CN1936583A - Method for detecting inhibition effect of curcumin to prostate cancer cells using specific target protein - Google Patents
Method for detecting inhibition effect of curcumin to prostate cancer cells using specific target protein Download PDFInfo
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- CN1936583A CN1936583A CN 200610016196 CN200610016196A CN1936583A CN 1936583 A CN1936583 A CN 1936583A CN 200610016196 CN200610016196 CN 200610016196 CN 200610016196 A CN200610016196 A CN 200610016196A CN 1936583 A CN1936583 A CN 1936583A
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Abstract
This invention is an application of detecting curcumin inhibition to prostate cancer by testing specific target protein, belongs to molecular biology field. This method detects curcumin inhibition to prostate cancer cells using AR specific target proteins and detecting the growth of prostate cancer cells, and the results are proved to be accurate. The use of immunoblotting in concrete application of this method can be proved reliable. The method is simple and easy to apply.
Description
Technical field
The invention belongs to biology field, especially a kind of using specific target protein detects curcumin to the inhibiting method of prostate gland cancer cell.
Background technology
Prostate cancer is the higher a kind of malignant tumour of deaths in men rate, and androgen and acceptor thereof play a significant role in the generation evolution of prostate cancer.Prostate cancer and prostate specific antigen (prostatespecific antigen, PSA) in close relations, most patients with prostate cancer are no matter early stage or the PSA continuous expression all arranged late period, height the expresser account for more than 90%, so PSA is as prostate cancer clinical diagnosis and one of sensitive indicator for the treatment of monitoring.The expression of PSA is regulated by androgen, androgen enter behind the prostate epithelial cell at first with nucleus in androgen receptor protein (androgen receptor, AR) combination, cause the AR conformational change, AR and heat shock protein dissociate subsequently, receptor phosphorylation forms dimer, and dimer combines with androgen response element (ARE) in the PSA promoter, induces the PSA gene transcription to express.The expression of some material scalable AR, for example growth factor makes five times of AR increases etc.Have that AR is very important molecule equally in the prostate cancer of discovering the non-dependence of androgen, its effect remains further research.
By above analysis as can be known, utilization induces the AR of PSA gene transcript expression to detect curcumin as specific target protein will have outstanding practical significance to the prostate gland cancer cell inhibiting effect, and the applicant does not also find by specific target protein oriented detection curcumin the inhibiting concrete grammar of prostate gland cancer cell at present.
Summary of the invention
The purpose of this invention is to provide a kind of using specific target protein and detect curcumin to the inhibiting method of prostate gland cancer cell, this method has that degree of accuracy height, applicability are wide, the advantage of high specificity.
Using specific target protein of the present invention detects curcumin the inhibiting method of prostate gland cancer cell is made up of following steps:
(1) handle the LNCap cell by curcumin, microscopically observation of cell metamorphosis also carries out image acquisition; Mtt assay detects LNCap cell growing state; Flow Cytometry FCM detects apoptosis rate;
(2) expression of usefulness Western blotting Western-blotting technology for detection androgen receptor AR:
1. the prostatic csarcinoma androgen-dependent cell of being handled by curcumin is collected behind the 24h at least, add cell pyrolysis liquid with after the PBS washing three times, centrifuging and taking supernatant after the lysis, the Bradford protein quantification is measured protein concentration behind the boiling water bath albuminous degeneration, and its suitable concn scope is 0.5-2 μ g/ μ L;
2. the gained total protein of cell carries out the SDS-PAGE electrophoresis after the cracking, gel behind electrophoresis shifts in the 60mA constant current, protein band is transferred to nitrocellulose filter, select recoverability Ponceaux dye liquor vibration dyeing for use, and compare band molecular weight size according to protein Marker and remove non-specific band, successively add one anti-and two anti-after, with the colour developing of ECL fluorescence western-blotting kit, develop, photographic fixing.
