CN116045878A - Vascular pipe diameter detection equipment - Google Patents
Vascular pipe diameter detection equipment Download PDFInfo
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- CN116045878A CN116045878A CN202310207081.1A CN202310207081A CN116045878A CN 116045878 A CN116045878 A CN 116045878A CN 202310207081 A CN202310207081 A CN 202310207081A CN 116045878 A CN116045878 A CN 116045878A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01B—MEASURING LENGTH, THICKNESS OR SIMILAR LINEAR DIMENSIONS; MEASURING ANGLES; MEASURING AREAS; MEASURING IRREGULARITIES OF SURFACES OR CONTOURS
- G01B21/00—Measuring arrangements or details thereof, where the measuring technique is not covered by the other groups of this subclass, unspecified or not relevant
- G01B21/10—Measuring arrangements or details thereof, where the measuring technique is not covered by the other groups of this subclass, unspecified or not relevant for measuring diameters
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01B—MEASURING LENGTH, THICKNESS OR SIMILAR LINEAR DIMENSIONS; MEASURING ANGLES; MEASURING AREAS; MEASURING IRREGULARITIES OF SURFACES OR CONTOURS
- G01B11/00—Measuring arrangements characterised by the use of optical techniques
- G01B11/08—Measuring arrangements characterised by the use of optical techniques for measuring diameters
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
Abstract
The invention discloses a vascular diameter detection device, which is used for fixing an isolated blood vessel and monitoring the diameter of the blood vessel corresponding to the isolated blood vessel infused with a sample to be detected; the vascular caliber detection device comprises: the isolated blood vessel incubation part is used for incubating the isolated blood vessel and the isolated blood vessel infused with the sample to be detected; the first end of the isolated blood vessel incubation part is provided with a liquid inlet; a liquid outlet is formed in the second end of the isolated blood vessel incubation part; the first pressure adjusting part is used for adjusting the pressure value of the first end of the isolated blood vessel incubation part; the first pressure regulating part is connected with the liquid inlet; the second pressure adjusting part is used for adjusting the pressure value of the second end of the isolated blood vessel incubation part; the second pressure regulating part is connected with the liquid outlet.
Description
Technical Field
The invention relates to the technical field of medical measurement equipment and methods, in particular to blood vessel diameter detection equipment, and a detection method and a detection system for blood vessel diameter change.
Background
The red blood cell substitute has the functions of carrying and releasing oxygen, has the advantages of no need of cross matching, no disease transmission risk and convenient storage and transportation, and has great significance in solving the dilemma faced by the traditional blood transfusion. Hemoglobin-based oxygen carriers (Hemoglobin-based oxygen carriers, HBOCs) are modified by crosslinking, microencapsulation, chemical modification and other methods based on Hemoglobin, and the obtained red blood cell substitute with oxygen carrying function. Up to now, various types of HBOCs products have been developed, representing products including glutaraldehyde polymeric hemoglobin and polyethylene glycol modified hemoglobin.
Since 1989, 7 HBOCs products have been FDA approved for clinical trials II-III. The results show that the incidence rate of hypertension after the patients infuse HBOCs is about 2 times of that of the control group, and the patients are one of side reactions restricting the clinical application of the HBOCs. Hypertension is caused by HBOCs induced vasoactivity (vessel activity), and two main hypotheses are that HBOCs cause vasoactivity, namely, NO is produced by vascular endothelial cells, is a good vasodilator in organisms, and free hemoglobin and HBOCs molecules with smaller particle size can penetrate gaps between endothelial cells and combine with NO to cause vasoconstriction; secondly, low oxygen affinity HBOCs results in premature release of oxygen before reaching hypoxic tissue, with increased oxygen supply to the local arterial wall, causing reflective arteriole contraction. Thus, the vascular activity assay is essential in the design and development phase of HBOCs products.
