CN103954571A - L-arginine-based improved method for detecting change of content of nitric oxide in cells - Google Patents
L-arginine-based improved method for detecting change of content of nitric oxide in cells Download PDFInfo
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- CN103954571A CN103954571A CN201410182084.5A CN201410182084A CN103954571A CN 103954571 A CN103954571 A CN 103954571A CN 201410182084 A CN201410182084 A CN 201410182084A CN 103954571 A CN103954571 A CN 103954571A
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Abstract
The invention discloses an L-arginine-based improved method for detecting the change of content of nitric oxide in cells. The method comprises the following steps: putting cultured cells in an exposure liquid, cracking the cells, then detecting absorbancy of the cells by a nitric oxide detection kit and further converting to obtain the change of synthetic amount of NO in the cells. The method is high in sensitivity, good in selectivity, simple and quick to operate and free of independence on large instruments and devices.
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 (Nitric Oxide Synthase, 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 factors, produces catalytic activity, could finally 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 processes in body, serves as in vivo important physiological regulatory action.Research shows that the biological effect of NO has duality, has important biological significance under normal operation, such as vasodilator, nerve conduction as the fast signal transmitter 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 and impel Apoptosis.Due to, the NO synthetic quantity in biosystem is conventionally all lower, after the more difficult reflection determinand of conventional method exposes, and the variation of NO synthetic quantity in biosystem.Therefore, for convenient research NO, the biological effect in each system and environmental contaminants, on the NOS signal path in biosystem and the synthetic impact of NO, are set up NO synthetic quantity detection method in a kind of simple, quick, highly sensitive cell and are just seemed under study for action particularly important in vivo.
Summary of the invention
The present invention is directed to prior art above shortcomings, propose a kind of based on L ?the detection method that changes of the cell intracellular nitric oxide NO synthetic quantity of arginine improvement, with L ?arginine be the substrate that NOS catalysis generates NO, the present invention is simple to operate quick, highly sensitive, can realize fast intracellular NO synthetic quantity and detect.
The present invention is achieved by the following technical solutions, and the present invention, by the cell after cultivating is placed in after exposure liquid, detects cell absorbance by nitrogen monoxide detection kit after cracking is processed, and further converts the variation that obtains the synthetic quantity of NO in cell.
Described cell refers to: human breast carcinoma MCF ?7 cells;
Described cultivation is that MCF ?7 cells are incubated to cell culture fluid and are placed in 37 ℃, 5%CO
2in incubator, cultivate.
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's Modified Eagle's Medium DMEM/HIGH Glucose, 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, expose liquid 1;
2) containing 1mM cupric chloride and 10mM L ?arginic cell culture fluid, expose liquid 2;
3) and for contrasting the cell culture fluid of contrasting, i.e. contrasting fluid.
Described exposure liquid by before exposure with containing 10mM L ?arginic nutrient solution pre-service 1h realize.
The duration of described exposure is 30min.
Described cracking refers to: to add in object to be measured as the cell pyrolysis liquid of 200 μ L (2%NP ?40,80mMNaCl, 100mM Tris ?HCl), to take out supernatant after centrifugal treating 3min under the environment of 12,000rpm, carry out NO synthetic quantity mensuration.
Described nitrogen monoxide detection kit is purchased from green skies biotechnology research institute.
Described absorbance refers to: the lysis supernatant after getting 50 μ L and respectively exposing liquid and process respectively adds Griess Reagent I and the II of 50 μ L, detects it and is positioned at the absorbance under 540nm.
Described conversion refers to:
wherein: A
0for cell culture fluid contrast absorbance, A
1for exposing liquid 1, process the absorbance in rear lysis supernatant, A
2for exposing liquid 2, process the absorbance in rear lysis supernatant, B is background value, and 50 μ L cell pyrolysis liquids directly add the Griess Reagent I of 50 μ L and the absorbance that II records without exposing.
This method proposes add L ?after arginine processes, the variation=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 variation 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 magnocell in the variation of NO synthetic quantity, the sensitivity that improves whole system, thus can reflect more directly, delicately the impact of tested material on NO synthetic quantity and NOS signal path.
Accompanying drawing explanation
Fig. 1 be cupric chloride in embodiment (Cu) and L ?arginine (L ?Arg) process after in cell NO synthetic quantity change schematic diagram;
In figure: on the same group, the relative synthetic quantity of NO of (contrast and add L ?Arg) exists the different capitalization (A, B) of use of significant difference (p<0.01) to represent, not on the same group between contrast, the relative synthetic quantity of NO that adds between L ?Arg exists the different lowercase (a, b) of use of significant difference (p<0.01) to represent.
Embodiment
Below embodiments of the invention are elaborated, the present embodiment is implemented take technical solution of the present invention under prerequisite, provided 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 ℃ of preservations, experiment before with nutrient solution, 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 ℃, 5%CO
2in incubator, cultivate.Cell culture fluid composition is: high sugared DMEM Dulbecco's Modified Eagle's Medium (DMEM/HIGH Glucose, 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, and 24h is adherent.
2) expose liquid composition:
3) cell Continuous Cultivation 30min in exposing liquid separately.
