JP2020060488A - Shear generating device quantitatively applying shear stress to blood, corpuscle, or coagulation related factor - Google Patents
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Abstract
Description
本発明は医療及び診断分野に関し、より詳しくは、血液、血球又は凝固関連因子へ定量的にずり応力を加える剪断応力発生装置およびそれを用いた血液、血球又は凝固関連因子の分析方法に関する。 The present invention relates to the fields of medicine and diagnosis, and more particularly, to a shear stress generator for quantitatively applying shear stress to blood, blood cells or coagulation-related factors and a method for analyzing blood, blood cells or coagulation-related factors using the same.
後天性フォン・ヴィレブランド症候群（以下、単に「ＡＶＷＳ」ともいう。）は種々の病因によりフォン・ヴィレブランド因子（以下、単に「ＶＷＦ」ともいう。）の量的／質的異常が引き起こされる稀な疾患であり、先天性フォン・ヴィレブランド病（以下、単に「ＶＷＤ」ともいう。）に類似した出血症状を呈する。その病因は多様で、主なものとして（１）ＶＷＦに対する自己抗体の産生、（２）ＶＷＦ高分子量マルチマーの減少、（３）ずり応力亢進によるＡＤＡＭＴＳ１３のＶＷＦ分解促進、（４）ＶＷＦの産生並びに分泌障害などが挙げられる。
これらの病因を引き起こす基礎疾患としては、（１）リンパ増殖性疾患や自己免疫疾患、（２）骨髄増殖性疾患（主に真性多血症並びに本態性血小板血症）やウイルムス腫瘍、（３）骨髄増殖性疾患、心臓弁膜症、補助人工心臓使用に伴う副作用、（４）甲状腺機能低下症などが挙げられる。
Acquired von Willebrand syndrome (hereinafter also simply referred to as “AVWS”) is a rare cause of various quantitative / qualitative abnormalities in von Willebrand factor (hereinafter simply referred to as “VWF”). The bleeding symptom is similar to congenital von Willebrand disease (hereinafter, also simply referred to as “VWD”). Its pathogenesis is diverse, and (1) production of autoantibodies against VWF, (2) reduction of VWF high molecular weight multimers, (3) acceleration of VWF degradation of ADAMTS13 by increased shear stress, (4) production of VWF and Examples include secretion disorders.
(1) lymphoproliferative disorders and autoimmune disorders, (2) myeloproliferative disorders (mainly polycythemia vera and essential thrombocythemia) and Wilms tumor, and (3) Examples include myeloproliferative diseases, valvular heart disease, side effects associated with the use of an assisted artificial heart, and (4) hypothyroidism.
ところで、最近の技術的進歩により、補助人工心臓の臨床応用が本邦でも数百例を超え、世界で最も良好な成績を誇っている。しかしながら、補助人工心臓症例の長期生存例が増加するに伴い、予測もしなかった新たな病態が発生するに至っている。例えば、非生理的な非常に高いずり応力が発生することにより、ＡＤＡＭＴＳ１３によるＶＷＦの切断が亢進し、ＡＶＷＳが発症する。人工心臓患者では、血液、血球、または凝固関連因子に、大動脈弁狭窄症と同様の高ずり応力が加わり、ＡＤＡＭＴＳ１３によるＶＷＦの過剰切断によりＶＷＦ高分子量マルチマーが減少して、出血傾向を起こすに至った、と推測されている。 By the way, due to recent technological progress, clinical application of the artificial heart has exceeded several hundred cases in Japan and boasts the best results in the world. However, as the number of long-term survivors of the assisted artificial heart increases, new and unexpected pathological conditions have occurred. For example, non-physiologically very high shear stress is generated, whereby the cleavage of VWF by ADAMTS13 is promoted and AVWS develops. In artificial heart patients, high shear stress similar to aortic valve stenosis is applied to blood, blood cells, or coagulation-related factors, and VWF high molecular weight multimers decrease due to excessive cleavage of VWF by ADAMTS13, leading to bleeding tendency. It is speculated that
ＡＤＡＭＴＳ１３によるＶＷＦ高分子量マルチマーの過剰切断の評価方法に関して、様々な提案がなされている。しかしながら、そもそも、血液中に、一定のずり応力を負荷する手段がなく、定量的な研究を不可能にしていた。 Various proposals have been made regarding the method for evaluating the excessive cleavage of VWF high molecular weight multimers by ADAMTS13. However, in the first place, there was no means for applying a constant shear stress in blood, making quantitative studies impossible.
生体における血管壁にはある種の応力がかかっている。静止流体では血管壁に対しての圧力だけであるが、血液が流れている時は、粘性により、垂直応力とずり応力が生じる。垂直応力は圧力に比べて無視できるくらい小さく、粘性の応力としてはずり応力のみを考慮すればよい。
ずり応力は、粘度と血流速度に比例し、血管径に逆比例する応力である。高ずり応力下では、従来の静止系や閉鎖撹拌実験系で構築された古典的概念とは全く異なるメカニズムで血小板粘着・凝集反応が進行するので、血栓形成メカニズムの解析には血流・ずり応力の計測や制御は必須であると考えられる。（非特許文献１，２）
しかしながら、血液や血球に一定のずり応力をかけて、血液、血球、または凝固関連因子に該ずり応力のみが与える作用を独立して計測できるシステムは存在しない。
Some sort of stress is applied to the blood vessel wall in the living body. In static fluid, there is only pressure on the blood vessel wall, but when blood is flowing, viscosity causes vertical stress and shear stress. The vertical stress is negligibly small compared to the pressure, and it is sufficient to consider only the shear stress as the viscous stress.