The component of described specificity lysate and concentration thereof are: glycerine 108mmol/L, Qu Latong (TritonX-100) 150mmol/L, sodium chloride 137mmol/L, sodium fluoride 10mmol/L, ethylene glycol bis (a-amino-ethyl) ether tetraacethyl (EGTA) 1mmol/L, ethylenediamine tetraacetic acid (EDTA) 5mmol/L, sodium pyrophosphate 1mmol/L, trishydroxymethylaminomethane-hydrochloric acid (Tris-HCl) 20mmol/L, former sodium vanadate 1mmol/L, SDS 3.7mmol/L, phenylmethylsulfonyl fluoride (PMSF) 1mmol/L, β-phosphoglycerol 100mmol/L, Aprotinin 3.6mmol/L, dithiothreitol (DTT) (DTT) 2.8mmol/L, bromophenol blue 1.5mmol/L.
Advantage of the present invention and beneficial effect are:
1. whether a kind of using specific target protein oriented detection curcumin disclosed in this invention has inhibiting method to prostatic csarcinoma androgen-dependent LNCaP cell, and its result proves that this method is utilized specific target protein AR, detected curcumin by growth of cancer cells situation and apoptosis rate is accurately to the prostate gland cancer cell inhibiting effect.Simultaneously, can prove that also immunoblot assay concrete application in the method also is reliable.
2. method of the present invention has outstanding advantage simple and that be easy to apply.
Description of drawings
Accompanying drawing 1LNCap idioblast attitude feature photo.
The LNCap cell morphological characteristic photo of accompanying drawing 2 after curcumin is handled.
Accompanying drawing 3MTT method is measured the cell survival rate statistical graph.
Accompanying drawing 4western blotting method is measured the expression of results photo of LNCap cell AR.
Accompanying drawing 5western blotting method is measured LNCap cell routine albumen, and (β-actin) is photo as a result.
Embodiment
Further set forth the present invention below in conjunction with specific embodiment.
Be to be understood that, these embodiment only are used to the present invention is described and are not used in restriction the scope of protection of present invention, unreceipted concrete experiment condition and method in the following example, usually according to normal condition as chief editors such as J. Sa nurse Brookers, Science Press, 1992, molecular cloning experiment guide (second edition); D.L. Spector etc., Science Press, 2001, cell experiment guide; Lv Hongsheng, Science Press, 1982, or connect the condition of advising according to manufacturer.
Embodiment:
One, cellular incubation
The LNCap cell is cultivated under the 5%CO2 condition at 37 ℃, and nutrient culture media is RPMI-1640, contains 10% NBCS and penicillin, each 100U/ml of streptomysin, and its morphological feature is shown in Fig. 1 photo.
Two, curcumin is to the influence of LNCap cellular morphology
The following operation of curcumin grouping carrying out with five kinds of concentration: in the LNCap cell of growth logarithmic phase, add the curcumin that concentration is 0 μ mol/L, 10 μ mol/L, 20 μ mol/L, 30 μ mol/L, 40 μ mol/L respectively, and respectively at observing the LNCap cellular morphology under 0h, 12h, 24h, the 48h inverted microscope, the laser scanning co-focusing microscope camera system is carried out image acquisition to each experimental group cellular morphology, and the cellular morphology photo as shown in Figure 2 after wherein curcumin concentration 40 μ mol/L organized 24h.
Photo shown in Figure 2 and Fig. 1 control group are relatively, in the clear LNCap cellular morphology that shows of microscopically obvious variation is arranged, apoptosis sign appears in the part cell, show as cellular contraction and become circle, cell space diminishes, and with out of touch on every side, after birth forms the blister projection, kytoplasm shrinks, and has typical apoptotic body to exist.
Three, mtt assay is surveyed cell growth curve
The LNCap cell is with 3.0 * 10
4Individual hole density is inoculated in the 96 porocyte culture plates, 37 ℃ of 5%CO
2Incubator is cultivated, the curcumin of 0 μ mol/L, 10 μ mol/L, 20 μ mol/L, 30 μ mol/L, 40 μ mol/L variable concentrations is handled 12h, 24h respectively, 36h, 48h stop cultivating, 4h adds 5mg/mLMTT10 μ l before stopping cultivating, final concentration is 0.5mg/mL, abandon supernatant behind the 4h, DMSO dissolving MTT crystallization, survey the OD value with BiotekMicroplate EJ309 enzyme mark analyzer, detect wavelength 570nm, reference wavelength is 630nm, each 12 holes of surveying of each group, triplicate is got average, calculates the cell relative survival rate with following formula:
Cell relative survival rate %=(OD experimental group/OD control group) * 100% (with the zeroing of blank group OD value)
The result carries out statistical analysis.Each group experiment all repeats 3 times.
