CN105238833A - Application of bullacta oligopeptide in resisting prostatic cancer - Google Patents
Application of bullacta oligopeptide in resisting prostatic cancer Download PDFInfo
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- CN105238833A CN105238833A CN201410277152.6A CN201410277152A CN105238833A CN 105238833 A CN105238833 A CN 105238833A CN 201410277152 A CN201410277152 A CN 201410277152A CN 105238833 A CN105238833 A CN 105238833A
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Abstract
The invention relates to application of bullacta oligopeptide in resisting prostatic cancer, in particular to application of bullacta oligopeptide in drugs for resisting prostatic cancer DH-145 tumor cells. The amino acid sequence of the bullacta oligopeptide is as shown in Gln-Pro-Pro-Gly-Leu, and the bullacta oligopeptide is prepared from bullacta by enzymatically hydrolyzing bullacta tissues with trypsin, and by separating and purifying in combination of gel chromatography and high performance liquid chromatography. The bullacta oligopeptide disclosed by the invention is significant in inhibitory effect on prostatic cancer DH-145 tumor cells, wherein an inhibition ratio on proliferation of DH-145 cells reaches 33.5-88.4% and affecting ratios on early apoptosis and late apoptosis of the prostatic cancer DH-145 tumor cells are respectively 20-22.27% and 28-30.66%, showing significant antineoplastic activity of the bullacta oligopeptide. The bullacta oligopeptide has excellent development prospect; and researches on the resistance of the bullacta oligopeptide on prostatic cancer DH-145 tumor cells offers theoretical support for researches on the development of prostatic cancer resistance drugs.
Description
Technical field
The present invention relates to the application of mud snail enzymolysis oligopeptide, be specifically related to the application of a kind of mud snail oligopeptides in antiprostate cancer.
Background technology
Mud snail (Bullactaexarata), be commonly called as " telling iron ", " yellow mud spiral shell ", " plum spiral shell " etc., belong to Mollusca, Gastropoda, Opisthobranchia, head Parapet order, A Di spiral shell section, mud snail genus, for the kind of west bank, Pacific Ocean seawater and salt-fresh water special product, be made up of shell and software two portions, containing compositions such as rich in protein, calcium, phosphorus, iron and VITAMIN, oligopeptides material abundance, being distributed widely in the Changjiang river of the East Sea and Huanghai Sea both sides in China, is a kind of common fishery products.
Malignant tumour is the disease of a class serious threat human health and life, and sickness rate is more and more higher, and the problems such as though current existing chemotherapeutics has certain curative effect to most of tumour, poor selectivity, toxic side effects are large, resistance still clearly.Therefore, searching is efficient, low toxicity, special strong antitumor drug are still imperative.Oligopeptides because its molecular weight is little, non-immunogenicity, activity is high, side effect is little, especially also has good inhibit activities to the tumor cell line of multi-medicine resistance.Marine organisms oligopeptides is a key areas of current marine biomaterial research, some bioactive oligopeptides are often present in protein with inactive state, these protein are through the hydrolytic action of proteolytic enzyme, the bioactive peptide be hidden in wherein is discharged, has likely given play to biological activity more more than protein.Such as: as Deng Wei etc. finds that the growth in vitro of Phycocyanins, C-enzymolysis oligopeptide to HeLa Cells strain has obvious restraining effect; The Mytilus edulis oligopeptides of the enzymolysis Mytilus edulis gained such as Yang Yongfang has specific inhibited proliferation to Human Prostate Cancer PC-3 Cell Line and DU-145 cell, and is better than the inhibited proliferation to PC-3 cell to the restraining effect of DU-145 cell; Yao is as forever waited research mud blood clam oligopeptides at 0.25-1.0gL
-1in scope, oligopeptides significantly can suppress the propagation of lung cell A549 and Ketr-3 cell, simultaneously can also the synthesis of T suppression cell protein, and in obvious dose-dependently.
