CN104306993B - Application of the circulation miR 505 in hypertension early diagnosis and target organ earlier damage forewarning function - Google Patents
Application of the circulation miR 505 in hypertension early diagnosis and target organ earlier damage forewarning function Download PDFInfo
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- CN104306993B CN104306993B CN201410546228.0A CN201410546228A CN104306993B CN 104306993 B CN104306993 B CN 104306993B CN 201410546228 A CN201410546228 A CN 201410546228A CN 104306993 B CN104306993 B CN 104306993B
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Abstract
The present invention relates to application of the expression of circulation miR 505 in hypertension early diagnosis and target organ earlier damage forewarning function.The invention has the advantages that：Result of study of the present invention shows：The expression of circulation miR 505 is raised with the rising of blood pressure, is that the early diagnosis of hypertension provides brand-new diagnosis basis；MiR 505 suppresses the tubulose of the propagation, migration and endothelial cell of endothelial cell to be formed；The expression of detection circulation miR 505 not only facilitates the early diagnosis of hypertension, early detection, the more importantly forewarning function to target organs of patients with essential hypertension Random early Detection, early diagnosis for hypertension and the infringement of early prediction target organ provide brand-new means, can had instructs clinical application, delay the infringement of target organ, improve the quality of life of patient.
Description
Technical field
The present invention relates to the new application of hprt minigene acid, specifically, is the expression for circulating miR-505 in hypertension
Application in early diagnosis and target organ earlier damage forewarning function.
Background technology
Hypertension（hypertension）Refer to that, under quiescent condition, systolic pressure and/or diastolic pressure increase（≥140mmHg/
90mmHg）, it is often accompanied by the target organ such as disorders of lipid metabolism and the heart, brain, kidney and retina function or organic change.Systolic pressure exists
Between 120 ~ 139mmHg and/or diastolic pressure is prehypertensive or normal high level between 80 ~ 89mmHg.
Hypertension is modal cardiovascular disease, is the great public health problem in global range.Epidemiology survey
Show, China 18 years old and above prevalence of hypertension rate are 18.8%, estimate national number of patients more than 1.6 hundred million.And blood pressure
Rising be multiple diseases fuse cord, increased the incidences of disease such as coronary heart disease, heart failure and kidney disease.However, part is high
Blood pressure patient has no obvious clinical symptoms, and hypertension is referred to as " the stealthy killer " of human health.Therefore, to high blood pressure
Early detection, early diagnosis, can effectively prevent and treat hypertension and develop, it is often more important that the morning to target organs of patients with essential hypertension infringement
Phase early warning, can effectively instruct clinical application, delay the infringement of target organ, improve the quality of life of patient.
Hprt minigene acid（MicroRNA, miRs）Be with well-conserved non-coding single stranded RNA, length about 18 ~
25 nucleotides.It was predicted that miRs regulates and controls in human genome 30 ~ 50% gene by the combination with target gene, life is participated in
A series of important processes during life, including early embryonic development, cell propagation, cell differentiation, Apoptosis, group hair-weaving
Educate, adjust physiology, the pathologic process such as die, and by regulating and controlling one group of gene and participating in adjusting the multiple diseases of body to develop, such as
Tumour, angiocardiopathy, aging etc..Research shows, in disease pathogenetic early stage, miRs generates reversible change, leads to
Cross the detection to miRs, can early prediction disease develop, so as to instruct clinic diagnosis.
Vascular endothelial cell is one layer of cell monolayer of blood vessel surface, directly contact blood constituent and tissue, plays regulation
The easypro contracting of blood vessel, prevent platelet adhesion reaction and thrombosis, the growth for adjusting VSMC and propagation, prevent inflammation thin
The infiltration of born of the same parents and harmful substance the effect such as penetrate.Endothelial Dysfunction is not only the pathologic of hypertension incidence earliest period and damages
Evil, the or " weather that the cardiocerebrovasculaevents events such as prediction sudden death, acute myocardial infarction AMI, unstable angina pectoris, palsy occur
Table ".Therefore, the function of early prediction endothelial cell, can effectively instruct clinical application, prevent and treat Endothelial Dysfunction, effectively
Prevent and treat generation development and the infringement of target organ of hypertension.
Content of the invention
The purpose of the present invention is for deficiency of the prior art, provides a kind of miR-505 and is preparing the high blood of early diagnosis
Application in pressure reagent.
Second object of the present invention is to provide a kind of miR-505 in the medicine equipment for preparing early diagnosis hypertension
Application.
Third object of the present invention is to provide a kind of miR-505 in prediction target organ earlier damage reagent is prepared
Application.