The survival rate of the LNCap cell that mtt assay is surveyed, the result as shown in Figure 3, as can be seen from the figure the curcumin of 10～40 μ mol/L can both suppress the propagation and the growth of cell, and inhibiting effect is all arranged more than 0h, wherein 40 μ mol/L effect 24h are the strongest, and cell survival rate is 40% of a control group.The tool clinical meaning of curcumin of 40 μ mol/L is described.
Four, FCM surveys cell growth cycle
Be to collect after the curcumin of 0 μ mol/L, 10 μ mol/L, 20 μ mol/L, 30 μ mol/L, 40 μ mol/L is handled LNCaP cell 24h with concentration respectively, the centrifugal 10min of 1000r/m, the PBS washing, 70% ethanol fixedly spends the night for 4 ℃, be stored in 4 ℃ standby; The LNCaP cell is washed twice, 500 order copper mesh with method and filtered, and is centrifugal, adds PI (propidium iodide propidium iodide) dyeing liquor, and 4 ℃ of lucifuges are spent the night.Cells were tested by flow cytometry DNA fluorescence intensity and scattered light parameter, excitation wavelength 488nm, and measure apoptosis rate and cell cycle distribution with apoptosis software.
Curcumin can be induced the LNCap Apoptosis, and concentration is that the apoptosis rate of 0,10,20,30,40 μ mol/L curcumin function cells is respectively 1.20%, 1.22%, 2.22%, 3.69% and 9.23%.40 μ mol/L curcumin most pronounced effects wherein, 10,20 μ mol/L curcumin effects are not obvious, and 30 μ mol/L curcumin effects are better than the curcumin of 10,20 μ mol/L.
Five, Western blotting western blotting detects the expression of androgen receptor AR
(1) be 0 μ mol/L with concentration, 10 μ mol/L, 20 μ mol/L, 30 μ mol/L, the curcumin of 40 μ mol/L is handled the LNCap cell respectively, collecting cell behind the 24h, the PBS washing, lysate cell lysis with following component and weight, lysate is formed: glycerine 108mmol/L, Qu Latong (TritonX-100) 150mmol/L, sodium chloride (NaCl) 137mmol/L, sodium fluoride (NaF) 10mmol/L, ethylene glycol bis (a-amino-ethyl) ether tetraacethyl (EGTA) 1mmol/L, ethylenediamine tetraacetic acid (EDTA) 5mmol/L, sodium pyrophosphate 1mmol/L, trishydroxymethylaminomethane-hydrochloric acid (Tris-HCl) 20mmol/L, former sodium vanadate 1mmol/L, SDS 3.7mmol/L, phenylmethylsulfonyl fluoride (PMSF) 1mmol/L, β-phosphoglycerol: 100mmol/L, Aprotinin 3.6mmol/L, dithiothreitol (DTT) DTT (DTT): 2.8mmol/L, bromophenol blue: 1.5mmol/L.
Centrifuging and taking supernatant behind the cell lysis is measured reagent Preliminary Determination protein concentration with the Bradford protein quantification behind the boiling water bath albuminous degeneration, and concentration is 1.5 μ g/ μ L.
(2) above-mentioned gained total protein of cell is done the SDS-PAGE electrophoretic analysis: the separation gel 10ml of preparation 8%, and behind the encapsulating, use the 0.1%SDS sealing, and leave standstill 30min, make its complete polymerization; Inclining SDS solution, with distilled water unpolymerized separation gel is dashed and removes, and behind the several, exhausts with filter paper repeatedly; Preparation 4% concentrated glue 5ml inserts encapsulating behind number comb, leave standstill 30min after, add electrophoretic buffer (runningbuffer:Tris-base 15.1g, Glycine 94g, SDS 5.0g is dissolved in and is settled to 1000ml in the distilled water), and unpolymerized concentrated Jiao Chong in the well removed; Add protein example 30 μ g in every well, under 100V voltage, carry out electrophoresis; Get glue and be coomassie brilliant blue staining 45min, the destainer decolouring of spending the night, scanning gel saving result.
