CN106319009A - Extracellular matrix protein peptide, as well as extraction method and application thereof - Google Patents
Extracellular matrix protein peptide, as well as extraction method and application thereof Download PDFInfo
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Abstract
The invention relates to the field of protein peptide extraction, and particularly relates to an extracellular matrix protein peptide, as well as an extraction method and application thereof. The method comprises the following steps: crushing beef tendons, and performing acid and alkali treatments; neutralizing, removing wastewater, homogenizing with water, and heating and cooking at a temperature of 70 DEG C or over to obtain colloid; and performing enzymolysis on the colloid to obtain the extracellular matrix protein peptide. The protein peptide prepared after treatment has an average molecular weight of 1200-1500 Daltons and a total protein extraction rate of more than or equal to 90 percent; in the extracted extracellular matrix protein peptide, the content of type-I collagen is 58-60 percent, the content of type-IV collagen is 30-32 percent, the content of glycoprotein and proteoglycan is 2.9-3.0 percent, and the content of L-hydroxyproline is 13-15 percent. The invention further provides application of the extracellular matrix protein peptide in preparation of medicines for treating cancers with a remarkable effect.
Description
Technical field
The present invention relates to protein peptide and extract field, in particular to a kind of extracellular matrix egg
White peptide and extracting method thereof and application.
Background technology
Fibroblast effect in neoplastic process has been carried out further by related basic research
Research, after research finds subcutaneous injection carcinogen, positive fibroblasts can be accumulated to carcinogen
Around, wrap up carcinogen by producing collagen, thus stop epithelial cell to occur DNA to damage
Wound.After utilizing mice specificity to eliminate positive fibroblasts, epithelial cell becomes carcinogen
, there is DNA damage, and cause the generation of epithelial tumor in target spot.Additionally, carcinogen processes
A part of mice, long-term " Tumor free ", and use the collagenase will parcel carcinogen
After fibrous capsule destroys, quickly there is tumor in mice.This result shows positive fibroblasts energy
By secretion collagen, wrap up carcinogen, thus stop epithelium to cancerate, the generation of suppression tumor.
This research illustrates the tumorigenic a kind of new mechanism of body fight.
Basic research confirms, tumor cell is not having and (or) lacking extracellular matrix (ECM)
There is in the presence of albumen multiplication capacity.This multiplication capacity reflects under the conditions of entirety, tumor
Cell is survived and growth by invasion and attack and transfer.The fracture of basement membrane, lack and reduce and destroy
The integrity of basement membrane so that tumor cell is prone to pass through substrate barrier.The change of ECM and weight
Mould the primary condition that (Remodeling) is tumor invasion and angiogenesis.In a word, come off,
Adhere to, degrade, mobile and hypertrophy is malignant tumor invasion and attack, the necessary process of transfer.
Extracellular matrix (ECM, extracellular matrix) albumen and Dan Baiduotang proteoglycan PG
(proteoglycan) rack (fyame-woyk) of basement membrane is constituted, mainly containing type i collagen
(ColI), IV collagen (ColIV), aminoglycan and Dan Baiduotang proteoglycan PG and elastin laminin.Certain
A little specific anatomical structures are with little or no by tumor cell invasion, such as tendon, aorta, cornea
Deng.This anti-invasion ability is considered as to wrap up suppression due to organizational structure feature and secretion to swell
The material of oncocyte invasion and attack.
Extracellular matrix protein peptide is to divide from the Ungula Bovis seu Bubali muscle without connective tissue of blood vessels that yak is the purest
The small molecular weight protein polypeptide of energy inhibitory enzyme degraded is confirmed from extraction, basis and preclinical study,
Actually it is compounded with multiple protein enzyme inhibitor, endothelial growth inhibitor and reinvent reparation
Substrate barrier and other composition.Tumor suppression can be wrapped up occur.It is that tumor causes changing of ECM
Become and reinvent (Remodeling) important target spot inhibitor.
In view of this, the special proposition present invention.
Summary of the invention
The first object of the present invention is to provide the extracting method of a kind of extracellular matrix protein peptide,
The method by by after broken for Ungula Bovis seu Bubali muscle, enzymolysis after acid-alkali treatment and obtain, gross protein extraction
Rate >=90%；Extract extracellular matrix protein peptide in, type i collagen content be 58%-60%,
IV collagen content is 30%-32%, glycoprotein (elastin laminin)+Dan Baiduotang proteoglycan PG is (with amino
Glucose meter) content is 2.9%-3.0%, L-hydroxyproline (precollagen) content 13%-15%,
Peptide mean molecule quantity 1200-1500 dalton.
The second object of the present invention is to provide carrying of a kind of described extracellular matrix protein peptide
Access method extracts the extracellular matrix protein peptide obtained.
The third object of the present invention is to provide carrying of a kind of described extracellular matrix protein peptide
Access method extracts the extracellular matrix protein peptide obtained application in preparation treatment cancer drug.
In order to realize the above-mentioned purpose of the present invention, spy by the following technical solutions:
The extracting method of a kind of extracellular matrix protein peptide, comprises the following steps:
Acid treatment and alkali process is carried out after broken for Ungula Bovis seu Bubali muscle；
Remove sewage after neutralized reaction, add water and carry out homogenizing, then in more than 70 DEG C heating
Boil, obtain colloid；
Described colloid is carried out enzymolysis, after enzyme denaturing, i.e. can get extracellular matrix protein peptide.
