阅读说明：本技术 一种细胞内络氨酸磷酸化水平的检测方法 (Method for detecting phosphorylation level of tyrosine in cell ) 是由 范子彦 熊巍 杨飞 陶晓秋 王颖 唐纲岭 卫民 庞夙 于 2020-04-24 设计创作，主要内容包括：本发明公开一种细胞内络氨酸磷酸化水平的检测方法,包括如下步骤：烟气冷凝物制备、细胞处理、免疫荧光标记与样品处理、检测。本发明通过研究烟气暴露后细胞内的活性氧浓度水平,用于评估烟气暴露对细胞的氧化应激反应的影响,对烟气成分的安全性进行生物学评价,反向为安全卷烟的研发和生产提供科学参考。(The invention discloses a method for detecting intracellular tyrosine phosphorylation level, which comprises the following steps: preparing a smoke condensate, processing cells, carrying out immunofluorescence labeling and sample processing, and detecting. The method is used for evaluating the influence of smoke exposure on the oxidative stress reaction of cells by researching the active oxygen concentration level in the cells after the smoke exposure, biologically evaluating the safety of smoke components, and reversely providing scientific reference for the research and development and production of safe cigarettes.)

1. A method for detecting the phosphorylation level of tyrosine in a cell, which is characterized by comprising the following steps:

step 1) preparation of flue gas condensate:

extracting a cigarette sample according to GB/T5606.1-2004, smoking by using a smoking machine, collecting total particulate matters in the smoke, and adding DMSO with a corresponding volume according to the mass of the total particulate matters in the smoke to obtain a total particulate matters stock solution in the smoke;

step 2) cell treatment: plating cells and culturing overnight; continuing the culture with the cell culture medium supplemented with the flue gas condensate for 1 hour;

step 3) immunofluorescence labeling and sample processing: the infected cells were washed with PBS; adding 70% ethanol solution for fixation, and standing overnight at low temperature; washing with PBS, permeabilizing with a PBS solution containing triton-X100, and washing with PBS; adding a fluorescent labeled phosphorylation antibody for reaction at 37 ℃, adding pancreatin for digestion, and stopping digestion by using a culture medium; separating out cells and dispersing again until the flow cytometry is used for determination;

step 4) detection, namely detecting the staining intensity of the fluorescent probe by using a flow cytometer, carrying out three-color fluorescence compensation before detection, setting a gate by using CD45-PerCP/SSC during detection, separating an abnormal cell group from a normal cell group, and capturing and analyzing each sample by 1 × 10 after the abnormal cell group is circled and gated⁴(ii) individual cells; alternatively, the staining intensity of the fluorescently labeled phosphorylated antibody is detected using a confocal laser microscope.

2. The detection method according to claim 1, wherein in the step 1), smoking is performed under both ISO and HCI smoking conditions by using a RM20H turntable type smoking machine.

3. The detection method according to claim 1, wherein in the step 1), the detection method is performed byAnd collecting the total particulate matters of the smoke by using a Cambridge filter.

4. The detection method according to claim 1, wherein in the step 2), the density of plated cells is 2.5 × 10⁵cells/mL。

5. The assay method according to claim 1, wherein in the step 2), the cells are separated by adding 1ml of LPBS solution after the digestion is terminated, blowing the bottom of the culture plate to detach the cells, centrifuging at 2500 rpm, and repeating the operation 3-5 times.

6. The detection method according to claim 1, wherein in the step 3), the fluorescently labeled phosphorylated antibody is a Cy 3-labeled phosphorylated tyrosine antibody.

7. The detection method according to claim 1, wherein in the step 4), the staining intensity of the fluorescent probe is detected under 488nm and 530nm by using flow cytometry.

8. The detection method according to claim 1, wherein the step 2) is carried out at a cell density of 2.5 × 10⁵Plating cells/mL, and culturing overnight; cell culture was performed for 1 hour with the addition of cell culture media of smoke condensates prepared separately under ISO and HCI suction conditions, respectively.

