CN105418741A - Method for separating and purifying leech polypeptide - Google Patents
Method for separating and purifying leech polypeptide Download PDFInfo
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Abstract
The invention discloses a method for separating and purifying leech polypeptide. The method comprises the following steps: adding a leech extract into a system capable of simulating human body enterocyte absorption for culturing; performing chromatographic separation on absorbed substances. By adopting the method, the problem of difficulty in separating and purifying the leech polypeptide is solved from different aspects. The leech extract is purified by Caco-2 cells firstly to simulate the digestive absorption process of leech serving as traditional Chinese medicine in a human body, and a large quantity of polypeptides which cannot be absorbed by human body enterocytes are separated out by simulation of the absorption functions of the enterocytes, so that a separating-purifying process is simplified. Then, polypeptides which can be absorbed by the human body can be separated and purified through anion exchange chromatography and gel filtration chromatography. The polypeptides obtained by separation and purification according to the method have the effect of restraining migration of vascular smooth muscle cells.
Description
Technical field
The invention belongs to field of medicaments, be specifically related to a kind of separation purification method of leech polypeptide.
Background technology
Atherosclerosis be due to lipid bulk deposition and the ductus arteriosus wall caused in arterial blood tube wall thicken, hardening, and then artery is followed the string, luminal stenosis.It is a kind of chronic inflammatory disease jointly participated in by various kinds of cell and cytokine.Along with deepening continuously to atherosclerotic lesion process study, the inflammatory factors such as the interaction of the interaction between atherosclerosis relevant cell and cytokine and cell are more and more studied.Participate in atherosclerotic cell mainly to comprise: vascular endothelial cell, vascular smooth muscle cell and immunocyte, wherein immunocyte is mainly scavenger cell, also has neutrophil leucocyte, mastocyte, dendritic cell and T cell etc. in addition.
The propagation of vascular smooth muscle cell (vascularsmoothmusclecell, VSMCs), move also play critical effect in the forming process of atherosclerotic lesion.VSMCs is the important composition composition of vessel wall, is mainly positioned at tunica media, under physiological condition, keeps balance, and can make its proliferation out of control under many pathological states, thus cause a series of changes of vessel wall between the proliferation and apoptosis of VSMCs.Only there is a small amount of VSMCs in tunica intima, its Main Function be by diastole and shrink change blood vessel diameter, make the blood stream pressure that blood vessel keeps suitable, VSMCs now shows as shrinkage type, its propagation and synthesis capability very low.Under the effect of multiple pathological factor, the phenotype of VSMCs can be converted into synthesis type by shrinkage type.The VSMCs of synthesis type can synthesize and secrete a large amount of extracellular matrix, thus changes pericellular microenvironment, and extracellular matrix can affect VSMCs propagation, stick and transition process.Inflammatory response can stimulate VSMCs to move, breed, and interacts with the inflammatory component in inflammatory lesion, and arterial wall is thickened.Monocytic accumulation, VSMCs propagation and fibrous tissue are formed and cause pathology to expand the fibrous cap of laying equal stress on and consisting of and covering on lipid core and necrotic tissue further, finally cause the formation of the compound pathology of patch.Therefore, VSMCs is also one of key cells promoting atherosclerotic process.
Leech (Leech) is used for the treatment of various diseases as traditional Chinese medicine, how on the books in Ancient Times in China medical book.Such as, call in Shennong's Herbal its " main by extravesated blood, hemostasis, close by the moon, broken blood disappears and gathers ... ".Leech pharmacological action is extensive, comprises anti-freezing, antithrombotic, antitumor, anti-inflammatory, endotheliocyte protection and anti-fibrosis etc.In cardiovascular and cerebrovascular diseases clinical treatment, Chinese traditional medicine leech and be widely used in the control of the diseases such as the disease such as coronary heart diseases and angina pectoris, Acute Myocardial Infarction, ischemia apoplexy (as cerebral thrombosis, cerebral infarction and sequela thereof), hypertension, hyperlipidemia, cerebral atherosclerosis, the sclerosis of artery congee and peripheral angiopathy containing the Chinese patent medicine of leech or compound preparation.Fundamental research in recent years about leech control cardiovascular and cerebrovascular diseases deepens continuously.The domestic report of the research to leech pharmacological action mainly comprises three aspects: anticoagulation, to the therapeutic action of cardiovascular and cerebrovascular diseases, to the Therapy study of diabetic retinopathy.But due to the complicacy of leech polypeptide and the difficulty of screening active ingredients, there is no the separation purification method of easy active leech polypeptide at present.
