CN109906979A - A kind of method of zebra fish high cholesterol model foundation - Google Patents
A kind of method of zebra fish high cholesterol model foundation Download PDFInfo
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Abstract
The present invention relates to a kind of construction methods of zebra fish high cholesterol model, it includes the following steps: that S1, zebra male and female adult fish are placed in mating box with the ratio of 1:2~2:3, post-coitum receives ovum, by the ovum of health in 28 DEG C of constant temperature incubations, selects healthy zebra fish juvenile fish after 5 days under microscope；S2, cholesterol is added in yolk powder make high cholesterol diet, will not add the yolk powder of cholesterol as normal diet, for raising the health zebra fish juvenile fish；S3, after zebra fish juvenile fish is continuously cultivated with high cholesterol diet, while with normal diet feed zebra fish juvenile fish as a control group, zebra fish blood vessel cholesterol accumulation conspicuousness be higher than control group when, zebra fish high cholesterol model is built up.The method for the zebra fish high cholesterol model that the present invention constructs has many advantages, such as economical, easy, stable, reliable, and zebra fish high cholesterol model can be used for the directions such as drug screening, toxicity evaluation and the Mechanism Study of hyperlipidemia and its complication.
Description
Technical field
The present invention relates to a kind of methods of zebra fish high cholesterol model foundation, belong to biomedicine technical field.
Background technique
Hyperlipemia is a kind of chronic disorders of lipid metabolism disease, is the Major Risk Factors that cardiovascular disease occurs.?
It is a kind of important risk factor of impaired glucose tolerance, hypertension, diabetes, it is hard can also result in fatty liver, hyperuricemia, liver
The diseases such as change, pancreatitis, cholelithiasis, fundus hemorrhage, peripheral vascular disease, blindness, limping.Cholesterol Model is established as cholesterol
The prevention and treatment of disease disease associated therewith and the research of pathogenesis provide important tool.
At present both at home and abroad about there are many method for building up of high blood lipid model, most common model animal is mouse and rabbit.Mouse
It is widely used, easily acquisition, easily raising, physiological structure and the mankind are very much like.Zhu Lei etc. is by SD feeding rats different formulations
High lipid food with confirm obese model establish the best approach, finally think 45% rouge energize high lipid food feeding 6 weeks be most
Economic modeling scheme.Chen Jianfeng etc. adds the hardness and edibility that bentonite improves high lipid food in high lipid food, it was demonstrated that
Bentonite high lipid food induction SD rat hyperlipemia is better than common high lipid food group after feeding 8 weeks.Rabbit is dynamic for phytophage
Object, and researchers very sensitive to high fat diet establish the object of high blood lipid model preference.Formula and feeding due to feed
Feeding method is different, and modeling length of time also varies with each individual.You Xiaoqing etc. thinks that new zealand rabbit induces 6 Zhou Kecheng through high lipid diet
Function establishes high blood lipid model, and Wu Yunhong etc. is fed 5 weeks through autogamy high lipid food using healthy male New Zealand rabbit and established
High blood lipid model.Pika respectively has a unique advantage in terms of high blood lipid model foundation, but its unavoidable period it is long, it is at high cost,
It is not easy the disadvantage of somatoscopy and large scale experiment.High lipid food is that high blood lipid model establishes Foods, for mammal
As long as will be created as over a period to come with high lipid food feeding high blood lipid model is carried out to its long-time for birds
Function, and its vital movement can't be seriously affected during testing.And zebra fish juvenile fish figure baby is fragile, and lives in food
In the water environment of object, the feeding volume and feeding time of food are possible to influence the healthy growth of juvenile fish.Although also having at present few
Number scholar establishes high blood lipid model using zebra fish, but there are drawbacks for method, are primarily present after detection, illustrate model foundation
Success, but zebra fish has been dead volume, it is impossible to be used in Subsequent pharmacological screening.The present invention mainly probes into a kind of reliable and stable spot
Horse fish high cholesterol method for establishing model is living body after model foundation detection, is drug screening, the poison of hyperlipidemia and its complication
Property assessment and Mechanism Study provide important tool.
