CN106955349A - Application of the PP1158 4 in protection endothelial barrier function - Google Patents
Application of the PP1158 4 in protection endothelial barrier function Download PDFInfo
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Abstract
The invention discloses application of the PP1158 4 in protection endothelial barrier function.The present invention discloses application of the PP1158 4 in the product for preparing protection and/or enhancing barrier cell function.Present invention demonstrates that PP1158 4 has the effect of protection endothelial barrier function, and diabetes myocardial ischemia-reperfusion injury can be mitigated.
Description
Technical field
The invention belongs to biological technical field, it is related to the answering in protection endothelial barrier function of PP1158 4
With.
Background technology
PP1158 4 (Angptl4) is secreting type glycoprotein, and in addition to Angptl5, Angptl1-8 exists
Found in people and Mice Body.People's Angptl4 genes are highly conserved, have 77% similar amino acid sequence with mouse.People
Angptl4 genes are located at 19p13.3, and coding molecule amount is 45-65Kda glycoprotein.It is similar to Angptl families, Angptl4
It is made up of N-terminal frizzled domain and C-terminal fibrin prodomain.At present, Angptl4 is considered as orphan ligand, mesh
It is preceding not yet to find its acceptor.However, Angptl4 is by known PPAR families, glucocorticoid receptor, PKC etc. activator institute
Activation, is suppressed by angiotensin receptor blocker, inhibin, leptin and insulin.Angptl4 is mainly expressed in Mice Body
In adipose tissue, liver, skeletal muscle, heart, skin and small intestine.However, human body Angptl4 is mainly expressed in liver, blood plasma, tire
Disk, small intestine, heart and adipose tissue.Angptl4 expression is regulated and controled by glucocorticoid receptor and nuclear receptors PPAR's s.
In human body and mouse, PPAR response elements can mediate Angptl4 expression.In addition, under anoxic conditions cardiac muscle, endothelial cell and
Angptl4 expression increase in tumor tissues.TGF-β can also promote Angptl4 expression in primary tumor cells and transfer stove.No
It is different with the function of Angptl4 in tissue.Originally find that Angptl4 works in lipid metaboli in liver, have now found that
Angptl4 also plays a role in vascular permeability, angiogenesis and inflammatory signals path.Sertoli cell is expressed under normal operation
Angptl4 levels are relatively low, and sertoli cell expression Angptl4 increases under pathological conditions.Research recently finds that sertoli cell is overexpressed
Angptl4 can promote the morbidity of nephrotic syndrome.Angptl4 is raised along with typical pathological change, including a large amount of selectivity
Albuminuria, glomerular basement membrane changes and sertoli cell podocytic process is merged extensively.These results prompting Angptl4 may be to glomerulus base
Counterdie has destruction.And utilizing Angptl4 to knock out and be overexpressed mouse, research finds that Angptl4 is permeated by modulating vascular
Property, suppress the transfer of Lewis lung cancer and B16F0 melanomas.However, another research finds what tumor tissues were produced
β 1 of connection protein integration element α 5/ that Angptl4 is connected and is adhesively joined with endothelial tight, VE-cadherin, claudin-5 knot
Close, cause endothelium to destroy.Glioma growth and angiogenesis can be suppressed by knocking out Angptl4.People are in Angptl4 knock-out mices
Found in research during retinal development and under pathological conditions, Angptl4 is adjusted by regulating and controlling Endothelial junction
Save vascular permeability and angiogenesis.These results prompting Angptl4 is important in modulating vascular permeability and angiogenesis
Effect.However, Angptl4 is unclear to the regulatory mechanism of vascular permeability, therefore it is in vascular permeability and metastases
Status full of dispute.In a word, numerous data show that Angptl4 possesses the regulating and controlling effect to vascular permeability and angiogenesis,
And the development with metastases, cardiovascular event and diseases associated with inflammation is relevant.
Microvascular endothelial barrier function refers to the endothelial cell and each component therebetween for constituting capilary structural pipe wall, control
Cell component and macromolecular substances in blood ooze out into the ability of vascular wall.Endothelial barrier is usually used as using microvascular permeability
The evaluation index of function.Research confirms that the connection between Endothelial junction and endothelial cell and basilar memebrane is to maintain micro- blood
The normal important feature of tube wall permeability and function basis.
The content of the invention
The technical problems to be solved by the invention are protection endothelial barrier functions, and mitigate diabetes myocardial ischemia-reperfusion
Damage.
Preparing protection in order to solve the above technical problems, the present invention provides PP1158 4 and/or strengthening thin
Application in the product of born of the same parents' barrier function；
The protection and/or enhancing barrier cell function embodiment are in terms of following (1)-(3) are at least any shown：
(1) cell permeability increase is suppressed；
(2) protection cytoskeleton and/or suppression Cytoskeleton；
(3) protect and/or promote Cell tracking.
It is protection and/or enhancing the barrier cell function, suppression cell permeability increase, described in above-mentioned application
Protection cytoskeleton and/or suppress Cytoskeleton and the protection and/or promote Cell tracking be respectively protection and/or
The high sugar of enhancing and/or oxygen-sugar-serum deprivation/reoxygenation (OGSD/R) stimulate lower barrier cell function, suppress high sugared and/or oxygen-
Sugar-serum deprivation/reoxygenation (OGSD/R) stimulates lower cell permeability increase, the high sugar of protection and/or oxygen-sugar-serum deprivation/again
Under the high sugar of cytoskeleton and/or suppression and/or oxygen-sugar-serum deprivation/reoxygenation (OGSD/R) under oxygen (OGSD/R) stimulation are stimulated
Cytoskeleton and protection and/or promote high sugar and/or oxygen-sugar-serum deprivation/reoxygenation (OGSD/R) stimulate under cell
Between connect；
Or,
The protection and/or enhancing barrier cell function, suppression cell permeability increase and the protection and/or rush
Barrier cell function under myocardial ischemia-reperfusion injury Wei not protected and/or strengthen, suppress myocardial ischemia by entering Cell tracking
Cell permeability increase and protection caused by reperfusion injury and/or the iuntercellular under promotion myocardial ischemia-reperfusion injury connect
Connect；
The myocardial ischemia-reperfusion injury is preferably diabetes myocardial ischemia-reperfusion injury.
In above-mentioned application, the cell is endothelial cell, preferably CMEC, is further preferably heart microvascular
Endothelial cell.
Preparing prevention in order to solve the above technical problems, the present invention also provides PP1158 4 and/or mitigating
And/or the application in the product for the treatment of myocardial ischemia-reperfusion injury.
In above-mentioned application, the myocardial ischemia-reperfusion injury damages for heart microvascular endothelial cell barrier function.
In above-mentioned application, the heart microvascular endothelial cell barrier function damage is the increase of myocardium at risk area, the heart
Dirty microvascular permeability increase, cardiac muscular tissue's myeloperoxidase expression of enzymes increase, cardiac muscular tissue's focal hemorrhage, in heart microvascular
Connection declines and/or heart microvascular endothelial cell apoptosis between chrotoplast.
In any of the above-described described application, the myocardial ischemia-reperfusion injury damages for diabetes myocardial ischemia-reperfusion
Wound.
In any of the above-described described application, the myocardial ischemia is the myocardial ischemia as caused by acute myocardial infarction AMI.
Preparing prevention in order to solve the above technical problems, the present invention also provides PP1158 4 and/or treating
Application in the product of myocardial infarction.
In above-mentioned application, the myocardial infarction is the cardiac muscle stalk after myocardial ischemia-reperfusion caused by acute myocardial infarction AMI
Extremely.
In above-mentioned application, the acute myocardial infarction AMI is Diabetic Acute myocardial infarction.
Preparing prevention in order to solve the above technical problems, the present invention also provides PP1158 4 and/or treating
Application in the product of disease or injury caused by hyperglycaemia.
In any of the above-described described application, the amino acid sequence such as SEQ ID No.1 institutes of the PP1158 4
Show.
