JP2020031538A - Method related to pathosis reconstruction of blood vessel disease, disease model and apparatus - Google Patents

Abstract

To provide a disease model capable of establishing a production method of ex vivo disease model which approximates to a pathosis of a blood vessel disease of a patient, which can be easily produced in a short term, by which a process until blood vessel tissues are varied from a normal state to a pathosis in time-variant, can be clarified, the disease model capable of being used for screening of medicines for preventing or treating the blood vessel disease.SOLUTION: There is provided an analysis method of a pathophysiology of blood vessel disease in ex vivo, the analysis method comprises: a step for applying a physical stress to an endothelial cell side surface of an incised blood vessel tissue of an extracted blood vessel which is arranged in a culture fluid, in a constant period; a step for observing a pathological change of a physical stress load portion of the extracted blood vessel or a peripheral portion thereof; and a step for analyzing the pathological change.SELECTED DRAWING: Figure 11

Description

  The present invention relates to a method for reconstructing a pathological condition of a vascular disease, in particular, a method for analyzing the pathophysiology of a vascular disease, a disease model, a method for producing the same, and a device for producing the same.

  Heart disease and stroke, which are the main causes of death and need for nursing care in Japan, are caused by aggravation of underlying diseases such as arteriosclerosis and aneurysms. These underlying diseases are vascular diseases, that is, vascular diseases, and have a difficulty in that once developed, their progress cannot be controlled. Vascular disease is treated by methods of reducing risk factors by medical techniques and suppressing the severity of the disease by surgical techniques. The number of patients in Japan reaches 3 million, and medical expenses reach 2.5 trillion yen annually (Non-Patent Document 1).

  Physiological factors explained by hemodynamics, such as wall stress due to blood pressure and wall shear stress due to blood flow, play an important role in the occurrence of vascular disease (Non-Patent Document 2).

  In addition, with the progress of magnetic resonance imaging tomography (MRI) and angiography, the detection of unruptured aneurysms triggered by consultation for a medical checkup or other diseases is increasing. The unruptured cerebral aneurysm is considered to be slightly more than 3% in adults over 30 years (Non-Patent Document 3). And it is said that about 0.95% of the carriers of the unruptured cerebral aneurysm rupture in one year (Non-Patent Document 3).

  In addition to the high prevalence of vascular disease, the economic impact, and the fact that it is often found asymptomatically by docking, etc., we will develop a treatment to prevent the onset of vascular disease from occurring. This is an urgent issue for Japan in a super-aging society.

  Therefore, in order to elucidate the mechanism of aneurysm generation or growth and to screen for a drug for its prevention or treatment, an attempt has been made to develop an in vitro or in vivo aneurysm model using cultured cells ( Patent Literatures 2 and 3 and Non-Patent Literature 4) are used for drug screening (Patent Literature 3). However, these in vitro disease models are hardly close to the pathological conditions observed in patients, and the in vivo disease models do not allow continuous observation of vascular pathological changes in one individual. However, since only observations can be made at one point in time, there is a problem that a lot of trouble and time are required.

JP 2009-107955 A JP 2003-052364 A JP 2012-080888 Gazette

Ministry of Health, Labor and Welfare, 2015 National Health Expenditure Mari Oshima, et al., Extracerebral Journal 23 (2014) 710-714 Japan Brain Dock Association Brain Dock New Guidelines Development Committee, “Brain Dock Guidelines 2014 [Revised, 4th Edition]” pp71-83, April 2014, Kyobunsha Co., Ltd., Sapporo Hashimoto N., et al., Surg Neurol. 10 (1978) 3-8.

  The present invention provides an ex vivo disease that approximates the pathological condition of a vascular disease of a patient, can be prepared easily and in a short period of time, and is capable of elucidating a process in which vascular tissue changes from a normal state to a pathological state over time. It is an object to establish a method for preparing a model and to provide a disease model that can be used for screening a drug for preventing or treating vascular disease.

  The present inventors have studied the relationship between cerebral aneurysms removed from humans and hemodynamics for many years, and based on the pathological findings during the initial stage of cerebral aneurysm-growth process, based on endothelial cell detachment and collapse of internal elastic plate. As is characterized by vascular wall thinning via vascular smooth muscle cell necrosis and vascular wall thickening via vascular smooth muscle cell proliferation, we have obtained the finding that vascular smooth muscle cell necrosis is triggered by apoptosis, The present inventors have intensively studied the establishment of an ex vivo disease model that expresses these characteristic findings, and completed the present invention.

Specifically, the present invention is an ex vivo method for analyzing the pathophysiology of vascular disease,
A step of applying physical stress to the endothelial cell side surface of the cut vascular tissue of the isolated blood vessel placed in the culture solution for a certain period of time,
Observing a pathological change in the site of physical stress loading of the isolated blood vessel or its surrounding area,
Analyzing the pathological changes,
An analysis method, comprising:

  In the analysis method of the present invention, the step of applying the physical stress may include a collision of a steady flow of the culture solution with vascular tissue.

  In the analysis method of the present invention, the pathological change may be selected from a morphological change, a biochemical change, or a change in gene expression.

  In the analysis method of the present invention, the pathological change is the presence or absence of endothelial cell detachment, thinning or thickening of the media, collapse of the internal elastic plate, change in the shape of vascular smooth muscle cells, proliferative change or apoptosis. It may be an observation.

  In the analysis method of the present invention, the observation may be a measurement of a blood vessel wall thickness or a measurement of the number of cells classified into properties selected from the presence or absence of proliferative change or apoptosis of vascular smooth muscle cells.

