CN105418741B - A kind of isolation and purification method of leech polypeptide - Google Patents
A kind of isolation and purification method of leech polypeptide Download PDFInfo
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Abstract
The invention discloses a kind of isolation and purification methods of leech polypeptide, are added to imitate hirudo extract in the system that human body enterocyte absorbs first and cultivate, absorbed substance is then carried out chromatography.What the present inventor solved the problems, such as leech polypeptide from different angles isolates and purifies male, by the way that hirudo extract is purified using Caco-2 cells first, imitated Chinese traditional medicine leech in vivo digest and assimilate process, by the absorption for simulating intestinal cell, it will largely cannot first be separated by the polypeptide that human body intestinal canal cell absorbs, the process of isolating and purifying is simplified, then can both isolate and purify to obtain the polypeptide that can be absorbed by the body and utilize using anion exchange chromatography and gel permeation chromatography.The polypeptide that method through the invention isolates and purifies has the function of inhibiting vascular smooth muscle cells migration.
Description
Technical field
The invention belongs to field of medicaments, and in particular to a kind of isolation and purification method of leech polypeptide.
Background technology
Atherosclerosis is that ductus arteriosus wall thickens, becomes caused by lipid bulk deposition in arterial blood tube wall
Firmly, and then artery is made to follow the string, luminal stenosis.It is a kind of chronic inflammatory participated in jointly by various kinds of cell and cell factor
Disease.With deepening continuously to atherosclerotic lesion process study, the phase interaction between atherosclerosis relevant cell
With and the inflammatory factors such as interaction of cell factor and cell more and more studied.Participate in the thin of atherosclerosis
Born of the same parents include mainly：Vascular endothelial cell, vascular smooth muscle cells and immunocyte, wherein immunocyte are mainly that macrophage is thin
Born of the same parents, in addition there are neutrophil leucocyte, mast cell, dendritic cells and T cells etc..
The proliferation of vascular smooth muscle cells (vascular smooth muscle cell, VSMCs) is migrated in artery congee
Critical effect is also played in the forming process of sample hardening lesion.VSMCs is the important composition ingredient of vascular wall, is predominantly located at
Tunicae media vasorum under physiological condition, keeps balance between the proliferation and apoptosis of VSMCs, and can make its increasing under many pathological states
Grow it is out of control, so as to cause a series of variations of vascular wall.There is only a small amount of VSMCs in endangium, main function is to pass through
Diastole and contraction change blood vessel diameter, make blood vessel that blood stream pressure appropriate, VSMCs at this time be kept to show as shrinkage type, increase
It grows and synthesis capability is very low.Under the action of a variety of pathological factors, the phenotype of VSMCs can be converted into synthesis type by shrinkage type.It closes
Molding VSMCs can synthesize and secrete a large amount of extracellular matrixs, to change the microenvironment of cell peripheral, and extracellular matrix
The proliferation of VSMCs can be influenced again, sticked and transition process.Inflammatory reaction can stimulate VSMCs that migration, proliferation, and and inflammatory occurs
Inflammatory component interaction in lesion, makes artery wall thickening.Accumulation, VSMCs proliferation and the fibr tissue of monocyte are formed
Cause lesion further to expand and reassemble into, to be covered in the fibrous cap on lipid core and slough, it is multiple to eventually lead to patch
The formation of mould assembly lesion.Therefore, VSMCs is also one of the key cells for pushing atherosclerotic process.
Leech (Leech) is used to treat a variety of diseases as traditional Chinese medicine, how on the books in Ancient Times in China medical book.Such as
《Sheng Nong's herbal classic》Middle meaning its " master closes by extravesated blood, hemostasis, the moon, blood-breaking disperse accumulation is poly- ... ".Leech pharmacological action is extensive, including anti-
Solidifying, antithrombotic, antitumor, anti-inflammatory, endothelial cell protection and anti-fibrosis etc..In cardiovascular and cerebrovascular disease clinical treatment, in
Liquid medicine leech and Chinese patent drug containing leech or compound preparation are widely used to coronary heart diseases and angina pectoris, acute myocardial infarction, ischemic
Property headstroke (such as cerebral thrombus, cerebral infarction and its sequelae), hypertension, hyperlipidemia, cerebral atherosclerosis disease, artery congee
The prevention of the diseases such as hardening and peripheral angiopathy.The basic research for preventing cardiovascular and cerebrovascular disease about leech in recent years is constantly deep
Enter.The country includes mainly three aspects to the research report of leech pharmacological action：Anticoagulation, the treatment to cardiovascular and cerebrovascular disease
Effect, to the Therapy study of diabetic retinopathy.But due to the difficulty of the complexity of leech polypeptide and screening active ingredients, mesh
The isolation and purification method of preceding not easy active leech polypeptide.