Prostate cancer is male sex's malignant tumour occurred frequently, and its mortality ratio is in the second of cancer mortality, is only second to lung cancer.In recent years, along with the continuous lifting of the change of people life style, the aging of population and diagnostic level, the sickness rate that I crosses prostate cancer is obviously in rising trend.Prostate cancer DU-145 cell is separated from prostate cancer brain metastes tumour, and differentiation degree is low, is the prostate cancer cell of Androgen-dependent, has powerful metastatic potential, lack the expression of endogenic androgen receptor.DU-145 cell completely adherent time is generally in 24h, has stronger invasion and attack and angiogenesis promoting ability.The restraining effect of research mud snail enzymolysis oligopeptide many prostate cancers DU-145 cell, significant to the theoretical investigation of prostate cancer medicine.
Summary of the invention
Technical problem to be solved by this invention is on the basis that enzymolysis mud snail tissue obtains mud snail oligopeptides, provides the application of a kind of mud snail oligopeptides in antiprostate cancer, especially anti-prostate cancer DU-145 tumour cell.
The present invention solves the problems of the technologies described above adopted technical scheme: the application of a kind of mud snail oligopeptides in anti-prostate cancer DU-145 cell drug, it is characterized in that: the aminoacid sequence of described mud snail oligopeptides is as described in SEQIDNO.1, and the specific implementation form of its Chinese traditional medicine can be tablet, capsule, granule or pill etc.
Above-mentioned mud snail oligopeptides is obtained by following steps: new fresh snail is cleaned and shelled, get the homogenate of mud snail tissue mashing, with hydrochloric acid soln and sodium hydroxide solution, the pH value of homogenate is adjusted to 8 ~ 9, solid-liquid ratio is 1:(3.8 ~ 4) add the trypsinase of 0.4 ~ 0.5% homogenate volume, 40 ~ 50 DEG C of insulation hydrolysis 7 ~ 9h, latter 95 ~ 100 DEG C are hydrolyzed, 10 ~ 15min carries out going out enzyme, at 4 DEG C, hydrolyzed solution is in the centrifugal 15 ~ 20min of 9500 ~ 10000r/min, get supernatant liquor, first component of 3kd is less than through 3kd ultra-filtration membrane ultrafiltration acquisition molecular weight, this first component is obtained second component through gel chromatography wash-out, finally this second component RPLC is separated and obtains described mud snail oligopeptides.
As preferably, described gel chromatography condition is as follows: the concentration of described first component is 48 ~ 50mg/mL, loading 3 ~ 4mL, speed are 2.5 ~ 3.5ml/min, and moving phase is ultrapure water, 280nm ultraviolet detection.
As preferably, described RP-HPLC condition is as follows: ZorbaxSB-C18 (4.6 × 250,5um); Column temperature is 20 DEG C; Moving phase is 1%TFA and acetonitrile; Gradient elution: from terminate to 30min, acetonitrile concentration changes to 40% from 0, and elution speed is 1.0mL/min; Sampling volume is that 50ul ultraviolet detection wavelength is respectively 214nm, 280nm.
Further, after mud snail oligopeptides described in 6 ~ 14mg/ml acts on DU-145 cell 24 ~ 36h, be 33.5 ~ 88.4% to the proliferation inhibition rate of prostate cancer DU-145 cell, and the concentration of proliferation inhibition rate and mud snail oligopeptides is proportionate.
Further again, after mud snail oligopeptides described in 6 ~ 14mg/ml acts on DU-145 cell 24h, 20 ~ 22.27% and 28 ~ 30.66% are respectively to the early apoptosis of prostate cancer H1299 cell and the contributive rate of late apoptic, and the concentration of proliferation inhibition rate and mud snail oligopeptides is proportionate
Compared with prior art, the invention has the advantages that: the present invention utilizes mud snail for raw material, by enzymolysis and extraction mud snail oligopeptides, mud snail is a kind of low value shellfish, extensively distributes in China, and source is wide, cost is low, and the gentle easily control of the extracting method of mud snail oligopeptides in the present invention, technique is simple, easy to operate.Mud snail oligosaccharides in the present invention significantly can suppress prostate cancer DU-145 tumour cell, wherein can reach 33.5 ~ 88.4% to the proliferation inhibition rate of DU-145 cell, 20 ~ 22.27% and 28 ~ 30.66% are respectively to the early apoptosis of prostate cancer DU-145 cell and the contributive rate of late apoptic, there is anti-tumor activity as seen significantly.Mud snail oligopeptides has good DEVELOPMENT PROSPECT, and the development research that the research of the anti-prostate cancer DU-145 tumour cell of mud snail oligosaccharides be can be to antiprostate cancer provides theories integration.
