JP2004251630A - Thrombus process observing apparatus, and thrombus process observation method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To observe the interaction with the dynamic form change of a platelet under an environment for simulating conditions in a living body. <P>SOLUTION: A monoclonal antibody to membrane glycoprotein GP IIb/IIIa existing on the surface of the platelet and GP Ibalpha is subjected to fluorescent labeling by an FITC, or the like. Sample blood mixed into blood in whole blood including a red corpuscle is flowed to a flow chamber 20 using a glass plate 22 that is subjected to a solid phase, for example, by coating the upper surface with matrix, such as collagen for simulating a damaged blood vessel wall and vWF (von Willebrand factor). At the same time, by using a confocal microscope device, the three-dimensional form of the platelet is imaged as a moving picture. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a technique for observing a thrombus formation process for evaluating the efficacy of an antithrombotic drug and the like.
[0002]
[Prior art]
Conventionally, the evaluation of the efficacy of antithrombotic drugs has been based on the release of granules in platelets induced when platelets are activated with an activating substance such as ADP or thrombin, the change in morphology of activated platelets, and the evaluation of activated platelets. It has been used as an index for the inhibitory effect on platelet aggregation and the like caused by stirring.
[0003]
However, the thrombus formation process in vivo does not occur as a result of platelet exposure to activators, but rather as a result of damage to the inner tube wall and exposure of the subendothelial matrix to the bloodstream. Under flow conditions, the platelets aggregate and interact with the platelets and the matrix to activate the platelets and proceed.
[0004]
Therefore, in order to evaluate the drug efficacy during the actual thrombus formation process, conventionally, it has been practiced to fix the platelet specimen that has interacted with the matrix and observe the specimen as a target for observation using a scanning electron microscope to observe the morphology of platelets. I have been.
[0005]
[Problems to be solved by the invention]
According to the technique of observing platelets by the scanning electron microscope, it is not possible to observe dynamic changes in platelet morphology and interaction with the matrix, because fixed platelet specimens are targeted for observation. For this reason, the effect and effect of the antithrombotic drug on platelet function under in vivo conditions could not be evaluated in detail.
[0006]
Therefore, an object of the present invention is to make it possible to observe in detail the dynamic morphological change of platelets and the interaction with the matrix under conditions equivalent to those in vivo.
[0007]
[Means for Solving the Problems]
In order to achieve the above object, the present invention provides a flow chamber having a flow channel in which a layer of collagen or vWF (von Willebrand factor) is provided on the flow channel wall surface, and a fluid for flowing blood through the flow channel of the flow chamber. Provided is a thrombus generation process observation device including a pump and a confocal microscope device for repeatedly and three-dimensionally imaging blood flowing through a flow channel of the flow chamber.
[0008]
According to such a thrombus formation process observation device, the three-dimensional morphology of platelets in the blood flow is determined by using a confocal microscope while the blood flow is passed through a channel simulating the damaged blood vessel wall by a layer of collagen or vWF. , Can be observed as a moving image with good spatial resolution and temporal resolution.
[0009]
Here, in such a thrombus generation process observation device, the flow channel formed in the flow chamber has a plurality of ranges in the flow channel direction having mutually different cross-sectional sizes. Is good.
By doing so, it is possible to observe the thrombus generation process due to different wall shear speeds and different flow channel widths only by changing the imaging position on the flow channel of the flow chamber by the confocal microscope device.
In order to achieve the above object, the present invention provides a method for observing a thrombus formation process, in which a flow channel in which a layer of collagen or vWF (von Willebrand factor) is provided on a channel wall surface is formed. The blood flowing through the flow chamber was passed through a flow channel of the flow chamber, and blood containing a fluorescently-labeled monoclonal antibody against platelet membrane glycoprotein GP IIb / IIIa was passed through the flow chamber. Provided is a method for observing a thrombus generation process in which repeated three-dimensional imaging is performed.
