CN107625770A - Applications of the Tempol in terms of as the medicine for suppressing cancer cell multiplication - Google Patents

Abstract

Tempol as suppress cancer cell multiplication medicine in terms of and as promotion cisplatin induction cancer cell-apoptosis medicine in terms of application.Cancer cell is ovarian cancer cell, breast cancer cell, pancreatic cancer cell, liver cancer cells, stomach cancer cell.Tempol suppresses the propagation of oophoroma OVCAR3 and SKOV3 cell, by disturbing nNOS to produce the glycolysis that NO reaches suppression cancer cell.Tempol promotes DDP to suppress cancer cell multiplication, the expression for reducing the cancer cell Bcl 2/Bax that DDP is induced, promote the cancer cell-apoptosis of DDP inductions by increasing intracellular ROS level.Present invention research draws applications of the Tempol in the application in terms of as the medicine for suppressing cancer cell multiplication and Tempol in terms of as the cancer cell-apoptosis medicine for promoting cisplatin induction, can be realized using Tempol performance and obtain efficient, less toxic antineoplastic.

Description

Applications of the Tempol in terms of as the medicine for suppressing cancer cell multiplication

Technical field

The present invention relates to cancer drug technical field, more particularly to Tempol as the medicine for suppressing cancer cell multiplication Application of the application and Tempol of aspect in terms of as the cancer cell-apoptosis medicine for promoting cisplatin induction.

Background technology

Cancer is that present society is difficult to one of illness for effectively administering, at present effective way of the medical field without the cancer of radical cure Footpath.The global people of cancer patient about 7,000,000 newly-increased every year, dead tumor patient number about 5,000,000, just has one in average every 6 seconds People dies from tumour.

At present, chemicals improvement is still the essential therapeutic arsenals of cancer patient, is played in treatment of cancer irreplaceable Effect.The effective percentage only 10-20% of clinical single medicine, the effective percentage of combined chemotherapy is also less than 35%, and the secondary work of combined chemotherapy poison With greatly enhancing, the clinical application effect of chemotherapeutics significantly limit.Therefore, research is efficient, the treating cancer of low toxicity Medicine, it is significant.

The content of the invention

It is an object of the invention to provide Tempol as suppress cancer cell multiplication medicine in terms of application and Applications of the Tempol in terms of as the cancer cell-apoptosis medicine for promoting cisplatin induction, so as to realize that acquisition is efficient, low toxicity is anti-swollen Tumor medicine.

An object of the present invention is realized by following technical measures.

Applications of the Tempol in terms of as the medicine for suppressing cancer cell multiplication.

Above-mentioned cancer cell is ovarian cancer cell, breast cancer cell, pancreatic cancer cell, liver cancer cells, stomach cancer cell.

Further, Tempol suppresses the propagation of oophoroma OVCAR3 and SKOV3 cell.

Further, Tempol is by disturbing nNOS to produce the glycolysis that NO reaches suppression cancer cell.

Further, Tempol suppress cancer cell glucose consumption and lactic acid generation, suppress cancer cell ATP generation, Suppress cancer cell NADPH generation, activation cancer cell AMPK phosphorylation, suppression cancer cell glycolysis key enzyme PFK1 and PKM2 Nitrosation, do not influence cancer cell glycolysis key enzyme PFK1 and PKM2 protein expression.

Another object of the present invention is realized by following technical measures.

Applications of the Tempol in terms of as the cancer cell-apoptosis medicine for promoting cisplatin induction.

Further, Tempol promotes DDP to suppress cancer cell multiplication.

Further, Tempol reduces the cancer cell Bcl-2/Bax of DDP inductions expression.

Further, Tempol promotes the cancer cell-apoptosis of DDP inductions by increasing intracellular ROS level.

Further, Tempol is by disturbing the glycolysis of nNOS function inhibitio cancer cells.

Present invention research show that applications and Tempol of the Tempol in terms of as the medicine for suppressing cancer cell multiplication are being made To promote the application in terms of the cancer cell-apoptosis medicine of cisplatin induction, it can realize that acquisition is efficient, low using Tempol performance Malicious antineoplastic.

Figure of description

Fig. 1 is Tempol structure chart；

Fig. 2 is that the 3.1.1 parts of present invention implementation 2 act on oophoroma by MTT experiment method detection Tempol The IC50 of OVCAR3 and SKOV3 cells result schematic diagram；

Fig. 3 be the present invention implement 2 3.1.1 parts by MTT experiment method detect Tempol to oophoroma OVCAR3 and The result schematic diagram of the effect of SKOV3 cells propagation；

Fig. 4 is that the 3.2.1 parts various concentrations Tempol of the embodiment of the present invention 2 combines DDP and acts on oophoroma respectively The result that OVCAR3 cells 48h, MTT technology for detection Tempol suppresses the influence of oophoroma OVCAR3 cells propagation to DDP is illustrated Figure；

Fig. 5 is that the 3.2.1 parts of the embodiment of the present invention 2 observe Tempol joint DDP effects in the case where being inverted luminescence microscope After oophoroma OVCAR3 cells, the variation diagram of cellular morphology quantity；

Fig. 6 is that Tempol promotes the oophoroma OVCAR3 Apoptosis knots of DDP inductions in 3.2.2 in the embodiment of the present invention 2 Fruit schematic diagram；

Fig. 7 is that Tempol reduces the oophoroma OVCAR3 cells Bcl-2/ of DDP inductions in 3.2.3 in the embodiment of the present invention 2 The result schematic diagram of Bax expression；

Fig. 8 is that Tempol promotes the oophoroma OVCAR3 Apoptosis of DDP inductions to lead in 3.2.4 in the embodiment of the present invention 2 Cross the result schematic diagram of increase intracellular ROS level；

Fig. 9 be the embodiment of the present invention 3 1.1 in detect three kinds of NOSs inhibitor L-NAME, NPLA and 1400W to oophoroma The result schematic diagram of the influence of OVCAR3 grape cells sugar consumption and lactic acid generation；

Figure 10 be the embodiment of the present invention 3 1.2 in Tempol nNOS protein levels intracellular to oophoroma OVCAR3 work Use result schematic diagram；

Figure 11 is in the bright implementation 3 of the present invention 3.2 Tempol suppression oophoroma OVCAR3 cell glycolysis key enzymes PFK1 With the result schematic diagram of PKM2 nitrosation.

Embodiment

The invention will be further described with the following Examples.

Embodiment 1.

Applications of the Tempol in terms of as the medicine for suppressing cancer cell multiplication.Cancer cell includes but not to be limited to oophoroma thin Born of the same parents, breast cancer cell, pancreatic cancer cell, liver cancer cells, stomach cancer cell.

Specifically, Tempol can suppress the propagation of oophoroma OVCAR3 and SKOV3 cell.Tempol is by disturbing nNOS Produce NO and reach the glycolysis for suppressing cancer cell, reach the propagation for suppressing cancer cell.The glucose that Tempol suppresses cancer cell disappears Consumption and lactic acid generation, suppress cancer cell ATP generation, the generation for suppressing cancer cell NADPH, activation cancer cell AMPK phosphoric acid Change, suppress cancer cell glycolysis key enzyme PFK1 and PKM2 nitrosation, do not influence cancer cell glycolysis key enzyme PFK1 and PKM2 protein expression.

It can be drawn from result above, Tempol can suppress the propagation of cancer cell, be suitable as suppressing cancer cell multiplication Medicine in terms of application.

Embodiment 2.

Applications of the Tempol in terms of as the cancer cell-apoptosis medicine for promoting cisplatin induction.Specifically, Tempol promotes DDP suppresses cancer cell multiplication.Tempol reduces the cancer cell Bcl-2/Bax of DDP inductions expression, promotes the cancer of DDP inductions thin Born of the same parents' apoptosis is by increasing intracellular ROS level.Tempol is by disturbing the glycolysis of nNOS function inhibitio cancer cells.

Present invention research draws applications of the Tempol in terms of as the cancer cell-apoptosis medicine for promoting cisplatin induction, can Realized using Tempol performance and obtain efficient, less toxic antineoplastic.

In order to verify the effect above, using Ovarian Cancer Cells OVCAR3 and SKOV3 as object, the effect of embodiment 2 is verified, Specific experiment process is as follows.

1. experiment material

1.1 cell line

Ovarian Cancer Cells OVCAR3 and SKOV3 are that institute of oncology of Nanfang Medical Univ preserves cell early stage.

1.2 major experimental reagents

1.2.1 cell culture medium DMEM and pancreatin Tyrisin is purchased from Gibco companies (USA), and hyclone is purchased from BI companies (UT, USA).

1.4- hydroxyl -2,2,6,6- tetramethyl piperidine oxides (Tempol, are abbreviated as：TPL) it is purchased from Tokyo and answers chemical industry Co., Ltd. (Tokyo, Japan).