  In the analysis method of the present invention, the vascular disease may be a disease caused by a hemodynamic factor.

  In the analysis method of the present invention, the disease caused by the hemodynamic factor may be selected from cerebral aneurysm, aortic aneurysm, carotid aneurysm, arteriosclerosis, or hypertension.

Further, the present invention is a method for preparing an ex vivo disease model for vascular disease,
A step of applying physical stress to the incised luminal vessel wall of the isolated blood vessel placed in the culture solution for a certain period of time,
There is provided a production method comprising:

  In the production method of the present invention, the vascular disease may be a disease caused by a hemodynamic factor.

  In the production method of the present invention, the disease caused by the hemodynamic factors may be selected from cerebral aneurysm, aortic aneurysm, carotid aneurysm, arteriosclerosis, or hypertension.

Further, the present invention is an ex vivo disease model for vascular disease,
Produced by a production method including a step of applying a physical stress to the incised luminal vessel wall surface of the isolated blood vessel placed in the culture solution for a certain period of time,
Provided is a disease model comprising at least one of vascular tissue from which vascular endothelial cells have been detached, a thinned or thickened media, a collapsed internal elastic plate, and vascular smooth muscle cells that have induced a proliferative change or apoptosis.

  In the disease model of the present invention, the vascular disease may be a disease caused by a hemodynamic factor.

  In the disease model of the present invention, the disease caused by the hemodynamic factors may be selected from cerebral aneurysms, aortic aneurysms, carotid aneurysms, arteriosclerosis, or hypertension.

Further, the present invention is a method for screening a drug for the prevention or treatment of vascular disease,
A step of applying a physical stress to the incised luminal vessel wall of the isolated blood vessel placed in the culture solution containing the test compound for a certain period of time,
Observing a pathological change in the site of physical stress loading of the isolated blood vessel or its surrounding area,
A screening method including a step of analyzing and evaluating the pathological change is provided.

  In the screening method of the present invention, the step of applying the physical stress may include the collision of a steady flow of the culture solution.

  In the screening method of the present invention, the vascular disease may be a disease caused by a hemodynamic factor.

  In the screening method of the present invention, the disease caused by the hemodynamic factors may be selected from cerebral aneurysms, aortic aneurysms, carotid aneurysms, arteriosclerosis, or hypertension.

Further, the present invention is an apparatus for producing an ex vivo disease model of vascular disease,
Provided is a manufacturing device including a tissue culture unit for culturing vascular tissue, a steady flow generating unit, and a nozzle unit for adjusting a collision pressure by a steady flow to vascular tissue.

  In the manufacturing apparatus of the present invention, the vascular disease may be a disease caused by a hemodynamic factor.

  In the manufacturing apparatus of the present invention, the disease caused by the hemodynamic factor may be selected from cerebral aneurysm, aortic aneurysm, carotid aneurysm, arteriosclerosis, or hypertension.

  According to the present invention, an ex vivo disease that can approximate a pathological condition of a vascular disease of a patient, can be prepared simply and in a short time, and can elucidate a process in which vascular tissue changes from a normal state to a pathological state over time. A method for preparing a model can be established to provide a disease model that can be used for screening a drug for preventing or treating vascular disease.

A schematic diagram (left) illustrating the relationship between the progression of vascular disease and the degree of vascular homeostasis disruption, and a photograph showing the presence of thinned and thickened parts during the occurrence and growth of a patient's cerebral aneurysm ( Right figure). The schematic diagram of a disease model production device. Left figure: schematic diagram of the entire device. Right: Enlarged view of the steady flow jet nozzle to the vascular tissue fixed to the fixed base. The figure which plotted the change of the maximum wall shear stress with respect to the change of the flow rate calculated by computer simulation. FIG. 3 is an optical micrograph showing that a section of a vascular tissue subjected to a collision flow for 3 days was stained with HE to observe whether endothelial cells were detached. (A) shows the collision area, and (B) shows the non-collision area. Endothelial cells are detached in the collision area, and endothelial cells can be confirmed in the non-collision area (arrowhead: ▼). Optical micrograph showing a section of a vascular tissue subjected to a collision flow for 3 days, stained with HE staining and TUNNEL staining, to observe a collision area and a non-collision area. The figure showing the result of having measured and compared the blood-vessel wall thickness of the collision area | region and the non-collision area | region of the vascular tissue which applied the collision flow for 3 days. Scanning electron micrograph of a section of a collision area of vascular tissue subjected to a collision flow for 3 days. Smooth muscle cell nuclei were enriched, indicating apoptotic smooth muscle cell death. The figure which represents the result of measuring the ratio of the number of TUNNEL-staining positive smooth muscle cells in the section of the vascular tissue subjected to the collision flow for 3 days, and comparing the ratio of the smooth muscle cell apoptosis between the collision region and the non-collision region. FIG. 4 is an optical micrograph showing observation of an elastic plate with HE staining in a section of vascular tissue subjected to a collision flow for 3 days. (A) shows the results immediately after extraction, and (B) shows the results in the collision area where the collision flow was applied for 3 days. FIG. 3 is a fluorescence photograph by a fluorescence microscope in which proliferating cells were observed by Ki67 staining in a section of vascular tissue subjected to a collision flow for 3 days. (A) is a Hoechst33342 stained diagram, (B) is a Ki67 stained diagram, and (C) is a superimposed diagram of Hoechst33342 and Ki67. Schematic diagram summarizing the results of the analysis of the pathological conditions obtained in this experiment. Upper figure: A figure showing the strength of wall shear stress and pressure (impact pressure) corresponding to the positions in the lower figure. Lower figure: A diagram schematically showing the distribution of normal cells, apoptosis-inducing cells and proliferating cells, and a pathological image of the inner elastic plate of endothelial cells and smooth muscle cells in the collision area, the non-collision area, and their boundary areas.

