CN106370833A - Method for evaluating effect of compound on angiogenesis in pathological state - Google Patents
Method for evaluating effect of compound on angiogenesis in pathological state Download PDFInfo
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Abstract
The invention relates to a method for evaluating the effect of a compound on angiogenesis in a pathological state. According to the invention, a vascular endothelial growth factor (VEGF) is used for treating young zebra fish in a specific period to simulate a pathological state, then complete subintestinal peripheral vein length is used as an evaluation index, so the evaluation method for the effect of a drug on angiogenesis in the pathological state is successively established. The method has the advantages of simple operation, rapidness, stability, reliability and good repeatability, is more scientific and objective, presents the generation condition of spinous process and improves accuracy of results.
Description
Technical field
The present invention relates to a kind of for evaluating the method to angiogenesis function under pathological state for the compound, belong to drug sieve
Select field.
Background technology
Angiogenesis are to be adjusted by complementary and complicated signal pathways many present in human body, with a lot of diseases
Occur relevant with treatment.Angiogenesis in addition to the generation that can affect tumor, in some other disease, such as retinopathy, rheumatism
Also play an important role in the generation of property arthritis, atherosclerosiss, endometriosis etc..Stop the blood under pathological state
Pipe generates the new way also having become as such disease.
Research shows, VEGF (vascular endothelial growth factor, vegf) can
Directly act on vascular endothelial cell, stimulating it that mitosiss occur, thus promoting the growth of new vesselses, being most important blood
Pipe forms the factor.Vegf is the specificity growth factor of endotheliocyte, and is that the most special and most critical angiogenesis stimulate
The factor, almost take part in each step that physiological and pathologic vessels are formed, can directly act on vascular endothelial cell, stimulate
There are mitosiss in it, thus promoting the growth of new vesselses.The malignant cells such as colorectal cancer, breast carcinoma, pulmonary carcinoma all have
The function of autocrine vegf, occurring the tumor cell of migration transfer can discharge vegf in local stimulates the formation of local vascular.
Vegf and its receptor are expressed in kinds of tumors, and less expression beyond angiogenesis enliven tissue (as tumor etc.).Therefore,
Under the pathological states such as tumor, vegf is high, and expression is of paramount importance pathological characteristicses.
Experimental model is the important step of medicament research and development.The effectiveness of experimental model, can determine the efficiency of drug discovery, fall
Low R＆D risk.The in vitro study model of angiogenesis at present, the method mainly using Cell culture invitro, carry out endotheliocyte
Culture, observes its impact to angiogenesis.The In vivo study model of angiogenesis mainly has animal (rat, rabbit etc.) cornea
Capsule (camera oculi anterior), Hamster Cheek Pouch, nude mice external ear be subcutaneous and the animal model such as chick chorioallantoic membrane.The external model of angiogenesis
Environment in residing in vivo complexity can not be reacted well, the accuracy of evaluation result is poor.The internal animal mould of angiogenesis
Type research is closer to human body, but complex operation, take long.
And emerging laboratory animal Brachydanio rerio embodies good application prospect.Brachydanio rerio has small volume, and egg laying amount is high,
Time of reaching maturity is short, and body surface is rich in permeability, rearing conditions simple it is easy to the features such as culture, such that it is able to shorten drug sieve
The experimental period of choosing, improve screening efficiency；Reduce the requirement to given the test agent in screening process, be conducive to micro guide's chemical combination
The screening active ingredients experiment of thing sample；Directly test medicine can be put in culture medium and be tested, can carry out multilevel many simultaneously
The screening of factor.Therefore, Brachydanio rerio is convenient and economical in the operation for drug screening.Brachydanio rerio has external fertilization simultaneously
With individual transparent feature.Using this feature, may be implemented in and under Laser Scanning Confocal Microscope, carry out viviperception, both dynamic observation
The impact that its fetal development is whole and exogenous material is to vascular development, can carry out the operation on gene level again.But lose
Regret, the angiogenesis model set up currently with zebra fish model is the direct blood vessel observing new life, is in physiology shape
The angiogenesiss of state, strictly speaking, can not effectively distinguish the blood vessel life under normal angiogenesis and pathological state
Become.
Content of the invention
For the deficiencies in the prior art, the present invention provides one kind to be used for evaluating compound angiogenesis under pathological state are made
Method, the method is set up under pathological state, can the accurately generation to blood vessel for the Fast Evaluation medicine.