Currently, methods for detecting HBOCs vascular activity are divided into two types, in vivo and in vitro. In vivo testing requires injection of HBOCs into the veins of animals, which react to vascular activity through arterial blood pressure. The in vitro detection method has the following advantages: (1) saving experimental animals; (2) the sample demand is small; (3) it is more advantageous to explore the mechanism of vascular activity. At present, two in-vitro detection methods are reported in literature, namely, the separation of rat glomeruli by Y, xiong and the like, and the microscopic perfusion and observation of the change of the caliber of an intraglomerular artery (confirmed scale) prove that the novel HBOCs-hemoglobin micro-nano particles have no vascular activity; secondly, the pulmonary artery and aortic vascular rings of the rats are isolated by H.W. Kim et al, and the process can be reversed by vasodilating drugs through perfusion and observation of tension change of the vascular rings, which proves that the vascular activity induced by hemoglobin has dose dependency.
In addition to arterioles and aorta, resistance blood vessels (resistance vessel) play an important role in regulating systemic vascular resistance, and have important significance for researching the vascular activity of HBOCs, but no in-vitro detection method for the vascular activity of HBOCs of resistance blood vessels exists at present.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems existing in the prior art. Therefore, the invention provides a detection device for the change of the vessel diameter; the device improves the existing DMT120CP microvascular pressure-diameter perfusion detection system based on the uniqueness of the product to be detected, so as to be used for evaluating the vasoconstrictor or diastole activity of macromolecular drugs such as HBOCs and the like which cannot permeate the vascular wall; meanwhile, an in-vitro detection method for the HBOCs vascular activity based on the resistance vascular caliber change is also established, and the in-vitro vascular activity detection is completed by recording the resistance vascular caliber change, so that a technical foundation is laid for reducing the vascular activity of the HBOCs and improving the safety of clinical application.
The first aspect of the application discloses a vascular diameter detection device, which is used for fixing the isolated blood vessel and monitoring the diameter of the blood vessel corresponding to the isolated blood vessel infused with a sample to be detected; the vascular caliber detection device comprises:
The isolated blood vessel incubation part is used for incubating the isolated blood vessel and the isolated blood vessel infused with the sample to be detected; the first end of the isolated blood vessel incubation part is provided with a liquid inlet; a liquid outlet is formed in the second end of the isolated blood vessel incubation part;
the first pressure adjusting part is used for adjusting the pressure value of the first end of the isolated blood vessel incubation part; the first pressure regulating part is connected with the liquid inlet;
the second pressure adjusting part is used for adjusting the pressure value of the second end of the isolated blood vessel incubation part; the second pressure regulating part is connected with the liquid outlet.
The isolated vessel incubation portion includes: a bath and a fixed tube; the fixed pipe is positioned in the bath; the two ends of the isolated blood vessel are respectively fixed on the side of the fixed tube;
optionally, the number of the fixing pipes is 2, and the fixing pipes are respectively fixed on the side walls of the first end and the second end of the bath.
The first pressure regulating part comprises a liquid collecting unit connected with the liquid inlet, and the liquid collecting unit is connected with a host to control the pressure value of the first end of the isolated blood vessel incubation part;
optionally, the first pressure adjusting part includes: a syringe pump, an injection unit, and a connector; the first end of the connector is connected with the injection pump, the second end of the connector is connected with the liquid collecting unit filled with buffer solution, and the third end of the connector is connected with the liquid inlet; the injection unit is arranged on the injection pump, a sample to be detected is arranged in the injection unit, and the sample to be detected in the injection unit is infused into the isolated blood vessel through the connector and the liquid inlet sequentially by using the injection pump; controlling the pressure value of the first end of the isolated blood vessel incubation part by changing the pump speed of the injection pump; optionally, the injection unit is configured as a syringe; the liquid collecting unit is arranged as a liquid collecting bottle; the connector is provided with a three-way valve;
Optionally, the second pressure adjusting part comprises a liquid collecting unit connected with the liquid outlet, and the liquid collecting unit is connected with the host to control the pressure value of the second end of the isolated blood vessel incubation part;
the blood vessel diameters corresponding to the isolated blood vessel infused with the sample to be measured comprise: the initial pipe diameter value corresponding to the isolated blood vessel infused with the sample to be tested and the pipe diameter value after incubation of the sample to be tested of the isolated blood vessel infused with the sample to be tested.