Owing to Ungula Bovis seu Bubali muscle quality is tight, high resilience, protein peptide therein is not easy to extract.This
The extracting method of the extracellular matrix protein peptide that invention provides, through test of many times, by Ungula Bovis seu Bubali muscle
After Po Sui, first pass through acid treatment, to be discharged by the extracellular matrix protein in Ungula Bovis seu Bubali muscle；
Process through alkali the most again, with the structure of the most loose Ungula Bovis seu Bubali muscle, be beneficial to next step and extract；
After neutralizing reaction, the Ungula Bovis seu Bubali muscle after processing carries out homogenizing, obtains the slurry of Ungula Bovis seu Bubali muscle, so
Boiled by heating afterwards, obtain colloid；Directly colloid is carried out enzymolysis, beneficially enzyme to play a role,
Albumen in colloid passes through sufficient enzymolysis, and the mean molecule quantity of the protein peptide obtained is
1200-1500 dalton.
The extracting method of the extracellular matrix protein peptide that the present invention provides, gross protein extraction ratio >=
90%；In the extracellular matrix protein peptide extracted, type i collagen content is 58%-60%, IV glue
Former content is 30%-32%, glycoprotein (elastin laminin)+Dan Baiduotang proteoglycan PG is (with glucosamine
Meter) content is 2.9%-3.0%, L-hydroxyproline (precollagen) content 13%-15%.
Specifically, described crushing is by after Ungula Bovis seu Bubali muscle roguing, puts into high-speed tissue mashing machine and blends
?.High-speed tissue mashing machine is bought, such as DS-1 high-speed tissue mashing machine by commercially available.
Ungula Bovis seu Bubali muscle after Po Sui is in granular form, and is beneficial to next step process.
Further, described acid treatment includes pickling and weary acid；
Described alkali processes and includes caustic dip and weary alkali.
Wherein, weary acid be by pickling after Ungula Bovis seu Bubali muscle extrusion, acid solution therein is extruded；
The Ungula Bovis seu Bubali muscle that weary acid obtains is directly placed in alkali liquor and soaks, and is extruded by Ungula Bovis seu Bubali muscle after immersion,
Alkali liquor therein is extruded, has i.e. carried out the step of weary alkali.
By pickling and weary acid, so that the extracellular matrix protein in Ungula Bovis seu Bubali muscle is fully discharged
Come；By caustic dip and weary alkali, with the structure of the most loose Ungula Bovis seu Bubali muscle, it is beneficial to the equal of next step
Matter.
In order to reach the effect of more preferable pickling and caustic dip, it is preferable that described pickling uses quality
Percent is the dilute hydrochloric acid of 10%～20%；
The lime water that described caustic dip uses mass percent to be 10%～20%.
Further, during described pickling, described dilute hydrochloric acid and the weight ratio of Ungula Bovis seu Bubali muscle
For 1:1～2, the time of described pickling is preferably 50-70min；
During described caustic dip, described lime water is 1:1～2 with the weight ratio of Ungula Bovis seu Bubali muscle, institute
The time stating caustic dip is preferably 50-70min.
Further, described homogenizing is: through neutralizing reacted Ungula Bovis seu Bubali muscle with pure water by weight
Than being 1: 2-3 to put into high speed dispersion homogenizer and carry out homogenizing；
Described more than 70 DEG C heating boil into: under stirring condition, at temperature 72 DEG C～80 DEG C
Reaction 5～6h, is then passed through filter and is concentrated to give described colloid.
Wherein, high speed dispersion homogenizer is bought, such as FJ-200 high speed dispersion by commercially available
Homogenizer.
Preferably, described filtration uses ionic adsorption pressing plate to carry out.Entered by ionic adsorption pressing plate
Row filters, simultaneously by Adsorption heavy metal ion therein such as cadmium, copper, ferrum etc.,
The colloid quality arrived is purer, and the final extracellular matrix protein peptide mouthfeel prepared is more preferable.
Further, first described colloid is carried out colloidal sol in 72-75 DEG C before enzymolysis.By heating
Colloidal sol, obtains the solution of liquid, is beneficial to the carrying out of enzymolysis.
In order to make the more abundant of enzymolysis, it is preferable that described enzymolysis is: add albumen water after colloidal sol
Solving enzyme, regulation temperature is 50～52 DEG C, and regulation pH is 8.7～9.2, reacts 2～3h；Wherein,
Described colloid is 5000: 1～10000: 1 with the weight ratio of described proteolytic enzyme.
Preferably, also include filtration, nanofiltration, sterilizing after enzyme denaturing, be spray-dried and pack i.e.
Available extracellular matrix protein peptide finished product.Wherein, filtration is to remove dividing of larger particles
Son, is refined further by nanofiltration, then through conventional sterilizing and spray drying, obtains cell
Extracellular matrix protein peptide finished product.The extracellular matrix protein peptide finished product that the present invention prepares is in good taste, clothes
With convenient, it is easy to absorption of human body.
Present invention also offers the extracellular matrix protein peptide prepared by said extracted method.This
The extracellular matrix protein peptide of bright offer, type i collagen content is that 58%-60%, IV collagen contains
Amount is 30%-32%, glycoprotein (elastin laminin)+Dan Baiduotang proteoglycan PG (in terms of glucosamine)
Content is 2.9%-3.0%, L-hydroxyproline (precollagen) content 13%-15%, nutritional labeling
Abundant.