Summary of the invention
Object of the present invention is exactly the separation purification method in order to provide a kind of leech polypeptide.
To achieve these goals, the present invention adopts following technical scheme:
A separation purification method for leech polypeptide, first adds Hirudo extract in the system of imitating the absorption of human body intestinal cells and cultivates, then absorbed material is carried out chromatographic separation.
Preferred: the extracting method of Hirudo extract is as follows:
The dry entirety of water intaking leech is ground into 20-150 order powder, adds the water of 6-16 times of weight, or the fresh and alive entirety of leech, adds the water of 1-6 times of weight, homogenate; Get homogenate, regulate pH to 7-9, the ratio adding 2000-20000U enzyme in the dry medicinal material of every 1g leech adds trypsinase, control temperature 40-60 DEG C, and enzymolysis 2-12 hour, is cooled to room temperature; After enzymolysis is complete, in above-mentioned enzymolysis solution, add acetone or the ethanol refrigeration precipitation 4-12 hour of 1-5 times of volume, filter, filtrate decompression is dry, obtains Hirudo extract.
Preferred: the system of imitating the absorption of human body intestinal cells is cover with/have the transwell cell of people's Colon and rectum gland cancer Caco-2 cell.
Preferred: described culture condition is at 37 DEG C, 5% carbonic acid gas, cultivate 0.5-10h (preferred 2h) under saturated humidity condition.
Preferred: before Hirudo extract adds the system of imitating the absorption of human body intestinal cells, use the PBS of pH value 7.2 to be configured to the solution that concentration is 0.5-5mg/ml.
Preferred: chromatographic separation is first through anion exchange chromatography (preferably: anion exchange chromatography medium is QSephrose), concentration is the NaCl solution linear elution of 0.5-2mol/L, collect the activeconstituents at each peak, intravascular smooth muscle cell migration screens, get the activeconstituents at an active the highest peak, concentrated and dry, then will concentrate and dried activeconstituents, dissolve with distilled water, again through gel permeation chromatography (preferably: the filler of described gel permeation chromatography post: separating ranges is the daltonian filler of 100-5000, more preferably: the filler of described gel permeation chromatography post is SephadexLH-20), distilled water wash-out, collect the activeconstituents at each peak, intravascular smooth muscle cell migration screens, get the activeconstituents at an active the highest peak, concentrated and dry, obtain the leech polypeptide after separation and purification.
The leech polypeptide that the present invention also protects above-mentioned separation purification method to obtain, and described leech polypeptide suppresses the application in vascular smooth muscle cells migration medicine in preparation.
Suppress a medicine for vascular smooth muscle cells migration, its main active ingredient is the leech polypeptide that aforesaid method separation and purification obtains.
The application of described leech polypeptide in preparation treatment atherosclerosis medicine.
One treats atherosclerotic medicine, and its main active ingredient is the leech polypeptide that aforesaid method separation and purification obtains.
Beneficial effect:
Hirudo extract is when using chromatography method to carry out separation and purification, mix because extract contains the large and kind of the amount of polypeptide, the net charge of ion between polypeptide, the electrical property difference difference of surface charge distribution is less, so when using the methods such as conventional chromatography to be directly separated, difficulty is larger, and be separated the polypeptide obtained possibly cannot be absorbed by the body, existing separation purification method is all generally the separation and purification realizing polypeptide by changing the kind of chromatography column or improving layer analysis method, the present inventor solves the problem of the separation and purification difficulty of leech polypeptide from different angles, purifying is carried out by first Hirudo extract being adopted Caco-2 cell, imitate Chinese traditional medicine leech absorption process in vivo, through the sorption of simulation intestinal cell, first a large amount of can not be separated by the polypeptide of human intestinal Cell uptake, simplify separation and purification process, and then through anion exchange chromatography and gel permeation chromatography, namely separation and purification the active leech polypeptide utilized that can be absorbed by the body can be obtained in conjunction with vascular smooth muscle migration screening model.The active leech polypeptide obtained by method separation and purification of the present invention has the effect suppressing vascular smooth muscle cells migration.