1. Ex vivo Method for Analyzing the Pathophysiology of Vascular Disease One embodiment of the present invention is a method for analyzing the pathophysiology of vascular disease ex vivo.

  The vascular disease targeted by the present invention is caused by a hemodynamic factor such as a wall stress due to pressure and a wall shear stress due to blood flow.

  Vascular disease begins with a deviation from vascular homeostasis and undergoes a state in which vascular transformation and repair are antagonized. After that, if the transformation succeeds in restoration, it will lead to the breakdown of homeostasis, and if the restoration overcomes the transformation, it will undergo a new transformation of homeostasis. FIG. 1 illustrates the pathological transformation of an example of a cerebral aneurysm. A cerebral aneurysm shows a state in which a blood vessel wall is uniformly thinned at the time of occurrence, but a thinned part and a thickened part coexist as it grows. That is, during the growth process, a part of the thin tissue turns into thickening, thereby acquiring new vascular homeostasis. In order to grasp the clues toward the control of vascular diseases for the purpose of prevention or treatment, it is necessary to be able to elucidate and control the conversion mechanism of this rupture repair. However, it is a life support system consisting of extremely complex life phenomena, that is, the interaction between cells and organs, and the principle and governing law of this conversion mechanism have not yet been elucidated.

  Understanding vascular disease began with the accumulation and insight of pathological information from human pathological samples and has been reinforced by animal experiments and cell culture. In order to elucidate the mechanism of pathological transformation, it is necessary to clarify the time-dependent process of onset of pathological conditions. However, human pathological samples and animal experiments are information (snapshots) at a single point in time, and have the drawback that they cannot visualize the chronological process. Cell culture is a method of culturing a single or a plurality of cells under an artificial material (such as collagen) as a method for compensating for this disadvantage. However, cell culture-based disease models aim to elucidate cell functions, but cannot reproduce the degeneration or breakdown of the extracellular matrix exhibited by vascular disease, and the interaction between extracellular matrix and vascular cells This is disadvantageous. As described above, there has been no experimental technique for elucidating a temporal process in which vascular disease progresses.

  One embodiment of the present invention is an analysis method using a disease model for elucidating a physiological state of a process in which vascular tissue changes from a normal state to a pathological state over time and the change thereof. That is, the present invention relates to the present invention, in which the vascular tissue is transformed by a hemodynamic factor to the extent that the physiological state of the blood vessel can be repaired, and thereafter the disease state progresses, and further, after a turning point, the disease state is stabilized or destabilized. To provide an analysis method for elucidating the pathophysiology of the process leading to and / or these processes and the mechanism thereof. In the vascular disease targeted by the present invention, in particular, in an aneurysm, the stabilization of the disease state turns into a vascular wall thickening due to the turning point of the blood vessel wall having an aneurysm-like shape through thinning of the artery. Refers to the stabilization of the aneurysm without rupture. Further, the instability of the disease state refers to a disease state in which the blood vessel wall continues to be thinned and progresses to aneurysm rupture without switching from thinning to thickening.

  The present invention provides experimental techniques for elucidating the process by which vascular tissue changes from normal to pathological conditions over time. The process of transition from normal to disease involves two contradictory changes: degenerative and proliferative. Here, it is an object of the present invention to develop a new experimental technique for reproducing a degenerative change and a proliferative change of a blood vessel outside a body by applying physical stress to a normal vascular tissue.

  The process of progressing from normal to pathological conditions can be classified into two types, degenerative changes and proliferative changes, based on the pathological image of an aneurysm removed from a patient. As used herein, the term “regressive change” refers to (1) detachment of endothelial cells, (2) apoptosis of smooth muscle cells, and (3) thinning of the blood vessel wall. (4) proliferation of endothelial cells and (5) proliferation of smooth muscle cells.

  The present invention includes a new culture technique for reproducing degenerative changes and proliferative changes of blood vessels in vitro by applying physical stress to normal vascular tissues extracted from animals. To elucidate the processes from / to the progress of the disease state and / or the mechanism thereof, and to establish a method for the prevention or treatment of vascular disease.

  Specifically, the present invention provides a disease model in an environment in which the type, degree, and ratio of stress can be adjusted ex vivo for a blood vessel extracted from an animal under an environment in which physical stress can be variably controlled. This is a method for preparing and analyzing the pathophysiology of this disease model. By applying the analysis method of the present invention, it is possible to elucidate the action points targeted by the prophylactic and / or therapeutic drugs against vascular diseases, to screen drug candidates against these targets, and to further verify the effects of the drugs. Can bring benefits.

  The animal from which the isolated blood vessel used for the analysis method of the present invention is collected is selected from humans, monkeys, dogs, cats, pigs, horses, cows, rabbits, rats, mice, guinea pigs or chickens, and is extracted from these animals. Blood vessels can be used. The isolated blood vessel is preferably an artery. The organ from which the blood vessel is removed is not particularly limited as long as the disease model of the present invention can be prepared.However, the removed blood vessel from a human, for example, is obtained after obtaining the consent of the subject in advance and undergoing a removed tumor tissue or vascular bypass surgery. Can be collected from the excised organs, cadaver, etc. In large or medium animals such as pigs, horses, cows, dogs, cats and rabbits, various arteries including cerebral arteries can be extracted and used in the analysis method of the present invention. In the case of small animals such as rats, mice, and guinea pigs, for example, aorta such as ascending aorta, descending aorta, and abdominal aorta can be used, but abdominal aorta is preferable because of ease of extraction.