1.2.3 cis-platinum (Cisplatin), tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO) (DMSO) and active oxygen detection probe DCFH-DA is purchased from Sigma-Aldrich companies (MO, USA).

1.2.4 AnnexinV-FITC cell apoptosis detection kits are purchased from BD Biosciences companies (Rockville, MD).

1.2.5 protein lysate RIPA, PMSF, protease inhibitor cocktail, albumen pre-dyed marker, 5 × loading Buffer, 20 × TBS, primary antibody internal reference GAPDH, secondary antibody HRP mark goat-anti rabbit peroxidase Conjugated Gaot Anti-Rabbit IgG (h+l) and sheep anti mouse peroxidase Conjugated Gaot anti-Mouse IgG (h+l) are equal It is purchased from Fu De bio tech ltd (Guangzhou, China).

1.2.6 BCA protein quantifications kit, 1.5M Tris-HCL (PH 8.8) buffer solution, 1M Tris-HCL (PH 6.8) buffer solution, Western primary antibodies dilution are purchased from green skies company (China).

1.2.7 pvdf membrane (0.22um) and ECL chemical luminescence for liquid are purchased from Millipore companies (USA).

1.2.8 primary antibody Bcl-2 and Bax is purchased from Cell Signaling Technology companies (CA, USA).Primary antibody HSP90 and nNOS is purchased from Abcam (CA, USA).

1.2.9 co-immunoprecipitation Pierce Co-Immunoprecipitation (Co-IP) Kit (Cat.No.26149) It is purchased from

Thermo Fisher companies (USA).

The configuration of 1.3 main agents

1.3.1 phosphate buffer (Phosphate Buffer Solution, PBS)：Weigh 8.0g Nacl, 0.2g Kcl, 1.44g Na2HPO4 and 0.24g KH₂PO₄, with 800ml deionized water dissolvings, with 1mol/l Hcl or 1mol/l NaOH PH to 7.4 is adjusted, is settled to 1000ml, normal temperature preserves.

Note：PBS used, which needs to be cooled to 4 DEG C after autoclaving, during cell culture saves backup.

1.3.2 10%FBS cell culture mediums：450ml DMEM culture medium+50ml FBS.

1.3.3 cells frozen storing liquid：Hyclone and DMSO press 9:1 volume ratio shifts to an earlier date 24H configurations, is put into 4 DEG C of refrigerators It is standby.

1.3.4 MTT configuration：0.5g MTT are weighed, are dissolved in 10ml phosphate buffer (PBS), with 0.22 μm of filter membrane Filtering is put 4 DEG C and is kept in dark place to remove the bacterium in solution.MTT is it is generally advisable to matching while using, and 4 DEG C of lucifuges are protected after filtering Deposit in two weeks effectively, or be configured to 5mg/ml and be stored in -20 DEG C long-term preservation, low dose packing, avoid multigelation.

1.3.5 10%SDS：Weigh 100g SDS to be slowly transferred in the beaker of the water containing about 0.9L, use magnetic stirring apparatus Stirring is settled to 1L with water, gives room temperature preservation, in half a year effectively, if being precipitated in preserving, water-bath is dissolved up to being completely dissolved Still it can be used afterwards.

1.3.6 10% ammonium persulfate：0.1g ammonium persulfates are weighed, add 1ml deionized waters, solid powder is thoroughly molten Solution, is stored in 4 DEG C.

Note：10% ammonium persulfate is preferably now with the current, and the solution prepared can be used 2 weeks or so in 4 DEG C of preservations.

1.3.7 30% acrylamide：By 14.5g acrylamides and 0.5gN, N '-methylene bisacrylamide is dissolved in 50ml and goes In ionized water, 4 DEG C are kept in dark place fully after dissolving, recover to room temperature and without precipitation during use.

1.3.8 1 × Tris- glycine running buffers：3.03gTris, 14.4g glycine and 1gSDS are weighed, is used 800ml deionized water dissolvings, until completely dissolved, it is standby to be settled to 1000ml room temperature preservations.

1.3.9 1 × transferring film buffer solution：3.03gTris and 14.4g glycine is weighed, with 700ml deionized water dissolvings, is treated 200ml methanol is added after being completely dissolved, is settled to 1000ml with deionized water, 4 DEG C of refrigerators save backup.

1.3.10 1 × TBST buffer solutions：25ml 20 × TBS and 500ul 20%Tween20 are measured, add deionized water 500ml is settled to, room temperature preservation is standby.

1.3.11 5%BSA confining liquids：Bovine serum albumin(BSA) 1.25g is weighed, it is abundant to add 25ml 1 × TBST buffer solutions Dissolving.

1.3.12 the configuration (gel thicknesses 10mm) of polyacrylamide concentration glue and separation gel：

1.3.13 the configuration of 0.01mol/l citrate buffers：0.1mol/L citric acid 9mL+0.1M citric acid trisodiums 41mL+ distilled water 450mL, room temperature preservation.

1.4 major experimental instruments

1.4.1 superclean bench is purchased from safe and sound company of Su Jing groups (China)

1.4.2 specification be lmL, 200 μ L, 100 μ L, 20 μ L, l0 μ L, 2.5 μ L liquid-transfering gun be purchased from Eppendorf companies (Germany)

1.4.3 UV detector is purchased from AlphaSpec companies (USA)

1.4.4 CO2 cell incubations case is purchased from Harris companies (USA)

1.4.5 low-temperature and high-speed centrifuge (Pharamcia, Sweden)

1.4.6 high sensitivity chemistry luminescence imaging system (Bio-RAD, the U.S.)

1.4.7 ELIASA (Bio-TEK, the U.S.)

1.4.8 inverted fluorescence microscope (Nikon, Japan)

1.4.9 ultra low temperature freezer (Thermo, Germany)

1.4.10 glue imager (Invitrogen, the U.S.)

1.4.11 electrophoresis apparatus (Bio-RAD, the U.S.)

1.4.12 Milli-Q water purifiors (Millipore companies, the U.S.)

2 experimentations

2.1 cell culture

2.1.1 cell recovery

The recovery requirement quickly dissolving of cell, can so ensure the rapid dissolving of extracellular crystallization, avoid because slow Slow dissolving makes water penetration be caused damage into the cell to cell.Preparing experiment platform, blake bottle, culture medium, 15ml before cell recovery Centrifuge tube and other items, and thermostat water bath is adjusted to 37 DEG C.Shaken as early as possible in 37 DEG C of water-baths after taking out cell from liquid nitrogen container It is dynamic that until melting completely, tube wall sprays alcohol disinfecting, and in superclean bench, cells frozen storing liquid is transferred in 15ml centrifuge tubes, 800-1000r/min centrifuges 2min, as far as possible draws upper strata frozen stock solution totally, adds 1ml RPMI1640 culture mediums by cell Precipitation fully mixes.Cell suspension is added to preprepared blake bottle, then adds 3-5ml RPMI1640 culture mediums, 8 Word shakes up method mixing.Blake bottle is transferred to 37 DEG C, and 24h is cultivated in 5%CO2 incubator, and whether observation cell state is adherent good It is good, and give the culture medium that cell more renews.

2.1.2 cell culture

Daily observation cell state, the change of the color and transparency of primary part observation nutrient solution.Contain in general nutrient solution Have phenol red as instruction composition and show the pH value of nutrient solution：The nutrient solution of fresh normal is pink, PH is about 7.2- 7.4.Add after cell is cultivated because cell metabolism produces acid product, medium pH value is declined and is caused lighter Turn yellow.Once it was found that nutrient solution turns yellow, illustrate that metabolite has been stacked into a certain amount of in nutrient solution, need to be changed at liquid or passage Reason.- as the stable cell of normal condition growth need 2-3d to change liquid 1 time.Cell culture a few days ago use 37 DEG C, CO2 incubators, this Sample makes pH value relatively stable, beneficial to cell growth.After passage, through suspension, it is adherent extend into incubation period, then start Grow into exponential phase cells and start amount reproduction, be gradually connected in flakes and cover with bottom of bottle.The cell of adherent growth is in length It should just be passed in time during full bottom of bottle 80%, otherwise cell can lack the accumulation with metabolite due to nutriment, into platform Phase simultaneously fails.Culture passage takes different methods according to different cells.The cell of adherent growth is passed on digestion method, portion Divide adherent growth but attach unstable cell and also can use directly piping and druming passage.The digestion method passage of attached cell：Absorb as far as possible Or old nutrient solution in bottle is outwelled, washed 1-2 times with PBS, remove PBS, 1ml digestive juices (trypsase) are added into bottle, are gently shaken Dynamic blake bottle, makes digestive juice flow through all cell surfaces, and blake bottle is placed microscope after 37 DEG C of incubators digest 2-5min Under observed, find kytoplasm retraction, space between cells increase after, digestion should be terminated immediately.1-2ml cultures are drawn to be based in bottle, Draw culture in glassware liquid, blow and beat bottle inner cell repeatedly, piping and druming process will carry out in order, since blake bottle bottom while to another While terminate, to ensure that all bottoms are all received.Action will not overexert softly during piping and druming, while not go out as far as possible Existing foam, these can all be caused to damage to cell.Cell suspension is formed after cell detachment bottle wall.Collect cell suspension to 15ml from Heart pipe, 800-1000r/min centrifugation 2min, is eliminated as much as supernatant, adds 1ml culture mediums and cell is mixed into individual cells Cell suspension.TCS is calculated, cell suspension is uniformly assigned in 3 or 4 blake bottles, 1 is carried out and passes 3 or 1 training for passing 4 Support.

