CN104450854A - Screening method of esophagus cancer markers by tea polyphenol in tea and golden camellia - Google Patents

Screening method of esophagus cancer markers by tea polyphenol in tea and golden camellia Download PDF

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CN104450854A
CN104450854A CN201410639293.8A CN201410639293A CN104450854A CN 104450854 A CN104450854 A CN 104450854A CN 201410639293 A CN201410639293 A CN 201410639293A CN 104450854 A CN104450854 A CN 104450854A
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tea
cell
polyphenol
esophageal cancer
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程金生
朱文娟
郑启祥
韦卓恒
陈信炎
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Abstract

The invention discloses a screening method of esophagus cancer markers by tea polyphenol in tea and golden camellia. The tea polyphenol in tea and golden camellia is extracted and prepared from different types of tea and golden camellia, and the tea polyphenol acts on human esophageal squamous cells and adenocarcinoma cell OE33 which are cultured in vitro; an anti-proliferative effect on esophagus cancer by the tea polyphenol is detected in an MTT method, and an inducing differentiation effect on the esophagus cancer by the tea polyphenol is detected by flow cytometry so as to observe cell cycles and apoptotic effects; cell morphological change is observed by using a fluorescence inversion microscope; protein expression change in the esophagus cancer cells is analyzed by means of immunohisochemistry, Real-time fluorescent quantitation RT-PCR and Westem-blot. Based on SPSS1 statistics, the detection results are analyzed, and differential expression proteins obtained by performing action on the esophagus cancer by the tea polyphenol in the tea and golden camellia are detected, and associated proteins are screened out according to database search and matching. In the invention, a mechanism of the function of tea polyphenol can be expounded in accordance with the research on marker molecules of the differential expression proteins acted by the tea polyphenol.

Description

In a kind of tea, Camellia nitidissima Chi, tea-polyphenol is to the screening method of esophageal cancer marker
Technical field
The present invention relates to biological technical field, specifically in a kind of tea, Camellia nitidissima Chi tea-polyphenol to the screening method of esophageal cancer marker.
Background technology
Tea-polyphenol (tea polyphenols, TP) tea tannin, tea tannin is again, be the general name of Polyphenols of Tea, comprise flavanol compound (catechin), anthocyanin class (anthocyanidin and leucoanthocyanidin), flavonoid, flavonols, phenolic acids and depside material etc.Tea-polyphenol is the chemical substance that a class contains polyphenol hydroxyl, and living radical in energy purged body, having very strong antioxygenation, is therefore a kind of up-and-coming antioxidant from natural food.BHT(antioxidant 264), BHA(butylated hydroxy anisole) etc., although they have many advantages, there is toxicity in various degree simultaneously.And TP not only has as natural antioxidants that security is high, the advantage such as have no side effect, and resistance of oxidation is also better than the similar natural antioxidants such as VE, VC, has therefore become the focus of people's research and development.The compositions such as the tea-polyphenol be rich in the plants such as tea (comprising as green tea, black tea, black tea, Leaf of Assam Tea, yellow tea, white tea, blue or green tea, oolong tea, camellia, rock tea etc.), Camellia nitidissima Chi can scavenging free radicals, alleviate the detrimental effect of radical pair human body, there is the effect recovering skin cell activity, smooth out wrinkles, skin maintenance.
Wang Xia etc. adopt ferrous tartrate method to measure polyphenol content.Result shows, Green Tea Polyphenols content exceeds 144.8% and 59.53% than black tea and oolong tea respectively, high-end green tea polyphenol content exceeds 13.01% and 23.20%(Wang Xia than middle-grade and low gear green tea respectively, Lai Suisheng. the comparison of several Tea Polyphenols in Tea content. Information, 2009,24,212-212), Song Jianhong carries out microwave extracting using ethyl acetate as solvent to tealeaves, with the relative merits crossing more different extraction process, propose, measure the tea-polyphenol calibration curve method extracted, result shows, higher by the percentage extraction of this method to tea-polyphenol, and the efficiency that saves time high (Song Jianhong. extract the process modification research of Tea Polyphenols in Tea with microwave extraction method. Zhejiang chemical industry, 2006,2,1-3), section red magnitude with Leaf of Assam Tea (ripe tea) for raw material, adopt dehydrated alcohol respectively, distilled water, different volumes mark aqueous ethanol is solvent extraction tea-polyphenol, at extraction temperature, extraction time, solid-liquid ratio, on the basis of different concns aqueous ethanol 4 single factor experiments, pass through orthogonal test, the contribution rate size of these 4 factor pair tea polyphenol extract of comparative analysis and otherness, and different solvents extraction Leaf of Assam Tea (ripe tea) tea-polyphenol massfraction is compared, result shows: with different volumes mark aqueous ethanol for solvent, tea polyphenol extract massfraction is the highest, be 13.86%, take distilled water as solvent, tea polyphenol extract massfraction takes second place, and is 9.67%, and be solvent with dehydrated alcohol, tea polyphenol extract massfraction is minimum, is 5.49%(section Red Star, Zhou Chune, Li Jiahua. different solvents is to the influence research of Leaf of Assam Tea (ripe tea) tea polyphenol extract. Yunnan Prov Agriculture University's journal: natural science edition, 2014,2,246-250).
Mostly be confined to conventional ultra-violet about tea polyphenols species activity composition Study in Camellia nitidissima Chi at present, chromatogram detects or extraction, membrane sepn etc., as: profound will China has carried out qualitative analysis to the chemical composition in Camellia nitidissima Chi leaf.Find that Camellia nitidissima Chi leaf polyphenol content accounts for the profound will China of 9.66%(. the Extraction and isolation [D] of flavone component in Camellia nitidissima Chi leaf. Guangxi Normal University, 2006); Peng Xiao etc. adopt repeatedly the method such as silica gel column chromatography, Sephadex LH-20 column chromatography, ODS column chromatography, repeatedly recrystallization to carry out separation and purification to the chemical composition of golden flower camellia, and carry out Structural Identification by methods such as physicochemical constant mensuration and Spectrum Analysis.Be separated from the ethanol extraction of golden flower camellia and obtain 13 compounds (Peng Xiao, Yu great Yong, Feng Baomin comprising tea polyphenols material, Tang Ling, Wang Yongqi, Shi Liying. the research of golden flower camellia chemical composition. GUIHAIA, 2011,31(04), 550-553).Patent of invention: camellia nitidissima tea polyphenols slow release microsphere particle and the production method (patent No.: 201010153006) then successfully make camellia nitidissima tea polyphenols slow release microsphere particle with Camellia nitidissima Chi leaf thereof.
Esophagus cancer is the common malignant tumours in some countries and regions, the world.The statistical information display that the World Health Organization announces, the mortality ratio of esophagus cancer is the highest with China, and male sex's esophagus cancer is the second of mortality of malignant tumors, is only second to cancer of the stomach.Women's esophagus cancer is then the 3rd, is only second to cancer of the stomach and cervical cancer.Annual about 300,000 people in the whole world die from esophagus cancer.China is the hotspot of esophagus cancer, every year because of esophagus cancer died about 150,000 people, accounts for whole mortality of malignant tumors nearly 1/4th.
The Infected regions otherness of China is very large, and the Wild jujube in Taihang Mountain Area had a common boundary with Henan, Hebei, Shanxi San Sheng is the highest with the sickness rate that Lin County, Henan Province, Ci County, Hebei province and Yangcheng County, Shanxi Province are representative.The regional sickness rate such as the north in Jiangsu is higher, and the sickness rate of 35 ~ 64 years old male sex's esophagus cancer in Lin County, Henan Province is current one of district occurred frequently in the world.In Guangdong, esophagus cancer ranks the Guangdong malignant tumour cause of the death the 4th, is more in national second in the mortality ratio in area, Nan'ao, East Guangdong.
In recent years, along with molecular biological progress, gene level is deep into the research of tumor aetiology and pathogenesis.The Proliferation and apoptosis of tumour cell and the abnormal expression of involved important gene and albumen, the activation of oncogene or the mutation deletes of cancer suppressor gene, the sudden change of DNA damage revision points and cell signalling exception etc. are that mediate tumor occurs, the important molecule basis of development.The research of tea polyphenolic compounds is shown to they anti-oxidant and to carcinogenic removing may relate to tumorigenic molecular events, have influence on each stage of the startup of tumour, promotion and progress, in the generation evolution of tumour, have vital role.Therefore, tea polyphenols material antitumor action in the plant such as tea, Camellia nitidissima Chi is furtherd investigate from cell levels, gene and protein expression level and inquired into, may be deeply illustrating of antitumor action molecular mechanism, new clue is provided.