Present invention demonstrates that PP1158 4 has the effect of protection endothelial barrier function, it is possible to mitigate glycosuria
Sick myocardial ischemia-reperfusion injury.
Brief description of the drawings
Fig. 1 detects to strike the permeability of low each group human cardiac microvascular endothelial cells in embodiment 1 without siRNA.
Fig. 2 is to strike the logical of each group human cardiac microvascular endothelial cells after low Angptl4 expression using siRNA in embodiment 1
Permeability is detected.
Fig. 3 is the comparison of each group endothelial cell skeleton structure in embodiment 1.
Fig. 4 is the expression of Western blot detection each group intercellular tight junction albumen in embodiment 1.
Fig. 5 is the expression of Western blot detection each group cell-cell adhesion connection albumen in embodiment 1.
Fig. 6 is the expression of Western blot detection each group cell-cell adhesion spot connection albumen in embodiment 1.
Fig. 7 compares cardiac muscular tissue's focal hemorrhage degree between each group for Hematoxylin-eosin decoration method in embodiment 2.
Fig. 8 is that immunohistochemical method compares cardiac muscular tissue MPO between each group and expressed in embodiment 2.
Fig. 9 is the Electronic Speculum testing result of each group rat heart microvascular endothelial cell in embodiment 2.
Figure 10 is the testing result of each group rat heart microvascular endothelial cell p120-catenin expression in embodiment 2.
Figure 11 is the apoptosis testing result of each group rat heart muscle CMEC in embodiment 2.
Figure 12 is the expression of Western blot detection each group rat heart muscle tissue JAM-A albumen in embodiment 2.
Figure 13 is the expression of Western blot detection each group rat heart muscle tissue VE-cadherin albumen in embodiment 2.
Figure 14 is the expression of the albumen of Western blot detection each group rat heart muscle tissue Integrin- α 5 in embodiment 2.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.
Below in conjunction with specific embodiment, the invention will be further described.It should be understood that following examples are merely to illustrate this
Invention is not for restriction the scope of the present invention.
Human cardiac microvascular endothelial cells (HCMEC) are U.S.'s Sciencell Products, and catalog number is 6000.
1% green grass or young crops/streptomysin twin antibiotic, i.e. penicillin/streptomycin solution (100X), are the production of Sciencell companies of the U.S.
Product, catalog number is 0503.
DMEM low sugar fluid nutrient medium is U.S.'s Hyclone Products, and catalog number is SH30021.01B.
The component of high glucose medium is as shown in table 1：
The high glucose medium component of table 1
Basal medium in table 1 is DMEM low sugar fluid nutrient mediums.
The preparation of insulin (1nM)：Insulin (Sigma Products, catalog number is I-5500) 10mg is weighed, plus
(2M hydrochloric acid conditioning solutions pH value=2.0, after after dry powder insulin dissolving, pH are finely tuned with 0.5M sodium hydroxide solutions to 5ml distilled water
To the storage liquid for 7.0) being made into 2mg/ml, 20 parts, every part of 0.25ml (insulin containing 0.5mg), -20 DEG C of guarantors are packed as with EP pipes
Deposit, take appropriate storage liquid to be added in culture medium when using, be diluted to aimed concn.
RhAngptl4 (1 μ g/ml) preparation：Taking 50 μ g Angptl4 dry powder, (R＆D Products, catalog number is
4487-AN-050), the PBS for adding 0.5mL is made into 100 μ g/ml storage liquid, is placed in -20 DEG C of preservations, appropriate storage is taken when using
Liquid storage is added in culture medium, is diluted to aimed concn.
Angptl4siRNA is U.S.'s Applied Biosystems Products, and catalog number is s169900, sequence
For 5 '-GGGCCGCUACUAUCCACUAtt-3 ' (SEQ ID No.2).
The preparation of Angptl4siRNA solution (60pmol)：5nmol Angptl4siRNA pulvis adds 50 μ l DEPC
Water, -20 DEG C of preservations.Take the above-mentioned solution of 1 μ l to add 9 μ l DEPC water, dilute 10 times to 10 μM, take the 6 μ l solution to add to 600 μ
L Opti-MEM I culture mediums (Invitrogen Products, catalog number be 51985034) in, stand 5min, obtain A
Liquid.6 μ l lipo-imax (Invitrogen Products, catalog number is 13778-100) reagents are taken to add to 600 μ l
In Opti-MEM I culture mediums, B liquid is obtained.By A liquid and B liquid by being well mixed, stand 20min at room temperature, then with 10 μ l Red
(Invitrogen Products, catalog number is 14750100) to be well mixed to Oligo, obtains 1.2ml transfection reagents, can use
In 4 holes of 24 orifice plates.
Negative siRNA (Select Negative Control#1siRNA) is Applied Biosystems companies of the U.S.
Product, catalog number is 4390843.
The preparation of negative siRNA solution (60pmol)：With Angptl4siRNA solution (60pmol) preparation, with 5nmol
Negative siRNA pulvis replaces Angptl4siRNA pulvis, can be configured to negative siRNA solution (60pmol), 4 for 24 orifice plates
Individual hole.
Diabetic mice (genotype：ZDF-Leprfa/fa) and the thin mouse (genotype of non-diabetic：ZDF-Leprfa/+) it is SPF grades
12 week old male ZDF rats, body weight：(325 ± 25) g, is Beijing Vital River Experimental Animals Technology Co., Ltd.'s product.
The amino acid sequence of PP1158 4 is as shown in SEQ ID No.1.
PP1158 4 under embodiment 1, high sugar and oxygen-sugar-serum deprivation/reoxygenation (OGSD/R) Co stituation
(Angptl4) to the influence of endothelium barrier function
Low endothelial cell Angptl4 expression is struck using siRNA, to confirm whether Angptl4 has protection endothelial cell
Function.
First, human cardiac microvascular endothelial cells (HCMEC) are cultivated in high sugared (18mM) (table 1) culture medium, by 5-6
Following each group is randomly divided into for cell, is cultivated respectively under conditions of following each group in T75 Tissue Culture Flasks, then distinguish
Give oxygen-sugar-serum deprivation/reoxygenation (OGSD/R) processing.
OGSD/R processing includes：(1) carry out changing liquid using corresponding culture medium, realize sugar-serum deprivation.Wherein, medicine
Intervene and OGSD/R interventions are shown in Table 2 with cell culture media component.(2) anaerobism box (BioM é rieux, Marcy are moulded by force using 2.5L
L ' Etoile, France, catalog number be 96127) and anaerobic gas generation bag (BioM é rieux, Marcy l ' Etoile, France,
Catalog number is 96124), anoxic to be carried out to the cell in blake bottle and intervenes 2h (the constant red tables of anoxic bar presentation
Show and reach anoxia condition), Tissue Culture Flask is transferred to incubator (37 DEG C, 5%CO from anoxic box2) reoxygenation intervention 2h is carried out,
Realize anoxia _ reoxygenation.So far OGSD/R processing is completed.
The pharmaceutical intervention of table 2 and OGSD/R intervention cell culture mediums
Basal medium in table 2 is DMEM low sugar fluid nutrient mediums.
