KR20140054622A - A method for providing mimetic environment in vitro of blood flow in vivo - Google Patents
A method for providing mimetic environment in vitro of blood flow in vivo Download PDFInfo
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Abstract
The present invention is a method of providing an environment that mimics blood flow in vitro in a test tube, the method comprising applying a shock wave having an energy of 0.005 to 0.07 mJ / mm 2 , a shock wave of 0.005 to 0.07 mJ / mm 2 energy Providing means; And a kit for confirming the expression of at least one gene selected from the group consisting of Akt, KLF2, KLF4, MT1-MMP and Nur77 and a kit for confirming the expression of at least one gene selected from the group consisting of Akt, KLF2, KLF4, MT1-MMP and Nur77 And a composition for confirming vascular regeneration or wound healing by shock waves including an agent for confirming expression of at least one selected gene.
Description
The present invention is in vitro method for within an environment which mimics the body, blood flow in, from 0.005 to 0.07 mJ / mm which is characterized by a second applying a shock wave having an energy method, from 0.005 to 0.07 mJ / mm provides shock waves of the second energy Way; And a kit for confirming the expression of at least one gene selected from the group consisting of Akt, KLF2, KLF4, MT1-MMP and Nur77 and a kit for confirming the expression of at least one gene selected from the group consisting of Akt, KLF2, KLF4, MT1-MMP and Nur77 And a composition for confirming vascular regeneration or wound healing by shock waves including an agent for confirming expression of at least one selected gene.
An extracorporeal shock wave (ESW) is defined as a series of single waves conducted through a specific single-wave generator. It is a single longitudinal pulse with a short duration of less than 1 ms and an amplitude of maximum pressure of 100 MPa. Since the 1980s, high energy extracorporeal shockwave (ESWT) has been commonly used in renal stone fracture. However, over the past few decades, low-energy ESWTs have been used for the treatment of a variety of diseases, such as bone and inflammatory tendon disease and muscle tension disorders, targeting specific sites at energy levels ranging from 0.005 to 0.32 mJ / mm 2 .
In particular, recent studies have confirmed that shock waves stimulate local stress on the endothelial cell membrane and improve angiogenesis similar to fluid shear stress in blood vessels. They also induced angiogenesis in myocardial infarction pig models and improved myocardial perfusion and clinical symptoms in patients with severe coronary artery disease. However, it has not been known whether SW mediates angiogenesis by any central mechanism.
On the other hand, the endothelium refers to a cell membrane forming the inner surface of blood vessels and lymphatic vessels. Thus forming the interface with the blood or lymph fluid of the lumen and the rest of the tube wall. The cells forming the endothelium are called endothelial cells. Endothelial cells in direct contact with blood are called vascular endothelial cells, and endothelial cells in direct contact with lymphatic fluid are called lymphatic endothelial cells.
Vascular endothelial cells form the entire circulatory system from the heart to the capillaries. These cells have very unique and specific functions that are most important in vascular biology. Such functions include body fluid filtration such as renal glomerulonephritis, vasoconstriction, hemostasis, neutrophil invasion and hormone tracing. The endocardium is called the endocardium.
These vascular endothelial cells play an important role in regulating vascular relaxation by continuously releasing a small amount of nitric oxide (NO), a blood vessel relaxation substance. To date, nitric oxide secreted from the endothelial cells by the basal response or stimulation has been shown to maintain the tightness of the blood vessels appropriately and to prevent leukocyte attachment, platelet aggregation and adhesion, and inhibition of arterial vascular smooth muscle proliferation And is involved in maintenance of blood vessel homeostasis.
Since the blood vessel endothelial cells come into direct contact with blood, they continuously receive shear stress due to the flow of blood, that is, blood flow in the living body, and thus, the formation of new blood vessels by controlling the expression of angiogenesis- When the vessel is damaged, it moves to the damaged area and heals the wound. The blood flow in the blood vessels is different in the types of blood vessels, i.e., veins, arteries or capillaries, but has a constant velocity in each case. Therefore, if the flow rate of the blood in the blood vessel is too fast or slow, if the blood flow is peeled off, the blood flow is shifted from the normal, and if the shear stress applied to the blood vessel endothelium changes, it causes various diseases in the blood vessel do.
Therefore, the present inventors have made intensive researches to mimic the vascular endothelium environment in vivo, which experiences fluid shear stress due to blood flow in studying vascular endothelium in a laboratory environment. As a result, Thereby providing the same effect as the fluid shear stress applied to the endothelium by the blood flow in the body, thereby promoting the expression of the whole blood vessel gene and inducing cell migration and tube formation into the wound site.