The second aspect of the application discloses a method for detecting the change of the vessel diameter, which is implemented by adopting the detection equipment in the first aspect to execute the following detection method:
determining a sample to be tested;
the sample to be detected is infused into the isolated blood vessel based on the blood vessel diameter detection equipment, and an initial pipe diameter value corresponding to the isolated blood vessel infused with the sample to be detected and a pipe diameter value after incubation of the sample to be detected of the isolated blood vessel infused with the sample to be detected are respectively obtained;
and determining the diameter change value of the isolated blood vessel according to the initial pipe diameter value and the pipe diameter value of the sample to be detected after incubation.
The step of obtaining the initial pipe diameter value comprises the following steps:
after the sample to be detected is incubated in the isolated blood vessel for 0 min, determining the uppermost edge line and the lowermost edge line of the target position of the isolated blood vessel, and determining the initial horizontal reference lines of the two isolated blood vessels;
Determining the initial vessel diameter perpendicular to the initial horizontal direction reference line at the target position of the isolated vessel, namely the initial vessel diameter value;
optionally, the step of obtaining the tube diameter value of the sample to be tested after incubation includes:
after the sample to be detected is incubated in the isolated blood vessel for N min, adding a vasoconstrictor solution to the periphery of the isolated blood vessel based on the blood vessel diameter detection equipment, and changing the concentration of the vasoconstrictor solution in the peripheral solution of the isolated blood vessel to a target concentration;
determining the uppermost and lowermost two edge lines of the target position of the isolated blood vessel based on the target concentration of the peripheral solution of the isolated blood vessel, and determining two horizontal reference lines after the treatment of the isolated blood vessel;
determining the diameter of the blood vessel after incubation of the sample to be detected, which is vertical to the treated horizontal direction reference line, at the target position of the isolated blood vessel, namely the diameter value of the blood vessel after incubation of the sample to be detected;
optionally, the vasoconstrictor solution comprises: NE;
optionally, the target concentration range includes: 1X 10 -6 -3×10 -6 M。
Optionally, the sample to be tested includes any one or several of the following: bHb solution, malPEG-bHb solution.
Optionally, the method for determining the diameter variation value of the isolated blood vessel comprises the following steps: and subtracting the pipe diameter value of the sample to be detected after incubation from the initial pipe diameter value.
The method further comprises the steps of: calculating a percent shrinkage of the isolated blood vessel based on the diameter change value; obtaining the activity of the isolated blood vessel infused with the sample to be tested based on the contraction percentage; wherein the shrinkage percentage is larger than or equal to a first threshold value, and a result of large activity of the isolated blood vessel infused with the sample to be detected is obtained; the shrinkage percentage is smaller than or equal to a first threshold value, and a result of small activity of the isolated blood vessel infused with the sample to be detected is obtained;
optionally, the calculation mode of the shrinkage percentage of the isolated blood vessel is as follows: (initial pipe diameter value-pipe diameter value after incubation of sample to be tested)/initial pipe diameter value is 100%.
Optionally, the method further comprises: obtaining a quality classification result of the sample to be detected based on the shrinkage percentage; wherein the shrinkage percentage is larger than or equal to a second threshold value, and a result of bad quality of the sample to be detected is obtained; and the shrinkage percentage is smaller than or equal to a second threshold value, and a result with good quality of the sample to be detected is obtained.
The isolated blood vessel is an isolated blood vessel meeting the requirements and is prepared by the following steps:
Fixing the isolated blood vessel at the fixing tube sides at the two ends in the bath, and pouring a PSS buffer solution into the bath and the isolated blood vessel;
adjusting the pressure values of the liquid inlet and the liquid outlet at two ends of the bath until reaching a target pressure value;
10ml KPSS solution is added into the bath tank to induce vasoconstriction, the stimulation is continued for M min, and the pipe diameter value of the isolated blood vessel and the percentage of the pipe diameter value relative to the initial pipe diameter value of the vascular ring are detected; repeating the operation, and if the percentage difference value of the pipe diameter values of the two adjacent isolated blood vessels is smaller than a third threshold value, obtaining the isolated blood vessels meeting the requirements; otherwise, the result that the isolated blood vessel is not in accordance with the requirement is obtained. The range of the third threshold value is 0-10%.