  The excised blood vessel is promptly dissected in the longitudinal direction (blood vessel running direction), and fixed so that a steady flow of the culture solution can collide with the lumen side of the blood vessel, that is, the vascular endothelial cell side. Collide. At this time, the blood vessel wall is extended and fixed to the length before extraction. Maximum pressure is applied to the area where the impinging flow directly impinges. The area around the collision area includes an area where the wall shear stress caused by the collision flow is maximum (see FIG. 11).

At this time, the steady flow is caused to collide with the blood vessel tissue as a constant pressure flow, and if the pressure is extremely strong, physical tissue damage such as cell detachment is caused to the blood vessel tissue. On the other hand, even if the level is lower than the level at which such physical tissue injury is caused, a physiological change may be caused in the vascular tissue. The degree of pressure applied to the vascular tissue at a constant pressure / steady flow is not particularly limited as long as it causes a physiological change in the vascular tissue. On the other hand, it is known in clinical practice that the maximum shear stress in a cerebral aneurysm is about 20 Pa (Miura Y. et al., Stroke 44: 519-521 (2013)). In addition, when cultured perfusion solution was used to impinge on the cultured cells at a pressure of 68 dynes / cm ² (6.8 Pa), the detachment of the endothelial cells was not induced, and 210 dynes / cm ² (21 Pa) was used. ), It has been reported that detachment of endothelial cells is caused by collision with a perfusate composed of a culture solution (Ostrowski MA et al., Biophys. J. 106: 366-374 (2014). In order to prepare a disease model of vascular tissue with detachment of endothelial cells approximated to clinical pathology and to analyze the pathophysiology, for example, the maximum value of shear stress is about 10 to 30 Pa, preferably In a mode of 15 to 25 Pa, more preferably about 20 Pa, by impinging a constant pressure / steady flow for a predetermined period, a disease model having a pathological condition accompanied by detachment of vascular endothelial cells similar to a clinical cerebral aneurysm is obtained. Can be made.

  The period during which the constant-pressure / steady-state flow impinges is, for example, 1, 2, 3, 4 or 5 days, and is preferably 3 days at which pathological findings similar to human vascular disease can be obtained. Further, the collision period can be appropriately changed and selected depending on the difference in the pressure of the collision flow with the vascular tissue.

  For a predetermined period, a vascular tissue created as a disease model by colliding a constant pressure / steady flow with an isolated blood vessel is then subjected to, for example, morphopathological observation, immunohistochemical observation, electron microscopic observation, biochemical change. , And the pathophysiology of vascular tissue can be analyzed by measuring changes in expressed genes, and the like.

  For morphopathological observation or immunohistochemical observation, for example, based on a method known to those skilled in the art, the collected vascular tissue is fixed with 4% formalin, 10% glutaraldehyde, or frozen. For example, after preparing a section by slicing to a thickness of several μm ~ 1 mm, general staining method, histochemical immunostaining method, or a special staining method or metal staining (Osmium staining, Ruthenium staining, uranium staining, silver plating staining, staining with a colloidal gold-labeled antibody) and observing with an optical microscope, a fluorescence microscope, or an electron microscope.

  General staining methods include, for example, hematoxylin and eosin (HE) staining, toluidine blue staining, Alcian blue staining, PAS staining, colloidal silver staining, Elastica Wangieson staining, Masson trichrome staining, Azan staining, Berlin blue staining, Papanichiro A typical general staining method such as staining can be used.

  The histochemical immunoassay uses an antibody against a biological component to be observed, and can be observed and analyzed by microscopic examination of changes in the biological component to be observed in a tissue section by a method well known to those skilled in the art. it can.

As a special staining method, for example, when staining cells in which apoptosis is induced, for example, Tunnel staining for specifically detecting DNA fragmentation due to apoptosis, and Hoechst ^R 33342 capable of specifically staining nuclei use ^{SYTO R 9, SYTO R 82,} SYTOX R Green, tO-PRO R -3, DRAQ5 TM, DRAQ7 TM, DAPI (4 ', 6-diamidino-2-phenylindole) staining method using stains such as can do.

  When cells in the proliferation phase are stained, for example, histochemical immunostaining using an anti-Ki67 antibody against the Ki67 protein, which is a cell proliferation marker, can be used. After Ki67 staining, for example, microscopic examination with an optical microscope, a fluorescence microscope, or a fluorescence microscope can observe morphological changes and physiological changes in the cultured vascular tissue compared to the normal tissue. it can.

  Other staining methods include tissue immunostaining using antibodies against various cytokines, chemokines, extracellular matrix components, intracellular signal transduction components, and other vascular tissue components, and various staining agents, and the pathology of disease models Observe and analyze your physiology.

  Observation by general staining method, histochemical immunostaining method and special staining method, for example, perform single staining or multiple staining, and count the number of cells positive or negative for the stain, cell density, cell shape, morphology, properties, etc. Observing the shape, morphology, properties, thickness, thinning, thickening, etc. of the intima, media, adventitia, or the entire vascular tissue, such as the shape, form, and properties of the extracellular matrix component or inner elastic plate in By analyzing the result of (1), the physiological change of the vascular tissue to be observed can be analyzed, but the present invention is not limited thereto.

