CN110393231A - A kind of purposes and preparation method thereof of roe polypeptide - Google Patents
A kind of purposes and preparation method thereof of roe polypeptide Download PDFInfo
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- CN110393231A CN110393231A CN201910684834.1A CN201910684834A CN110393231A CN 110393231 A CN110393231 A CN 110393231A CN 201910684834 A CN201910684834 A CN 201910684834A CN 110393231 A CN110393231 A CN 110393231A
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- Prior art keywords
- roe
- polypeptide
- cell
- preparation
- fish
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
- A23J1/04—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from fish or other sea animals
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/04—Animal proteins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/02—Peptides of undefined number of amino acids; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/30—Extraction; Separation; Purification by precipitation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Analytical Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Nutrition Science (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Zoology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Immunology (AREA)
- Epidemiology (AREA)
- Mycology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Water Supply & Treatment (AREA)
- Marine Sciences & Fisheries (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present invention relates to a kind of Anticancer Food field, espespecially a kind of purposes and preparation method thereof of roe polypeptide, purposes is the application for preparing roe polypeptide in antitumor and prophylactic treatment tumour food, and preparation method includes: 1) to collect fish egg cell；2) supernatant → retention removal of impurities → detection processing is precipitated → taken by clean → cell homogenates → freeze thawing → to fish egg cell；3) roe polypeptide stoste is prepared；4) preparation of roe polypeptide powder, mother liquor or solution；The present invention, to melanoma, the inhibiting effect of tumour, plays antitumor, anti-cancer function by fish-egg, and the present invention efficiently utilizes fish by-product in medicinal and food, creates social benefit for economic development；And the present invention is compared with traditional anti-tumor drug preparation, since fish-egg extracts naturally occurring biologically active peptide in obtained roe polypeptide stoste, its ingredient has certain safety, and when as anti-tumor drug or health food, reliable degree is higher than traditional anti-tumor drug preparation.
Description
Technical field
The present invention relates to a kind of Anticancer Food field, espespecially a kind of purposes and preparation method thereof of roe polypeptide.
Background technique
In recent years, anti-tumor drug is the life topic that modern compares concern, and R&D work is also paid close attention to by the common people,
It is well known that malignant tumour seriously threatens the life and health and safety of the mankind, but current most of anti-tumor drug is endless
Can resist virus entirely, and there are low selectivity, toxic side effect is strong, is also easy to produce the disadvantages of drug resistance, therefore research and develop
New anti-tumor drug is always the research and development center of gravity of biological field.Currently, it has been investigated that antibacterial peptide has broad spectrum antibiotic activity,
Can quick killing target, and be much wherein pure natural peptide, it made to rapidly become potential therapeutic agent, antibacterial peptide
Therapeutic domain are as follows: gramnegative bacterium, gram-positive bacterium, fungi, helminth, tumour cell etc., and antibacterial peptide is one
Kind has the small peptide of antibacterial activity, induces generation by cell specific gene coding, through external condition, is widely present in living nature,
It is the important component of natural immune system.
Many antibacterial peptides in addition to having an antibacterial effect, also show the activity of antiviral and antitumor.Many has antitumor
Active antibacterial peptide has carried out clinical trial, and wherein lactoferrin (Lactoferrin) has been used for lung non-small cell carcinoma
Treatment；Nevertheless, same peptide is not all effective to all tumour cells due to the diversity of tumour cell type, furthermore,
Some antineoplastic polypeptides specificity and in terms of it is also undesirable, and there are toxic side effects such as hemolytics, therefore Ying Ji
The screening of the continuous polypeptide for reinforcing having anti-tumor function；In addition, being directed to the detection of anti-tumor experiment, testing result is simultaneously non-ideal
Precisely, it is also necessary to reinforce the operability and data levels of precision of experiment.