Term illustrates:
Completely outer rim vein length under intestinal: the spinous process length bearing on outer rim vein under outer rim vein and intestinal under intestinal total
With see Fig. 1.
Hpf (hours post fertilization): biology proprietary term, refer to the time of after fertilization, such as 48hpf refers to
The after fertilization embryo of 48 hours.
Technical scheme is as follows:
Summary of the invention:
The present invention adopts VEGF (vegf) to process specific period Brachydanio rerio juvenile fish, simulates pathological state,
Then using " outer rim vein length under complete intestinal " as evaluation index, it has been successfully established medicine to pathological state angiogenesis function
Evaluation methodology, have the advantages that simple to operate, quick, reliable and stable and reproducible.
Detailed Description Of The Invention:
A kind of for evaluate compound to the method for angiogenesis function under pathological state it is characterised in that include step
As follows:
(1) normal for the growth of after fertilization 48h transgenic zebrafish embryo is moved in culture hole, add vascular endothelial growth
The factor is processed, and obtains vegf and processes zebrafish embryo；By normal for the growth of after fertilization 48h zebrafish embryo without blood vessel endothelium
Somatomedin is processed as normal zebrafish embryo；
(2) one groups of vegf process zebrafish embryo successive administration and are incubated to 72hpf, as dosing group, another set vegf
Processing zebrafish embryo adds the culture water of equivalent to be incubated to 72hpf, and as vegf group, normal zebrafish embryo adds culture water
It is incubated to 72hpf, as Normal group；
(3) Brachydanio rerio of anesthesia dosing group, vegf group and Normal group, gathers every Brachydanio rerio subintestinal vein (sivs)
The fluorescence microscope images in region, outer rim vein length under the complete intestinal of every group of every fish of measurement；
(4) quantitative analyses
Under the complete intestinal of every group of Brachydanio rerio obtaining outer rim vein length withRepresent, com-parison and analysis Normal group,
The significance of difference between vegf group, dosing group；
When dosing group Brachydanio rerioLess than vegf group Brachydanio rerioAnd reach significance level statistically
(p < 0.05), illustrates that the angiogenesis that medicine to be measured processes Brachydanio rerio to vegf are inhibited.
The stability and the reliability that are provided for evaluation experimental material of Normal group of the present invention.
According to currently preferred, in step (1), described zebrafish embryo is the fluorescently-labeled transgenic zebrafish of blood vessel
Embryo.Blood vessel fluorescently-labeled transgenic zebrafish embryo can adopt this area ordinary commercial products, such as blood vessel fluorescent transgenic
Brachydanio rerio tg (vegfr2:gfp), Shandong Scientific Research Academy Brachydanio rerio drug screening center provides.
According to currently preferred, in step (1), the concentration of culture fluid VEGF is 1-10ng/ml,
Preferably, the concentration of culture fluid VEGF is 5-10ng/ml.
According to currently preferred, in step (1), need zebrafish embryo is carried out before VEGF addition
De- egg membrane treatment.
It is further preferred that de- egg membrane treatment is specially the chain pheron that zebrafish embryo is added to concentration 1-2mg/ml
2-5min is kept, you can complete de- egg membrane treatment in enzyme e solution.
According to currently preferred, in step (1), the concrete processing method that vegf processes zebrafish embryo is, by blood vessel
Endothelial cell growth factor (ECGF) aqueous solution is added in culture hole, makes hole concentration of vascular endothelial growth factor reach 1-10ng/ml, jointly
Incubation 4-12h, causes the pathological state of the paraplasm of new vesselses, then removes vegf；Preferably, common incubation time is
4-6h.
It is further preferred that the concentration of VEGF aqueous solution is 90～100ng/ml.
According to currently preferred, in step (2), the zebrafish embryo of dosing group, vegf group and Normal group is in 28
DEG C control optical culture be incubated to 72hpf, every group of Brachydanio rerio 10-20 bar.
According to currently preferred, in step (3), the tricaine that the anesthesia of Brachydanio rerio is is 0.3 ‰ using mass concentration
Soak 40～90s to be anaesthetized.
According to currently preferred, in step (3), collection image is by zebrafish embryo fixing side Postural immobilization,
Fluorescence microscopy Microscopic observation, take pictures, obtain the side bit image of zebrafish embryo.