1st, normal glucose OGSD/R groups：By final concentration of 5 × 105/cm2Cell under the conditions of normal glucose (5.5mM) (table 1)
24h is cultivated, followed by liquid is changed with 5.5mM culture mediums (table 2), OGSD/R processing is given immediately；
2nd, high sugar OGSD/R groups：By final concentration of 5 × 105/cm2Cell cultivated under the conditions of high sugared (18mM) (table 1)
24h, followed by changes liquid with 18mM culture mediums (table 2), OGSD/R processing is given immediately；
3rd, insulin group (1nM)：1h before anoxic, by final concentration of 5 × 105/cm2Cell 18mM culture mediums (table 2, its
The middle final concentration of 1nM of insulin) middle culture 1h, followed by gives OGSD/R processing；
4th, Angptl4 groups (1 μ g/ml)：1h before anoxic, by final concentration of 5 × 105/cm2Cell in 18mM culture medium (tables
The final concentration of 1 μ g/ml of 2, wherein Angptl4) middle culture 1h, followed by gives OGSD/R processing；
5th, Angptl4+ feminine genders siRNA groups：It is 4 per hole density when the cell cultivated in 24 orifice plates reaches 80% fusion
×104/cm2Cell, which is added, changes high sugared (18mM) (table after 300 μ l transfection reagents (60pmol feminine gender siRNA solution), transfection 6h
1) fluorescence microscopy Microscopic observation transfection after culture medium, transfection 24h and 48h, after after stable growth, the 1h before anoxic will eventually
Concentration is 5 × 105/cm2Cell in the 18mM culture mediums (the final concentration of 1 μ g/ml of table 2, wherein Angptl4) culture 1h, followed by
Give OGSD/R processing；
6th, Angptl4+Angptl4siRNA groups：Transfection reagent is only changed to 60pmol by processing method with the 5th group
Angptl4siRNA solution, the 1h before anoxic, by final concentration of 5 × 105/cm2Cell 18mM culture mediums (table 2, wherein
The final concentration of 1 μ g/ml of Angptl4) middle culture 1h, followed by gives OGSD/R processing.
Above each group is all provided with 6 repetitions.
2nd, fluorogenic quantitative detection each group individual layer endothelial permeability
(1) comparison of low each group endothelial barrier function is struck without siRNA
The total serum IgE of each group cell in extraction step one, and application HiFi-MMLVcDNA the first chain synthetic agent box (Beijing
Cwbio Products, catalog number is cw0744) reverse transcription is carried out, obtain the cDNA of each group.Respectively using the cDNA of each group as
Template, using Power SYBR green PCR Master Mix kits, (American AB I Products, catalog number is
4367659), using GAPDH as house-keeping gene, Real-time PCR testing goal Gene As ngptl4 expression is carried out.
Real-time PCR primers are as follows：
GAPDH sense primers：5’-ACATCGCTCAGACACCATG-3’(SEQ ID No.3)；
GAPDH anti-sense primers：5’-TGTAGTTGAGGTCAATGAAGGG-3’(SEQ ID No.4).
Angptl4 sense primers：5’-AGACACAACTCAAGGCTCAG-3'(SEQ ID No.5)；
Angptl4 anti-sense primers：5’-CTCATGGTCTAGGTGCTTGTG-3'(SEQ ID No.6).
Real-time PCR reaction conditions are as shown in table 3.
Real-time PCR the reaction conditions of table 3
95 DEG C, 10min；95 DEG C, 15 seconds, 60 DEG C, this collection step fluorescence signal of 1min, 40 circulations, after the completion of reaction
With melt curve analysis analysis is carried out at 60-95 DEG C, 1 DEG C is raised within every 10 seconds.
According to Real-time PCR testing results, using 2-ΔΔctMethod is to detecting that the differential expression of gene is carried out in each sample
Relative quantitative assay.2-ΔΔctRelative quantitative assay computing formula is as follows：
F=2- { [the to be measured group of house-keeping gene Average Ct values of target gene Average Ct values-to be measured group]-[control group target gene Average Ct values-control group house-keeping gene Average Ct values] }
Using human heart capilary normal single layer endothelial cell as control, to normal glucose OGSD/R groups, height sugar OGSD/R groups, pancreas
The monolayer endothelial cell of island element group (1nM) and Angptl4 groups (1 μ g/ml) carries out permeability detection, utilizes extracorporeal blood vessel permeability
Detection kit (U.S.'s Millipore Chemicon Products, catalog number is ECM640) carries out each group fluorescence and determined
Amount, light microscopic testing result and fluorogenic quantitative detection result are respectively as shown in Fig. 1 and table 4.
In Fig. 1, A：Normal single layer endothelial cell；B：Normal glucose OGSD/R groups；C：High sugar OGSD/R groups；D：
Note：★★P<0.01vs. human heart capilary normal single layer endothelial cell groups；**P<0.01vs. normal glucoses OGSD/R
Group；▲▲P<0.01vs. height sugar OGSD/R groups.
Microvascular endothelial barrier function refers to the endothelial cell and each component therebetween for constituting capilary structural pipe wall, control
Cell component and macromolecular substances in blood ooze out into the ability of vascular wall.Endothelial barrier is usually used as using microvascular permeability
The evaluation index of function.
Fig. 1 and table 4 show, compared with human heart capilary normal single layer endothelial cell group, normal glucose OGSD/R groups and height
The permeability of sugared OGSD/R groups human cardiac microvascular endothelial cells significantly increases that (P is equal<0.05), and high sugar OGSD/R groups ratio just
The permeability of normal sugar OGSD/R group human cardiac microvascular endothelial cells significantly increases (P<0.05).Compared with high sugar OGSD/R groups,
The permeability of the human cardiac microvascular endothelial cells of Angptl4 groups significantly reduces (P<, and insulin group and Angptl4 0.05)
Quite (P is equal for the effect of group>0.05).Illustrate that Angptl4 can protect the permeability of human cardiac microvascular endothelial cells, suppress thin
The increase of born of the same parents' permeability.
(2) each group endothelial barrier function after low Angptl4 expression is struck using siRNA to compare
Following each group is set, then gives OGSD/R processing of the following each group with step one respectively.
1st, the negative siRNA groups of monolayer endothelial cell：ECM culture mediums (U.S.'s Sciencell Products, catalogue
Number for 6000) in 24 orifice plates cultivate human cardiac microvascular endothelial cells (HCMEC) reach 80% fusion when, per hole density be 4
×104/cm2Cell add 300 μ l transfection reagents (60pmol feminine gender siRNA solution), transfection 6h after change high sugar (18mM)
Fluorescence microscopy Microscopic observation transfection after (table 1) culture medium, transfection 24h and 48h, treats stable growth；
2nd, the Angptl4siRNA groups of monolayer endothelial cell：Transfection reagent is only changed to 60pmol by processing method with the 1st group
Angptl4siRNA solution；
3rd, high sugar OGSD/R negative siRNA groups：With high sugared (18mM) (table 1) culture medium human heart is cultivated in 24 orifice plates
It is 4 × 10 per hole density when CMEC (HCMEC) reaches 80% fusion4/cm2Cell add 300 μ l transfection examination
Fluorescence microscopy Microscopic observation after complete medium, transfection 24h and 48h is changed after agent (60pmol feminine gender siRNA solution), transfection 6h
Transfection, treats stable growth；
4th, high sugar OGSD/R Angptl4siRNA groups：Transfection reagent is only changed to 60pmol by processing method with the 3rd group
Angptl4siRNA solution；
5th, Angptl4 negative siRNA groups：The cell cultivated with high sugared (18mM) (table 1) culture medium in 24 orifice plates reaches
It is 4 × 10 per hole density to during 80% fusion4/cm2Cell add 300 μ l transfection reagents (60pmol feminine gender siRNA solution),
Fluorescence microscopy Microscopic observation transfection after complete medium, transfection 24h and 48h is changed after transfection 6h, after stabilization grows,
1h gives Angptl4 (final concentration of 1 μ g/ml) interventions before anoxic；
6th, Angptl4 Angptl4siRNA groups：Transfection reagent is only changed to 60pmol by processing method with the 5th group
1h gives Angptl4 (final concentration of 1 μ g/ml) interventions before Angptl4siRNA solution, anoxic；
Above each group is all provided with 8 repetitions.
By the negative siRNA groups and Angptl4siRNA groups of monolayer endothelial cell obtained above, high sugar OGSD/R the moon
Property siRNA groups and Angptl4siRNA groups and Angptl4 negative siRNA groups and Angptl4siRNA groups.To this 6 groups of list
Layer endothelial cell progress permeability detection, light microscopic testing result and fluorogenic quantitative detection result are respectively as shown in Fig. 2 and table 5.