Shear stress is a stress that is proportional to viscosity and blood flow velocity and inversely proportional to blood vessel diameter. Under high shear stress, the platelet adhesion / aggregation reaction proceeds by a mechanism that is completely different from the classical concept established in conventional static systems and closed stirring experimental systems. It is considered that measurement and control of is essential. (Non-patent documents 1 and 2)
However, there is no system capable of independently measuring the effect of only the shear stress on blood, blood cells, or coagulation-related factors by applying a constant shear stress to blood or blood cells.
上記のように、従来の技術では、血液や血球や凝固関連因子に一定のずり応力をかけて、血液、血球、凝固関連因子に該ずり応力のみが与える作用を独立して計測できるシステムは存在しなかった。
本発明は、血液、血球、凝固関連因子へのずり応力の作用を定量診断的に、解析・研究する剪断応力発生装置を提供することを課題とする。
本発明はまた、圧力と独立に、血液、血球、凝固関連因子へのずり応力の作用だけを解析することができる剪断応力発生装置を提供することを課題とする。
As described above, in the conventional technology, there is a system capable of independently measuring the effect of only the shear stress on blood, blood cells, and coagulation-related factors by applying a constant shear stress to blood, blood cells and coagulation-related factors. I didn't.
It is an object of the present invention to provide a shear stress generator that analyzes and studies the action of shear stress on blood, blood cells, and coagulation-related factors quantitatively and diagnostically.
It is another object of the present invention to provide a shear stress generator capable of analyzing only the action of shear stress on blood, blood cells, and coagulation-related factors independently of pressure.
本発明の剪断応力発生装置は、
第一の血液収容部、
第二の血液収容部、
該第一および第二の血液収容部それぞれの流入口に接続されて第一および第二の血液収容部を連通する狭窄部、並びに、
該第一および第二の血液収容部に収容された血液を狭窄部に対し、気密に流出入させるための第一及び第二の可動部、を含み、
該第一の可動部が狭窄部に近づく方向に移動するときは第二の可動部は狭窄部から離れる方向に移動し、該第一の可動部が狭窄部から離れる方向に移動するときは第二の可動部は狭窄部に近づく方向に移動し、該第一および第二の可動部が往復運動することで、該血液を該狭窄部に往復させて、血液又は血液成分にずり応力を負荷することを特徴とする。
The shear stress generator of the present invention is
The first blood container,
The second blood container,
A narrowing portion connected to the inflow ports of the first and second blood storage portions to communicate the first and second blood storage portions, and
The first and second movable parts for airtightly flowing in and out the blood contained in the first and second blood containing parts with respect to the narrowed part,
When the first movable part moves in the direction approaching the stenosis, the second movable part moves in the direction away from the stenosis, and when the first movable part moves in the direction away from the stenosis, The second movable part moves toward the stenosis, and the first and second movable parts reciprocate to reciprocate the blood to the stenosis and load shear stress on the blood or blood components. It is characterized by doing.
また、本発明の、剪断応力による血液中のＶＷＦ高分子量マルチマーの分解を解析するためのシステムは、
本発明の剪断応力発生装置、および
ＶＷＦ高分子量マルチマー解析装置を含むことを特徴とする。
Further, the system for analyzing the degradation of VWF high molecular weight multimer in blood by the shear stress of the present invention comprises:
It is characterized by including the shear stress generator of the present invention and a VWF high molecular weight multimer analyzer.
さらに、本発明の、剪断応力による血液中のＶＷＦ高分子量マルチマーの分解を解析する方法は、
本発明の剪断応力発生装置を用いてインビトロで血液に剪断応力を負荷する工程、および
剪断応力が負荷された血液を用いてＶＷＦ高分子量マルチマーの分解を解析する工程、を含むことを特徴とする。
Furthermore, the method of analyzing the degradation of VWF high molecular weight multimer in blood by shear stress according to the present invention comprises:
And a step of applying a shear stress to blood in vitro using the shear stress generator of the present invention, and a step of analyzing the decomposition of the VWF high molecular weight multimer using the blood subjected to the shear stress. .
本発明に係る装置によれば、可動部を駆動させて血液を狭窄部に往復させることで、血液にインビトロで定量的に圧力及び／又はずり応力を負荷することができ、さらに、可動部を同期することにより、圧力と独立に、血液、血球、凝固関連因子へのずり応力の作用だけを解析することができる。
本発明に係る装置は、新しい治療法評価試験装置であり、血液試料に対して流体力学的剪断負荷を与え、剪断ストレスによって活性化する血液成分の変化プロセスを阻害する薬理学的有効性評価を可能とする。
According to the device of the present invention, by driving the movable part to reciprocate the blood to and from the stenosis, it is possible to quantitatively apply pressure and / or shear stress to the blood in vitro. By synchronizing, only the effect of shear stress on blood, blood cells, and coagulation-related factors can be analyzed independently of pressure.
The device according to the present invention is a new therapeutic method evaluation test device, which applies a hydrodynamic shear load to a blood sample and evaluates the pharmacological efficacy of inhibiting the process of changing blood components activated by shear stress. It is possible.