Summary of the invention
(1) technical problems to be solved
In order to solve the above problem of the prior art, the present invention provides a kind of side of zebra fish high cholesterol model foundation
Method.
(2) technical solution
In order to achieve the above object, the main technical schemes that the present invention uses include:
A kind of method of zebra fish high cholesterol model foundation comprising following steps:
S1, zebra fish male and female adult fish are placed in mating box with the ratio of 1:2~2:3, and post-coitum receives ovum, by the ovum of health 28
DEG C constant temperature incubation, selects healthy zebra fish juvenile fish later under microscope；
S2, cholesterol production high cholesterol diet is added in yolk powder, will not add the yolk powder of cholesterol as general
Logical feed, for raising the healthy zebra fish juvenile fish；
S3, after zebra fish juvenile fish is continuously cultivated with high cholesterol diet, while with normal diet feed zebra fish children
As a control group, when zebra fish blood vessel cholesterol accumulation conspicuousness is higher than control group, zebra fish high cholesterol model is built up fish.
Wherein, zebra fish blood vessel cholesterol accumulation conspicuousness be higher than control group when, refer to t detection statistics analysis P <
0.05。
Be preferably carried out in scheme one, in step sl, time of the mating be 1.5h after receive ovum, the culture
Time is 5 days.
It is preferably carried out in scheme one, in step s 2, cholesterol is in yolk powder in the high cholesterol diet
Additive amount is mass percent 2.5%~12.5%.
It is preferably carried out in scheme one, in step s 2, the daily feeding volume of the high cholesterol diet is
1.4mg/ tail~3.2mg/ tail.
Most preferably, additive amount of the cholesterol in yolk powder is mass percent 5%, institute in the high cholesterol diet
The daily feeding volume for stating high cholesterol diet is 2mg/ tail.
It is preferably carried out in scheme one, in step s 2, cholesterol tracer is also added in the high cholesterol diet
Fluorescence cholesteryl ester is similar to object addition than being 10 μ g/g.
Further, the cholesterol tracer fluorescence cholesteryl ester is CHOLESTERYL BODIPY 524/ similar to object
563C11 is that 524,563nm is detected in wavelength, generates red fluorescence, is 10 μ g/g in feed addition ratio, ether is organic
Cosolvent, be protected from light down makes ether volatilize completely in vent cabinet ventilation process 20h.
Be preferably carried out in scheme one, in step s3, it is described it is continuous culture for 28 DEG C constant temperature incubation 8~10 days.
It is preferably carried out in scheme one, it is glimmering using the cholesterol tracer added with 10 μ g/g is only fed in step s3
Light cholesteryl ester is control group similar to the yolk powder of object, and burnt or Stereo fluorescence microscope is copolymerized after culture and observes juvenile fish tail portion in real time
Blood vessel cholesterol accumulation, if its tail veins cholesterol accumulation is substantially less than High cholesterol diet group (P < 0.05), explanation
High cholesterol diet induction juvenile fish blood lipid increases successfully.
It is preferably carried out in scheme one, in step s 2, the yolk powder is that Fresh Egg is cooked, yolk is taken out,
Freeze-dryer handles to obtain yolk dry powder after crushing.
(3) beneficial effect
The beneficial effects of the present invention are:
The method of zebra fish high cholesterol model foundation provided by the invention, using AB system wild type and transgenosis Fli1-
For EGFP zebra fish juvenile fish as experimental animal, the cholesterol that different weight percentage is added into pure yolk dry powder is configured to high cholesterol
Feed, using copolymerization is burnt or Stereo fluorescence microscope observe in real time with the intracorporal physiological and biochemical index of juvenile fish as hyperlipemia model
Evaluation measures.Research, which confirms to increase cholesterol percentage in feed, can cause juvenile fish blood lipid to increase, and increases feed in right amount
Amount can accelerate juvenile fish vascular lipid deposition；5% High cholesterol diet model group feeds 30 tail zebras in 70ml culture dish
Fish feeds 30mg every time, and high cholesterol zebra fish model can be successfully established by feeding 8~10 days.