The extracellular matrix protein peptide that the present invention prepares can directly be taken, it is possible to according to the market demand
Add adjuvant make various pharmaceutical dosage form, as injection, injection, tablet, electuary, granule,
Pill, capsule, suspending agent, Emulsion, unguentum, cream, transdermal patch etc., in order to clothes
With and preserve.
Present invention also offers the extracellular matrix protein peptide prepared by said extracted method in system
Application in standby treatment cancer drug.
The Therapeutic Method of cancer, including the extracellular matrix protein peptide giving experimenter's effective dose.
Administering mode can be oral, intravenous injection or transdermal penetration mode, puts on and needs
Patient to be treated, specifically can be determined by doctor according to the state of an illness of patient, age etc..
The extracellular matrix protein peptide that the present invention provides, after patient takes, repairs base in vivo
Film, parcel tumor, and DNA plerosis damage, reach to limit tumor migration and propagation
Purpose, and can progressively there is apoptosis in tumor cell.
Compared with prior art, the invention have the benefit that
(1) extracting method of the extracellular matrix protein peptide that the present invention provides, by by Ungula Bovis seu Bubali
After muscle is broken, enzymolysis after acid-alkali treatment and obtain, gross protein extraction ratio >=90%；Wherein,
Type i collagen content be 58%-60%, IV collagen content be 30%-32%, glycoprotein (elastic
Albumen)+Dan Baiduotang proteoglycan PG (in terms of glucosamine) content as 2.9%-3.0%, L-hydroxyl dried meat ammonia
Acid (precollagen) content 13%-15%, peptide mean molecule quantity 1200-1500 dalton.
(2) present invention has also selected specific bronsted lowry acids and bases bronsted lowry, with specific concentration, processes
The specific time, it is beneficial to extract protein peptide therein.
(3) present invention is also boiled after Ungula Bovis seu Bubali muscle homogenizing, is then passed through filter with dense
Contracting obtain colloid, to colloid use specific condition carry out enzymolysis so as to get peptide mean molecule
Amount 1200-1500 dalton.
(4), in process of the present invention, filter and use ionic adsorption pressing plate to carry out, remove wherein
Heavy metal ion so as to get extracellular matrix protein peptide safety, and in good taste.
(5) the extracellular matrix protein peptide that the present invention provides is after cancer patient takes, at body
Interior reparation basement membrane, parcel tumor, and DNA plerosis damage, reach limit tumor migration with
And the purpose of propagation, and can progressively there is apoptosis in tumor cell.
Accompanying drawing explanation
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below
The accompanying drawing used required in embodiment or description of the prior art will be briefly described.
The extracting method schematic flow sheet of the extracellular matrix protein peptide that Fig. 1 provides for the present invention；
Fig. 2 is the work that in experimental example 1 of the present invention, tumor tissues in tumor model group invades peplos
Inspection microgram；
Fig. 3 is the rule of the borderline tumor in test group in experimental example 1 of the present invention, and peplos is complete
Biopsy microgram；
Fig. 4 is that in experimental example 1 of the present invention, tumor tissues in tumor model group invades periphery fiber
The biopsy microgram of connective tissue；
Fig. 5 is that in experimental example 1 of the present invention, tumor boundary clear in test group has complete peplos
Biopsy microgram；
Fig. 6 is the CT of the concrete typical clinical example patient 2011-8-11 in experimental example 2 of the present invention
Figure；
Fig. 7 is the concrete typical clinical example patient 2011-11-28 in experimental example 2 of the present invention
CT schemes.
Detailed description of the invention
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but ability
Field technique personnel are it will be appreciated that the following example is merely to illustrate the present invention, and are not construed as limit
The scope of the present invention processed.Unreceipted actual conditions person in embodiment, according to normal condition or manufacture
The condition of business's suggestion is carried out.Agents useful for same or instrument unreceipted production firm person, be and can lead to
Cross the conventional products of commercially available acquisition.
The extracting method schematic flow sheet of the extracellular matrix protein peptide that Fig. 1 provides for the present invention,
It is described in detail especially by following example.
Embodiment 1
The extracting method of a kind of extracellular matrix protein peptide, comprises the following steps:
After Ungula Bovis seu Bubali muscle roguing, put into DS-1 high-speed tissue mashing machine and blend, the cattle after crushing
The animal limb tendons immerse in the dilute hydrochloric acid that mass percent is 10%, soak 70min；
Carry out after pickling extruding weary acid, be then placed in the lime water that mass percent is 10%,
The time of caustic dip is 50-70min, and caustic dip extrudes weary alkali after completing；
Ungula Bovis seu Bubali muscle acid adding after weary alkali is neutralized, and removes sewage, add pure water after neutralizing reaction
Putting into FJ-200 high speed dispersion homogenizer and carry out homogenizing, Ungula Bovis seu Bubali muscle is 1 with the weight ratio of pure water:
2；
After homogenizing under agitation, at temperature 72 DEG C, 6h is reacted, then through ionic adsorption
Pressing plate filters, and is then concentrated to give colloid；
In 72 DEG C, colloid being carried out colloidal sol, adds proteolytic enzyme after colloidal sol, regulation temperature is
50 DEG C, regulation pH is 8.7, reacts 3h；Wherein, colloid and the weight ratio of proteolytic enzyme
It is 10000: 1；
After colloid enzymolysis completes, through enzyme denaturing, filtration, nanofiltration, sterilizing, it is spray-dried and wraps
Dress i.e. obtains extracellular matrix protein peptide finished product.