Invention content
The purpose of the present invention is just to provide for a kind of isolation and purification method of leech polypeptide.
To achieve the goals above, the present invention adopts the following technical scheme that：
A kind of isolation and purification method of leech polypeptide, first by hirudo extract be added imitate human body enterocyte absorb be
It is cultivated in system, absorbed substance is then subjected to chromatography.
It is preferred that：The extracting method of hirudo extract is as follows：
Water intaking leech drying entirety is ground into 20-150 mesh powder, and the water or the fresh and alive entirety of leech of 6-16 times of weight is added,
The water of 1-6 times of weight, homogenate is added；Homogenate is taken, pH to 7-9 is adjusted, 2000-20000U enzymes are added by per the dry medicinal material of 1g leech
Ratio be added trypsase, control 40-60 DEG C of temperature, digest 2-12 hours, be cooled to room temperature；After having digested, to above-mentioned enzyme
It solves the acetone of 1-5 times of volume of addition or ethyl alcohol refrigeration in liquid to precipitate 4-12 hours, filtering, filtrate decompression drying obtains leech and carries
Take object.
It is preferred that：It is to cover with/have people's Colon and rectum gland cancer Caco-2 cells to imitate the system that human body enterocyte absorbs
The cells transwell.
It is preferred that：The condition of culture is 5% carbon dioxide at 37 DEG C, and culture 0.5-10h is (preferably under the conditions of saturated humidity
2h)。
It is preferred that：Before the system for imitating the absorption of human body enterocyte is added in hirudo extract, it is configured to using the PBS of pH value 7.2
The solution of a concentration of 0.5-5mg/ml.
It is preferred that：Chromatography first through anion exchange chromatography (preferably：Anion exchange chromatography medium is Q
Sephrose), the NaCl solution linear elution of a concentration of 0.5-2mol/L, collects the active constituent at each peak, and intravascular is smooth
Myocyte migrates screening, takes the active constituent at the highest peak of activity, is concentrated and dried, then by the work after being concentrated and dried
Property ingredient, with distillation water dissolution, (preferably using gel permeation chromatography：The filler of the gel permeation chromatography column：Separating ranges
For the filler of 100-5000 dalton, more preferably：The filler of the gel permeation chromatography column is Sephadex LH-20), distillation
Water elution, collects the active constituent at each peak, and the screening of intravascular smooth muscle cell migration takes the activity at the highest peak of activity
Ingredient is concentrated and dried to get the leech polypeptide after isolating and purifying.
The present invention also protects the leech polypeptide that above-mentioned isolation and purification method obtains and the leech polypeptide to press down preparing
Application in vascular smooth muscle cells migration drug processed.
A kind of drug inhibiting vascular smooth muscle cells migration, main active is that the above method isolates and purifies to obtain
Leech polypeptide.
The leech polypeptide is preparing the application in treating atherosclerosis drug.
A kind of drug for treating atherosclerosis, main active are the leech that the above method isolates and purifies
Polypeptide.
Advantageous effect：
Hirudo extract using chromatography method when being isolated and purified, since extract contains the amount of polypeptide greatly and type
Miscellaneous, the electrical property difference difference of the net charge, surface charge distribution of ion is smaller between polypeptide, so using the side such as conventional chromatography
When method is directly separated, difficulty is larger, and isolated polypeptide possibly can not be absorbed by the body, existing isolation and purification method
Isolating and purifying for polypeptide, the present inventor are typically realized by the type or improvement chromatography method of change chromatographic column
Solve the problems, such as leech polypeptide from different angles isolates and purifies hardly possible, by using Caco-2 thin first hirudo extract
Born of the same parents purify, and have imitated the absorption process of Chinese traditional medicine leech in vivo, will be a large amount of by simulating the absorption of intestinal cell
It cannot first be separated by the polypeptide that human body intestinal canal cell absorbs, the process of isolating and purifying be simplified, then using anion
Column chromatography and gel permeation chromatography are exchanged, can isolate and purify to obtain in conjunction with vascular smooth muscle migration screening model can be by human body
The active leech polypeptide being absorbed and utilized.The active leech polypeptide that method through the invention isolates and purifies, which has, inhibits blood vessel
The effect of smooth muscle cell migration.