Compared with prior art, the high cholesterol zebra fish model of foundation of the invention can be in living body, for dropping
The experiment of rouge drug is observed in real time；The present invention can control height according to control feed cholesterol percentage composition and feedstuff feeding amount
Pionemia models the time, and then probes into or environmental contaminants are acute or subacute sudden and violent for acute or subacute fat-reducing medicament
To the assessment of lipid metaboli poisonous effect in organism when dew.
Detailed description of the invention
Fig. 1 is transgenosis Fli1-EGFP zebra fish juvenile fish tail veins lipidosis situation；Wherein, A. normal diet pair
According to the confocal images of group (Control) and 5% High cholesterol diet model group (5%HCD)；B. zebra fish juvenile fish exists
It adds after being raised 10 days under cholesterol (2.5%, 5%, 7.5%, 10% and 12.5%) feed of different weight percentage, region of interest
Relative value (n=3) of the interior red fluorescence OD average to normal diet control group；C is control under Stereo fluorescence microscope
Group (Control) and 5% Cholesterol Model group (5%HCD) juvenile fish tail veins lipidosis, red are region of interest away from frame；
D. phase of the red fluorescence OD average to normal diet control group in 5% Cholesterol Model group region of interest in figure C
To value (n=25)；
Fig. 2A be 5dpf transgenosis Fli1-EGFP zebra fish juvenile fish through different feeding volumes 5% high cholesterol diet (7,
21,35,49,63 and 77mg) after nursing 1 day, the relative value of red fluorescence OD average in region of interest；
Fig. 2 B be 5dpf wild type AB system zebra fish juvenile fish through different feeding volumes 5% high cholesterol diet (7,21,35,
49,63 and 77mg) feed 10 days after survival rate；
Fig. 3 is the 5% High cholesterol diet influence schematic diagram wide to young fish length body；Wherein, A be normal diet group and
The average body of 5% Cholesterol Model group juvenile fish is long, and B is wide for the average body of normal diet group and 5% Cholesterol Model group juvenile fish, C
For measurement method (n=15)；
Fig. 4 is influence schematic diagram of 5% High cholesterol diet to biochemical indicator in juvenile fish body.
Specific embodiment
In order to preferably explain the present invention, in order to understand, with reference to the accompanying drawing, by specific embodiment, to this hair
It is bright to be described in detail.
Embodiment 1
A kind of method of zebra fish high cholesterol model foundation, specifically with the following method:
1. zebra fish selects
Healthy AB system male and female zebra adult fish is placed in mating box with the ratio of 2:1, is separated with partition.8 points of morning next day takes
Partition is opened, receives ovum after the 1.5h that mates.The ovum for choosing health under microscope is placed in 28 DEG C of constant temperature incubations in 1 × E3 culture solution, daily
A water is changed, selects healthy zebra fish juvenile fish for testing under microscope after 5 days.
It tests transgenosis Fli1-EGFP zebra fish juvenile fish used to obtain:, fertilization identical with fish method as wild-type zebrafish
The zebra fish of (48hpf, hours post fertilization) picks out blood vessel tool with Stereo fluorescence microscope after 48 hours
There is the healthy zebra fish juvenile fish of egfp expression to be placed in 28 DEG C of constant incubators to continue to cultivate.After being fertilized 5 days
Healthy transgenic zebrafish juvenile fish sub-elects for testing.
2. prepared by high cholesterol diet
Fresh Egg is cooked, yolk is taken out, crushing is dispensed into 50mL centrifuge tube, and freeze-dryer is handled 3 days and obtained
Yolk dry powder adds the cholesterol of certain mass percent (2.5%, 5%, 7.5%, 10% and 12.5%) in yolk dry powder
It makes high cholesterol diet (HCD, high cholesterol diet), pure yolk dry powder is normal diet, and ether is hydrotropy
Agent, high cholesterol diet and normal diet all pass through ether volatilization processing.Height used in transgenosis Fli1-EGFP zebra fish juvenile fish
Cholesterol feed adds a kind of similar object CHOLESTERYL of cholesterol tracer red fluorescence cholesteryl ester with normal diet
BODIPY 524/563C11 (Invitrogen), adding proportion are added by 10 μ g/g, and ether is organic cosolvent.Logical
Magnetic agitation 45min, ventilation process make ether volatilize completely in wind cupboard.