In Fig. 2, A, E：Normal single layer endothelial cell；B、F：High sugar OGSD/R groups；C、G：Angptl4 groups (1 μ g/ml)；A-
C：Negative siRNA groups；E-G：Angptl4siRNA groups.
Influence (X ± s, n=8) of the different pharmaceutical of table 5 to monolayer endothelial cell permeability
Note：In each row★★P<0.01vs. monolayer endothelial cells；**P<0.01vs. height sugar OGSD/R groups.
Fig. 2 and table 5 show, compared with human heart capilary normal single layer endothelial cell, and the endothelium of high sugar OGSD/R groups is thin
Born of the same parents' permeability significantly increases (P<0.05).Compared with high sugar OGSD/R groups, Angptl4 negative siRNA groups endothelial permeability is equal
Significantly reduce (P<0.05).Compared with Angptl4 negative siRNA groups, Angptl4 Angptl4siRNA group endothelial permeabilities
Nothing significantly changes (P>0.05).As a result show, the defencive function of endothelial cell barrier can be blocked by striking low Angptl4 expression,
But the effect of protection barrier function of endothelial cells can still be played by striking exogenous supplement Angptl4 after low Angptl4.
3rd, the comparison of each group endothelial cell skeleton structure
Using human heart capilary normal single layer endothelial cell as control, observation of steps one under laser scanning co-focusing microscope
Middle normal glucose OGSD/R groups, height sugar OGSD/R groups, insulin group, Angptl4 groups, Angptl4+ feminine gender siRNA groups and Angptl4
The cytoskeleton of+Angptl4siRNA groups.
The method of above-mentioned laser scanning co-focusing microscope detection cytoskeleton is specific as follows：
(1) preparation of cell climbing sheet
Using(Denmark's Thermo NUNC Products, catalog number is II chamber slides system
154534) cell climbing sheet of each group is prepared.
(2) fixation of cell climbing sheet
1st, the culture medium supernatant in the small chamber of chamber slide system is discarded, small chamber is embathed 3 times with PBS, each 3min notes
Action must be soft, to prevent damaging cells.The liquid in each small chamber is exhausted when last time is rinsed.
2nd, at room temperature, cell 15min is fixed with 4% paraformaldehyde, embathed 3 times with PBS, each 3min.
(3) rupture of membranes
0.1%Triton X-100/PBS room temperature ruptures of membranes 5min, PBS embathe cell 3 times, each 3min.
(4) close
(Wuhan doctor's moral bioengineering is limited for 10% Normal Goat Serum that dropwise addition PBS is prepared in small chamber after exhaustion PBS
Products, catalog number is AR0009), 40min is closed at room temperature.
(5) cytoskeleton is contaminated
5 μ l FITC- phalloidines storage liquid (Sigma Co., USA's product, catalog number is P5282) adds 150 μ l
Working solution (5 μ g/ml) is made into PBS and to contaminate each group cell, room temperature dyes 30-60min in wet box.
(6) core and mounting are redyed
PBS embathes each group cell climbing sheet 3 times, after each 3min, the liquid in each small chamber of exhaustion, with the envelope containing DAPI
Piece liquid (Beijing biotech company of Zhong Shan Golden Bridge product, catalog number is ZLI-9557) mounting, and pasted carefully with nail polish
Born of the same parents' creep plate surrounding.The careful spot for cleaning creep plate surface.
(7) IMAQ
Randomly select every group of 5 visuals field progress observation under laser scanning co-focusing microscope to take pictures, with swashing for 405nm wavelength
Light device excitated blue fluorescence, green fluorescence is excited with the laser of 488nm wavelength, is observed and is compared each group cell.
As a result it is as shown in Figure 3.
In Fig. 3, A：Normal single layer endothelial cell；B：Normal glucose OGSD/R groups；C：High sugar OGSD/R groups；D：Insulin group；
E：Angptl4 groups；G：Angptl4+ feminine gender siRNA groups；I：Angptl4+Angptl4siRNA groups.
Fig. 3 shows, compared with human heart capilary normal single layer endothelial cell, normal glucose OGSD/R groups and high sugar OGSD/R
The stress myofilament of group endothelial cell is dramatically increased, and the linear cytoskeleton of endothelial cell rule disappears, and myofilament is to cell film transfer
Increase, cell retraction, Intercellular Bridge increase.Compared with high sugar OGSD/R groups, insulin group and Angptl4 groups endothelial cell should
Power myofilament is reduced, and Intercellular Bridge is reduced, and insulin group is suitable with the effect of Angptl4 groups, points out Angptl4 to protect
Endothelial cell skeleton is protected, suppresses Cytoskeleton or scattered.
4th, influence of each group to intercellular tight junction protein expression
Using JAM-A antibody (Abcam Products, catalog number is Britain AB52647), Occludin antibody
(Abcam Products, catalog number is Britain AB31721) and GAPDH antibody (U.S.'s CST Products, catalog number
For 2118), normal glucose OGSD/R groups, height sugar OGSD/R groups, insulin group, Angptl4 in Western blot detecting steps one
The expression of group, Angptl4+ feminine gender siRNA groups and Angptl4+Angptl4siRNA group endothelial cells Occludin and JAM-A,
Integrality for evaluating tight connecting device between endothelial cell.
As a result it is as shown in Figure 4.
In Fig. 4,1：Normal glucose OGSD/R groups；2：High sugar OGSD/R groups；3：Insulin group；4：Angptl4 groups；5：
Angptl4+ feminine gender siRNA groups；6：Angptl4+Angptl4siRNA groups；A：Occludin protein expressions, B：JAM-A albumen tables
Reach, n=6；★★P<0.01vs. normal glucose OGSD/R groups；**P<0.01vs. height sugar OGSD/R groups.
Fig. 4 shows, compared with normal glucose OGSD/R groups, and high sugar OGSD/R group endothelial cells Occludin expression is without significance difference
Not (P>0.05), and JAM-A expression significantly reduce (P<0.05).Compared with high sugar OGSD/R groups, insulin group and Angptl4 groups
Without significantly changing, (P is equal for endothelium Occludin expression>0.05), and two groups of endothelium JAM-A expression significantly increases that (P is equal<
0.05), and insulin suitable with Angptl4 effect (P is equal>0.05).Illustrate that Angptl4 can promote tight junction protein
JAM-A expression, strikes the expression that low Angptl4 can prevent endothelial cell tight to connect albumen.
5th, influence of each group to cell-cell adhesion connexin expression
Using VE-cadherin antibody (U.S.'s Santa Cruz Products, catalog number is Sc-28644),
(CST companies of the U.S. produce for p120-catenin antibody (Abcam Products, catalog number is AB92514) and GAPDH antibody
Product, catalog number is 2118) normal glucose OGSD/R groups, height sugar OGSD/R groups, pancreas islet in Western blot detecting steps one
Plain group, Angptl4 groups, Angptl4+ feminine gender siRNA groups and Angptl4+Angptl4siRNA group endothelial cells VE-cadherin
With p120-catenin expression, the integrality for evaluating adhesion junction structure between endothelial cell.
As a result it is as shown in Figure 5.
In Fig. 5,1：Normal glucose OGSD/R groups；2：High sugar OGSD/R groups；3：Insulin group；4：Angptl4 groups；5：
Angptl4+ feminine gender siRNA groups；6：Angptl4+Angptl4siRNA groups；A：The expression of VE-cadherin albumen, B：p120-
The expression of catenin albumen, n=6；★★P<0.01vs. normal glucose OGSD/R groups, * * P<0.01vs. height sugar OGSD/R groups.