以下、本発明の剪断応力発生装置を、図１を用いてより具体的に説明するが、本発明は以下の説明によって何ら限定されるものではない。 Hereinafter, the shear stress generator of the present invention will be described more specifically with reference to FIG. 1, but the present invention is not limited to the following description.
図１は、本発明の剪断応力発生装置の一実施態様を示す模式図である。
図１の剪断応力発生装置は、第一の血液収容部１と、第二の血液収容部２と、第一および第二の血液収容部１，２それぞれの流入口に接続されて第一および第二の血液収容部１，２を連通する狭窄部３と、第一および第二の血液収容部１，２に収容された血液をそれぞれ狭窄部３に気密に流出入させるための第一及び第二の可動部４，５を備える。
第一の血液収容部１および第二の血液収容部２の少なくとも一方には血液が収容される。第一の可動部４が狭窄部３に近づく方向に移動するときは第二の可動部５は狭窄部３から離れる方向に移動し、第一の可動部４が狭窄部３から離れる方向に移動するときは第二の可動部５は狭窄部３に近づく方向に移動し、該第一の可動部４および第二の可動部５が往復運動することで、血液収容部１，２内の血液が狭窄部３を往復し、血液又は血液成分にずり応力が負荷される。すなわち、図１の場合、第一および第二の可動部がそろって左方向へ、次いで右方向へと移動し、往復運動することで、血液が狭窄部内を往復移動し、血液又は血液成分にずり応力が負荷される。
FIG. 1 is a schematic view showing an embodiment of the shear stress generator of the present invention.
The shear stress generator of FIG. 1 is connected to the first blood container 1, the second blood container 2, and the inlets of the first and second blood containers 1 and 2, respectively, and The narrowed portion 3 that communicates the second blood containing portions 1 and 2, and the first and second portions for allowing the blood contained in the first and second blood containing portions 1 and 2 to flow into and out of the narrowed portion 3 in an airtight manner, respectively. The second movable parts 4 and 5 are provided.
Blood is contained in at least one of the first blood container 1 and the second blood container 2. When the first movable portion 4 moves toward the narrowed portion 3, the second movable portion 5 moves away from the narrowed portion 3, and the first movable portion 4 moves away from the narrowed portion 3. When moving, the second movable portion 5 moves in a direction approaching the narrowed portion 3, and the first movable portion 4 and the second movable portion 5 reciprocate, so that the blood in the blood containing portions 1 and 2 is reciprocated. Reciprocates through the narrowed portion 3, and shear stress is applied to blood or blood components. That is, in the case of FIG. 1, the first and second movable parts are aligned to the left and then to the right, and reciprocally move so that blood reciprocates in the stenosis and blood or blood components are generated. Shear stress is applied.
第一及び第二の可動部４，５の移動は上記のような移動方向であれば特に限定されず、別個独立に移動を制御することができる。これにより、圧力や、血液に負荷される種々の応力やずり応力を自在に制御することが可能となる。一方、上記のような移動方向で移動速度を同期することもでき、それにより、血液に圧力とは独立に一定のずり応力を負荷することができ、生体内での血流下のずり応力をより精度よく再現することができる。
狭窄部３は第一および第二の血液収容部１，２を気密に連通する細管であるが、その内径は、血液に負荷するずり応力の大きさに応じて、任意に設計することができる。これにより、様々な圧力やずり応力を血液に負荷することができる。狭窄部３の内径は、好ましくは０．１〜１．０ｍｍであり、より好ましくは０．２〜０．８ｍｍである。内径を変化させることで様々なずり応力を負荷することができる。なお、狭窄部の内径は一様でもよいし、途中で変化してもよい。狭窄部の長さは特に制限はないが、例えば、１ｍｍ〜１０ｃｍである。
The movements of the first and second movable portions 4 and 5 are not particularly limited as long as they are in the movement directions described above, and the movements can be controlled independently. This makes it possible to freely control the pressure and various stresses and shear stresses applied to blood. On the other hand, it is also possible to synchronize the moving speed in the moving direction as described above, whereby a constant shear stress can be applied to the blood independently of the pressure, and the shear stress under the blood flow in the living body can be reduced. It can be reproduced more accurately.
The narrowed portion 3 is a thin tube that air-tightly connects the first and second blood containing portions 1 and 2, and the inner diameter thereof can be arbitrarily designed according to the magnitude of shear stress applied to blood. . This makes it possible to apply various pressures and shear stress to blood. The inner diameter of the narrowed portion 3 is preferably 0.1 to 1.0 mm, more preferably 0.2 to 0.8 mm. Various shear stresses can be applied by changing the inner diameter. The inner diameter of the narrowed portion may be uniform or may change in the middle. The length of the narrowed portion is not particularly limited, but is, for example, 1 mm to 10 cm.
また、狭窄部３の内面は、表面粗さＲａが１〜１００μｍとなるように処理されていてもよい。このように狭窄部３の内面が処理されていることで、狭窄部３の内面粗さを適宜調節し、血液に負荷される圧力やずり応力を調整することができる。このような狭窄部内面の処理は、例えば、プラズマ処理や紫外線処理、化学処理などによる官能基の導入などによって行うことができる。 Further, the inner surface of the narrowed portion 3 may be treated so that the surface roughness Ra becomes 1 to 100 μm. By treating the inner surface of the stenosis 3 in this manner, the inner surface roughness of the stenosis 3 can be appropriately adjusted, and the pressure and shear stress applied to blood can be adjusted. Such treatment of the inner surface of the narrowed portion can be performed by, for example, introducing a functional group by plasma treatment, ultraviolet treatment, chemical treatment, or the like.
