CN104483493A - Method for screening drug action target position of mizoribine in immunized T cell - Google Patents

Abstract

The invention relates to a method for utilizing a proteomic technology to screen a drug action target position of mizoribine in an immunized T cell. According to the method for screening the drug action target position of mizoribine in the immunized T cell, the proteomic technology is used for screening the drug action target position of mizoribine in the immunized T cell and the method comprises the following steps: step 1) establishing sample groups of the action of mizoribine immunizing the T cell; step 2) preparing two-dimensional electrophoresis protein samples of the immunized T cell; step 3) carrying out two-dimensional gel electrophoresis; step 4) collecting images and analyzing to obtain the result. The method for screening the drug action target position of mizoribine in the immunized T cell, disclosed by the invention, is clear and definite in operation through, and true and reliable in result, overcomes the defects that with adoption of the conventional screening method, the screening result is high in error and low in accuracy as the analysis data are huge and complicated, and provides the detection result with pharmacologic significance and guiding function for clinical medication.

Description

A kind of mizoribine that screens is in the method for immune t-cell Chinese traditional medicine target site

Technical field

The present invention relates to a kind of method utilizing proteomic techniques to screen in immune t-cell Chinese traditional medicine target site mizoribine.

Background technology

Mizoribine (Mizoribine, MZ, bredinin) mizoribine is the imidazoles microbiotic that Japanese Asahi Chemical Industry obtains from the culturing filtrate of soil mould.As immunodepressant, in Dec, 1991 rises and applies in Japan Clinic kidney transplant.Mizoribine is suppressed medicine as the routine immunization after kidney transplant by Japan many clinical transplantation centers.China also it can be used as kidney transplant anti-repulsion medicine for clinical in recent years.Mizoribine compared with similar drugs imuran, hepatotoxicity wind agitation and bone marrow inhibition little, its main adverse reaction is gastrointestinal reaction, hematological system obstacle and allergic symptom, accidental Inhibitory effect of marrow function and acute renal failure.。At present, clinically for individual sufferer, mizoribine cannot be determined in immune t-cell Chinese traditional medicine target site always, and conventional screening means are too huge and complicated owing to analyzing data, cause the selection result error larger, accuracy rate is low, and being difficult to provides the testing result with pharmacology meaning and directive function when clinical application.

Summary of the invention

The technical problem to be solved in the present invention is the above-mentioned defect how overcoming prior art, provides a kind of method utilizing proteomic techniques to screen in immune t-cell Chinese traditional medicine target site mizoribine.

For solving the problems of the technologies described above, this screening mizoribine utilizes proteomic techniques in the method for immune t-cell Chinese traditional medicine target site, and screen in immune t-cell Chinese traditional medicine target site mizoribine, its concrete grammar comprises the following steps:

Step 1): will be cultured to quintan immune t-cell, and be divided into the control group of equivalent and some test group, add the mizoribine of variable concentrations in test group respectively, the overall mizoribine that formed is to the drug effect sample cohort of immune t-cell model;

Step 2): by step 1) described in the drug effect sample cohort of mizoribine to immune t-cell model be cultured to exponential phase; Discard nutrient solution, scraping cells collecting cell is to the centrifuge tube of some correspondences; Cell is rinsed 5 times with phosphate buffer after collection completes, residual phosphoric acid damping fluid in centrifuge tube is blotted after rinsing, then add equal-volume 10mol/L urea, 50mmol/L DTT, 5% 3-[(3-courage amido propyl)-diethylamine 1-propane sulfonic acid, 30mmol/L trishydroxymethylaminomethane formed mixed solution, multigelation cell 5 ~ 8 times; Put into cryosel bath and leave standstill 30min; Then centrifugal 2h under being warming up to the condition of 10 DEG C; Centrifugal complete after get supernatant and measure protein concentration, and according to the protein compression scale designation recorded, packing, be transferred in the insulation can of 70 DEG C store store for subsequent use;