2.1.3 cell cryopreservation

Slow gradient freezing is wanted during freeze-stored cell.Because cell be not added with it is any it is protectant in the case of directly freezed when, The moisture of intraor extracellular can all quickly form ice crystal, and the formation of ice crystal will cause a series of adverse reaction.In cell cryopreservation ICW is equably reduced as far as possible, and the formation for reducing intracellular ice crystal is to reduce the key of cellular damage.At present Protective agent is made using glycerine or dimethyl sulfoxide (DMSO) more.Both materials after cryogenic freezing to cell without overt toxicity, and molecule Measure small, solubility is big, easy penetration cell, can decline intracellular ice point, improves after birth leading into property to water；Plus slow freezing Method can make outside intracellular moisture emigrated cell, in extracellular formation ice crystal, reduce the formation of intracellular ice crystal, so as to reduce by Caused cellular damage is formed in ice crystal.

It may be used to freeze from proliferation period to the culture cell formed before fine and close cell monolayer, but be preferably selected pair Number growth period cells, survival rate can preferably change 1 time than relatively low, therefore freezing the previous day after the cryopreservation covered with Nutrient solution.The cell dissociation of monolayer growth is got off with trypsase, according to propagating method the cell digested be collected in from Heart pipe simultaneously counts, and centrifuges 1000r/min, 2min.Remove trypsase and old nutrient solution, add prepare freeze nutrient solution (containing 10%DMSO or glycerine), the final densities of cell are l06/ml of (5-10) * in frozen stock solution.Gently being blown and beaten with pipette tips makes Cell is uniform, is then distributed into sterile cryopreservation tube.Every 1-1.5ml of cryopreservation tube liquid feeding.Cryopreservation tube must screw ensure it is close Envelope.Write the title of cell on cryopreservation tube exactly, freeze the information such as time.- 80 DEG C are transferred in freezing storing box afterwards overnight, are taken within second day Go out to be transferred in liquid nitrogen and preserve for a long time.

2.2 MTT detect cytoactive

2.2.1 cell is prepared：First grope cell before experiment and the optimal cell number that need to plant plate is covered with special time.Will growth Logarithmic phase cell dissociation in good condition, centrifugation, takes 4000 cell per wells to be seeded in 96 orifice plates, cross, which shakes up method, makes cell Bed board is uniform, is placed in 37 DEG C, 5%CO2 incubator culture 24h, adds and gives directions the medicine of concentration to act on the specific time.

2.2.2 MTT is incubated：Culture medium is removed, and is washed one time with PBS.By the low μ l+MTT of blood serum medium 100 in every hole (5mg/ml) 10 μ L proportional arrangement MTT mixed liquors, addition have in cell culture well and 3 acellular blank control wells, and 37 DEG C lucifuge is incubated 2-4 hours, it is seen that purple crystal precipitation first a ceremonial jade-ladle, used in libation is blue in hole forms.It is careful to exhaust liquid in hole, and add 100 μ Per hole to dissolve purple crystal, lucifuge is placed in 15min on shaking table and detects 490nM exciting lights by Bio-tek ELIASAs lDMSO Under OD values.

2.2.3 cytoactive is calculated：Pass through formula：Cytoactive (%)=(experimental group OD values-blank group OD values)/(right According to a group OD values-blank group OD values) * 100%.

2.3 inverted fluorescence microscopes observe cellular morphology

2.3.1 cell is prepared：By the good logarithmic phase cell dissociation of growth conditions, centrifugation, 20*104/ holes cell per well is taken It is seeded in 6 orifice plates, cross, which shakes up method, makes plating cells uniform, is placed in 37 DEG C, 5%CO2 incubator culture 24h, is separately added into Final concentration of 1.5mM Tempol, 3 μM of DDP, 1.5mM Tempol+3 μM of DDP processing 48h.

2.3.2 inverted fluorescence microscope observes cellular morphology：Different groups of medicines are observed under inverted fluorescence microscope respectively The change of cellular morphology and number after effect, and take pictures.

2.4 Annexin V-FITC/PI dyeing detection Apoptosis and death

2.4.1 vitellophag：Bed board on request, after medicine function cells certain time, remove culture medium, PBS washing patches Parietal cell once, adds the trypsin digestion cell 3-5min for being free of EDTA in right amount, adds 2-3 times of pancreatin volume of culture medium and neutralizes, Piping and druming, is transferred in centrifuge tube, and 1000g is centrifuged 5 minutes, abandons supernatant, collects cell.

2.4.2 cell is washed：PBS mixes cleaning cell, centrifugation, removes supernatant, counts cell.

2.4.3 dye marker：The cell for taking 5-10*104 to be resuspended, 1000g are centrifuged 5 minutes, abandon supernatant, are added in kit Cell is resuspended in the μ l of bindingbuffer 500.5 μ lAnnexin V-FITC are added, are gently mixed.Add 10 μ l propidium iodides dye Color liquid (PI), is gently mixed.Lucifuge is carried out using aluminium-foil paper, is incubated at room temperature 5-15 minutes, is subsequently placed in ice bath.It was incubated Cell is resuspended in journey 2-3 times to improve Color.2.4.4 detection：Flow cytomery, Annexin V- are carried out immediately FITC is green fluorescence, and propidium iodide (PI) is red fluorescence, detector output image, and draws the cell of two kinds of dye markers Rate of dyeing, finally calculate apoptosis and the death rate.

2.5 Western Blot detect protein expression level

2.5.1 reagent prepares

(1) 10%SDS:L.0g SDS+10mL deionized waters, dissolved under 60 DEG C of water-baths, give room temperature preservation, have in half a year Effect, is such as precipitated in preservation, and water-bath still can be used after dissolving；1.3.510% Ammonium Persulfate 98.5：0.1g Ammonium Persulfate 98.5s (AP)+ L.0mL deionized water, 4 DEG C of preservations after being completely dissolved, in 1-2 weeks effectively；

(2) 30% acrylamides：Double propylene phthalein amine (the Bic)+50mL deionizations of 14.5g propylene phthalein amine (Acr)+0.5g methenes 4 DEG C are kept in dark place after water, fully dissolving, recover to room temperature and without precipitation during use；

(3) 5 × Tris- glycine running buffers (mother liquor)：15.15g Tris+93.85g glycine+5.0g SDS+ 600mL deionized waters, after being completely dissolved, it is settled to 1000ml room temperature preservations；

(4) 10 × transferring film buffer solutions (mother liquor)：58g Tris+29g glycine+3.7g SDS+700ml deionized waters are abundant After dissolving, room temperature preservation after 1000ml is settled to.

(5) transferring film buffer solution：100ml10 × transferring film buffer solution+700ml deionized water+200m1 methanol, 4 degree of refrigerators are protected Deposit, it is recyclable to reuse 2-3 times；

(6) TBST buffer solutions：20 × TBS 25ml+20%Tween20 500ul+ deionized waters 475ml；

(7) 5%BSA：Bovine serum albumin(BSA) 1.25g+TBST buffer solutions 25ml；

2.5.2 protein concentration extracts

Oophoroma OVCAR3 cells are inoculated into 6 orifice plates by 20*104/ holes, 37 DEG C, are cultivated in 5%CO2 incubator 24h, whether next day observation cell state is adherent completely, in good condition.Added into culture medium final concentration of 1.5mM Tempol, After 3 μM of DDP, 1.5mM Tempol+3 μM of DDP processing 48h, cell is collected.Prepare to extract intracellular total protein, and survey albumen Concentration.

6 orifice plates are taken out from incubator, are placed on ice.The RIPA lysates containing protease inhibitors are pre-configured with, with 990ulRIPA lysates add the ratio of 10 μ l protease inhibitors PMSF and 10 μ l protein phosphatase inhibitor mixtures to prepare, Add 50-150 μ l lysate per hole, crack 30min on ice.Prepare 1.5ml centrifuge tube precoolings, open 4 DEG C of centrifuge precoolers Device.Period shakes 6 orifice plates every 5min, makes lysate that ware bottom cell be completely covered.Cell is scraped with cell, it is as complete as possible Full collection cell lysate is into 1.5ml centrifuge tubes, 4 DEG C of centrifugation 15min.Sample is taken out, collects supernatant to advance precooling In 1.5ml centrifuge tubes.Protein concentration is surveyed by the method for BCA determination of protein concentration kits, and is recorded.By 4:1 ratio adds 5 × SDS-PAGE albumen sample-loading buffers, boil 5-10min in metal bath, are placed in cooled on ice, can be stored in-80 DEG C of refrigerators Preserve.