上記の、血液収容部と可動部とを備える機構としては、収容部と可動部とを備えるものであれば特に制限されない。収容部と可動部とを備える機構の具体例としては、例えば、注射器および注射器状のピストン機構を挙げることができる。
また、狭窄部としては、これを介して該機構を組み合わせることができるものであれば、特に制限されない。狭窄部の具体例としては、所定のゲージの注射針を挙げることができる。
図２に、本発明の剪断応力発生装置の簡易的な一実施形態とその使用態様の模式図を示す。図２は、２本の注射器を１本の注射針で組み合わせ、ピストンの移動により血液を注
射針内に往復させているものである。
The above-mentioned mechanism including the blood storage unit and the movable unit is not particularly limited as long as it includes the storage unit and the movable unit. Specific examples of the mechanism including the housing portion and the movable portion include a syringe and a syringe-like piston mechanism.
Further, the narrowed portion is not particularly limited as long as the mechanism can be combined through the narrowed portion. A specific example of the narrowed portion is an injection needle of a predetermined gauge.
FIG. 2 shows a schematic view of a simple embodiment of the shear stress generator of the present invention and a usage mode thereof. In FIG. 2, two syringes are combined with one injection needle, and blood is reciprocated in the injection needle by movement of the piston.
図３は、本発明の装置の他の実施態様を示す模式図である。
図３の剪断応力発生装置は、第一の血液収容部１と、第二の血液収容部２と、狭窄部３と、第一及び第二の可動部４，５を備え、さらに、該第一及び第二の可動部を駆動する駆動部６、および該駆動部を制御する制御部７を含む。
第一及び第二の可動部４，５は駆動部６に接続され、駆動部６は制御部７に接続されている。
また、狭窄部３は、その内部を血液が流通自在となるように設けられており、該血液収容部１から血液収容部２へ、または該血液収容部２から血液収容部１へと、該狭窄部３を経由して血液が流通する。血液が狭窄部３を流通する際に、該狭窄部３において血液にずり応力が加えられる。
血液収容部１，血液収容部２は、狭窄部３の一端および他端それぞれに接続されている。可動部４，可動部５はそれぞれ駆動部６に接続され、駆動部６は制御部７に接続されている。例えば、血液収容部１から狭窄部３に血液が流入するときには、可動部４は狭窄部３に近づく方向に移動し、可動部５は狭窄部３から離れる方向に移動し、血液は狭窄部３を通過して血液収容部２内に流入する。一方、血液収容部２から狭窄部３に血液が流入するときには、可動部５は狭窄部３に近づく方向に移動し、可動部４は狭窄部３から離れる方向に移動し、血液は狭窄部３を通過して血液収容部１内に流入する。このように、可動部４，可動部５は互いに反転位相となるように制御部で制御される。
FIG. 3 is a schematic view showing another embodiment of the device of the present invention.
The shear stress generator shown in FIG. 3 includes a first blood container 1, a second blood container 2, a stenosis 3, and first and second movable parts 4 and 5, and further, A drive unit 6 that drives the first and second movable units, and a control unit 7 that controls the drive units are included.
The first and second movable parts 4 and 5 are connected to a drive part 6, and the drive part 6 is connected to a control part 7.
Further, the narrowed portion 3 is provided so that blood can flow through the inside of the narrowed portion 3, and from the blood containing portion 1 to the blood containing portion 2, or from the blood containing portion 2 to the blood containing portion 1, Blood circulates through the narrowed portion 3. When blood circulates in the narrowed portion 3, shear stress is applied to the blood in the narrowed portion 3.
The blood containing portion 1 and the blood containing portion 2 are connected to one end and the other end of the narrowed portion 3, respectively. The movable section 4 and the movable section 5 are connected to a drive section 6, and the drive section 6 is connected to a control section 7. For example, when blood flows from the blood container 1 into the stenosis 3, the movable part 4 moves toward the stenosis 3, the movable part 5 moves away from the stenosis 3, and the blood flows into the stenosis 3. To flow into the blood container 2. On the other hand, when blood flows from the blood container 2 into the stenosis 3, the movable part 5 moves toward the stenosis 3, the movable part 4 moves away from the stenosis 3, and the blood flows into the stenosis 3. To flow into the blood container 1. In this way, the control part controls the movable part 4 and the movable part 5 so that they are in opposite phases to each other.
可動部４、可動部５の変位は完全逆位相となるように制御部７で制御され、狭窄部３に流入する血液に対して、血液収容部内筒の加速や減速などの速度変化に伴う内圧変動を抑制する、すなわちゼロプレッシャーとなることが好ましい。
すなわち、該制御部７は、前記第一及び第二の可動部４，５の移動速度を同期するように制御することが好ましい。第一及び第二の可動部４，５の移動速度が同期することで、狭窄部３を往復して通過する血液に圧力とは独立に一定のずり応力を負荷することができ、生体内での血流下のずり応力をより精度よく再現することができる。
The displacements of the movable part 4 and the movable part 5 are controlled by the control part 7 so as to have completely opposite phases, and the internal pressure of the blood flowing into the stenosis part 3 due to the speed change such as acceleration or deceleration of the inner cylinder of the blood storage part. It is preferable that the fluctuation is suppressed, that is, there is zero pressure.