Accompanying drawing explanation
Fig. 1: transwell cell explanation;
Fig. 2: QSephrose anion-exchange chromatography post is to the separation and purification result of room leech polypeptide under transwell cell;
Fig. 3: SephadexLH-20 gel permeation chromatography is to the separating resulting of leech polypeptide;
Fig. 4: do not adopt Caco-2 cell purification Hirudo extract through QSephrose anion-exchange chromatography post separating resulting;
Fig. 5: leech polypeptide (HEE) is on the impact of vascular smooth muscle cells migration ability, and X-coordinate is dosing grouping, and LPS (+) is 1.0 μ g/ml, Simvastatin (SIM) (+) is 10 μMs of i.e. 4.1856 μ g/ml; Ordinate zou is vascular smooth muscle cells migration number;
#p < 0.05vs blank,
##p < 0.01vs blank,
*p < 0.05vs modeling group,
*p < 0.01vs modeling group,
△p < 0.05vs Simvastatin group,
△ △p < 0.01vs Simvastatin group; Bar is 50 μm;
Fig. 6: active leech polypeptide (HEE) is on the impact of vascular smooth muscle cell inflammatory factor, chemokine, adhesion molecule protein expression level, X-coordinate is dosing grouping, LPS (+) is 1.0 μ g/ml, and Simvastatin (SIM) (+) is 10 μMs of i.e. 4.1856 μ g/ml; Ordinate zou is vascular smooth muscle cells migration number;
#p < 0.05vs blank,
##p < 0.01vs blank,
*p < 0.05vs modeling group,
*p < 0.01vs modeling group,
△p < 0.05vs Simvastatin group,
△ △p < 0.01vs Simvastatin group;
Fig. 7: each component is to vascular smooth muscle transporting action result after QSephrose anion-exchange column is separated;
Fig. 8: each component is to vascular smooth muscle transporting action result after LH-20 gel column is separated.
Embodiment
Below in conjunction with accompanying drawing and embodiment, the invention will be further described.
Embodiment 1: the extraction of Hirudo extract
The dry all 1kg of water intaking leech are ground into 60 order powder, add 10kg distilled water water, homogenate; Get homogenate, regulate pH to 8.5, the ratio adding 10000U enzyme in the dry medicinal material of every 1g leech adds trypsinase, control temperature 50 DEG C, and enzymolysis 6h, is cooled to room temperature; After enzymolysis is complete, in above-mentioned enzymolysis solution, add the ethanol refrigeration precipitation 8h of 4 times of volumes, filter, filtrate decompression is dry, obtains Hirudo extract 100g.(or using the method for ZL200810138936.5 to obtain Hirudo extract)
Embodiment 2: the separation and purification of leech polypeptide
People's Colon and rectum gland cancer Caco-2 is carefully inoculated in transwell cell, and adopt DMEM substratum, containing 20%FBS, 100U/mL penicillin, 100 μ g/mL Streptomycin sulphates, 2mmol/LL-glutamine, culture condition is 5%CO
2, 37 DEG C, inoculum density is 450,000 cells/well, changes weekly liquid 3 times, cultured continuously two weeks.The integrity of Caco-2 cellular layer detects TEER value by cell resistance instrument and determines, TEER value is greater than 500 Ω/cm
2can be used for experimental study.
The Hirudo extract extracted in embodiment 1 is dissolved in PBS (pH value 7.2) damping fluid, is mixed with the Hirudo extract solution that concentration is 1mg/mL.Before experiment starts, remove substratum by transwell cell, cultivate cell twice with PBS cleaning.Each upper indoor add 0.3mL Hirudo extract solution, and lower room adds 1.2mLPBS damping fluid.After effect 2h, PBS solution in lower room is all collected, for subsequent use.
By above-mentioned lower room solution through QSephrose anion exchange chromatography, 2mol/LNaCl linear wash-out, collect the activeconstituents at each peak, intravascular smooth muscle cell migration screens, and gets the activeconstituents at an active the highest peak, obtains four peaks in this experiment, the activity at the 3rd peak is the highest (refers to accompanying drawing 2, the 3rd from left to right), collect active ingredient herein, concentrated and dry;
Above-mentioned the 3rd peak component distilled water is dissolved, through LH-20 gel permeation chromatography, distilled water wash-out, collects the activeconstituents at each peak, intravascular smooth muscle cell migration screens, get the activeconstituents at an active the highest peak, obtain two peaks in this experiment, get first peak (see accompanying drawing 3, first from left to right), collect active ingredient herein, concentrated and dry, obtain HIRULOG.