Fig. 5 shows, compared with normal glucose OGSD/R groups, and high sugar OGSD/R group endothelial cells VE-cadherin expression shows
Write reduction (P<0.05), and p120-catenin expression without significantly changing (P>0.05).Compared with high sugar OGSD/R groups, insulin
The VE-cadherin expression of group endothelial cell significantly increases (P<0.05), Angptl4 group endothelial cells VE-cadherin expression
Have and increase trend (P>0.05).Insulin group and Angptl4 group endothelial cells p120-catenin expression are without significantly changing (P
>0.05).Illustrate that Angptl4 can promote endothelial cell adhesion to connect albumen VE-cadherin expression, strike low Angptl4
Endothelial cell adhesion connexin expression can be prevented.
6th, influence of each group to cell-cell adhesion spot connexin expression
Resisted using the antibody of Integrin- α 5 (Abcam Products, catalog number is AB150361), Integrin- β 1
(U.S.'s Cell Signaling Technology Products, catalog number is (CST 4706s) and GAPDH antibody to body
(U.S.'s CST Products, catalog number is 2118) normal glucose OGSD/R groups, height in Western blot detecting steps one
Sugared OGSD/R groups, insulin group, Angptl4 groups, Angptl4+ feminine gender siRNA groups and Angptl4+Angptl4siRNA group endotheliums
Cell integrin- α 5 and integrin- β 1 expression, the integrality for evaluating focal adhension attachment structure between endothelial cell.
As a result it is as shown in Figure 6.
In Fig. 6,1：Normal glucose OGSD/R groups；2：High sugar OGSD/R groups；3：Insulin group；4：Angptl4 groups；5：
Angptl4+ feminine gender siRNA groups；6：Angptl4+Angptl4siRNA groups；A：The protein expressions of integrin- α 5, B：
The protein expressions of integrin- β 1, n=6；★★P<0.01vs normal glucose OGSD/R groups；**P<0.01vs height sugar OGSD/R groups.
Fig. 6 shows, is compared with normal glucose OGSD/R groups, height sugar OGSD/R group integrin- α 5 and integrin- β 1 table
(P is significantly changed up to equal nothing>0.05).Compared with high sugar OGSD/R groups, insulin group and Angptl4 group endothelium integrin- α 5
Expression significantly increase that (P is equal<0.05), and insulin suitable with Angptl4 effect (P is equal>0.05), but in this two groups
Without significantly changing, (P is equal for the expression of integrin- β 1 of chrotoplast>0.05).Prompting Angptl4 can promote focal adhension to connect albumen
Expression.
The result of above-mentioned steps four to six shows that PP1158 4 can promote Cell tracking.
In summary, normal glucose and height sugar OGSD/R cause the barrier function of heart microvascular endothelial cell to damage, and
The latter's damage is heavier.And insulin and Angptl4 can protect the heart microvascular endothelial barrier work(under high sugar OGSD/R states
Energy.
Embodiment 2, PP1158 4 (Angptl4) mitigate diabetes cardiac muscle by protecting endothelial barrier function
Ischemical reperfusion injury
Myocardial infarction after acute myocardial infarction AMI (AMI) Reperfu- sion occurs respectively to verify the thin mouse of non-diabetic, diabetic mice
Scope and the degree of reperfusion injury, the influence to Microvascular Endothelial Cellss apoptosis and to myocardial microvascular endothelial barrier
Function has not damaged, and can PP1158 4 reduce myocardial infarct size and Reperfu- sion after diabetic mice AMI Reperfu- sions
Damage, Microvascular Endothelial Cellss apoptosis and protection myocardial microvascular endothelial barrier function, are tested as follows：
First, by 12 week old male ZDF rats 56, it is randomly divided into following each group：
1st, diabetic mice sham-operation group：Diabetic mice LAD (left anterior descending branch (blood vessel)) threading but do not ligature.
2nd, diabetic mice MI control groups：It is not administered before and after diabetic mice LAD threading.
3rd, the thin mouse MI control groups of non-diabetic：It is not administered before and after the thin mouse LAD threading of non-diabetic.
4th, insulin group (5U/100g body weight)：Diabetic mice 1h before following coronary artery occlusion descending anterior branch starts intravenous injection
Insulin (5U/100g body weight), it is 10mmol/L to monitor and maintain blood glucose.
5th, Angptl4 groups (10 μ g/kg body weight)：Diabetic mice 1h intravenous injection recombined humans Angptl4 before ligation LAD
(rhAngptl4,10 μ g/kg body weight).
6th, Angptl4+ feminine genders siRNA groups：48h, myocardial injection transfection reagent feminine gender siRNA before diabetic mice LAD ligation
(80 μ g/kg body weight)：
1) N/P ratios (key of influence transfection efficiency) are calculated
N/P ratios are the electrically charged measurements to JetPEI complexs.It refers to the electronegative phosphate of DNA molecular
Group and the mol ratio of N residues in JetPEI, are the parameters for weighing nucleic acid and transfection reagent ratio.Theoretically, JetPEI/
The electroneutral of DNA complex is in N/P=2-3, but this experiment, N/P=6 transfections is best.
2) 5nM siRNA add the glucose solutions of 4.5 μ l 10% (μ g/ μ l of final concentration 8), freeze in -20 DEG C of Refrigerator stores.Take 3
μ l store liquid, and 8 μ l are diluted to 10% glucose solution.I.e.：The μ l of+7 μ l deionized waters of 5 μ l, 10% glucose solutions+3 are negative
SiRNA, obtains 1/2 volume injected (glucose final concentration 5%), mixes and stands 5min.
3) the μ l in vivo-jetPEI reagents (Polyplus- of+4 μ l deionized waters of 8 μ l, 10% glucose solutions+3
Transfection Products, catalog number is 201-10G), 1/2 volume injected (glucose final concentration 5%) is obtained, is mixed
It is even.
4) by step 2) and the above two liquid 1 that 3) obtains：1 mixing, obtains the transfection examination of 1 volume injected (30 μ l)
Agent (0.8 μ g/ μ l, N/P=6), is mixed, and is incubated at room temperature 15min, for a rat injection.
5) obtained transfection reagent JetPEI-DNA complex solutions are injected to cardiac muscle, specific method is as follows：
1. anaesthetize：Inject in yellow Jackets (1%, 35mg/kg) abdominal cavity；
2. preserved skin, sterilization, fixed animal；
3. the left side intercostal of chest the 5th is cut off, and cuts pericardium, exposure heart；
4. 32G micro syringes injection transfection reagent, point 3 points of injection left ventricle antethecas, often locate 10 μ l of injection.
5. chest is closed, rat normally raises 48h and is followed by by ischemia/reperfusion.
This group of animal 1h before ligation LAD starts intravenous injection rhAngptl4 (10 μ g/kg body weight).
7th, Angptl4+Angptl4siRNA groups：48h, myocardial injection transfection reagent before diabetic mice LAD ligation
1h gives rhAngptl4 (10 μ g/kg body weight) intravenous injections before Angptl4siRNA (method is with the 6th group), ligation LAD.
Totally 7 groups, every group 8.
By the 2nd, 3,4,5,6,7 groups open chest following coronary artery occlusion 45min, then open 180min, set up AMI Reperfu- sion moulds
Type.
2nd, between each group microvascular permeability comparison
Hazardous area (including non-ischemic region and Reperfu- sion area) cardiac muscle FITC- dextrans content is detected to assess the micro- blood of heart
Endothelial cell barrier function.
(1) judgement of dangerous area and materials
Weigh after heart gross mass, excision left atrium and right ventricle weigh left room gross mass.Then left room is cut into uniformly
Thin slice, thickness is slightly larger than 2mm, weighs the quality of each myocardium piece.Take pictures, photo imports computer, measure dangerous area, calculate
Hazardous area volume and quality.
Observe the non-ischemic region hazardous area scope of terminal each group as shown in table 6.
Observe terminal each group Reperfu- sion area hazardous area scope as shown in table 7.