It is further preferred that in step (3), the image of collection is to obtain under same amplification, picture format
For jpg form.
According to currently preferred, in step (3), measurement length is the figure to collection using image processing software image j
As measuring, first calibrate scale before measurement, obtain accurate length.
According to currently preferred, the medicine to be measured that the method according to the invention filters out is Rhizoma Cyperi volatile oil, Rhizoma Cyperi
Alcohol or paeonol.
Under the complete intestinal of 72hpf Brachydanio rerio, outer rim vein length and the effect of Drug inhibition pathological state angiogenesis are in positive
Close, be shown in Table 1.
Evaluation index has subintestinal vein length, subintestinal vein area, radical of spinous process etc..Subintestinal vein length is all intestinal
The length summation (not comprising spinous process) of lower vein (including outer rim blood vessel and internal blood vessel), Shortcomings are that subintestinal vein is in itself
The netted space structure of one basketball, the technology by the image acquisition of two dimensional surface is limited, when Brachydanio rerio is side position, left and right
Two vascellum laterales state in an overlapping, by depth of field undertreatment to exclude left and right sides blood vessel completely and all display or all
Do not display, cause uncertainty and the inaccuracy measured.Subintestinal vein area is that whole subintestinal vein area is covered
Region area (with outer rim blood vessel as boundary), deficiency be when blood vessel attenuate or internal blood vessel disappearance when, with whole region
Area e insufficient to embody the effect of suppression.The radical of spinous process is only to grow the radical of spinous process as index, to spinous process
The length of growth is simultaneously not concerned with, and there is also obvious deficiency.The present invention to evaluate compound using outer rim vein length under complete intestinal
Angiogenesis function under pathological state be can be used in reflect the effect of the angiogenesis to morbid state for the medicine, clinical meaning is more
Significantly, the result of evaluation is more nearly disease time of day, has novelty, actual meaning.
The feature of the present invention and beneficial effect:
1st, the present invention using outer rim vein length under complete for Brachydanio rerio intestinal as medicine to angiogenesis function under morbid state
The index evaluated, compared with existing subintestinal vein length (total lengths of all subintestinal veins), more science, objective, embodiment
The generation situation of spinous process, improves the accuracy of result.
2nd, Brachydanio rerio subintestinal vein is three-dimensional basketball network structure, and existing index such as subintestinal vein area etc. is easily subject to
The impact of the depth of field of taking pictures, the also impact of easy receptor site.Under the complete intestinal of the present invention, outer rim vein length is to calculate outer most edge
Length of vessel, not receptor position influence, accuracy is high.
3rd, Brachydanio rerio subintestinal vein area is big, is calculated using its profile, easy to implement, simple.
4th, the method that the present invention sets up simulates pathological state using vegf, with existing direct sight on live body Brachydanio rerio
Examine subintestinal vein to compare, evaluation result is reliable.
Brief description
Fig. 1 is the fluorescence photo of outer rim vein under the complete intestinal of Brachydanio rerio；In figure, white dashed line is shown as of the present invention
Outer rim vein length under intestinal completely；
Fig. 2 is that the fluorescence of outer rim vein under the complete intestinal of subintestinal vein of Brachydanio rerio after normal Brachydanio rerio and vegf are processed shines
Piece, in figure:
A is the fluorescence photo of outer rim vein under the complete intestinal of normal Brachydanio rerio subintestinal vein；A is the partial enlarged drawing of figure a；
B is the fluorescence photo of outer rim vein under the complete intestinal of subintestinal vein of Brachydanio rerio after vegf process；B is the local of figure b
Enlarged drawing.
Specific embodiment
With reference to embodiment and accompanying drawing, technical scheme is further elaborated, but institute of the present invention protection domain
Not limited to this.