That is, it is preferable that the control unit 7 controls the moving speeds of the first and second movable units 4 and 5 so as to synchronize with each other. Since the moving speeds of the first and second movable parts 4 and 5 are synchronized, a constant shear stress can be applied to the blood passing back and forth through the narrowed part 3 independently of the pressure, and in vivo It is possible to more accurately reproduce the shear stress under the blood flow.
さらに、第一および第二の血液収容部１，２の少なくとも一方は、血液貯蔵部（図示せず）と直接的または間接的に接続された圧調節バルブ８を備えていてもよい。圧調節バルブ８により血液収容部１，２の内圧を適宜変更し、血液貯蔵部から血液収容部１，２内に試料となる血液を導入してもよい。 Furthermore, at least one of the first and second blood storage units 1 and 2 may include a pressure control valve 8 that is directly or indirectly connected to a blood storage unit (not shown). It is also possible to appropriately change the internal pressure of the blood containing units 1 and 2 by the pressure control valve 8 and introduce the sample blood from the blood storage unit into the blood containing units 1 and 2.
また、本発明によれば、収容部と可動部とを備える機構を二つ以上組み合わせた、可変式剪断応力発生システムを提供することができる。
さらにまた、本発明によれば、収容部と可動部とを備える機構を二つ以上組み合わせ、該機構を、同期し、または、独立にコントロールすることで、様々な圧力とずり応力を血液に付加することができる、剪断応力発生システムを提供することができる。
さらにまた、本発明によれば、収容部と可動部とを備える機構を二つ以上、狭窄部を介して組み合わせ、該機構を、同期し、または、独立にコントロールし、かつ、狭窄部の内径を適宜設計することで、様々な圧力とずり応力を血液に付加することができる、剪断応力発生システムを提供することができる。
Further, according to the present invention, it is possible to provide a variable shear stress generating system in which two or more mechanisms each including a housing portion and a movable portion are combined.
Furthermore, according to the present invention, various pressures and shear stresses are added to blood by combining two or more mechanisms each having a housing portion and a movable portion, and controlling the mechanisms in synchronization or independently. It is possible to provide a shear stress generating system.
Furthermore, according to the present invention, two or more mechanisms each including a housing portion and a movable portion are combined through a narrowed portion, the mechanisms are synchronized or independently controlled, and the inner diameter of the narrowed portion is controlled. By appropriately designing, it is possible to provide a shear stress generation system capable of applying various pressures and shear stress to blood.
本発明のシステムは、剪断応力による血液中のＶＷＦ高分子量マルチマーの分解を解析するためのものであって、本発明の剪断応力発生装置、および、ＶＷＦ高分子量マルチマー解析装置を含むものとすることができる。 The system of the present invention is for analyzing the degradation of VWF high molecular weight multimers in blood due to shear stress, and can include the shear stress generator of the present invention and a VWF high molecular weight multimer analyzer. .
上記ＶＷＦ高分子量マルチマー解析装置は、ＶＷＦ高分子量マルチマーを解析できる装置であれば特に制限されず、一の装置でもよく、二以上の装置を組み合わせてＶＷＦ高分
子量マルチマーを解析できるようとしたものであってもよい。該装置の具体例としては、例えば、ゲルの電気泳動装置と、抗ＶＷＦ抗体を用いたウェスタンブロッティング装置と、を組み合わせたものが挙げられる。
The VWF high molecular weight multimer analyzer is not particularly limited as long as it is an apparatus capable of analyzing the VWF high molecular weight multimer, and may be one apparatus, or a combination of two or more apparatuses to analyze the VWF high molecular weight multimer. It may be. Specific examples of the device include a combination of a gel electrophoresis device and a western blotting device using an anti-VWF antibody.
本発明の、剪断応力による血液中のＶＷＦ高分子量マルチマーの分解を解析する方法は、本発明の剪断応力発生装置を用いてインビトロで血液に剪断応力を負荷する工程、および、剪断応力が負荷された血液を用いてＶＷＦ高分子量マルチマーの分解を解析する工程、を含む。 The method of analyzing the decomposition of VWF high molecular weight multimers in blood by shear stress of the present invention includes a step of applying a shear stress to blood in vitro using the shear stress generator of the present invention, and a step of applying a shear stress to blood. Analyzing the degradation of the VWF high molecular weight multimer using blood.
本発明の方法によれば、微量（例えば、０．０１μｌ〜１ｍｌ程度）の血液を用いてインビトロでＶＷＦ高分子量マルチマーの解析を行うことができる。
以下、本発明の方法によるＶＭＦ高分子量マルチマーの解析方法の一例を説明するが、以下の方法に限定されるものではない。
According to the method of the present invention, a VWF high molecular weight multimer can be analyzed in vitro using a trace amount (for example, about 0.01 μl to 1 ml) of blood.
Hereinafter, an example of a method for analyzing a VMF high molecular weight multimer according to the method of the present invention will be described, but the method is not limited to the following method.
