CN103954571B - Based on the detection method of the cell intracellular nitric oxide content of L-arginine improvement - Google Patents
Based on the detection method of the cell intracellular nitric oxide content of L-arginine improvement Download PDFInfo
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- CN103954571B CN103954571B CN201410182084.5A CN201410182084A CN103954571B CN 103954571 B CN103954571 B CN 103954571B CN 201410182084 A CN201410182084 A CN 201410182084A CN 103954571 B CN103954571 B CN 103954571B
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Abstract
A kind of based on L arginine improvement cell intracellular nitric oxide NO synthetic quantity change detection method, after the cell after cultivation is placed in exposure liquid, after cracking process, detect cell absorbance by nitrogen monoxide detection kit, and conversion obtains the change of the synthetic quantity of NO in cell further.The present invention is highly sensitive, selectivity good, simple and efficient to handle, do not rely on large-scale instrument and equipment.
Description
Technical field
What the present invention relates to is a kind of method of technical field of biological, specifically a kind of based on L ?the detection method of cell intracellular nitric oxide content of arginine improvement.
Background technology
Nitrogen monoxide (NO) be nitric oxide synthetase (NitricOxideSynthase, NOS) catalytic oxygen and L ?the product that generates of arginine.NOS is the general name of a class isoenzymes, and the NOS of monomeric form forms homodimer by the effect of a lot of accessory factor, produces catalytic activity, finally could generate NO.Because NO is a kind of soluble gas, as signaling molecule, penetration capacity is strong, and conduction of velocity is fast, participates in numerous physiology and pathologic process in body, serves as important physiological regulatory action in vivo.Research shows that the biological effect of NO has duality, has important biological significance under normal operation, such as vasodilator, nerve conduction as the rapid signal conduction material in body.In the research of oxidative stress, NO has Cell protection, prevents apoptotic effect.But then, there is again report NO to participate in oxidative stress simultaneously impel Apoptosis.Due to, the NO synthetic quantity in biosystem is usually all lower, after the more difficult reflection determinand of conventional method exposes, and the change of NO synthetic quantity in biosystem.Therefore, conveniently study the impact on the NOS signal path in biosystem and NO synthesis of the biological effect of NO in vivo in each system and environmental contaminants, set up NO synthetic quantity detection method in a kind of simple, quick, highly sensitive cell and just seem particularly important under study for action.
Summary of the invention
The present invention is directed to prior art above shortcomings, propose a kind of based on L ?arginine improvement cell intracellular nitric oxide NO synthetic quantity change detection method, with L ?arginine be that NOS catalysis generates the substrate of NO, the present invention is simple to operate quick, highly sensitive, can realize intracellular NO synthetic quantity fast and detect.
The present invention is achieved by the following technical solutions, and the present invention exposes after liquid by being placed in by the cell after cultivating, and detect cell absorbance, and conversion obtains the change of the synthetic quantity of NO in cell further after cracking process by nitrogen monoxide detection kit.
Described cell refers to: human breast carcinoma MCF ?7 cells;
Described cultivation is that MCF ?7 cell chulture is placed in 37 DEG C, 5%CO in cell culture fluid
2cultivate in incubator.
The component of described cell culture fluid is: 100 μ L/mL penicillin, 100 μ g/mL streptomysins, 10%v/v hyclone, surplus is high sugared DMEM (Dulbecco'sModifiedEagle'sMediumDMEM/HIGHGlucose, Hyclone).
Described hyclone brand is Chinese holly, purchased from Tian Hang bio tech ltd, Zhejiang.
Described exposure liquid refers to:
1) containing 10mML ?arginic cell culture fluid, namely expose liquid 1;
2) containing 1mM cupric chloride and 10mML ?arginic cell culture fluid, namely expose liquid 2;
3) and for contrasting the cell culture fluid contrasted, i.e. contrasting fluid.
Described exposure liquid by before exposure with containing 10mML ?arginic nutrient solution pre-service 1h realize.
The duration of described exposure is 30min.
Described cracking refers to: in object to be measured, add the cell pyrolysis liquid (2%NP ?40,80mMNaCl, 100mMTris ?HCl) as 200 μ L, carry out NO synthetic quantity mensuration to take out supernatant after centrifugal treating 3min under the environment of 12,000rpm.
Described nitrogen monoxide detection kit is purchased from green skies biotechnology research institute.
Described absorbance refers to: get 50 μ L respectively expose liquid process after lysis supernatant respectively add GriessReagentI and II of 50 μ L, detect its be positioned at 540nm under absorbance.