Step 3): by step 2) in store supernatant for subsequent use take out two groups, often organize each 50ug, with equivalent 10mol/L urea, 5% 3-[(3-courage amido propyl)-diethylamine]-propane sulfonic acid, 20mmol/L dithiothreitol (DTT) 0.5% IPG damping fluid composition mixing material mixing; Adding IPG after mixing holds in glue groove, and the glue of IPG immobilized ph gradient strip faces down and puts into groove, and in its surface smear one deck humatite oil, and IPG holds glue groove and adds a cover to be placed on IPGphor horizontal cataphoresis apparatus battery lead plate and carry out isoelectric focusing; After isoelectric focusing terminates, IPG immobilized ph gradient strip balances 30min in equilibrium liquid; Moved on SDS-PAGE separation gel by IPG immobilized ph gradient strip after having balanced, and with agarose sealing, glass plate is placed in electrophoresis tank and carries out electrophoresis with current constant mode, to electrophoresis tank, bromophenol blue forward position extends to and stops electrophoresis apart from 5mm place, gel base, obtains gel;

Step 4): will by step 3) obtain gel-colored, and use gel scanner transmission scan; Image PDQUEST software will be obtained analyze, image analysis process comprises the shearing of collection of illustrative plates, the detection of protein spots, the coupling between different gel, the normalization of amount and interior relative molecular weight and the isoelectric point marking protein site in the standard scores of 2DMARKER amount and isoelectric point determination glue of utilization; With the Two-dimensional Gel Electrophoresis of immune t-cell protein under normal condition for reference glue, carry out proportioning with the Two-dimensional Gel Electrophoresis of the immune t-cell protein cultivated under mizoribine effect, obtain the differential expression protein that mizoribine acts on immune t-cell;

Step 5): will by step 4) differential expression protein that obtains after in-gel digestion, by mass spectrum, measure differential expression protein; Compare with mass spectral database, filter out differential expression protein; After specifying differential protein, its biological function is determined, is verified by Western blot, obtains mizoribine in immune t-cell Chinese traditional medicine target site.

The present invention screens mizoribine in the method for immune t-cell Chinese traditional medicine target site, operation thinking is clear and definite, real result is reliable, overcome conventional screening means too huge and complicated owing to analyzing data, cause the selection result error larger, the defect that accuracy rate is low, for clinical application provides the testing result with pharmacology meaning and directive function.

Embodiment

This will be cultured to quintan immune t-cell, be divided into the control group of equivalent and some test group, add the mizoribine of variable concentrations in test group respectively, and the overall mizoribine that formed is to the drug effect sample cohort of immune t-cell model;

Step 2): by step 1) described in the drug effect sample cohort of mizoribine to immune t-cell model be cultured to exponential phase; Discard nutrient solution, scraping cells collecting cell is to the centrifuge tube of some correspondences; Cell is rinsed 5 times with phosphate buffer after collection completes, residual phosphoric acid damping fluid in centrifuge tube is blotted after rinsing, then add equal-volume 10mol/L urea, 50mmol/L DTT, 5% 3-[(3-courage amido propyl)-diethylamine 1-propane sulfonic acid, 30mmol/L trishydroxymethylaminomethane formed mixed solution, multigelation cell 5 ~ 8 times; Put into cryosel bath and leave standstill 30min; Then centrifugal 2h under being warming up to the condition of 10 DEG C; Centrifugal complete after get supernatant and measure protein concentration, and according to the protein compression scale designation recorded, packing, be transferred in the insulation can of 70 DEG C store store for subsequent use;

Step 3): by step 2) in store supernatant for subsequent use take out two groups, often organize each 50ug, with equivalent 10mol/L urea, 5% 3-[(3-courage amido propyl)-diethylamine]-propane sulfonic acid, 20mmol/L dithiothreitol (DTT) 0.5% IPG damping fluid composition mixing material mixing; Adding IPG after mixing holds in glue groove, and the glue of IPG immobilized ph gradient strip faces down and puts into groove, and in its surface smear one deck humatite oil, and IPG holds glue groove and adds a cover to be placed on IPGphor horizontal cataphoresis apparatus battery lead plate and carry out isoelectric focusing; After isoelectric focusing terminates, IPG immobilized ph gradient strip balances 30min in equilibrium liquid; Moved on SDS-PAGE separation gel by IPG immobilized ph gradient strip after having balanced, and with agarose sealing, glass plate is placed in electrophoresis tank and carries out electrophoresis with current constant mode, to electrophoresis tank, bromophenol blue forward position extends to and stops electrophoresis apart from 5mm place, gel base, obtains gel;