(3) gel behind the SDS-PAGE electrophoresis shifts in the 60mA constant current, transfer time: conventional albumen β-actin is 45min, and AR is 1.5h, and protein band is transferred on the nitrocellulose filter, and detailed process is:
Separation gel is peeled off from electrophoresis apparatus, be placed on prior with electrophoretic buffer [trishydroxymethylaminomethane (Tris-base): 5.8g, glycocoll (Glycine): 2.9g, lauryl sodium sulfate (SDS): 0.37g, methyl alcohol 200ml is dissolved in and is settled to 1000ml in the distilled water] on the filter paper that soaks into, and cover nitrocellulose filter and filter paper successively, thoroughly soak into and the removal bubble with electrophoretic buffer then; Separation gel and nitrocellulose filter after handling are put into the electrotransfer groove, shift 1h with 100V voltage; After electrotransfer finished, available Ponceaux dye liquor vibration dyeing 4～5min took out nitrocellulose filter, cleans with distilled water and observes, and removes non-specific band.
Treat that albumen is transferred on the nitrocellulose filter, film cleaned earlier that 5% the skim milk of containing with the TBST preparation seals 1h again with TBST (a kind of mixing washing lotion: T represents trishydroxymethylaminomethane, and B represents hydrochloric acid, and S represents sodium chloride, and T represents Tween-20); Take out film, film is put into working fluid vibration hybridization 2～3h, working fluid is mixed by TBST, skim milk, mouse-anti people AR monoclonal antibody and mouse-anti people β-actin antibody and forms, and its weight ratio is 1000: 5: 1: 0.5.After suction removes to contain an anti-working fluid, again with TBST washing nitrocellulose filter three times, each 10min, anti-to remove unnecessary one; Afterwards film is put into another working fluid vibration hybridization 1h, working fluid is by TBST, skim milk and contain goat anti-mouse IgG and form, and its weight ratio is 2000: 5: 1.After suction removes to contain two anti-working fluids, again with TBST washing nitrocellulose filter three times, each 10min, anti-to remove unnecessary two; With PBS cleansing solution (Na
2HPO
4: 1.15g/L, KH
2PO
4: 0.2g/L, NaCl:8g/L, KCl:0.2g/L PH:7.4) soaks, washes film 2～3min, and Tween-20 (Tween-20) is to the influence of fluorescence reaction among the removal TBST.
(4) colour developing of the ECL Western-blotting of system kit is strengthened in chemiluminescence
Preservative film of clip is placed on the washed nitrocellulose filter of TBST on the preservative film.With radioimmunoassay fluorescent reagent Rumi sodium solution A (Luminol Reagent SolutionA) in the kit and radioimmunoassay fluorescent reagent Rumi sodium solution B (Luminol Reagent SolutionB) equal proportion mixing, according to 0.125ml/cm
2Evenly be layered on the protein powder of film, behind the reaction 1min, that preservative film is folding under the room temperature, bring the darkroom into together with the target protein film and comprise operations such as sensitization, development, photographic fixing.
After handling LNCap cell 24h with the curcumin of variable concentrations respectively, Western-blot detects AR and expresses, and the results are shown in Figure 4.AR protein immunoblot band from left to right is to be the result of the curcumin processing LNCap cell of 40 μ mol/L, 30 μ mol/L, 20 μ mol/L, 10 μ mol/L, 0 μ mol/L through over-richness successively among Fig. 4.Fig. 4 is clear to show that curcumin can suppress the expression of AR, and the inhibition degree that AR expresses is depended on the concentration of curcumin, and it is best to suppress effect when wherein curcumin concentration is 40 μ mol/L.