Description of the drawings
Fig. 1：The cells transwell explanation；
Fig. 2：Q Sephrose anion exchange chromatography isolates and purifies knot to room leech polypeptide under the cells transwell
Fruit；
Fig. 3：Separating resulting of the Sephadex LH-20 gel permeation chromatographies to leech polypeptide；
Fig. 4：It does not use Caco-2 cell purifications hirudo extract to detach through Q Sephrose anion exchange chromatography to tie
Fruit；
Fig. 5：Influence of the leech polypeptide (HEE) to vascular smooth muscle cells migration ability, abscissa are grouped for dosing, LPS
(+) is 1.0 μ g/ml, and Simvastatin (SIM) (+) is 10 μM i.e. 4.1856 μ g/ml；Ordinate is vascular smooth muscle cells migration
Number；#P < 0.05vs blank controls,##P < 0.01vs blank controls,*P < 0.05vs modeling groups,**P < 0.01vs modeling groups
,△P < 0.05vs Simvastatin groups,△△P < 0.01vs Simvastatin groups；Bar is 50 μm；
Fig. 6：Active leech polypeptide (HEE) is to vascular smooth muscle cells inflammatory factor, chemotactic factor (CF), adhesion molecule albumen table
Up to horizontal influence, abscissa is grouped for dosing, and LPS (+) is 1.0 μ g/ml, and Simvastatin (SIM) (+) is 10 μM i.e. 4.1856
μg/ml；Ordinate is vascular smooth muscle cells migration number；#P < 0.05vs blank controls,##P < 0.01vs blank controls,*P
< 0.05vs modeling groups,**P < 0.01vs modeling groups,△P < 0.05vs Simvastatin groups,△△P < 0.01vs Simvastatin groups；
Fig. 7：Each component is to vascular smooth muscle migration result after Q Sephrose anion exchange post separations；
Fig. 8：Each component is to vascular smooth muscle migration result after LH-20 gel post separations.
Specific implementation mode
The invention will be further described with embodiment below in conjunction with the accompanying drawings.
Embodiment 1：The extraction of hirudo extract
Water intaking leech drying entirety 1kg is ground into 60 mesh powder, and 10kg distilled water water, homogenate is added；Homogenate is taken, pH is adjusted
To 8.5, trypsase is added in the ratio that 10000U enzymes are added per the dry medicinal material of 1g leech, controls temperature 50 C, digests 6h, it is cooling
To room temperature；After having digested, the ethyl alcohol refrigeration precipitation 8h of 4 times of volumes, filtering are added into above-mentioned enzymolysis liquid, filtrate decompression is dried,
Obtain hirudo extract 100g.(or obtaining hirudo extract using the method for ZL200810138936.5)
Embodiment 2：Leech polypeptide isolates and purifies
People's Colon and rectum gland cancer Caco-2 is carefully inoculated into the cells transwell, using DMEM culture mediums, contains 20%
FBS, 100U/mL penicillin, 100 μ g/mL streptomysins, 2mmol/L L-Glutamines, condition of culture 5%CO2, 37 DEG C, connect
Kind density is 450,000 cells/wells, changes liquid weekly 3 times, continuous culture two weeks.The integrality of Caco-2 cellular layers passes through cell electricity
Resistance instrument detects TEER values and determines, TEER values are more than 500 Ω/cm2It can be used for experimental study.
The hirudo extract extracted in embodiment 1 is dissolved in PBS (pH value 7.2) buffer solution, is configured to a concentration of
The hirudo extract solution of 1mg/mL.Before experiment starts, culture medium will be removed in the cells transwell, it is small to clean culture with PBS
Room is twice.Each indoor addition 0.3mL hirudo extract solution, 1.2mL PBS buffer solution is added in lower room.After acting on 2h,
PBS solution in lower room is all collected, it is spare.