It is an object of the present invention to provide a method for applying an impact wave having an energy of 0.005 to 0.07 mJ / mm < 2 > in a method for providing an environment that imitates blood flow in vivo in vitro.
Another object of the present invention is to provide a shock wave providing means of 0.005 to 0.07 mJ / mm 2 energy; And a kit for confirming the expression of at least one gene selected from the group consisting of Akt, KLF2, KLF4, MT1-MMP and Nur77.
It is still another object of the present invention to provide a composition for confirming vascular regeneration or wound healing by shock waves comprising an agent for confirming expression of at least one gene selected from the group consisting of Akt, KLF2, KLF4, MT1-MMP and Nur77.
As one aspect for solving the above problems, the present invention provides a method for providing an environment that imitates blood flow in the body in a test tube, comprising applying a shock wave having an energy of 0.005 to 0.07 mJ / mm 2 to provide.
The term "shock wave" of the present invention is a type of progressive disturbance that carries energy like a normal wave and is transmitted through a medium such as a solid, liquid, gas or plasma, It is. These shock waves may be different depending on the medium that transmits them. As the shock wave progresses, pressure, temperature, and / or density in the path can rapidly increase. These shock waves are also used as alternatives to the treatment of musculoskeletal disorders. Shock waves are applied from the outside to induce regeneration by stimulating the affected part, so that the disease or damage of the joint or tendon can be treated without surgery. This is called extracorporeal shock wave therapy.
Blood continues to circulate through the blood vessels from the heart to the end of the body, from the arteries through the capillaries, back through the veins back to the heart. Such blood flow is caused by the pressure generated by the cardiac contraction, and since such a flow advances while applying pressure to the inner wall of the blood vessel, the surface of the inner wall of the blood vessel forming the lumen, which is in direct contact with the proceeding blood, Chemical and mechanical stimuli. The stimulus that the vascular endothelium receives by the body blood flow is called fluid shear stress. Thus, the environment that imitates the body blood flow may be one that provides fluid shear stress that the vascular endothelium is subject to by the body blood flow. Such a fluid shear stress may preferably be a layered shear stress caused by a laminar flow.
According to a specific embodiment of the present invention, when shock waves were applied to the cultured endothelial cells, phosphorylation of proangiogenic factors such as Akt, eNOS and
The term "Akt" of the present invention is also known as serine / threonine-specific protein kinase and also protein kinase B and plays an important role in various cellular processes such as glucose metabolism, apoptosis, cell proliferation, . The term " eNOS (endothelial NOS) "of the present invention is an enzyme known as NOS3 (nitric oxide synthase 3) or cNOS (constitutive NOS), and is a nitric oxide synthase involved in NO production in blood vessels. Inhibits smooth muscle contraction and platelet aggregation, and is involved in vasospasm regulation. The term " Erk (extracellular-signal regulated kinase) 1/2 "of the present invention is an enzyme involved in cell division in differentiating cells. It is also used as a synonym for mitogen-activated protein kinase (MAPK). These enzymes are phosphorylated and show activity.
Therefore, the method of the present invention can provide the same environment in which the angiogenesis factor is phosphorylated when the layered shear stress is applied by the blood flow to the vascular endothelial cells in the body.
The method of the present invention can provide an environment for mimicking blood flow in the body by applying the shock wave to cells or floating cells attached on a substrate in a culture medium. In addition, after the separated blood vessels are allowed to stand in the culture solution, the shock waves can be applied to provide an environment for imitating blood flow in the body.
Preferably, shock waves having a low energy of 0.005 to 0.07 mJ / mm < 2 > may be used in the method of the present invention.
According to a specific embodiment of the present invention, there is no cell damage when applying a shock wave having an energy of 0.01 to 0.04 mJ / mm 2. However, when a shock wave having an energy of 0.09 mJ / mm 2 or more is applied, (Fig. 1).
Therefore, it is preferable to use a shock wave having a low energy of 0.005 to 0.07 mJ / mm < 2 > so as to provide an environment for mimicking blood flow in the body without causing cell damage during shock wave application. More preferably, a shock wave having an energy of 0.01 to 0.07 mJ / mm 2 or 0.01 to 0.045 mJ / mm 2 can be used. The frequency and frequency of the shock wave to be applied in addition to the energy of the shock wave can be adjusted and used.
In addition, other elements may be further included as long as they do not interfere with imitation of body blood flow in the test tube of the present invention. For example, the shock wave of the present invention can be simultaneously applied by using a substance promoting blood vessel regeneration or a material known to be helpful for wound healing, thereby achieving a synergistic action, thereby exhibiting faster blood vessel regeneration and wound healing effects.