[0010]
According to such a method of observing a thrombus formation process, the three-dimensional morphology of platelets in the bloodstream is determined using a confocal microscope while the bloodstream is passed through a channel simulating the damaged blood vessel wall by a layer of collagen or vWF. , Can be observed as a moving image with good spatial resolution and temporal resolution. Therefore, it is possible to more closely grasp the dynamic morphological change of platelets and the interaction with the matrix, and to perform detailed evaluation of antithrombotic drugs and the like. Further, by imaging the morphology of platelets via a fluorescently labeled monoclonal antibody against GP IIb / IIIa, the morphology of platelets can be favorably imaged without being disturbed by red blood cells in the bloodstream.
[0011]
Similarly, the present invention provides a method for observing a thrombus formation process in which a layer of collagen or vWF (von Willebrand factor) is formed on a flow channel wall in which a flow channel is formed. A thrombus which repeatedly and three-dimensionally images the blood flowing through the flow channel of the flow chamber by a confocal microscope apparatus while flowing blood mixed with a fluorescently labeled monoclonal antibody against the platelet membrane glycoprotein GP Ibalpha in the flow channel. Provide a method for observing the generation process.
[0012]
According to such a method of observing a thrombus formation process, the distribution of GP Ibalpha of platelets in the bloodstream is determined using a confocal microscope while the bloodstream is passed through a channel simulating a damaged blood vessel wall by a layer of collagen or vWF. Therefore, it can be observed as a moving image with good spatial resolution and time resolution. Here, GP Ibalpha exhibits a phenomenon of moving and concentrating from the vicinity of the membrane to the inside of the platelet due to the activation of the platelet. Therefore, according to the present method, the function change of the platelet is grasped more finely, and the antithrombotic drug is obtained. Can be evaluated in detail. In addition, by performing imaging via a fluorescently labeled monoclonal antibody against GP Ibalpha, good imaging can be realized without being disturbed by red blood cells in the bloodstream.
[0013]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, embodiments of the present invention will be described.
FIG. 1 shows the configuration of the thrombus generation process monitoring apparatus according to the present embodiment.
The thrombus generation process observation device includes an in-vivo condition simulation device and a confocal microscope device.
The in-vivo condition simulator includes a flow chamber 20 in which a flow path simulating a blood vessel is formed, and a blood flow pump 30 that supplies a blood flow to the flow chamber 20. 1. A stage 2 on which a flow chamber 20 is mounted, a laser light source 3, a confocal scanner unit 4, a high-speed camera 5 such as a 66-frame / second double-speed camera conforming to the IEEE 1394 standard, and an image enhancement and high-speed shutter function for the camera 5. An image intensifier 6, an objective lens 7, an actuator 8 for moving the objective lens 7 in the optical axis direction, a Z-axis scanning control device 9, an image processing device 10, such as a computer having a video capture interface, and a display. Device 11.
[0014]
Here, the principle of the confocal microscope device will be described.
As schematically shown in FIG. 2, the basic configuration of the confocal microscope apparatus includes a microlens array 101 for condensing laser light on a sample 50, a pinhole array 102, an objective lens 7, An actuator 8 for moving the objective lens 7 in the optical axis direction (Z direction in the figure), a camera 5 having a condenser lens 103 and an image sensor 104, and a sample 50 having passed through the objective lens 7 and the pinhole array 102 And a beam splitter 105 that changes the course of the reflected light from the camera 5 in the direction of the camera 5.
[0015]
In such a configuration, the Z coordinate of the focal point of the laser beam is controlled by the position of the objective lens 7 in the Z direction, and the focal point of the laser beam is rotated by rotating the microlens array 101 and the pinhole array 102. Are controlled. That is, the scanning point in the sample 50 photographed by the camera 5 can be three-dimensionally controlled by the position of the objective lens 7 in the Z direction and the rotation angles of the microlens array 101 and the pinhole array 102.
[0016]
The confocal scanner unit 4 in FIG. 1 includes a microlens array 101, a pinhole array 102, a beam splitter 105, and a rotation control unit that drives the microlens array 101 and the pinhole array 102 shown in FIG. It is what contained. In the present embodiment, the sample 50 becomes a blood flow flowing inside the flow chamber 20.
[0017]
Further, the confocal microscope apparatus includes an illumination light source 12, and the illumination light source 12 and the optical system housed in the main body 1 function as an optical microscope apparatus.