Gross protein extraction ratio is 92%；In the extracellular matrix protein peptide extracted, type i collagen
Content is 58%, IV collagen content is 32%, glycoprotein (elastin laminin)+Dan Baiduotang proteoglycan PG (with
Glucosamine meter) content is 2.9%, L-hydroxyproline (precollagen) content 15%；Carefully
The mean molecule quantity of extracellular matrix protein peptide is 1500 dalton.
Embodiment 2
The extracting method of a kind of extracellular matrix protein peptide, comprises the following steps:
After Ungula Bovis seu Bubali muscle roguing, put into DS-1 high-speed tissue mashing machine and blend, the cattle after crushing
The animal limb tendons immerse in the dilute hydrochloric acid that mass percent is 20%, soak 50min；
Carry out after pickling extruding weary acid, be then placed in the lime water that mass percent is 20%,
The time of caustic dip is 50min, and caustic dip extrudes weary alkali after completing；
Ungula Bovis seu Bubali muscle acid adding after weary alkali is neutralized, and removes sewage, add pure water after neutralizing reaction
Putting into FJ-200 high speed dispersion homogenizer and carry out homogenizing, Ungula Bovis seu Bubali muscle is 1 with the weight ratio of pure water:
3；
After homogenizing under agitation, at temperature 80 DEG C, 5h is reacted, then through ionic adsorption
Pressing plate filters, and is then concentrated to give colloid；
In 75 DEG C, colloid being carried out colloidal sol, adds proteolytic enzyme after colloidal sol, regulation temperature is
52 DEG C, regulation pH is 9.2, reacts 2h；Wherein, colloid and the weight ratio of proteolytic enzyme
It is 5000: 1；
After colloid enzymolysis completes, through enzyme denaturing, filtration, nanofiltration, sterilizing, it is spray-dried and wraps
Dress i.e. obtains extracellular matrix protein peptide finished product.
Gross protein extraction ratio is 95%；In the extracellular matrix protein peptide extracted, type i collagen
Content is 60%, IV collagen content is 30%, glycoprotein (elastin laminin)+Dan Baiduotang proteoglycan PG (with
Glucosamine meter) content is 3.0%, L-hydroxyproline (precollagen) content 13%；Carefully
The mean molecule quantity of extracellular matrix protein peptide is 1300 dalton.
Embodiment 3
The extracting method of a kind of extracellular matrix protein peptide, comprises the following steps:
After Ungula Bovis seu Bubali muscle roguing, put into DS-1 high-speed tissue mashing machine and blend, the cattle after crushing
The animal limb tendons immerse in the dilute hydrochloric acid that mass percent is 15%, and dilute hydrochloric acid with the weight ratio of Ungula Bovis seu Bubali muscle is
1:1, soaks 60min；
Carry out after pickling extruding weary acid, be then placed in the lime water that mass percent is 15%,
Lime water is 1:1 with the weight ratio of Ungula Bovis seu Bubali muscle, and the time of caustic dip is 60min, after caustic dip completes
Extrude weary alkali；
Ungula Bovis seu Bubali muscle acid adding after weary alkali is neutralized, and removes sewage, add pure water after neutralizing reaction
Putting into FJ-200 high speed dispersion homogenizer and carry out homogenizing, Ungula Bovis seu Bubali muscle is 1 with the weight ratio of pure water:
2.5；
After homogenizing under agitation, at temperature 75 DEG C, 5h is reacted, then through ionic adsorption
Pressing plate filters, and is then concentrated to give colloid；
In 73 DEG C, colloid being carried out colloidal sol, adds proteolytic enzyme after colloidal sol, regulation temperature is
51 DEG C, regulation pH is 9.0, reacts 2h；Wherein, colloid and the weight ratio of proteolytic enzyme
It is 8000: 1；
After colloid enzymolysis completes, through enzyme denaturing, filtration, nanofiltration, sterilizing, it is spray-dried and wraps
Dress i.e. obtains extracellular matrix protein peptide finished product.
Gross protein extraction ratio is 94%；In the extracellular matrix protein peptide extracted, type i collagen
Content is 60%, IV collagen content is 31%, glycoprotein (elastin laminin)+Dan Baiduotang proteoglycan PG (with
Glucosamine meter) content is 3.0%, L-hydroxyproline (precollagen) content 14%；Carefully
The mean molecule quantity of extracellular matrix protein peptide is 1200 dalton.
Embodiment 4
The extracting method of a kind of extracellular matrix protein peptide, comprises the following steps:
After Ungula Bovis seu Bubali muscle roguing, put into DS-1 high-speed tissue mashing machine and blend, the cattle after crushing
The animal limb tendons immerse in the dilute hydrochloric acid that mass percent is 15%, and dilute hydrochloric acid with the weight ratio of Ungula Bovis seu Bubali muscle is
1:2, soaks 60min；
Carry out after pickling extruding weary acid, be then placed in the lime water that mass percent is 15%,
Lime water is 1:2 with the weight ratio of Ungula Bovis seu Bubali muscle, and the time of caustic dip is 60min, after caustic dip completes
Extrude weary alkali；
Ungula Bovis seu Bubali muscle acid adding after weary alkali is neutralized, and removes sewage, add pure water after neutralizing reaction
Putting into FJ-200 high speed dispersion homogenizer and carry out homogenizing, Ungula Bovis seu Bubali muscle is 1 with the weight ratio of pure water:
3；
After homogenizing under agitation, at temperature 76 DEG C, 5h is reacted, then through ionic adsorption
Pressing plate filters, and is then concentrated to give colloid；
In 74 DEG C, colloid being carried out colloidal sol, adds proteolytic enzyme after colloidal sol, regulation temperature is
52 DEG C, regulation pH is 9.0, reacts 2h；Wherein, colloid and the weight ratio of proteolytic enzyme
It is 7000: 1；
After colloid enzymolysis completes, through enzyme denaturing, filtration, nanofiltration, sterilizing, it is spray-dried and wraps
Dress i.e. obtains extracellular matrix protein peptide finished product.