There is no any document at present and relate to tea polyphenols material in the plants such as tea (comprising the different varietiess such as black tea, green tea, oolong tea, camellia), Camellia nitidissima Chi in relevant reports such as the molecular biology suppressed in esophagus cancer or epidemiology.But part document to relate in other plant tea-polyphenol active ingredient substance suppresses the correlative study such as esophagus cancer molecular biology or epidemiology, inquire into tea-polyphenol (TP) as: Liu is round, effect and mechanism thereof that taxol (Paclitaxel) suppresses esophagus cancer Eca-109 Growth of Cells, cell death inducing.Found by the Eca-109 cell research in vegetative period of taking the logarithm, different concns, difference TP group action time, Paclitaxel group cell proliferation inhibition rate difference have significance (F=134.46 ~ 423.75, P0.05) (Liu is round. and tea-polyphenol taxol affect esophagus cancer Eca-109 Cell apoptosis and proliferation. University Of Qingdao's Master's thesis, 2013); Zhang Qiang etc. adopt mtt assay and flow cytometry, identify 3 kinds of flavones (luteolin, chrysin, apigenin) and the inhibited proliferation of 3 kinds of flavonol (Quercetin, kaempferol, ampelopsin) to 2 strain people esophageal cancer cells (squama cancer KYSE-510 and gland cancer OE33) and the inducing action of G2/M cycle arrest.Result shows, p21waf1, GADD45 β, 14-3-3 σ and cyclin B1 is the target gene (Zhang Qiang of mediation flavones and flavonol induction KYSE-510 and OE33 cell G2/M cycle arrest, Zhao Xinhuai. the molecular mechanism of flavones and flavonol induction people esophageal cancer cell cycle arrest. Progress in Biochemistry and Biophysics, 2008,9,1031-1038).
Summary of the invention
To the object of the present invention is to provide in a kind of tea, Camellia nitidissima Chi tea-polyphenol to the screening method of esophageal cancer marker, to solve the problem proposed in above-mentioned background technology.
For achieving the above object, the invention provides following technical scheme:
In tea, Camellia nitidissima Chi, tea-polyphenol is to a screening method for esophageal cancer marker, and concrete steps comprise:
1) apply XDA macroporous resin adsorption method of purification and prepare tea-polyphenol by extracting in different types of tea, Camellia nitidissima Chi, its step comprises: tea, Camellia nitidissima Chi → pulverized 30 mesh sieves → with 30% alcohol immersion 20min → microwave extracting 20min → extracting solution through Middle hollow fiber membrane → rotary evaporation concentrated → cross macroporous absorption resin XDA-200 post → water elution removal of impurities → 10% ethanol elution removal of impurities → 30% ethanol gradient elution → elutriant respectively rotary evaporation concentrate → lyophilize → obtain dried powder; Polyphenol content measures by " detection method of GB/T 8313-2008 Tea Polyphenols in Tea and catechin content " method, polyphenol content=(in sample powder tea-polyphenol quality/sample mass) × 100%;
2) Human esophageal squamous cell cancer cell KYSE-510, EC9706, KYSE-510 of vitro culture, EC9706, EC8712, EC8733, EC9706, Eca109 and adenocarcinoma cell OE33 are model, the equal adherent growth of above-mentioned cell in RPMI 1640 substratum, at 37 DEG C, the CO of 5% 2, Secondary Culture under saturated humidity;
Experiment is divided into tea-polyphenol group, positive controls, solvent control group and blank group, and wherein in tea-polyphenol group, each dosage component of tea-polyphenol is not 10,20,40,60 and 80mg/m1; In positive controls, Fluracil dosage is 0.25mg/m1; Solvent control group adds the DMSO that final concentration is 0.1%; Blank group only adds equal-volume nutrient solution, does not add medicine and cell;
3) tea-polyphenol is detected to the effect of esophageal cancer cell
A) mtt assay is adopted to detect tea-polyphenol to the inhibited proliferation of esophageal cancer cell, concrete steps comprise Human esophageal squamous cell cancer cell KYSE-510, EC9706, KYSE-510, EC9706, EC8712, EC8733, EC9706, the Eca109 and adenocarcinoma cell OE33 in vegetative period that take the logarithm, be the trysinization of 0.25% through mass percent concentration, be inoculated in 96 well culture plates, 5 × 10 3individual cells/well, preculture 24h, the whole supernatant of sucking-off after cell attachment, empirically grouping adds the various tested materials of different concns, cumulative volume 200 μ l/ hole, and every dose group establishes 4 multiple holes; Cultivate 24,48,72 and 96h respectively, it is the MTT of 5mg/L that every hole adds 20 μ l concentration, continue to cultivate 4h, discard nutrient solution, every hole adds the DMSO of 200 μ l, and plate shaker shaking 10min, returns to zero with DMSO, measure each hole light absorption value A with enzyme-linked immunosorbent assay instrument with 490nm wavelength, calculate average A-value, proliferation rate PR and inhibiting rate CIR; Proliferation rate PR=(experimental group A value/solvent control group A value) × 100%; Inhibiting rate CIR=(1-test group average A-value/control group average A-value) × 100%; Use curvilinear regression analysis, obtain dose-response relationship equation and regression coefficient;
B) according to the result of MTT colorimetry, suitable concentration is chosen according to experimental design process cell; The esophageal cancer cell of taking the logarithm vegetative period, adjustment density 2.5 × 10 4individual/ml, puts 28cm 2in culturing bottle, cultivate 24h after cell attachment, add different concns process factor, stop cultivating in 96h; Get EC9706, KYSE-510 or OE33 cell, discard nutrient solution, mass percent concentration is the trysinization of 0.25%, and the centrifugal l0min of 2000r/min, abandons supernatant liquor, is resuspended in by cell precipitation in PBS liquid, adjustment cell density to 1 × 10 6the centrifugal l0min of individual/m1,2000r/min, abandons supernatant liquor; Add 75% ethanol 1ml of 4 DEG C of precoolings fast, make cell resuspended, at 4 DEG C, be incubated 10-15h; Get single cell suspension lml, add the RNA inhibitor 5 μ l of fluorescence dye propidium iodide 10 μ l and l0mg/ml of l0 μ g/ml, under slight oscillatory, room temperature, lucifuge hatches 20min, filters with 400 eye mesh screens, obtained cell suspension, under 488nm wavelength, carries out cell DNA content detection with flow cytometer; Obtain data through Muticycle AV software processes, each phase distribution percentage of cell cycle is analyzed, and calculates G in the cell cycle o/ G 1phase, S phase and G 2the per-cent that/M phase cell is shared in sum, represents the proliferation activity of cell with proliferation index PI;
C) fluorescence inverted microscope observation of cell morphological change is adopted: be inoculated in by each cell in 96 orifice plates, 5 × 10 3individual cells/well, preculture 24h, each experimental group add respectively 10,20,40,60 and 80mg/m1 tea in tea-polyphenol activity extract, cultivate 24,48,72 and 96h respectively in solvent control group, positive controls, observe under fluorescence inverted microscope after hematoxylin-eosin staining method stain smear;
D) protein expression change in immunohistochemical methods, Real-time fluorescence quantitative RT-RCR, Westem-blot analysis esophageal cancer cell is applied, three strain esophageal cancer cells are inoculated in 9 porocyte culture plates, cultivate 24h, after the tea-polyphenol effect 24h of 80 μm of ol/L, every 5-10 × 10 6cell adds the Trizol reagent of 1ml, and 15-30 DEG C of effect 15min, abandons supernatant liquor, add 0.5ml Virahol/ml Trizol reagent in throw out, 15-30 DEG C of effect 10min, 2-8 DEG C, and 12000 leave heart 15min, abandon supernatant liquor; Add 75% ethanol of 1ml/mlTrizol reagent wash RNA, 2-8 DEG C again, 7500 leave heart 5min, abandon supernatant liquor, and the sample obtained is preserved at-80 DEG C;
Use ABI/9700 system to carry out each gene PCR amplified reaction, response procedures is as follows: 95 DEG C, 10s; Denaturation: 95 DEG C, 5s; 60 DEG C, 34s; 40 circulations; The melting curve analysis of amplified production is all carried out in the PCR reaction of various gene; Each genetic expression more all adopts β-actin as reference gene;
Follow-up employing Western-Blot analyzes protein expression change in esophageal cancer cell, three strain esophageal cancer cells are inoculated in 9 porocyte culture plates, cultivate 24h, after the tea-polyphenol effect 24h of 80 μm of ol/L, PBS liquid washs three times, add cold cell pyrolysis liquid and act on 30min on ice, scraping cells on Eppendorf tube, 4 DEG C, 14000 leave heart 15min, Aspirate supernatant, ultraviolet spectrophotometer measures each sample total protein concentration, and regulates concentration consistent; Sample, after 10-12% polyacrylamide gel electrophoresis 4h, is transferred on nitrocellulose filter, 100mA, 20-60min; Nitrocellulose filter is closed after 3h through confining liquid, TBS buffer solution three times, in the first antibody working fluid of Dilution ratio 1:500-1:2000, hatch 2h; TBS buffer solution 3 times, the second antibody working fluid of the horseradish peroxidase mark of thinning ratio 1:2000 hatches 2h, TBS buffer solution 3 times, and super quick luminescence is aobvious light also, after photosensitive X-ray, developing fixing, β-actin albumen is as internal reference albumen;
4) adopt SPSS12.0 statistical software to analyze the result in step 3), compare with one-way analysis of variance between multiple sample mean; Compare between two and check with LSD; Dose-response relationship curvilinear regression and correlation analysis, inspection level α=0.05;
5) have detected the differentially expressed protein after tea-polyphenol effect esophageal cancer cell in tea, Camellia nitidissima Chi, obtain two-dimensional electrophoresis difference expression atlas, identify the peptide fingerprinting spectrum of differential protein, mate through database retrieval, filter out associated protein, described associated protein has comprised heterogeneous nuclear ribonucleoprotein H1, ubiquitin, microtubule gone steady albumen, PKC to disturb albumen 1, heterogeneous nuclear ribonucleoprotein, high mobility albumen, tubulin molecule companion, aldolase A.