(2) Miles methods microvascular permeability is determined
Heart microvascular endothelial permeability is according to " Hultstrom D, Malmgren L, Gilstring D, et
al.Fitc-dextrans as tracers for macromolecular movements in the nervous
system.A freeze-drying method for dextrans of various molecular sizes
injected into normal animals[J].Acta Neuropathol,1983,59(1):Side disclosed in 53-62. "
Method, cardiac muscular tissue extract fluorescein isothiocynate (FITC) concentration is determined using ELIASA.30min tail veins are noted after Reperfu- sion
Penetrate progress capilary after 0.2ml FITC-dextran containing 200mg/ml (70kDa) normal saline solution, cardiac muscle materials penetrating
Property detection.Cardiac muscular tissue's materials are carried out after rat is put to death after 2.5h.(weight in wet base 100mg) is drawn materials in rat left ventricle myocardium at risk,
After cold PBS is rinsed, it is placed in the solution (pH=7.4) of ammonium acetate and 150mmol/l sodium chloride of the 1ml containing 50mmol/l and is homogenized,
15min is centrifuged in 12000g at 4 DEG C.Supernatant is transferred in new EP pipes, and 1: 100 dilution proportion, storage are pressed with above-mentioned buffer solution
Preserved in -70 DEG C of refrigerators, according to (Lowry OH, Rosebrough NJ, Farr AL, the et al.Protein such as Lowry
measurement with the folin phenol reagent[J].J Biol Chem,1951,193(1):265-275)
Method, using BSA as standard items, utilize multi-function microplate reader detect FITC-dextran concentration.
Observe the non-ischemic region microvascular permeability Parameters variation of terminal each group as shown in table 6.
Observe terminal each group Reperfu- sion area microvascular permeability Parameters variation as shown in table 7.
Table 6 observes the non-ischemic region microvascular permeability Parameters variation (X ± s, n=8) of terminal each group
Note：Compared with diabetic mice sham-operation group,★P<0.05；
The observation terminal each group Reperfu- sion area's microvascular permeability Parameters variation of table 7 (X ± s, n=8)
Note：Compared with diabetic mice MI control groups, * * P<0.01；
Table 7 shows, compared with diabetic mice sham-operation group, diabetic mice MI control groups and the thin mouse MI control groups of non-diabetic
Myocardium FITC- dextrans content dramatically increases that (P is equal<, and diabetic mice MI control groups mouse MI thinner than non-diabetic 0.05)
Control group cardiac muscle FITC- dextran contents dramatically increase (P<0.05).Compared with diabetic mice MI control groups, insulin group and
Angptl4 groups cardiac muscle FITC- dextran contents significantly reduce that (P is equal<, and insulin is suitable with Angptl4 effect 0.05)
(P is equal>0.05), illustrate that Angptl4 is capable of the permeability of cardioprotection CMEC, suppress the increase of its permeability.
3rd, between each group cardiac muscular tissue's focal hemorrhage degree comparison
Hematoxylin-eosin (H-E) is dyed and cardiac muscular tissue's focal hemorrhage degree scoring, is comprised the following steps that：
1st, 5 μm of paraffin sections, are placed in 60 DEG C of constant temperature roasters, toast 1h；
2nd, dimethylbenzene dewaxes, 20-30min；
3rd, graded ethanol aquation：100%, 95%, 90%, 80% and 70% is followed successively by from high to low；
4th, flowing water is rinsed well, adds haematoxylin dye liquor 5min, washing；
5th, flowing water is rinsed well, adds 1% hydrochloride alcohol differentiation liquid 2-3 seconds；
6th, clear water rinses 2min, and warm water returns indigo plant；
7th, 1% Yihong ethanol solution 20s is added；
8th, gradient alcohol dehydration：It is 70%, 80%, 90%, 95% and 100% from low to high；
9th, dimethylbenzene is transparent：Each immersion 10min in dimethylbenzene I and dimethylbenzene II；
10th, neutral gum mounting；
11st, light Microscopic observation cardiac muscular tissue focal hemorrhage degree and score：Every slice, thin piece selects 4 visuals field at random, × 100
The extravasation degree of light Microscopic observation red blood cell, and added their confirmation under × 400 mirrors.Standards of grading (with reference to " Barrabes JA,
Garcia-Dorado D,Gonzalez MA,et al.Regional expansion during myocardial
ischemia predicts ventricular fibrillation and coronary reocclusion[J].Am J
Physiol,1998,274(5Pt2):H17671775”)：0 point：Have no erythrocyte extravasation；1 point：Red blood cell slightly exosmoses；2 points：
Red blood cell largely exosmoses between tissue；3 points：The red blood cell region property fusion of extravasation, forms hemotoncus.
Hematoxylin-eosin (H-E) coloration result is as shown in fig. 7, cardiac muscular tissue's focal hemorrhage scoring is as shown in table 8.
In Fig. 7, A：Diabetic mice sham-operation group；B：Diabetic mice MI control groups；C：The thin mouse MI control groups of non-diabetic；D：
Insulin group；E：Angptl4 groups；G：Angptl4+ feminine genders siRNA；I：Angptl4+Angptl4siRNA groups.
Fig. 7 and table 8 show, compared with diabetic mice sham-operation group, diabetic mice MI control groups and the thin mouse MI of non-diabetic
Control group cardiac muscle FITC- dextrans content, focal hemorrhage, MPO expression dramatically increase that (P is equal<, and diabetic mice MI 0.05)
The focal hemorrhage of control group mouse MI control groups thinner than non-diabetic dramatically increases that (P is equal<0.05).With diabetic mice MI control group ratios
Significantly reduce that (P is equal compared with, insulin group and Angptl4 groups cardiac muscle FITC- dextrans content, focal hemorrhage, MPO expression<
0.05), and insulin suitable with Angptl4 effect (P is equal>0.05), illustrate that Angptl4 can reduce myocardium FITC- dextrorotation
Sugared acid anhydride content, focal hemorrhage, MPO expression, protect CMEC.
4th, the influence that each group is expressed cardiac muscular tissue's myeloperoxidase (MPO)
Detection hazardous area cardiac muscle MPO expresses to assess heart microvascular endothelial barrier function.
MPO is dyed
1st, piece is baked：Paraffin-embedded tissue section 3-4 μ m thicks, section to be done are placed in slide holding frame, in 60 DEG C of thermostatic roastings
1h is at least baked in case；
2nd, dewax：Section is put into 3 times (i.e. dimethylbenzene I, II, III) of dewaxing, each 10min in the container for fill dimethylbenzene；
3rd, aquation：Section is through graded ethanol aquation, absolute ethyl alcohol 5min, 95% ethanol 2 times (each 2min), 85% ethanol
2min；75% ethanol 2min, running water is rinsed, ddH2O washes 2 × 2min；
4th, antigen retrieval：Enzymic digestion repairing method, section TBS is rinsed, and pepsin or trypsase, 37 is organizationally added dropwise
DEG C be incubated 20~30min after TBS washing (2 × 2min).
5th, close：Confining liquid is added dropwise, i.e. (Wuhan doctor's moral bioengineering is limited for 10% Normal Goat Serum that PBS is prepared
Products, catalog number is AR0009), 37 DEG C of wet box are incubated 30min；
6th, primary antibody is added：MPO antibody (ProSci Products, catalog number is 70-900) is added dropwise, 37 DEG C of wet box are incubated
Educate 2h；
7th, wash：TBS-T washs (3 × 5min)；
8th, close：Confining liquid is added dropwise, i.e. 10% Normal Goat Serum that PBS is prepared, 37 DEG C of wet box are incubated 10min；
9th, secondary antibody is added：Biotinylation secondary antibody (biotin labeling goat anti-rabbit igg (the H+ that dropwise addition antibody diluent dilutes
L), green skies biotech company product, catalog number is A0277), 30min is incubated in 37 DEG C of wet box；
10th, wash：TBS-T washs (3 × 5min)；
11st, close：20,37 DEG C of wet box of reagent Tween are added dropwise and are incubated closing 20min；
12nd, HRP-SA (horseradish peroxidase-labeled Streptavidin, green skies biotech company product, production are added
Product catalog number (Cat.No.) is A0303)：Dropwise addition antibody diluent dilutes (1：50~200,5~20nM of final concentration), it is incubated in 37 DEG C of wet box
30min；
13rd, wash：TBS-T washs (3 × 5min), TBS washings (2 × 5min)；
14th, develop the color：Developed the color using DAB solution；
15th, redye：Running water is fully rinsed, and is redyed, dehydration, transparent；
16th, mounting：After tissue specimen is dry, glycerine mounting medium mounting is buffered with reagent.