Summary of the invention
To solve the above problems, the present invention is directed to disclose a kind of Anticancer Food field, espespecially a kind of use of roe polypeptide
Way and preparation method thereof, prepare stoste by obtaining roe polypeptide, and be prepared as antineoplastic health product, drug etc..
To achieve the above object, the technical solution adopted by the present invention is that:
A kind of purposes of roe polypeptide, which is characterized in that the purposes is to prepare antitumor and prophylactic treatment tumour food
Application in product, health care product or drug.
Preferably, the effective component of the roe polypeptide is fish-egg extract.
A kind of preparation method of roe polypeptide, which is characterized in that the preparation method mainly comprises the steps that
1) firstly, collecting fish egg cell；
2) secondly, fish egg cell is successively precipitated → taken supernatant → retention by clean → cell homogenates → freeze thawing → and remove
The processing of miscellaneous → detection；
3) roe polypeptide stoste thirdly, is obtained after filtration sterilization；
4) roe polypeptide stoste is subjected to freeze-drying concentration to be prepared as roe polypeptide powder；
5) roe polypeptide high concentration mother liquor is configured to by roe polypeptide powder；
6) the roe polypeptide solution of various concentration is diluted to by roe polypeptide high concentration mother liquor.
Preferably, the fish egg cell of the step 1) is obtained out of deep-sea sturgeon roe.
Preferably, the molecular cut off of step 2) the retention removal of impurities is 10000 dalton.
The beneficial effects of the present invention are embodied in: the present invention extracts roe polypeptide stoste using fish egg cell, is applied to anti-swollen
In tumor and prophylactic treatment tumour, and its function and effect is verified by external and experiment in vivo.
The present invention is by the application of roe polypeptide extract, in the inhibiting tumor cell proliferation function data and card for obtaining roe polypeptide
According to support and animal and clinical trial safety assurance under the premise of, by roe polypeptide switch to cancer clinical treatment adjuvant
Object provides treatment medication or rehabilitation medication for tumor patient, mitigates the physical pain of patient and progressively reach healing curative effect.
The antitumor action for the roe polypeptide stoste extracted from fish-egg can be achieved in the present invention, and passes through a series of experiments method,
As cell proliferation experiment, flow cytometry Annexin V/PI double-staining, Western Blot, animal model for tumour are built
Vertical, immunohistochemistry etc., studies the antitumor action of roe polypeptide from experiment in vitro to experiment in vivo.
The present invention, to melanoma, the inhibiting effect of tumour, plays antitumor, anti-cancer function, while this hair by fish-egg
Bright that fish by-product is utilized, avoiding the problem that usual fish by-product utilized rate is not high leads to the wasting of resources, and the present invention will
Fish by-product efficiently utilizes in medicinal and food, lays the foundation for food, medicine trade in the future to the development and utilization of fish-egg,
Social benefit is created for economic development；And the present invention is compared with traditional anti-tumor drug preparation, due to fish-egg extract it is obtained
Naturally occurring biologically active peptide in roe polypeptide stoste, ingredient have certain safety, thus as anti-tumor drug or
When health food, reliable degree is higher than traditional anti-tumor drug preparation.
Detailed description of the invention
Fig. 1 is cell proliferation inhibition rate chart of the roe polypeptide to MCF-7.Note: * indicates the statistics meaning compared with for 24 hours
Justice, * p < 0.05, * * * p < 0.0001, # indicate statistical significance of the 48h compared with 72h, #p < 0.05.
Fig. 2 is influence chart of the fish-egg extract to SD rat BM-MSCs cell Proliferation.Note: * is indicated compared with for 24 hours
Statistical significance, * * * p < 0.0001, # indicate statistical significance of the 48h compared with 72h, #p < 0.05.
Fig. 3 is that the fish-egg extract of various concentration acts on the different degrees of apoptosis figure occurred after MCF-7 cell.
Fig. 4 is MCF-7 intracellular mitochondrial film potential comparative diagram.