3. zebra fish juvenile fish confocal fluorescent or stereoscopic fluorescence are taken pictures
(1) experimental method
Fli1-EGFP zebra fish juvenile fish is randomly divided into normal diet group (cholesterol 0%) and High cholesterol diet (contains
2.5%, 5%, 7.5%, 10% and 12.5% cholesterol) model group, every group of 30 tail fishes, 70mL1 × E3 culture, each feeding
30mg, daily feeding twice, 28 DEG C of constant temperature incubations.After continuous feed 10 days, normal diet is fed with methyl cellulose gel
The zebra fish flat sides supported in group or High cholesterol diet model group are fixed on glass slide, aobvious with copolymerization coke or stereoscopic fluorescence
Micro mirror carries out living body Image Acquisition to juvenile fish tail portion, and with imageJ software or Zeiss ZEN analysis software to specific caudal artery
The red fluorescence optical density of region of interest carries out data processing.
(2) experimental result
(a), High cholesterol diet accumulates zebra fish juvenile fish caudal artery vascular lipid and influences
It is as shown in Figure 1A normal diet control group (Control) and 5% High cholesterol diet model group (5%HCD)
Confocal microscopy image, wherein EGFP indicates that reinforced green fluorescent protein tracer blood vessel, Dil indicate red fluorescence cholesterol
Tracer tracer blood vessel inner cholesterol, Merge indicate the two fluorescent marker blending image.Figure 1B data are from Confocal Images
Processing, being expressed as High cholesterol diet model group, (mass percent containing cholesterol is 2.5%, 5%, 7.5%, 10% and
12.5%) with the ratio of normal diet group (0%) juvenile fish caudal artery blood vessel region of interest red fluorescence OD average, scheme
Middle a, b, c indicate significance otherness, P < 0.05.The results show that compared with normal diet group, high cholesterol diet model
The extremely significant property (p < 0.01) of the relative value of group OD average increases, and with the cholesterol added in high cholesterol diet
The increase of ratio, OD average are consequently increased.
(b), the 5dpf transgenosis Fli1-EGFP zebra fish juvenile fish of health simultaneously, is randomly divided into normal diet control group
(Control) it is selected with 5% cholesterol diet model group (5%HCD), every group of 30 tails, 70mL1 × E3 culture, each feeding volume
30mg, daily feeding is twice.It is deposited after continuous feed 10 days using vascular lipid of the fluorophor microscope to zebra fish juvenile fish
Amount has carried out Image Acquisition, and as a result as shown in Figure 1 C, this figure indicates control group (Control) and 5% Cholesterol Model group (5%
HCD) juvenile fish tail veins lipidosis, green fluorescence come from vascular endothelial cell, and red fluorescence comes from vascular lipid, it is red away from
Frame is region of interest.It can intuitively observe that 5%HCD red fluorescence optical density is greater than in the region of interest of zebra fish tail portion in figure
Control group.Fig. 1 D data are expressed as 5% model group and normal diet group children from Stereo fluorescence microscope image procossing
The ratio of fish tail portion arteries region of interest red fluorescence OD average, focusing results are similar together for result, model
Group juvenile fish caudal artery blood vessel region of interest red fluorescence OD average is 1.2 times of normal diet group, and shows pole
Significant difference (p < 0.01, n=25).The above result shows that 5% High cholesterol diet can promote zebra fish vascular lipid product
It is tired.