Fourth object of the present invention is to provide a kind of miR-505 in the Medical treatment device for preparing prediction target organ earlier damage
Application in tool.
5th purpose of the present invention is to provide a kind of miR-505 and is screening hypotensive and target organ as drug target
Application in the medicine of protection.
6th purpose of the present invention is to provide a kind of miR-505 antagonist or inhibitor and is preparing hypotensive and target device
Application in the medicine of official's protection.
For achieving the above object, the present invention is adopted the technical scheme that：MiR-505 is preparing early diagnosis anti-hypertensive agent
In application, during described early diagnosis anti-hypertensive agent is circulated by detection, the expression of miR-505 is carried out to hypertension
Early diagnosis.
For realizing above-mentioned second purpose, the present invention is adopted the technical scheme that：MiR-505 is preparing the high blood of early diagnosis
Application in the medicine equipment of pressure, the medicine equipment of described early diagnosis hypertension circulated by detection in miR-505 table
Reach level to early diagnose hypertension.
For realizing above-mentioned 3rd purpose, the present invention is adopted the technical scheme that：MiR-505 is preparing prediction target organ morning
Application in phase Damaging agent, described prediction target organ earlier damage reagent circulated by detection in miR-505 expression water
The flat infringement to target organ carries out early prediction.
For realizing above-mentioned 4th purpose, the present invention is adopted the technical scheme that：MiR-505 is preparing prediction target organ morning
Application in the medicine equipment that phase damages, the medicine equipment of described prediction target organ earlier damage circulated by detection in miR-
Infringement of 505 expression to target organ carries out early prediction.
Described target organ is the heart, brain, kidney, peripheral vascular.
For realizing above-mentioned 5th purpose, the present invention is adopted the technical scheme that：MiR-505 is as drug target in screening
Application in the medicine of hypotensive and target-organ protection.
For realizing above-mentioned 6th purpose, the present invention is adopted the technical scheme that：MiR-505 antagonist or inhibitor are in system
The application that makes preparation for dropping in the medicine of blood pressure and target-organ protection.
The invention has the advantages that：
1st, the present invention collects normotensive, prehypertensive patient, Plasma of Patients with Hypertension, to miR-505 in circulation
Expression studied, result of study shows：The expression of circulation miR-505 is raised with the rising of blood pressure, is high blood
The early diagnosis of pressure provides brand-new diagnosis basis；
2nd, the present invention studies the influence of miR-505 Human Umbilical Vein Endothelial Cells function, result of study using different endothelial cells
Show：The tubulose of the propagation, migration and endothelial cell of miR-505 suppression endothelial cell is formed；
3rd, the expression of detection circulation miR-505 not only facilitates the early diagnosis of hypertension, early detection, more important
It is the forewarning function to target organs of patients with essential hypertension Random early Detection, is that the early diagnosis of hypertension and the infringement of early prediction target organ are carried
For brand-new means, can had instructs clinical application, delays the infringement of target organ, improves the quality of life of patient.
Description of the drawings
Fig. 1. the expression of circulation miR-505（Compared with Healthy Control, *p<0.05）.SYBG dye method
RT-PCR detection normotensive, prehypertensive patient, the expression of hyperpietic circulation miR-505：Fig. 1 result shows
Show, prehypertensive patient, the expression of hyperpietic circulation miR-505 are all significantly raised（p<0.05）.
Fig. 2. the expression of circulation miR-505（Compared with Healthy Control, *p<0.05）.Sonde method RT-
PCR detection normotensive, hyperpietic's circulation miR-505 expression：Fig. 2 result shows that hyperpietic is circulated
The expression of miR-505 is significantly raised（p<0.05）.
Fig. 3. the impact that miR-505 is acted on to different endothelial cell proliferations（Compared with normal group, *p<0.05）.miR-
The propagation of 505 suppression endothelial cells：Retinal endothelial cell is cultivated respectively using the DMEM culture medium containing 10% hyclone
（HRECs）, Human Brain Microvascular Endothelial（HBMECs）, Human umbilical vein endothelial cells（HUVECs）, it is inoculated in 3000/hole
In 96 orifice plates, 20nM miR-505 mimic and inhibitor is transfected respectively（RNAiMax transfection reagent）After 48h, MTT is carried out
Experiment.Fig. 3 result shows that miR-505 mimic significantly inhibits the propagation of retinal endothelial cell（p<0.05）.