Gross protein extraction ratio is 95%；In the extracellular matrix protein peptide extracted, type i collagen
Content is 59%, IV collagen content is 30%, glycoprotein (elastin laminin)+Dan Baiduotang proteoglycan PG (with
Glucosamine meter) content is 3.0%, L-hydroxyproline (precollagen) content 14%；Carefully
The mean molecule quantity of extracellular matrix protein peptide is 1300 dalton.
Experimental example 1
Nonsmall-cell lung cancer (Non-Small Cell Lung Cancer, NSCLC) be clinic
Common malignant tumor, Most patients is already belonging to late period when clinical diagnosis, loses operation and controls
Treat chance, after the failure of platiniferous class medication combined chemotherapy regimen, then lack effective Therapeutic Method.
Therefore find effective Therapeutic Method and become the focus of NSCLC research.Through basic research table
Bright, the extracellular matrix protein peptide that the present invention provides is to NSCLC growth and various malignant entity
Tumor has good inhibitory action.NSCLC clinical efficacy may be improved, carried out for this with
Lower experiment.
1. material
1.1 animals and cell line
BALB/C-nu nude mice 40,6～8 week old, SPF level, male and female half and half, by China
Laboratory animal room of tumour hospital of Academy of Medical Sciences institute of oncology breeds and provides.Animal productiong closes
Lattice card number: capital is dynamic is betrothed to (2013) No. 016；The animal quality certification number: SCXK capital 2007
-0005, raise in animal experimental center shielding harness is aided with clean Streamline cabinet environment.People's lung
Adenocarcinoma (A549) cell line, conventional by inspection center of Tumour Inst., Chinese Medical Academy
Preserve and provide.
1.2 medicine
The extracellular matrix protein peptide that the embodiment of the present invention prepares.
1.3 instrument
The grand auspicious purification Science and Technology Ltd. in superclean bench system Suzhou produces (model SCW-CJ)；
Experiment centrifuge system Jiangsu Heng Rui pharmaceutical machine company limited's production (model LS150)；It is inverted
NIKON Co., Ltd. of Japan of microscope system produces (model TS100)；China of slide gauge system
Hangzhou Tool and Measuring Meas Co., Ltd produces (model HGL-18-1)；Ao Haosi precise electronic balance
It is U.S.'s OHAUS Products (model ARA520)；Forma constant incubator is beautiful
State's Forma Products (model 3110).
2. method
2.1 cells are cultivated and inoculation
People source portability adenocarcinoma of lung (A549) cell line DMEM containing 10% calf serum
Culture fluid is cultivated, and cell use 0.25% trypsinization after covering with monolayer, culture fluid blows and beats,
Centrifugal, to collect cell PSB and dilute, concentration is adjusted to 1 × 107Individual cell/ml, every mice
It is inoculated in right axil with 0.2ml subcutaneous, treats that tumor length is about 1000mm to volume3Shi Yizhi.
2.2 modelings and packet
Growth selection is good, tumor ties the tumor bearing nude mice without ulceration, breaks cervical vertebra and puts to death, and is purifying
In workbench, completely strip tumor knot under aseptic condition, wash away bloodstain with normal saline, cut off tumor
Knot, removes both central necrotic tissue, and abundant homogenate is prepared tumor cell suspension, added normal saline dilution
Cell number is to 1 × 107Individual cell/mL.Only take 0.2ml/, be inoculated in the right axillary fossa of nude mice subcutaneous,
Complete in being seeded in 30min.After 10 days, gross tumor volume is about at 100-300mm3Between (16
Nude mice modeling all successes) time, it is layered according to sex, is randomly divided into by gross tumor volume size
Tumor model group and test group, often group 8, label of weighing.
2.3 dosages and method
Dosage all uses people to determine with animal dose lonvestion method.1. tumor model group, physiology
Saline 0.2ml/10g every day gavage；2. test group includes test group 1-4, and correspondence is edible respectively
The extracellular matrix protein peptide that embodiment 1-4 prepares, in each test group, extracellular matrix egg
White peptide is pressed 7.875g/kg/d (conversion of adult treatment amount 63g/70kg/d) and is administered, normal saline
Configuration concentration is 5.625mg/ml, every day 0.2ml/10g gavage；It is grouped and started the same day to be administered,
Once a day, continuous 14 days.
2.4 computational methods
Forward and backward every 4 days of drug treating millimeter vernier caliper measurement tumor length and width maximum perpendicular
Diameter.It is calculated as follows gross tumor volume: gross tumor volume (mm3)=tumor major diameter × swollen
Tumor minor axis 2/2.Medication terminates to put to death nude mice in latter 2 days, takes out tumor, weighs.By following public
Formula calculating tumor control rate: tumour inhibiting rate (%)=(matched group average tumor average tumor of weight-treatment group
Weight)/matched group average tumor weight × 100%.
