CN107976545B - Method for screening cell autophagy regulator in vitro - Google Patents
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- CN107976545B CN107976545B CN201711264034.1A CN201711264034A CN107976545B CN 107976545 B CN107976545 B CN 107976545B CN 201711264034 A CN201711264034 A CN 201711264034A CN 107976545 B CN107976545 B CN 107976545B
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Abstract
The invention discloses a method for screening a cell autophagy regulator in vitro, which is characterized in that an autophagy regulator to be detected is used as an intervention factor, a HEK293 cell in vitro evaluation model for stably expressing an HA-SOD1A4V mutant is processed, a cell lysate containing Triton X-100 is used for cracking the model, then the model is evaluated in vitro, a Triton X-100 insoluble part is separated, and the regulation effect of the autophagy regulator on cell autophagy is judged according to the expression change of the HA-SOD1A4V mutant in the detected Triton X-100 insoluble part. The method is simple, visual, accurate and reliable, and can accurately screen the cell autophagy regulator.
Description
Technical Field
The invention belongs to the technical field of screening of autophagy regulators, and relates to an experimental method for screening the autophagy regulators in vitro.
Background
Autophagy is a key physiological process in maintaining metabolic balance in eukaryotic cells. It consists of a number of successive steps: formation of phagocytic vesicles; formation of autophagosomes; fusing autophagosomes and lysosomes to form autophagosomes; degradation of the substrate, etc. The autophagy disorder is closely related to various human diseases, so that screening of high-efficiency autophagy regulators is an important way for searching for drugs for treating diseases related to the autophagy disorder.
Detection of autophagosome marker L C3 by Western blot
But the application of this method is highly uncertain and can easily lead to misjudgment of the result, for example, induction or inhibition of autophagy can cause L C3
The increase in expression was not even altered. Therefore, there is a need to develop a method for intuitively and accurately screening modulators of autophagy.
Disclosure of Invention
The invention aims to provide a method for screening a cell autophagy regulator in vitro so as to intuitively and accurately screen the cell autophagy regulator.
The method for screening the cell autophagy regulator in vitro adopts HEK293 cells stably expressing an HA-SOD1A4V mutant to establish an in vitro evaluation model for screening the autophagy regulator, treats the in vitro evaluation model by using an autophagy regulating factor to be detected as an intervention factor, then uses cell lysate containing Triton X-100 to crack the treated in vitro evaluation model, separates out a Triton X-100 insoluble part in the cell lysate, judges the regulating effect of the autophagy regulating factor on cell autophagy according to the expression change of the HA-SOD1A4V mutant in the Triton X-100 insoluble part, and screens out the potential cell autophagy regulator.
Wherein, the cell lysate preferably contains 1% Triton X-100.
Specifically, if the autophagy-regulating factor can inhibit accumulation of the HA-SOD1A4V mutant in the insoluble fraction of Triton X-100, the autophagy-regulating factor is an autophagy inducer; on the other hand, the autophagy regulator promoting accumulation of HA-SOD1A4V mutant in insoluble fraction of Triton X-100 is an autophagy inhibitor.
The method for screening the cell autophagy regulator in vitro adopts HEK293 cells as a cell model, and the HEK293 cells are commercialized human embryonic kidney cell strains, have the immortalization characteristic, are simple in culture method and rapid in proliferation, and are the best choice for establishing an in vitro detection model.
The method for screening the cell autophagy regulator in vitro specifically adopts a centrifugal separation mode to separate out the insoluble part of Triton X-100. Triton X-100 is a nonionic surfactant, commonly used to increase the solubility of proteins. The invention utilizes the characteristic that the SOD1A4V mutant is insoluble in Triton X-100 and the difference of the sedimentation coefficients of the soluble part and the insoluble part of the Triton X-100, and adopts a centrifugal separation method to separate the insoluble part of the Triton X-100.
More specifically, the method for screening the cell autophagy regulator in vitro adopts Western hybridization to detect the expression change of the HA-SOD1A4V mutant in the insoluble part of Triton X-100, and further screens the cell autophagy regulator in vitro. Western blotting is widely used to detect protein level expression.