A third aspect of the present application discloses a detection system for vascular caliber variation, comprising:
an acquisition unit for determining a sample to be measured;
the first processing unit infuses the sample to be detected into the isolated blood vessel based on the blood vessel diameter detection equipment of the first aspect, and respectively obtains an initial pipe diameter value corresponding to the isolated blood vessel infused with the sample to be detected and a pipe diameter value after incubation of the sample to be detected of the isolated blood vessel infused with the sample to be detected;
The second processing unit is used for determining the diameter change value of the isolated blood vessel according to the initial pipe diameter value and the pipe diameter value of the sample to be detected after incubation.
A fourth aspect of the present application discloses a detection device for vascular caliber variation, the device comprising: a memory and a processor;
the memory is used for storing program instructions; the processor is configured to invoke program instructions, which when executed, are configured to perform the method for detecting a change in vessel diameter according to the second aspect.
The application has the following beneficial effects:
1. the application creatively discloses a vascular caliber detection device, which improves the existing DMT120CP microvascular pressure-diameter perfusion detection system based on the uniqueness of a product to be detected, tests a free hemoglobin sample and a polyethylene glycol modified hemoglobin sample, and completes in-vitro vascular activity detection by recording resistance vascular caliber change; the improved device can be used for evaluating vasoconstrictor or diastolic activity of macromolecular drugs such as HBOCs and the like which cannot permeate the vascular wall;
2. the application discloses an innovative detection device for vascular tube diameters based on improvement, and establishes an in vitro detection method for HBOCs vascular activity based on resistance vascular tube diameter change, and by recording resistance vascular tube diameter change, in vitro vascular activity detection is completed, thereby laying a technical foundation for reducing vascular activity of HBOCs and improving safety of clinical application. In the process of detecting the vascular activity in vitro, a method of adding NE is adopted to increase the variation amplitude of the vascular diameter, amplify the vascular activity, facilitate observation and measurement and increase the detection sensitivity. In addition to afferent arterioles and aorta, resistance vessels (resistance vessel) also play an important role in regulating systemic vascular resistance; it is predicted that there must be resistance to vascular involvement in vascular activity by HBOCs. Thus, the present study was intended to establish an in vitro method for evaluating HBOCs-induced vascular activity based on resistance vascular diameter changes. The method is expected to provide a simple method for in vitro evaluation of the HBOCs-induced vascular activity and lay a guarantee for promoting clinical application of the HBOCs.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is apparent that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic view of a vessel diameter detection apparatus according to a first aspect of an embodiment of the present invention;
FIG. 2 is a schematic flow chart of a method for detecting a change in vessel diameter according to a second aspect of the present invention;
FIG. 3 is a schematic flow chart of a system for detecting changes in vessel diameter according to a third aspect of an embodiment of the present invention;
FIG. 4 is a schematic diagram of a detecting device for vascular diameter variation according to a fourth aspect of the present invention;
FIG. 5 is a schematic diagram of a prior art DMT120CP microvascular pressure-diameter perfusion detection system according to an embodiment of the present invention;
FIG. 6 is a graph showing the distribution of vessel diameters before and after equilibrium of mesenteric artery provided by the example of the present invention;
FIG. 7 is a schematic representation of KPSS-stimulated vasoconstrictor restorative activity provided by an embodiment of the present invention;
FIG. 8 is a schematic representation of the enhancement of vasoconstriction by varying concentrations of NE provided by an embodiment of the present invention;
FIG. 9 is a graph showing the molecular size distribution of dynamic light scattering analysis roar for molecular sizes of bHb and MalPEG-bHb provided in the examples of the present invention;
FIG. 10 is a graph of size exclusion chromatography and oxygen saturation dissociation provided by an embodiment of the present invention;
FIG. 11 is a graph showing the variation of vessel diameter with time when PSS, malPEG-bHb and bHb samples are infused into the vessel, respectively, in an in vitro vasoconstrictor activity assay provided by an embodiment of the present invention;
FIG. 12 is a graph showing the variation of the diameter of an isolated blood vessel with the concentration of NE according to an embodiment of the present invention;
in the figure, 11, ex vivo vessels; 12. a liquid inlet; 13. a liquid outlet; 14. a bath; 15. a fixed tube; 21. a syringe pump; 22. a syringe; 23. a three-way valve; 24. a liquid collecting bottle; 3. a microscope; 4. differential adjusting rule.