例えば、図１に示す装置を用いて、血液を狭窄部に往復させることにより血液にずり応力を加えることができる。往復の回数としては血液に目的のずり応力を負荷できる回数であれば特に制限されない。血液に加えるずり応力としては、検査等の目的にもよるが、１００ｄｙｎｅ／ｃｍ２以上であることが好ましく、２００ｄｙｎｅ／ｃｍ２以上であることがより好ましい。
ずり応力の値は、狭窄部の内径や血液の流速などにより調節することができる。
For example, by using the device shown in FIG. 1, it is possible to apply shear stress to blood by reciprocating the blood through the stenosis. The number of reciprocations is not particularly limited as long as the target shear stress can be applied to blood. The shear stress applied to the blood, depending on the purpose of inspection, is preferably 100dyne / cm 2 or more, and more preferably 200dyne / cm 2 or more.
The value of the shear stress can be adjusted by the inner diameter of the narrowed portion, the flow velocity of blood, and the like.
血液としては被検対象から単離された血液であればよく、ｖＷＦマルチマーを含む限り全血でも血漿でもよいが、剪断応力を負荷した後に電気泳動を行うという解析のしやすさにおいては血漿を用いることが好ましい。
血液試料は希釈されてもよいし、抗凝固処理されたものでもよい。
血液試料はまた、薬剤が添加されたものとすることができる。薬剤が添加されたものとされていないものとを共に解析することで、剪断応力によるＶＷＦ高分子量マルチマーの分解に対する薬剤の効果を評価することができる。
The blood may be blood isolated from the test subject, and may be whole blood or plasma as long as it contains the vWF multimer. However, in view of ease of analysis in which electrophoresis is performed after applying shear stress, plasma is selected. It is preferable to use.
The blood sample may be diluted or may be anticoagulated.
The blood sample can also be spiked with a drug. The effect of the drug on the degradation of the VWF high molecular weight multimer due to shear stress can be evaluated by analyzing both the drug added and the drug not added.
被検対象は特に制限されないが、ｖＷＦ高分子量マルチマーの保持率に基づく病態を予測・解析するという観点からは、心臓病患者または心臓病が疑われる被検者が好ましく、補助人工心臓の適用が必要とされる心臓病患者がより好ましい。
単離される血液試料は特にどの血管から単離されたものでもよいが、試料の入手しやすさから、末梢血を用いることが好ましい。
血液試料は単離後すぐに剪断応力負荷試験に供してもよいし、血漿として凍結保存したのち、解凍して剪断応力負荷試験に供してもよい。
The subject to be examined is not particularly limited, but from the viewpoint of predicting / analyzing the pathological condition based on the retention rate of vWF high molecular weight multimers, a heart disease patient or a subject suspected of having heart disease is preferable, and the application of an auxiliary artificial heart is preferable. More preferred are those patients with heart disease that are needed.
The blood sample to be isolated may be one isolated from any blood vessel, but peripheral blood is preferably used because the sample is easily available.
The blood sample may be subjected to a shear stress load test immediately after isolation, or may be frozen and stored as plasma and then thawed and subjected to a shear stress load test.
ずり応力を負荷したサンプルに対し、評価を行う。評価方法としては、例えば以下の方法が挙げられるが、この方法に限定されるものではない。 Evaluation is performed on the sample that is subjected to shear stress. The evaluation method includes, for example, the following methods, but is not limited to this method.
ｖＷＦは、分子量約２５万の分子であるが、そのＮ末端およびＣ末端が結合し、２〜８０量体のマルチマーとして存在する。ｖＷＦのマルチマーパターンは、例えば、後述の実施例で示すようなアガロースゲル電気泳動およびＥＬＩＳＡ法で解析することができる。
このうち、ＶＷＦ高分子量マルチマーとしては、ウェスタンブロッティングで得られるｖＷＦのマルチマーのバンドパターンにおいて、最も低分子量側から数えて１１番目以上のバンドとして検出されるマルチマーと定義することができる。ここで、当該１１番目のバンドはｖＷＦの２２量体である。
Although vWF is a molecule having a molecular weight of about 250,000, its N-terminal and C-terminal are bound to each other, and it exists as a multimer of 2 to 80 mer. The vWF multimer pattern can be analyzed by, for example, agarose gel electrophoresis and an ELISA method as shown in Examples described later.
Among them, the VWF high molecular weight multimer can be defined as a multimer detected as the eleventh or more band counted from the lowest molecular weight side in the vWF multimer band pattern obtained by western blotting. Here, the 11th band is a 22-mer of vWF.
ｖＷＦの高分子量マルチマーの存在比は、例えば、上記のような１１番目以上のバンドとして検出されるＶＷＦ高分子量マルチマーの全ｖＷＦマルチマーに対する割合に基づい
て算出することができるが、この方法に限定されるものではない。
高分子量マルチマーの全ｖＷＦマルチマーに対する割合（高分子量マルチマーの存在比）は、例えば、血液試料をアガロースゲル電気泳動で分離した後に、タンパク質をＰＶＤＦ膜やニトロセルロース膜に転写し、抗ｖＷＦ抗体を用いたウエスタンブロットによりｖＷＦを染色して得られるｖＷＦマルチマーのバンドパターンを画像解析することによって算出することができる。
具体的には、ＩｍａｇｅＪ（ＮＩＨから入手可能）などの画像処理ソフトを使用して、１１番目以上のバンドの濃さの合計と、全てのバンドの濃さの合計と、をそれぞれ算出し、前者を後者で除することによって、ＶＷＦ高分子量マルチマーの全ｖＷＦマルチマーに対する割合を得ることができる。
The abundance ratio of high molecular weight multimers of vWF can be calculated based on, for example, the ratio of the VWF high molecular weight multimers detected as the 11th or more band as described above to the total vWF multimers, but is not limited to this method. Not something.