The invention utilizes the constructed HEK293 cell strain which stably expresses the HA-SOD1A4V mutant to evaluate the influence of the autophagy regulatory factor of a treatment group on autophagy activity under in vitro conditions by comparing the change of the expression level of the HA-SOD1A4V mutant in the insoluble part of Triton X-100 in the treatment group and a blank control group. The method is simple, visual, accurate and reliable, and can accurately screen the cell autophagy regulator.
Drawings
FIG. 1 is a graph of the effect of rapamycin on the expression level of HA-SOD1A4V mutant in the insoluble fraction of Triton X-100 in example 1.
FIG. 2 is a graph showing the effect of trehalose on the expression level of the HA-SOD1A4V mutant in the insoluble fraction of Triton X-100 in example 2.
FIG. 3 is a graph showing the effect of 3-methyladenine on the expression level of the HA-SOD1A4V mutant in the insoluble fraction of Triton X-100 in example 3.
FIG. 4 is a graph of the effect of bafilomycin A1 on the expression level of the HA-SOD1A4V mutant in the insoluble fraction of Triton X-100 in example 4.
FIG. 5 is a graph of the effect of dimethyl sulfoxide on the expression level of the HA-SOD1A4V mutant in the insoluble fraction of Triton X-100 in example 5.
Detailed Description
The following examples are only preferred embodiments of the present invention and are not intended to limit the present invention in any way. Various modifications and alterations to this invention will become apparent to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Unless otherwise indicated, the percentage contents in the invention are all mass percentage contents.
Example 1.
Rapamycin is an inhibitor of mammalian target protein of rapamycin kinase (mTOR) and has a significant effect of inducing activation of autophagy in cells.
This example demonstrates the feasibility of the screening method of the present invention to evaluate rapamycin as a modulator of autophagy in cells, using rapamycin as the subject of the study.
Rapamycin used in this example was purchased from Solebao technologies, Inc. and dissolved in DMSO to make a 20mM stock solution.
PCR amplification of human SOD1A4V mutantVariant genes, insertion of the PCR amplification products into the plasmid vector pCMV-C-HAEcoR 
AndBamH 
double enzyme cutting sites.
The constructed plasmid is used for transfecting HEK293 cells, cell strains which stably express the pCMV-C-HA-SOD1A4V mutant are screened out through G418, the stable expression of the HA-SOD1A4V mutant is identified through an HA antibody, and the HEK293 cell strains which successfully and stably express the HA tag SOD1A4V mutant are constructed.
Treating the cells with rapamycin 2 × 10524 hours after the inoculation of one/ml in the six-well plate, rapamycin was added to a final concentration of 50nM, 100nM as treatment group 1 and treatment group 2, respectively. A blank control was also set, replacing rapamycin with the corresponding DMSO.
And collecting 3 test group cells after 24 hours of rapamycin treatment, adding cell lysate containing 1% Triton X-100, collecting the treated cells, carrying out ultrasonic crushing, centrifuging at 500 × g, taking supernatant, filtering through a 0.45 mu m filter membrane, and determining the protein concentration.
Cell lysates with the same amount of protein were taken from 3 test groups according to the protein concentration to be measured, centrifuged at 10000 × g for 50 minutes, the supernatant was discarded, the pellet was broken by ultrasonication, 2 × SDS loading buffer was added and mixed well, heated at 95 ℃ for 5 minutes to denature the protein, and subjected to SDS-PAGE electrophoresis, then the protein was transferred to nitrocellulose membrane, Western blot detection was performed using actin as a loading control, HA antibody was used to detect HA-SOD1A4V mutant expression, chemiluminescence kit was used to develop protein bands, and Image J was used to quantify the intensity of the corresponding bands.
Based on the intensity of each band, the expression level of Triton X-100 insoluble HA-SOD1A4V mutant in the blank control group without intervention was compared. The experimental data are expressed as mean ± sem (
SD) represents (n =3),tthe assay was subjected to statistical analysispDifferences were considered significant < 0.05.
As shown in figure 1, after the cells of the treatment group 1 and the treatment group 2 were treated with rapamycin, the expression level of the HA-SOD1A4V mutant in the insoluble part of Triton X-100 was significantly reduced, which indicates that rapamycin as an autophagy inducer can promote the degradation of the HA-SOD1A4V mutant in the insoluble part of Triton X-100.
Example 2.
Trehalose promotes fusion of autophagosomes with lysosomes through an mTOR independent signal transduction pathway, and induces autophagy. In this example, trehalose was used as a subject to verify the feasibility of the screening method of the present invention in evaluating trehalose as a modulator of autophagy.
