molecule in a high expression vector by a molecular cloning method, and placing the cDNA and the cDNA at the N end or the C end of the EGFP according to requirements;
(2) transfecting the plasmid into tested tumor cells by an electrotransfection and virus transfection method;
(3) collecting cells after 24 hours or 48 hours, staining the cells by using Annexin V marked by APC (activated carbon), and collecting fluorescence intensity information of EGFP or APC or EGFP and APC in the tumor cells by using a flow cytometer, thereby collecting fluorescence intensity information of EGFP at the critical point of tumor cell tolerance apoptosis-promoting molecules;
(4) when a sample is collected, simultaneously collecting fluorescence intensity information of a certain number of gradient FITC standard fluorescent microspheres under the same working condition by using a flow cytometer, and making a linear relation graph of FITC fluorescence intensity and number;
(5) EGFP protein is purified in vitro, and the linear relation between the number of standard FITC fluorescent microspheres and the emission light intensity is determined and calculated by a fluorescence spectrophotometer and a flow cytometer; measuring and calculating the linear relation between the number of purified EGFP protein molecules and the emission light intensity by a fluorescence spectrophotometer and a flow cytometer, thereby calculating the conversion relation between the standard FITC fluorescent microspheres and the number of EGFP proteins when the emission light intensity is the same;
(6) and converting the fluorescence intensity information of the EGFP at the critical point of the tumor cell tolerance apoptosis-promoting molecules into the molecular number of the EGFP, thereby obtaining the absolute numerical value of the tolerance apoptosis-promoting molecules.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009085216A2 (en) * | 2007-12-20 | 2009-07-09 | Squicor | Compositions and methods for detecting or elimninating senescent cells to diagnose or treat disease |
| CN102382893A (en) * | 2011-11-23 | 2012-03-21 | 博生吉医药科技(苏州)有限公司 | Specificity quantitive detection for cell apoptosis |
| CN103712967A (en) * | 2013-12-27 | 2014-04-09 | 首都医科大学附属北京友谊医院 | Method for detecting activity of NK (Natural Killer) cell |
| CN104833625A (en) * | 2015-06-04 | 2015-08-12 | 武汉友芝友生物制药有限公司 | Early-stage evaluation method of stability of CHO cell line of expression recombinant antibody |
| CN105445248A (en) * | 2015-12-14 | 2016-03-30 | 上海应用技术学院 | Method for rapid determination of cyanobacteria cell apoptosis rate |
-
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009085216A2 (en) * | 2007-12-20 | 2009-07-09 | Squicor | Compositions and methods for detecting or elimninating senescent cells to diagnose or treat disease |
| CN102382893A (en) * | 2011-11-23 | 2012-03-21 | 博生吉医药科技(苏州)有限公司 | Specificity quantitive detection for cell apoptosis |
| CN103712967A (en) * | 2013-12-27 | 2014-04-09 | 首都医科大学附属北京友谊医院 | Method for detecting activity of NK (Natural Killer) cell |
| CN104833625A (en) * | 2015-06-04 | 2015-08-12 | 武汉友芝友生物制药有限公司 | Early-stage evaluation method of stability of CHO cell line of expression recombinant antibody |
| CN105445248A (en) * | 2015-12-14 | 2016-03-30 | 上海应用技术学院 | Method for rapid determination of cyanobacteria cell apoptosis rate |
Non-Patent Citations (2)
| Title |
|---|
| "人fadd基因cDNA的克隆和诱导舌癌细胞凋亡的研究";刘大庆等;《实用口腔医学杂志》;20020930;第18卷(第5期);第401-404页 * |
| "诱导pcDNA3.1-Egr1-EGFP-Omi/HtrA2对人眼脉络膜黑色素瘤细胞OCM-1凋亡的影响";余天;《中国优秀硕士学位论文全文数据库》;20120215(第2期);正文第9-14页 * |
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