The velocity of blood flow in the body varies depending on the type of blood vessels. For example, an aneurysm is faster than the blood flow in other blood vessels, the blood velocity through the capillary is the slowest, and the varicose is faster than the flow in the capillary but is still somewhat slower than the aneurysm. Accordingly, various blood flow environments can be realized in the test tube by adjusting the energy level of the applied shock wave, or adjusting the frequency or application frequency, within the above energy range.
In another aspect, the present invention provides a shock wave generator comprising: means for providing a shock wave of 0.005 to 0.07 mJ / mm 2 energy; And a kit for confirming expression of at least one gene selected from the group consisting of Akt, KLF2, KLF4, MT1-MMP and Nur77.
Confirmation of the gene expression can be achieved by Western blot or RT-PCR, and a kit for confirming gene expression for this purpose may preferably include an antibody or a primer for the proteins.
According to a specific embodiment of the present invention, the endothelial cells isolated from blood vessels to which shock waves having an energy of 0.012 or 0.045 mJ / mm 2 are applied, or Akt, KLF2, KLF4, MT1-MMP and Nur77 were increased (Fig. 6). In addition, it was confirmed that the cells were significantly increased in the ability to move to the wound site and the tube formation ability was also improved (FIG. 7). Therefore, it was confirmed that the cells with increased expression of Akt, KLF2, KLF4, MT1-MMP and Nur77 enhanced the ability to regenerate blood vessels and treat wound by applying low energy shock waves.
Accordingly, the device for regenerating or wounding blood vessels of the present invention is provided with a shock wave of 0.005 to 0.07 mJ / mm 2 energy to a site or cell where blood vessel regeneration or wound treatment is required and is composed of Akt, KLF2, KLF4, MT1-MMP and Nur77 ≪ / RTI > to induce vascular regeneration or wound healing.
In another embodiment, the present invention provides a composition for confirming vascular regeneration or wound healing by shock waves comprising an agent for confirming the expression of at least one gene selected from the group consisting of Akt, KLF2, KLF4, MT1-MMP and Nur77 .
The above-mentioned Akt, KLF2, KLF4, MT1-MMP and Nur77 are all angiogenic genes. The term "Akt" of the present invention is a gene encoding the above-mentioned Akt enzyme. "KLF2" and "KLF4" are involved in cell proliferation, differentiation and survival as a Kruppel-like factor. "MT1-MMP (
The term "agent for confirming gene expression" of the present invention means a substance capable of confirming the gene expression, that is, the product of synthesizing a protein by reading information from the gene. Specifically, the gene expression includes transcription, ie, a process of synthesizing mRNA from DNA and a process of translation, ie, synthesis of a target protein from the mRNA. Therefore, confirmation of the expression of the gene can be performed by detecting a protein, which is an end product of gene expression, or by detecting mRNA as an intermediate product. Therefore, the agent for confirming gene expression may preferably be an antibody specific to the gene or a specific primer for amplifying the gene.
According to a specific embodiment of the present invention, inhibition of VEGFR-2, PECAM-1 or VE-cadherin, which is known as a gene involved in angiogenesis using siRNA, Whereas in the experimental group treated with shock wave, the expression of the genes was increased (FIG. 6). In addition, it was confirmed that the cells in which the expression of the pre-angiogenic gene was treated by treating these shock waves had improved ability to move to the injured site and tube formation ability (FIG. 7). This means that gene expression such as Akt, KLF2, KLF4, MT1-MMP and Nur77 can act as a marker for confirming the effect of vascular regeneration or wound healing. Accordingly, a composition containing an agent for confirming expression of one or more of the above genes can be used as a composition for confirming blood vessel regeneration or wound healing effect by shock waves.
In the present invention, in studying vascular-related disorders and / or diseases, a shock wave having a low energy enough not to damage a cell is applied to a cell cultured in a test tube to imitate blood flow to the vascular endothelium by blood flow The environment can be provided. Since vascular endothelial cells in vivo are influenced by gene expression and the like due to fluid shear stress due to blood flow, the method of the present invention realizes the vascular endothelial cell environment in vivo, Vascular endothelial growth factor and the like.
Figure 1 shows the effect of SW treatment on cell morphology and survival in HUVEC. (A) is an image of an aluminum thin film in a focus region. The distance between the small bars is 0.5 cm. (B) is a schematic view of the shockwave impact. (C) shows HUVECs exposed to low energy level SW at various energy levels indicated.