Embodiment 3: do not adopt Caco-2 cell purification Hirudo extract through QSephrose anion-exchange chromatography column separating purification
The Hirudo extract extracted in embodiment 1 is dissolved in PBS damping fluid, is mixed with the Hirudo extract solution that concentration is 1mg/mL.Through QSephroseFastFlow anion exchange chromatography, 2mol/LNaCl linear wash-out, elution curve is shown in accompanying drawing 4: do not adopt Caco-2 cell purification Hirudo extract through QSephroseFastFlow anion-exchange chromatography post separating resulting.
Contrast as can be seen from accompanying drawing 4 and accompanying drawing 2, after Caco-2 cell purification, the separated portion of Hirudo extract obviously reduces, and is conducive to later stage separation and purification.
Embodiment 4: active leech polypeptide suppresses the Effect study of vascular smooth muscle migration
1. with containing 0.4%FBS (foetal calf serum) DMEM substratum gradient dilution embodiment 2 in obtain leech polypeptide, concentration gradient is followed successively by 50 μ g/ml, 100 μ g/ml, 200 μ g/ml, 400 μ g/ml;
2. vascular smooth muscle cell trysinization, centrifugal and after counting inoculation 200 μ l on Transwell in room, cell concn 5 × 10
5individual/ml, lower room adds the leech polypeptide of different concns successively, the DMEM substratum of blank 0.4%FBS, and positive controls is Simvastatin group, and 24 porocyte culture plates are placed in incubator 37 DEG C together with cell, 5%CO
2cultivate;
3. cultured continuously absorbs the remaining vascular smooth muscle cell in room after 12 hours; PBS cleans film bottom surface, room cell on Transwell, and methyl alcohol fixes 10min, violet staining 10min;
4., after PBS rinsing, cell is placed in observation on slide glass, takes pictures, ImageJ counts, and the results are shown in accompanying drawing 5.
Leech enzymatic extract affects result as shown in Figure 5 to rat VSMCs transfer ability, X-coordinate is dosing grouping, ordinate zou is vascular smooth muscle migration number, compared with blank, LPS (lipopolysaccharides) model group mobility increases, and Simvastatin group anti-migration effect is very remarkable; HEE (referring to that the inventive method extracts the leech polypeptide obtained) 200 μ g/ml, 400 μ g/ml groups all show very strong anti-migratory activity (P < 0.05) compared with LPS model group, wherein quite (P < 0.05), the anti-migratory activity of HEE400 μ g/ml group and the anti-migratory activity of 200 μ g/ml groups do not have significant difference to the anti-migratory activity of the anti-migratory activity of HEE200 μ g/ml group and the Simvastatin group of 10 μMs.
Embodiment 5: active leech polypeptide is to the influence research of vascular smooth muscle correlation factor
(1) protein sample preparation
1. remove cell culture medium and use PBS rinsing cell; Add 100-150 μ l cell pyrolysis liquid (1:100 adds 100mmol/LPMSF), be placed in cracking 15-20min on ice chest;
2. cell pyrolysis liquid and cell debris are moved in EP pipe, 4 DEG C of centrifugal 10min of 12000r/min;
3. Coomassie brilliant blue G250 measures the concentration of protein in the albumen supernatant liquor of centrifugal segregation precipitation;
4. albumen centrifuged supernatant: 5 × sample-loading buffer=1:4 mixing, boiling water bath 5min;
5. the centrifugal 10min of 12000r/min, obtains supernatant and is desirable proteins sample ,-80 DEG C of preservations;
(2) protein electrophoresis
1. SDS-PAGE gel formula table preparation offset plate is pressed, resolving gel concentration 12%, concentrated gum concentration 5%; Preparation electrophoretic buffer also assembles electrophoresis accessory;
2. the protein sample that albumen Marker and boiling water treating cross is added in loading hole;
3. deposition condition is constant voltage 70V, 30min extremely two-layer glue line of delimitation, and 100V electrophoresis has just been run out of can stop electrophoresis to tetrabromophenol sulfonphthalein;
(3) transferring film
1. prepare transferring film liquid, and activate pvdf membrane with methyl alcohol; The protein electrophoresis band of needs is extracted according to albumen Marker mark;
2. clip is assembled according to " sponge-filter paper-glue-pvdf membrane-filter paper-sponge "; Assembling membrane-transferring device, 100V constant voltage ice bath transferring film 2h;
(4) antibody incubation
1. moved to by film in the confining liquid containing 5% skimmed milk, on shaking table, room temperature is closed 2h or 4 DEG C and is left standstill closed spending the night;
2. containing the TBST solution dilution antibody (primary antibodie, two resists) of 5% skimmed milk;
3. pvdf membrane incubated at room primary antibodie diluent 2h;
4. TBST shaking table rinsing pvdf membrane;
5. the anti-diluent 2h of pvdf membrane incubated at room two;
6. TBST shaking table rinsing pvdf membrane;
(5) chemoluminescence, development, fixing
Dark room operation: luminescence reagent A and B two kinds of reagent equal-volume mixing; Luminescence reagent hatches pvdf membrane; X-ray exposure after development, fixing;
(6) gel images analysis
Carried out scanning or taking pictures by film, ImageJ analyzes optical density value, the results are shown in accompanying drawing 6.
