CN107881226A - The application of SCAD genes or SCAD albumen in treatment of vascular reconstruct and important vascular disease medicament is prepared - Google Patents
The application of SCAD genes or SCAD albumen in treatment of vascular reconstruct and important vascular disease medicament is prepared Download PDFInfo
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Abstract
The invention discloses a kind of application of SCAD genes or SCAD albumen in vascular remodeling, specifically discloses the application of the expression accelerator or activator of SCAD genes, SCAD albumen or SCAD albumen in the medicine for the treatment of of vascular reconstruct and related significant vascular diseases is prepared.The structure that the present invention passes through animal model, important function of the SCAD in Vascular Remodeling in Hypertension is disclosed first, new drug development for vascular remodeling and related significant vascular diseases is provided fundamental basis and feasibility foundation, is used as vascular remodeling and the new drug target of related significant vascular diseases.
Description
Technical field
The invention belongs to biomedicine technical field, more particularly it relates to which SCAD genes or albumen are anti-in preparation
Control the application in the medicine of vascular remodeling, and the application in the medicine for preparing preventing and treating important vascular disease.
Background technology
Hypertension is one of common cardiovascular diseases for seriously endangering human health, and hypertension can cause blood vessel structure and work(
The change of energy, i.e. Vascular Remodeling in Hypertension, hypertension progress and cardiovascular disability rate and the death rate can be promoted to raise.Important vascular
The generation of disease and Vascular Remodeling in Hypertension are closely related.
For a long time, vascular remodeling is attributed to the secondary complications of hypertension and other angiocardiopathies by we.However,
Current research result shows that vascular remodeling and hypertension exist side by side, is observing 3 to 4 week old spontaneous hypertensive rat blood vessels
Shi Faxian, for vascular remodeling even earlier than blood pressure change, vascular remodeling is likely to one of key factor to cause elevation of the blood pressure；Its
It is secondary, vascular remodeling or the continuous worsening architecture basics of hypertension.Therefore, early prevention or reverse vascular remodeling will be for height
The prevention and treatment of blood pressure produce very important positive effect.
Pharmaceutical intervention is carried out for the key link that vascular remodeling is formed, potentially contributes to control Vascular Remodeling in Hypertension
Development.However, existing antihypertensive drugs only rests on the aspect of reducing blood pressure, the layer for directly improving vascular remodeling it is not related to
Face, and vascular remodeling occurs for hypertension, and into vicious circle blood pressure will be made further to raise, vascular remodeling further aggravates.
Therefore, the study of incident mechanism of Vascular Remodeling in Hypertension is deeply carried out, vascular remodeling and important vascular disease are directed to seek
Novel target sites for drug action, improve the prognosis of hypertension, it will be the important research direction in this area.
The content of the invention
Based on this, in order to overcome above-mentioned prior art to vascular remodeling and important vascular disease research and treatment technology not
Foot, the invention provides the application of SCAD genes or SCAD albumen in vascular remodeling and important vascular disease.Short chain acyl is auxiliary
Enzyme A dehydrogenases (short chain acyl-CoA dehydrogenase, SCAD) gene is one related to vascular remodeling
Gene, basis is provided for the Study on Molecular Mechanism of vascular remodeling.
In order to realize foregoing invention purpose, this invention takes following technical scheme：
SCAD genes, the expression accelerator of SCAD albumen or SCAD albumen or activator are preparing treatment of vascular reconstruct
Application in medicine.
In wherein some embodiments, the vascular remodeling is induced by spontaneous hypertensive rat to be occurred.
The expression accelerator of SCAD genes, SCAD albumen or SCAD albumen or activator are preparing preventing and treating important vascular disease
Application in the medicine of disease.
