The specific embodiment
Below in conjunction with Figure of description and embodiment, the present invention is further elaborated, but protection scope of the present invention is not limited to this.
The Brachydanio rerio initialism of being correlated with
After fertilization hourage: hpf-hours postfertilization
Back of the body major axis blood vessel: DLAV-dorsal longitudinal anastomotic vessel
Blood vessel between body segment: ISV-intersegmental vessel
Dorsal aorta: DA-dorsal aorta
Posterior cardinal vein: PCV-posterior cardinal vessel
Subintestinal vein blood vessel: SIV-subintestinal vessel
Green fluorescent protein: GFP-green fluorescent protein
embodiment 1the inhibition of qualitative observation mesnaum to blood vessel (ISV) generation model between the Brachydanio rerio body segment
Brachydanio rerio:
The Brachydanio rerio that the present embodiment is used is blood vessel transgenic green fluorescence Brachydanio rerio (a kind of gene of being expressed by the Brachydanio rerio endothelial-cell specific drives green fluorescent protein at Brachydanio rerio vascular endothelial cell specifically expressing as driven element), and raising and Application standard strictly carry out with reference to U.S.'s management of laboratory animal and the requirement of using committee (IACUC).
The water (Fish water) of breeding fish:
Compound method: 1L reverse osmosis water (reverse osmosis (RO) water) adds 0.3g sea salt (Instant Ocean salts).
Dimethyl sulfoxide (DMSO, analytical pure):
Buy in Shanghai Jing Chun Industrial Co., Ltd. (article No. #1095515, lot number #30573).0.1% DMSO solution (negative control) preparation: during use, with the water of breeding fish, be mixed with the working solution that concentration is 0.1%, now with the current.
Lovastatin (positive control):
Buy the U.S. logical sequence in Dalian, purity is greater than 98% (HPLC method).During use, with 0.1% DMSO solution preparation, become the required concentration of experiment, in this experiment, the working concentration of positive control drug is 10 μ M.
Mesnaum is bought the company in Sigma-Aldrich, becomes the mesnaum solution of variable concentrations during use with 0.1% DMSO solution preparation, and working concentration is 10 μ M.
The Brachydanio rerio vascular endothelial cell sprouts from after fertilization 20hpf, and the 30-31hpf left and right forms blood vessel network between main body segment, and as blood vessel (ISV) between back of the body major axis blood vessel (DLAV) and body segment, 48hpf forms complete axon blood vessel network basically
[30], blood vessel (ISV) between high-visible complete body segment now.Between complete body segment, blood vessel mainly refers to connect that section blood vessel between (DLAV) between dorsal aorta (DA) and back of the body major axis blood vessel, sees
fig. 1(vascular pattern between 48hpf blood vessel transgenic fluorescence Brachydanio rerio body segment).Experimental technique is as follows:
(1) experiment grouping and embryo process: get 45 well-developed zebrafish embryos, during fetal development, be after fertilization 23hpf (hour-postfertilization mutually, hpf), be divided at random 3 groups of (negative control group, the mesnaum processed group, positive controls), every group of embryo's quantity is 15.During operation by embryo's uniform distribution to 48 porocyte culture plate (Greiner, Germany), 15, every hole embryo, every hole embryo raises water 1ml.
(2) drug treating: the medicinal liquid that will prepare in advance rapidly with pipettor (range 100~1000 μ l, Eppendorf) adds Zhong,Mei hole, the hole 1ml that 48 porocyte culture plates are corresponding.Before adding medicinal liquid, with pipettor (range 10~1000 μ l, Eppendorf), the raising water of hatching the embryo in 48 orifice plates is shifted out as possible, this operation needs to complete in advance at short notice, to prevent embryo's drying.The experimental situation temperature is controlled at 28.5 ℃ of left and right, relative humidity 40~70%.Then with masking foil, 48 orifice plates are wrapped, carry out the experiment labelling, be positioned over rapidly in the Brachydanio rerio incubator and continue to cultivate 24h (the incubator temperature is controlled at 28.5 ± 0.5 ℃).
(3) Phenotypic Observation and statistics: observe each hole embryo's phenotype under Stereo microscope, observation index: observe the impact of medicine on aspects such as fetal development, blood circulation and heartbeats.Then, the affected embryo of blood circulation being placed under body formula fluorescence microscope (Nikon AZ100 body formula fluorescence microscope) and further observing and take pictures, is 48hpf mutually while taking pictures, and to confirm angiogenesis, suppresses phenotype.