2.5.3 BCA determines protein concentration

(1) dilution of standard items：BSA standard items are diluted with PBS；

(2) BCA mixed liquors are prepared：According to testing protein sample size, reagent A and reagent B are pressed 50:1 volume ratio is matched somebody with somebody BCA mixed liquors processed, fully mix.

(3) micro-pipe assay method：

1) 2 μ lBSA titers and testing sample are taken respectively, is added in 96 orifice plates, then add 18 μ l PBS in every hole Buffer solution, gently vibration mix.

2) per adding the fresh μ l of BCA working solutions 200 configured in hole, in gently waving abundant mixing on shaking table.

3) 30min is incubated in 37 DEG C of incubators, its absorbance is detected at ELIASA 562nm.

5) testing sample protein concentration is calculated according to standard curve

2.5.4 SDS- polyacrylamide gel electrophoresises

2.5.4.1 cleaning：Glass system offset plate is cleaned, baking box is placed in and dries；

2.5.4.2 leak hunting：Gel maker is assembled, is placed it on horizontal desktop, clear water is filled it up between glue plate, is stood Whether 10min, observation liquid level decline；

2.5.4.3 glue：8%-12% separation gels 10ml is prepared in glue ratio, 5%, which concentrates glue 6ml, (is not added with TEMED)；

2.5.4.4 separation gel is filled：Overturn and shake up 20 times or so after addition TEMED in separation gel, pay attention to produce greatly Bubble is measured, pours into separation gel immediately, clear water covering separation gel is carefully added into after pouring at once, to flatten separation glue liquid surface, and hinders Only inhibitory action of the oxygen in air to gel polymerisation；

2.5.4.5 fill concentration glue：Horizontal rest 30min, after gelling to be separated is solid, the water on upper strata is poured out, by residual moisture Blot, whether observation separation gel plane is horizontal, then concentrates addition TEMED in glue and shakes up encapsulating,

Comb is horizontally inserted after filling it up with；

2.5.4.6 after gelling to be concentrated is solid, the both sides that two hands pinch comb respectively are gently pulled out straight up, by electricity Swimming groove inside groove fills it up with fresh electrophoresis liquid, and electrophoresis tank water jacket can add recovery electrophoresis liquid；

2.5.4.7 loading：Sample applied sample amount is calculated according to the concentration of measure and adds sample (total egg of the 1mm thickness glue per hole Lean type product is usually no more than 30 μ l, 1.5mm thickness glue and is no more than 40 μ l), the first hole is left blank, and Marker is added in the hole of left side second 2-3μl；

2.5.4.8 electrophoresis：The slow electrophoresis of constant pressure 80V makes every PFP be gathered in same horizontal line (about 30min), treats Marker is separated, and albumen is changed to constant pressure 100-120V (about 40-60min) into separation gel, and glue bottom has just been run out of i.e. to bromjophenol blue Electrophoresis can be terminated.

2.5.5 transferring film

2.5.5.1 make transferring film folder：Wear PE gloves clips it is sizeable it is clean filter paper 4 is opened, foam-rubber cushion 2 is opened, special turn Film packing paper 4 is opened.A clean pallet is taken, transferring film liquid is poured into disk.Transferring film clip is put into pallet, black side is placed on lower floor, successively Pad a foam-rubber cushion, transferring film packing paper, clean filter paper.It is fixed on the other hand, several times are rolled back and forth with roller on the other hand to drive the bubble of the inside away, Clip another side is also such；

2.5.5.2 glue is cut：After electrophoresis terminates, clamping plate is removed, glass exposure gel is pried open, according to destination protein and internal reference egg White molecular size range, band where band where destination protein and internal reference albumen is cut respectively, glue is placed in the filter of clip black flour On paper, pay attention to keeping the moistening of glue；

2.5.5.3 clean gloves are worn, are cut into pvdf membrane by the size of cut gel band correspondingly sized, one jiao is cut off and does Good mark, it is placed in methanol and soaks 10 seconds, be put into 2-3min of cleaning and dipping in transferring film liquid, then pvdf membrane is covered on adhesive tape, according to The secondary filter paper and foam-rubber cushion for covering another side, clip and fixed；

2.5.5.4 transferring film：Clip is put into transfer groove (black flour of clip must be made to the black flour of groove, clip it is transparent In face of the red face of groove), pour into transferring film liquid.Transferring film instrument is placed in transferring film on ice, constant current 300mA, about 1.5-2h is (according to protein molecular Measure size to determine)；

2.5.6 closing, primary antibody and secondary antibody immune response, scanning development

2.5.6.1 closing：TBST is eluted 1 time, 5 minutes/time, the pvdf membrane after transferring film is put into 5%BSA, is placed in and shakes 1-2h is closed on bed, TBST is eluted 1 time, 5 minutes/time；

2.5.6.2 it is incubated primary antibody：Clean 15mL centrifuge tubes are taken, will with primary antibody dilution by the suggestion of antibody specification Primary antibody is diluted to respective concentration respectively, and often pipe about 6mL, pvdf membrane is inserted, and good seal is put into 4 DEG C of refrigerator overnights (12-15h). Pvdf membrane is taken out, is placed on shaking table and is eluted 4 times with TBST, 5 minutes/time；

2.5.6.3 it is incubated secondary antibody：Clean 15mL centrifuge tubes are taken, are diluted to secondary antibody by the suggestion of secondary antibody specification corresponding dense Degree (1:5000-8000) purpose pvdf membrane, is put into secondary antibody, shaking table is placed at room temperature and is incubated 1 hour；Pvdf membrane is taken out, used TBST is eluted 4 times, 5 minutes/time.

2.5.6.4 develop (ECL methods)：ECL A liquid：B liquid=1:1, it is imaged under high sensitivity chemistry luminescence imaging system and claps According to.

2.6 active oxygen ROS probes DCFH-DA：

2.6.1 probe is configured：According to 1:2000 dilute DCFH-DA (20mM) with serum-free medium, make final concentration of 10 Micromoles per liter.

2.6.2 cell is collected：Cell culture fluid is removed, PBS washings two digest to three times, centrifugation, remove supernatant, collect thin Born of the same parents, and washed twice with serum free medium.

2.6.3 probe is loaded：The DCFH-DA that proper volume has diluted is added, adds what is diluted generally for six orifice plates DCFH-DA is no less than 1ml/ holes, is incubated 30 minutes in 37 DEG C of cell culture incubators, every the reverse mixings of 3-5min once, makes probe Fully contacted with cell.

2.6.4 detection：Cell is washed with serum-free cell culture medium 2 times, and intracellular DCFH- is introduced into abundant removal DA.The sample flow cytomery of probe is loaded after collection cell.Parameter setting：Use 488nm excitation wavelengths, 525nm Launch wavelength, the power of fluorescence before and after stimulating in real time or by time point detection.DCF fluorescence spectrum and FITC is closely similar, can To detect DCF with FITC parameter setting.

The detection of 2.5 protein sulfhydryl nitrosation modification

2.5.1 cell is collected when cell growth state is good, the careful culture medium removed in cell, with the washing of precooling Three times, last time scraping cells are in EP pipes, 1000 × g, 5min for buffer solution washing cell.

2.5.2 supernatant is carefully removed, often pipe adds 500 μ L Buffer A containing Blocking Reagent, 4 DEG C of shaking tables are incubated 30min.

2.5.3 EP pipes are transferred in high speed freezing centrifuge, 12000 × g, 4 DEG C centrifugation 10 minutes it is broken with sedimentation cell Piece.

2.5.4 supernatant is transferred in a new microcentrifugal tube, adds the acetone of 4 times of volumes (2mL), mixed, -20 DEG C place 1h.

2.5.5 EP pipes are transferred in high speed freezing centrifuge, 3000 × g, 4 DEG C centrifuge 10 minutes.

2.5.6 supernatant is removed, adds 500 μ L Buffer B containing Reducing and Labeling Reagent, room temperature place 1h.

2.5.7 the acetone of 4 times of volumes (2mL) is added, is mixed, -20 DEG C of placement 1h.

2.5.8 EP pipes are transferred in high speed freezing centrifuge, 3000 × g, 4 DEG C centrifuge 10 minutes.

2.5.9 remove supernatant, add the lavation buffer solution of 100 μ L precoolings, be resuspended albumen carry out determination of protein concentration and SDS-PAGE electrophoresis.

2.6 Western Immunos are co-precipitated Co-IP

2.6.1 antibody immobilization

Pay attention to：Following operating process is applied to 10-65 μ g of crosslinking affinity purification antibody, can not contain other amine in solution Base molecule (such as Tris, glycine) or carrier protein.