2 high cholesterol diet feeding volume of embodiment, which changes, influences zebra fish juvenile fish body vessel lipid level
In the present embodiment, the transgenosis Fli1-EGFP spot of (5dpf, days post fertilization) after fertilization 5 days
Horse fish juvenile fish is randomly divided into 6 groups, and every group of 30 tail fishes, every group feeds 5% high cholesterol diet, 7,21,35,49,63 and every time respectively
77mg.Twice, 1 × E3 of 70mL is cultivated daily feeding.It is as shown in Figure 2 A different feeding volume juvenile fish caudal arteries after nursing one day
The ratio of blood vessel region of interest red fluorescence OD average and minimum feeding volume (7mg) group.As seen from the figure, with feeding volume
Increase, the relative value of juvenile fish caudal artery blood vessel region of interest red fluorescence OD average first increases to tend towards stability afterwards,
And high feeding volume (35,49,63 and 77mg) organizes extremely significant property and is higher than low feeding volume group (7 and 21mg) (p < 0.01), but height is fed
There is no significant difference (p > 0.05, n=17) between amount group (49,63 and 77mg)；Fig. 2 B is each feeding volume after feeding 10 days
After the average survival time rate of group, the bigger survival rate of feeding volume is smaller as seen from the figure, and feeding volume is greater than 49mg, increasing feeding volume can be bright
The aobvious survival rate (P < 0.05, n=3) for reducing zebra fish juvenile fish.The result shows that spot can be accelerated by increasing high cholesterol diet feeding volume
The accumulation of horse fish juvenile fish vascular lipid, but feeding volume is excessively high will affect its survival rate.
The 3 wide measurement of zebra fish children's fish length body of embodiment
The 5dpf wild type AB system zebra fish juvenile fish of health is randomly divided into normal diet group and 5% High cholesterol diet model
Group, every group of 30 tail fishes, 70mL1 × E3 culture, each feeding volume select 30mg, and daily feeding is twice.After continuous feed 10 days,
It is wide with ImageJ software/Zeiss ZEN analysis software measurement long body of body.Body length is the length that end is kissed from zebra fish juvenile fish to tail fin base portion
Degree, body is wide be perpendicular to rear portion mainline and cloaca before the 4th to the 5th segment position from back top to mainline
The width of hypoblast.
Image Acquisition is integrally carried out to normal diet group and the random selected juvenile fish of model group using Stereo fluorescence microscope,
As shown in Figure 3 C.Average body as shown in Figure 3A for normal diet group and model group juvenile fish is long.Compared with normal diet group juvenile fish,
Model group body length increased, but not have significant difference (p > 0.05, n=15).Fig. 3 B is normal diet group and model group children
The average body of fish is wide, is higher than normal diet group (p < 0.01, n=15) to the extremely significant property of model group body width, C is measurement method, note:
Control/0 in figure: normal diet control group；5%HCD/5:5% High cholesterol diet group；`X ± SE, with Control group phase
Than P < 0.01 * *.The above result shows that High cholesterol diet can promote zebra fish juvenile fish lateral growth, but do not have shadow to body length
It rings.
The detection of 4 zebra fish juvenile fish blood lipid biochemical indicator of embodiment
5dpf wild type AB system zebra fish juvenile fish is randomly divided into normal diet group and 5% High cholesterol diet model group, often
30 tails of group, 28 DEG C of constant temperature incubations of 70mL1 × E3 culture solution, each feeding volume select 30mg, and daily feeding is twice.Continuously feeding
10 days, sample is received after empty stomach 36h, is saved it in -80 DEG C of ultra low temperature freezers when until using and is taken out.Tissue samples processing method:
Every juvenile fish weight is calculated with 1mg, by weight (g): volume (mL)=1:9 ratio be added physiological saline, ice water homogenate in
Be centrifuged 10min under 2500rpm, take supernatant, for total cholesterol (TC, total cholesterol), total triglycerides (TG,
Total glyceride) and low density lipoprotein cholesterol (LDL-C, low-density lipoprotein
Cholesterol measurement).Supernatant is taken to be diluted to the total protein concentration that 1% homogenised tissue is used to detect each sample, for biochemistry
The standardization of index.
Experimental result
TC, TG and the LDL-C detected in juvenile fish global tissue is horizontal, as shown in Figure 4.The results show that model group juvenile fish group
It knits the horizontal extremely significant property of middle TC and TG and is higher than normal diet group (p < 0.01), also conspicuousness is higher than normal diet group to LDL-C level
(p<0.05).The above result shows that High cholesterol diet can promote the horizontal significant raising of juvenile fish body lipid.