As the further scheme of the present invention: described tea bag draws together green tea, black tea, black tea, Leaf of Assam Tea, yellow tea, white tea, blue or green tea, oolong tea, camellia, rock tea.
As the further scheme of the present invention: containing 10% standard foetal calf serum, 100 U/ml penicillin and 100 μ g/ml Streptomycin sulphates in RPMI 1640 substratum.
As the further scheme of the present invention: concentrated glue 80V, separation gel 120V in polyacrylamide gel electrophoresis.
As the further scheme of the present invention: confining liquid comprises 5% skimming milk and 0.05% Tween 20, pH7.6.
As the further scheme of the present invention: this method is also applicable to other leaf, flower, fruit, rhizome containing tea polyphenols material.
Compared with prior art, the invention has the beneficial effects as follows: in the present invention, the change of correlative protein expression points out tea-polyphenol activeconstituents in the plant such as Camellia nitidissima Chi, tea can act on the number of ways such as cellular metabolism, signal transduction, oxidative stress, makes to have had the mechanism of its multipath effect to understand more deeply.The site of tea-polyphenol effect is disclosed from the change of protein level, the albumen of these differential expressions can as the marker molecules of the tea-polyphenol effect be derived from the plant such as Camellia nitidissima Chi, tea, by clearly can illustrate the mechanism of its effect to their further investigation, these albumen are likely as the target molecule of clinical treatment esophageal cancer medicine effect simultaneously.
Accompanying drawing explanation
Fig. 1 is techniqueflow chart of the present invention.
Fig. 2 is the impact that tea-polyphenol is expressed in vain on KYSE-510 (left side) and OE33 (right side) cell differentiation related protein; Wherein swimming lane 1 is tea-polyphenol group, and swimming lane 2 is 1% DMSO group.
Fig. 3 is EC9706 cell morphological change figure under different treatment factor; Wherein (A): tea-polyphenol 40mg/ml, cell comparatively obviously reduces quantity before process, and more elongated, has more cell detachment or swims in nutrient solution; (B): Fluracil group: 0.25mg/ml, cell quantity reduces, and cell diminishes, and becomes circle; (C): Fluracil hematoxylin-eosin staining method (HE) dyeing group: 0.25mg/ml, a small amount of engrain of nucleus, most of core and endochylema red coloration, endochylema after birth dissolves and breaks, and display Fluracil has lethal effect to cell; (D): tea-polyphenol HE dyeing group: 20mg/ml, showed cell core is little, concentrated, limit collection, and nuclear-cytoplasmic ratio reduces.
Embodiment
Below in conjunction with the embodiment of the present invention, be clearly and completely described the technical scheme in the embodiment of the present invention, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
The change occurred at gene and protein level after the object of the invention is to act on esophageal cancer cell, filters out exogenous tea-polyphenol and suppresses the biomarker that esophagus cancer activity is relevant.Particular by tea polyphenols material in Camellia nitidissima Chi extract as catechin, anthocyanidin, flavones, the separation and purification of flavonol etc., to act on Human esophageal squamous cell cancer cell KYSE-510 and EC9706 of vitro culture, and adenocarcinoma cell OE33 is model, use laser co-focusing inverted microscope, differential disply inverse transcription polymerase chain reaction technology (DDRT-PCR), proteomics methodology and Matrix-assisted flight time mass spectrum (MALDI-TOF MS) technology etc., inquire into tea polyphenols material in Camellia nitidissima Chi extract and suppress esophageal cancer cell propagation, apoptosis-induced, and in the change that gene and protein level occur after acting on esophageal cancer cell, detection and localization Human esophageal squamous cell cancer KYSE-510 and EC9706 cell and gland cancer OE33 cell differen-tiation markers CK8, cyclin D1 albumen and c-erbB-2 gene, Camellia nitidissima Chi is disclosed from the change of protein level, effective site of tea polyphenols material effect in the plants such as tea, find above-mentioned tea, the esophageal cancer cell growth-inhibiting caused after tea-polyphenol composition effect in Camellia nitidissima Chi etc., contact potential between the form of apoptosis-induced and cell and phenotype, mate through database retrieval, select and suppress the active relevant ubiquitin (ubiauitin) of esophagus cancer to tea-polyphenol, microtubule removes steady albumen (stathmin 1), PKC disturbs albumen 1 (PKC-interactingprotein, PKC1), high mobility albumen (High mobility group protein B1, HMG), aldolase A (Aidolase A), the biomarkers such as heterogeneous nuclear ribonucleoprotein (Heterogeneous nuclear ribonucleoprotein HI), the target molecule that screening exogenous tea-polyphenol activeconstituents works, explore its antibumor molecules mechanism.
Embodiment 1
In the Dan Congcha of Meizhou, tea polyphenol extract is separated: XDA macroporous resin adsorption method of purification prepares tea-polyphenol by extracting in the Dan Congcha of Meizhou, and its key step comprises: Meizhou Dan Congcha preferably weighed → pulverized 30 mesh sieves → with 30% alcohol immersion 20min → microwave extracting 20min → extracting solution through hollow-fibre membrane (Mr=10000) separations → rotary evaporation concentrated → macroporous absorption resin XDA-200 post → water elution removal of impurities → 10% ethanol elution removal of impurities → 30% ethanol gradient elution → elutriant respectively rotary evaporation concentrate → lyophilize → obtain dried powder.Polyphenol content measures by " detection method of GB/T 8313-2008 Tea Polyphenols in Tea and catechin content " method, polyphenol content=(in sample powder tea-polyphenol quality/sample mass) × 100%, this tea-polyphenol separation purification method of the present invention, polyphenol content is more than 85%.
Human esophageal squamous cell cancer cell KYSE-510 vitro culture: Human esophageal squamous cell cancer cell KYSE-510 is provided by Tumor Hospital Attached to Zhongshan Univ., the equal adherent growth of this cell is (containing 10% standard foetal calf serum, 100 U/ml penicillin and 100 μ g/ml Streptomycin sulphates) in RPMI 1640 substratum, at 37 DEG C, the CO of 5% 2, routine passage is cultivated under saturated humidity.
Experiment grouping: experiment is divided into tea-polyphenol group, positive controls, solvent control group and blank group, wherein each dosage component of tea-polyphenol is not 10,20,40,60 and 80mg/m1; Positive controls Fluracil dosage is 0.25mg/m1; Solvent control group adds the DMSO that final concentration is 0.1%.Blank group only adds equal-volume nutrient solution, does not add medicine and cell.
MTT (tetrazolium bromide) method detects the inhibited proliferation concrete steps of tea-polyphenol to esophageal cancer cell and comprises the KYSE-510 in vegetative period that takes the logarithm, and through 0.25% (g/v) trysinization, is inoculated in 96 well culture plates interior (5 × 10 3individual cells/well), preculture 24h, the whole supernatant of sucking-off after cell attachment, empirically grouping adds the various tested materials (cumulative volume 200 μ l/ hole) of different concns, and every dose group establishes 4 multiple holes.Cultivate 24,48,72 and 96h respectively, it is the MTT of 5mg/L that every hole adds 20 μ l concentration, continues to cultivate 4h, discards nutrient solution, every hole adds DMSO 200 μ l, plate shaker shaking 10min, with DMSO zeroing, measures each hole light absorption value A with enzyme-linked immunosorbent assay instrument with 490nm wavelength, calculate average A-value and proliferation rate (proliferation rate, and inhibiting rate (cell, inhibitory rate, CIR) PR).Proliferation rate (PR)=(experimental group A value/solvent control group A value) × 100%; Inhibiting rate (CIR)=(1-test group average A-value/control group average A-value) × 100%.Use curvilinear regression analysis, obtain dose-response relationship equation and regression coefficient.
Flow cytometer monitoring apoptosis and cell cycle: according to the result of MTT colorimetry, choose suitable concentration according to experimental design process cell.The KYSE-510 esophageal cancer cell of taking the logarithm vegetative period, adjustment density 2.5 × 10 4individual/ml, puts 28cm 2in culturing bottle, cultivate 24h after cell attachment, add different concns process factor, stop cultivating in 96h.Get KYSE-510 cell, discard nutrient solution, the trysinization of 0.25%, the centrifugal l0min of 2000r/min, abandons supernatant liquor, is resuspended in by cell precipitation in PBS liquid, adjustment cell density to 1 × 10 6individual/m1, centrifugal, abandon supernatant liquor.Add 75% ethanol 1ml of 4 DEG C of precoolings fast, make cell resuspended, 4 DEG C are spent the night.Get fluorescence dye propidium iodide (the Propidium iodide that single cell suspension lml adds l0 μ g/ml, PI) the RNA inhibitor 5 μ l of 10 μ l and l0mg/ml, under slight oscillatory, room temperature, lucifuge hatches 20min, filter with 400 eye mesh screens, get appropriate cell suspension under 488nm wavelength, carry out cell DNA content detection with flow cytometer.Obtain data through Muticycle AV software processes, each phase distribution percentage of cell cycle is analyzed, and calculates G in the cell cycle o/ G 1phase, S phase and G 2/ M phase cell percentage in sum, represents cell-proliferation activity with proliferation index (proliferation index, PI).