4) cracking of cell, add 200 μ L cell pyrolysis liquid (2%NP ?40,80mMNaCl, 100mM Tris ?HCl), cracking is complete, the centrifugal 3min of 12,000rpm.
5) collect in the centrifuge tube that cell pyrolysis liquid supernatant to is new, respectively get in 50 μ L sample to 96 orifice plates, nitrogen monoxide detection kit detects NO, the Griess Reagent I and the II that in every hole, add successively 50 μ L, 540nm measures absorbance, calculates after exposure, and in cell, NO synthetic quantity changes.
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 values)/(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: 50 μ L cell pyrolysis liquids respectively add the Griess Reagent I of 50 μ L and absorbance that II records is background value.This method proposes add L ?after arginine processes, after cupric chloride exposes, in cell, the variation=ratio 1 of NO synthetic quantity deducts ratio 2.Add in an embodiment the comparative example of 1mM cupric chloride cell culture fluid, detect do not add L ?arginine process, the variation of NO synthetic quantity in cell after cupric chloride exposes, with content contrast of the present invention, shows content of the present invention added L ?advantage after arginine processing.In actual content application of the present invention, do not need to add the comparative example of 1mM cupric chloride cell culture fluid.
Result shows: contrasting of processing with separate cell nutrient solution compared, add after the exposure of 1mM cupric chloride, in cell, NO synthetic quantity is down to 60.7 ± 4.2% (ratios 3) of comparative example, in comparing cell with control group, NO synthetic quantity has declined 39.3%, do not add L ?arginine process, the variation of NO synthetic quantity in cell after cupric chloride exposes; Nutrient solution add 10mM L ?after arginine processes, in cell, NO synthetic quantity is increased to 160.1 ± 12.2% (ratios 1) that separate cell nutrient solution is processed; With nutrient solution add 10mML ?contrasting of processing of arginine compare, 1mM cupric chloride and 10mM L ?after arginine exposes altogether, in cell, NO synthetic quantity is down to 70.0 ± 4.4% (ratios 2), in cell, NO synthetic quantity has declined 90.1%, be this method propose add L ?after arginine processes, the variation of NO synthetic quantity in cell after cupric chloride exposes.
In proof this method with 10mM L ?after arginine processes, cupric chloride expose inhibiting effect that NO is generated by do not add L ?arginine process 39.3% rise to 10mM L ?arginine 90.1% after processing.L ?arginine process the sensitivity can improve whole system, the variation by NO synthetic quantity in cell more directly, sensitiveer reflect tested material expose after the variation of NO synthetic quantity.
Claims (8)
1. the detection method that in the cell based on the improvement of L ?arginine, NO synthetic quantity changes, it is characterized in that, by the cell after cultivating is placed in and is exposed after liquid, after cracking is processed, by nitrogen monoxide detection kit, detect cell absorbance, and further convert the variation that obtains the synthetic quantity of NO in cell;
Described exposure liquid refers to:
1) containing 10mML ?arginic cell culture fluid, expose liquid 1;
2) containing 1mM cupric chloride and 10mM L ?arginic cell culture fluid, expose liquid 2;
3) and for contrasting the cell culture fluid of contrasting, i.e. contrasting fluid;
Described conversion refers to:
wherein: A
0for cell culture fluid contrast absorbance, A
1for exposing liquid 1, process the absorbance in rear lysis supernatant, A
2for exposing liquid 2, process the absorbance in rear lysis supernatant, 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 cells are incubated to cell culture fluid and are placed in 37 ℃, 5%CO
2in incubator, cultivate.
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, described exposure liquid by before exposure with containing 10mM L ?arginic nutrient solution pre-service 1h realize.
5. method according to claim 1, is characterized in that, the duration of described exposure is 30min.
6. method according to claim 1, it is characterized in that, described cracking refers to: to add in object to be measured as the cell pyrolysis liquid 2%NP of 200 μ L ?40,80mMNaCl, 100mM Tris ?HCl, to take out supernatant after centrifugal treating 3min under the environment of 12,000rpm, carry out NO synthetic quantity mensuration.
7. method according to claim 1, is characterized in that, described absorbance refers to: the lysis supernatant after getting 50 μ L and respectively exposing liquid and process respectively adds Griess Reagent I and the II of 50 μ L, detects it and is positioned at the absorbance under 540nm.
8. method according to claim 1, is characterized in that, described background value refers to: 50 μ L cell pyrolysis liquids directly add the Griess Reagent I of 50 μ L and the absorbance that II records without exposing.
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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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2014
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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 |
Non-Patent Citations (3)
| Title |
|---|
| D.MARMOLINO 等: "Pregabalin Antagonized Copper－Induced Toxicity in Brain:In vitro and in vivo Studies", 《NEUROSIGNALS》 * |
| WANG LU 等: "Nitric oxide synthase and vascular endothelial growth factor expression in hepatocellular carcinoma and the correlation with angiogenesis", 《CHINESE JOURNAL OF CANCER RESEARCH》 * |
| 刘怡晟 等: "一氧化氮在全肠外营养所致胆汁淤积中的作用", 《肠外与肠内营养》 * |
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