Accompanying drawing explanation
Fig. 1 is the gel chromatography collection of illustrative plates of the first component in embodiment 1;
Fig. 2 is the high-efficient liquid phase chromatogram of second component in embodiment 1;
Fig. 3 be in embodiment 4 mud snail oligopeptides to the inhibited proliferation of prostate cancer DU-145 tumour cell and action time and activity relation broken line graph;
Fig. 4 is the histogram of Fig. 3;
Fig. 5 be the mud snail oligopeptides of different concns in embodiment 4 act on DU-145 cell after HE colored graph, A: control group, B:6mg/ml, C:10mg/ml, D:14mg/ml;
Fig. 6 be the mud snail oligopeptides of different concns in embodiment 4 act on DU-145 cell after AO/EB two contaminate fluorogram, A: control group, B:6mg/ml, C:10mg/ml, D:14mg/ml;
Fig. 7 be the mud snail oligopeptides of different concns in embodiment 4 act on DU-145 cell after AnnexinV-FITC/PI coloration result figure, A-1: control group, A-2:6mg/ml, A-3:10mg/ml, A-4:14mg/ml.
Embodiment
Below in conjunction with accompanying drawing embodiment, the present invention is described in further detail.
Embodiment 1: the preparation of mud snail oligopeptides
(1.1) Feedstock treating: get new fresh snail, mud snail is shelled, to drain rear homogenate for subsequent use.
(1.2) mud snail enzymolysis process
With high-speed tissue mashing machine by the homogenate of mud snail tissue mashing, precision takes homogenate, with the hydrochloric acid soln of 0.1mol/L and the NaOH solution adjust pH of 0.1mol/L, add trypsin digestion a few hours, enzymatic hydrolysis condition is hydrolysis temperature 45 DEG C, and pH is 8.7, solid-liquid ratio 1:4, enzymolysis time 8h, enzyme concentration 0.48%.Go out at 100 DEG C enzyme 15min, centrifugal 15min (10000r/min) at 4 DEG C, and it is for subsequent use to get supernatant concentration.
(1.3) separation and purification of mud snail polypeptide
First ultrafiltration process is selected to be separated mud snail polypeptide enzymolysis solution, ultra-filtration membrane selects 10kd respectively, 5kd, 3kd, be trapped into molecular weight 10-5kd, 5-3kd respectively and be less than 3kd tri-components, be made into the concentration of 20mg/mL after lyophilize respectively, measure the inhibiting rate of each component to prostate cancer DU-145 with mtt assay.Select the highest active the first component (molecular weight is less than 3kd), select SephadexG-25 gel chromatography, with deionized water balance after dress post, concentration is 50mg/mL, loading 4mL, speed is 3ml/min, and moving phase is ultrapure water, 280nm ultraviolet detection, gel chromatography curve as shown in Figure 1, collect the highest elution peak lyophilize powdered, detect the proliferation inhibition rate to DU-145 cell, and curve plotting draws IC
50, second component the highest for activity is collected in a large number lyophilize and carries out efficient liquid phase chromatographic analysis acquisition mud snail oligopeptides.
By the water dissolution of second component 0.06%TFA good for lyophilize in the centrifuge tube of 0.6ml, at 12000rpm, centrifugal 10min gets supernatant liquor.High performance liquid phase condition: ZorbaxSB-C18 (4.6 × 250,5um); Column temperature is 20 DEG C; Moving phase is 1%TFA and acetonitrile; Gradient elution: from terminate to 30min, acetonitrile concentration changes to 40% from 0, and elution speed is 1.0mL/min; Sampling volume is that 50ul ultraviolet detection wavelength is respectively 214nm, 280nm.High-efficient liquid phase chromatogram as described in Figure 2, is collected elution peak A respectively, is obtained mud snail oligopeptides.