Above-mentioned experimental data illustrates to feed zebra fish juvenile fish using 5% High cholesterol diet model group in the present invention, establish
Zebra fish high cholesterol model, addition cholesterol tracer fluorescence cholesteryl ester illustrates zebra similar to object in high cholesterol diet
Fish high cholesterol model can be used for the research of subsequent fat-reducing medicament experiment, and Stereo fluorescence microscope can be used under its condition of living body
The research of its lipid-loweringing is observed in real time.
The research of Li Chunqi et al. is by the pure yolk dry powder to zebra fish feeding various dose concentration in the prior art
Zebra fish hyperlipemia model is established, but feeding volume is excessively high, is unfavorable for the health existence of zebra fish juvenile fish, and the present invention is in egg
The content of cholesterol, can be in relatively low forage feed amount situation as high lipid food in increase feed on yellow dry powder basis
Under achieve the effect that same, and this method is stable, reliable, easy.
The above described is only a preferred embodiment of the present invention, being not the limitation for doing other forms to the present invention, appoint
What those skilled in the art can use the equivalence enforcement that technology contents disclosed above were changed or be modified as equivalent variations
Example.But without departing from the technical solutions of the present invention, according to the technical essence of the invention to the above embodiments
What simple modification, equivalent variations and remodeling, still falls within the protection scope of technical solution of the present invention.
Claims (10)
1. a kind of method of zebra fish high cholesterol model foundation, which is characterized in that it includes the following steps:
S1, zebra male and female adult fish are placed in mating box with the ratio of 1:2~2:3, and post-coitum receives ovum, and healthy ovum is trained in 28 DEG C of constant temperature
It supports, selects healthy zebra fish juvenile fish under microscope later；
S2, cholesterol production high cholesterol diet is added in yolk powder, the yolk powder that will do not add cholesterol is raised as common
Material, for raising the healthy zebra fish juvenile fish；
S3, after zebra fish juvenile fish is continuously cultivated with high cholesterol diet, while with normal diet feed zebra fish juvenile fish make
For control group, when zebra fish blood vessel cholesterol accumulation conspicuousness is higher than control group, zebra fish high cholesterol model is built up.
2. the method as described in claim 1, which is characterized in that in step sl, time of the mating be 1.5h after receive ovum,
The time of the culture is 5 days.
3. the method as described in claim 1, which is characterized in that in step s 2, cholesterol exists in the high cholesterol diet
Additive amount in yolk powder is mass percent 5%~12.5%.
4. the method as described in claim 1, which is characterized in that in step s 2, the daily of the high cholesterol diet feeds
The amount of supporting is 1.4mg/ tail~3.2mg/ tail.
5. the method as described in claim 1, which is characterized in that in step s 2, cholesterol exists in the high cholesterol diet
Additive amount in yolk powder is that mass percent is 5%, and the daily feeding volume of the high cholesterol diet is 2mg/ tail.
6. the method as described in claim 1, which is characterized in that in step s 2, also add gallbladder in the high cholesterol diet
For sterol tracer fluorescence cholesteryl ester similar to object, additive amount is 10 μ g/g.
7. method as claimed in claim 6, which is characterized in that in step s3, the cholesterol tracer fluorescence cholesterol
Ester analogs are 524/563 C11 of CHOLESTERYL BODIPY, using ether as organic cosolvent.
8. the method as described in claim 1, which is characterized in that in step s3, the continuous culture is in 28 DEG C of constant temperature training
It supports 8~10 days.
9. the method as described in claim 1, which is characterized in that solid using the gallbladder added with 10 μ g/g is only fed in step s3
Alcohol tracer fluorescence cholesteryl ester is control group similar to the yolk powder of object, and burnt or Stereo fluorescence microscope is copolymerized after culture and is seen in real time
It examines.
10. the method as described in claim 1, which is characterized in that in step s 2, the yolk powder is to boil Fresh Egg
It is ripe, yolk is taken out, freeze-dryer handles to obtain yolk dry powder after crushing.
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| CN113243466A (en) * | 2021-07-01 | 2021-08-13 | 常州鼠一鼠二生物科技有限公司 | Zebra fish hyperlipidemia model feed and preparation method and application thereof |
| CN114667955A (en) * | 2020-12-25 | 2022-06-28 | 浙江省化工研究院有限公司 | Method for constructing zebra fish obesity model |
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