  As described above, the measurement of biochemical changes for analyzing the pathophysiology of vascular disease using the above disease model can be performed by histochemical immunostaining using an antibody against a biological component to be measured. In addition, as a method other than the histochemical immunostaining method, a tissue piece of a site to be measured of a test vascular tissue is collected, the tissue is pretreated by a method well known to those skilled in the art, and then subjected to high performance liquid chromatography (HPLC). Using gas chromatography (GLC), mass spectrometry, or immunoassay, changes in the biological component to be measured due to the disease state can be observed.

  In order to analyze changes in gene expression, for example, analysis can be performed using a method well known in the art. For example, a part of the tissue to be observed is collected from the vascular tissue cultured by colliding a constant pressure and a steady flow, dissolved in a tissue lysing agent, and then used with a PCR probe for a gene encoding the protein to be observed. , RT-PCR, or by using a commercially available microarray, and analyzing by comparing with a normal tissue.

  Diseases to be analyzed by the present invention are diseases caused by hemodynamic factors, specifically vascular diseases. More specifically, for example, cerebral aneurysms, aortic aneurysms, carotid aneurysms, arteriosclerosis Disease or hypertension.

  The analysis method by the above method, and the result of the analysis by the above method, for example, elucidation of the mechanism leading to the development and further breakdown of vascular disease, a method for prevention or treatment of vascular disease, prevention of vascular disease Alternatively, it can be used for an action point targeted for treatment, or a method for screening a drug for preventing or treating vascular disease.

2. Method for Producing Ex Vivo Disease Model for Vascular Disease Another embodiment of the present invention is a method for producing an ex vivo disease model for the above vascular disease. Embodiments of the method for producing a disease model of the present invention have been described in detail in “1. Method for ex vivo analysis of pathophysiology of vascular disease” above.

  Briefly, using a blood vessel isolated from an animal, the blood vessel is incised, and the culture solution is subjected to a predetermined pressure at a predetermined pressure for a predetermined period in the disease model producing apparatus, and a constant pressure / steady flow is applied to the lumen side, that is, the vascular endothelial cell side. By applying the wall stress due to the pressure of the collision flow and the wall shear stress caused by the collision flow liquid after the collision, the disease model of the present invention can be produced.

  In the method for producing a disease model of the present invention, the target disease is a disease caused by hemodynamic factors, more specifically, a vascular disease, and more specifically, for example, cerebral aneurysm, aortic aneurysm , Carotid aneurysm, arteriosclerosis or hypertension.

  The disease model produced by the production method of the present invention is, for example, for elucidation of the mechanism leading to the onset and progress of vascular disease, further breakdown, a method for preventing or treating vascular disease, and for preventing or treating vascular disease. It can be used as a target action point or a method for screening a drug for preventing or treating vascular disease.

3. Disease Model Another embodiment of the present invention is an ex vivo disease model for the vascular disease. The details thereof have been described above in “1. Method for ex vivo analysis of pathophysiology of vascular disease”.

  Briefly, using a blood vessel isolated from an animal, the blood vessel is incised, and the culture solution is subjected to a predetermined pressure at a predetermined pressure for a predetermined period in the disease model producing apparatus, and a constant pressure / steady flow is applied to the lumen side, that is, the vascular endothelial cell side. By applying the wall stress due to the pressure of the collision flow and the wall shear stress caused by the collision flow liquid after the collision, the disease model of the present invention can be produced.

  In the disease model of the present invention, the target disease is a disease caused by hemodynamic factors, more specifically a vascular disease, and more specifically, for example, cerebral aneurysm, aortic aneurysm, carotid artery It can be selected from aneurysm, arteriosclerosis or hypertension.

  The disease model of the present invention is, for example, elucidation of the mechanism leading to the development and further breakdown of vascular disease, a method for prevention or treatment of vascular disease, an action point targeted for prevention or treatment of vascular disease, Alternatively, it can be used for a method for screening a drug for preventing or treating vascular disease, and the like.

4. Method for Screening Drug for Prevention or Treatment of Vascular Disease One embodiment of the present invention is a method for screening a drug for prevention or treatment of vascular disease. For example, under the culture conditions described in the method for producing the disease model, before the collision flow collides with the vascular tissue, during the collision period or after the collision, the test substance to be screened is placed in the culture solution and / or in the collision flow. Morphological changes, biochemical changes or changes in gene expression described in “1. Method for ex vivo analysis of the pathophysiology of vascular disease”, and targeting the experimental group to which no test substance is added. The test substance can be screened by comparing and analyzing the presence / absence of the effect of the test substance on the pathophysiology of the vascular tissue and / or the strength of the action, and selecting candidate compounds for the drug.

5. Apparatus for Manufacturing Ex Vivo Disease Model of Vascular Disease Another embodiment of the present invention is an apparatus for manufacturing an ex vivo disease model of vascular disease. More specifically, the manufacturing apparatus of the present invention includes a tissue culture unit for culturing vascular tissue, a steady flow generating unit, and a nozzle for adjusting a collision pressure by a steady flow to the vascular tissue.

  In the production apparatus of the present invention, a vascular tissue extracted from an animal is immersed in a culture solution and fixed, and under a culture condition, a constant pressure / steady flow collides with the lumen side of the vascular tissue, that is, the vascular endothelial cell side. It has a mechanism (see FIG. 2).

  As the tissue culture unit, a commercially available tissue culture device can be obtained and used.

  For the steady flow generation unit, for example, an apparatus described in a known document (Sakaguchi K. et al., Scientific Reports 3: 1316, 1-7 (2013)) can be obtained or manufactured and used. This device creates a steady flow by using a repetitive movement of two pairs of syringes and a three-way valve.