Described conversion refers to:
wherein: A
0for cell culture fluid contrast absorbance, A
1the absorbance in rear lysis supernatant is processed, A for exposure liquid 1
2process absorbance in rear lysis supernatant for exposing liquid 2, B is background value, and namely 50 μ L cell pyrolysis liquids are without the absorbance exposing GriessReagentI and II that directly add 50 μ L and record.
What this method proposed add L ?after arginine process, the change=X of NO synthetic quantity in cell after cupric chloride exposes
1-X
2.
Technique effect
Compared with prior art, the present invention adopt add L ?arginic method, utilize human breast carcinoma MCF ?7 cells, simple, fast, the change of NO synthetic quantity in high-sensitivity detection cell, its principal feature and advantage have: the method is simple to operate, quick.L ?arginine process can the change of NO synthetic quantity in magnocell, improve the sensitivity of whole system, thus the impact of tested material on NO synthetic quantity and NOS signal path can be reflected more directly, delicately.
Accompanying drawing explanation
Fig. 1 be in embodiment cupric chloride (Cu) and L ?NO synthetic quantity change schematic diagram in cell after arginine (L ?Arg) process;
In figure: there is representing with different capitalizations (A, B) of significant difference (p<0.01) with the NO of (contrast and add L ?Arg) in group relative to synthetic quantity, between different group contrasts, add NO between L ?Arg and there is (a, b) representing with different lowercase of significant difference (p<0.01) relative to synthetic quantity.
Embodiment
Elaborate to embodiments of the invention below, the present embodiment is implemented under premised on technical solution of the present invention, give detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Embodiment 1
The present embodiment determinand: nitrogen monoxide; Model organism: human breast carcinoma MCF ?7 cells; Reagent: L ?arginine (sigma), cupric chloride (standard items be dissolved in sterilized water be mixed with 0.1mol/L mother liquor Bing Yu ?20 DEG C of preservations, test front nutrient solution and be diluted to desired concn), nitrogen monoxide detection kit (green skies biotechnology research institute, Jiangsu).
The present embodiment concrete steps are as follows:
1) human breast carcinoma MCF ?the cultivation of 7 cells: cell is at 37 DEG C, 5%CO
2cultivate in incubator.Cell culture fluid composition is: high sugared DMEM Dulbecco'sModifiedEagle'sMedium (DMEM/HIGHGlucose, Hyclone), add 100u/mL penicillin, 100 μ g/mL streptomysins, 10% Chinese holly hyclone (Tian Hang bio tech ltd, Zhejiang).Before experiment, cell is inoculated in 35mm double dish, 24h, adherent.
2) liquid composition is exposed:
3) cell is exposing Continuous Cultivation 30min in liquid separately.
4) cracking of cell, add the cell pyrolysis liquid (2%NP ?40,80mMNaCl, 100mMTris ?HCl) of 200 μ L, cracking is complete, the centrifugal 3min of 12,000rpm.
5) collect in the new centrifuge tube of cell lysate supernatant liquid to one, respectively get in 50 μ L sample to 96 orifice plates, nitrogen monoxide detection kit detects NO, GriessReagentI and II of 50 μ L is added successively in every hole, 540nm measures absorbance, calculates after exposing, NO synthetic quantity change in cell.
Computation process:
Ratio 1=(exposing liquid 1 absorbance subtracting background value)/(cell culture fluid contrast absorbance subtracting background value);
Ratio 2=(exposing liquid 2 absorbance subtracting background value)/(cell culture fluid contrast absorbance subtracting background value);
Ratio 3=(comparative example absorbance subtracting background value)/(cell culture fluid contrast absorbance subtracting background value);
Wherein: the absorbance that GriessReagentI and II that 50 μ L cell pyrolysis liquids respectively add 50 μ L records is background value.What this method proposed add L ?after arginine process, after cupric chloride exposes, in cell, the change=ratio 1 of NO synthetic quantity deducts ratio 2.Add the comparative example of 1mM cupric chloride cell culture fluid in an embodiment, detect do not add L ?arginine process, after cupric chloride exposes, the change of NO synthetic quantity in cell, contrasts with content of the present invention, show content of the present invention added L ?advantage after arginine process.In actual content application of the present invention, do not need the comparative example adding 1mM cupric chloride cell culture fluid.