Specific embodiment
The following detailed description of a specific embodiment of the invention:
A kind of purposes of roe polypeptide, the purposes be prepare antitumor and prophylactic treatment tumour food, health care product or
Application in drug, the effective component of the roe polypeptide are fish-egg extract.
A kind of preparation method of roe polypeptide, the preparation method mainly comprise the steps that
1) firstly, collecting fish egg cell；Fish egg cell can be obtained out of deep-sea sturgeon roe；
2) secondly, fish egg cell is successively precipitated → taken supernatant → retention by clean → cell homogenates → freeze thawing → and remove
The processing of miscellaneous → detection；The molecular cut off of retention removal of impurities is 10000 dalton；
3) roe polypeptide stoste thirdly, is obtained after filtration sterilization；
4) roe polypeptide stoste is subjected to freeze-drying concentration to be prepared as roe polypeptide powder；
5) roe polypeptide high concentration mother liquor is configured to by roe polypeptide powder；
6) the roe polypeptide solution of various concentration is diluted to by roe polypeptide high concentration mother liquor.
One, the present embodiment can application experiment method include:
The experiment of the applicable detection antitumor activity energy of the present embodiment is as follows: cell proliferation experiment (MTT Assay), CCK-
8 methods, Apoptosis dyeing, flow cytometry (Flow Cytometry), immunoblotting (Western blot), tumour are dynamic
The foundation and immunohistochemistry of object model:
Wherein, cell proliferation experiment (MTT Assay): testing principle is the succinate dehydrogenase energy in living cells mitochondria
Be that exogenous MTT is reduced to the bluish violet crystallization of water-insoluble and first a ceremonial jade-ladle, used in libation (Formazan) and is deposited in cell, and dead cell without
This function；Dimethyl sulfoxide (DMSO) can dissolve the first a ceremonial jade-ladle, used in libation in cell, its absorption value is measured at 4900nm wavelength with microplate reader,
Within the scope of certain cell number, it is directly proportional to cell number that MTT crystallizes the amount to be formed；Judged according to the absorbance value measured living thin
Born of the same parents' quantity, OD value is bigger, and cell activity is stronger；It should be noted that: in cell proliferation experiment, the first caused by MTT is restored
A ceremonial jade-ladle, used in libation product is not soluble in water, could detect after need to being dissolved.
Contain water-soluble tetrazolium salts WST-8, the entitled 2- (2- of chemistry in Cell Counting Kit-8:CCK-8 kit
Methoxyl group -4- nitre phenyl) -3- (4- nitre phenyl) -5- (2,4- disulfobenzene) -2H- tetrazolium monosodium salt, it is deposited in electronics coupled reagent
In case, water-soluble strong yellow formazan substance can be reduced to by the dehydrogenase in living cells mitochondria；WST-8 is generated
Crystallization be it is water-soluble, solubility is larger, can save subsequent dissolving step, reduces error, does not need lytic cell, can
Colorimetric is directly carried out, the depth for testing color is directly proportional to the proliferation degree of cell, is inversely proportional with cytotoxicity degree, can use enzyme
Mark instrument measures its absorbance at wavelength 450nm, to reflect living cells quantity.
AO/EB dyeing: acridine orange (AO) can penetrate the complete cell of after birth, be embedded in nucleus DNA, be allowed to what sending became clear
Green fluorescence, ethidium bromide are only capable of the cell being damaged through after birth, are embedded in core DNA, issue Chinese red fluorescence；The cell of apoptosis is in
It is now dyeing enhancing, fluorescence is more bright, uniform round shape or pyknosis shape, crumb structure；Non- apoptotic nucleus is presented
The deep mixed structure like feature of fluorescence；The two form is totally different, easily differentiates very much, in fluorescence microscopy microscopic observation, it is seen that four kinds thin
Born of the same parents' form: living cells (VN), nuclear chromatin green and be in normal configuration；Viable apoptotic cell (VA), nuclear chromatin green
In pyknosis shape or round bead shape；The dead cell (NVN) of non-apoptosis, nuclear chromatin Chinese red and be in normal configuration；Late apoptic
Cell (NVA), nuclear chromatin are Chinese red and pyknosis shape or round bead shape.