FIG. 2 is a graph showing the PI3K / Akt / eNOS phosphorylation stimulation effect by SW in HUVEC. FIG. (A and B) represent phosphorylation and expression of PI3K, Akt and eNOS in cell extracts of HUVECs exposed to SW (1000 times, 0.012 and 0.045 mJ / mm 2 ) for various times indicated. Specific methods are described in the following examples. (N = 4), which is a standardized quantification result for representative immunoblot and actin levels. *, # And &, p <0.05 compared to values from control without SW treatment. The error bar represents the standard deviation.
3 is a
Figure 4 shows the role of flow shear stress (FSS) -induced receptors VEGFR2 / PECAM-1 / VE-cadherin on SW-mediated Akt / eNOS phosphorylation and
Figure 5 shows the recovery effect of SW on impaired flow-induced down-regulation of Akt / Erk / eNOS phosphorylation in HUVEC. (A and B) show that Akt, eNOS, and
Fig. 6 shows the recovery effect of SW on the damage flow (low-energy vibration shear stress) -induced downward regulation of Akt / Erk / eNOS phosphorylation in HUVEC. (A and B) were transfected with scrambled siRNA (30 nM, control) or siRNA (30 nM) of VEGFR-2, PECAM-1 and VE-cadherin, 2 , PECAM-1, VE-cadherin, Akt, eNOS, KLF2, KLF4, MT1-MMP, MMP10, Nur77 and GADPH (internal control) as the primers of VEGFR-2, FIG. 5 shows the result of performing RT-PCR. FIG. Specific methods are described in the following examples. Representative images and quantitative results were shown (n = 4). (C) shows mRNA extracted from carotid endothelial cells of C57 / BL6 mice treated with SW (1000 times, 0.045 mJ / mm 2 ) on partially ligated carotid arteries and noncirculating carotid arteries. Specific methods are described in the following examples. (D) shows the result of performing RT-PCR with primers of Akt, eNOS, KLF2 and GADPH (internal control group). Representative images and quantitative results were shown (n = 4).
FIG. 7 shows angiogenesis and cell migration stimulation of SW according to an in vivo blood vessel formation assay. (A and B) are the results of measurement of cell migration in response to VEGF in the wound closure analysis of HUVEC exposed to the control and SW (1000 times, 0.045 mJ / mm 2 ). (C and D) were obtained by measuring the in vitro vascularization of SW by SWV (1000 times, 0.045 mJ / mm 2 ) and HUVEC tube formation assay (n = 4 ). The length of the tube-like structure was quantified. *, p < 0.05 compared with control values.
Figure 8 shows SW-induced Akt / eNOS phosphorylation and angiogenic gene expression mediated through the formation of a mechanical sensory complex. SW-guided angiogenesis. ≪ / RTI >
Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.
Example
1: cell culture, flow (
flow
) And shock wave (
shock
wave
;
SW
) Experiment
Human umbilical vein endothelial cells (HUVECs) were isolated from fresh human umbilical veins and cultured in medium containing 5% fetal bovine serum and low-serum growth supplement (LSGS, Cascade Biologics Inc) 200 (medium 200). Confluent cells cultured in 60 mm dishes were exposed to fluid shear stress. Cells were exposed to flow in a conical and plate viscometer. Unidirectional steady flow (
Example
2: Antibody
Phosphorylated-Akt antibody (Ser-473, p-Akt), anti-phosphorylated-eNOS antibody (Ser-1177, p-eNOS), anti-phosphorylated-
Example
3:
SDS
-
PAGE
And
Western
Blot
analysis
Cells were lysed in lysis buffer (0.5% Triton X-100, 0.5% Nonidet P40, 10 mM Tris, pH 7.5, 2.5 mM KCl, 150 mM NaCl, 30 mM glycerophosphate, 50 mM NaF, 1 mM Na 3 VO 4 ) Recovered in a protease inhibitor mixture (Sigma) and clarified by centrifugation. The protein concentration in the lysate was determined using the Bradford method (Bio-Rad). Protein complexes were separated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel) electrophoresis, transferred to nitrocellulose membrane, and incubated with appropriate primary antibody. After washing and incubation with secondary antibody, the immune response protein was visualized using an ECL detection system (Amersham Biosciences). The membrane was peeled off and re-labeled with another antibody. Densitometric analysis of the immunoblot by the National Institutes of Health Image was performed. The results were normalized by adjusting the density of the control cells to an arbitrary value of 1.0.