Experimental result is shown in
fig. 2: with negative control, compare, 25 μ M mesnaum can suppress the generation of blood vessel between the Brachydanio rerio body segment (ISV), show as part intersegmental blood vessel disappearance.
embodiment 2the inhibition of qualitative observation mesnaum to blood vessel (SIV) generation model under the Brachydanio rerio intestinal
Blood vessel under the Brachydanio rerio intestinal (SIV, subintestinal vessel) is grown in the yolk sac both sides, and its shape is like one basket, and blood vessel under intestinal (SIV) is about 50~100 μ m by the body segment veutro to the length of downward-extension
[15-16].See
fig. 3(vascular pattern under 72hpf blood vessel transgenic fluorescence Brachydanio rerio intestinal).Experimental technique is as follows:
(1) experiment grouping and embryo process: get 45 well-developed zebrafish embryos, during fetal development, be after fertilization 48hpf (hour-postfertilization mutually, hpf), be divided at random 3 groups of (negative control group, the mesnaum processed group, positive controls), every group of embryo's quantity is 15.During operation by embryo's uniform distribution to 48 porocyte culture plate (Greiner, Germany), 15, every hole embryo, every hole embryo raises water 1ml.
(2) drug treating: see the experimental technique operating procedure (2) in embodiment 1.
(3) Phenotypic Observation and statistics: observe each hole embryo's phenotype under Stereo microscope, observation index: observe medicine to fetal development, blood circulation, the impact of the aspects such as heartbeat.Then, the affected embryo of blood circulation being placed under body formula fluorescence microscope (Nikon AZ100 body formula fluorescence microscope) and further observing and take pictures, is 72hpf mutually while taking pictures, and to confirm angiogenesis, suppresses phenotype.
Experimental result is shown in
fig. 4: with negative control, compare, 25 μ M mesnaum significantly suppress the generation of blood vessel under the Brachydanio rerio intestinal (SIV), show as blood vessel area under intestinal and reduce.
embodiment 3the inhibition of quantitative assessment mesnaum to blood vessel (SIV) generation model under the Brachydanio rerio intestinal
experimental technique:
(1) experiment grouping and embryo process: getting 240 well-developed zebrafish embryos, is after fertilization 48hpf (hour-postfertilization, hpf) mutually during fetal development, is divided at random 8 groups, sees the following form:
30 of every group of zebrafish embryo quantity.During operation by embryo's uniform distribution to 48 porocyte culture plate (Greiner, Germany), 15, every hole embryo, each drug level is processed 30 embryos, every hole embryo raises water 1ml.
(2) drug treating: see the experimental technique operating procedure (2) in embodiment 1.
(3) Phenotypic Observation and quantitative statistics: the embryo after each drug level is processed observes and takes pictures under body formula fluorescence microscope (Nikon AZ100 body formula fluorescence microscope), while taking pictures, be 72hpf mutually, impact blood vessel (SIV) under the Brachydanio rerio intestinal generated to analyze each drug level.Get at random 10 embryos from each experimental group and carry out quantitative statistics, statistical indicator is as follows:
1. blood vessel area (SIV area) under intestinal: utilize Nikon AZ100 body formula fluorescence microscope configuration
NIS-Elements 3.1 softwares are calculated
Utilize GraphPad Prism software to add up mapping, and calculate mesnaum and suppress the IC that under the Brachydanio rerio intestinal, blood vessel (SIV) generates
50, experimental result is shown in
fig. 5: the suppression ratio that mesnaum generates blood vessel (SIV) under the Brachydanio rerio intestinal presents step increase along with the rising of concentration, the suppression ratio that each concentration mesnaum group generates blood vessel (SIV) under the Brachydanio rerio intestinal is respectively: 0.25 μ M(6.9%), 0.5 μ M(18%), 1 μ M(39%), 2.5 μ M(48%), 10 μ M(69%), 25 μ M(85%).
embodiment 4brachydanio rerio Human colon cancer (Colo320) transplantation model is estimated the antitumor drug effect of mesnaum
The growth of entity tumor and diffusion depend on the formation of neovascularity in tumor, and obtain nutrient by new vessels; The formation of neovascularity and growth, promoted the transfer of tumor cell.The present embodiment is for illustrating that mesnaum can suppress growth and the migration of tumor.Experimental technique is as follows:
(1) experiment grouping and embryo process: getting the zebrafish embryo that 150 transplanting have Human colon cancer (Colo320) cell, is after fertilization 2dpf (day-postfertilization, dpf) mutually during fetal development, is divided at random 5 groups, sees the following form:
30 of every group of zebrafish embryo quantity.During operation by embryo's uniform distribution to 6 porocyte culture plate (Greiner, Germany), 30, every hole embryo, each drug level is processed 30 embryos, every hole embryo raises water 3ml.