Laboratory animal: using blood vessel fluorescently-labeled transgenic zebrafish flk (purchased from Shandong Scientific Research Academy Brachydanio rerio medicine
Screening center).Male and female Brachydanio rerio is separately raised under illumination 14h/ dark 10h, 28 DEG C of standard conditions, and timing is fed with graininess bait
Material and shrimps.When using ovum, take healthy sexually matured Brachydanio rerio, put in copulation cylinder in the ratio of male and female 1/1 or 1/2, middle
Place dividing plate, be placed in dark surrounds, before next day bright light, pump dividing plate, light stimulation makes it ovulate, and after half an hour drags for adult fish
Go out, so that ovulation period is controlled within half an hour, during next day 9～10 obtain germ cell, collect germ cell, be transferred to culture water in
28 DEG C of control optical cultures are to 48hpf.
Raw materials used in embodiment it is conventional commercial products, wherein: VEGF (vegf) cas is
127464-60-2, the used time is dissolved as solution for standby with sterile distilled water.Rhizoma Cyperi volatile oil, limited purchased from Jiangxi BAICAO Pharmaceutical
Company, outward appearance is amber to brown color glop；Main composition component contains cyperene, cyperol, cyperone；Refractive index:
1.4980～1.5280；Optical rotation: -11 °～+35.5 °；Relative density: 0.960～0.992, molten with dimethyl sulfoxide (dmso)
Solution is prepared.Paeonol, molecular weight is 166.17, and No. cas is 552-41-0, is dissolved with dimethyl sulfoxide (dmso) and prepares.Reaction
Stop, chemistry entitled (+)-Thalidomide, molecular weight be 258.23, No. cas be 50-35-1, with dimethyl sulfoxide (dmso) dissolving
Prepare.Curcumenol, molecular weight is 236.35, and No. cas is 4871-97-0, is dissolved with dimethyl sulfoxide (dmso) and prepares.Radix Curcumae carries
Take thing, be brown color fine powder, specification is 10:1, is dissolved with dimethyl sulfoxide (dmso) and prepares.
Embodiment 1, for evaluating the method to angiogenesis function under pathological state for the compound
(1) normal for the growth of after fertilization 48h transgenic zebrafish embryo is moved in culture hole, add various dose
Vegf is added to after processing 4h in culture hole, then removes vegf, obtains vegf and processes zebrafish embryo；Sending out after fertilization 48h
Educate normal zebrafish embryo and be processed as normal zebrafish embryo without VEGF；
(2) one groups of vegf process zebrafish embryo successive administration and are incubated to 72hpf, as dosing group, another set vegf
Processing zebrafish embryo adds the culture water of equivalent to be incubated to 72hpf, and as vegf group, normal zebrafish embryo adds culture water
It is incubated to 72hpf, as Normal group；
(3) Brachydanio rerio of anesthesia dosing group, vegf group and Normal group, gathers every Brachydanio rerio subintestinal vein (sivs)
The fluorescence microscope images in region, outer rim vein length under the complete intestinal of every group of every fish of measurement；
(4) quantitative analyses
Under the complete intestinal of every group of Brachydanio rerio obtaining outer rim vein length withRepresent, com-parison and analysis Normal group,
The significance of difference between vegf group, dosing group；The results are shown in Table 1.
Table 1: outer rim vein length under various dose vegf complete intestinal to 72hpf Brachydanio rerio
| Group | Dosage (ng/ml) | Outer rim vein length (μm) under intestinal completely |
| Normal group | - | 646.5±83.1 |
| Vegf group | 1 | 663.9±88.9 |
| 2 | 716.2±91.1* | |
| 5 | 745.4±103.7** | |
| 8 | 796.6±154.3** | |
| 10 | 850.5±126.3** |
Note: *: vs Normal group, p < 0.05；*: vs Normal group, p < 0.01.
Result shows, processes after Brachydanio rerio using the vegf in 1-10ng/ml dosage range, outer rim vein under its complete intestinal
Length is gradually increased, and in the range of 2ng/ml, under the complete intestinal of Brachydanio rerio, outer rim vein length, compared with matched group, assumes significance poor
Different (p < 0.05)；In the range of 5-10ng/ml, under the complete intestinal of Brachydanio rerio, outer rim vein length, compared with matched group, presents extremely aobvious
Write sex differernce (p < 0.01).