2.5 statistical method
SPSS13.0 statistical software is used to carry out data process.Mouse Weight before and after therapeutic intervention
And gross tumor volume is with sx ± represent, and use t to check, between group, compare employing F inspection, when
P < is considered statistically significant when 0.05；Tumor control rate represents with percentage rate.
3. result
3.1 General observation
Before experiment, each group nude mice general status is good, and at the end of experiment, 40 nude mices all survive.
Dynamically it has been observed that model control group nude mice is looked for food, drinks water normally, but movable minimizing, reaction
More blunt；Test group nude mice looks for food, drinks water, movable etc. in order.
3.2 gross tumor volume changes
Starting to experiment to terminate from packet medication, transplanted tumor change in volume is shown in Table 1.
The dynamically change (sx ±) of each group of gross tumor volume tested by table 1
With tumor model group than P ＜ 0.01
It can be seen in table 1 that from the beginning of the 4th day, test group gross tumor volume compared with model group,
Statistically significant (P ＜ 0.01), and there is obvious inhibitory action.
3.2 tumor control rate
Different group tumor weights and calculated tumor control rate are as shown in table 2.
Table 2 tests each group of tumor weight and tumour inhibiting rate (sx ±)
| Packet | N | Tumor weight g | Tumor control rate (%) |
| Tumor model group | 8 | 1.59±0.45 | - |
| Test group 1 | 8 | 0.88±0.33 | 44.65 |
| Test group 2 | 8 | 0.90±0.34 | 43.40 |
| Test group 3 | 8 | 0.89±0.32 | 44.02 |
| Test group 4 | 8 | 0.88±0.28 | 44.65 |
With model group than P ＜ 0.01.
When extracellular matrix protein peptide is used alone, inhibitory rate is to more than 40%, with model group
Comparing, statistically significant (P ＜ 0.01), explanation extracellular matrix protein peptide is to A549 simultaneously
Growth has obvious inhibition.
4. peplos evaluation under biopsy mirror
The tumor of different groups is carried out biopsy observation, result such as Fig. 2-Fig. 5 under microscope
Shown in.Fig. 2 is the biopsy microgram that the tumor tissues in tumor model group invades peplos；Fig. 3
For the borderline tumor rule in test group, the biopsy microgram that peplos is complete；Fig. 4 is tumor mould
Tumor tissues in type group invades the biopsy microgram of periphery fibrous connective tissue；Fig. 5 is test
Tumor boundary clear in group has the biopsy microgram of complete peplos.
In different tests group, the biopsy microgram result of tumor is consistent.From Fig. 2-5 it can be seen that
In extracellular matrix protein peptide intervention group, borderline tumor rule, tumor boundary clear, have complete
Peplos；And the tumor tissues in matched group invades peplos and infiltration periphery fibrous connective tissue.
Illustrate extracellular matrix protein peptide that the present invention provides to human lung adenocarcinoma growth inhibited effect, for entering
One step further investigation extracellular matrix protein peptide antitumor and reconstruction tumor stroma barrier provide and depend on
According to.
Nonsmall-cell lung cancer belongs to common malignant tumor occurred frequently, poor to Concurrent Chemoradiotherapy Sensitivity, is mesh
The emphasis of front research.The extracellular matrix protein peptide that the present invention provides may be by rebuilding tumor base
Matter barrier, it is suppressed that the propagation of cancerous cell, the invasive ability such as divide a word with a hyphen at the end of a line, lowers VEGF, rise
The expression of TIMP-2, the generation of suppression tumor-microvessel, reduces tumor microvessel density；Shadow
Ring the propagation of endotheliocyte, reduce the blood supply of tumor cell.
This test example by people source portability adenocarcinoma of lung (A549) cell line after Secondary Culture,
Carry out tumour transplatation with BALB/c-nu Mus, and intervene with extracellular matrix protein peptide
Property treatment, evaluate alone extracellular matrix protein peptide by observing peplos under tumour inhibiting rate and biopsy mirror
To A549 growth of xenografted inhibitory action.Laboratory nude mice model test find, treatment group with
Matched group compares, and Subcutaneous Tumor Growth is the most suppressed, and Lung metastases is suppressed, and can substantially suppress root
Controlling the relapse and metastasis after excision, pathology shows that tumor capsule is complete, smooth.Tumor vessel is obvious
Suppressed, apoptosis is the most more.Visible, that the present invention provides extracellular matrix protein peptide tool
There are growth and the effect of transfer of well suppression cancer.
Experimental example 2
The extracellular matrix protein peptide finished product that embodiment 1-4 prepares can directly eat, it is possible to logical
Cross and add pharmaceutically acceptable adjuvant and be processed into other drug dosage form, as injection, injection,
Tablet, electuary, granule, pill, capsule, suspending agent, Emulsion, unguentum, cream, thoroughly
Skin patch etc..
In this experimental example, face with the extracellular matrix protein peptide finished product that embodiment 1-4 prepares
The checking of bed food therapy effect.
1. case inclusion criteria: be 354 example cancers in April, 2008 in March, 2014
Patient has carried out extracellular matrix protein peptide dietetic therapy, each 10g, every day 5-6 time；And by this
A little cases are added up.
2. clinical data: follow-up rate reaches 97.7%, follow-up time 47.7 months (25.0～
83.9 months).Wherein male accounts for 82.8% (293 example)；Women accounts for 17.2% (61 example),
49.3 years old mean age (14～71 years old).