Fluorescence inverted microscope observation of cell morphological change: each cell to be inoculated in 96 orifice plates (5 × 10 3individual cells/well), preculture 24h, each experimental group adds the camellia nitidissima tea polyphenols activity extract of 10,20,30,50,90 μm of ol/L respectively, cultivate 24,48,72 and 96h respectively in solvent control group (0.1%DMSO), Fluracil group (0.25mg/ml), observe under Olympus IX-71 fluorescence inverted microscope.As described herein, after tea-polyphenol induction KYSE-510 cytodifferentiation, cell there will be flattening, and volume increases, and cell density reduces, core neat appearance, and core/slurry waits the typical morphological feature relevant to cytodifferentiation than reducing.
Real-time fluorescence quantitative RT-RCR analyzes protein expression change in cancer cells: two strain esophageal cancer cells are inoculated in 6 porocyte culture plates, cultivate 24h, after tea-polyphenol (80 μm of ol/L) acts on 24h, and every 5-10 × 10 6cell, add 1ml Trizol reagent, 15-30 DEG C of effect 15min, inhales colorless supernatant liquid, add 0.5ml Virahol/ml Trizol reagent, 15-30 DEG C of effect 10min, 2-8 DEG C, 12000 leave heart 15min, add 1ml75% ethanol/ml Trizol reagent wash RNA, 2-8 DEG C, 7500 leave heart 5min, and sample retention is in-80 DEG C.Use ABI/9700 system to carry out each gene PCR amplified reaction, response procedures is as follows: 95 DEG C, 10s; Denaturation: 95 DEG C, 5s; 60 DEG C, 34s; 40 circulations.The melting curve analysis of amplified production is all carried out in the PCR reaction of various gene, to determine specificity and the purity of each amplified production.Each genetic expression more all adopts β-actin as reference gene.
Western-Blot analyzes protein expression change in esophageal cancer cell, three strain esophageal cancer cells are inoculated in 9 porocyte culture plates, cultivate 24h, after tea-polyphenol (80 μm of ol/L) acts on 24h, PBS liquid washs three times, add cold cell pyrolysis liquid and act on 30min on ice, scraping cells on Eppendorf tube, 4 DEG C, 14000 leave heart 15min, Aspirate supernatant, ultraviolet spectrophotometer measures each sample total protein concentration, and regulates concentration consistent.Sample, after 10-12% polyacrylamide gel electrophoresis (concentrated glue 80V, separation gel 120V) 4h, is transferred to (100mA, 20-60min) on nitrocellulose filter.Nitrocellulose filter is after confining liquid (5% skimming milk, 0.05% Tween 20, pH7.6) closes 3h, TBS damping fluid is (commercially available, 20mmol/L, pH7.6) wash three times, in first antibody working fluid (Dilution ratio 1:500-1:2000), hatch 2h.TBS washs 3 times, and 2h is hatched in the second antibody work that horseradish peroxidase marks also (thinning ratio 1:2000), and TBS washs 3 times, and super quick luminescence is aobvious light also, and after photosensitive X-ray, developing fixing, β-actin albumen is as internal reference albumen.
SPSS 12.0 statistical software analyzes result, compares with one-way analysis of variance between multiple sample mean; Compare between two and check with LSD; Dose-response relationship curvilinear regression and correlation analysis, inspection level α=0.05.
The biomarker relevant to tea-polyphenol anticarcinogenic effect screens: adopt DDRT-PCR technology to act on the experimental group after esophageal cancer cell to tea-polyphenol and control group difference expression gene detects, key step comprises: continue to cultivate 48h, extract mRNA respectively, and carry out the qualification of mRNA integrity, become cDNA with anchor primer reverse transcription, performing PCR of going forward side by side increases; Upstream primer is random primer, downstream primer is the anchor primer that the band fluorescent substance identical with reverse transcriptase primer series marks, carry out DDRT-PCR reaction, by denaturing polyacrylamide electrophoresis and separation differential disply fragment, realized the Cloning and sequence analysis of differential disply fragment by technology such as image analysis, differential band recovery, differential band increase again.
Differential expression finger printing is obtained by above-mentioned technology; Differentially expressed protein after adopting proteomic techniques to detect tea-polyphenol effect, obtains the two dimensional electrophoresis of differentially expressed protein.Matrix solid-dispersion flight time mass spectrum (MALDI-TOF-MS) is used to identify the peptide fingerprinting spectrum of a series of differential protein subsequently, major experimental design is as follows: matrix solid-dispersion time-of-flight mass spectrometer is the Autoflex of Bruker company, sample is dissolved in 0.1% trifluoroacetic acid, get 1 μ l and saturated CCA (alpha-cyano-4-hydroxycinnamic acid saturated solution, α-CCA) stromal supernatant mixing, get 1 μ l point on Scorce 384 target, after crystallization to be evaporated, send in ion source and detect.Adopt reflection detection mode; Pancreatin is cut; Wavelength 337nm: mixed peptide standard corrects instrument.By MALDI-TOF-MS mass spectroscopy, the quality of polypeptide mixture after mensuration in-gel digestion, obtains the peptide mass fingerprinting spectrum detecting sample.Select the species intending inquiry, peptide mapping fingerprinting data and other parameter, use the Mascot:Peptide Mass Fingerprint protein database search program that provides of http://www.Matrixscience.com website, at the Protein database search peptide hydrolysis albumen that can match with it in theory.Through database retrieval coupling, from cellular metabolism, signal transduction, cytoskeleton composition, oxidative stress, immunity, cell proliferation and each angle sieve such as apoptosis and molecular chaperones have selected some associated protein, as tubulin molecule companion, aldolase A etc.
Embodiment 2
In Camellia nitidissima Chi, tea polyphenol extract is separated: XDA macroporous resin adsorption method of purification prepares tea-polyphenol by extracting in Camellia nitidissima Chi, and its key step comprises: Camellia nitidissima Chi dried flower preferably weighed → pulverized 30 mesh sieves → with 30% alcohol immersion 20min → microwave extracting 20min → extracting solution through hollow-fibre membrane (Mr=10000) separations → rotary evaporation concentrated → macroporous absorption resin XDA-200 post → water elution removal of impurities → 10% ethanol elution removal of impurities → 30% ethanol gradient elution → elutriant respectively rotary evaporation concentrate → lyophilize → obtain dried powder.Polyphenol content measures by " detection method of GB/T 8313-2008 Tea Polyphenols in Tea and catechin content " method, polyphenol content=(in sample powder tea-polyphenol quality/sample mass) × 100%, this tea-polyphenol separation purification method of the present invention, polyphenol content is more than 85%.
Human esophageal squamous cell cancer cell EC9706 vitro culture: Human esophageal squamous cell cancer cell EC9706 is provided by Tumor Hospital Attached to Zhongshan Univ., the equal adherent growth of this cell is (containing 10% standard foetal calf serum, 100 U/ml penicillin and 100 μ g/ml Streptomycin sulphates) in RPMI 1640 substratum, at 37 DEG C, the CO of 5% 2, routine passage is cultivated under saturated humidity.
Experiment grouping: experiment is divided into tea-polyphenol group, positive controls, solvent control group and blank group, wherein each dosage component of tea-polyphenol is not 10,20,40,60 and 80mg/m1; Positive controls Fluracil dosage is 0.25mg/m1; Solvent control group adds the DMSO that final concentration is 0.1%.Blank group only adds equal-volume nutrient solution, does not add medicine and cell.
MTT (tetrazolium bromide) method detects tea-polyphenol to the inhibited proliferation of esophageal cancer cell, and concrete steps comprise the EC9706 in vegetative period that takes the logarithm, and through 0.25% (g/v) trysinization, is inoculated in 96 well culture plates interior (5 × 10 3individual cells/well), preculture 24h, the whole supernatant of sucking-off after cell attachment, empirically grouping adds the various tested materials (cumulative volume 200 μ l/ hole) of different concns, and every dose group establishes 4 multiple holes.Cultivate 24,48,72 and 96 h respectively, it is the MTT of 5mg/L that every hole adds 20 μ l concentration, continue to cultivate 4h, discard nutrient solution, every hole adds DMSO 200 μ l, and plate shaker shaking 10 min, returns to zero with DMSO, measure each hole light absorption value (A) with enzyme-linked immunosorbent assay instrument with 490nm wavelength, calculate average A-value and proliferation rate (PR) and inhibiting rate (CIR).Proliferation rate (PR)=(experimental group A value/solvent control group A value) × 10096 inhibiting rates (CIR)=(1-test group average A-value/control group average A-value) × 100%.Use curvilinear regression analysis, obtain dose-response relationship equation and regression coefficient.
Flow cytometer monitoring apoptosis and cell cycle: according to the result of MTT colorimetry, choose suitable concentration according to experimental design process cell.The EC9706 esophageal cancer cell of taking the logarithm vegetative period, adjustment density 2.5 × 10 4individual/ml, puts 28cm 2in culturing bottle, cultivate 24 h after cell attachment, add different concns process factor, stop cultivating in 96h.Get EC9706 cell, discard nutrient solution, the trysinization of 0.25%, the centrifugal l0min of 2000r/min, abandons supernatant liquor, is resuspended in by cell precipitation in PBS liquid, adjustment cell density to 1 × 10 6individual/m1, centrifugal, abandon supernatant liquor.Add 75% ethanol 1ml of 4 DEG C of precoolings fast, make cell resuspended, 4 DEG C are spent the night.Get the RNA inhibitor 5 μ l that single cell suspension l ml adds fluorescence dye propidium iodide (PI) 10 μ l and l0mg/ml of l0 μ g/ml, under slight oscillatory, room temperature, lucifuge hatches 20min, filter with 400 eye mesh screens, get appropriate cell suspension under 488nm wavelength, carry out cell DNA content detection with flow cytometer.Obtain data through Muticycle AV software processes, each phase distribution percentage of cell cycle is analyzed, and calculates G in the cell cycle o/ G 1phase, S phase and G 2the per-cent that/M phase cell is shared in sum, represents the proliferation activity of cell with proliferation index (PI).