2.6.1.1 reinforced AminoLink binding resins and reagent are balanced to room temperature.

2.6.1.2 with ultra-pure water dilute 20 × cross-linking buffer to 1 ×, each IP reactions prepare 2 milliliter 1 × crosslinking buffering Liquid.

2.6.1.3 the bottle of reinforced AminoLink binding resins is gently revolved, to obtain homogeneous suspension.Use big mouth Footpath truncates pipette tips by 20 μ L resin grout liquids addition Pierce centrifugal columns.Pillar is placed in a microcentrifugal tube, 1000 × g is centrifuged 1 minute, is discarded and is flowed through liquid.

2.6.1.4 resin is washed with 200 μ l 1 × cross-linking buffer, centrifuges and discard and flow through liquid.

2.6.1.5 the bottom of centrifugal column is patted on paper handkerchief to remove remaining liquid, inserts bottom.

2.6.1.6 with enough ultra-pure water and 20 × cross-linking buffer, antibody (10-75 μ g) liquor capacity is adjusted

To 200 μ l, buffer concentration is 1 ×.For example, for the antibody that 10 μ l concentration are 1 μ g/ μ l, the μ l 20 of addition 10 × The ultra-pure water of cross-linking buffer and 180 μ l.Directly the antibody of ultra-pure water, 20 × cross-linking buffer and affinity purification is added to and contained Have in the centrifugal column of resin.

2.6.1.7 in fume hood, 3 μ l sodium cyanoborohydride solution is added into every 200 μ l reaction system.

2.6.1.8. screw lid is covered, at ambient temperature in incubation 90-120 minutes on circulator or vortex mixer, it is ensured that Slurries are in suspended state during incubation.

2.6.1.9. unload and retain bottom plug, unscrew screw-cap.Centrifugal column is placed and in collecting pipe and centrifuged.Preserve stream Liquid is worn to verify antibody binding efficiency.

2.6.1.10. remove screw lid, in post plus 200 μ l 1 × cross-linking buffer, centrifuge and discard and flow through liquid. Repeat aforesaid operations once.

2.6.1.11. plus 200 μ l quenching buffers are to centrifugal column, centrifuge and discard and flow through liquid.

2.6.1.12. centrifugation column bottom is patted on paper handkerchief to remove remaining liquid, inserts bottom.Added into resin 200 μ l quenching buffers.

2.6.1.13. in fume hood, 3 μ l sodium cyanoborohydride solutions is added into centrifugal column and cover screw-cap.Gently Jog moves or mixing of turning upside down, and is incubated 15 minutes.

2.6.1.14. unloading bottom plug, unscrew screw-cap.Centrifugal column is put back in collecting pipe, centrifuges and discards and flow through liquid.

2.6.1.15. screw lid is removed, resin is washed twice with 200 μ 1 × cross-linking buffers of l, it is equal after washing every time Need to centrifuge.

2.6.1.16. wash resin six times with 150 μ l lavation buffer solutions, be both needed to centrifuge after washing every time.

2.6.2. attached cell cracks

2.6.2.1. the culture medium in cell is carefully removed.

2.6.2.2. with modified form Du Shi PBSs cell once.

2.6.2.3. 500 μ l IP cracking of precooling on ice/lavation buffer solution is added in cell.5 minutes are incubated on ice simultaneously Periodically mix.

2.6.2.4. cell pyrolysis liquid is transferred in microcentrifugal tube, 13000g, 4 DEG C, 10min it is broken with sedimentation cell Piece.

1.2.7 statistical analysis

Statistical analysis is carried out using the softwares of SPSS 20.0.The comparison of two sample averages is examined using the t of two independent samples (Independent-Sample T Test) or single-sample t-test, sample average more than two compare using completely randomized design The variance analysis (One-wayANOVA) of data, homogeneity test of variance use Levene'sTest.P values<0.05 thinks there is statistics Learn meaning.

3. experimental result

3.1 Tempol promote the ovarian cellular apoptosis of cisplatin induction

3.1.1 Tempol suppresses the propagation of oophoroma OVCAR3 and SKOV3 cell

For effects of the detection Tempol to human epithelial ovarian carcinoma cells proliferation, we act on ovum with the Tempol of various concentrations respectively Nest cancer OVCAR3 and SKOV3 cell, and Tempol is calculated by MTT experiment method oophoroma OVCAR3 and SKOV3 cell are increased Grow the drug concentration (IC50) during inhibiting rate 50%.Suitable Tempol activities then are selected according to IC50, it is real by MTT The effect that proved recipe method detection Tempol breeds to oophoroma OVCAR3 and SKOV3 cell.First with various concentrations Tempol (2,4, 6th, 8,10mM) processing oophoroma OVCAR3 and SKOV3 cell 48h, MTT experiment method detection Tempol act on oophoroma The IC50 of OVCAR3 and SKOV3 cells.Experimental result is as shown in Figure 2：Tempol IC50 is about in oophoroma OVCAR3 cells It is about 2.32mM in oophoroma SKOV3 cells for 3.72mM.It is to detect Tempol to oophoroma OVCAR3 and SKOV3 cell The influence of propagation, we handle SKOV3 cells, action time with 1.5mM Tempol processing OVCAR3 cells and 1mM Tempol It is 5 days, the effect bred by MTT experiment method detection Tempol to oophoroma OVCAR3 and SKOV3 cell.Experimental result As shown in Figure 3：When Tempol handled OVCAR3 and SKOV3 cells to the 3rd day, relative to control group, Tempol groups significantly press down Oophoroma OVCAR3 cells (P processed<And SKOV3 cells (P 0.0097)<0.0286) propagation.This part of test results shows Tempol suppresses oophoroma OVCAR3 and SKOV3 cell propagation, prompts Tempol to have the work for suppressing tumor cell proliferation With.

3.2 Tempol promote the oophoroma OVCAR3 Apoptosis of cisplatin induction

3.2.1 Tempol promotes the inhibitory action that cis-platinum is bred to oophoroma OVCAR3 cells

Cis-platinum (DDP) is clinically conventional chemotherapy in ovarian cancer medicine, but its side effect can not be ignored, therefore is probed into more It is the direction for having always a demand for making great efforts in oncotherapy approach that adduction, which manages suitable drug combination method,.It is to study Tempol to DDP Suppress the influence of oophoroma OVCAR3 cells propagation, therefore it is thin to act on oophoroma OVCAR3 with MTT experiment method detection DDP first The IC50 of born of the same parents.Various concentrations DDP handles OVCAR3 cell 48h, and the IC50 of OVCAR3 cells is then acted on MTT detections DDP. DDP IC50 is about 4.71 μM in oophoroma OVCAR3 cells.Again oophoroma is acted on various concentrations Tempol joints DDP OVCAR3 cells, and suppress the influence that oophoroma OVCAR3 cells are bred to DDP by MTT technology for detection Tempol.First not Combine DDP (3 μM) respectively with concentration Tempol (Tempol-1 1.5mM and Tempol-2 2mM) and act on oophoroma OVCAR3 Cell 48h, MTT technology for detection Tempol suppresses the influence of oophoroma OVCAR3 cells propagation to DDP.Experimental result such as Fig. 4 institutes Show：Relative to DDP groups, Tempol-1 (1.5mM) joint DDP groups (P<0.0139) and Tempol-2 (2mM) combines DDP groups (P< 0.0136) reduction of oophoroma OVCAR3 cell survival rates is made, and Tempol-2 (2mM) combines DDP groups relative to Tempol-1 (1.5mM) joint DDP group cell survival rates further reduce, P<0.0136, there is significant difference.Further to prove Tempol suppresses the humidification of oophoroma OVCAR3 cells propagation to DDP, and we are used with luminescence microscope observation joint is inverted The change of cellular morphology quantity after medicine.Tempol joints DDP is observed in the case where being inverted luminescence microscope and acts on oophoroma OVCAR3 After cell, the change of cellular morphology quantity.Experimental result is as shown in Figure 5：Tempol (1.5mM) joint DDP (3 μM) groups act on After oophoroma OVCAR3 cells 48h, relative to DDP groups, cell quantity is significantly reduced, and cellular morphology is rounded, and is no longer close to Culture dish surface.This part of test results shows that Tempol promotes the inhibitory action that DDP breeds to oophoroma OVCAR3 cells, And with the increase of Tempol concentration, it is stronger to promoting DDP to suppress oophoroma OVCAR3 cel l proliferations.