Next, as shown in FIG. 3, the blood flow pump 30 of the in-vivo condition simulator simulates a syringe 31 for injecting the sample blood into the supply pipe and a rod of the syringe 31 at a set speed with respect to the cylinder. An injection device 32 having a linear motion mechanism for pushing the sample blood into the supply pipe 33 by the blood flow pump 30 forms a blood flow through a flow path provided inside the flow chamber 20. And discharged to a discharge pipe 34 and collected in a collection container 35.
[0018]
Next, the configuration of the flow chamber 20 will be described with reference to FIG.
FIG. 4A is a perspective view of the flow chamber 20, and FIG. 4B is a cross-sectional view of a vertical plane passing through the center line of the flow chamber 20.
As shown in the figure, the flow chamber 20 includes a metal base 21 and a glass plate 22 provided on the upper surface thereof with a layer 221 on which a matrix simulating a damaged blood vessel wall, such as collagen or vWF (von Willebrand factor), is immobilized. (Slide glass), a flow path side wall forming sheet 23 made of silicon having a thickness of about 220 μm, and a glass block 24 in which a blood supply tunnel 241 to the flow path and a blood discharge tunnel 242 from the flow path are formed. Then, as shown in FIG. 4C, the components are stacked from the bottom in the order of the description and are fastened in the vertical direction by four screws 25. However, here, the direction in which the laser beam enters when the flow chamber 20 is installed in the confocal microscope apparatus is set to the upward direction.
[0019]
The flow path simulating the blood vessel described above has a lower wall made of collagen or vWF immobilized on the glass plate 22, a side wall of a hole provided in the flow path side wall forming sheet 23 as a side wall, and a glass block 24. Is formed as an upper wall. In such a configuration, the sample blood injected from the supply pipe 33 into the blood supply tunnel 241 provided in the glass block 24 flows into the flow path through the blood supply tunnel 241 and passes through the flow path. Is discharged to the discharge pipe 34 through the blood discharge tunnel 242.
[0020]
Here, the shape of the holes provided in the flow path side wall forming sheet 23, and thus the shape of the flow path of the sample blood as viewed from above and below, is as shown in FIG. It has a shape that narrows toward the side. The width of the channel is, for example, 6 mm? The length is about 2 mm, and the length is about 30 mm.
[0021]
Now, observation by such a thrombus formation process observation device is performed as follows.
First, when observing mainly the change in platelet morphology, a monoclonal antibody to the membrane glycoprotein GP IIb / IIIa present on the platelet surface is fluorescently labeled with FITC or the like, and the fluorescently labeled monoclonal antibody is used for whole blood cells including erythrocytes. It is mixed with blood and used as sample blood. On the other hand, when observing mainly changes in platelet function, a monoclonal antibody against the membrane glycoprotein GP Ibalpha, which exhibits a phenomenon of movement and concentration from the vicinity of the membrane to the inside of the platelet due to platelet activation, is fluorescently labeled with FITC or the like. Then, a fluorescently labeled monoclonal antibody is mixed into whole blood including red blood cells to obtain a sample blood.
[0022]
Then, the injector 31 filled with the sample blood is set in the injector 32, and the blood pump 30 is provided with a constant blood shear rate in the flow chamber 20 (for example, with respect to the flow direction of the flow chamber 20). The confocal microscope apparatus performs imaging by the scanning sequence shown in FIG. 5 while supplying the blood in the injector 31 to the flow chamber 20 at a speed of 1500 -1 at the center of the confocal microscope.
[0023]
The scanning sequence of the confocal microscope device shown in FIG. 5 is started by the TRIGER signal output from the Z-axis scanning control device 9 shown in FIG. 5A. The camera 5 inputs the TRIGER signal as an external trigger signal, synchronizes the vertical synchronization signal with the TRIGER signal as shown in FIG. 5B, and starts shooting.
[0024]
The vertical synchronizing signal V SYNC of the camera 5 is output to the confocal scanner unit 4 and the Z-axis scanning controller 9. Then, the rotation control unit of the confocal scanner unit 4 performs the micro lens array 101 and the pinhole array so as to perform one XY scan every imaging period of each video frame in synchronization with the input vertical synchronizing signal VSYNC. 102 is driven to rotate. Here, the imaging period refers to a period excluding at least the vertical synchronization signal period in one video frame period. Note that the imaging period may not include a horizontal synchronization signal period, or a period in which pixels before and after the horizontal synchronization signal period and the vertical synchronization signal period are not imaged.