  For the nozzle part for adjusting the collision pressure by the steady flow to the vascular tissue, for example, a mechanism having microtubes manufactured by stereolithography can be used. Through this mechanism, the flow created by the steady flow generating section can be accelerated, and a collision load by a constant pressure / steady flow can be applied to the vascular tissue fixed in the culture section. The collision load can continuously or intermittently apply the collision flow to the vascular tissue for a predetermined period at a constant or changed predetermined collision pressure by controlling the generation of the steady flow.

  The adjustment of the collision pressure can be controlled by adjusting the diameter of the ejection port of the collision pressure adjustment nozzle with respect to the flow velocity of the fluid created by the steady flow generation unit in a manner that can obtain a desired collision pressure. That is, when the diameter of the injection port of the nozzle portion is constant, the collision pressure of the collision flow can be increased by increasing the flow rate of the steady flow. In addition, when the flow rate of the steady flow is constant, the smaller the diameter of the injection term of the nozzle portion, the more the collision pressure can be increased.

  In the preparation of the disease model of the present invention, for example, by causing a steady flow to collide with the vascular tissue as a constant pressure flow, it is possible to bring about a physiological change to the vascular tissue without causing physical tissue damage. it can. The level of the pressure applied to the vascular tissue at a constant pressure / steady flow is not particularly limited as long as it causes a physiological change in the vascular tissue.

On the other hand, it is known in clinical practice that the maximum shear stress in a cerebral aneurysm is about 20 Pa (Miura Y. et al., Stroke 44: 519-521 (2013)). In addition, when cultured perfusion solution was used to impinge on the cultured cells at a pressure of 68 dynes / cm ² (6.8 Pa), the detachment of the endothelial cells was not induced, and 210 dynes / cm ² (21 Pa) was used. ), It has been reported that detachment of endothelial cells is induced when the culture medium is collided (Ostrowski MA et al., Biophys. J. 106: 366-374 (2014). Using a manufacturing apparatus, to create a disease model of vascular tissue accompanied by detachment of endothelial cells approximated to the clinical pathology, to analyze the pathophysiology or to screen for drugs for the prevention or treatment of vascular disease, For example, in a mode in which the maximum value of the shear stress is about 10 to 30 Pa, preferably 15 to 25 Pa, and more preferably about 20 Pa, clinical cerebral aneurysms are caused by impinging a constant pressure / steady flow for a predetermined period. Can create a disease model with a pathological condition involving vascular endothelial cell detachment that approximates .

  For example, when a steady flow of 20 mL / min is generated in the steady flow generating section, the impinging stream having a jet pressure of 20 Pa is generated by injecting the impinging stream from the nozzle having a nozzle diameter of 0.7 mm. Can act on vascular tissue.

  By using the apparatus for producing an ex vivo disease model of vascular disease, an ex vivo disease model of vascular disease can be prepared. This production apparatus can elucidate the mechanism of the occurrence and progression of the pathological condition of vascular disease. It can be used for screening of drugs for preventing or treating vascular diseases.

  All documents mentioned herein are incorporated by reference in their entirety. The examples described herein are illustrative of embodiments of the present invention and should not be construed as limiting the scope of the invention.

1. Reagents and equipment Hematoxylin and eosin (HE) staining was performed using New Hematoxylin Type M (Cat. No. 30141) and Pure Eosin solution (Cat. No. 32043) of Mutoh Chemical Co., Ltd. (Tokyo). For TUNNEL staining, an in situ Apoptosis Detection Kit MK500 from Takara Bio Inc. (Shiga) was used. Ki67 staining, as a primary antibody rabbit anti--Ki67 polyclonal antibody (Abcam, Inc. catalog No.ab15580, UK), sheep as a secondary antibody anti-rabbit ^{IgG H & L (Alexa Fluor R} 488) ( same, ab150077) was used. For nuclear staining, Hoechst 33342 (Thermofisher, Catalog No. H1399, USA) was used.
The culture media used for tissue culture were DMEM (Fuji Film Wako Pure Chemical Co., Ltd., Osaka, catalog No. 043-30085), 10% FBS (Japan Bioseala, Hiroshima, catalog No. S1650-500) and 1 % penicililin (Fuji Film Wako Pure Chemical Industries, Ltd., catalog No. 168-23191) was used.
The optical microscope was manufactured by Olympus Corporation (Tokyo) (Model BX51), the fluorescence microscope was manufactured by Olympus Corporation (Model FV1200), and the transmission electron microscope was manufactured by Hitachi, Ltd. (Tokyo) (Model H7650).

  The disease model preparation device is operated by a perfusion-culture system (Sakaguchi K. et al., Scientific Reports 3: 1316, 1-7 (2013)) equipped with a micro syringe pump (KD Scientific No. KDS270, USA). Used as a flow making machine and manufactured by the inventor.

2. Experimental method This study was approved by the Animal Experimentation Ethics Committee of Waseda University (approval number 2018-A109). Male SD rats (8-12 weeks old, N = 3) were used. General anesthesia was performed with 2.7% isoflurane (50 mg / kg · BW) and euthanized under anesthesia. Immediately, the abdomen was incised, and the abdominal aorta (2 mm in diameter) was removed. After washing the remaining blood and removing the adipose tissue of the outer wall of the blood vessel, the blood vessel is incised in the longitudinal direction, and the blood vessel lumen side in a planar shape, that is, the endothelial cell side is the upper side, DMEM, 10% FBS and 1% In a culture dish containing a culture medium of Penicililin, the blood vessels were restored to their original state before extraction and fixed.