By above-mentioned lower room solution through Q Sephrose anion exchange chromatographies, 2mol/L NaCl solution linear elutions are received
Collect the active constituent at each peak, the screening of intravascular smooth muscle cell migration takes the active constituent at the highest peak of activity, this reality
Four peaks are obtained in testing, the active highest (referring to attached drawing 2, from left to right third) at third peak collects active component herein, dense
It contracts drying；
The distillation water dissolution of above-mentioned third peak component is distilled into water elution through LH-20 gel permeation chromatographies, is collected
The active constituent at each peak, intravascular smooth muscle cell migration screening, takes the active constituent at the highest peak of activity, this experiment
In obtain two peaks, take first peak (see attached drawing 3, first from left to right), collect active component herein, be concentrated and dried, i.e.,
Obtain hirudin.
Embodiment 3：Do not use Caco-2 cell purifications hirudo extract through Q Sephrose anion exchange chromatography point
From purifying
The hirudo extract extracted in embodiment 1 is dissolved in PBS buffer solution, is configured to the leech of a concentration of 1mg/mL
Extract solution.Through Q Sephrose Fast Flow anion exchange chromatographies, 2mol/L NaCl solution linear elutions are washed
De- curve is shown in attached drawing 4：Do not use Caco-2 cell purifications hirudo extract through Q Sephrose Fast Flow anion exchanges
Chromatographic column separating resulting.
From attached drawing 4 and the comparison of attached drawing 2 as can be seen that the separation component of hirudo extract is bright after Caco-2 cell purifications
Aobvious to reduce, being conducive to the later stage isolates and purifies.
Embodiment 4：Active leech polypeptide inhibits the Effect study of vascular smooth muscle migration
1. leech polypeptide is obtained in the DMEM culture medium gradient dilutions embodiment 2 containing 0.4%FBS (fetal calf serum),
Concentration gradient is followed successively by 50 μ g/ml, 100 μ g/ml, 200 μ g/ml, 400 μ g/ml；
2. the digestion of vascular smooth muscle cells pancreatin centrifuges and is inoculated with 200 μ l after counting in Transwell upper chambers, carefully
Born of the same parents' concentration 5 × 105A/ml, lower room sequentially add the leech polypeptide of various concentration, the DMEM cultures of blank control 0.4%FBS
Base, positive controls are Simvastatin group, and 24 porocyte culture plates are placed in incubator 37 DEG C together with cell, 5%CO2Culture；
3. continuously culture absorbs the remaining vascular smooth muscle cells of upper chamber after 12 hours；PBS cleans Transwell upper chamber films
Bottom surface cell, methanol fix 10min, violet staining 10min；
4. after PBS rinsings, cell is placed on glass slide and observes, take pictures, ImageJ is counted, and as a result sees attached drawing 5.
Leech enzymatic extract is as shown in Fig. 5 to the influence result of rat VSMCs transfer abilities, and abscissa is dosing point
Group, ordinate are that vascular smooth muscle migrates number, and compared with blank control, LPS (lipopolysaccharides) model group mobility increases, and pungent
It is very notable to cut down statin group anti-migration effect；HEE (refers to that the leech that the method for the present invention is extracted is more compared with LPS model groups
Peptide) 200 μ g/ml, 400 μ g/ml groups show very strong anti-migratory activity (P < 0.05), wherein 200 μ g/ml groups of HEE
Anti-migratory activity is suitable with 10 μM of the anti-migratory activity of Simvastatin group (P < 0.05), the anti-migration of 400 μ g/ml groups of HEE
The anti-migratory activity of activity and 200 μ g/ml groups does not have significant difference.