The present inventor, which studies, to be found, the key enzyme short chain acyl coa dehydrogenase (short- of fatty acid beta oxidation
Chain acyl-CoA dehydrogenase, SCAD) in the sustainer of spontaneous hypertensive rat, its mRNA and albumen table
Reach, enzymatic activity significantly reduces, sustainer free fatty acid content substantially increases, and ROS generations increase, and shows spontaneous high blood
The decline of rat aorta SCAD protein expressions and enzymatic activity is pressed, the decline of aortic fatty acid beta-oxidation ability, fat may be result in
Fat acid-utilising is reduced, so as to cause the increase of free fatty acid content.The consequence that a large amount of free fatties stimulate is that ROS generations increase
It is more, so as to start oxidative stress, cause the generation of Vascular Remodeling in Hypertension.Compared with prior art, the present invention has
Following beneficial effect：
Structure of the invention by animal model, important function of the SCAD in Vascular Remodeling in Hypertension is disclosed first,
New drug development for vascular remodeling and related significant vascular diseases is provided fundamental basis and feasibility foundation, is used as blood vessel
Reconstruct and the new drug target of related significant vascular diseases.
Brief description of the drawings
Fig. 1 is the change of different week old spontaneous hypertensive rat blood pressures in the embodiment of the present invention 1；
Fig. 2 is each group rat chest aorta morphological change in the embodiment of the present invention 2；
Fig. 3 is mRNA, SCAD protein expression and SCAD enzyme activity of each group rat chest aorta SCAD in the embodiment of the present invention 3
Property change；
Fig. 4 is free fatty in ATP in each group rat chest aorta in the embodiment of the present invention 4 and aorta pectoralis and serum
The change of content；
Fig. 5 is the change of each group rat chest aorta ROS contents in the embodiment of the present invention 5.
Embodiment
Further illustrate the present invention below in conjunction with the drawings and specific embodiments, but embodiment the present invention is not done it is any
The restriction of form.Unless stated otherwise, the reagent of the invention used, method and apparatus is the art conventional reagent, methods
And equipment.
Unless stated otherwise, following examples agents useful for same and material are purchased in market.
Vascular remodeling rat used in following examples of the present invention is spontaneous hypertensive rat, and Wistar used is big
Mouse is normal control rat.
The abbreviation of term used is defined as follows in following examples and Figure of description：
SHR：Spontaneous hypertensive rat (spontaneously hypertensive rats)；
ROS：Active oxygen (reactive oxygen species)
SCAD：Short chain acyl coa dehydrogenase (short chain acyl-CoA dehydrogenase)；
α-tubulin：Alpha-tubulin；
Experimental method used in following examples is as follows：
1st, experimental animal is grouped
Experiment is divided into four groups：(1) normal group：The normal 15 week old Wistar rats of male 9 weeks of raising 8.(2) normal trip
Swimming group：The 8 week old Wistar rats of male 15 examination swimming exercises carry out swimming endurance training after 1 week, the examination swimming exercise stage
Time progressively increases to 5d 60min from 1d 15min, and persistently swam 60min since the 2nd week during every morning 9, often
6 swimming instructions of Zhou Jinhang, swimming pool volume and the depth of water are respectively 100cm × 60cm × 60cm, and water temperature is controlled to 30~35 DEG C,
Maintain this amount of exercise 8 weeks.(3) SHR groups：The normal 15 week old spontaneous hypertensive rat of male 9 weeks of raising 8.(4) SHR is swum
Swimming group：Male 15 week old spontaneous hypertensive rat 8, examination swimming exercise carry out swimming endurance and trained 8 weeks after 1 week, swimming is resistance to
Power training method is the same as normal swimming group.
2nd, the measure of rat-tail systolic pressure
Each group rat tail artery systolic pressure is determined using the rat tail artery indirect method of measurement, every two weeks measure once, directly
Terminate to swimming, U.S. Ken rat blood pressures non-invasive blood pressure instrument (CODA Monitor) is purchased from U.S. Kent company.
3rd, the measure of serum and sustainer free fatty acid content
After yellow Jackets (45mg/kg) anesthetized animal, abdominal cavity exposure abdominal aorta is opened, immediately with vacuum blood collection tube abdomen master
Arterial blood extracting about 5mL, 3500r/min centrifugation 10min, careful collection serum are simultaneously dispensed into EP pipes, -80 DEG C of ultralow temperature ices
Case preserves serum.Take after blood with the abundant perfused hearts of pre- cold saline, be rapidly separated rat aorta tissue, peel off sustainer
Surrounding superabundant fats tissue, physiological saline clean up, and -80 DEG C of preservation progress subsequent experimental free fatties contain after liquid nitrogen flash freezer
It is fixed to measure.Serum and sustainer free fatty acid content measure are carried out in strict accordance with free fatty acid determination reagent kit specification,
The content of free fatty in serum and sustainer is calculated according to absorbance.