Embodiment 2: the mensuration of mud snail oligopeptides aminoacid sequence
The amino acid sequence analysis of target polypeptides adopts N-terminal amino acid degradation detection method to measure.Criterion amino acid collection of illustrative plates: utilize kilnitamin standard substance (PTH-AA), runs generation standard substance color atlas under normal conditions, corrects the retention time of mixed amino acidity scale product, generates standard method file.Sample pre-treatments: for subsequent use by getting supernatant liquor after centrifugal for pure sample product; Polybrene (Polybrene) 15ul is added to the upper nitrogen of glass fibre membrane (GlassFiberDisk) to dry up; Upper machine is by glass fibre membrane pre-treatment, and namely run 5 circulations and enough pure sample spot be added on pretreated glass fibre membrane, nitrogen dries up.Upper machine testing: be sealed and placed in the reactor of Protein Sequencer PPSQ-31A by the glass fibre membrane PTFE filter membrane having added sample, setting detects amino acid no and other parameters.
In the present embodiment, collect the mud snail oligopeptides be separated through high performance liquid chromatography, the aminoacid sequence of this peptide is as described in SEQIDNO.1 after testing.
The cultivation of embodiment 3:DU-145 cell with go down to posterity
(3.1) cell recovery
The DU-145 cell strain deposited is taken out from liquid nitrogen container, put into 37 DEG C of thermostat water baths fast to melt, after melting, enter aseptic working spaces operate, cell strain is sucked centrifuge tube by the suction pipe good with sterilizing, add F12 nutritive medium or the RPMI1640 nutritive medium of 2mL, the centrifugal 10min of 1000rpm, removes supernatant liquor, add the nutritive medium of 4ml, piping and druming makes cell become individual cells repeatedly.Then with suction pipe, cell is drawn in the culturing bottle of 2 25mL uniformly, puts into 5%CO
2, the constant incubator of 37 DEG C cultivates, change liquid next day and outwell not adherent dead cell.
(3.2) cell cultures
Human Prostate Cancer Cells DU-145 is inoculated in the F12 and 1640 nutritive mediums containing 10% foetal calf serum (volume fraction) FBS and dual anti-(penicillin G 100IU/mL, Streptomycin sulphate 100IU/mL) respectively, is positioned over 37 DEG C, 5%CO
2constant incubator in cultivate, cell attachment grow, within every 1 day, change liquid once, go down to posterity when cell covers with about 80% of culturing bottle.Go down to posterity according to the ratio of 1: 2, collect logarithmic phase cell and test.
(3.3) passage
First the culturing bottle covering with cell is taken out from constant incubator and be put on aseptic operating platform.When going down to posterity, first outwell the nutritive medium in bottle, remove the dead cell of not adherent growth, with 0.25% trypsinase/0.02%EDTA Digestive system mixture slaking, the time of different cell dissociation is different, and general digestion time is 3-5min; Basis of microscopic observation cell, when intercellular substance obviously becomes large and cell rounding brightens, illustrates that cell has digested complete, removes digestive ferment liquid.In culturing bottle, add the nutritive medium of about 2.5mL, repeatedly blow and beat the attached cell digested and make it to become individual cells, generalized case next bottle of cell passes 2 bottles, the cell gone down to posterity is placed in 37 DEG C, 5%CO
2incubator in cultivate.
(3.4) cell cryopreservation
Basis of microscopic observation, when cell grows to about 80% of culturing bottle, select cellular form good carry out frozen, remove nutrient solution, add Digestive system to digest cell, digestive ferment is outwelled when Microscopic observation intercellular substance is obvious, in culturing bottle, add the nutritive medium of about 3mL again, piping and druming makes it to become individual cells repeatedly, sucks in centrifuge tube, 1000rpm, centrifugal 10min, nutrient solution is abandoned in careful suction, adds containing 10%DMSO foetal calf serum 1mL, with suction pipe repeatedly blow and beat cell evenly after, be drawn in aseptic cryopreservation tube.After first putting into 4 DEG C of refrigerator 30min, then put into liquid nitrogen position of bottleneck 2h, finally put into liquid nitrogen bottle and carry out frozen.