The ratio of the high molecular weight multimers to the total vWF multimers (abundance ratio of the high molecular weight multimers) is, for example, after separating a blood sample by agarose gel electrophoresis, transferring the protein to a PVDF membrane or a nitrocellulose membrane, and using an anti-vWF antibody. The band pattern of vWF multimers obtained by staining vWF by Western blotting can be calculated by image analysis.
Specifically, image processing software such as ImageJ (available from NIH) is used to calculate the sum of the darknesses of the 11th and higher bands and the sum of the darknesses of all the bands, respectively. Can be divided by the latter to obtain the ratio of VWF high molecular weight multimers to total vWF multimers.
健常人血液を用いたｖＷＦの高分子量マルチマーの存在比の解析結果と比べて、ｖＷＦの高分子量マルチマーの存在比が低下している場合、その被検者はｖＷＦの高分子量マルチマーが高ずり応力によって分解しやすく、ＡＶＷＳの危険性が高いと判定することができる。したがって、補助人工心臓を採用するかの判断の参考とすることができる。
また、薬剤を添加した血液を用いて本発明の装置を用いてずり応力を負荷し、ｖＷＦの高分子量マルチマー解析を行い、薬剤を添加しない場合の結果と比較することにより、当該薬剤がＶＷＦマルチマーの分解を抑え、ＡＶＷＳの治療薬となりうるかの指標とすることができる。
Compared with the analysis result of the abundance ratio of the high molecular weight multimer of vWF using the blood of a healthy person, when the abundance ratio of the high molecular weight multimer of vWF is decreased, the subject has a high shear stress of the high molecular weight multimer of vWF. Therefore, it can be determined that it is easy to disassemble and the risk of AVWS is high. Therefore, it can be used as a reference for determining whether to use the auxiliary artificial heart.
In addition, shear stress was applied to the drug-added blood using the device of the present invention, high molecular weight multimer analysis of vWF was performed, and the results were compared with the results in the case where no drug was added. It can be used as an index of whether it can be used as a therapeutic drug for AVWS.
以下に、実施例を挙げて本発明を具体的に説明するが、本発明はこれらの実施例に制限されるものではない。 Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.
以下に示す方法により、血液に煎断応力を負荷し、血液中のＶＷＦマルチマーの解析を行った。 By the method described below, the blood was subjected to a decoupling stress, and VWF multimers in the blood were analyzed.
図２に示すように、テルモ社製１ｍｌシリンジ２つを、１８ゲージ（内径０．４７ｍｍ）の注射針（テルモ社製）で連結した。一方のシリンジに血液試料を導入し、該試料を、注射針内を往復して通過させる操作を行った（二秒間に一往復で６分間、合計１８０往復）。 As shown in FIG. 2, two Terumo 1 ml syringes were connected by an 18-gauge (inner diameter 0.47 mm) injection needle (Terumo). A blood sample was introduced into one of the syringes, and the sample was reciprocally passed through the injection needle (one reciprocation every 2 seconds for 6 minutes, a total of 180 reciprocations).
ここで、ずり応力は、以下の式で計算した。
ずり応力（τ）＝４μＶ／ｒ
μ：粘度、Ｖ：平均流速、ｒ：内径
Here, the shear stress was calculated by the following formula.
Shear stress (τ) = 4μV / r
μ: viscosity, V: average flow velocity, r: inner diameter
なお、サンプルはクエン酸処理血液、またはクエン酸処理血液＋ＡＤＡＭＴＳ１３抗体Ａ１０（Ａ１０抗体最終濃度：５０μｇ／ｍＬ）を用い、それぞれ実験を行った。
なお、クエン酸処理血液、および、クエン酸処理血液＋ＡＤＡＭＴＳ１３抗体Ａ１０の粘度は、いずれも２．０ｍＰａ・ｓであった。
流速、粘度、および内径から計算したずり応力は２１６ｄｙｎｅ／ｃｍ２であった。
The samples were citrate-treated blood or citrate-treated blood + ADAMTS13 antibody A10 (A10 antibody final concentration: 50 μg / mL), and the experiments were carried out.
The citrate-treated blood and the citrate-treated blood + ADAMTS13 antibody A10 all had a viscosity of 2.0 mPa · s.
The shear stress calculated from the flow velocity, viscosity, and inner diameter was 216 dyne / cm 2 .
上記のようにしてずり応力を負荷した後、それぞれのサンプルおよびずり応力を負荷しないサンプルを用いてアガロースゲル電気泳動を行った。電気泳動後、タンパク質をアガロースゲルからＰＶＤＦ膜に転写し、抗ＶＷＦ抗体（ウサギ抗ＶＷＦポリクローナル抗体、ＤａｋｏＣｙｔｏｍａｔｉｏｎ製）を用いたウエスタンブロットによりＶＷＦを染色した。ＩｍａｇｅＪを使用して、低分子量側から数えて１１番目以上のバンドの濃さの合計と、全てのバンドの濃さの合計と、をそれぞれ算出し、前者を後者で除して、ＶＷＦ高分子量マルチマーの全ＶＷＦマルチマーに対する割合を調べた。 After the shear stress was applied as described above, agarose gel electrophoresis was performed using each sample and the sample not applied with the shear stress. After electrophoresis, the protein was transferred from an agarose gel to a PVDF membrane, and VWF was stained by Western blotting using an anti-VWF antibody (rabbit anti-VWF polyclonal antibody, manufactured by DakoCytomation). Using ImageJ, the sum of the darknesses of the 11th and higher bands counted from the low molecular weight side and the sum of the darknesses of all bands were calculated, and the former was divided by the latter to obtain the VWF high molecular weight. The ratio of multimers to total VWF multimers was investigated.