3. group technology: all cases are divided into six groups:
It was divided into for three phases according to Tumor size:
1. in early days, lump diameter ＜ 3 centimetres；
2. mid-term, lump diameter ＞ 3 centimetres, occur or be likely to occur lymphatic metastasis；
3. late period, lump diameter ＞ 3 centimetres, lymph node and other organ metastasis have occurred；
According to treating situation in the past, it is divided into two classes for each issue:
1. without operation, radiotherapy, chemotherapy person, referred to as A.
2. through the therapist of one of operation, radiotherapy, chemotherapy, referred to as B.
Statistics survival rate, result is as shown in table 3.
Table 3 survival rate
| Clinical statistics | Number of cases | 1 year | 3 years | 5 years |
| A in early days | 53 | 100% | 100% | 100% |
| B in early days | 90 | 100% | 97.7% | 94.4% |
| Mid-term A | 62 | 100% | 96.8% | 80.6% |
| Mid-term B | 114 | 96.5% | 85% | 74.5% |
| Late period A | 26 | 92.3% | 68.8% | 53.8% |
| Late period B | 9 | 66.7% | 22.2% | 11.1% |
| Total case | 354 | 97.5% | 89.8% | 87.3% |
From table 3 it can be seen that the food therapy effect of early stage patient, it is better than the cancer in mid-term, late period
Patient；No matter early stage, mid-term, the cancer patient in late period, without operation, radiotherapy, chemotherapy
Person, uses the effect of extracellular matrix protein peptide, is better than through operation, radiotherapy, patients undergoing chemotherapy.
Concrete typical clinical example:
Adenocarcinoma of lung companion's patients with pleural
Women, 34 years old.Patient is chest pain on the right side of occurring without obvious inducement in July, 2011,
In persistent ache, tight without heating night sweat and gas uncomfortable in chest, without dizzy headache, without nausea and vomiting and
He is uncomfortable.Once local hospital treatment (concrete treatment is not quite clear), symptom is without being clearly better.In
20011-7-9 to 2011-7-13 carries out breast CT and shows right pleural effusion.Examination of hydrothorax shows:
See adenocarcinoma cell.In proceeding to 2011-7-15 day in certain tumour hospital's row thoracic puncture drainage and row
One journey chemotherapy, chemotherapy regimen is: pemetrexed+cisplatin；Chemotherapy process is smooth.In 2011-8-11
Carrying out the second journey chemotherapy, improve coherence check after being admitted to hospital, breast CT is shown: superior lobe of right lung is shown in one
Irregular moderate strengthening shadow, a large amount of hydrops in thoracic cavity, right side, lymph node of lesser curvature of stomach enlargement；Row thoracic cavity
Tapping and to the ill Supporting Therapy, give gemcitabine+cisplatin row two journey chemotherapy, suffers from
Person's a good appetite suddenly appearing in a serious disease degree anorexia and tired outside, other symptoms take a turn for the better, leave hospital in 2011-8-25.
There is again rest pain and involve sense, to local county hospital breast in two weeks remaining right breasts of leaving hospital
Chamber X-ray is swept and is shown: a large amount of hydrops in thoracic cavity, right side.Then the 3rd journey chemotherapy is abandoned, in 2011-9-18
The extracellular matrix protein peptide that starting the edible present invention provides carries out dietetic therapy and controls hydrothorax and the state of an illness.
Owing to patient is tired, become thin, anorexia, cough, alopecia, right breast rest pain involve sense
Substantially, carry out in the following ways: one, with fresh oral ascending stomaches of squeezing the juice such as Fructus Mali pumilae+white turnips
Gas, strengthens digestive and absorptive functions, activates phagocyte activity；Two, 60 gram bases are administered orally every day
The extracellular matrix protein peptide that invention provides rebuilds substrate barrier (ECM, the ring of encirclement), repairs
The pleura of invasion and attack ulceration and tuberosity；Three, drink the coarse grain such as milled congee, do not eat afternoon, keep gasification
And absorption；Four: drink Cyprinus carpio steamed beef soup and strengthen nutrition；Five: strictly observe dietetic contraindication etc..20
After Yu Tian, symptom takes a turn for the better.CT examination is carried out in 2011-11-28 after adhering to about 70 days；Fig. 6
CT for 2011-8-11 schemes；Fig. 7 is the CT figure of 2011-11-28, and both understand in contrast:
Patient's superior lobe of right lung tile shadow reduces earlier above, shallow, mediastinum window size about 2.5*1.4cm, its
Peripheral branch tracheaectasy, and see that patch shape obscures shadow distribution.Superior lobe of right lung apical segment mediastinum is other visible
One lesser tubercle, the most slightly reduces.The a little streak shadow of middle lobe of right lung, border is clear.Double lung marking
Thick random, the bright degree of lung increases.Double hilus pulumonis have no increase.Enlarged lymph node is had no in vertical diaphragm.Right
Side hydrothorax absorbs, and thoracic cavity, left side has no hydrops.Achieve the phenomenon that significantly takes a turn for the better.