(2) drug treating: with pipettor, rapidly pre-configured medicinal liquid is added to Zhong,Mei hole, the hole 3ml that 6 porocyte culture plates are corresponding.Then with masking foil, 6 orifice plates are wrapped, carry out the experiment labelling, be positioned in the Brachydanio rerio incubator and continue to cultivate 4d (the incubator temperature is controlled at 35.5 ± 0.5 ℃).
(3) Phenotypic Observation and quantitative statistics: the embryo after each concentration drug treating is observed and takes pictures under body formula fluorescence microscope (Nikon AZ100 body formula fluorescence microscope), while taking pictures, be 6dpf mutually, to analyze the inhibitory action of each drug level to Brachydanio rerio Human colon cancer (Colo320) transplantation model.Get at random 10 embryos from each experimental group and carry out quantitative statistics, statistical indicator is as follows:
1. the inhibitory action of qualitative evaluation mesnaum to neoplasm metastasis;
2. the inhibitory action of quantitative assessment mesnaum to tumor growth: utilize Nikon NIS-Elements 3.1 computed in software tumor cell fluorescence intensities (S), the statistical procedures result means with mean ± SE; Mesnaum is as follows to the inhibition computing formula of tumor growth:
Utilize GraphPad Prism software to add up mapping, experimental result is shown in
fig. 6~Fig. 7: mesnaum presents step increase to the suppression ratio of mankind's carcinoma transplanted Growth of Cells along with the rising of concentration, three concentration mesnaum group suppression ratio are respectively: 1 μ M(10.3%), 2.5 μ M(21.2%), 10 μ M(38.9%).
embodiment 5the quantitative assessment mesnaum is looked the therapeutical effect of degeneration of macula to moist old age
Look degeneration of macula moist old age main because choroidal artery generates extremely, seepage appears in newborn invalid blood capillary, the liquid of vascular leakage and then destruction macula lutea.Cobaltous chloride can be induced Brachydanio rerio retina choroid plexus blood vessel hyperplasia, visual cell degeneration, is similar to the change that the mankind look degeneration of macula moist old age
[31].The present embodiment is for illustrating that mesnaum has therapeutic effect to looking degeneration of macula moist old age.Experimental technique is as follows:
(1) experiment grouping and embryo process: getting 150 well-developed zebrafish embryos, is after fertilization 1dpf (day-postfertilization, dpf) mutually during fetal development, is divided at random 5 groups, sees the following form:
30 of every group of zebrafish embryo quantity.During operation by embryo's uniform distribution to 6 porocyte culture plate (Greiner, Germany), 30, every hole embryo, every hole embryo raises water 3ml.
(2) drug treating: add DMSO in negative control group, making its final concentration is 0.1%; Add cobaltous chloride in model group, making its final concentration is 1 mg/ml; Mesnaum, by the administration of microinjection mode, is all injected 30 embryos for every group, after injection, the embryo is put into respectively to the raising water that 3ml contains 1 mg/ml cobaltous chloride by group.
(3) Phenotypic Observation and quantitative statistics: the embryo after each dose drug is processed observes and takes pictures under body formula fluorescence microscope (Nikon AZ100 body formula fluorescence microscope), while taking pictures, be 5dpf mutually, to analyze the inhibitory action of each drug dose to Brachydanio rerio eye choroidal abnormalities hypertrophy blood vessel.Get at random 10 embryos from each experimental group and carry out quantitative statistics, statistical indicator is as follows:
1. the inhibitory action of qualitative evaluation mesnaum to eye choroidal abnormalities hypertrophy blood vessel;
2. the inhibitory action of quantitative assessment mesnaum to choroidal abnormalities hypertrophy blood vessel: utilize NIS-Elements 3.1 computed in software choroidal abnormalities hypertrophy blood vessel fluorescence intensities (S), the statistical procedures result means with mean ± SE; Mesnaum is as follows to the inhibition computing formula of choroidal abnormalities hypertrophy blood vessel:
Utilize GraphPad Prism software to add up mapping, experimental result is shown in
fig. 8~Fig. 9: mesnaum presents step increase to the paraplasm suppression ratio of choroidal artery along with the rising of dosage, three mesnaum dosage group suppression ratio are respectively: 0.16 μ g(10.5%), 0.55 μ g(25.7%), 1.64 μ g(34.3%).
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