The culture dish was set in a disease model preparation device (FIG. 2). The disease model production apparatus used in this experiment had two pairs of syringes and one valve, and created a steady flow of culture solution (flow rate 20 ml / min) by operating the syringes. The steady flow is accelerated through a nozzle with a diameter (φ) of 0.7 mm, and controlled by computer simulation to control the shear stress to 20 Pa in the region where the shear stress on the wall due to the collision is maximum (Fig. 3). ), And collided with the extracted vascular tissue for 3 days, which is the culture period. The culture was performed in an incubator (temperature: 37 ° C., CO ₂ concentration: 5.0%).

The shape used in the above simulation was created using 3D CAD software (SOLIDWORKS) manufactured by Dassault Systems (France). The simulation used ANSYS CFX, a general-purpose thermal fluid analysis software manufactured by Ansys (USA). The fluid was Newtonian fluid (density 1000 Kg / m ³ , viscosity 0.001 Pas). The analysis was performed under a steady flow. The inlet was under velocity boundary conditions and the outlet was under pressure boundary conditions. The velocity boundary condition was defined for each flow rate, and the pressure boundary was set to 0 Pa.

  After fixing the cultured tissue with 4% formalin, a 2 μm-thick section is prepared, hematoxylin and eosin (HE) staining, Tunnel staining for apoptosis, anti-Ki67 antibody for nuclear staining with Hoechst33342 and cell proliferation phase Was performed, and observation was performed with an optical microscope to evaluate degenerative changes and proliferative changes.

3. Experimental result
(1) Endothelial cell exfoliation After excision of rat abdominal aortic tissue, incision was performed in the longitudinal direction, and a constant pressure / steady flow of 20 Pa was applied to the vascular endothelial cells for 3 days under culture conditions to apply physical stress. After fixing the tissue, a slice having a thickness of 2 μm was prepared, stained with hematoxylin and eosin (HE staining), and the cross section of the blood vessel was observed with an optical microscope. The result is shown in FIG.

  (A) shows a constant pressure / steady flow collision area, and (B) shows a non-collision area. Endothelial cells were exfoliated in the collision area as in the case of clinical vascular disease, while endothelial cells (▼) remained in the non-collision area.

(2) Thinning of blood vessel wall thickness and apoptosis of smooth muscle cells Physical stress was applied for 3 days at the above-mentioned constant pressure and steady flow at a pressure of 20 Pa. The cells were stained with TUNNEL stain, and the pathological image and the blood vessel wall thickness were compared between the collision area and the non-collision area (FIG. 5-1). The measurement of the blood vessel wall thickness was performed on sub-continuous sections (six pieces, 100 μm intervals), each section was divided into regions of 200 μm, and the length was calculated from the length of the bisector in each region. The area within 0.7 mm in diameter from the collision position was defined as the collision area, and the area 1 mm away from the collision area was defined as the non-collision area. The average value and standard deviation of the wall thickness were calculated for each area. The ratio of smooth muscle cell apoptosis was determined by the ratio of the number of TUNNEL-staining positive cells to the total number of cells in each region.

  In the collision area, the blood vessel wall thickness was smaller than the non-collision area (Student's t-test P <0.01), and the thinning of the media was progressing (FIG. 5: upper and middle rows; HE staining, lower row; TUNNEL staining, FIG. 6; measurement result of wall thickness).

  From TUNNEL staining, many cell deaths (white inverted triangles: ▽) of smooth muscle cells were observed in the collision area (FIG. 5, lower panel). Apoptosis of smooth muscle cells was observed only in the collision area, and it was confirmed that degenerative changes in vascular tissue were reproduced.

  Furthermore, observation of this specimen with a transmission electron microscope revealed that the smooth muscle cell nuclei were concentrated and that smooth muscle cell death was apoptosis (FIG. 7).

  The percentage of smooth muscle cells that induced apoptosis significantly increased in the collision area compared to the non-collision area (t-test P <0.01) (FIG. 8).

(3) Collapse of inner elastic plate
The inner elastic plate was observed by HE staining (FIG. 9). FIG. 9 (A) shows a pathological image immediately after the extraction, and FIG. 9 (B) shows a pathological image of a collision area where physical stress was applied by collision with a constant pressure / steady flow of 20 Pa for 3 days. Immediately after the extraction, five layers of elastic plate were confirmed, but it was recognized that the collapse of the inner elastic plate was progressing due to the application of physical stress for three days.

(4) Observation of cells in the proliferative phase Histochemical immunostaining using anti-Ki67 protein antibody, which is a cell proliferation marker, for a sample of vascular tissue subjected to the above-mentioned 20 Pa constant pressure and steady flow for 3 days and subjected to physical stress The observation of proliferating cells was performed using a fluorescence microscope (FIG. 10).

  (A) is a Hoechst33343 stain, (B) is a Ki67 stain, and (C) is a fluorescence microscope image of Hoechst33342 and Ki67 superimposed. The observation region is a boundary region from the collision region to the non-collision region. Ki67-positive cells were observed in the border zone (B). Ki67-positive cells were endothelial cells and smooth muscle cells. From these results, the proliferative change of the vascular tissue could be reproduced in the disease model.

  Endothelial cells detached in the collision area, and endothelial cells appeared from the boundary area. Also, Ki67-positive smooth muscle cells were found to be concentrated near the intima. From these observations, it was understood that endothelial cell proliferation and smooth muscle cell proliferation were in the cell proliferation phase while interacting.

Small activity Fig. 11 summarizes the observation results of the degenerative change and the proliferative change associated with different observation sites in the collision area, the non-collision area, and the peripheral area located in the middle of the collision flow. The method and apparatus of the present invention can provide a disease model that can reproduce two types of degenerative changes and proliferative changes in vascular tissue at a specified location by utilizing physical stress due to collision of blood flow.