Example
4:
siRNA
(
small
interference
RNA
) And its transfection
SiRNA duplexes and scrambled siRNA controls (non-target siRNA mix) targeting VEGFR2, PECAM-1, VE-cadherin (vascular endothelial-cadherin) were purchased from Integrated DNA Technologies (IDT). The siRNA sequence for human VEGFR2 is sense 5'-ACAAUGACUAUAAGACAUGCUAUGG (SEQ ID NO: 1) and antisense 5'-CCAUAGCAUGUCUUAUAGUCAUUGUUC (SEQ ID NO: 2). The siRNA sequence for human PECAM-1 is sense 5'-AUAGUACCAAGAACUCAAAUGAUCC (SEQ ID NO: 3) and antisense 5'-GGAUCAUUUGAGUUCUUGGUACUAUUC (SEQ ID NO: 4). The siRNA sequence for human VE-cadherin is sense 5'-GCAAUAGACAAGGACAUAACACCAC (SEQ ID NO: 5) and antisense 5'-GUGGUGUUAUGUCCUUGUCUAUUGCGG (SEQ ID NO: 6). siRNA transfection was performed using lipofectamine 2000 according to the manufacturer ' s protocol. Mechanical stimulation was performed 48 hours after siRNA transfection.
Example
5:
RNA
detach,
Reverse transcription
PCR
Total RNA was isolated from ECs cultured using a total RNA isolation kit. First strand cDNA was synthesized with SuperScript III first strand synthesis system. The cDNA was amplified by 30 cycles of polymerase chain reaction (PCR).
In the present invention, the following oligonucleotide primers were used: human VEGFR-2, sense 5'-gtgaccaacatggagtcgtg-3 '(SEQ ID NO: 7) and antisense 5'-TGCTTCACAGAAGACCATGC-3' (SEQ ID NO: 8); Human PECAM-1, sense 5'-gcaaaatgggaagaacctga-3 '(SEQ ID NO: 9) and antisense 5'-CACTCCTTCCACCAACACCT-3' (SEQ ID NO: 10); Mouse PECAM-1, sense 5'-tgcaggagtccttctccact-3 '(SEQ ID NO: 11) and antisense 5'-ACGGTTTGATTCCACTTTGC-3' (SEQ ID NO: 12); Human VE-cadherin, sense 5'-cctaccagcccaaagtgtgt-3 '(SEQ ID NO: 13) and antisense 5'-GACTTGGCATCCCATTGTCT-3' (SEQ ID NO: 14); Human KLF2, sense 5'-cctccaaactgtgactggt-3 '(SEQ ID NO: 15) and antisense 5'-ACTCGTCAAGGAGGATCGTG-3' (SEQ ID NO: 16); Mouse KLF2, sense 5'-gcctgtgggttcgctataaa-3 '(SEQ ID NO: 17) and antisense 5'-AAGGAATGGTCAGCCACATC-3' (SEQ ID NO: 18); Human KLF4, sense 5'-cccacacaggtgagaaacct-3 '(SEQ ID NO: 19) and antisense 5'-ATGTGTAAGGCGAGGTGGTC-3' (SEQ ID NO: 20); Human eNOS, sense 5'-tgatgcattggatctttgga-3 '(SEQ ID NO: 21) and antisense 5'-CCATGTTACTGTGCGTCCAC-3' (SEQ ID NO: 22); Mouse eNOS, sense 5'-gaccctcaccgctacaacat-3 '(SEQ ID NO: 23) and antisense 5'-CTGGCCTTCTGCTCATTTTC-3' (SEQ ID NO: 24); Human Akt, sense 5'-catcacaccacctgaccaag-3 '(SEQ ID NO: 25) and antisense 5'-CTCAAATGCACCCGAGAAAT-3' (SEQ ID NO: 26); Mouse Akt, sense 5'-cccttctacaaccaggacca-3 '(SEQ ID NO: 27) and antisense 5'-ATACACATCCTGCCACACGA-3' (SEQ ID NO: 28); Human Nur77 (NR4A1), sense 5'-ggcatggtgaaggaagttgt-3 '(SEQ ID NO: 29) and antisense 5'-CGGAGAGCAGGTCGTAGAAC-3' (SEQ ID NO: 30); Human MT1-MMP, sense 5'-CGCTACGCCATCCAGGGTCTCAAA-3 '(SEQ ID NO: 31) and antisense 5'-CGGTCATCATCGGGCAGCACAAAA-3' (SEQ ID NO: 32); Human MMP10, sense 5'-GTCACTTCAGCTCCTTTCCT-3 '(SEQ ID NO: 33) and antisense 5'-ATCTTGCGAAAGGCGGAACT-3' (SEQ ID NO: 34); Human GAPDH, sense 5'-gagtcaacggatttggtcgt-3 '(SEQ ID NO: 35) and antisense 5'-TTGATTTTGGAGGGATCTCG-3' (SEQ ID NO: 36); Mouse GAPDH, sense 5'-aactttggcattgtggaagg-3 '(SEQ ID NO: 37) and antisense 5'-ACACATTGGGGGTAGGAACA-3' (SEQ ID NO: 38). The experiment was repeated 3 times.