Fluorescence inverted microscope observation of cell morphological change: each cell to be inoculated in 96 orifice plates (5 × 10 3individual cells/well), preculture 24h, each experimental group adds the camellia nitidissima tea polyphenols activity extract of 10,20,30,50,90 μm of ol/L respectively, cultivate 24,48,72 and 96h respectively in solvent control group (0.1%DMSO), Fluracil group (0.25mg/ml), observe under Olympus IX-71 fluorescence inverted microscope.
Real-time fluorescence quantitative RT-RCR analyzes protein expression change in cancer cells: two strain esophageal cancer cells are inoculated in 6 porocyte culture plates, cultivate 24h, after tea-polyphenol (80 μm of ol/L) acts on 24h, and every 5-10 × 10 6cell, add 1ml Trizol reagent, 15-30 DEG C of effect 15min, inhales colorless supernatant liquid, add 0.5ml Virahol/ml Trizol reagent, 15-30 DEG C of effect 10min, 2-8 DEG C, 12000 leave heart 15min, add 1ml 75% ethanol/ml Trizol reagent wash RNA, 2-8 DEG C, 7500 leave heart 5min, and sample retention is in-80 DEG C.Use ABI/9700 system to carry out each gene PCR amplified reaction, response procedures is as follows: 95 DEG C, 10s; Denaturation: 95 DEG C, 5s; 60 DEG C, 34s; 40 circulations.The melting curve analysis of amplified production is all carried out in the PCR reaction of various gene, to determine specificity and the purity of each amplified production.Each genetic expression more all adopts β-actin as reference gene.
Western-Blot analyzes protein expression change in esophageal cancer cell, three strain esophageal cancer cells are inoculated in 9 porocyte culture plates, cultivate 24h, after tea-polyphenol (80 μm of ol/L) acts on 24h, PBS liquid washs three times, add cold cell pyrolysis liquid and act on 30min on ice, scraping cells on Eppendorf tube, 4 DEG C, 14000 leave heart 15min, Aspirate supernatant, ultraviolet spectrophotometer measures each sample total protein concentration, and regulates concentration consistent.Sample, after 10-12% polyacrylamide gel electrophoresis (concentrated glue 80V, separation gel 120V) 4h, is transferred to (100mA, 20-60min) on nitrocellulose filter.Nitrocellulose filter is after confining liquid (5% skimming milk, 0.05% Tween 20, pH7.6) closes 3h, TBS damping fluid is (commercially available, 20mmol/L, pH7.6) wash three times, in first antibody working fluid (Dilution ratio 1:500-1:2000), hatch 2h.TBS washs 3 times, and 2h is hatched in the second antibody work that horseradish peroxidase marks also (thinning ratio 1:2000), and TBS washs 3 times, and super quick luminescence is aobvious light also, and after photosensitive X-ray, developing fixing, β-actin albumen is as internal reference albumen.
SPSS 12.0 statistical software analyzes result, compares with one-way analysis of variance between multiple sample mean; Compare between two and check with LSD; Dose-response relationship curvilinear regression and correlation analysis, inspection level α=0.05.
The biomarker relevant to tea-polyphenol anticarcinogenic effect screens: adopt DDRT-PCR technology to act on the experimental group after esophageal cancer cell to tea-polyphenol and control group difference expression gene detects, key step comprises: continue to cultivate 48h, extract mRNA respectively, and carry out the qualification of mRNA integrity, become cDNA with anchor primer reverse transcription, performing PCR of going forward side by side increases; Upstream primer is random primer, downstream primer is the anchor primer that the band fluorescent substance identical with reverse transcriptase primer series marks, carry out DDRT-PCR reaction, by denaturing polyacrylamide electrophoresis and separation differential disply fragment, realized the Cloning and sequence analysis of differential disply fragment by technology such as image analysis, differential band recovery, differential band increase again.Differential expression finger printing is obtained by above-mentioned technology; Differentially expressed protein after adopting proteomic techniques to detect tea-polyphenol effect, obtains the two dimensional electrophoresis of differentially expressed protein.Matrix solid-dispersion flight time mass spectrum (MALDI-TOF-MS) is used to identify the peptide fingerprinting spectrum of a series of differential protein subsequently.
Main technical procedures is as follows: 1) instrument and reagent are: BIFLEX III type MALDI TOF mass spectrograph (Bruker company); Nitrogen laser, wavelength 337 nm, adopt ion to postpone to draw the mode of operation of (delayed extraction) and reflection (reflectorn), positive ion detects; Data separate Flexcontrol 2.2, the data analysis software Flex Analysis 2.4 gathered, mass spectrum imaging software Flex Imaging 1.0, variance analysis software Clin software processes such as Protools 2.0; 2) on carrier, graphene nanometer sheet is prepared (with renewable resourcess such as bagasse, straw, corn stalks for carbon source, standby by modified version Hummers legal system, Cheng Jinsheng et al. CrystEngComm, 2012,14 (2), 397-400) coating, gas dries up for subsequent use; 3) carrier is fixed on MALDI target; 4), after rear for the dispersion of N, N-solvent dimethylformamide MF59 sample solution being added drop-wise to Graphene coatingsurface, making sample and substrate formed mixed crystal secondary crystal, after solvent evaporates, namely obtain analytic sample to be measured, send in ion source and detect.Adopt reflection detection mode; Pancreatin is cut; Wavelength 337nm: mixed peptide standard corrects instrument.By MALDI-TOF-MS mass spectroscopy, the quality of polypeptide mixture after mensuration in-gel digestion, obtains the peptide mass fingerprinting spectrum detecting sample.Select the species intending inquiry, peptide mapping fingerprinting data and other parameter, the Mascot:Peptide Mass Fingerprint protein database search program that http://www.Matrixscience.com website is provided, at the NCBInr Protein database search peptide hydrolysis albumen that can match with it in theory.Through database retrieval coupling, from cellular metabolism, signal transduction, cytoskeleton composition, oxidative stress, immunity, cell proliferation and each angle sieve such as apoptosis and molecular chaperones have selected some associated protein, as ubiquitin, microtubule remove steady albumen etc.
Embodiment 3
In the western rock camellia of Dabu, tea polyphenol extract is separated: XDA macroporous resin adsorption method of purification prepares tea-polyphenol by extracting in the western rock camellia in Dabu, and its key step comprises: the western rock camellia in Dabu preferably weighed → pulverized 30 mesh sieves → with 30% alcohol immersion 20min → microwave extracting 20min → extracting solution through hollow-fibre membrane (Mr=10000) separations → rotary evaporation concentrated → macroporous absorption resin XDA-200 post → water elution removal of impurities → 10% ethanol elution removal of impurities → 30% ethanol gradient elution → elutriant respectively rotary evaporation concentrate → lyophilize → obtain dried powder.Polyphenol content measures by " detection method of GB/T 8313-2008 Tea Polyphenols in Tea and catechin content " method, polyphenol content=(in sample powder tea-polyphenol quality/sample mass) × 100%, this tea-polyphenol separation purification method of the present invention, polyphenol content is more than 85%.
People's esophageal adenocarcinoma cells OE33 vitro culture: adenocarcinoma cell OE33 is provided by Tumor Hispital Attached to Fudan Univ, the equal adherent growth of this cell is (containing 10% standard foetal calf serum, 100 U/ml penicillin and 100 μ g/ml Streptomycin sulphates) in RPMI 1640 substratum, at 37 DEG C, the CO of 5% 2, routine passage is cultivated under saturated humidity.
Experiment grouping: experiment is divided into tea-polyphenol group, positive controls, solvent control group and blank group, and wherein each dosage component of tea-polyphenol is not 10,20,40,60 and 80 mg/m1; Positive controls Fluracil dosage is 0.25mg/ m1; Solvent control group adds the DMSO that final concentration is 0.1%.Blank group only adds equal-volume nutrient solution, does not add medicine and cell.
MTT (tetrazolium bromide) method detects tea-polyphenol to the inhibited proliferation of esophageal cancer cell, and concrete steps comprise the adenocarcinoma cell OE33 in vegetative period that takes the logarithm, and through 0.25% (g/v) trysinization, is inoculated in 96 well culture plates interior (5 × 10 3individual cells/well), preculture 24h, the whole supernatant of sucking-off after cell attachment, empirically grouping adds the various tested materials (cumulative volume 200 μ l/ hole) of different concns, and every dose group establishes 4 multiple holes.Cultivate 24,48,72 and 96h respectively, it is the MTT of 5mg/L that every hole adds 20 μ l concentration, continue to cultivate 4h, discard nutrient solution, every hole adds DMSO 200 μ l, and plate shaker shaking 10min, returns to zero with DMSO, measure each hole light absorption value (A) with enzyme-linked immunosorbent assay instrument with 490nm wavelength, calculate average A-value and proliferation rate (PR) and inhibiting rate (CIR).Proliferation rate (PR)=(experimental group A value/solvent control group A value) × 100%; Inhibiting rate (CIR)=(1-test group average A-value/control group average A-value) × 100%.Use curvilinear regression analysis, obtain dose-response relationship equation and regression coefficient.