Fig. 4. the impact that miR-505 is acted on to different endothelial cell migrations（Multiplication factor：100 ×, with Control group phase
Than *p<0.05）.The migration of miR-505 suppression endothelial cell：With containing 10% hyclone DMEM culture medium difference HRECs,
HBMECs, HUVECs, are inoculated in 96 orifice plates with 3000/hole, transfect 20nM miR-505 mimic and inhibitor respectively
（RNAiMax transfection reagent) after 48h, carry out Cell migration assay respectively.Fig. 4 A, B result shows that miR-505 mimic shows
Write the migration of suppression HRECs, HBMECs, HUVECs（p<0.05）.
Fig. 5. influence of the miR-505 to different endothelial cell tube formation（Multiplication factor：40 ×, with
Control group is compared, *p<0.05）.MiR-505 suppression endothelial cell tubulose is formed：Trained with the DMEM containing 10% hyclone
Foster base distinguishes HRECs, HUVECs, HBMECs, is inoculated in 96 orifice plates with 3000/hole, transfects 20nM miR-505 respectively
Mimic (RNAiMax transfection reagent）After 48h, little tube formation assay is carried out respectively.Fig. 5 A, B result shows, miR-505
Mimic significantly inhibits the tubulose of HRECs, HUVECs, HBMECs and is formed（p<0.05）.
Specific embodiment
Below in conjunction with the accompanying drawings the specific embodiment that the present invention is provided is elaborated.
Embodiment 1
In the present embodiment, we are respectively adopted the research meanses of SYBG dye method, sonde method RT-PCR, inquire into and bright
Expression and blood pressure the being proportionate property of miR-505 are really circulated.Also, in the present embodiment, we are thin using retinal endothelial
Born of the same parents（HRECs）, Human Brain Microvascular Endothelial（HBMECs）, Human umbilical vein endothelial cells（HUVECs）Research clear and definite miR-
505 overexpression significantly inhibits propagation, migration and the tubulose of endothelial cell and is formed, and this research discloses circulation miR-505 first
Expression not only react blood pressure level, and anticipated that the function of endothelial cell, be hypertension early stage target organ damage
Play forewarning function.
First, method
1. clinical research sample collection
The court all is from for the clinical sample of the present invention, and all research objects are all conscientiously read and signed in the know
Letter of consent, meets ethical standard.Research object is the male sex, and the age is between 30-60 year.Not using antihypertensive drugs
In the case of, non-three measurements on the same day, systolic pressure≤120mmHg and diastolic pressure≤90mmHg is normal arterial pressure；Systolic pressure >=
140mmHg and/or diastolic pressure >=90mmHg is hypertension；120 ~ 139mmHg of systolic pressure and/or diastolic pressure 80 ~
89mmHg is prehypertensive.All research objects previously no other angiocardiopathies, serious liver and kidney dysfunction, glycosuria
Disease, the communicable disease such as hepatitis, tuberculosis.All research objects are all in early 8：00-9:00, on an empty stomach, using K2-EDTA anticoagulant tube
Venous collection 2ml blood.3000 revs/min, be centrifuged 10 minutes, upper plasma taken for this research.
2. clinical sample circulates the extraction of microRNA
The Standard Operating Procedure provided according to miRNeasy Mini Kit (QIAGEN) and according to production firm is carried out
Plasma sample microRNA is extracted, standby.
3. RT-PCR detection clinical sample circulates the expression of miR-505
According to miScript Reverse Transcription Kit (cat218061, QIAGEN) and its operation rule
Model carries out reverse transcription 20ul reaction system, be respectively adopted SYBR Green PCR Master Mix (cat218073,
QIAGEN), probe（Life technology）, according to manufacturers specifications flow operations, using Roche LightCycler
480 II carry out RT-PCR detection：95℃,15min；94 DEG C, 15s, 40 circulations；55 DEG C, 30s；70℃ , 30 s.
4. miR-505 significantly inhibits the propagation of endothelial cell：DMEM culture medium containing 10% hyclone is cultivated respectively
Human umbilical vein endothelial cells（HUVECs）Retinal endothelial cell（HRECs）, Human Brain Microvascular Endothelial（HBMECs）, with
3000/hole is inoculated in 96 orifice plates, transfects 20nM miR-505 mimic and inhibitor respectively（RNAiMax transfection examination
Agent）, in 37 DEG C, 5%CO2After incubator culture 48h, row MTT experiment, detects its light absorption value under 490nm wavelength.As Fig. 3 result shows
Show, compared with transfection Negative control, after transfection miR-505 mimic, the propagation to HUVECs, HBMECs has no bright
Aobvious inhibitory action, but significantly inhibit the propagation of HRECs（P<0.05）, transfection miR-505 Inhibitor after, cell propagation with
Transfection Negative control is compared, and cell propagation is showed no notable difference.