3.2.2 Tempol promotes the oophoroma OVCAR3 Apoptosis of DDP inductions

Give Tempol (1.5mM), DDP (3 μM) and Tempol joint DDP processing oophoroma OVCAR3 cell 48h, Annexin V-FITC cell apoptosis detection kits are marked, and Annexin V-FITC (+) are apoptotic cell, and PI (+) is Dead cell, the positive cell of FCM analysis instrument count tag simultaneously calculate positive rate, testing result such as Fig. 6 A and Fig. 6 B It is shown：Tempol joint DDP group inducing ovarian cancer OVCAR3 early apoptosis of cells rates (Annexin+/PI-) for 74.1 ± 2.9%, compared to the 59.52 ± 1.58% of DDP groups, dramatically increase (P<0.0121)；Tempol combines DDP group inducing ovarians Cancer OVCAR3 cell mortalities (Annexin+/PI+) are 5.43 ± 0.58%, are close with the 7.29 ± 2.3% of DDP groups.This Part of test results shows, Tempol promotes the oophoroma OVCAR3 Apoptosis of DDP inductions, and using increase early apoptosis as It is main.

3.2.3 Tempol reduces the oophoroma OVCAR3 cells Bcl-2/Bax of DDP inductions expression

After acting on oophoroma OVCAR3 cells with Western blot technology for detection Tempol joints DDP, OVCAR3 is thin Intracellular anti-apoptotic proteins Bcl-2, pro apoptotic protein Bax and Bcl-2/Bax expression.Give Tempol (1.5mM), DDP (3 μM) and Tempol joint DDP processing oophoroma OVCAR3 cell 24h, with the intracellular Bcl-2 of Western blot technology for detection each groups and Bax expression.Experimental result is as shown in Figure 7：Relative to DDP groups, Tempol joint DDP group OVCAR3 cell Bcl-2 and Bax tables Up to having declined, and Bcl-2/Bax expression ratio significantly reduces (P<0.0228).This part of test results shows, Tempol Reduce the oophoroma OVCAR3 cells Bcl-2/Bax expression of DDP inductions.

3.2.4 Tempol promotes the oophoroma OVCAR3 Apoptosis of DDP inductions by increasing intracellular ROS level

To oophoroma OVCAR3 intracellular ROS levels after being acted on ROS probes labelling experiment detection Tempol joints DDP Influence.Oophoroma OVCAR3 cell 12h are handled with Tempol (1.5mM), DDP (3 μM) and Tempol joints DDP first, then Intracellular ROS is marked with ROS probes DCFH-DA, then with the intracellular ROS of FCM analysis instrument detection oophoroma OVCAR3 It is horizontal.Experimental result is as shown in Figure 8：Compared to DDP groups, Tempol joint DDP group oophoroma OVCAR3 intracellular ROS levels are bright Aobvious increase (P<0.0035).This experiment shows that Tempol promotes the oophoroma OVCAR3 Apoptosis of DDP inductions thin by increasing Intracellular ROS is horizontal.

Embodiment 3.

In order to verify the effect above, using Ovarian Cancer Cells OVCAR3 and SKOV3 as object, the effect of embodiment 1 is verified, Specific experiment process is as follows.

1 experiment material

1.1 major experimental reagents

L-NAME, N-PLA, 1400W, DETA-NONO are purchased from sigma companies；ATP reagents are purchased from promega companies； XTT/PMS is purchased from sigma companies；Hyclone is purchased from BI；DMEM culture mediums, pancreatin are purchased from Gibco companies；96 hole cell culture Plate, culture plate are clean special Products；Nitrate/Nitrite Fluorometric Assay Kit kits are purchased from Cayman companies；Glucose determination reagent box and lactate acid detection kit are Biovision Products.

2 experimental methods

2.1 NO Concentration Testings

NO is detected using the Nitrate/Nitrite FluorometricAssay Kit of Cayman companies.The first step, first Nitrate ion is changed into nitrite ion with nitrate reductase；Second step, by nitrite and fluorescence probe DAN (2,3- diaminonaphthalene) combines, while NaOH can strengthen the yield of fluorescence, and the fluorescence intensity finally measured is with total NO yield into just It is related.

2.1.1 reagent prepares

(1) NO2-/NO3-Aassay buffer are prepared：The Aassay of 1 volume is dissolved with 100 μ l ultra-pure water buffer；

(2) NO3- reductases：Dissolved, put on ice during operation, best matching while using with 1.2ml Aassay buffer；

(3) cofactors：Dissolved, put on ice during operation, best matching while using with 1.2ml Aassay buffer；

(4) NO3- standard items：After carefully removing lid, sample loss is avoided, adds 1ml Aassay buffer dissolvings, Vortex sample is to being completely dissolved；

(5) NO2- standard items：After carefully removing lid, sample loss is avoided, adds 1ml Aassay buffer dissolvings, Vortex sample is to being completely dissolved；

(6) fluorometric reagent DAN and NaOH.

2.1.2 experimental procedure；

(1) NO2- standard curves：

1) 0.9ml Aassay buffer are added in a clean 1.5ml centrifuge tube, add 0.1ml NO2- Standard items, fully mix, that is, be made into 200 μM of storing liquid；7 1.5ml centrifuge tubes separately are taken, draw 950 μ l's respectively Aassay buffer take 500 μ l Aassay buffer into pipe 2-7 into pipe 1, take 50 μ l storing liquid into pipe 1, Fully mix, then take 500 μ l into pipe 2 from pipe 1, fully mix, successively into pipe 7, that is, be made into concentration be respectively 10,5, 2.5th, 1.25,0.625,0.313,0.156 μM of NO2- solution.

2) 96 orifice plates of a printing opacity are taken, add 80 μ l Aassay buffer to blank well, then add 30 μ l to remaining standard In curved slot, add 50 μ l standard items；

3) sample detection, add 10-20 sample μ l product into sample well, add Aassay buffer to adjust volume to 80 μ l；

4) 10 μ l cofactors is added per hole；

5) 10 μ lNO3- reductases are added per hole；

6) lucifuge incubation at room temperature 1h；

7) it is incubated after terminating and adds 10 μ l DAN, then is incubated 10min；

8) 20 μ l NaOH are added；

9) with 375nm excitation wavelengths, 417nm launch wavelengths detection absorbance on multi-function microplate reader.

(2) Griess methods detect the content of NO in oophoroma OVCAR3 cell culture medium supernatants

The oophoroma OVCAR3 cells in exponential phase are taken, 96 orifice plates are inoculated in 5 × 103/hole cell, each Experimental group sets three secondary orifices to carry out cell inoculation, and Tempol (1.5mM), L-NAME are separately added into after cell attachment (1mmol/l), NPLA (100 μm of ol/l) and 1400W (100 μm of ol/l) carry out intervention processing, while set up and be not added with medicine and suppression The control group of preparation, OVCAR3 cells are continued after cultivating 24h, stay cell culture supernatant in 96 orifice plates standby：

(1) a 96 new orifice plates are taken, every group takes 20 μ l nutrient solutions supernatants to add 96 orifice plates per hole, while every group adds per hole 60μl Assay Buffer。

(2) every group adds 10 μ l Enzyme Cofactor Mixture per hole.

(3) every group adds 10 μ l Nitrate Reductase Mixture per hole.

(4) liquid in 96 orifice plates is uniformly mixed, is incubated at room temperature 1h.

(5) every group adds 10 μ l DAN Reagent per hole, and liquid in 96 orifice plates is uniformly mixed, and is incubated at room temperature 10min.

(6) every group adds 20 μ l NAOH per hole, liquid in 96 orifice plates is uniformly mixed, multi-function microplate reader 365/430nm Place's measure fluorescent value, the content of NO in sample is calculated according to standard curve.

2.2 glucose consumption

Using BioVision glucose colorimetric method detection kit.2.2.1 reagent prepares

(1) glucose substrate mixture：Dissolved, be kept in dark place with 220 μ l glucose Assay Buffer；

(2) glucolase mixture：Dissolved with 220 μ l pure water, the used time puts on ice.

2.2.2 experimental procedure

(1) standard curve prepares

Take 10ul dextrose standard sample to be added to 990ul glucose Assay Buffer, make 1nmol/ μ l's Solution, it is well mixed；0,2,4,6,8,10 μ l standard items are once added to the standard curve hole of 96 orifice plates respectively, use glucose Assay Buffer adjust volume to 50 μ l.

(2) preparation of samples

According to requirement of experiment, cell is spread with 96 orifice plates, after next day adds Tempol processing 48h, takes supernatant detection glucose dense Degree.The detection architecture of glucose is 50 μ l；

(3) glucose response system

The reaction system for preparing 50 μ l includes：46 μ l glucose Assay Buffer, 2 μ l glucolases mixtures, 2 μ l Portugals Grape sugar substrate mixture；

(4) add μ l2 medium supernatant, add 48 μ l reaction system, be well mixed；

(5) lucifuge reaction 30min；

(6) 450nm wavelength detecting absorbances are used

(7) calculate the standard curve of glucose, and according to sample group absorbance, calculate containing for glucose in sample Amount, control group subtract the amount that experimental group concentration of glucose is exactly the glucose consumed.

2.3 lactic acid produce

Using BioVision lactic acid colorimetric determination kit, lactic acid is by can be with one after lactic dehydrogenase oxydasis Individual probe, which combines, produces color, compares the content of lactic acid by measuring absorbance.