Flow cytometer monitoring apoptosis and cell cycle: according to the result of MTT colorimetry, choose suitable concentration according to experimental design process cell.The adenocarcinoma cell OE33 taken the logarithm vegetative period, adjustment density 2.5 × 10 4individual/ml, puts 28cm 2in culturing bottle, cultivate 24h after cell attachment, add different concns process factor, stop cultivating in 96h.Get adenocarcinoma cell OE33, discard nutrient solution, the trysinization of 0.25%, the centrifugal l0min of 2000r/min, abandons supernatant liquor, is resuspended in by cell precipitation in PBS liquid, adjustment cell density to 1 × 10 6individual/m1, centrifugal, abandon supernatant liquor.Add 75% ethanol 1ml of 4 DEG C of precoolings fast, make cell resuspended, 4 DEG C are spent the night.Get the RNA inhibitor 5 μ l that single cell suspension lml adds fluorescence dye propidium iodide (PI) 10 μ l and l0mg/ml of l0 μ g/ml, under slight oscillatory, room temperature, lucifuge hatches 20min, filter with 400 eye mesh screens, get appropriate cell suspension under 488nm wavelength, carry out cell DNA content detection with flow cytometer.Obtain data through Muticycle AV software processes, each phase distribution percentage of cell cycle is analyzed, and calculates G in the cell cycle o/ G 1phase, S phase and G 2the per-cent that/M phase cell is shared in sum, represents the proliferation activity of cell with proliferation index (PI).
Fluorescence inverted microscope observation of cell morphological change: each cell to be inoculated in 96 orifice plates (5 × 10 3individual cells/well), preculture 24h, each experimental group adds the camellia nitidissima tea polyphenols activity extract of 10,20,30,50,90 μm of ol/L respectively, cultivate 24,48,72 and 96h respectively in solvent control group (0.1%DMSO), Fluracil group (0.25mg/ml), observe under Olympus IX-71 fluorescence inverted microscope.As described herein, after tea-polyphenol induction OE33 cytodifferentiation, cell there will be flattening, and volume increases, and cell density reduces, core neat appearance, and core/slurry waits the typical morphological feature relevant to cytodifferentiation than reducing.Real-time fluorescence quantitative RT-RCR analyzes protein expression change in cancer cells: two strain esophageal cancer cells are inoculated in 6 porocyte culture plates, cultivate 24h, after tea-polyphenol (80 μm of ol/L) acts on 24h, and every 5-10 × 10 6cell, add 1ml Trizol reagent, 15-30 DEG C of effect 15min, inhales colorless supernatant liquid, add 0.5ml Virahol/ml Trizol reagent, 15-30 DEG C of effect 10min, 2-8 DEG C, 12000 leave heart 15min, add 1ml 75% ethanol/ml Trizol reagent wash RNA, 2-8 DEG C, 7500 leave heart 5min, and sample retention is in-80 DEG C.Use ABI/9700 system to carry out each gene PCR amplified reaction, response procedures is as follows: 95 DEG C, 10s; Denaturation: 95 DEG C, 5s; 60 DEG C, 34s; 40 circulations.The melting curve analysis of amplified production is all carried out in the PCR reaction of various gene, to determine specificity and the purity of each amplified production.Each genetic expression more all adopts β-actin as reference gene.
Western-Blot analyzes protein expression change in esophageal cancer cell, three strain esophageal cancer cells are inoculated in 9 porocyte culture plates, cultivate 24h, after tea-polyphenol (80 μm of ol/L) acts on 24h, PBS liquid washs three times, add cold cell pyrolysis liquid and act on 30min on ice, scraping cells on Eppendorf tube, 4 DEG C, 14000 leave heart 15min, Aspirate supernatant, ultraviolet spectrophotometer measures each sample total protein concentration, and regulates concentration consistent.Sample, after 10-12% polyacrylamide gel electrophoresis (concentrated glue 80V, separation gel 120V) 4h, is transferred to (100mA, 20-60min) on nitrocellulose filter.Nitrocellulose filter is after confining liquid (5% skimming milk, 0.05% Tween 20, pH 7.6) closes 3h, TBS damping fluid is (commercially available, 20mmol/L, pH7.6) wash three times, in first antibody working fluid (Dilution ratio 1:500-1:2000), hatch 2h.TBS washs 3 times, and 2h is hatched in the second antibody work that horseradish peroxidase marks also (thinning ratio 1:2000), and TBS washs 3 times, and super quick luminescence is aobvious light also, and after photosensitive X-ray, developing fixing, β-actin albumen is as internal reference albumen.
SPSS 12.0 statistical software analyzes result, compares with one-way analysis of variance between multiple sample mean; Compare between two and check with LSD; Dose-response relationship curvilinear regression and correlation analysis, inspection level α=0.05.
The biomarker relevant to tea-polyphenol anticarcinogenic effect screens: adopt DDRT-PCR technology to act on the experimental group after esophageal cancer cell to tea-polyphenol and control group difference expression gene detects, key step comprises: continue to cultivate 48h, extract mRNA respectively, and carry out the qualification of mRNA integrity, become cDNA with anchor primer reverse transcription, performing PCR of going forward side by side increases; Upstream primer is random primer, downstream primer is the anchor primer that the band fluorescent substance identical with reverse transcriptase primer series marks, carry out DDRT-PCR reaction, by denaturing polyacrylamide electrophoresis and separation differential disply fragment, realized the Cloning and sequence analysis of differential disply fragment by technology such as image analysis, differential band recovery, differential band increase again.
Differential expression finger printing is obtained by above-mentioned technology; Differentially expressed protein after adopting proteomic techniques to detect tea-polyphenol effect, obtains the two dimensional electrophoresis of differentially expressed protein.Matrix solid-dispersion flight time mass spectrum (MALDI-TOF-MS) is used to identify the peptide fingerprinting spectrum of a series of differential protein subsequently, major experimental design is as follows: matrix solid-dispersion time-of-flight mass spectrometer is the Voyager DE-STR mass spectrograph of Applied biosystems, sample is dissolved in 0.1% trifluoroacetic acid, get 1 μ l to mix with saturated alpha-CCA stromal supernatant, get 1 μ l point on Scorce384 target, after crystallization to be evaporated, send in ion source and detect.Adopt reflection detection mode; Pancreatin is cut; Wavelength 337nm: mixed peptide standard corrects instrument.By MALDI-TOF-MS mass spectroscopy, the quality of polypeptide mixture after mensuration in-gel digestion, obtains the peptide mass fingerprinting spectrum detecting sample.Select the species intending inquiry, peptide mapping fingerprinting data and other parameter, use the Mascot:Peptide Mass Fingerprint protein database search program that provides of http://www.MatrixScience.com website, at the NCBInr Protein database search peptide hydrolysis albumen that can match with it in theory.Through database retrieval coupling, from cellular metabolism, signal transduction, cytoskeleton composition, oxidative stress, immunity, cell proliferation and each angle sieve such as apoptosis and molecular chaperones have selected some associated protein, as PKC disturbs albumen, high mobility albumen etc.
Embodiment 4
In yunnan puer tea, tea polyphenol extract is separated: XDA macroporous resin adsorption method of purification prepares tea-polyphenol by extracting in yunnan puer tea, and its key step comprises: yunnan puer tea preferably weighed → pulverized 30 mesh sieves → with 30% alcohol immersion 20min → microwave extracting 20min → extracting solution through hollow-fibre membrane (Mr=10000) separations → rotary evaporation concentrated → macroporous absorption resin XDA-200 post → water elution removal of impurities → 10% ethanol elution removal of impurities → 30% ethanol gradient elution → elutriant respectively rotary evaporation concentrate → lyophilize → obtain dried powder.Polyphenol content measures by " detection method of GB/T 8313-2008 Tea Polyphenols in Tea and catechin content " method, polyphenol content=(in sample powder tea-polyphenol quality/sample mass) × 100%, this tea-polyphenol separation purification method of the present invention, polyphenol content is more than 85%.
Human esophageal squamous cell cancer cell Eca109 vitro culture: squamous cell carcinoma Eca109 is buied by Shanghai Sheng Gong biotechnology company limited, the equal adherent growth of this cell is (containing 10% standard foetal calf serum, 100U/ml penicillin and 100 μ g/ml Streptomycin sulphates) in RPMI 1640 substratum, at 37 DEG C, the CO of 5% 2, routine passage is cultivated under saturated humidity.
Experiment grouping: experiment is divided into tea-polyphenol group, positive controls, solvent control group and blank group, wherein each dosage component of tea-polyphenol is not 10,20,40,60 and 80mg/m1; Positive controls Fluracil dosage is 0.25mg/m1; Solvent control group adds the DMSO that final concentration is 0.1%.Blank group only adds equal-volume nutrient solution, does not add medicine and cell.