Embodiment 4: mud snail oligopeptides anti-prostate cancer DU-145 cell activity assays
(4.1) on the impact of DU-145 cell inhibitory effect
The cell of taking the logarithm vegetative period makes suspension, is seeded to 96 orifice plates, every hole 200 μ L, if 5 parallel holes, in 5%CO
2, 37 DEG C of adherent 16-48h, observe under inverted microscope, discard nutrient solution, are dissolved in respectively in nutrient solution by mud snail enzymolysis solution with different concns simultaneously.Then add each hole respectively, establish the control group not adding sample simultaneously, put 5%CO
2, hatch 36h in 37 DEG C of incubators, rinse 2 times with PBS, add the nutritive medium containing MTT, continue to cultivate 4h.Stop cultivating, carefully suck nutrient solution in hole.Add DMSO, put low-speed oscillation 10min on shaking table, adopt enzyme-linked immunosorbent assay instrument to survey absorbance (OD value) in 490nm.Calculate cell inhibitory effect index (IR, Inhibitionrate), i.e. proliferation inhibition rate, by following formulae discovery:
Mud snail oligopeptides obtained in embodiment 1 is set to 5 concentration groups, is respectively 6mg/mL, 8mg/mL, 10mg/mL, 12mg/mL and 14mg/mL.Act on after 24,36 hours, MTT detection is carried out to DU-145 cell proliferation inhibition rate.Result shows: after effect 36h, the proliferation inhibition rate of mud snail oligopeptides to DU-145 cell reaches 88.4%, as shown in table 1, and, to concentration and the action time of the positive correlation respectively of DU-145 cell proliferation inhibition rate and mud snail oligopeptides, as shown in Figure 3, Figure 4.
Table 1 mud snail oligopeptides to the proliferation inhibition rate of DU-145 cell (
) (n=3)
Note: ﹡ compared with the minimum cell inhibitory rate of each component, P<0.05
(4.2) on the apoptotic impact of DU-145
(4.2.1) HE dyeing
Cover glass is through poly-lysine acidifying 24h, tap water 20 times, distilled water immersion 24h, the method process such as dry rear autoclaving, careful is laid in six well culture plates, the Digestive system of the cancer cells 0.25% in culturing bottle is digested, when intercellular substance is obvious and change circle brightens, sop up Digestive system, nutritive medium adds 10% foetal calf serum, repeatedly blow and beat to individual cells with dropper, with nutrient solution, cell concn is adjusted to 1 × 10
5mL
-1left and right.Careful is inoculated on cover glass, and every hole drips about 2mL, then puts in incubator and cultivates, treat that the cell on cover glass covers with about 80%, adds the different concns medicine group for preparing with nutritive medium and control group is cultivated.
After cell climbing sheet terminates, careful cover glass to be taken out, gently wash 2 times with PBS, each about 1min, then 95% alcohol fixation 15min, gently wash 3 times with PBS, each 3min.Add haematoxylin dyeing 8min, tap water embathes 30s, and hydrochloride alcohol breaks up, and tap water embathes.When nucleus becomes blue, then add eosin stain and redye 30s, tap water embathes.Use 50%, 75%, 95% and dehydrated alcohol serial dehydration respectively, dimethylbenzene is transparent, each 5min, totally 3 times.With resinene, the slide that dye is lustful is carried out mounting, have the one side of cell to be fitted on slide glass downwards.Observe form under an optical microscope and take the photograph sheet.
As shown in Figure 5, the control group A figure of normal DU-145 cell, observes cellular form comparatively regular full, and tenuigenin dye distribution is comparatively even, and kernel number is more.B figure is form under 6mg/mL concentration, and feature has cell appearance to change, and cytolemma becomes irregular, and nuclear targeting is denseer, concentration phenomenon that tenuigenin is slight.C figure is the cell under 10mg/mL drug level, occurs comparatively significantly karyopyknosis, occurs cavity, and cytolemma becomes irregular further compared with B figure, and tenuigenin dyeing is in grumeleuse shape, and the corpusculum present situation of apoptosis appears in cell; D figure is the cellular form under 14mg/mL drug level, and obvious apoptosis feature appears in cell, occurs the features such as cell is irregular, after birth comes off.Visible, after mud snail oligopeptides effect 24h, along with the increase gradually of drug dose, comparatively obvious to the suppression phenomenon of DU-145 cell, cytoplasmic condensati, the number of nucleus kernel reduces gradually, nucleus color deepens gradually, and cytolemma comes off, and occurs cavity, the volume of cell diminishes gradually, and the metamorphosis of cell is particularly evident.