Example
6: Wound suture cell migration (
wound
closure
cell
migration
)
To measure cell migration during the wound suturing process, the ECs were dispensed into 6-well plates coated with gelatin and cultured to confluence at least 80% of the cells on the plates. The cell monolayer was then separated into 1 to 2 mm cell scrapers and photographed at 0 and 12 hours SW stimulation with an optical microscope equipped with a digital camera (Olympus DP11) (Olympus CKX41). The number of endothelial cells migrated to the wound site was counted.
Example
7: Capillary-like tube formation analysis for in vitro vascular formation
capillary
-
like
tube
formation
assay
)
For the intravascular formation analysis test tubes, divided by 2.5 × 10 5 cells / concentration of the well in a 6-well frame rate, including the 1% FBSEC medium the EC on Matrigel (BD Biosciences) thin film and incubated for 12 hours at 37 ℃ Respectively. The capillary-like tube structure was visualized with an optical microscope (Olympus CKX41) at different time points and taken with a digital camera (Olympus DP11). The total length of tube-like structures per field was measured with an ImageJ analyzer. Ten random fields per dish were measured to calculate the total length per field of view.
Example
8: partial carotid artery
Ligature
Animal models and
SW
Exposure to
The animal experiment of the present invention was carried out according to the animal experiment regulations of the animal experiment ethics committee of Ewha Womans University. To confirm the function of SW in HUVEC under disturbed flow, a model with damaged flow in the mouse carotid artery was prepared by partial ligation. Six-week-old male and female mice were ligated. C57 / BL6 mice were purchased from a central laboratory animal. Partial carotid ligation mice induced viable endothelial dysfunction and atherosclerosis by inducing damaged flow with low vibrational shear stress. All mice received solid feed and drinking water ad libitum until partial ligation. Partial ligation was performed on the left carotid artery (LCA) in a known manner. The mice were then treated with SW (0.045 mJ / mm 2 , 1000 times) for partially ligated LCA and noncontact LCA for SW studies.
Example
9: Endothelial cells from the carotid artery
RNA
detach
Mice were killed by inhalation of CO2 according to the animal experiment protocol of Ewha Womans University and heparin (10 U / ml) was injected through the left ventricle after pressure was applied after severing the inferior vena cava And then perfused with saline containing water. Partially ligated LCA and noncontact control LCA were separated and extraneuronal fats were carefully cleaned by known methods. Briefly, the LCA lumen was quickly washed with a 200 μl QIAzol lysis reagent (QIAGEN) in a microfused tube using a 29 gauge insulin injection for several seconds. The eluate was used for RNA isolation of endothelial cells using miRNAeasy mini kits.
Experimental Example
1: Low energy level for endothelial cell morphology and survival
SW
Effect of
We used AR2 to provide a stable electromagnetic SW with 1 cm diameter and 4 cm effective length as a single-sonic generator (Dornier MedTech, Germany) (FIG. 1A). Various SWs of the indicated energy levels were applied to the aluminum film and the effect of the recess formation due to erosion on the aluminum film was observed (FIG. 1B). To determine the range of SW energy levels for treatment with HUVECs in vitro, we first measured the adverse effects of SW with various energy levels on endothelial cells (Fig. 1C). It was stimulated with SW of energy level indicated HUVEC. There was no change in HUVEC cell morphology at low energy levels of 0.01 to 0.04 mJ / mm 2 per 1000 cycles. However, at 0.09 mJ / mm 2 , a high mortality rate was observed for EC, and when the maximum reached 0.16 mJ / mm 2 , almost all of the ECs disappeared from the bottom of the culture dish.