Embodiment 2, for evaluating the method to angiogenesis function under pathological state for the compound
(1) normal for the growth of after fertilization 48h transgenic zebrafish embryo is moved in culture hole, the concentration of preparation is
The VEGF aqueous solution of 100ng/ml is added in culture hole, so that hole concentration of vascular endothelial growth factor is reached
10ng/ml, is jointly incubated 6h, then removes vegf, obtains vegf and processes zebrafish embryo；By normal for the growth of after fertilization 48h speckle
Horse fish embryo is processed as normal zebrafish embryo without VEGF；
(2) one groups of vegf process zebrafish embryo and add various dose Rhizoma Cyperi volatile oil sample to be incubated to 72hpf, as
Dosing group, another set vegf processes zebrafish embryo and adds the culture water of equivalent to be incubated to 72hpf, as vegf group, normally
Zebrafish embryo adds the culture water of equivalent to be incubated to 72hpf, as Normal group；
(3) Brachydanio rerio of anesthesia dosing group, vegf group and Normal group, gathers every Brachydanio rerio subintestinal vein (sivs)
The fluorescence microscope images in region, outer rim vein length under the complete intestinal of every group of every fish of measurement；
(4) quantitative analyses
Under the complete intestinal of every group of Brachydanio rerio obtaining outer rim vein length withRepresent, com-parison and analysis Normal group,
The significance of difference between vegf group, dosing group；
When dosing group Brachydanio rerioLess than matched group Brachydanio rerioAnd reach significance level statistically
(p < 0.05), illustrates that medicine to be measured is inhibited to the angiogenesis of pathological state.The addition pair of Rhizoma Cyperi volatile oil
Under Brachydanio rerio pathological state, the effect of angiogenesis is as shown in table 2 below.
Table 2: the effect to angiogenesis under Brachydanio rerio pathological state for the Rhizoma Cyperi volatile oil
| Group | Dosage | Outer rim vein length (μm) under intestinal completely |
| Normal group | - | 664.5±85.2 |
| Vegf group | 10ng/ml | 873.2±71.8## |
| Rhizoma Cyperi volatilization line of oils | 0.01μg/ml | 868.9±90.4 |
| 0.1μg/ml | 608.9±80.4** | |
| 1μg/ml | 555.3±100.5** |
Note: ##:vs Normal group, p < 0.01；*: vs vegf group, p < 0.05；*: vs vegf group, p < 0.01.
Embodiment 3, for evaluating the method to angiogenesis function under pathological state for the compound
(1) normal for the growth of after fertilization 48h transgenic zebrafish embryo is moved in culture hole, concentration is 100ng/ml
VEGF aqueous solution be added in culture hole, make hole concentration of vascular endothelial growth factor reach 5ng/ml,
Common incubation 12h, then removes vegf, obtains vegf and processes zebrafish embryo；By normal for the growth of after fertilization 48h zebrafish embryo
It is processed as normal zebrafish embryo without VEGF；
(2) one groups of vegf process zebrafish embryo and add various dose paeonol sample incubation to 72hpf, as dosing
Group, another set vegf processes zebrafish embryo and adds the culture water of equivalent to be incubated to 72hpf, as vegf group, normal zebra
Fish embryo adds the culture water of equivalent to be incubated to 72hpf, as Normal group；
(3) Brachydanio rerio of anesthesia dosing group, vegf group and Normal group, gathers every Brachydanio rerio subintestinal vein (sivs)
The fluorescence microscope images in region, outer rim vein length under the complete intestinal of every group of every fish of measurement；
(4) under the complete intestinal of every group of Brachydanio rerio that quantitative analyses obtain outer rim vein length withRepresent, com-parison and analysis are just
The significance of difference between normal matched group, vegf group, dosing group；
When dosing group Brachydanio rerioLess than matched group Brachydanio rerioAnd reach significance level statistically
(p < 0.05), illustrates that medicine to be measured is inhibited to the angiogenesis of pathological state.The addition of paeonol is to Brachydanio rerio
Under pathological state, the effect of angiogenesis is as shown in table 3 below.
Table 3: the effect to angiogenesis under Brachydanio rerio pathological state for the paeonol
| Group | Dosage | Outer rim vein length (μm) under intestinal completely |
| Normal group | - | 681.5±95.2 |
| Vegf group | 10ng/ml | 888.0±100.4## |
| Paeonol group | 0.01μg/ml | 859.4±90.8 |
| 0.1μg/ml | 689.4±93.8** | |
| 1μg/ml | 439.4±102.7** |
Note: ##:vs Normal group, p < 0.01；*: vs vegf group, p < 0.05；*: vs vegf group, p < 0.01.