5. miR-505 significantly inhibits the migration of endothelial cell：DMEM culture medium containing 10% hyclone is cultivated respectively
Human umbilical vein endothelial cells（HUVECs）Retinal endothelial cell（HRECs）, Human Brain Microvascular Endothelial（HBMECs）, with
3000/hole is inoculated in 96 orifice plates, transfects 20nM miR-505 mimic and inhibitor respectively（RNAiMax transfection examination
Agent）After 48h, prepared using the DMEM containing 1%FBS and contain 2 × 105The cell suspension of cell/mL, adds Transwell cell
In, lower room is the DMEM culture medium of 20% hyclone, 5% CO2In incubator after culture 24h, 4% poly methyl alcohol is fixed, and
Carry out 0.1% violet staining, observation of cell migration situation.As Fig. 4 A, B result shows, after transfection miR-505 inhibitor,
Human Umbilical Vein Endothelial Cells migration has no notable difference, but after transfection miR-505 mimic, significantly inhibit HUVECs, HRECs,
The migration of HBMECs（p<0.05）.
6. miR-505 significantly inhibits the tubulose of endothelial cell and is formed：Distinguished using the DMEM culture medium of 10% hyclone
HRECs, HUVECs, HBMECs, are inoculated in 96 orifice plates with 3000/hole, transfect 20nM miR-505 mimic respectively
(RNAiMax transfection reagent）After 48h, Matrigel polymeric gel, transfection 2 × 10 is prepared4/ ml endothelial cell, 37 DEG C of 5% CO2
After incubator culture 24h, formed using the tubulose of 6.0 software analysis endothelial cell of Image-Pro Plus.As Fig. 5 A, B institute
Show, the tubulose for significantly inhibiting endothelial cell after transfecting miR-505 mimic is formed（p<0.05）.
4. statistical analysis
Measurement data is represented with mean ± standard deviation, meets normal distribution and homogeneity of variance adopts one-way analysis of variance
The variance analysis of one-way classification（one-way-classification ANOVA）, compare two-by-two and checked using t, just such as do not meet
State property and homogeneity of variance are then represented with M, min, max, compare and checked with Kruslal-Wallis H between group.
2nd, result
1. SYBG dye method RT-PCR detects sample clinical elementary information
As shown in table 1, normotensive, prehypertensive patient, hyperpietic's average age, BMI, third turn of ammonia of paddy
Enzyme, creatinine, urea, uric acid, fasting blood-glucose, triglyceride, cholesterol, HDL, low-density lipoprotein are showed no bright
Significant difference is different, and prehypertensive patient, hyperpietic's systolic pressure, diastolic pressure are all remarkably higher than normotensive（p<0.05）.
1. SYBG dye method RT-PCR of table detects sample clinical elementary information
Note：Compared with normal group, *p<0.05.
2. prehypertensive patient, the notable rise of hyperpietic's circulation miR-505 expression（p<0.05）
As shown in figure 1, the expression of prehypertensive patient, hypertension circulation miR-505 is all significantly raised, with statistics
Meaning（p<0.05）.
3. sonde method RT-PCR detects sample clinical elementary information
As shown in table 2, normotensive, prehypertensive patient's average age, BMI, glutamic-pyruvic transaminase, urea, creatinine,
The equal no significant difference of uric acid, fasting blood-glucose, triglycerides, cholesterol, prehypertensive patient's systolic pressure, diastolic pressure are all significantly high
In normotensive.
2. sonde method RT-PCR of table detects sample clinical elementary information
Note：Compared with normal person, *p<0.05.
4. hyperpietic's circulation miR-505 expression is notable raises（p<0.05）
As shown in Fig. 2 compared with normotensive, the expression of hyperpietic circulation miR-505 is significantly raised, with system
Meaning learned by meter（p<0.05）.
5. miR-505 suppresses the propagation of retinal endothelial cell（p<0.05）
DMEM culture medium containing 10% hyclone cultivates Human umbilical vein endothelial cells respectively（HUVECs, Fig. 3 A）View
Film endothelial cell（HRECs, Fig. 3 B）, Human Brain Microvascular Endothelial（HBMECs, Fig. 3 C）, 96 holes are inoculated in 3000/hole
In plate, 20nM miR-505 mimic and inhibitor is transfected respectively（RNAiMax transfection reagent）After 48h, MTT experiment result
Show, compared with transfection Negative control, after transfection miR-505 mimic, the propagation to HUVECs, HBMECs has no
Obvious inhibitory action, but significantly inhibit the propagation of HRECs（P<0.05）, after transfection miR-505 Inhibitor, cell is bred
Compared with transfection Negative control, cell propagation is showed no notable difference.