(4.2.2) AO/EB fluorescent dye
The prostate cancer DU-145 tumour cell of taking the logarithm vegetative period, adds appropriate pancreatin and digests, and is made into suspension with the foetal calf serum nutrient solution containing 10%, cover glass is tiled on 6 porocyte culture plates, add cell suspension 2mL to every hole, cell plate are placed on 37 DEG C, containing 5%CO
2incubator in cultivate, after cell covers with about 80% of slide take out.Liquid is added in each hole respectively, then cell plate are put in incubator cultivate, after 24h, take out Tissue Culture Plate.Take out slide glass, add 1 mixing fluorescent dye liquid: 100 μ g/mL acridine oranges (AO), 100 μ g/mL smell second pyridine (EB) mixing, drop on slide glass, the cover glass of climbing full cell is pressed, observing apoptosis cellular form under fluorescent microscope.
Fluorescence colour is different with cell membrane integrity according to nuclear structures, makes acridine orange (AO) and Ethidium Bromide (EB) mixing colouring agent dye different colours, carrys out discriminate between cells and whether apoptosis occurs.Fig. 6 is the effect of DU-145 cell under different pharmaceutical concentration, and wherein A figure is normal cell, without obvious apoptotic cell under fluorescent microscope, cell size, distribution all very even.B figure is the cellular form of cell under the effect of lower concentration medicine (6mg/mL), and cytolemma is comparatively complete but occur cavity; C figure is the cell under the drug influence of middle concentration (10mg/mL), nucleus be AO dyeing in yellow-green fluorescence, be densely polymerized to particulate state, be positioned at the side of cell, viable apoptotic cell starts to increase; D figure is that under high density (14mg/mL), along with the increase of drug level, non-viable apoptotic cell increases, and nuclear chromatin sends out fluorescent red-orange.
(4.2.3) FCM analysis of cells apoptosis
Digested by cultured DU-145 cell 0.25% pancreatin/0.02%EDTA mixed solution, the cell PBS digested cleans 1-2 time, and the trysinization time is not long, in order to avoid cause false positive.The cell that digested is sucked in centrifuge tube, and centrifugal at 4-6 DEG C (1200r/min) 5min, sucks supernatant liquor, add 400ul in conjunction with liquid, piping and druming repeatedly gently makes cell become individual cells, and cell concn is 1 × 10
6about cells/mL, in cell suspending liquid, add the FITC staining fluid mixing of 5ul, at 4-6 DEG C, carry out lucifuge hatch 15min, then add the PI dye liquor of 10ul, mix gently and hatch 5min at 4-6 DEG C, the cell processed is put into FACS immediately and detects.
Flow cytometry (FCM) can by normal in sample, downright bad and apoptotic cell etc. separately.As shown in Figure 7, in each collection of illustrative plates of A-1, A-2, A-3, A-4, cell is divided into four districts: what left upper quadrant represented is physical abuse cell; What left lower quadrant represented is normal cell; Right upper quadrant is the cell of late apoptic or necrosis; What right lower quadrant represented is viable apoptotic cell.The visible increase along with peptide concentration, early apoptosis and the late apoptic of DU-145 cell are also increasing gradually, and when mud snail oligopeptide concentration is 14mg/mL, early withering is 22.27%, and withering evening is 30.66%.
The key instrument used in each embodiment above and material as follows:
Key instrument
Super clean bench ZHJM-C12090, Shanghai ZHICHENG Anaiytical Instrument Manufacturing Co., Ltd.;
Forma3111 cell culture incubator, Bao Cheng bio tech ltd, Hangzhou;
Microplate reader (U.S. BIO-RAD); Bao Cheng bio tech ltd, Hangzhou;
FACSCalbur flow cytometer, BD company;
OLYMPUSCKX41 inverted phase contrast microscope, Japanese OLYMPUS company;
Bole 680 microplate reader (U.S.), Bao Cheng bio tech ltd, Hangzhou;
OLYMPUS microscope 5,000,000 pixel Pro-MicroScanCCD, Japanese OLYMPUS company;
Reagent
Bovine serum (Hangzhou folium ilicis chinensis biotechnology company limited);
MTT (SIGMA company of the U.S.);
F12 powder culture medium (SIGMA company of the U.S.);
RPMI1640 substratum, Gibco company;
DMSO; (SIGMA company of the U.S.)