Trehalose was purchased from Sigma and dissolved in physiological saline to prepare a stock solution at a concentration of 1M.
PCR amplifying human SOD1A4V mutant gene, inserting PCR amplification product into plasmid carrier pCMV-C-HAEcoR 
AndBamH 
double enzyme cutting sites.
The constructed plasmid is used for transfecting HEK293 cells, cell strains which stably express the pCMV-C-HA-SOD1A4V mutant are screened out through G418, the stable expression of the HA-SOD1A4V mutant is identified through an HA antibody, and the HEK293 cell strains which successfully and stably express the HA tag SOD1A4V mutant are constructed.
Treating the cells with trehalose by exposing the cells to 2 × 105After 24 hours of inoculation of each/ml in a six-well plate, trehalose was added to a final concentration of 20mM, 40mM as treatment group 1 and treatment group 2, respectively. Meanwhile, a blank control group is set, and corresponding normal saline is used for replacing trehalose.
And collecting 3 test group cells after trehalose treatment for 72 hours, adding cell lysate containing 1% Triton X-100, collecting the treated cells, crushing by ultrasonic waves, centrifuging by 500 × g, taking supernatant, filtering by a filter membrane of 0.45 mu m, and determining the protein concentration.
Cell lysates with the same amount of protein were taken from 3 test groups according to the protein concentration to be measured, centrifuged at 10000 × g for 50 minutes, the supernatant was discarded, the pellet was broken by ultrasonication, 2 × SDS loading buffer was added and mixed well, heated at 95 ℃ for 5 minutes to denature the protein, and subjected to SDS-PAGE electrophoresis, then the protein was transferred to nitrocellulose membrane, Western blot detection was performed using actin as a loading control, HA antibody was used to detect HA-SOD1A4V mutant expression, chemiluminescence kit was used to develop protein bands, and Image J was used to quantify the intensity of the corresponding bands.
Based on the intensity of each band, the expression level of Triton X-100 insoluble HA-SOD1A4V mutant in the blank control group without intervention was compared. The experimental data are expressed as mean ± sem (
SD) represents (n =3),tthe assay was subjected to statistical analysispDifferences were considered significant < 0.05.
As shown in FIG. 2, the expression level of the HA-SOD1A4V mutant in the insoluble fraction of Triton X-100 was significantly reduced after the cells of treatment group 1 and treatment group 2 were treated with trehalose, indicating that trehalose as an autophagy inducer promotes the degradation of the HA-SOD1A4V mutant in the insoluble fraction of Triton X-100.
Example 3.
Specific inhibition of PI3K by 3-methyladenine
Blocking the formation of autophagosomes.
In this example, 3-methyladenine was used as a study target to verify the feasibility of the screening method of the present invention in evaluating 3-methyladenine as an autophagy modulator.
3-methyladenine used in this example was purchased from Calbiochem and prepared in fresh medium just before use.
PCR amplifying human SOD1A4V mutant gene, inserting PCR amplification product into plasmid carrier pCMV-C-HAEcoR 
AndBamH 
double enzyme cutting sites.
The constructed plasmid is used for transfecting HEK293 cells, cell strains which stably express the pCMV-C-HA-SOD1A4V mutant are screened out through G418, the stable expression of the HA-SOD1A4V mutant is identified through an HA antibody, and the HEK293 cell strains which successfully and stably express the HA tag SOD1A4V mutant are constructed.
Treating cells with 3-methyladenine by treating the cells with 2 × 105After 24 hours of inoculation of each/ml in a six-well plate, 3-methyladenine was added to a final concentration of 2.5mM and 5mM, respectively, as treatment group 1 and treatment group 2. A blank control was set up with medium instead of 3-methyladenine.
3-methyladenine treatment is carried out for 48 hours, 3 test group cells are collected, cell lysate containing 1% Triton X-100 is added, the treated cells are collected, ultrasonic crushing is carried out, 500 × g is carried out for centrifugation, supernatant is taken, 0.45 mu m filter membrane filtration is carried out, and protein concentration is measured.