Comparative example 1
With described in embodiment 2 for evaluating the method to angiogenesis function under pathological state for the compound, difference
It is:
By concentration, the VEGF aqueous solution for 100ng/ml is added in culture hole step (1), makes in hole
VEGF concentration reaches 10ng/ml, is jointly incubated 4h, then removes vegf, obtains vegf and processes zebrafish embryo；
Step (2) dosing group adds the reaction of various dose to stop sample incubation to 72hpf, and other are with embodiment 2.
Compare outer rim vein length two indices under subintestinal vein total length and complete intestinal and suppression disease is stopped for evaluation response
The difference of the effect of reason state angiogenesis.The results are shown in Table 4.
Table 4: reaction stops suppressing the effect of Brachydanio rerio subintestinal vein
Note: ##:vs Normal group, p < 0.01；*: vs vegf group, p < 0.05；*: vs vegf group, p < 0.01.
Result shows, stops processing after Brachydanio rerio using 1-25 μ g/ml reaction, finds for Brachydanio rerio subintestinal vein total length
Do not show the difference of significance；But obvious dose-effect relationship is then shown for outer rim vein length under complete intestinal, instead
The dosage that should stop is bigger, and inhibitory action is stronger, and after 5-25 μ g/ml dosage group is processed, under complete intestinal, outer rim vein length shows
Go out obvious difference (p < 0.05).
Comparative example 2
With described in embodiment 2 for evaluating the method to angiogenesis function under pathological state for the compound, difference
It is:
By concentration, the VEGF aqueous solution for 100ng/ml is added in culture hole step (1), makes in hole
VEGF concentration reaches 10ng/ml, is jointly incubated 4h, then removes vegf, obtains vegf and processes zebrafish embryo；
Step (2) dosing group adds the curcumenol sample incubation of various dose to 72hpf, and other are with embodiment 2.
Compare outer rim vein length two indices under subintestinal vein area and complete intestinal and suppress pathology for evaluating curcumenol
The difference of the effect of state angiogenesis.The results are shown in Table 5.
Table 5: curcumenol suppresses the effect of Brachydanio rerio subintestinal vein
Note: ##:vs Normal group, p < 0.01；*: vs vegf group, p < 0.05；*: vs vegf group, p < 0.01.
Result shows, is processed after Brachydanio rerio using 0.1-2.5 μ g/ml curcumenol, finds for Brachydanio rerio subintestinal vein face
The long-pending difference not showing significance；But obvious dose-effect relationship is then shown for outer rim vein length under complete intestinal,
The dosage of curcumenol is bigger, and inhibitory action is stronger, after 0.5-2.5 μ g/ml dosage group is processed, outer rim vein length under complete intestinal
Show obvious difference (p < 0.05).
Comparative example 3
With described in embodiment 2 for evaluating the method to angiogenesis function under pathological state for the compound, difference
It is:
By concentration, the VEGF aqueous solution for 100ng/ml is added in culture hole step (1), makes in hole
VEGF concentration reaches 10ng/ml, is jointly incubated 4h, then removes vegf, obtains vegf and processes zebrafish embryo；
Step (2) dosing group adds the Radix Curcumae extract of various dose to be incubated to 72hpf, and other are with embodiment 2.
Compare outer rim vein length two indices under subintestinal vein area and complete intestinal to suppress for evaluating Radix Curcumae extract
The difference of the effect of pathological state angiogenesis.The results are shown in Table 6.
Table 6: Radix Curcumae extract suppresses the effect of Brachydanio rerio subintestinal vein
| Group | Dosage | Spinous process quantity (root) | Outer rim vein length (μm) under intestinal completely |
| Normal group | - | 0 | 661.5±75.2 |
| Vegf group | 10ng/ml | 2.8±0.3 | 869.2±121.4## |
| Radix Curcumae extract group | 10μg/ml | 2.8±0.4 | 848.4±90.5 |
| 50μg/ml | 2.7±0.3 | 785.6±80.5* | |
| 100μg/ml | 2.6±0.4 | 435.6±100.7** |
Note: ##:vs Normal group, p < 0.01；*: vs vegf group, p < 0.05；*: vs vegf group, p < 0.01.
Result shows, is processed after Brachydanio rerio using 10-100 μ g/ml Radix Curcumae extract, finds for Brachydanio rerio subintestinal vein
Spinous process does not show the difference of significance；But outer rim vein length under complete intestinal is then shown that obvious dose-effect closes
System, the dosage of Radix Curcumae extract is bigger, and inhibitory action is stronger, and after 50-100 μ g/ml dosage group is processed, under complete intestinal, outer rim is quiet
Arteries and veins length shows obvious difference (p < 0.05).