[0025]
On the other hand, as shown in FIG. 5C, the Z-axis scanning control device 9 transmits a pulse of a predetermined cycle to a predetermined number of actuators during the imaging period of each video frame to the movement control signal in synchronization with the input vertical synchronization signal VSYNC. Output as CNT. Further, the input vertical synchronization signal V SYNC is counted, and when a predetermined count is reached, the pulse output is stopped and a reset signal RST is output to the actuator 8. Then, when the next vertical synchronizing signal VSYNC is input, a predetermined number of pulses of a predetermined cycle are transferred to the actuator 8 as a movement control signal CNT during a predetermined number of video frame periods and an imaging period of each video frame in the same manner as described above. The sequence consisting of the stage 2 for outputting and the stage 2 for stopping the pulse output for one video frame period and outputting the reset signal RST to the actuator 8 is repeated.
[0026]
The actuator 8 integrates the pulse of the movement control signal CNT input from the Z-axis scanning control device 9, and as shown in FIGS. 5D and 5E, maintains the constant value during the period of the vertical synchronization signal V SYNC and sets the period during which the image frame is captured. Generates a drive signal which increases uniformly, and moves the objective lens 7 in the Z direction by this drive signal. Here, it is assumed that the amount of movement of the objective lens 7 is proportional to the magnitude of the drive signal. If the amount of movement of the objective lens 7 is not linearly proportional to the magnitude of the drive signal, the objective lens 7 does not move during the period of the vertical synchronization signal V SYNC and moves uniformly during the image frame imaging period. In this way, a drive signal is generated. As a mechanism for moving the objective lens 7, a mechanism using a piezo element or the like can be used.
[0027]
Then, when the reset signal RST is input from the Z-axis scanning control device 9, the actuator 8 returns the drive signal to the initial value.
By the above operation, the relationship between the scanning point in the sample 50 and each video frame is as shown in FIG. 5G. That is, in each of the video frame periods (T1 to T8 in FIG. 5), an image in a different Z coordinate range is captured for each video frame period. Here, in the present embodiment, the objective lens 7 is moved in the Z direction only during the imaging period of the video frame as described above. Therefore, the occurrence of a Z coordinate range in which no XY coordinates are captured at all is small. In the present embodiment, considering that the diameter of platelets is about 2 μm, by moving the objective lens 7 in the Z coordinate direction as described above, the focal position for each frame is changed in the Z coordinate direction. In the vicinity of the lower road wall, for example, it is moved by 0.2 μm. In addition, the number of video frames to be imaged in one Z-axis scan (the number of video frames between reset signals RST and -1 in the scanning sequence of FIG. 5) is about 10 to several tens of frames.
[0028]
Now, the image processing device 10 repeats the process of capturing and storing each video frame output from the camera 5 and combining and displaying the frames on the display device 11. Here, a timing signal indicating the timing of the TRIGER signal is supplied from the Z-axis scanning control device 9 to the image processing device 10, and the image processing device 10 converts each video frame and its video frame according to the timing signal. The three-dimensional image of the sample 50 is generated by combining or arranging the video frames in accordance with the recognized order, and recognizes the correspondence with the order of the imaged sample 50 in the scanning plane in the Z direction.
[0029]
As a result of the above operations, three-dimensional functional imaging including platelet morphological imaging and membrane protein reconstruction as moving images under conditions equivalent to in-vivo conditions has been realized.
FIG. 6 shows a display example of a three-dimensional image imaged in this manner. FIG. 6a shows platelets in a flow channel viewed from a direction at an angle of 10 degrees with respect to the matrix, and FIG. Fig. 3 shows platelets in a flow channel viewed from the direction. However, in practice, the movement or morphological change of platelets is recorded as a moving image or displayed in real time.