Fig. 5 is that western blotting method is measured LNCap cell routine albumen β-actin photo as a result, and the curcumin that variable concentrations is described is to not influence of LNCap cell routine albumen β-actin.
The curcumin dosage range is 0～50 μ mol/L in the foregoing description, and 50 μ mol/L are maximum non-toxic dosage, and above dosage range is to not influence of testing result.
In the foregoing description, curcumin (Curcurmin) is available from Sigma company, and prostate gland cancer cell LNCap is preserved by the applicant unit.The western-blotting of ECL system kit is purchased the biotech firm in Beijing Zhong Shan, nitrocellulose filter is available from Beijing ancient cooking vessel state biotech development center, and the antibody (sheep anti mouse) of AR monoclonal antibody (mouse-anti people) horseradish peroxidase mark is available from Beijing Zhong Shan company.
Claims (2)
1. a using specific target protein detects curcumin to the inhibiting method of prostate gland cancer cell, it is characterized in that being made up of following steps:
(1) handles LNCap cell, microscopically observation of cell metamorphosis by curcumin; Mtt assay detects LNCap cell growing state; Flow Cytometry FCM detects apoptosis rate;
(2) expression of usefulness Western blotting Western-blotting technology for detection androgen receptor protein AR:
1. the prostatic csarcinoma androgen-dependent cell of being handled by curcumin is collected behind the 24h at least, add cell pyrolysis liquid with after the PBS washing three times, centrifuging and taking supernatant after the lysis, the Bradford protein quantification is measured protein concentration behind the boiling water bath albuminous degeneration, and its suitable concn scope is 0.5-2 μ g/ μ L;
2. the gained total protein of cell carries out the SDS-PAGE electrophoresis after the cracking, gel behind electrophoresis shifts in the 60mA constant current, protein band is transferred to nitrocellulose filter, select recoverability Ponceaux dye liquor vibration dyeing for use, and compare band molecular weight size according to protein Marker and remove non-specific band, successively add one anti-and two anti-after, with the colour developing of ECL fluorescence western-blotting kit, develop, photographic fixing.
2. method according to claim 1 is characterized in that the component of specificity lysate and concentration thereof are:
Glycerine 108mmol/L, Qu Latong (TritonX-100) 150mmol/L, sodium chloride (NaCl) 137mmol/L, sodium fluoride (NaF) 10mmol/L, ethylene glycol bis (a-amino-ethyl) ether tetraacethyl (EGTA) 1mmol/L, ethylenediamine tetraacetic acid (EDTA) 5mmol/L, sodium pyrophosphate 1mmol/L, trishydroxymethylaminomethane-hydrochloric acid (Tris-HCl) 20mmol/L, former sodium vanadate 1mmol/L, SDS3.7mmol/L, phenylmethylsulfonyl fluoride (PMSF) 1mmol/L, β-phosphoglycerol: 100mmol/L, Aprotinin 3.6mmol/L, dithiothreitol (DTT) DTT (DTT): 2.8mmol/L, bromophenol blue: 1.5mmol/L.
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| CN106868148A (en) * | 2017-03-08 | 2017-06-20 | 中国科学院苏州生物医学工程技术研究所 | Coherent condition is controllable and the preparation method of nucleus that can preserve for a long time |
| CN109444094A (en) * | 2018-09-25 | 2019-03-08 | 江苏大学 | Urazole-gold nanoclusters compound and its preparation method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102323423A (en) * | 2011-05-31 | 2012-01-18 | 昆明理工大学 | Method for detecting influence of toltrazuril to expression of Trx-1 protein in MCF (Macrophage Chemotactic Factor)-7 cells |
| CN106868148A (en) * | 2017-03-08 | 2017-06-20 | 中国科学院苏州生物医学工程技术研究所 | Coherent condition is controllable and the preparation method of nucleus that can preserve for a long time |
| CN109444094A (en) * | 2018-09-25 | 2019-03-08 | 江苏大学 | Urazole-gold nanoclusters compound and its preparation method and application |
| CN109444094B (en) * | 2018-09-25 | 2021-08-03 | 江苏大学 | Urazole-gold nanocluster compound and preparation method and application thereof |
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