Flow cytometry (Flow Cytometry): be using flow cytometer carry out a kind of Single cell quantitative analysis and
Sorting technology；The height such as flow cytometry teacher's monoclonal anticancer and immunocytochemical technique, laser and electronic computer science
The high-tech product of development and comprehensive utilization.Flow cytometry with it quickly, accurate, high sensitivity the advantages that, be widely used in
Biological cell period and dynamic (dynamical) analysis, detection Apoptosis, are exempted from various tumprigenicity antigens, tumprigenicity albumen, oncogene
The theoretical research and clinical diagnosis of epidemiology；It should be noted that: cell is detected using flow cytometry Annexin V/PI double-staining
Apoptosis, PI dye liquor (propidium iodide) is toxic, can be absorbed by skin, and to the irritating effect of eyes, when use need to wear gloves；It is real
If testing with attached cell, it is improper to be digested with pancreatin, can cause false positive.
Immunoblotting (Western blot): the basic principle is that processed to gel electrophoresis by specific antibody
Cell or biological tissue samples coloured, by analysis coloring position and color depth obtain specific protein dividing
The information of expression in the cell or tissue of analysis；It should be noted that: Western Blot experiment indirect method has cross reaction to cause
Non-specific band, the secondary antibody that primary antibody incubation time is long, additional is incubated for.
The foundation of animal model for tumour: induction property animal model for tumour is (researcher's chemical carcinogens, radioactive ray, carcinogenic
The tumour of viral-induced animal, such as diethylnitrosamine DEN induce big white mouse liver cancer), spontaneous tumor animal model (without
Artificial processing, abiogenous animal model for tumour, such as spontaneous mammary cancer mouse model, C3H mouse, CBA mouse, A system are small
Mouse etc.)；It should be noted that: induction property animal model for tumour usually require long-term, a large amount of administrations could it is carcinogenic, have apparent kind poor
It is different；Spontaneous tumor animal model: in same strain, disease incidence is more stable, and difference is very big between different lines, each mouse disease
Cheng Buyi, after Spontaneous Tumor is made a definite diagnosis, when experiment, should match grouping.
Immunohistochemistry: applied immunology basic principle-antigen-antibody reaction makes the aobvious of labelled antibody by chemically reacting
Toner (fluorescein, enzyme, isotope, metal ion) develops the color to determine histocyte endoantigen, is positioned, is formed and phase to it
To determining quantifier elimination.
Two, the detection operating process of the present embodiment are as follows:
1, according to above-mentioned experimental method, the antitumor performance detection of present invention combination roe polypeptide to integrate experimentation,
Roe polypeptide stoste is subjected to extracorporeal anti-tumor research and internal antitumor detection respectively, to test from experiment in vitro to experiment in vivo
Demonstrate,prove the antitumor action of roe polypeptide；As extracorporeal anti-tumor research by cell proliferation experiment, CCK-8 method, Apoptosis dyeing,
Flow cytometry Apoptosis and western blot detect Wnt signal path；Antitumor research is dynamic by tumour in vivo
The foundation and immunohistochemistry of object model；Obtain roe extract polypeptide inhibiting tumor cell proliferation function data and evidence support with
And under the premise of animal and clinical trial safety assurance, roe polypeptide can be converted into cancer clinical treatment ancillary drug, realize one
Determine health care, prevent, treat function.
2, by different preparation methods prepare roe polypeptide stoste, roe polypeptide powder, roe polypeptide high concentration mother liquor,
Roe polypeptide solution.