As can be seen from accompanying drawing 6, the impact of HEE on vascular smooth muscle cell inflammatory factor, chemokine, adhesion molecule albumen mark level has concentration dependent, inhibition is all the more obvious with the increase of HEE activity, especially remarkable to the inhibition of MCP-1, ICAM-1, VCAM-1, MCP-1, ICAM-1, VCAM-1 can be down to normal level by HEE400 μ g/ml group substantially, and the protein expression inhibition of HEE400 μ g/ml group to MCP-1 is better than SIM, to the inhibition of ICAM-1, VCAM-1 albumen table slightly not as good as SIM.
The HEE of 400 μ g/ml groups also can reduce the expressing quantity of inflammatory factor TLR4, TNF α, iNOS, and reduction amplitude is respectively 50%, 75%, 33%, SIM and is followed successively by 125%, 135%, 133% to the reduction amplitude of the expression amount of above-mentioned three kinds of albumen.The Be very effective of HEE resisting vascular smooth muscle cell chemotactic factor, adhesion molecule expression, part may be realized by anti-inflammatory action.
Embodiment 6: each component is to the activity research of vascular smooth muscle transporting action after QSephrose anion-exchange column is separated
Through QSephrose anion exchange chromatography, 2mol/LNaCl linear wash-out, collects the activeconstituents at each peak, obtains four peaks (see Fig. 2) in this experiment, behind these four peaks respectively desalination, carries out activity experiment by the method in embodiment 4.
1. by gradient dilution four the peak components respectively of the DMEM substratum containing 0.4%FBS (foetal calf serum), concentration gradient is followed successively by 100 μ g/ml, 200 μ g/ml, 400 μ g/ml;
2. vascular smooth muscle cell trysinization, centrifugal and after counting inoculation 200 μ l on Transwell in room, cell concn 5 × 10
5individual/ml, lower room adds the peak component of different concns successively, the DMEM substratum of blank 0.4%FBS, and positive controls is Simvastatin group, and 24 porocyte culture plates are placed in incubator 37 DEG C together with cell, 5%CO
2cultivate;
3. cultured continuously absorbs the remaining vascular smooth muscle cell in room after 12 hours; PBS cleans film bottom surface, room cell on Transwell, and methyl alcohol fixes 10min, violet staining 10min;
4., after PBS rinsing, cell is placed in observation on slide glass, takes pictures, ImageJ counts, and the results are shown in accompanying drawing 7.
From Fig. 7, we can find out that same concentrations, the 3rd peak activity is the highest from left to right, therefore select the 3rd Peak Activity to proceed to be separated.
Embodiment 7: each component is to the activity research of vascular smooth muscle transporting action after LH-20 gel column is separated
Through LH-20 gel permeation chromatography, distilled water wash-out, collects the activeconstituents at each peak, obtains two peaks, (see Fig. 3) in this experiment, behind these two peaks respectively desalination, carries out activity experiment by the method in embodiment 4.
1. by gradient dilution two the peak components respectively of the DMEM substratum containing 0.4%FBS (foetal calf serum), concentration gradient is followed successively by 100 μ g/ml, 200 μ g/ml, 400 μ g/ml;
2. vascular smooth muscle cell trysinization, centrifugal and after counting inoculation 200 μ l on Transwell in room, cell concn 5 × 10
5individual/ml, lower room adds the peak component of different concns successively, the DMEM substratum of blank 0.4%FBS, and positive controls is Simvastatin group, and 24 porocyte culture plates are placed in incubator 37 DEG C together with cell, 5%CO
2cultivate;
3. cultured continuously absorbs the remaining vascular smooth muscle cell in room after 12 hours; PBS cleans film bottom surface, room cell on Transwell, and methyl alcohol fixes 10min, violet staining 10min;
4., after PBS rinsing, cell is placed in observation on slide glass, takes pictures, ImageJ counts, and the results are shown in accompanying drawing 8.