  The upper diagram of FIG. 11 shows the intensity of pressure and wall shear stress caused by the impinging flow. It is known that the pressure is strongest at the collision center, while the shear stress is strongest around the collision center.

  The lower diagram of FIG. 11 is a schematic diagram summarizing the pathological images observed in the above experiment. In the collision zone, the collision flow causes detachment of endothelial cells, apoptosis of vascular smooth muscle cells, collapse of the internal elastic lamina, and thinning of the media. The non-collision zone represents a region where no change is caused by the collision flow. In a boundary region between the collision region and the non-collision region where strong shear stress acts, the endothelial cells and smooth muscle cells are stimulated to proliferate, thereby causing thickening. The pathophysiology brought about by these impinging flows approximates the pathological features seen in patients with vascular disease, including cerebral aneurysms.

Summary According to the present invention, it was possible to reproduce two types of degenerative change and proliferative change of vascular tissue in the process of developing vascular disease by applying physical stress to vascular tissue ex vivo in a collision flow. . The two different types of vascular responses are likely to produce different morphological changes depending on the type and rate of these changes. For example, in a patient, the pathology of an aneurysm progresses so that the blood vessel wall becomes thinner due to degenerative changes in the blood vessel and projects morphologically outward. In arteriosclerosis, the disease state progresses such that the blood vessel wall is thickened due to the proliferative change of the blood vessel and morphologically protrudes inward. In other words, a pathological image similar to a clinical one observed by colliding a vascular tissue with a constant pressure / steady flow in the present invention is a simple and short-term, ex vivo system for generating and developing vascular diseases in vitro. Indicates that it could be reproduced.

  Further, the present invention can be applied not only to elucidating the pathological mechanism of the occurrence and progress of vascular disease, but also to the development of a drug for suppressing the occurrence, progress and breakdown of vascular disease. It can be used as a platform for a screening method for prevention or treatment of vascular diseases, including verification of drug effects. In addition, by reproducing the degenerative and proliferative changes by physical stress, a new concept of drug discovery that takes into account the magnitude and magnitude of physical stress and the type and stage of vascular disease in accordance with it. Can be. The process of escalation of vascular disease is associated with physical stress due to blood flow. However, in vivo animal experiments, which have been the main focus of drug efficacy verification methods, there is a problem that physical stress cannot be actively controlled. In other words, physical stress in an in vivo animal experiment environment is the result of the organism reacting at the individual level to an artificial operation to reproduce the disease state, and changing the content and degree of the stress. There is a problem that can not be. On the other hand, the present invention has an advantage that drug effect verification can be performed in an environment in which the type, degree, and ratio of stress can be adjusted under an environment in which physical stress can be variably controlled.

Claims (12)

An ex vivo method for analyzing the pathophysiology of vascular disease,
A step of applying physical stress to the endothelial cell side surface of the cut vascular tissue of the isolated blood vessel placed in the culture solution for a certain period of time,
Observing a pathological change in the site of physical stress loading of the isolated blood vessel or its surrounding area,
Analyzing the pathological changes,
An analysis method, comprising:   The analysis method according to claim 1, wherein the step of applying the physical stress includes collision of a steady flow of the culture solution with vascular tissue.   The analysis method according to claim 1, wherein the pathological change is selected from a morphological change, a biochemical change, or a change in gene expression.   The pathological change is characterized by endothelial cell detachment, thinning or thickening of the media, collapse of the internal elastic plate, shape change of vascular smooth muscle cells, proliferative change or observation of the presence or absence of apoptosis. The analysis method according to claim 3, wherein the analysis is performed.   The observation according to claim 4, wherein the observation is a measurement of a blood vessel wall thickness or a measurement of the number of cells classified into properties selected from the presence or absence of proliferative change or apoptosis of vascular smooth muscle cells. analysis method.   The method according to any one of claims 1 to 5, wherein the vascular disease is a disease caused by a hemodynamic factor.   The analysis method according to claim 6, wherein the disease caused by the hemodynamic factor is selected from cerebral aneurysm, aortic aneurysm, carotid aneurysm, arteriosclerosis, or hypertension. A method for preparing an ex vivo disease model for vascular disease,
A step of applying physical stress to the incised luminal vessel wall of the isolated blood vessel placed in the culture solution for a certain period of time,
A production method comprising: An ex vivo disease model for vascular disease,
Produced by a production method including a step of applying a physical stress to the incised luminal vessel wall surface of the isolated blood vessel placed in the culture solution for a certain period of time,
A disease model characterized by containing at least one of vascular tissue from which vascular endothelial cells have been detached, a thinned or thickened media, a collapsed internal elastic plate, and vascular smooth muscle cells that have induced a proliferative change or apoptosis. . A method for screening a drug for prevention or treatment of vascular disease,
A step of applying a physical stress to the incised luminal vessel wall of the isolated blood vessel placed in the culture solution containing the test compound for a certain period of time,
Observing a pathological change in the site of physical stress loading of the isolated blood vessel or its surrounding area,
A screening method, comprising a step of analyzing and evaluating the pathological change.   11. The screening method according to claim 10, wherein the step of applying the physical stress includes collision of a steady flow of the culture solution. An apparatus for producing an ex vivo disease model of vascular disease,
A manufacturing apparatus comprising: a tissue culture unit for culturing a vascular tissue; a steady flow generating unit; and a nozzle unit for adjusting a collision pressure by a steady flow to the vascular tissue.