3, extracorporeal anti-tumor detects:
Cell proliferation experiment: concentration is lyophilized in roe polypeptide stoste first, powdered roe polypeptide is configured to height
The mother liquor of concentration, various concentration roe polypeptide solution needed for being diluted to cell with mother liquor；Human breast cancer cell line Bcap-37 is through difference
After concentration roe polypeptide stoste inoculated and cultured different time, roe propolypeptide is detected using MTT kit or CCK-8 kit
Liquid is to the proliferation function of tumour cell, and whether observation is inhibited to its cell Proliferation, i.e. cytotoxic effect；Set up sky
White control and cell controls are using the MCF-7 cell without the processing of roe polypeptide as blank control, SD rat marrow mesenchyma
Stem cell is cell controls；
Flow cytometry: apoptosis morphology is observed by AO/EB dyeing, using flow cytometry
Annexin V/PI double-staining detects Apoptosis situation, and detects active oxygen, superoxides, mitochondria in MCF-7 cell
Level of membrane potential, the influence in conjunction with oxidative stress situation assessment roe polypeptide stoste to MCF-7 cell；
Western Blot:Western Blot method detects roe polypeptide to Wnt signal path correlation egg in tumour cell
The influence of white expression.
4, antitumor detection in vivo:
The foundation of transplanted human breast carcinoma nude mice model: SPF grades of BALB/c-n/n nude mices are raised in SPF grades of animal centers,
Drinking-water and feed are by the free feeding of animal after high pressure sterilization, and human breast cancer cell line Bcap-37 is containing 10% fetal calf serum
It is cultivated in DMEM culture medium, passage amplification is collected logarithmic growth phase cell and washed 2 times after trypsin digestion with PBS, adjusted
Whole cell concentration is 107/ml, and subsequent experimental carries out under SPF environment；In the right dorsal sc inoculating cell suspension of every mouse
0.2ml, after inoculation 7 days, it is seen that it is hard that small protrusion, matter occurs in inoculation position, and is gradually increased, and tumor average volume reaches
145.32 (100~200) mm3When, it is believed that modeling success starts grouping administration.Model nude mice is randomly divided into solvent pair by weight
According to group, treatment group and positive controls, treatment group is respectively with the grouping of various concentration roe polypeptide, using docetaxel as positive
Control group；Administration mode is intraperitoneal injection；Solvent group and treatment group are totally 21 days 1 time a day, and positive controls are every 7 days 1
It is secondary, totally 21 days；Sample to effective treatment concentration of breast cancer solid tumor really timing first choose several low middle high dose concentration into
Row preliminary experiment；
Prepared by the paraffin section of transplanted tumor in nude mice: the tumor tissues fixed being taken out, are rinsed to wash away fixer, through complete
Tissue is made into transparent processing, slice, mounting after waxdip and embedding after automatic dehydrator serial dehydration.
The HE of nude mice model tumor tissue is dyed: the paraffin on slice sloughed, makees rehydration processing through graded concentrations alcohol,
After Harris haematoxylin dyeing, slice be dehydrated again, transparent processing, neutral gum mounting.
The TUNEL of nude mice model tumor tissue is dyed: being operated by TUNEL kit operating instruction.
Immunohistochemistry: using tachysynthesis group method (MaxVisionTM kit) detection roe polypeptide stoste to swollen
The influence of the expression of primary tumor Wnt GAP-associated protein GAP in tumor animal model；
Three, specific embodiment:
The present embodiment is to be used for roe polypeptide in breast cancer detection and treatment, and roe polypeptide acts on the portion of MCF-7 cell
Divide experimental result:
1, CCK-8 method surveys MCF-7 cell proliferative conditions
There are dose-effect relationships for cell proliferation inhibition rate of the roe polypeptide to MCF-7, with the raising of its concentration, cell
The trend incrementally increased is also presented in proliferation inhibition rate, compared with processing group for 24 hours, at 200,250,300,350,400 μ g/ml of concentration
The result for managing 48h and 72h has significant statistical significance (p < 0.0001), and inhibiting rate highest is close to 40%；For 24 hours, 48h and
The cell proliferation inhibition rate of each dosage of 72h is lower than positive control cis-platinum group, and difference has statistical significance.