From Fig. 8, we can find out that same concentrations, first peak activity is the highest from left to right, and this is be separated the active leech polypeptide obtained.
By reference to the accompanying drawings the specific embodiment of the present invention is described although above-mentioned; but not limiting the scope of the invention; one of ordinary skill in the art should be understood that; on the basis of technical scheme of the present invention, those skilled in the art do not need to pay various amendment or distortion that creative work can make still within protection scope of the present invention.
Claims (10)
1. a separation purification method for leech polypeptide, is characterized in that: first added by Hirudo extract in the system of imitating the absorption of human body intestinal cells and cultivate, then absorbed material is carried out chromatographic separation.
2. separation purification method as claimed in claim 1, is characterized in that: the extracting method of Hirudo extract is as follows:
The dry entirety of water intaking leech is ground into 20-150 order powder, adds the water of 6-16 times of weight, or the fresh and alive entirety of leech, adds the water of 1-6 times of weight, homogenate; Get homogenate, regulate pH to 7-9, the ratio adding 2000-20000U enzyme in the dry medicinal material of every 1g leech adds trypsinase, control temperature 40-60 DEG C, and enzymolysis 2-12 hour, is cooled to room temperature; After enzymolysis is complete, in above-mentioned enzymolysis solution, add acetone or the ethanol refrigeration precipitation 4-12 hour of 1-5 times of volume, filter, filtrate decompression is dry, obtains Hirudo extract.
3. separation purification method as claimed in claim 1, is characterized in that: the system that described imitation human body intestinal cells absorbs is the transwell cell or cover with people's Colon and rectum gland cancer Caco-2 cell.
4. separation purification method as claimed in claim 1, is characterized in that: described culture condition is that 5% carbonic acid gas, cultivates 0.5-10h under saturated humidity condition at 37 DEG C.
5. separation purification method as claimed in claim 1, is characterized in that: before described Hirudo extract adds the system of imitating the absorption of human body intestinal cells, uses the PBS of pH value 7.2 to be configured to the solution that concentration is 0.5-5mg/ml.
6. separation purification method as claimed in claim 1, it is characterized in that: described chromatographic separation is first through anion exchange chromatography, concentration is the NaCl solution linear elution of 0.5-2mol/L, collect the activeconstituents at each peak, intravascular smooth muscle cell migration screens, get the activeconstituents at an active the highest peak, concentrated and dry, then will concentrate and dried activeconstituents, dissolve with distilled water, again through gel permeation chromatography, the filler of described gel permeation chromatography post: separating ranges is the daltonian filler of 100-5000, distilled water wash-out, collect the activeconstituents at each peak, intravascular smooth muscle cell migration screens, get the activeconstituents at an active the highest peak, collect active ingredient herein, .
7. the leech polypeptide that obtains of separation purification method separation and purification according to claim 6.
8. leech polypeptide according to claim 7 suppresses the application in vascular smooth muscle cells migration medicine in preparation.
9. suppress a medicine for vascular smooth muscle cells migration, it is characterized in that: main active ingredient is leech polypeptide according to claim 7.
10. the application of leech polypeptide according to claim 7 in preparation treatment atherosclerosis medicine.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110066318A (en) * | 2019-05-13 | 2019-07-30 | 山东大学 | A kind of hirudin and its application |
| CN113583092A (en) * | 2021-09-27 | 2021-11-02 | 渤海水产股份有限公司 | Leech peptide and application and product thereof |
| CN113621565A (en) * | 2021-10-08 | 2021-11-09 | 渤海水产股份有限公司 | Application of polypeptide in regulating and controlling fibroblast in vitro culture |
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| CN101332211A (en) * | 2008-08-06 | 2008-12-31 | 山东大学 | Leech extract and preparation method and use thereof |
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| CN101332211A (en) * | 2008-08-06 | 2008-12-31 | 山东大学 | Leech extract and preparation method and use thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110066318A (en) * | 2019-05-13 | 2019-07-30 | 山东大学 | A kind of hirudin and its application |
| CN110066318B (en) * | 2019-05-13 | 2022-02-15 | 山东大学 | Leech peptide and application thereof |
| CN113583092A (en) * | 2021-09-27 | 2021-11-02 | 渤海水产股份有限公司 | Leech peptide and application and product thereof |
| CN113621565A (en) * | 2021-10-08 | 2021-11-09 | 渤海水产股份有限公司 | Application of polypeptide in regulating and controlling fibroblast in vitro culture |
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