Step 4): will by step 3) obtain gel-colored, and use gel scanner transmission scan; Image PDQUEST software will be obtained analyze, image analysis process comprises the shearing of collection of illustrative plates, the detection of protein spots, the coupling between different gel, the normalization of amount and interior relative molecular weight and the isoelectric point marking protein site in the standard scores of 2DMARKER amount and isoelectric point determination glue of utilization; With the Two-dimensional Gel Electrophoresis of immune t-cell protein under normal condition for reference glue, carry out proportioning with the Two-dimensional Gel Electrophoresis of the immune t-cell protein cultivated under mizoribine effect, obtain the differential expression protein that mizoribine acts on immune t-cell;

Step 5): will by step 4) differential expression protein that obtains after in-gel digestion, by mass spectrum, measure differential expression protein; Compare with mass spectral database, filter out differential expression protein; After specifying differential protein, its biological function is determined, is verified by Western blot, obtains mizoribine in immune t-cell Chinese traditional medicine target site.

The present invention includes but be not limited to above-mentioned embodiment, any product meeting these claims and describe, all falls within protection scope of the present invention.

Claims (1)

1. screen mizoribine in a method for immune t-cell Chinese traditional medicine target site, it is characterized in that: this method utilizes proteomic techniques, screen in immune t-cell Chinese traditional medicine target site mizoribine, its concrete grammar comprises the following steps:

Step 1): will be cultured to quintan immune t-cell, and be divided into the control group of equivalent and some test group, add the mizoribine of variable concentrations in test group respectively, the overall mizoribine that formed is to the drug effect sample cohort of immune t-cell model;

Step 2): by step 1) described in the drug effect sample cohort of mizoribine to immune t-cell model be cultured to exponential phase; Discard nutrient solution, scraping cells collecting cell is to the centrifuge tube of some correspondences; Cell is rinsed 5 times with phosphate buffer after collection completes, residual phosphoric acid damping fluid in centrifuge tube is blotted after rinsing, then add equal-volume 10mol/L urea, 50mmol/L DTT, 5% 3-[(3-courage amido propyl)-diethylamine 1-propane sulfonic acid, 30mmol/L trishydroxymethylaminomethane formed mixed solution, multigelation cell 5 ~ 8 times; Put into cryosel bath and leave standstill 30min; Then centrifugal 2h under being warming up to the condition of 10 DEG C; Centrifugal complete after get supernatant and measure protein concentration, and according to the protein compression scale designation recorded, packing, be transferred in the insulation can of 70 DEG C store store for subsequent use;

Step 3): by step 2) in store supernatant for subsequent use take out two groups, often organize each 50ug, with equivalent 10mol/L urea, 5% 3-[(3-courage amido propyl)-diethylamine]-propane sulfonic acid, 20mmol/L dithiothreitol (DTT) 0.5% IPG damping fluid composition mixing material mixing; Adding IPG after mixing holds in glue groove, and the glue of IPG immobilized ph gradient strip faces down and puts into groove, and in its surface smear one deck humatite oil, and IPG holds glue groove and adds a cover to be placed on IPGphor horizontal cataphoresis apparatus battery lead plate and carry out isoelectric focusing; After isoelectric focusing terminates, IPG immobilized ph gradient strip balances 30min in equilibrium liquid; Moved on SDS-PAGE separation gel by IPG immobilized ph gradient strip after having balanced, and with agarose sealing, glass plate is placed in electrophoresis tank and carries out electrophoresis with current constant mode, to electrophoresis tank, bromophenol blue forward position extends to and stops electrophoresis apart from 5mm place, gel base, obtains gel;

Step 4): will by step 3) obtain gel-colored, and use gel scanner transmission scan; Image PDQUEST software will be obtained analyze, image analysis process comprises the shearing of collection of illustrative plates, the detection of protein spots, the coupling between different gel, the normalization of amount and interior relative molecular weight and the isoelectric point marking protein site in the standard scores of 2DMARKER amount and isoelectric point determination glue of utilization; With the Two-dimensional Gel Electrophoresis of immune t-cell protein under normal condition for reference glue, carry out proportioning with the Two-dimensional Gel Electrophoresis of the immune t-cell protein cultivated under mizoribine effect, obtain the differential expression protein that mizoribine acts on immune t-cell;

Step 5): will by step 4) differential expression protein that obtains after in-gel digestion, by mass spectrum, measure differential expression protein; Compare with mass spectral database, filter out differential expression protein; After specifying differential protein, its biological function is determined, is verified by Western blot, obtains mizoribine in immune t-cell Chinese traditional medicine target site.