9. The detection method according to claim 1, wherein the step 3) is specifically: treated cells were washed three times with PBS for three minutes each; adding 70% ethanol solution for fixation, and standing overnight at 4 deg.C; after three washes with PBS, permeabilized for 10 minutes with PBS solution containing 1% triton-100, washed three times with PBS; adding a fluorescent labeled phosphorylation antibody, reacting for 1h at 37 ℃, adding pancreatin for digestion, and stopping digestion by using a culture medium; adding 1mL of PBS solution, blowing and beating the bottom of the culture plate to enable cells to fall off, centrifuging at 2500 rpm, repeating the operation for 3 times, and redispersing for determination by a flow cytometer.

10. The detection method according to claim 1, wherein in the step 3), the goat serum is blocked at 37 ℃ for 30 minutes before the fluorescent-labeled phosphorylated antibody is added.

Technical Field

The invention belongs to the technical field of biochemical analysis of intracellular phosphorylated glycine, and relates to a method for detecting intracellular phosphorylated glycine.

Background

Smoking causes multiple physiological reactions such as gene damage, abnormal expression of protein, abnormal metabolism of exogenous compounds and the like, and the appearance is reflected by the occurrence of multiple biological effects, such as oxidative stress, inflammatory reaction, intracellular tyrosine phosphorylation and the like.

Flow Cytometry (FCM) is a cellular analysis technique that integrates multiple subjects and techniques such as optics, electronics, hydrodynamics, cytochemistry, immunology, and computer science, and is not affected by abnormalities in molecular biology, and can intuitively and accurately quantitatively analyze intracellular phosphorylated tyrosine.

Smoking causes multiple physiological reactions such as gene damage, abnormal expression of protein, abnormal metabolism of exogenous compounds and the like, and the appearance reflection is the occurrence of multiple biological effects, such as oxidative stress, inflammatory reaction, abnormal metabolism of exogenous substances and the like. The inventor hopes to evaluate the influence of smoke exposure on the oxidative stress reaction of cells by researching the active oxygen concentration level in the cells after the smoke exposure, and carry out biological evaluation on the safety of smoke components, thereby providing scientific reference for the research and development and production of safe cigarettes. Currently, there is no relevant research.

Disclosure of Invention

In view of the above situation, the present invention provides a method for detecting the phosphorylation level of tyrosine in a cell, comprising the following steps:

step 1) preparation of flue gas condensate:

extracting a cigarette sample according to GB/T5606.1-2004, smoking by using a smoking machine, collecting total particulate matters in the smoke, and adding DMSO with a corresponding volume according to the mass of the total particulate matters in the smoke to obtain a total particulate matters stock solution in the smoke;

step 2) cell treatment: plating cells and culturing overnight; continuing the culture with the cell culture medium supplemented with the flue gas condensate for 1 hour;

step 3) immunofluorescence labeling and sample processing: the infected cells were washed with PBS; adding 70% ethanol solution for fixation, and standing overnight at low temperature; washing with PBS, permeabilizing with a PBS solution containing triton-X100, and washing with PBS; adding a fluorescent labeled phosphorylation antibody for reaction at 37 ℃, adding pancreatin for digestion, and stopping digestion by using a culture medium; separating out cells and dispersing again until the flow cytometry is used for determination;

step 4) detection, namely detecting the staining intensity of the fluorescent probe by using a flow cytometer, carrying out three-color fluorescence compensation before detection, setting a gate by using CD45-PerCP/SSC during detection, separating an abnormal cell group from a normal cell group, and capturing and analyzing each sample by 1 × 10 after the abnormal cell group is circled and gated⁴(ii) individual cells; alternatively, the staining intensity of the fluorescently labeled phosphorylated antibody is detected using a confocal laser microscope.

According to a further embodiment of the method for detecting the phosphorylation level of intracellular tyrosine according to the present invention, in the step 1), the smoking is performed under both ISO and HCI smoking conditions using a RM20H turntable type smoking machine.

According to a further embodiment of the method for detecting the phosphorylation level of tyrosine in cells of the present invention, in step 1), the method comprisesAnd collecting the total particulate matters of the smoke by using a Cambridge filter.

According to a further embodiment of the method for detecting the phosphorylation level of tyrosine in cells of the present invention, in the step 2), the density of plated cells is 2.5 × 10⁵cells/mL。

According to a further embodiment of the method for detecting the phosphorylation level of tyrosine in cells of the present invention, in the step 2), the cells are separated by adding 1ml of PBS solution after the digestion is terminated, blowing the bottom of the culture plate to allow the cells to fall off, centrifuging at 2500 rpm, and repeating the operation for 3-5 times.