その結果、図４に示すように、ずり応力を負荷したときにＶＷＦ高分子量マルチマーの割合が減少し、負荷したずり応力に応じてＶＷＦ高分子量マルチマー分解が亢進しており、生体内で起こる高剪断応力によるＶＷＦ高分子量マルチマーの減少がインビトロで再現できることが分かった。
なお、ＡＤＡＭＴＳ１３の活性を阻害するＡＤＡＭＴＳ１３抗体を加えた場合には、ＶＷＦ高分子量マルチマーの分解が起こらなかったため、剪断応力でＶＷＦの分解部位が露出し、ＡＤＡＭＴＳ１３によって分解を受けるという生体内メカニズムが確認できた。このことは、ＡＤＡＭＴＳ１３の活性を阻害する薬剤など、ＶＷＦ高分子量マルチマーの分解を阻害する薬剤の評価やスクリーニングに本発明の装置及び方法が使用できることを示している。
As a result, as shown in FIG. 4, when the shear stress was applied, the proportion of the VWF high molecular weight multimer decreased, and the VWF high molecular weight multimer decomposition was enhanced in response to the applied shear stress. It was found that the reduction of VWF high molecular weight multimers due to shear stress was reproducible in vitro.
When an ADAMTS13 antibody that inhibits the activity of ADAMTS13 was added, the degradation of VWF high molecular weight multimer did not occur, so the degradation site of VWF was exposed by shear stress, and the in vivo mechanism of degradation by ADAMTS13 was confirmed. did it. This indicates that the device and method of the present invention can be used for the evaluation and screening of agents that inhibit the degradation of VWF high molecular weight multimers, such as agents that inhibit the activity of ADAMTS13.
本発明に係る、血液、血球、凝固関連因子へのずり応力を定量的に負荷することができる剪断応力発生装置は、他の血液疾患や、人工心臓、人工弁の開発における、血栓、溶血、その治療手段の開発など様々な展開が考えられる。 Shear stress generating device according to the present invention, which can quantitatively load shear stress to blood, blood cells, coagulation-related factors, other blood diseases, artificial hearts, in the development of artificial valves, thrombosis, hemolysis, Various developments are conceivable, such as the development of therapeutic means.
１：第一の血液収容部
２：第二の血液収容部
３：狭窄部
４：第一の可動部
５：第二の可動部
６：駆動部
７：制御部
８：圧調節バルブ
1: First blood storage part 2: Second blood storage part 3: Stenosis part 4: First movable part 5: Second movable part 6: Drive part 7: Control part 8: Pressure control valve
Claims (8)
第二の血液収容部、
該第一および第二の血液収容部それぞれの流入口に接続されて第一および第二の血液収容部を連通する狭窄部、並びに、
該第一および第二の血液収容部に収容された血液を狭窄部に対し、気密に流出入させるための第一及び第二の可動部、を含み、
該第一の可動部が狭窄部に近づく方向に移動するときは第二の可動部は狭窄部から離れる方向に移動し、該第一の可動部が狭窄部から離れる方向に移動するときは第二の可動部は狭窄部に近づく方向に移動し、該第一および第二の可動部が往復運動することで、該血液を該狭窄部に往復させて、血液又は血液成分にずり応力を負荷することを特徴とする、剪断応力発生装置。 The first blood container,
The second blood container,
A narrowing portion connected to the inflow ports of the first and second blood storage portions to communicate the first and second blood storage portions, and
The first and second movable parts for airtightly flowing in and out the blood contained in the first and second blood containing parts with respect to the narrowed part,
When the first movable part moves in the direction approaching the stenosis, the second movable part moves in the direction away from the stenosis, and when the first movable part moves in the direction away from the stenosis, The second movable part moves toward the stenosis, and the first and second movable parts reciprocate to reciprocate the blood to the stenosis and load shear stress on the blood or blood components. A shear stress generator, characterized by:
ＶＷＦ高分子量マルチマー解析装置を含む、
剪断応力による血液中のＶＷＦ高分子量マルチマーの分解を解析するためのシステム。 A shear stress generator according to any one of claims 1 to 5, and a VWF high molecular weight multimer analyzer.
A system for analyzing the degradation of VWF high molecular weight multimers in blood by shear stress.
剪断応力が負荷された血液を用いてＶＷＦ高分子量マルチマーの分解を解析する工程、を含む、剪断応力による血液中のＶＷＦ高分子量マルチマーの分解を解析する方法。 A step of applying a shear stress to blood in vitro using the shear stress generator according to any one of claims 1 to 5, and an analysis of the decomposition of VWF high molecular weight multimer using the blood subjected to the shear stress. And a step of analyzing the degradation of VWF high molecular weight multimers in blood due to shear stress.
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