Experimental Example
2: In endothelial cells
SW
On by
PI3K
,
Akt
,
eNOS
And
Erk
Activation of 1/2
Fluid shear stress has been reported to activate PI3K (phosphatidylinositol 3-kinase), Akt and eNOS in EC. To determine whether SW plays a role in mimicking blood flow in EC, we first examined the activation of PI3K, Akt and eNOS in SW in EC. HUVEC was exposed to low energy levels of SW (1000 times, 0.012 and 0.045 mJ / mm 2 ) for various times and phosphorylation of PI3K, Akt and eNOS was measured in the cell eluate by Western blot using phospho-specific antibody. Phosphorylation of PI3K and Akt occurred within 5 minutes by SW stimulation of 0.012 and 0.045 mJ / mm 2 and lasted for 4 hours (Figure 1A top and 1B). The phosphorylation of eNOS was induced within 5 minutes after SW stimulation, and was maximal at 15 minutes, lasting for 1 hour and recovering to baseline at 4 hours (lower part of FIG. 1A and 1B). In HUVEC, both Akt and eNOS were phosphorylated in a time-dependent manner in response to SW. The flow also stimulated Erk1 / 2 phosphorylation in a time-dependent manner in HUVECs (Figures 3A and 3B). The time-course for
Experimental Example
3:
siRNA
Mechanical Sensory Complexes by
mechanosensory
complex
) Of the knockdown
Flow shear stress modulates various EC functions through stimulation of a mechanical sensory complex consisting of VEGFR-2, PECAM-1, and VE-cadherin leading to rapid activation of Akt, eNOS and
Experimental Example
4:
HUVEC
in
SW
Vibration by
Shear stress (damage flow)
Akt,
eNOS
And
Erk
Activation recovery effect of 1/2
Recent studies have confirmed that vibration shear stress generally upregulates atherogenic genes and proteins and inhibits the activation of angiogenic genes. Endothelial cells were exposed to vibratory shear stress for 24 hours and stimulated with SW for 30 minutes (FIGS. 5A and 5B) to support SW's important role in flow-regulated Akt, eNOS and
Experimental Example
5:
SW
On by
Anterior vascularization
(
pro
-
angiogenic
) Gene Expression Stimulation
Since the fact that Akt, eNOS and
Furthermore, the present inventors have studied the biological role of SW-induced gene expression in vivo. To confirm the recovery effect of SW on angiogenic gene expression including Akt, eNOS and KLF2 under vibrational shear stress condition, a partial carotid artery ligation model with continuous layered blood flow damage was used in C57 / BL6 mice. SW (1000 times, 0.045 mJ / mm < 2 >) was exposed to the mice in the partially ligated CA and the full CA portion. Total RNA was isolated from endothelial cells and confirmed by RT-PCR for endothelium-specific marker PECAM-1 and smooth muscle actin (SMA), which is a wild-type muscle cell marker (FIG. 6C).
In the present invention, mRNA expression of Akt, eNOS and KLF2 was significantly increased by SW treatment. It was also confirmed that the inhibitory effect of the vibrational shear force on gene expression was greatly restored by SW treatment in endothelial cells.
Experimental Example
6: low energy level
SW
On by
EC
Induce movement and tube formation
Next, we confirmed whether SW stimulation is involved in the angiogenesis process in vitro. It was confirmed that low energy level SW (1000 times, 0.045 mJ / mm 2 ) induced significant EC migration using cell migration analysis during wound closure (FIGS. 7A and 7B). The capillary-like tube structure-forming ability of the primary ECs was studied by culturing on Matrigel in the in vivo angiogenesis analysis, and the length of the capillary-like tube structure was measured and quantified. Capillary-like tube formation in Matrigel was significantly increased by SW stimulation (Figs. 7C and 7D).