4th, ROS assays
Take after blood with the abundant perfused hearts of pre- cold saline, be rapidly separated rat aorta tissue, peel off sustainer week
Superabundant fats tissue is enclosed, physiological saline cleans up, and -80 DEG C preserve progress subsequent experimental, remainder after a part of liquid nitrogen flash freezer
Divide frozen section, dihydro second pyridine (dihydroethidium, DHE) dyeing, fluorescence microscope and shooting image, analyze ROS
In the production of aorta pectoralis.
5th, Aortic Morphology is studied
The abundant perfused hearts of physiological saline of precooling after blood are taken, are rapidly separated rat aorta tissue, peel off sustainer week
Superabundant fats tissue is enclosed, physiological saline is placed in 4% paraformaldehyde after cleaning up and fixed, and is cut into slices after FFPE, utilizes Soviet Union
H ＆ E dyeing (HE dyeing) carries out follow-up Aortic Morphology analysis, passes through image analyzer under an optical microscope
Lumen diameter, vascular wall intima-media thickness are measured, calculates intima-media thickness and lumen diameter ratio.
6th, real-time fluorescence quantitative PCR detection sustainer SCAD mRNA expression
It is purple under 260nm, 280nm wavelength in strict accordance with total serum IgE in Trizol kit specifications extraction aortic tissue
Outer spectrophotometric determination RNA sample absorbance, detect RNA purity and calculate RNA concentration.With reference to RT-PCR kit explanation
Book carries out reverse transcription reaction, and two-step method carries out pcr amplification reaction, and fluorescence dye is added according to SYBR Green specifications reaction system
Real-time PCR reactions are carried out after material, primer and RT products in Bio-Rad CFX96 PCR instruments.Response procedures are：95℃
10s；95℃ 5s；60 DEG C of 30s, circulate 40 times.Using threshold method, i.e. 2- △ △ Ct method result of calculations is compared, experiment repeats
3 times.Primer is synthesized by Shanghai life work, and SCAD sense primer is 5 '-CCAGTCTGTGG AACTACCTGAG-3 ', anti-sense primer
For 5 '-CCCTTCTTCTTCACCTGCGA-3 '.Internal reference GAPDH sense primers are 5 '-AGGAGTAAGAAACCCTGGAC-3 ',
Anti-sense primer is 5 '-CTGGGATGGAATTGTGAG-3 '.
7th, Western blot methods detection SCAD protein expressions
Each group aortic tissue total protein is extracted, protein content is detected using BCA kits, with suitable after Quantitative Western concentration
When concentration packing be placed in -20 DEG C it is standby.The concentration of separation gel and concentration glue is respectively 10% and 5%, with progress electricity after glue
Swim, by albumen transferring film to pvdf membrane (Bio-Rad) after electrophoresis, close 2h with BSA afterwards, add I anti-(SCAD) 4 DEG C of refrigerator mistakes
Night.1h is incubated under II anti-room temperature condition corresponding to being added after rinsing, chemical illuminating reagent is incubated appropriate time, tabletting, exposure, shown
Shadow, it is fixed, as a result band is analyzed using ImageJ image analysis systems, experiment is repeated 3 times.
8th, SCAD Enzyme assays
The detection of SCAD enzymatic activitys is carried out in strict accordance with SCAD activity colorimetric determination measure kit specification, cracking master
Arterial tissue, with the quantitative Supernatant protein of BCA protein reagents box, SCAD enzyme activity in sustainer is detected using ELIASA determination method
Property.
9th, ATP assays
ATP contents are determined in strict accordance with kit specification.ATP detection kits are according to firefly luciferase
(firefly luciferase) produces chemiluminescence needs the principle of ATP offer energy when producing fluorescence.Work as firefly luciferin
When enzyme and all excessive fluorescein, the generation of fluorescence is directly proportional with ATP concentration in certain concentration range, can be with sensitivity
Detect the ATP concentration in solution.