Cell lysates with the same amount of protein were taken from 3 test groups according to the protein concentration to be measured, centrifuged at 10000 × g for 50 minutes, the supernatant was discarded, the pellet was broken by ultrasonication, 2 × SDS loading buffer was added and mixed well, the protein was denatured by heating at 95 ℃ for 5 minutes, and subjected to SDS-PAGE electrophoresis, then the protein was transferred to nitrocellulose membrane, Western blot detection was performed using actin as a loading control, HA antibody was used to detect the expression of HA-SOD1A4V mutant, protein bands were developed with chemiluminescence kit, and Image J was used to quantify the intensity of the corresponding bands.
Based on the intensity of each band, the expression level of Triton X-100 insoluble HA-SOD1A4V mutant in the blank control group without intervention was compared. The experimental data are expressed as mean ± sem (
SD) represents (n =3),tcheck inLine statistical analysispDifferences were considered significant < 0.05.
As shown in FIG. 3, the expression level of the HA-SOD1A4V mutant in the insoluble fraction of Triton X-100 was significantly increased after the cells of treatment group 1 and treatment group 2 were treated with 3-methyladenine, indicating that 3-methyladenine acts as an autophagy inhibitor and blocks the degradation of the HA-SOD1A4V mutant in the insoluble fraction of Triton X-100.
Example 4.
The bafilomycin A1 can inhibit hydrogen ions ATPase on a vacuolar membrane, so that lysosome cavity can not be acidified, and the fusion of autophagosome and lysosome is interfered.
This example demonstrates the feasibility of the screening method of the present invention to evaluate bafilomycin a1 as a modulator of autophagy in a bafilomycin a1 study.
The bavlomycin A1 used in this example was purchased from Calbiochem and dissolved in DMSO to prepare a stock solution with a concentration of 100. mu.M.
PCR amplifying human SOD1A4V mutant gene, inserting PCR amplification product into plasmid carrier pCMV-C-HAEcoR 
AndBamH 
double enzyme cutting sites.
The constructed plasmid is used for transfecting HEK293 cells, cell strains which stably express the pCMV-C-HA-SOD1A4V mutant are screened out through G418, the stable expression of the HA-SOD1A4V mutant is identified through an HA antibody, and the HEK293 cell strains which successfully and stably express the HA tag SOD1A4V mutant are constructed.
Treating the cells with bafilomycin A1 by exposing the cells to 2 × 105After 24 hours of inoculation of each/ml in a six-well plate, bafilomycin A1 was added to a final concentration of 20nM, 40nM respectively as treatment 1 and treatment 2. A blank control was also set, substituting the corresponding DMSO for bafilomycin a 1.
Treating the bafilomycin A1 for 24 hours, collecting 3 test group cells, adding cell lysate containing 1% Triton X-100, collecting the treated cells, carrying out ultrasonic disruption, centrifuging at 500 × g, taking supernatant, filtering with a 0.45 mu m filter membrane, and determining the protein concentration.
Cell lysates with the same amount of protein were taken from 3 test groups according to the protein concentration to be measured, centrifuged at 10000 × g for 50 minutes, the supernatant was discarded, the pellet was broken by ultrasonication, 2 × SDS loading buffer was added and mixed well, heated at 95 ℃ for 5 minutes to denature the protein, and subjected to SDS-PAGE electrophoresis, then the protein was transferred to nitrocellulose membrane, Western blot detection was performed using actin as a loading control, HA antibody was used to detect HA-SOD1A4V mutant expression, chemiluminescence kit was used to develop protein bands, and Image J was used to quantify the intensity of the corresponding bands.
Based on the intensity of each band, the expression level of Triton X-100 insoluble HA-SOD1A4V mutant in the blank control group without intervention was compared. The experimental data are expressed as mean ± sem (
SD) represents (n =3),tthe assay was subjected to statistical analysispDifferences were considered significant < 0.05.
As shown in FIG. 4, the expression level of the HA-SOD1A4V mutant in the insoluble fraction of Triton X-100 was significantly reduced after treatment of the cells in treatment groups 1 and 2 with Barfosycin A1, indicating that Barfosycin A1 acts as an autophagy inhibitor and blocks the degradation of the HA-SOD1A4V mutant in the insoluble fraction of Triton X-100.
Example 5.
Dimethyl sulfoxide (DMSO) is used as a common polar aprotic solvent and has wide application. In this example, DMSO was used as a study to verify the feasibility of DMSO as an autophagy modulator in the screening method of the present invention.
DMSO used in this example was purchased from Sigma.