Embodiment 5：Influence research of the active leech polypeptide to vascular smooth muscle correlation factor
(1) prepared by protein sample
1. removing cell culture medium and rinsing cell with PBS；100-150 μ l cell pyrolysis liquids (1 are added:100 are added
100mmol/L PMSF), it is placed on ice chest and cracks 15-20min；
2. cell pyrolysis liquid and cell fragment are moved in EP pipes, 4 DEG C of 12000r/min centrifuge 10min；
3. Coomassie brilliant blue G250 measures the concentration of protein in the albumen supernatant that centrifugation removal precipitates；
4. albumen centrifuged supernatant：5 × sample-loading buffer=1:4 mixing, boiling water bath 5min；
5. 12000r/min centrifuges 10min, it is required protein sample, -80 DEG C of preservations to obtain supernatant；
(2) protein electrophoresis
1. matching plastic plate by PAGE gel formula table, resolving gel concentration 12% concentrates gum concentration 5%；Prepare electrophoresis
Buffer solution simultaneously assembles electrophoresis accessory；
2. the protein sample that albumen Marker and boiling water treating are crossed is added in loading hole；
3. deposition condition be constant pressure 70V, 30min to two layers of glue line of demarcation, 100V electrophoresis to bromophenol blue has just been run out of can be whole
Only electrophoresis；
(3) transferring film
1. preparing transferring film liquid, methanol is used in combination to activate pvdf membrane；The protein electrophoresis of needs is extracted according to albumen Marker marks
Band；
2. assembling clip according to " sponge-filter paper-glue-pvdf membrane-filter paper-sponge "；Assemble membrane-transferring device, 100V constant pressure ice
Bathe transferring film 2h；
(4) antibody incubation
1. moving to film in the confining liquid containing 5% skim milk, room temperature is closed 2h or 4 DEG C of standing and was closed on shaking table
Night；
2. the TBST solution dilution antibody (primary antibody, secondary antibody) containing 5% skim milk；
3. pvdf membrane is incubated at room temperature primary antibody dilution 2h；
4. TBST shaking tables rinse pvdf membrane；
5. pvdf membrane is incubated at room temperature secondary antibody diluent 2h；
6. TBST shaking tables rinse pvdf membrane；
(5) chemiluminescence, development, fixing
Dark room operation：Two kinds of reagents of luminescence reagent A and B mix in equal volume；Luminescence reagent is incubated pvdf membrane；X-ray exposes
Develop afterwards, be fixed；
(6) gel images are analyzed
Film is scanned or is taken pictures, ImageJ analyzes OD value, as a result sees attached drawing 6.
From attached drawing 6 as can be seen that HEE is to vascular smooth muscle cells inflammatory factor, chemotactic factor (CF), adhesion molecule albumen mark water
Flat influence has concentration dependent, and inhibition is with the increase of HEE activities and all the more apparent, especially to MCP-1,
The inhibition of ICAM-1, VCAM-1 are notable, and MCP-1, ICAM-1, VCAM-1 can be down to normally by HEE400 μ g/ml groups substantially
Level, and HEE400 μ g/ml groups are better than SIM to the protein expression inhibition of MCP-1, to ICAM-1, VCAM-1 albumen table
Inhibition is slightly not as good as SIM.
The HEE of 400 μ g/ml groups can also reduce the expressing quantity of inflammatory factor TLR4, TNF α, iNOS, reduce amplitude
Respectively 50%, 75%, 33%, SIM 125% is followed successively by the reduction amplitude of the expression quantity of above-mentioned three kinds of albumen, 135%,
133%.The significant effect of HEE resisting vascular smooth muscles cell chemotactic factor, adhesion molecule expression, partly may be by anti-inflammatory work
With realization.
Embodiment 6：Work of each component to vascular smooth muscle migration after Q Sephrose anion exchange post separations
Journal of Sex Research
Through Q Sephrose anion exchange chromatographies, 2mol/L NaCl solution linear elutions collect the activity at each peak
Ingredient obtains four peaks (see Fig. 2) in this experiment, after this four peak difference desalinations, activity is carried out by the method in embodiment 4
Experiment.
1. with containing 0.4%FBS (fetal calf serum) DMEM culture mediums distinguish four peak components of gradient dilution, concentration gradient according to
It is secondary be 100 μ g/ml, 200 μ g/ml, 400 μ g/ml；
2. the digestion of vascular smooth muscle cells pancreatin centrifuges and is inoculated with 200 μ l after counting in Transwell upper chambers, carefully
Born of the same parents' concentration 5 × 105A/ml, lower room sequentially add the peak component of various concentration, the DMEM culture mediums of blank control 0.4%FBS,
Positive controls are Simvastatin group, and 24 porocyte culture plates are placed in incubator 37 DEG C together with cell, 5%CO2Culture；
3. continuously culture absorbs the remaining vascular smooth muscle cells of upper chamber after 12 hours；PBS cleans Transwell upper chamber films
Bottom surface cell, methanol fix 10min, violet staining 10min；
4. after PBS rinsings, cell is placed on glass slide and observes, take pictures, ImageJ is counted, and as a result sees attached drawing 7.
From Fig. 7 we can see that third peak activity highest from left to right under same concentrations, therefore select third Peak Activity
Continue to detach.