10th, statistical analysis
All data are represented with mean ± standard deviation (mean ± SD), are handled using SPSS13.0 statistical softwares, between group
Compare and use one-way analysis of variance, and examine with Bonferroni t and to carry out comparing two-by-two between group, with P<0.05 is poor
It is different that there is conspicuousness.
The different week old spontaneous hypertensive rats of embodiment 1 are in hypertension stationary phase
1st, the present embodiment tests the systolic pressure situation of change of 16,18,20,22,24 week old spontaneous hypertensive rats.
2nd, result is as shown in figure 1, different week old spontaneous hypertensive rats are in hypertension stationary phase.Instructed by swimming
After practicing 8 weeks, spontaneous hypertensive rat swimming group blood pressure substantially reduces, and shows that swimming instruction has some improvement to hypertension tool
Effect.
The morphologic change of each group rat aorta of embodiment 2
1st, the present embodiment test morphologic situation of change of each group rat aorta.
2nd, Fig. 2 is each group rat aorta HE coloration results.Fig. 2A represents media thickness, and Fig. 2 B represent lumen of vessels
Diameter, Fig. 2 C represent the ratio of media thickness and lumen of vessels diameter.As a result as shown in Fig. 2 spontaneous hypertensive rat blood
Pipe intima-media thickness significantly increases, and lumen of vessels diameter is obviously reduced, and media thickness substantially increases with lumen of vessels ratio, and blood vessel is fine
Arrangement disorder is tieed up, layering is unclear and phenomena such as fracture, vascellum endometrial hyperplasia comes off substantially, and endangium smooth degree is obvious
Decline, obvious vascular remodeling occur.However, after spontaneous hypertensive rat is trained 8 weeks by swimming exercise, rat aorta
Intima-media thickness is obviously reduced, and lumen of vessels diameter substantially increases, and media thickness is obviously reduced with lumen of vessels diameter ratio, blood vessel
Endometrial hyperplasia comes off reduction, and the rough degree of endangium mitigates, and vascular remodeling is obviously improved.
The each group rat aorta SCAD of embodiment 3 mRNA, protein level and SCAD enzymatic activitys change
1st, the change of the present embodiment test each group rat aorta SCAD mRNA, protein level and SCAD enzymatic activitys.
2nd, Fig. 3 A represent the change of SCAD enzymatic activitys, and Fig. 3 B represent the change of SCAD mRNA level in-sites, and Fig. 3 C represent SCAD eggs
The change of white level.As a result as shown in figure 3, the SCAD mRNA and protein expression of exercise rats sustainer are substantially raised,
The increase of SCAD enzymatic activitys；And the SCAD mRNA and protein expression of SHR group sustainers are substantially lowered, SCAD enzymatic activitys decline.This
Outside, after SHR was by swimming instruction 8 weeks, the SCAD mRNA and protein expression of sustainer are substantially raised, and SCAD enzymatic activitys increase
Add.
Experimental result shows that SCAD expression is lowered may be closely related with the occurrence and development of Vascular Remodeling in Hypertension.Swimming
Training may be by raising SCAD expression, so as to improve Vascular Remodeling in Hypertension.
In each group rat aorta of embodiment 4 in ATP, sustainer and serum free fatty acid content change
1st, free fatty acid content in ATP, aorta pectoralis and serum in the present embodiment test each group rat chest aorta
Change.
2nd, Fig. 4 A represent the change of free fatty acid content in serum, and Fig. 4 B represent free fatty acid content in aorta pectoralis
Change, Fig. 4 C represent the change of ATP contents in aorta pectoralis.As a result as shown in figure 4, swimming exercise group rat aorta ATP
Content significantly increases, and serum and sustainer free fatty acid content significantly reduce；And spontaneous hypertensive rat group sustainer ATP
Content significantly reduces, and serum and sustainer free fatty acid content substantially increase.After SHR rats'swimmings, the ATP contents of sustainer
Significantly increase, the free fatty acid content of serum and sustainer significantly reduces.
Experimental result is shown, the expression of exercise rats sustainer SCAD albumen and the increase of enzymatic activity, may result in
Aortic fatty acid beta-oxidation ability strengthens, and so as to cause ATP synthesis increases, serum and sustainer free fatty acid content are reduced.