2.3.1 reagent prepares

(1) lactic acid substrate mixture：Dissolved, be kept in dark place with 220 μ l lactic acid Assay Buffer；

(2) lactic acid enzymatic mixture：Dissolved with 220 μ l pure water, the used time puts on ice.

2.3.2 experimental procedure

(1) standard curve prepares

Take 10 μ l lactate standard product to be added to 990 μ l lactic acid Assay Buffer, make 1mM solution, mix Uniformly；0,2,4,6,8,10 μ l standard items are once added to the standard curve hole of 96 orifice plates respectively, with glucose Assay Buffer adjusts volume to 50 μ l.

(2) preparation of samples

According to requirement of experiment, cell is spread with 96 orifice plates, after next day adds Tempol processing 48h, takes supernatant to detect lactic acid concn. The detection architecture of lactic acid is 50 μ l；

(3) lactic acid reaction system

The reaction system for preparing 50 μ l includes：46 μ l lactic acid Assay Buffer, 2 μ l lactic acid enzymatic mixtures, 2 μ l lactic acid bottoms Thing mixture；

(4) add 5 μ l medium supernatant, add 45 μ l reaction system, be well mixed；

(5) lucifuge reaction 30min；

(6) 450nm wavelength detecting absorbances are used

(7) calculate the standard curve of lactic acid, and according to sample group absorbance, calculate the content of lactic acid in sample.

2.4 ATP test experiences

Take the logarithm the OVCAR3 cells in growth period, be inoculated in 1.5 × 104/hole cell in 96 porocyte culture plates, in Cultivated in 5%CO2,37 DEG C of saturated humidity incubators.When cell growth to 70% is converged, it is handled as follows：Cell It is divided to two groups, respectively control group and Tempol groups.Cell continues after cultivating 6h, and 100 μ lATP detection reagents, room temperature are added per hole Lucifuge is incubated 10min, and the number of photons in each hole is detected at multi-function microplate reader light emitting module.Every group of three multiple holes, experiment repeat three It is secondary.

2.5 NADPH test experiences

Take the logarithm the OVCAR3 cells in growth period, be inoculated in 1.0 × 104/hole cell in 96 porocyte culture plates, in 37 DEG C, cultivated in 5%CO2 saturated humidity incubators.When cell growth to 50% is converged, it is handled as follows：Cell Divide 2 groups, respectively control group and Tempol groups.Cell continues after cultivating 24h, and the good XTT of 100 μ l Fresh is added per hole (0.625mM)/PMS (0.025mM) reaction system solution, 37 DEG C of lucifuges are incubated 3h, multi-function microplate reader wavelength 450nm, reference The OD values in each hole are detected at wavelength 650nm.Every group of three multiple holes, experiment is in triplicate.

The detection of 2.5 protein sulfhydryl nitrosation modification

2.5.1 cell is collected when cell growth state is good, the careful culture medium removed in cell, with the washing of precooling Three times, last time scraping cells are in EP pipes, 1000 × g, 5min for buffer solution washing cell.

2.5.2 supernatant is carefully removed, often pipe adds 500 μ l Buffer A containing Blocking Reagent, 4 DEG C of shaking tables are incubated 30min.

2.5.3 EP pipes are transferred in high speed freezing centrifuge, 12000 × g, 4 DEG C centrifugation 10 minutes it is broken with sedimentation cell Piece.

2.5.4 supernatant is transferred in a new microcentrifugal tube, adds the acetone of 4 times of volumes (2ml), mixed, -20 DEG C place 1h.

2.5.5 EP pipes are transferred in high speed freezing centrifuge, 3000 × g, 4 DEG C centrifuge 10 minutes.

2.5.6 supernatant is removed, adds 500 μ l Buffer B containing Reducing and Labeling Reagent, room temperature place 1h.

2.5.7 the acetone of 4 times of volumes (2ml) is added, is mixed, -20 DEG C of placement 1h.

2.5.8 EP pipes are transferred in high speed freezing centrifuge, 3000 × g, 4 DEG C centrifuge 10 minutes.

2.5.9 remove supernatant, add the lavation buffer solution of 100 μ l precoolings, be resuspended albumen carry out determination of protein concentration and SDS-PAGE electrophoresis.

3. experimental result

1 Tempol suppresses the function that oophoroma OVCAR3 cells nNOS produces NO

1.1 nNOS promote the glycolysis of oophoroma OVCAR3 cells

NOSs in tumour cell shifts electronic catalytic L- with mercaptides combination heme (HEME) for activated centre Nitrogen groups on arginine guanidine radicals end aoxidize, and produce NO.Tried with GlucoseAssay kits and LactateAssay Agent box detects influence of the NOSs inhibitor to oophoroma OVCAR3 cell glycolysis.First respectively with three kinds of NOSs inhibitor (NOS Broad spectrum activity inhibitor L-NAME, nNOS inhibitor NPLA, iNOS inhibitor 1400W) processing oophoroma OVCAR3 cells 48h, three The activity of kind of inhibitor is respectively L-NAME (1mmol/l), NPLA (100 μm of ol/l), 1400W (100 μm of ol/l), then With Glucose Assay kits and Lactate Assay kits detect three kinds of NOSs inhibitor L-NAME, NPLA and The influence that 1400W generates to oophoroma OVCAR3 grape cells sugar consumption and lactic acid.Experimental result is as shown in Figure 9：Relative to right According to group, L-NAME, NPLA and 1400W group reduce oophoroma OVCAR3 grape cells sugar consumption, wherein L-NAME with NPLA has significant difference (P=0.002 and P=0.048), and 1400W does not have significant difference (P=0.395), thus It can be seen that L-NAME inhibitory action is most strong, NPLA takes second place, and 1400W inhibitory action is most weak.Relative to control group, L-NAME Group reduces the secretory volume of oophoroma OVCAR3 cell lactic acid with NPLA groups, and has significant difference (P=0.000 and P= 0.038), and 1400W groups do not have significant difference (P=0.762), wherein L-NAME inhibitory action is most strong, NPLA suppress Effect is weaker.Thus illustrate that NOSs can promote oophoroma glycolysis, and prompt the nNOS important function that can promote glycolysis.

1.2 Tempol suppress oophoroma OVCAR3 cells nNOS function and expression

Tempol carries single electronics, can not only oxidation reaction occur but also reduction reaction can occur, so as to interfering material Electron transfer process.Therefore we want to inquire into function and expression that whether Tempol disturbs nNOS.In order to inquire into Tempol The influence of function and expression to oophoroma OVCAR3 cells nNOS, we train after being acted on first with Griess methods detection Tempol Support the concentration of oophoroma OVCAR3 Hemapoiesis NO in base supernatant.Oophoroma OVCAR3 cell 24h are handled with 1.5mM Tempol, Detect cell culture medium supernatant NO concentration.Griess method experimental results are shown, compared with control group, Tempol group oophoromas NO concentration substantially reduces (P caused by OVCAR3 cells<0.0291), test result indicates that Tempol suppression oophoromas OVCAR3 is thin Born of the same parents NO generation.Tempol nNOS protein levels intracellular to oophoroma OVCAR3 are have detected with Western blot experimental techniques Influence.Oophoroma OVCAR3 cell 48h are acted on 1.5mM Tempol first, then cell lysis extraction intracellular is total on ice Albumen, carry out the effect of Western blot experiment detection Tempol nNOS protein levels intracellular to oophoroma OVCAR3.It is real It is as shown in Figure 10 to test result：Compared to control group, Tempol groups can reduce the intracellular nNOS protein levels of oophoroma OVCAR3.This Part Experiment collectively show that Tempol suppresses the intracellular nNOS's of oophoroma OVCAR3

Function and expression.

2 Tempol suppress the glycolysis of oophoroma OVCAR3 cells

2.1 Tempol suppress glucose consumption and the lactic acid generation of oophoroma OVCAR3 cells

There is document to point out that NO is one of positivity Control factors of oophoroma glycolysis, we also show NOS energy at previous experiments Enough promote the glycolysis of oophoroma OVCAR3 cells.Because Tempol, which influences oophoroma OVCAR3 cells nNOS, produces NO, still Want to inquire into influences of the Tempol to oophoroma OVCAR3 cell glycolysis.With Glucose Assay kits and Lactate Assay kits detect the influence that Tempol generates to oophoroma OVCAR3 grape cells sugar consumption and lactic acid.We give first Oophoroma OVCAR3 cell 48h are handled with 1.5mM Tempol, then with Glucose Assay kits and Lactate Assay kits detect culture medium supernatant glucose residual concentration and lactic acid generation concentration.Glucose consumption and lactic acid generation are real Test result to show, compare and control group, the consumption of Tempol group oophoroma OVCAR3 grape cells sugar have reduced (P< 0.0068), the growing amount of lactic acid has also reduced (P<0.0445).This is test result indicates that Tempol suppresses oophoroma OVCAR3 Glucose consumption and the lactic acid generation of cell.