PCR amplifying human SOD1A4V mutant gene, inserting PCR amplification product into plasmid carrier pCMV-C-HAEcoR 
AndBamH 
double enzyme cutting sites.
The constructed plasmid is used for transfecting HEK293 cells, cell strains which stably express the pCMV-C-HA-SOD1A4V mutant are screened out through G418, the stable expression of the HA-SOD1A4V mutant is identified through an HA antibody, and the HEK293 cell strains which successfully and stably express the HA tag SOD1A4V mutant are constructed.
Treatment of cells with DMSO the cells were washed with 2 × 105After 24 hours of inoculation of each/ml in a six-well plate, 0.25% and 0.5% DMSO was added by volume as treatment group 1 and treatment group 2, respectively. A blank control was set up, DMSO was replaced with the corresponding medium.
And (3) collecting cells of the test group after 24 hours of DMSO treatment, adding cell lysate containing 1% Triton X-100, collecting the treated cells, carrying out ultrasonic crushing, centrifuging at 500 × g, taking supernatant, filtering through a 0.45 mu m filter membrane, and determining the protein concentration.
Cell lysates with the same amount of protein were taken from 3 test groups according to the protein concentration to be measured, centrifuged at 10000 × g for 50 minutes, the supernatant was discarded, the pellet was broken by ultrasonication, 2 × SDS loading buffer was added and mixed well, heated at 95 ℃ for 5 minutes to denature the protein, and subjected to SDS-PAGE electrophoresis, then the protein was transferred to nitrocellulose membrane, Western blot detection was performed using actin as a loading control, HA antibody was used to detect HA-SOD1A4V mutant expression, chemiluminescence kit was used to develop protein bands, and Image J was used to quantify the intensity of the corresponding bands.
Based on the intensity of each band, the expression level of Triton X-100 insoluble HA-SOD1A4V mutant in the blank control group without intervention was compared. The experimental data are expressed as mean ± sem (
SD) represents (n =3),tthe assay was subjected to statistical analysispDifferences were considered significant < 0.05.
As shown in FIG. 5, the expression level of HA-SOD1A4V mutant in the insoluble fraction of Triton X-100 was not significantly changed compared to the blank control after DMSO treatment in the cells of treatment groups 1 and 2, indicating that DMSO did not have the effect of regulating autophagy activity.
The foregoing is only a preferred embodiment of the present invention. The method can also be applied to other factors with autophagy regulating activity, and is used for screening the autophagy regulator of cells under in vitro conditions by analyzing the change of the expression quantity of the HA-SOD1 mutant of the insoluble part of Triton X-100.
Claims (6)
1. An in vitro screening method of a cell autophagy regulator is characterized in that an in vitro evaluation model for screening the autophagy regulator is established by adopting HEK293 cells stably expressing an HA-SOD1A4V mutant, the in vitro evaluation model is processed by taking an autophagy regulator to be detected as an intervention factor, then a cell lysate containing Triton X-100 is used for cracking the processed in vitro evaluation model, a Triton X-100 insoluble part in the cell lysate is separated, and the regulation effect of the autophagy regulator on cell autophagy is judged according to the expression change of the HA-SOD1A4V mutant in the Triton X-100 insoluble part.
2. The method of claim 1, wherein said cell lysate contains 1% Triton X-100.
3. The method of claim 1, wherein the autophagy modulating factor that inhibits accumulation of the HA-SOD1A4V mutant in the insoluble fraction of Triton X-100 is an autophagy inducer.
4. The method of claim 1, wherein the autophagy modulating factor that promotes accumulation of the HA-SOD1A4V mutant in the insoluble fraction of Triton X-100 is an autophagy inhibitor.
5. The method of claim 1, wherein the insoluble fraction of Triton X-100 is separated by centrifugation.
6. The method as set forth in claim 1, wherein the change in expression of the HA-SOD1A4V mutant in the insoluble fraction of Triton X-100 is detected by Western blotting.
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| Degradation of amyotrophic lateral sclerosis-linked mutant Cu,Zn-Superoxide Dismutase Proteins by Macroautophagy and the Proteasome;Tomohiro Kabuta，et al;《J Biol. Chem.》;20060818;第281卷(第41期);摘要，第30525页左栏第2段-右栏第4段，第30530页左栏第2段-右栏第1段，图3，图8 * |
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