AO/EB dye, Hao Tian Bioisystech Co., Ltd
AnnexinV-FITC/PI apoptosis test kit; Bei Bo biotech firm
Penicillin, Streptomycin sulphate; The anti-medical limited-liability company in Shandong
Cell strain
Human Prostate Cancer Cells DU-145 is purchased from Chinese Academy of Sciences's Shanghai cytobiology cell bank.
Claims (7)
1. the application of mud snail oligopeptides in anti-prostate cancer DU-145 cell drug.
2. the application of mud snail oligopeptides in anti-prostate cancer DU-145 cell drug as claimed in claim 1, is characterized in that: the aminoacid sequence of described mud snail oligopeptides is as described in SEQIDNO.1.
3. the application of mud snail oligopeptides in anti-prostate cancer DU-145 cell drug as claimed in claim 2, it is characterized in that described mud snail oligopeptides is obtained by following steps: new fresh snail is cleaned and shelled, get mud snail tissue mashing, add distilled water homogenate, solid-liquid ratio is 1:(3 ~ 4), with hydrochloric acid soln and sodium hydroxide solution, the pH value of homogenate is adjusted to 8 ~ 9, add the trypsinase of 0.4 ~ 0.5% homogenate volume, 40 ~ 50 DEG C of insulation hydrolysis 7 ~ 9h, latter 95 ~ 100 DEG C are hydrolyzed, 10 ~ 15min carries out going out enzyme, at 4 DEG C, hydrolyzed solution is in the centrifugal 15 ~ 20min of 9500 ~ 10000r/min, get supernatant liquor, first component of 3kd is less than through 3kd ultra-filtration membrane ultrafiltration acquisition molecular weight, this first component is obtained second component through gel chromatography wash-out, finally this second component RPLC is separated and obtains described mud snail oligopeptides.
4. the application of mud snail oligopeptides in anti-prostate cancer DU-145 cell drug as claimed in claim 3, it is characterized in that: described gel chromatography condition is as follows: the concentration of described first component is 48 ~ 50mg/mL, applied sample amount is 3 ~ 4mL, speed is 2.5 ~ 3.5ml/min, moving phase is ultrapure water, 280nm ultraviolet detection.
5. the application of mud snail oligopeptides in anti-prostate cancer DU-145 cell drug as claimed in claim 3, is characterized in that: described RP-HPLC condition is as follows: select ZorbaxSB-C18 (4.6 × 250,5um); Column temperature is 20 DEG C; Moving phase is 1%TFA and acetonitrile; Gradient elution: from terminate to 30min, acetonitrile concentration changes to 40% from 0, and elution speed is 1.0mL/min; Sampling volume is that 50ul ultraviolet detection wavelength is respectively 214nm, 280nm.
6. the application of the mud snail oligopeptides as described in claim arbitrary in Claims 1 to 5 in anti-prostate cancer DU-145 cell drug, it is characterized in that: after mud snail oligopeptides described in 6 ~ 14mg/ml acts on DU-145 cell 24 ~ 36h, be 33.5 ~ 88.4% to the proliferation inhibition rate of prostate cancer DU-145 cell, and the concentration of proliferation inhibition rate and mud snail oligopeptides is proportionate.
7. the application of the mud snail oligopeptides as described in claim arbitrary in Claims 1 to 5 in anti-prostate cancer DU-145 cell drug, it is characterized in that: after mud snail oligopeptides described in 6 ~ 14mg/ml acts on DU-145 cell 24h, 20 ~ 22.27% and 28 ~ 30.66% are respectively to the early apoptosis of prostate cancer DU-145 cell and the contributive rate of late apoptic, and the concentration of proliferation inhibition rate and mud snail oligopeptides is proportionate.
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