MTT (tetrazolium bromide) method detects tea-polyphenol to the inhibited proliferation of esophageal cancer cell, and concrete steps comprise the squamous cell carcinoma Eca109 in vegetative period that takes the logarithm, and through 0.25% (g/v) trysinization, is inoculated in 96 well culture plates interior (5 × 10 3individual cells/well), preculture 24h, the whole supernatant of sucking-off after cell attachment, empirically grouping adds the various tested materials (cumulative volume 200 μ l/ hole) of different concns, and every dose group establishes 4 multiple holes.Cultivate 24,48,72 and 96h respectively, it is the MTT of 5mg/L that every hole adds 20 μ l concentration, continue to cultivate 4h, discard nutrient solution, every hole adds DMSO 200 μ l, and plate shaker shaking 10 min, returns to zero with DMSO, measure each hole light absorption value (A) with enzyme-linked immunosorbent assay instrument with 490nm wavelength, calculate average A-value and proliferation rate (PR) and inhibiting rate (CIR).Proliferation rate (PR)=(experimental group A value/solvent control group A value) × 100%; Inhibiting rate (CIR)=(1-test group average A-value/control group average A-value) × 100%.Use curvilinear regression analysis, obtain dose-response relationship equation and regression coefficient.
Flow cytometer monitoring apoptosis and cell cycle: according to the result of MTT colorimetry, choose suitable concentration according to experimental design process cell.The squamous cell carcinoma Eca109 taken the logarithm vegetative period, adjustment density 2.5 × 10 4individual/ml, puts 28cm 2in culturing bottle, cultivate 24 h after cell attachment, add different concns process factor, stop cultivating in 96h.Get squamous cell carcinoma Eca109, discard nutrient solution, the trysinization of 0.25%, the centrifugal l0min of 2000r/min, abandons supernatant liquor, is resuspended in by cell precipitation in PBS liquid, adjustment cell density to 1 × 10 6individual/m1, centrifugal, abandon supernatant liquor.Add 75% ethanol 1ml of 4 DEG C of precoolings fast, make cell resuspended, 4 DEG C are spent the night.Get the RNA inhibitor 5 μ l that single cell suspension lml adds fluorescence dye propidium iodide (PI) 10 μ l and l0mg/ml of l0 μ g/ml, under slight oscillatory, room temperature, lucifuge hatches 20min, filter with 400 eye mesh screens, get appropriate cell suspension under 488nm wavelength, carry out cell DNA content detection with flow cytometer.Obtain data through Muticycle AV software processes, each phase distribution percentage of cell cycle is analyzed, and calculates G in the cell cycle o/ G 1phase, S phase and G 2/ M phase cell percentage in sum, represents cell-proliferation activity with proliferation index (PI).
Fluorescence inverted microscope observation of cell morphological change: each cell to be inoculated in 96 orifice plates (5 × 10 3individual cells/well), preculture 24h, each experimental group adds the camellia nitidissima tea polyphenols activity extract of 10,20,30,50,90 μm of ol/L respectively, cultivate 24,48,72 and 96h respectively in solvent control group (0.1% DMSO), Fluracil group (0.25mg/ml), observe under Olympus IX-71 fluorescence inverted microscope.As described herein, after tea-polyphenol induction Eca109 cytodifferentiation, cell there will be flattening, and volume increases, and cell density reduces, core neat appearance, and core/slurry waits the typical morphological feature relevant to cytodifferentiation than reducing.Real-time fluorescence quantitative RT-RCR analyzes protein expression change in cancer cells: two strain esophageal cancer cells are inoculated in 6 porocyte culture plates, cultivate 24h, after tea-polyphenol (80 μm of ol/L) acts on 24h, and every 5-10 × 10 6cell, add 1ml Trizol reagent, 15-30 DEG C of effect 15min, inhales colorless supernatant liquid, add 0.5ml Virahol/ml Trizol reagent, 15-30 DEG C of effect 10min, 2-8 DEG C, 12000 leave heart 15min, add 1ml 75% ethanol/ml Trizol reagent wash RNA, 2-8 DEG C, 7500 leave heart 5min, and sample retention is in-80 DEG C.Use ABI/9700 system to carry out each gene PCR amplified reaction, response procedures is as follows: 95 DEG C, 10s; Denaturation: 95 DEG C, 5s; 60 DEG C, 34s; 40 circulations.The melting curve analysis of amplified production is all carried out in the PCR reaction of various gene, to determine specificity and the purity of each amplified production.Each genetic expression more all adopts β-actin as reference gene.
Western-Blot analyzes protein expression change in esophageal cancer cell, three strain esophageal cancer cells are inoculated in 9 porocyte culture plates, cultivate 24h, after tea-polyphenol (80 μm of ol/L) acts on 24h, PBS liquid washs three times, add cold cell pyrolysis liquid and act on 30min on ice, scraping cells on Eppendorf tube, 4 DEG C, 14000 leave heart 15min, Aspirate supernatant, ultraviolet spectrophotometer measures each sample total protein concentration, and regulates concentration consistent.Sample, after 10-12% polyacrylamide gel electrophoresis (concentrated glue 80V, separation gel 120V) 4h, is transferred to (100mA, 20-60min) on nitrocellulose filter.Nitrocellulose filter is after confining liquid (5% skimming milk, 0.05% Tween 20, pH7.6) closes 3h, TBS damping fluid is (commercially available, 20mmol/L, pH7.6) wash three times, in first antibody working fluid (Dilution ratio 1:500-1:2000), hatch 2h.TBS washs 3 times, and 2h is hatched in the second antibody work that horseradish peroxidase marks also (thinning ratio 1:2000), and TBS washs 3 times, and super quick luminescence is aobvious light also, and after photosensitive X-ray, developing fixing, β-actin albumen is as internal reference albumen.
SPSS 12.0 statistical software analyzes result, compares with one-way analysis of variance between multiple sample mean; Compare between two and check with LSD; Dose-response relationship curvilinear regression and correlation analysis, inspection level α=0.05.
The biomarker relevant to tea-polyphenol anticarcinogenic effect screens: adopt DDRT-PCR technology to act on the experimental group after esophageal cancer cell to tea-polyphenol and control group difference expression gene detects, key step comprises: continue to cultivate 48h, extract mRNA respectively, and carry out the qualification of mRNA integrity, become cDNA with anchor primer reverse transcription, performing PCR of going forward side by side increases; Upstream primer is random primer, downstream primer is the anchor primer that the band fluorescent substance identical with reverse transcriptase primer series marks, carry out DDRT-PCR reaction, by denaturing polyacrylamide electrophoresis and separation differential disply fragment, realized the Cloning and sequence analysis of differential disply fragment by technology such as image analysis, differential band recovery, differential band increase again.
Differential expression finger printing is obtained by above-mentioned technology; Differentially expressed protein after adopting proteomic techniques to detect tea-polyphenol effect, obtains the two dimensional electrophoresis of differentially expressed protein.Matrix solid-dispersion flight time mass spectrum (MALDI-TOF-MS) is used to identify the peptide fingerprinting spectrum of a series of differential protein subsequently, major experimental design is as follows: matrix solid-dispersion time-of-flight mass spectrometer is the Voyager DE-STR mass spectrograph of Applied biosystems, sample is dissolved in 0.1% trifluoroacetic acid, get 1 μ l to mix with saturated alpha-CCA stromal supernatant, get 1 μ l point on Scorce384 target, after crystallization to be evaporated, send in ion source and detect.Adopt reflection detection mode; Pancreatin is cut; Wavelength 337nm: mixed peptide standard corrects instrument.By MALDI-TOF-MS mass spectroscopy, the quality of polypeptide mixture after mensuration in-gel digestion, obtains the peptide mass fingerprinting spectrum detecting sample.Select the species intending inquiry, peptide mapping fingerprinting data and other parameter, use the Mascot:Peptide Mass Fingerprint protein database search program that provides of http://www.MatrixScience.com website, at the NCBInr Protein database search peptide hydrolysis albumen that can match with it in theory.Through database retrieval coupling, from cellular metabolism, signal transduction, cytoskeleton composition, oxidative stress, immunity, cell proliferation and each angle sieve such as apoptosis and molecular chaperones have selected some associated protein, as high mobility albumen etc.
To those skilled in the art, obviously the invention is not restricted to the details of above-mentioned one exemplary embodiment, and when not deviating from spirit of the present invention or essential characteristic, the present invention can be realized in other specific forms.Therefore, no matter from which point, all should embodiment be regarded as exemplary, and be nonrestrictive, scope of the present invention is limited by claims instead of above-mentioned explanation, and all changes be therefore intended in the implication of the equivalency by dropping on claim and scope are included in the present invention.
In addition, be to be understood that, although this specification sheets is described according to embodiment, but not each embodiment only comprises an independently technical scheme, this narrating mode of specification sheets is only for clarity sake, those skilled in the art should by specification sheets integrally, and the technical scheme in each embodiment also through appropriately combined, can form other embodiments that it will be appreciated by those skilled in the art that.