Comparative example 4
(1) normal for the growth of after fertilization 48h transgenic zebrafish embryo is moved in culture hole, concentration is 100ng/ml
VEGF aqueous solution be added in culture hole, make hole concentration of vascular endothelial growth factor reach 10ng/ml,
Common incubation 4h, then removes vegf, obtains vegf and processes zebrafish embryo；By normal for the growth of after fertilization 48h zebrafish embryo
It is processed as normal zebrafish embryo without VEGF；
(2) one groups of vegf process zebrafish embryo and add the curcumin sample incubation of various dose to 72hpf, as dosing
Group, another set vegf processes zebrafish embryo and adds the culture water of equivalent to be incubated to 72hpf, and as vegf group, one group normal
Zebrafish embryo adds the culture water of equivalent to be incubated to 72hpf, as Normal group；The normal zebrafish embryo of another set adds
The curcumin entering equivalent is incubated to 72hpf, as normal experimental group；
(3) anesthesia dosing group, the Brachydanio rerio of vegf group, Normal group and normal experimental group, gather every Brachydanio rerio intestinal
The fluorescence microscope images in lower vein (sivs) region, outer rim vein length under the complete intestinal of every group of every fish of measurement；
(4) quantitative analyses
Under the complete intestinal of calculated every group of Brachydanio rerio outer rim vein length withRepresent, com-parison and analysis normal control
The significance of difference between group, vegf group, normal experimental group, Pathological experiment group.The results are shown in Table 6.
Utilize the effect of the suppression endotheliocyte of endotheliocyte description of test curcumin itself simultaneously.
Experiment is as follows: well-grown endotheliocyte (huvec) is with the mixing of 0.25% trypsin and 0.02%edta
Digestion.It is 2 × 10 with culture fluid adjustment cell density9Individual/l, be seeded on the coverslip in 6 well culture plates and 48 orifice plates in,
In culture plate after culture 24-48h, existing 80% cell is in converging state, changes serum-free m199 synchronization 24h, and huvec is divided
For 6 groups, add curcumin, be divided into blank control group, curcumin group.Using mtt method, observe curcumin to huvecs multiplication capacity
Impact, using crystal violet staining assay detection huvecs sticked and transfer ability impact.The results are shown in Table 7.
Table 6: using the experimental result contrast after vegf before processing
| Group | Dosage | Outer rim vein length (μm) under intestinal completely |
| Normal group | - | 661.5±75.2 |
| Vegf group | 10ng/ml | 869.2±121.4## |
| Normal experimental group | 6.25μg/ml | 634.7±80.6 |
| 25μg/ml | 630.3±90.6 | |
| 100μg/ml | 623.1±77.8 | |
| Pathological experiment group | 6.25μg/ml | 848.4±90.5 |
| 25μg/ml | 785.6±80.5* | |
| 100μg/ml | 635.6±100.7** |
Note: ##:vs Normal group, p < 0.01；*: vs vegf group, p < 0.05；*: vs vegf group, p < 0.01.
Table 7: the impact that under corresponding dosage, curcumin endothelial cell proliferation sticks and migrates
| Group | Dosage | Propagation (od value) | Stick 30min (od) | Migration 1d (od) |
| Normal group | - | 0.221±0.019 | 0.085±0.003 | 0.149±0.017 |
| Curcumin group | 6.25μg/ml | 0.220±0.027 | 0.088±0.02 | 0.147±0.015 |
| 25μg/ml | 0.203±0.015** | 0.081±0.007* | 0.137±0.018* | |
| 100μg/ml | 0.198±0.015** | 0.074±0.0038** | 0.113±0.006** |
Note: *: vs vegf group, p < 0.05；*: vs vegf group, p < 0.01.
Result shows, in 6.25-100 μ g/ml test dose, 25-100 μ g/ml curcumin is thin for classical endothelium
Born of the same parents show obvious inhibition of endothelial cell proliferation, the effect sticked and migrate.But in Brachydanio rerio experiment, it is being not added with vegf
Under the normal Brachydanio rerio state processing, same dose of curcumin does not show the effect of obvious inhibition of angiogenesis, but
Being in adding the zebra fish model that vegf was processed it is shown that significantly suppressing the activity of angiogenesis, further illustrating
The reliability of the evaluation methodology of the present invention and accuracy.