The cell of pulmonary carcinoma is divided into two large-scale: small cell lung cancer and nonsmall-cell lung cancer, non-small cell
Pulmonary carcinoma (NSCLC) can be divided into again the hypotypes such as scale cancer, adenocarcinoma, maxicell.Minicell lung
The feature of cancer is common young males, and growth is fast, and once, chemicotherapy is very in multiplication in 18 days
Sensitivity, drug withdrawal is easily recurred, and is very easy to brain metastes, and chemotherapy must be accomplished more than 12 times and heal
After the best.It is unfortunate for obtaining pulmonary carcinoma, but obtaining adenocarcinoma of lung is the misfortune in misfortune, adenocarcinoma of lung
Feature be common women and the male not smoked, little adenocarcinoma, shift greatly, transfer early, recurrence
Hurry up, brain metastes, Bone tumour, it is very easy to that hydrothorax occurs, chemicotherapy is insensitive, poor prognosis,
Mortality rate is high.
This patient tapping pathological diagnosis is adenocarcinoma of lung, the existing substantial amounts of carcinous breast of pleura metastasis
Water.Twice tapping twice chemotherapy, and a large amount of hydrothorax fail to control that (chemotherapy is unwise repeatedly
Sense), and occur that pulmonary parenchyma damages (chronic bronchitis, emphysema sign, middle lobe of right lung
A few fibres stove etc.) side effect.Clinic shows the extracellular matrix protein peptide pair that the present invention provides
The substrate barrier of malignant solid tumor rebuilds (parcel is repaired), and mass reduction, as benign tumor one
Sample the smooth of the edge, clear-cut, significant to symptom management progress.
Pathology shows, when cancer occurs, the positive expression of hole wall collagen and laminin is obvious
Strengthen, and lack in lamellar in various degree or disappear, the brightest in poor differentiated carcinoma tissue
Aobvious.The fracture of basement membrane, lack and reduce and destroy the integrity as barrier so that tumor
Cell is prone to pass through the circulation of basement membrane barrier intravasation and attack other organs.The present invention provides
Extracellular matrix protein peptide after cancer patient takes, repair basement membrane in vivo, parcel is swollen
Tumor, and DNA plerosis damage, reach to limit tumor migration and the purpose of propagation, and
Can progressively there is apoptosis in tumor cell.
Although illustrate and describing the present invention with specific embodiment, but it will be appreciated that
May be made that without departing from the spirit and scope of the present invention many other change and
Amendment.It is, therefore, intended that include in the following claims belonging in the scope of the invention
All such changes and modifications.
Claims (10)
1. the extracting method of an extracellular matrix protein peptide, it is characterised in that include with
Lower step:
Acid treatment and alkali process is carried out after broken for Ungula Bovis seu Bubali muscle；
Remove sewage after neutralized reaction, add water and carry out homogenizing, then in more than 70 DEG C heating
Boil, obtain colloid；
Described colloid is carried out enzymolysis, after enzyme denaturing, i.e. can get extracellular matrix protein peptide.
Extracting method the most according to claim 1, it is characterised in that described broken
It is by after Ungula Bovis seu Bubali muscle roguing, puts into high-speed tissue mashing machine and blend.
Extracting method the most according to claim 1, it is characterised in that at described acid
Reason includes pickling and weary acid；
Described alkali processes and includes caustic dip and weary alkali.
Extracting method the most according to claim 3, it is characterised in that described pickling
The dilute hydrochloric acid using mass percent to be 10%～20%；
The lime water that described caustic dip uses mass percent to be 10%～20%.
Extracting method the most according to claim 4, it is characterised in that in described leaching
During acid, the weight ratio of described dilute hydrochloric acid and Ungula Bovis seu Bubali muscle is 1:1～2, described pickling time
Between be preferably 50-70min；
During described caustic dip, described lime water is 1:1～2 with the weight ratio of Ungula Bovis seu Bubali muscle, institute
The time stating caustic dip is preferably 50-70min.
Extracting method the most according to claim 1, it is characterised in that described homogenizing
For: it is 1: 2-3 to put at a high speed point by weight through neutralizing reacted Ungula Bovis seu Bubali muscle and pure water
Scattered homogenizer carries out homogenizing；
Described more than 70 DEG C heating boil into: under stirring condition, at temperature 72 DEG C～80 DEG C
Reaction 5～6h, is then passed through filter and is concentrated to give described colloid；
Preferably, described filtration uses ionic adsorption pressing plate to carry out.
Extracting method the most according to claim 1, it is characterised in that before enzymolysis first
Described colloid is carried out colloidal sol in 72-75 DEG C.
Extracting method the most according to claim 1, it is characterised in that described enzymolysis
For: adding proteolytic enzyme after colloidal sol, regulation temperature is 50～52 DEG C, and regulation pH is
8.7～9.2, react 2～3h；Wherein, described colloid with the weight ratio of described proteolytic enzyme is
5000: 1～10000: 1；
Preferably, also include filtration, nanofiltration, sterilizing after enzyme denaturing, be spray-dried and pack i.e.
Available extracellular matrix protein peptide finished product.
9. the extracellular matrix egg that the extracting method described in any one of claim 1-8 prepares
White peptide.
10. the extracellular matrix egg that the extracting method described in any one of claim 1-8 prepares
The application in preparation treatment cancer drug of the white peptide.
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| US20210198713A1 (en) * | 2017-10-11 | 2021-07-01 | Shuang Liu | Method for preparing protein peptide based on connective tissue and prepared protein peptide and use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20210198713A1 (en) * | 2017-10-11 | 2021-07-01 | Shuang Liu | Method for preparing protein peptide based on connective tissue and prepared protein peptide and use thereof |
| US11884953B2 (en) * | 2017-10-11 | 2024-01-30 | Shuang Liu | Method for preparing protein peptide based on connective tissue and prepared protein peptide and use thereof |
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