Claims (6)

1. in tea, Camellia nitidissima Chi, tea-polyphenol is to a screening method for esophageal cancer marker, and it is characterized in that, concrete steps comprise:
1) apply XDA macroporous resin adsorption method of purification and prepare tea-polyphenol by extracting in different types of tea, Camellia nitidissima Chi, its step comprises: tea, Camellia nitidissima Chi → pulverized 30 mesh sieves → with 30% alcohol immersion 20min → microwave extracting 20min → extracting solution through Middle hollow fiber membrane → rotary evaporation concentrated → cross macroporous absorption resin XDA-200 post → water elution removal of impurities → 10% ethanol elution removal of impurities → 30% ethanol gradient elution → elutriant respectively rotary evaporation concentrate → lyophilize → obtain dried powder; Polyphenol content measures by " detection method of GB/T 8313-2008 Tea Polyphenols in Tea and catechin content " method, polyphenol content=(in sample powder tea-polyphenol quality/sample mass) × 100%;
2) Human esophageal squamous cell cancer cell KYSE-510, EC9706, KYSE-510 of vitro culture, EC9706, EC8712, EC8733, EC9706, Eca109 and adenocarcinoma cell OE33 are model, the equal adherent growth of above-mentioned cell in RPMI 1640 substratum, at 37 DEG C, the CO of 5% 2, Secondary Culture under saturated humidity;
Experiment is divided into tea-polyphenol group, positive controls, solvent control group and blank group, and wherein in tea-polyphenol group, each dosage component of tea-polyphenol is not 10,20,40,60 and 80mg/m1; In positive controls, Fluracil dosage is 0.25mg/m1; Solvent control group adds the DMSO that final concentration is 0.1%; Blank group only adds equal-volume nutrient solution, does not add medicine and cell;
3) tea-polyphenol is detected to the effect of esophageal cancer cell
A) mtt assay is adopted to detect tea-polyphenol to the inhibited proliferation of esophageal cancer cell, concrete steps comprise Human esophageal squamous cell cancer cell KYSE-510, EC9706, KYSE-510, EC9706, EC8712, EC8733, EC9706, the Eca109 and adenocarcinoma cell OE33 in vegetative period that take the logarithm, be the trysinization of 0.25% through mass percent concentration, be inoculated in 96 well culture plates, 5 × 10 3individual cells/well, preculture 24h, the whole supernatant of sucking-off after cell attachment, empirically grouping adds the various tested materials of different concns, cumulative volume 200 μ l/ hole, and every dose group establishes 4 multiple holes; Cultivate 24,48,72 and 96h respectively, it is the MTT of 5mg/L that every hole adds 20 μ l concentration, continue to cultivate 4h, discard nutrient solution, every hole adds the DMSO of 200 μ l, and plate shaker shaking 10min, returns to zero with DMSO, measure each hole light absorption value A with enzyme-linked immunosorbent assay instrument with 490nm wavelength, calculate average A-value, proliferation rate PR and inhibiting rate CIR; Proliferation rate PR=(experimental group A value/solvent control group A value) × 100%; Inhibiting rate CIR=(1-test group average A-value/control group average A-value) × 100%; Use curvilinear regression analysis, obtain dose-response relationship equation and regression coefficient;
B) according to the result of MTT colorimetry, suitable concentration is chosen according to experimental design process cell; The esophageal cancer cell of taking the logarithm vegetative period, adjustment density 2.5 × 10 4individual/ml, puts 28cm 2in culturing bottle, cultivate 24h after cell attachment, add different concns process factor, stop cultivating in 96h; Get EC9706, KYSE-510 or OE33 cell, discard nutrient solution, mass percent concentration is the trysinization of 0.25%, and the centrifugal l0min of 2000r/min, abandons supernatant liquor, is resuspended in by cell precipitation in PBS liquid, adjustment cell density to 1 × 10 6the centrifugal l0min of individual/m1,2000r/min, abandons supernatant liquor; Add 75% ethanol 1ml of 4 DEG C of precoolings fast, make cell resuspended, at 4 DEG C, be incubated 10-15h; Get single cell suspension lml, add the RNA inhibitor 5 μ l of fluorescence dye propidium iodide 10 μ l and l0mg/ml of l0 μ g/ml, under slight oscillatory, room temperature, lucifuge hatches 20min, filters with 400 eye mesh screens, obtained cell suspension, under 488nm wavelength, carries out cell DNA content detection with flow cytometer; Obtain data through Muticycle AV software processes, each phase distribution percentage of cell cycle is analyzed, and calculates G in the cell cycle o/ G 1phase, S phase and G 2the per-cent that/M phase cell is shared in sum, represents the proliferation activity of cell with proliferation index PI;
C) fluorescence inverted microscope observation of cell morphological change is adopted: be inoculated in by each cell in 96 orifice plates, 5 × 10 3individual cells/well, preculture 24h, each experimental group add respectively 10,20,40,60 and 80mg/m1 tea in tea-polyphenol activity extract, cultivate 24,48,72 and 96h respectively in solvent control group, positive controls, observe under fluorescence inverted microscope after hematoxylin-eosin staining method stain smear;
D) protein expression change in immunohistochemical methods, Real-time fluorescence quantitative RT-RCR, Westem-blot analysis esophageal cancer cell is applied, three strain esophageal cancer cells are inoculated in 9 porocyte culture plates, cultivate 24h, after the tea-polyphenol effect 24h of 80 μm of ol/L, every 5-10 × 10 6cell adds the Trizol reagent of 1ml, and 15-30 DEG C of effect 15min, abandons supernatant liquor, add 0.5ml Virahol/ml Trizol reagent in throw out, 15-30 DEG C of effect 10min, 2-8 DEG C, and 12000 leave heart 15min, abandon supernatant liquor; Add 75% ethanol of 1ml/mlTrizol reagent wash RNA, 2-8 DEG C again, 7500 leave heart 5min, abandon supernatant liquor, and the sample obtained is preserved at-80 DEG C;
Use ABI/9700 system to carry out each gene PCR amplified reaction, response procedures is as follows: 95 DEG C, 10s; Denaturation: 95 DEG C, 5s; 60 DEG C, 34s; 40 circulations; The melting curve analysis of amplified production is all carried out in the PCR reaction of various gene; Each genetic expression more all adopts β-actin as reference gene;
Follow-up employing Western-Blot analyzes protein expression change in esophageal cancer cell, three strain esophageal cancer cells are inoculated in 9 porocyte culture plates, cultivate 24h, after the tea-polyphenol effect 24h of 80 μm of ol/L, PBS liquid washs three times, add cold cell pyrolysis liquid and act on 30min on ice, scraping cells on Eppendorf tube, 4 DEG C, 14000 leave heart 15min, Aspirate supernatant, ultraviolet spectrophotometer measures each sample total protein concentration, and regulates concentration consistent; Sample, after 10-12% polyacrylamide gel electrophoresis 4h, is transferred on nitrocellulose filter, 100mA, 20-60min; Nitrocellulose filter is closed after 3h through confining liquid, TBS buffer solution three times, in the first antibody working fluid of Dilution ratio 1:500-1:2000, hatch 2h; TBS buffer solution 3 times, the second antibody working fluid of the horseradish peroxidase mark of thinning ratio 1:2000 hatches 2h, TBS buffer solution 3 times, and super quick luminescence is aobvious light also, after photosensitive X-ray, developing fixing, β-actin albumen is as internal reference albumen;
4) adopt SPSS12.0 statistical software to analyze the result in step 3), compare with one-way analysis of variance between multiple sample mean; Compare between two and check with LSD; Dose-response relationship curvilinear regression and correlation analysis, inspection level α=0.05;
5) have detected the differentially expressed protein after tea-polyphenol effect esophageal cancer cell in tea, Camellia nitidissima Chi, obtain two-dimensional electrophoresis difference expression atlas, identify the peptide fingerprinting spectrum of differential protein, mate through database retrieval, filter out associated protein, described associated protein has comprised heterogeneous nuclear ribonucleoprotein H1, ubiquitin, microtubule gone steady albumen, PKC to disturb albumen 1, heterogeneous nuclear ribonucleoprotein, high mobility albumen, tubulin molecule companion, aldolase A.
2. in tea according to claim 1, tea-polyphenol, to the screening method of esophageal cancer marker, is characterized in that, described tea bag draws together green tea, black tea, black tea, Leaf of Assam Tea, yellow tea, white tea, blue or green tea, oolong tea, camellia, rock tea.
3. in tea according to claim 1, Camellia nitidissima Chi, tea-polyphenol, to the screening method of esophageal cancer marker, is characterized in that, containing 10% standard foetal calf serum, 100 U/ml penicillin and 100 μ g/ml Streptomycin sulphates in described RPMI 1640 substratum.
4. in tea according to claim 1, Camellia nitidissima Chi, tea-polyphenol, to the screening method of esophageal cancer marker, is characterized in that, concentrated glue 80V, separation gel 120V in polyacrylamide gel electrophoresis.
5. in tea according to claim 1, Camellia nitidissima Chi, tea-polyphenol, to the screening method of esophageal cancer marker, is characterized in that, described confining liquid comprises 5% skimming milk and 0.05% Tween 20, pH7.6.
6. in tea according to claim 1, Camellia nitidissima Chi, tea-polyphenol, to the screening method of esophageal cancer marker, is characterized in that, this method is also applicable to other leaf, flower, fruit, rhizome containing tea polyphenols material.
CN201410639293.8A 2014-11-13 2014-11-13 Screening method of esophagus cancer markers by tea polyphenol in tea and golden camellia Pending CN104450854A (en)

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CN104887839A (en) * 2015-06-23 2015-09-09 广西北部湾海皇生物科技有限公司 Golden camellia extract for preventing cancer and inhibiting cancer growth and preparation method thereof
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CN111154910A (en) * 2020-03-03 2020-05-15 武夷学院 Primer, kit and method for identifying Wuyi narcissus tea
CN111154910B (en) * 2020-03-03 2022-05-10 武夷学院 Primer, kit and method for identifying Wuyi narcissus tea
CN111662999A (en) * 2020-07-03 2020-09-15 广东省农业科学院环境园艺研究所 Rhododendron and camellia different tissue organ fluorescence quantitative internal reference gene and application

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