<110> Ewha University - Industry Collaboration Foundation <120> A method for providing mimetic environment in vitro of blood flow in vivo <130> PA120787 / KR <160> 38 <170> Kopatentin 2.0 <210> 1 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> siRNA against human VEGFR2, sense <400> 1 acaaugacua uaagacaugc uaugg 25 <210> 2 <211> 27 <212> RNA <213> Artificial Sequence <220> <223> siRNA against human VEGFR2, antisense <400> 2 ccauagcaug ucuuauaguc auuguuc 27 <210> 3 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> siRNA against human PECAM-1, sense <400> 3 auaguaccaa gaacucaaau gaucc 25 <210> 4 <211> 27 <212> RNA <213> Artificial Sequence <220> <223> siRNA against human PECAM-1, antisense <400> 4 ggaucauuug aguucuuggu acuauuc 27 <210> 5 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> siRNA against human VE-cadherin, sense <400> 5 gcaauagaca aggacauaac accac 25 <210> 6 <211> 27 <212> RNA <213> Artificial Sequence <220> <223> siRNA against human VE-cadherin, antisense <400> 6 gugguguuau guccuugucu auugcgg 27 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for human VEGFR-2, sense <400> 7 gtgaccaaca tggagtcgtg 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for human VEGFR-2, antisense <400> 8 tgcttcacag aagaccatgc 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for human PECAM-1, sense <400> 9 gcaaaatggg aagaacctga 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for human PECAM-1, antisense <400> 10 cactccttcc accaacacct 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for mouse PECAM-1, sense <400> 11 tgcaggagtc cttctccact 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for mouse PECAM-1, antisense <400> 12 acggtttgat tccactttgc 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for human VE-cadherin, sense <400> 13 cctaccagcc caaagtgtgt 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for human VE-cadherin, antisense <400> 14 gacttggcat cccattgtct 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for human KLF2, sense <400> 15 cctcccaaac tgtgactggt 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for human KLF2, antisense <400> 16 actcgtcaag gaggatcgtg 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for mouse KLF2, sense <400> 17 gcctgtgggt tcgctataaa 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for mouse KLF2, antisense <400> 18 aaggaatggt cagccacatc 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for human KLF4, sense <400> 19 cccacacagg tgagaaacct 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for human KLF4, antisense <400> 20 atgtgtaagg cgaggtggtc 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for human eNOS, sense <400> 21 tgatgcattg gatctttgga 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for human eNOS, antisense <400> 22 ccatgttact gtgcgtccac 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for mouse eNOS, sense <400> 23 gaccctcacc gctacaacat 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for mouse eNOS, antisense <400> 24 ctggccttct gctcattttc 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for human Akt, sense <400> 25 catcacacca cctgaccaag 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for human Akt, antisense <400> 26 ctcaaatgca cccgagaaat 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for mouse Akt, sense <400> 27 cccttctaca accaggacca 20 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for mouse Akt, antisense <400> 28 atacacatcc tgccacacga 20 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for human Nur77 (NR4A1), sense <400> 29 ggcatggtga aggaagttgt 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for human Nur77 (NR4A1), antisense <400> 30 cggagagcag gtcgtagaac 20 <210> 31 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer for human MT1-MMP, sense <400> 31 cgctacgcca tccagggtct caaa 24 <210> 32 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer for human MT1-MMP, antisense <400> 32 cggtcatcat cgggcagcac aaaa 24 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for human MMP10, sense <400> 33 gtcacttcag ctcctttcct 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for human MMP10, antisense <400> 34 atcttgcgaa aggcggaact 20 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for human GAPDH, sense <400> 35 gagtcaacgg atttggtcgt 20 <210> 36 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for human GAPDH, antisense <400> 36 ttgattttgg agggatctcg 20 <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for mouse GAPDH, sense <400> 37 aactttggca ttgtggaagg 20 <210> 38 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for mouse GAPDH, antisense <400> 38 acacattggg ggtaggaaca 20
Claims (9)
Wherein a shock wave having an energy of 0.005 to 0.07 mJ / mm < 2 > is applied.
Wherein the environment mimicking the body blood flow provides a fluid shear stress that the vascular endothelium is subjected to by in vivo blood flow.
Wherein said fluid shear stress is a layer shear stress caused by a laminar flow.
Wherein the shock wave is applied to cells or floating cells attached on a substrate in a culture medium to provide an environment for mimicking blood flow in the body.
Wherein the separated blood vessel is allowed to stand in a culture solution, and then the shock wave is applied to provide an environment for mimicking blood flow in the body.
Wherein confirmation of gene expression is achieved by Western blot or RT-PCR.
Wherein the kit for confirming gene expression comprises an antibody or primer for the proteins.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101719569B1 (en) * | 2016-01-20 | 2017-03-27 | 이화여자대학교 산학협력단 | The novel method for secretion of extracellular vesicles from cells and tissues using shockwave |
| WO2020105800A1 (en) * | 2018-11-22 | 2020-05-28 | (주)엑솔런스바이오테크놀로지 | Apparatus for delivering target material using extracorporeal shock-waves |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101719569B1 (en) * | 2016-01-20 | 2017-03-27 | 이화여자대학교 산학협력단 | The novel method for secretion of extracellular vesicles from cells and tissues using shockwave |
| US10125360B2 (en) | 2016-01-20 | 2018-11-13 | Ewha University-Industry Collaboration Foundation | Method for secretion of extracellular vesicles from cells and tissues using shock wave |
| WO2020105800A1 (en) * | 2018-11-22 | 2020-05-28 | (주)엑솔런스바이오테크놀로지 | Apparatus for delivering target material using extracorporeal shock-waves |
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