Myeloperoxidase (MPO) coloration result is as shown in Figure 8.
Carried out with the histochemical analysis module in Med 6.0 digital Pathologic image analysis system under graphical analysis, high power lens
10 visuals field are randomly choosed, the Positive Objects surface density of MPO in each visual field are calculated respectively, and take average as various albumen tables
The relative amount reached, cardiac muscular tissue's MPO levels are as shown in table 8.
In Fig. 8, A：Diabetic mice sham-operation group；B：Diabetic mice MI control groups；C：The thin mouse MI control groups of non-diabetic；D：
Insulin group；E：Angptl4 groups；G：Angptl4+ feminine genders siRNA；I：Angptl4+Angptl4siRNA groups.
Fig. 8 and table 8 show, compared with diabetic mice sham-operation group, diabetic mice MI control groups and the thin mouse MI of non-diabetic
Control group cardiac muscle MPO expression dramatically increases that (P is equal<, and diabetic mice MI control groups mouse MI thinner than non-diabetic controls 0.05)
The MPO expression of group cardiac muscle dramatically increases (P<0.05).Compared with diabetic mice MI control groups, insulin group and Angptl4 groups cardiac muscle
MPO expression significantly reduces that (P is equal<0.05), and insulin suitable with Angptl4 effect (P is equal>0.05) Angptl4, is illustrated
Myocardium MPO expression can be suppressed, endothelial barrier is protected.
5th, influence of each group tight connecting device Microvascular Endothelial Cellss under Electronic Speculum
Transmission electron microscope observing endothelial cell tight connects ultra microstructure.
According to " Phinikaridou A, Andia ME, Protti A, et al.Noninvasive magnetic
resonance imaging evaluation of endothelial permeability in murine
atherosclerosis using an albumin-binding contrast agent[J].Circulation,2012,
126(6):Method disclosed in 707-719 ", utilizes transmission electron microscope observing myocardial microvascular ultra microstructure.Myocardium at risk sample
2h in 2% glutaraldehyde solution is fixed on after (every group 2 pieces) materials, followed by 2h is rinsed with phosphate buffer, then with 1% 4
Somuum oxide fixes 2h.Every cardiac muscular tissue is divided into three parts (from ischemia margin zones to ischemic core), and then every part of tissue makes 3
To 4 sections, every animal makes 9-12 section altogether.It is coated with the epoxy after the serial dehydration that sample passes through ethanol.
Semithin section (0.2 μm) with after Toluidine blue staining in light Microscopic observation, the region abundant to determine capilary.In 150 purposes
Slice (0.09 μm) is carried out on copper mesh, and with uranyl acetate and the double dyes of lead citrate, finally using transmission electron microscope observing.
As a result it is as shown in Figure 9.
In Fig. 9, A：Diabetic mice sham-operation group；B：Diabetic mice MI control groups；C：The thin mouse MI control groups of non-diabetic；D：
Insulin group；E：Angptl4 groups；G：Angptl4+ feminine genders siRNA；I：Angptl4+Angptl4siRNA groups.
Fig. 9 shows, compared with diabetic mice sham-operation group, diabetic mice MI control groups and the thin mouse MI control groups of non-diabetic
Myocardium at risk endothelial cell tight connection width is dramatically increased, and MI pairs of diabetic mice MI control groups mouse thinner than non-diabetic
Dramatically increased according to a group myocardium at risk endothelial cell tight connection width.Compared with diabetic mice MI control groups, insulin group and
Angptl4 groups myocardium at risk endothelial cell (myocardium at risk refers to ligature area), close connection width was significantly reduced, and pancreas
Island element is suitable with Angptl4 effect, further illustrates that Angptl4 can protect tight connecting device between endothelial cell.
6th, the influence that each group is expressed CMEC adhesion junction albumen p120-catenin
Hazardous area cardiac muscle p120-catenin expression is detected to assess myocardium endothelial barrier structural intergrity.
Confocal microscopy endothelial cell specific connection albumen p120-catenin expression, myocardium frozen tissue is cut
Piece carries out immunofluorescence analysis, with 5 μ m thick serial section, comprises the following steps that：
1st, frozen section is taken out from -80 DEG C of refrigerators, 30min is air-dried in fume hood.
2nd, the fixed 10min of 4 DEG C of cold acetone.
3rd, PBS is rinsed, 3 times × 3min.
4th, the 10% Normal Goat Serum incubation 30min that PBS is prepared is added dropwise.
5th, inhale and abandon serum, do not wash, anti-p120-catenin antibody (the Abcam Products, product mesh of respective concentration are added dropwise
Record number is AB92514) 100 μ l, 4 DEG C are overnight.
6th, PBS is rinsed, 3 times × 3min.
7th, the goat anti-rabbit igg (EarthOx Products, catalog number is E031230) of FITC marks, 37 is added dropwise
DEG C, it is incubated 30min.
8th, PBS is rinsed, 3 times × 3min.
9th, PBS is blotted, with the mounting fluid-tight piece containing DAPI.
10th, taken pictures immediately in observation under laser scanning co-focusing microscope.Also it can be positioned in -20 DEG C of refrigerators, in one week
Observation is taken pictures.
Confocal microscopy result is as shown in Figure 10.
Carried out with the histochemical analysis module in Med 6.0 digital Pathologic image analysis system under graphical analysis, high power lens
10 visuals field are randomly choosed, the Positive Objects surface density of p120-catenin in each visual field are calculated respectively, and take average conduct
The relative amount of various protein expressions, endothelial cell p120-catenin expressions are as shown in table 8.
In Figure 10, A：Diabetic mice sham-operation group；B：Diabetic mice MI control groups；C：The thin mouse MI control groups of non-diabetic；
D：Insulin group；E：Angptl4 groups；G：Angptl4+ feminine genders siRNA；I：Angptl4+Angptl4siRNA groups.
Figure 10 and table 8 show, compared with diabetic mice sham-operation group, diabetic mice MI control groups and the thin mouse MI of non-diabetic
Control group cardiac muscle p120-catenin expression significantly reduces that (P is equal<, and diabetic mice MI control groups compare non-diabetic 0.05)
Thin mouse MI control groups cardiac muscle p120-catenin expression significantly reduces (P<0.05).Compared with diabetic mice MI control groups, pancreas
The expression of island element group and Angptl4 groups cardiac muscle p120-catenin dramatically increases that (P is equal<, and insulin and Angptl4 0.05)
Effect quite (P is equal>0.05).
7th, influence of each group to AMI Reperfu- sions area Microvascular Endothelial Cellss apoptosis
The double dye detection endothelial cell apoptosis of TUNEL-CD34.
According to document " de Resende MM, Amaral SL, Munzenmaier DH, et al.Role of
endothelial cell apoptosis in regulation of skeletal muscle angiogenesis
during high and low salt intake[J].Physiol Genomics,2006,25(2):325335. " reports
After method, (8 μm) coatings of cardiac muscular tissue's frozen section, air-dried 30min, 5min, acetone-chloroform (volume ratio are fixed in cold acetone
1：1) fixed 5min, the solid 5min of acetone.Sample is washed 3 times with PBS (pH=7.2), according to " Tedjarati S, Baker CH,
Apte S,et al.Synergistic therapy of human ovarian carcinoma implanted
orthotopically in nude mice by optimal biological dose of pegylated
interferon alpha combined with paclitaxel[J].Clin Cancer Res,2002,8(7):2413-
Method disclosed in 2422. " carries out the double dyes of CD34-TUNEL, and section carries out DAPI dyeing to calculate total cell core number.200 ×
Fluorescence microscopy Microscopic observation shoots cell picture.Endothelial cell red coloration, normal cell core blueness, apoptotic nucleus and blueness
And green (being in cyan).The ratio that the apoptosis endothelial cell number under every square millimeter of visual field accounts for endothelial cell sum is calculated, is used for
Quantitative assessment endothelial cell apoptosis situation.