[0030]
In this embodiment, as described above, the shape of the flow path of the sample blood in the flow chamber 20 has a shape that narrows from the blood inflow side to the blood discharge side in the left-right direction as shown in FIG. 4D. Therefore, only by changing the installation position of the flow chamber 20 in the confocal microscope apparatus, it is possible to observe the thrombus generation process due to different wall shear rates and different flow channel widths.
[0031]
Moreover, since the morphology of platelets is imaged via the fluorescently labeled monoclonal antibody against GP IIb / IIIa or GP Ibalpha, the morphology of platelets can be favorably imaged without being disturbed by red blood cells in the bloodstream. .
As described above, according to the present embodiment, the three-dimensional form of platelets in the blood flow is determined using a confocal microscope while the blood flow is passed through the flow path simulating the in-vivo conditions configured using the flow chamber 20. Therefore, it can be observed as a moving image with good spatial resolution and time resolution. Therefore, it is possible to more closely grasp the dynamic morphological change of platelets and the interaction with the matrix, and to perform detailed evaluation of antithrombotic drugs and the like.
[0032]
【The invention's effect】
As described above, according to the present invention, a dynamic morphological change of platelets and an interaction with a matrix can be observed in detail under conditions equivalent to in vivo conditions.
[Brief description of the drawings]
FIG. 1 is a diagram showing a configuration of a thrombus generation process observation device according to an embodiment of the present invention.
FIG. 2 is a diagram schematically showing a basic configuration of a confocal microscope device according to an embodiment of the present invention.
FIG. 3 is a diagram showing a configuration of an in-vivo condition simulation apparatus according to an embodiment of the present invention.
FIG. 4 is a view showing another scanning sequence of the flow chamber according to the embodiment of the present invention;
FIG. 5 is a diagram showing a scanning sequence of the confocal microscope device according to the embodiment of the present invention.
FIG. 6 is a diagram showing an example of three-dimensional imaging of platelets in a bloodstream according to an embodiment of the present invention.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 ... Body part, 2 ... Stage, 3 ... Laser light source, 4 ... Confocal scanner unit, 5 ... Camera, 6 ... Image intensifier, 7 ... Objective lens, 8 ... Actuator, 9 ... Axis scanning control device, 10 ... Image processing device, 11 Display device, 12 Illumination light source, 20 Flow chamber, 21 Base, 22 Glass plate, 23 Flow channel side wall forming sheet, 24 Glass block, 25 Screw, 30 Blood flow pump Reference numeral 31, 31 injector, 32 injector, 33 supply pipe, 34 discharge pipe, 35 collection container, 50 sample, 101 microlens array, 102 pinhole array, 103 condensing lens, 104 Image sensor 105, beam splitter 241 blood supply tunnel 242 blood drain tunnel.

Claims (4)

A flow chamber formed with a flow channel in which a layer of collagen or vWF (von Willebrand factor) is provided on the flow channel wall surface;
A fluid pump for flowing blood into the flow channel of the flow chamber,
A thrombus generation process observation device, comprising: a confocal microscope device that repeatedly and three-dimensionally images blood flowing through the flow channel of the flow chamber. The thrombus generation process observation device according to claim 1,
The thrombus formation process observation device, wherein the flow channel formed in the flow chamber has a plurality of ranges in the flow channel direction where the cross-sectional sizes of the flow channels are different from each other. A method for observing a thrombus formation process for observing a thrombus formation process,
A fluorescently labeled monoclonal antibody against the membrane glycoprotein GP IIb / IIIa of platelets is placed in a flow channel of a flow chamber in which a layer of collagen or vWF (von Willebrand factor) is provided on the channel wall surface. A method for observing a thrombus formation process, wherein blood flowing through a flow path of the flow chamber is repeatedly three-dimensionally imaged by a confocal microscope device while flowing mixed blood. A method for observing a thrombus formation process for observing a thrombus formation process,
A fluorescence-labeled monoclonal antibody against the platelet membrane glycoprotein GP Ibalpha was mixed into the flow channel of a flow chamber in which a layer of collagen or vWF (von Willebrand factor) was provided on the channel wall surface. A method for observing a thrombus formation process, wherein blood flowing through the flow channel of the flow chamber is repeatedly three-dimensionally imaged by a confocal microscope while flowing blood.

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