According to a further embodiment of the method for detecting the phosphorylation level of tyrosine in cells of the present invention, in the step 3), the fluorescent-labeled phosphorylated antibody is a Cy 3-labeled phosphorylated tyrosine antibody.

According to a further embodiment of the method for detecting the phosphorylation level of tyrosine in cells of the present invention, in the step 4), the staining intensity of the fluorescent probe is detected at 488nm and 530nm by using a flow cytometer.

According to a further embodiment of the method for detecting the intracellular tyrosine phosphorylation level, the step 2) is carried out by specifically using the method with the cell density of 2.5 × 10⁵Plating cells/mL, and culturing overnight; cell culture was performed for 1 hour with the addition of cell culture media of smoke condensates prepared separately under ISO and HCI suction conditions, respectively.

According to a further embodiment of the method for detecting the phosphorylation level of tyrosine in cells of the present invention, in the step 3), the goat serum is blocked at 37 ℃ for 30 minutes before the fluorescent-labeled phosphorylated antibody is added.

According to a further embodiment of the method for detecting the phosphorylation level of tyrosine in cells of the present invention, the step 3) is specifically: treated cells were washed three times with PBS for three minutes each; adding 70% ethanol solution for fixation, and standing overnight at 4 deg.C; after three washes with PBS, permeabilized for 10 minutes with PBS solution containing 1% triton-100, washed three times with PBS; adding a fluorescent labeled phosphorylation antibody, reacting for 1h at 37 ℃, adding pancreatin for digestion, and stopping digestion by using a culture medium; adding 1ml PBS solution, blowing and beating the bottom of the culture plate to make the cells fall off, centrifuging at 2500 rpm, repeating the operation for 3 times, and redispersing for determination by a flow cytometer.

According to the inventionIn a further embodiment of the method for detecting intracellular tyrosine phosphorylation level, the step 4) is specifically to detect the staining intensity of the fluorescent probe under 488nm and 530nm conditions by using a flow cytometer, carry out trichromatic fluorescence compensation before detection, set a gate by using CD45-PerCP/SSC during detection, separate abnormal cell groups from normal cell groups, and capture and analyze 1 × 10 per sample after the abnormal cell groups are circled out and set the gate⁴And (4) cells.

The detection method provided by the invention has the advantages of simple and convenient operation steps and short detection period, can complete detection only in a few hours, greatly shortens the detection period, is favorable for accelerating the progress of related research, and saves the time cost.

In one embodiment of the detection method, the phosphorylation tyrosine in the cell under different exposure conditions is further obtained by carrying out fluorescence labeling on the phosphorylation tyrosine in the cell by using a Cy3 labeled specific antibody, and observing the change of an intracellular fluorescence signal in real time by using a laser confocal microscope to realize monitoring in the phosphorylation process.

Drawings

FIG. 1 is a graph showing intracellular tyrosine phosphorylation levels under stimulation by different concentrations of smoke condensate obtained by flow cytometry for different aspiration modes.

FIG. 2 shows typical cell morphology of KB oral cell line (485 nm in the upper panel and 505nm in the lower panel).

FIG. 3 is a visual measurement of intracellular tyrosine phosphorylation levels after exposure of smoke condensate prepared in the deep draw Canada mode (condensate concentration 40 μ g/mL, upper panel CY3 detector, lower panel CY5 detector).

FIG. 4 is a visual measurement of intracellular tyrosine phosphorylation levels following exposure to smoke condensate prepared in ISO aspirate mode (condensate concentration 40 μ g/mL, upper panel CY3 detector, lower panel CY5 detector).

FIG. 5 is a visual measurement of intracellular tyrosine phosphorylation levels after exposure of smoke condensate prepared in the deep draw Canada mode (condensate concentration 80 μ g/mL, upper panel CY3 detector, lower panel CY5 detector).

FIG. 6 is a visual measurement of intracellular tyrosine phosphorylation levels following exposure to smoke condensate prepared in ISO suction mode (condensate concentration 80 μ g/mL, top panel CY3 detector, bottom panel CY5 detector).

Detailed Description

The technical solution of the present invention will be further explained with reference to the accompanying drawings and specific embodiments. Unless otherwise indicated, the drugs, reagents, and instruments used in the following examples are commercially available.