As a result as shown in Figure 11 and table 8.
In Figure 11, A：Diabetic mice sham-operation group；B：Diabetic mice MI control groups；C：The thin mouse MI control groups of non-diabetic；
D：Insulin group；E：Angptl4 groups；G：Angptl4+ feminine genders siRNA；I：Angptl4+Angptl4siRNA groups.
Figure 11 and table 8 show that the detection of such as hazardous area Microvascular Endothelial Cellss apoptosis is shown：Done evil through another person with diabetic mice
Art group is compared, diabetic mice MI control groups and the thin mouse MI control groups myocardium at risk CMEC apoptosis ratio of non-diabetic
Example dramatically increases that (P is equal<, and diabetic mice MI control groups mouse MI control groups myocardium at risk endothelium thinner than non-diabetic 0.05)
Apoptosis ratio dramatically increases (P<0.05).Compared with diabetic mice MI control groups, insulin group and Angptl4 groups cardiac muscle danger
Danger zone endothelial cell apoptosis ratio significantly reduces that (P is equal<0.05), and insulin suitable with Angptl4 effect (P is equal>
0.05), illustrate that Angptl4 can suppress CMEC apoptosis.
The each group endothelial barrier structural intergrity parameter (X ± s, n=6) of table 8
Note：Compared with diabetic mice sham-operation group,★P<0.05,★★P<0.01；Compared with diabetic mice MI control groups, * P<
0.05, * * P<0.01.
8th, influence of each group connexin expression the Microvascular Endothelial Cellss of AMI hazardous areas
(1) close connection
Hazardous area cardiac muscle JAM-A expression is detected to assess myocardium endothelial barrier structural intergrity.Method is with the step for implementing 1
Rapid four, as a result as shown in figure 12.
In Figure 12,1：Diabetic mice sham-operation group；2：Diabetic mice MI control groups；3：The thin mouse MI control groups of non-diabetic；
4：Insulin group；5：Angptl4 groups；6：Angptl4+ feminine genders siRNA；7：Angptl4+Angptl4siRNA groups.
Figure 12 shows, compared with diabetic mice sham-operation group, and diabetic mice MI control groups cardiac muscle JAM-A expression is significantly dropped
It is low that (P is equal<, and diabetic mice MI control groups mouse MI control group cardiac muscle JAM-A thinner than non-diabetic expression is significantly reduced 0.05)
(P<0.05).Compared with diabetic mice MI control groups, the expression of insulin group and Angptl4 groups cardiac muscle JAM-A is dramatically increased
(P is equal<0.05), and insulin suitable with Angptl4 effect (P is equal>0.05).
2nd, adhesion junction
Hazardous area cardiac muscle VE-cadherin expression is detected to assess myocardium endothelial barrier structural intergrity.Method is with real
The step of applying 1 five, as a result as shown in figure 13.
In Figure 13,1：Diabetic mice sham-operation group；2：Diabetic mice MI control groups；3：The thin mouse MI control groups of non-diabetic；
4：Insulin group；5：Angptl4 groups；6：Angptl4+ feminine genders siRNA；7：Angptl4+Angptl4siRNA groups.
Figure 13 shows, compared with diabetic mice sham-operation group, diabetic mice MI control groups and the thin mouse MI controls of non-diabetic
Group cardiac muscle VE-cadherin expression significantly reduces that (P is equal<, and diabetic mice MI control groups mouse thinner than non-diabetic 0.05)
MI control groups cardiac muscle VE-cadherin expression significantly reduces (P<0.05).Compared with diabetic mice MI control groups, insulin group
Expression with Angptl4 groups cardiac muscle VE-cadherin dramatically increases that (P is equal<0.05), and insulin and Angptl4 effect
Quite (P is equal>0.05).
3rd, focal adhension is connected
Hazardous area cardiac muscle Integrin- α 5 expression is detected to assess myocardium endothelial barrier structural intergrity.Method is with real
The step of applying 1 six, as a result as shown in figure 14.
In Figure 14,1：Diabetic mice sham-operation group；2：Diabetic mice MI control groups；3：The thin mouse MI control groups of non-diabetic；
4：Insulin group；5：Angptl4 groups；6：Angptl4+ feminine genders siRNA；7：Angptl4+Angptl4siRNA groups.
Figure 14 shows, compared with diabetic mice sham-operation group, diabetic mice MI control groups cardiac muscle Integrin- α 5 expression
Significantly reduce that (P is equal<0.05), and diabetic mice MI control groups mouse MI control group Integrin- α 5 thinner than non-diabetic expression
Significantly reduce (P<0.05).Compared with diabetic mice MI control groups, Angptl4 groups cardiac muscle Integrin- α 5 expression is notable
(P is equal for increase<0.05), and insulin group cardiac muscle Integrin- α 5 expression is without significantly changing (P>0.05).
Result above shows, causes myocardial ischemia-reperfusion to damage after the thin mouse MI of non-diabetic and diabetic mice MI Reperfu- sions
Wound, heart microvascular endothelial cell apoptosis and endothelial barrier function damage, and diabetic mice damage is heavier.Insulin and
Angptl4 can mitigate the myocardial ischemia-reperfusion injury after diabetic mice MI Reperfu- sions, suppress heart microvascular endothelial cell
Apoptosis and mitigation endothelial barrier function damage.
Claims (10)
1. application of the PP1158 4 in the product for preparing protection and/or enhancing barrier cell function.
2. application according to claim 1, it is characterised in that：The cell is endothelial cell, preferably microvascular endothelial
Cell, is further preferably heart microvascular endothelial cell.
3. PP1158 4 is in the product for preparing prevention and/or mitigation and/or treatment myocardial ischemia-reperfusion injury
Application.
4. application according to claim 3, it is characterised in that：The myocardial ischemia-reperfusion injury is in heart microvascular
Epithelial cell barrier function damage.
5. application according to claim 4, it is characterised in that：The heart microvascular endothelial cell barrier function is damaged
Myocardium at risk area increase, the increase of heart microvascular permeability, cardiac muscular tissue's myeloperoxidase expression of enzymes increase, cardiac muscular tissue
Connection declines and/or heart microvascular endothelial cell apoptosis between focal hemorrhage, heart microvascular endothelial cell.
6. according to any described applications of claim 3-5, it is characterised in that：The myocardial ischemia-reperfusion injury is diabetes
Myocardial ischemia-reperfusion injury.
7. application of the PP1158 4 in the product for preparing prevention and/or treatment myocardial infarction.
8. application according to claim 7, it is characterised in that：The myocardial infarction is cardiac muscle caused by acute myocardial infarction AMI
Myocardial infarction after ischemia-reperfusion.
9. application according to claim 8, it is characterised in that：The acute myocardial infarction AMI is Diabetic Acute cardiac muscle stalk
Extremely.
10. the answering in the product of disease or injury caused by prevention and/or treatment hyperglycaemia is prepared of PP1158 4
With.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102690780A (en) * | 2011-03-22 | 2012-09-26 | 上海市肿瘤研究所 | Method for regulating expression of vascular cell adhesion molecule-1 in vascular endothelial cell |
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|---|---|---|---|---|
| CN102690780A (en) * | 2011-03-22 | 2012-09-26 | 上海市肿瘤研究所 | Method for regulating expression of vascular cell adhesion molecule-1 in vascular endothelial cell |
| CN103782177A (en) * | 2011-08-08 | 2014-05-07 | 南洋理工大学 | Angiopoietin-like 4 and its use in modulating cell leakiness |
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