And the decline of spontaneous hypertensive rat sustainer SCAD protein expressions and enzymatic activity, aortic fatty acid β oxygen may be result in
Change ability declines, so as to cause ATP synthesis to reduce, serum and the increase of sustainer free fatty acid content.
The change of each group rat aorta ROS contents of embodiment 5
1st, the present embodiment tests the change of ROS contents in each group rat aorta.
2nd, as shown in figure 5, the red fluorescence of SHR group sustainers is remarkably reinforced, average fluorescent strength increases result, shows
SHR groups high free fatty acid stimulates the ROS for causing sustainer to generate increase.And the red fluorescence of SHR swimming group sustainers is obvious
Weaken, average fluorescent strength reduces, and shows swimming exercise training by reducing free fatty acid content, so as to reduce ROS production
It is raw.
Above example 1 uses spontaneous hypertensive rat and control rats to embodiment 5 in animal level, it was demonstrated that SCAD
Effect of the gene in Vascular Remodeling in Hypertension model.The achievement in research mainly obtained is as follows：
There is obvious vascular remodeling in (1) 24 week old spontaneous hypertensive rat, SCAD eggs in vascular remodeling tissue
In vain, being decreased obviously occur in mRNA expression and enzymatic activity；
(2) spontaneous hypertensive rat by swimming exercise train 8 weeks after, vascular remodeling be improved significantly, SCAD eggs
In vain, mRNA expression and enzymatic activity, which decline, has also obtained a certain degree of reverse.
(3) effect the invention firstly discloses SCAD genes in Vascular Remodeling in Hypertension, acted on as new medicine
Target spot, SCAD genes answering in the medicine for preparing prevention, alleviating and/or treating vascular remodeling and related significant vascular diseases
With, including prepare by SCAD gene upstream and downstream target spots the effect in vascular remodeling and the medicine of related significant vascular diseases.
Structure of the invention by animal model, important function of the SCAD in Vascular Remodeling in Hypertension is disclosed first,
New drug development for vascular remodeling and related significant vascular diseases is provided fundamental basis and feasibility foundation, is used as blood vessel
Reconstruct and the new drug target of related significant vascular diseases.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
God any modification, equivalent substitution and improvements made etc., should be included in the scope of the protection with principle.
Embodiment described above only expresses the several embodiments of the present invention, and its description is more specific and detailed, but simultaneously
Can not therefore it be construed as limiting the scope of the patent.It should be pointed out that come for one of ordinary skill in the art
Say, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection of the present invention
Scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (3)
1.SCAD genes, the expression accelerator of SCAD albumen or SCAD albumen or activator are preparing the medicine for the treatment of of vascular reconstruct
Application in thing.
2. application according to claim 1, it is characterised in that the vascular remodeling is induced by spontaneous hypertensive rat and sent out
It is raw.
The expression accelerator of 3.SCAD genes, SCAD albumen or SCAD albumen or activator are preparing preventing and treating important vascular disease
Medicine in application.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1045918A4 (en) * | 1998-01-12 | 2003-10-08 | Univ Georgetown Med Center | G protein-related kinase mutants in essential hypertension |
| CN106540272A (en) * | 2016-11-07 | 2017-03-29 | 广东药科大学 | The application of SCAD genes or SCAD albumen |
-
2017
- 2017-10-30 CN CN201711039664.9A patent/CN107881226A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1045918A4 (en) * | 1998-01-12 | 2003-10-08 | Univ Georgetown Med Center | G protein-related kinase mutants in essential hypertension |
| CN106540272A (en) * | 2016-11-07 | 2017-03-29 | 广东药科大学 | The application of SCAD genes or SCAD albumen |
Non-Patent Citations (3)
| Title |
|---|
| JINXIAN HUANG ET.AL,: "Changes in short-chain acyl-coA dehydrogenase during rat cardiac development and stress", 《J CELL MOL MED》 * |
| 周四桂等: "短链酰基辅酶A脱氢酶在大鼠心脏发育中的表达及其与心肌肥厚的关系", 《中国病理生理杂志》 * |
| 王要军等: "《高级临床医学》", 30 September 2008, 内蒙古科学技术出版社 * |
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