Embodiment 7：Activity research of each component to vascular smooth muscle migration after LH-20 gel post separations
Through LH-20 gel permeation chromatographies, water elution is distilled, the active constituent at each peak is collected, two is obtained in this experiment
After desalination is distinguished at the two peaks, activity experiment is carried out by the method in embodiment 4 by peak, (see Fig. 3).
1. with containing 0.4%FBS (fetal calf serum) DMEM culture mediums distinguish two peak components of gradient dilution, concentration gradient according to
It is secondary be 100 μ g/ml, 200 μ g/ml, 400 μ g/ml；
2. the digestion of vascular smooth muscle cells pancreatin centrifuges and is inoculated with 200 μ l after counting in Transwell upper chambers, carefully
Born of the same parents' concentration 5 × 105A/ml, lower room sequentially add the peak component of various concentration, the DMEM culture mediums of blank control 0.4%FBS,
Positive controls are Simvastatin group, and 24 porocyte culture plates are placed in incubator 37 DEG C together with cell, 5%CO2Culture；
3. continuously culture absorbs the remaining vascular smooth muscle cells of upper chamber after 12 hours；PBS cleans Transwell upper chamber films
Bottom surface cell, methanol fix 10min, violet staining 10min；
4. after PBS rinsings, cell is placed on glass slide and observes, take pictures, ImageJ is counted, and as a result sees attached drawing 8.
From Fig. 8 we can see that first peak activity highest from left to right under same concentrations, this is isolated active water
Leech polypeptide.
Above-mentioned, although the foregoing specific embodiments of the present invention is described with reference to the accompanying drawings, not protects model to the present invention
The limitation enclosed, those skilled in the art should understand that, based on the technical solutions of the present invention, those skilled in the art are not
Need to make the creative labor the various modifications or changes that can be made still within protection scope of the present invention.
Claims (3)
1. a kind of isolation and purification method of leech polypeptide, it is characterized in that：Hirudo extract is added first and imitates human body enterocyte
It is cultivated in the system of absorption, absorbed substance is then subjected to chromatography；
Wherein, the extracting method of hirudo extract is as follows：
Water intaking leech drying entirety is ground into 20-150 mesh powder, and the water or the fresh and alive entirety of leech of 6-16 times of weight is added, is added
The water of 1-6 times of weight, homogenate；Homogenate is taken, pH to 7-9 is adjusted, by the ratio that 2000-20000U enzymes are added per the dry medicinal material of 1g leech
Trypsase is added in example, controls 40-60 DEG C of temperature, digests 2-12 hours, is cooled to room temperature；After having digested, to above-mentioned enzymolysis liquid
The acetone of middle 1-5 times of volume of addition or ethyl alcohol refrigeration precipitate 4-12 hours, filtering, and filtrate decompression drying obtains hirudo extract；
It is described that imitate the system that human body enterocyte absorbs be to have or the transwell of long Manchu's Colon and rectum gland cancer Caco-2 cells
Cell；
The chromatography first through anion exchange chromatography, receive by the NaCl solution linear elution of a concentration of 0.5-2mol/L
Collect the active constituent at each peak, the screening of intravascular smooth muscle cell migration takes the active constituent at the highest peak of activity, concentration
And it is dry, then by the active constituent after being concentrated and dried, with distillation water dissolution, using gel permeation chromatography, the gel
The filler of filtration chromatography column is Sephadex LH-20, distills water elution, collects the active constituent at each peak, intravascular smooth muscle
Cell migration is screened, and is taken the active constituent at the highest peak of activity, is collected active component herein, you can.
2. isolation and purification method as described in claim 1, it is characterized in that：The condition of culture is 5% titanium dioxide at 37 DEG C
Carbon cultivates 0.5-10h under the conditions of saturated humidity.
3. isolation and purification method as described in claim 1, it is characterized in that：The hirudo extract, which is added, imitates human body enterocyte
Before the system of absorption, the solution of a concentration of 0.5-5mg/ml is configured to using the PBS of pH value 7.2.
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| 水蛭酶解提取物抑制脂多糖诱导的大鼠血管平滑肌细胞ICAM-1、VCAM-1及MCP-1的表达;成龙;《中国学位论文全文数据库》;20151012;摘要、第三章第1-2段及第3.3节、第五章第3.2节至第5节、图2-5及图2-9 * |
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