2.2 Tempol suppress the generation of oophoroma OVCAR3 cell ATPs

Atriphos (ATP) is the energy currency in cell, and it is a kind of high energy phosphate compound, with adenosine diphosphate (ADP) (ADP) mutual conversion realizes energy storage and exoergic.Produced in normal cell ATP mainly by the glycolysis that is carried out in cytosol and Two kinds of approach of the oxidative phosphorylation carried out in mitochondria produce.Tumour cell is often in deficiency states, in order to meet itself The demand of rapid growth breeding, tumour cell usually needs be metabolized reprogramming to produce more ATP, therefore the energy of tumour cell Amount metabolism is usually based on glycolysis.Because Tempol suppresses glucose consumption and the lactic acid generation of oophoroma OVCAR3 cells, The glycolysis of oophoroma OVCAR3 cells is disturbed, so we want further to inquire into Tempol to caused by glycolysis ATP Influence.We are with ATP detection reagents detection Tempol on influence caused by oophoroma OVCAR3 cell ATPs.We give first 1.5mM Tempol handle oophoroma OVCAR3 cell 6h, and the OD values in each hole are then detected with multi-function microplate reader.Experimental result Show：Compared with control group, Tempol group oophoroma OVCAR3 cell ATP yields have been reduced, and difference has statistics Meaning (P<0.034).This part of test results shows that Tempol suppresses the generation of oophoroma OVCAR3 cell ATPs.

2.3 Tempol suppress the generation of oophoroma OVCAR3 cellular NADPHs

NADP (NADPH), is called reduced Coenzyme II, and the chemistry in many organisms is anti- The effect of Ying Zhongqi hydrogen carriers, the anabolic reaction of a variety of macromolecular substances is participated in, such as the conjunction of lipid, aliphatic acid and nucleotides Into.NADPH is needed as reducing agent, the donor of hydride ion in these reactions, and NADPH is NADP+ reduction form.Tumour is thin Born of the same parents are often in deficiency states, and in order to meet the needs of itself rapid growth breeding, tumour cell usually needs to be metabolized Reprogramming produces more NADPH.For further influences of the clear and definite Tempol to oophoroma OVCAR3 cell glycolysis, we It has detected the effect that Tempol produces NADPH to oophoroma OVCAR3 cells.Tempol is detected to ovary with NADPH test experiences Influenceed caused by cancer OVCAR3 cellular NADPHs.We handle oophoroma OVCAR3 cell 24h with 1.5mM Tempol first, after And the OD values in each hole are detected with multi-function microplate reader.Experimental result is shown：Compared with control group, Tempol group oophoromas OVCAR3 The yield of cellular NADPH decreases, and difference has statistical significance (P<0.0244).This is test result indicates that Tempol Suppress the generation of oophoroma OVCAR3 cellular NADPHs.

2.4 Tempol activation oophoroma OVCAR3 cells AMPK phosphorylation

Adenylate cyclase (AMPK) is the switch of intracellular energy, for cell, it is desirable to intracellular to keep At a relatively high ATP：AMP ratios could survive, and intracellular ATP：AMP ratios have reacted the state of intracellular energy.AMP：ATP The rising of ratio can start the cascading protein kinase reaction in downstream of AMPK activation.When intracellular energy is using hindered When, AMPK can receive the prompting of emergency parachute at once, activate the expression of autophosphorylation, and then activate the various signals in downstream and lead to The transduction on road, carry out the various reactions of promotion ability synthesis.In order to which clear and definite Tempol is opened oophoroma OVCAR3 intracellular energies AMPK influence is closed, we have detected expressional functions of the Tempol to oophoroma OVCAR3 cell AMPK phosphorylations.1.5mM first Tempol handles oophoroma OVCAR3 cell 48h, and the intracellular total protein of cell lysis extraction on ice, carries out Westernblot afterwards Influences of the experiment detection Tempol to oophoroma OVCAR3 Intracellular phosphorylations AMPK (P-AMPK) protein expression. Westernblot experimental results are shown：Compared with control group, the intracellular P-AMPK protein levels of Tempol group oophoromas OVCAR3 Expression is substantially increased.This experiment shows that Tempol can activate the intracellular P-AMPK of oophoroma OVCAR3, may suppress with Tempol The generation of oophoroma OVCAR3 cell ATPs is relevant.

3. Tempol suppresses oophoroma OVCAR3 cell glycolysis key enzymes PFK1 and PKM2 nitrosation

3.1 Tempol do not influence oophoroma OVCAR3 cell glycolysis key enzymes PFK1 and PKM2 protein expression

Seminar early stage, verified nNOS promoted two key enzyme phosphofructokinases 1 (PFK1) of glycolysis and pyruvic acid Kinases M2 (PKM2) nitrosation, and then promote glycolysis.Therefore we want to inquire into two sugar that Tempol acts on nNOS The influence of glycolysis key enzyme.1.5mM Tempol first handle oophoroma OVCAR3 cell 48h, and cell lysis extracts on ice afterwards Intracellular total protein, Westernblot experiment detections Tempol is carried out to oophoroma OVCAR3 endocellular sugar glycolysis key enzymes PFK1 With PKM2 protein expression.Experimental result shows that Tempol does not interfere with glycolysis key enzyme PFK1 and PKM2 in protein level Expression.

3.2 Tempol suppress oophoroma OVCAR3 cell glycolysis key enzymes PFK1 and PKM2 nitrosation

Verified nNOS modifies glycolysis key enzyme PFK1 and PKM2 to seminar's previous experiments by producing NO, nitrosation Function, and then promote oophoroma glycolysis.Tempol suppresses nNOS and produces NO, therefore our the desired Tempol that inquire into can be no logical Cross the effect for suppressing nNOS and then two glycolysis key enzymes are had an impact.1.5mM Tempol first handle oophoroma OVCAR3 cell 48h, then carry out nitrosation detection according to the experimental procedure of nitrosification agent box.Experimental result such as Figure 11 institutes Show, the nitrosation that Tempol suppresses oophoroma OVCAR3 cell glycolysis key enzymes PFK1 and PKM2 is horizontal.Thus prompt, Tempol may produce NO by suppressing nNOS, and then suppress NO nitrosation modification glycolysis key enzyme PFK1 and PKM2, suppress The glycolysis of oophoroma.

Finally it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than the present invention is protected The limitation of scope, although being explained in detail with reference to preferred embodiment to the present invention, one of ordinary skill in the art should manage Solution, can modify or equivalent substitution to technical scheme, without departing from technical solution of the present invention essence and Scope.

Claims (10)

1. Applications of the 1.Tempol in terms of as the medicine for suppressing cancer cell multiplication.
2. 2. applications of the Tempol according to claim 1 as the medicine for suppressing cancer cell multiplication, it is characterised in that：It is described Cancer cell is ovarian cancer cell, breast cancer cell, pancreatic cancer cell, liver cancer cells, stomach cancer cell.
3. 3. applications of the Tempol according to claim 1 as the medicine for suppressing cancer cell multiplication, it is characterised in that： Tempol suppresses the propagation of oophoroma OVCAR3 and SKOV3 cell.
4. 4. applications of the Tempol according to claim 1 as the medicine for suppressing cancer cell multiplication, it is characterised in that： Tempol is by disturbing nNOS to produce the glycolysis that NO reaches suppression cancer cell.
5. 5. applications of the Tempol according to claim 4 as the medicine for suppressing cancer cell multiplication, it is characterised in that： Tempol suppresses the glucose consumption of cancer cell and lactic acid generates, suppressed cancer cell ATP generation, suppresses cancer cell NADPH's Cancer cell AMPK phosphorylation is produced, activated, suppresses cancer cell glycolysis key enzyme PFK1 and PKM2 nitrosation, influence cancer Cell glycolysis key enzyme PFK1 and PKM2 protein expression.
6. Applications of the 6.Tempol in terms of as the cancer cell-apoptosis medicine for promoting cisplatin induction.
7. 7. applications of the Tempol according to claim 6 as the cancer cell-apoptosis medicine for promoting cisplatin induction, its feature It is：Tempol promotes DDP to suppress cancer cell multiplication.
8. 8. applications of the Tempol according to claim 7 as the cancer cell-apoptosis medicine for promoting cisplatin induction, its feature It is：Tempol reduces the cancer cell Bcl-2/Bax of DDP inductions expression.
9. 9. applications of the Tempol according to claim 8 as the cancer cell-apoptosis medicine for promoting cisplatin induction, its feature It is：Tempol promotes the cancer cell-apoptosis of DDP inductions by increasing intracellular ROS level.
10. 10. applications of the Tempol according to claim 9 as the cancer cell-apoptosis medicine for promoting cisplatin induction, its feature It is：Tempol is by disturbing the glycolysis of nNOS function inhibitio cancer cells.