6. miR-505 significantly inhibits the migration of endothelial cell（p<0.05）
DMEM culture medium containing 10% hyclone cultivates HUVECs, HRECs, HBMECs respectively, is connect with 3000/hole
Plant in 96 orifice plates, respectively after transfection 20nM miR-505 mimic and inhibitor48h, Cell migration assay result shows
（Fig. 4 A, B）, after transfection miR-505 inhibitor, Human Umbilical Vein Endothelial Cells migration has no notable difference, but in transfection miR-505
After mimic, the migration of HUVECs, HRECs, HBMECs is significantly inhibited（p<0.05）.
7. miR-505 significantly inhibits endothelial cell tubulose and is formed
Difference Cultured endothelial cell（HRECs, HBMECs and HUVECs）Carry out little tube formation assay.Experimental result shows
（Fig. 5 A, B）, after transfection miR-505mimic, the tubulose for all significantly inhibiting HRECs, HBMECs and HUVECs is formed.
The present invention collects normotensive, prehypertensive, hypertension blood plasma, and research and probe circulates the expression of miR-505
Situation.Result of study is disclosed, and circulates the up-regulated of miR-505 and the being proportionate property of level of blood pressure.And the present invention is in cell
Learn in experiment and disclose, the overexpression of miR-505 significantly inhibits the tubular shape of the propagation of endothelial cell, migration and endothelial cell
Become.This research discloses the expression of circulation miR-505 first and not only reacts blood pressure level, and anticipated that endothelial cell
Function, be that clinical early prevention and treatment hypertension provides brand-new diagnosis and treatment thinking, it is brand-new to be that hypertension early stage target organ damage is provided
Early warning means.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
Member, on the premise of without departing from the inventive method, can also make some improvement and supplement, and these improve and supplement also should be regarded as
Protection scope of the present invention.
Claims (2)
- Application of the detection reagent of 1.miR-505 in early diagnosis anti-hypertensive agent is prepared, described early diagnosis hypertension During reagent is circulated by detection, the expression of miR-505 is early diagnosed to hypertension.
- Application of the detection reagent of 2.miR-505 in the medicine equipment for preparing early diagnosis hypertension, described early diagnosis During the medicine equipment of hypertension is circulated by detection, the expression of miR-505 is early diagnosed to hypertension.
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| WO2019227262A1 (en) * | 2018-05-26 | 2019-12-05 | 深圳市博奥康生物科技有限公司 | Method for mediating mirna overexpression based on mammalian virus |
| CN111286531B (en) * | 2018-12-06 | 2022-10-21 | 北京大学 | Application of circulating nucleic acid as marker of hypertension, diabetes and hyperlipidemia |
| CN111286532B (en) * | 2018-12-06 | 2022-08-26 | 国家心血管病中心 | Application of circulating nucleic acid as marker of hypertension and coronary heart disease |
| CN111718991A (en) * | 2020-07-03 | 2020-09-29 | 西安交通大学医学院第一附属医院 | Application of plasma miRNA molecular marker in diagnosis of metabolic syndrome |
| CN113332434B (en) * | 2021-05-25 | 2023-09-05 | 大连医科大学 | Application of miR-451a regulator in preparation of blood pressure regulating and controlling preparation |
| CN113384595B (en) * | 2021-07-13 | 2022-08-02 | 浙江中医药大学 | Application of miR-674-3p in preparation of medicine for preventing or treating stress hypertension |
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| MicroRegulators come of age in senescence;Myriam Gorospe et al.;《Trends in Genetics》;20110630;第27卷(第6期);第234页图1，第237页表1 * |
| MicroRNA (miRNA)-mediated Interaction between Leukemia/Lymphoma-related Factor (LRF) and Alternative Splicing Factor/Splicing Factor 2 (ASF/SF2) Affects Mouse Embryonic Fibroblast Senescence and Apoptosis;Lorena Verduci et al.;《THE JOURNAL OF BIOLOGICAL CHEMISTRY》;20101004;第285卷(第50期);摘要 * |
| The splicing factor SRSF1 as a marker for endothelial senescence;Francisco J. Blanco et al.;《Vascular Physiology》;20120328;第3卷;摘要 * |
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| CN103667459A (en) | 2014-03-26 |
| CN104306993A (en) | 2015-01-28 |
| CN104306992A (en) | 2015-01-28 |
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