Therefore, roe polypeptide goes out obvious rush to MCF-7 cells show in 50 μ g/ml and 100 μ g/ml concentration processing groups and increases
The effect of growing, wherein the 100 μ g/ml processing groups of 48h have significant statistical significance (p < 0.0001) compared with for 24 hours, are specifically shown in
In Fig. 1,2, Fig. 1: * indicates that the statistical significance compared with for 24 hours, * p < 0.05, * * * p < 0.0001, # indicate 48h compared with 72h
Statistical significance, #p < 0.05.；In Fig. 2: * indicates statistical significance with for 24 hours compared with, * * * p < 0.0001, # indicate 48h and
The statistical significance that 72h compares, #p < 0.05..
2, AO/EB method observes apoptosis morphology
After acting on MCF-7 cell for 24 hours with the fish-egg extract of various concentration, there is different degrees of apoptosis, AO/EB in cell
Visible cell apoptosis morphological change after dyeing is shown in Fig. 3, and cell is mostly normal live cells when 50 μ g/ml low dosage concentration are handled,
Nuclear chromatin iridescent, and be in normal configuration；Visible cell apoptosis early stage state when the processing of 200 μ g/ml concentration, nuclear chromatin
Iridescent pyknosis shape or round bead shape；See that non-viable apoptotic cell, nuclear chromatin are iridescent and tie when the processing of 400 μ g/ml concentration
Structure is in pyknosis shape or round bead shape (arrow is signified).
3, mitochondrial membrane potential
Mitochondria is the important organelle for promoting cellular energy conversion, participating in Apoptosis, the decline of mitochondrial membrane potential
When Apoptosis early stage a hallmark events, with the fish-egg extract of various concentration effect MCF-7 cell for 24 hours after, observation
It is reduced to intracellular mitochondrial film potential, as a result sees Fig. 4, be MCF-7 intracellular mitochondrial film potential comparative diagram, the line of processing group
Mitochondrial membrane potential decline degree is above control group, respectively 19.2%, 16.5%, 16.9%, 22.5%, 22.9%,
22.8%, 22.5%, 22.9%, mitochondrial membrane potential descension theory clear-cells is in Apoptosis early stage.
The above is only presently preferred embodiments of the present invention, is not intended to limit the scope of the present invention, current row
The technical staff of industry can make some deformations and modification, all technologies according to the present invention under the inspiration of the technical program
Essence still falls within the range of technical solution of the present invention to any modification, equivalent variations and modification made by above embodiment
It is interior.
Claims (5)
1. a kind of purposes of roe polypeptide, which is characterized in that the purposes be prepare antitumor and prophylactic treatment tumour food,
Application in health care product or drug.
2. a kind of purposes of roe polypeptide according to claim 1, which is characterized in that the effective component of the roe polypeptide
For fish-egg extract.
3. a kind of preparation method of roe polypeptide, which is characterized in that the preparation method mainly comprises the steps that
1) firstly, collecting fish egg cell；
2) secondly, to fish egg cell successively through cleaning → cell homogenates → freeze thawing → precipitate → take the removal of impurities of supernatant → retention →
The processing of detection；
3) roe polypeptide stoste thirdly, is obtained after filtration sterilization；
4) roe polypeptide stoste is subjected to freeze-drying concentration to be prepared as roe polypeptide powder；
5) roe polypeptide high concentration mother liquor is configured to by roe polypeptide powder；
6) the roe polypeptide solution of various concentration is diluted to by roe polypeptide high concentration mother liquor.
4. a kind of preparation method of roe polypeptide according to claim 3, which is characterized in that the fish-egg of the step 1) is thin
Born of the same parents obtain out of deep-sea sturgeon roe.
5. a kind of preparation method of roe polypeptide according to claim 3, which is characterized in that step 2) the retention removal of impurities
Molecular cut off be 10000 dalton.
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