Result shows: compared with the contrast of separate cell nutrient solution process, after adding the exposure of 1mM cupric chloride, in cell, NO synthetic quantity is down to 60.7 ± 4.2% (ratios 3) of comparative example, in cell, NO synthetic quantity have dropped 39.3% compared with control group, namely do not add L ?arginine process, the change of NO synthetic quantity in cell after cupric chloride exposes; Nutrient solution add 10mM L ?after arginine process, in cell, NO synthetic quantity is increased to 160.1 ± 12.2% (ratios 1) of separate cell nutrient solution process; With nutrient solution add 10mML ?arginine process contrast compared with, 1mM cupric chloride and 10mML ?after arginine exposes altogether, in cell, NO synthetic quantity is down to 70.0 ± 4.4% (ratios 2), in cell, NO synthetic quantity have dropped 90.1%, what namely this method proposed add L ?after arginine process, the change of NO synthetic quantity in cell after cupric chloride exposes.
Prove in this method with 10mML ?after arginine process, cupric chloride expose inhibiting effect that NO is generated by do not add L ?arginine process 39.3% rise to 10mML ?90.1% after arginine process.L ?arginine process can improve the sensitivity of whole system, by the change of NO synthetic quantity in cell more directly, sensitiveer reflect tested material expose after the change of NO synthetic quantity.
Claims (7)
1. the detection method based on NO synthetic quantity change in the cell of L ?arginine improvement, it is characterized in that, after the cell after cultivation is placed in exposure liquid, after cracking process, detect cell absorbance by nitrogen monoxide detection kit, and conversion obtains the change of the synthetic quantity of NO in cell further;
Described exposure liquid refers to:
1) containing 10mML ?arginic cell culture fluid, namely expose liquid 1;
2) containing 1mM cupric chloride and 10mML ?arginic cell culture fluid, namely expose liquid 2;
3) and for contrasting the cell culture fluid contrasted, i.e. contrasting fluid;
Described conversion refers to:
wherein: A
0for cell culture fluid contrast absorbance, A
1the absorbance in rear lysis supernatant is processed, A for exposure liquid 1
2process the absorbance in rear lysis supernatant for exposure liquid 2, B is background value; Described synthetic quantity be changed to X
1-X
2.
2. method according to claim 1, is characterized in that, described cultivation is that human breast carcinoma MCF ?7 cell chulture is placed in 37 DEG C, 5%CO in cell culture fluid
2cultivate in incubator.
3. method according to claim 1 and 2, is characterized in that, the component of described cell culture fluid is: 100 μ L/mL penicillin, 100 μ g/mL streptomysins, 10%v/v hyclone, surplus is high sugared DMEM.
4. method according to claim 1, is characterized in that, the duration of described exposure is 30min.
5. method according to claim 1, it is characterized in that, described cracking refers to: add in object to be measured 200 μ L cell pyrolysis liquid 2%NP ?40,80mMNaCl, 100mMTris ?HCl, NO synthetic quantity mensuration is carried out to take out supernatant after centrifugal treating 3min under the environment of 12,000rpm.
6. method according to claim 1, is characterized in that, described absorbance refers to: get 50 μ L respectively expose liquid process after lysis supernatant respectively add GriessReagentI and II of 50 μ L, detect its be positioned at 540nm under absorbance.
7. method according to claim 1, is characterized in that, described background value refers to: the absorbance that GriessReagentI and II that 50 μ L cell pyrolysis liquids directly add 50 μ L without exposure records.
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Citations (2)
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| CN102617467A (en) * | 2012-02-21 | 2012-08-01 | 大连理工大学 | Ultrahigh-sensitivity fluorescent probe for detecting nitrogen monoxide |
| CN103063634A (en) * | 2012-12-25 | 2013-04-24 | 华南师范大学 | Flow cytometry detection method of nitric oxide level of shrimp blood corpuscles |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102617467A (en) * | 2012-02-21 | 2012-08-01 | 大连理工大学 | Ultrahigh-sensitivity fluorescent probe for detecting nitrogen monoxide |
| CN103063634A (en) * | 2012-12-25 | 2013-04-24 | 华南师范大学 | Flow cytometry detection method of nitric oxide level of shrimp blood corpuscles |
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| Nitric oxide synthase and vascular endothelial growth factor expression in hepatocellular carcinoma and the correlation with angiogenesis;wang lu 等;《Chinese Journal of Cancer Research》;20010630;第13卷(第2期);124－127 * |
| Pregabalin Antagonized Copper－Induced Toxicity in Brain:In vitro and in vivo Studies;D.Marmolino 等;《Neurosignals》;20101230;210－222 * |
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