Claims (10)
1. a kind of for evaluate compound to the method for angiogenesis function under pathological state it is characterised in that include step such as
Under:
(1) normal for the growth of after fertilization 48h transgenic zebrafish embryo is moved in culture hole, add VEGF
Processed, obtain vegf and process zebrafish embryo；By normal for the growth of after fertilization 48h zebrafish embryo without vascular endothelial growth
Factor treatment is normal zebrafish embryo；
(2) one groups of vegf process zebrafish embryo successive administration and are incubated to 72hpf, as dosing group, the process of another set vegf
Zebrafish embryo adds the culture water of equivalent to be incubated to 72hpf, and as vegf group, normal zebrafish embryo adds culture water incubation
To 72hpf, as Normal group；
(3) Brachydanio rerio of anesthesia dosing group, vegf group and Normal group, gathers every Brachydanio rerio subintestinal vein (sivs) region
Fluorescence microscope images, measurement every group of every fish complete intestinal under outer rim vein length；
(4) quantitative analyses
Under the complete intestinal of every group of Brachydanio rerio obtaining outer rim vein length withRepresent, com-parison and analysis Normal group, vegf group,
The significance of difference between dosing group；
When dosing group Brachydanio rerioLess than vegf group Brachydanio rerioAnd reach significance level statistically (p <
0.05), illustrate that the angiogenesis that medicine to be measured processes Brachydanio rerio to vegf are inhibited.
2. according to claim 1 for evaluating the method to angiogenesis function under pathological state for the compound, its feature
It is, in step (1), described zebrafish embryo is blood vessel fluorescently-labeled transgenic zebrafish embryo.
3. according to claim 1 for evaluating the method to angiogenesis function under pathological state for the compound, its feature
It is, in step (1), the concentration of culture fluid VEGF is 1-10ng/ml it is preferred that culture fluid medium vessels
The concentration of endothelial cell growth factor (ECGF) is 5-10ng/ml.
4. according to claim 1 for evaluating the method to angiogenesis function under pathological state for the compound, its feature
It is, in step (1), VEGF needs before adding zebrafish embryo is carried out with de- egg membrane treatment.
5. according to claim 4 for evaluating the method to angiogenesis function under pathological state for the compound, its feature
It is, de- egg membrane treatment is specially to be added to zebrafish embryo in the pronase e solution of concentration 1-2mg/ml and keeps 2-
5min, you can complete de- egg membrane treatment.
6. according to claim 1 for evaluating the method to angiogenesis function under pathological state for the compound, its feature
It is, in step (1), the concrete processing method that vegf processes zebrafish embryo is to add VEGF aqueous solution
Enter in culture hole, make hole concentration of vascular endothelial growth factor reach 1-10ng/ml, be jointly incubated 4-12h, cause new hemopoietic
The pathological state of the paraplasm of pipe, then removes vegf；Preferably, common incubation time is 4-6h；It is further preferred that blood
The concentration of endothelial tube somatomedin aqueous solution is 90～100ng/ml.
7. according to claim 1 for evaluating the method to angiogenesis function under pathological state for the compound, its feature
Be, in step (2), the zebrafish embryo of dosing group, vegf group and Normal group in 28 DEG C of control optical cultures be incubated to
72hpf, every group of Brachydanio rerio 10-20 bar.
8. according to claim 1 for evaluating the method to angiogenesis function under pathological state for the compound, its feature
It is, in step (3), the anesthesia of Brachydanio rerio is to soak 40～90s using the tricaine that mass concentration is 0.3 ‰ to be anaesthetized；
In step (3), collection image is by zebrafish embryo fixing side Postural immobilization, in fluorescence microscopy Microscopic observation, takes pictures, and obtains
The side bit image of zebrafish embryo；It is further preferred that in step (3), the image of collection is to obtain under same amplification
, picture format is jpg form.
9. according to claim 1 for evaluating the method to angiogenesis function under pathological state for the compound, its feature
It is, in step (3), measurement length is using image processing software image j, the image of collection to be measured, before measurement first
Calibration scale, obtains accurate length.
10. the medicine to be measured that the method described in claim 1 filters out is Rhizoma Cyperi volatile oil, cyperol or paeonol.
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