Detailed Description
In order to enable those skilled in the art to better understand the present invention, the following description will make clear and complete descriptions of the technical solutions according to the embodiments of the present invention with reference to the accompanying drawings. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments according to the invention without any creative effort, are within the protection scope of the invention.
In some of the flows described in the specification and claims of the present invention and in the foregoing figures, a plurality of operations occurring in a particular order are included, but it should be understood that the operations may be performed out of order or performed in parallel, with the order of operations such as 101, 102, etc., being merely used to distinguish between the various operations, the order of the operations themselves not representing any order of execution. In addition, the flows may include more or fewer operations, and the operations may be performed sequentially or in parallel. It should be noted that, the descriptions of "first" and "second" herein are used to distinguish different messages, devices, modules, etc., and do not represent a sequence, and are not limited to the "first" and the "second" being different types.
Fig. 1 is a schematic diagram of a vessel diameter detection device provided in a first aspect of the present invention, and fig. 5 is a schematic diagram of an existing DMT120CP microvascular pressure-diameter perfusion detection system provided in the embodiment of the present invention, specifically, the first aspect discloses a vessel diameter detection device for fixing an isolated vessel and monitoring a vessel diameter corresponding to the isolated vessel into which a sample to be measured has been infused; the vascular caliber detection device comprises:
The isolated blood vessel incubation part is used for incubating the isolated blood vessel and the isolated blood vessel infused with the sample to be detected; the first end of the isolated blood vessel incubation part is provided with a liquid inlet; a liquid outlet is formed in the second end of the isolated blood vessel incubation part; the first pressure adjusting part is used for adjusting the pressure value of the first end of the isolated blood vessel incubation part; the first pressure regulating part is connected with the liquid inlet; the second pressure adjusting part is used for adjusting the pressure value of the second end of the isolated blood vessel incubation part; the second pressure regulating part is connected with the liquid outlet.
The isolated vessel incubation portion includes: a bath and a fixed tube; the fixed pipe is positioned in the bath; the two ends of the isolated blood vessel are respectively fixed on the side of the fixed tube;
optionally, the number of the fixing pipes is 2, and the fixing pipes are respectively fixed on the side walls of the first end and the second end of the bath. The fixed tube is a glass tube;
the first pressure regulating part comprises a liquid collecting unit connected with the liquid inlet, and the liquid collecting unit is connected with a host to control the pressure value of the first end of the isolated blood vessel incubation part; optionally, the first pressure adjusting part includes: a syringe pump, an injection unit, and a connector; the first end of the connector is connected with the injection pump, the second end of the connector is connected with the liquid collecting unit filled with buffer solution, and the third end of the connector is connected with the liquid inlet; the injection unit is arranged on the injection pump, a sample to be detected is arranged in the injection unit, and the sample to be detected in the injection unit is infused into the isolated blood vessel through the connector and the liquid inlet sequentially by using the injection pump; by varying the injection The pump speed of the jet pump controls the pressure value of the first end of the isolated blood vessel incubation part; optionally, the injection unit is configured as a syringe; the liquid collecting unit is arranged as a liquid collecting bottle; the connector is provided with a three-way valve; a light source is arranged at the upper end of the bath, and gas (95% O) is continuously introduced into the bath 2 +5%CO 2 ) The bath temperature was set at 37 ℃.
Optionally, the second pressure adjusting part comprises a liquid collecting unit connected with the liquid outlet, and the liquid collecting unit is connected with the host to control the pressure value of the second end of the isolated blood vessel incubation part;
the blood vessel diameters corresponding to the isolated blood vessel infused with the sample to be measured comprise: the initial pipe diameter value corresponding to the isolated blood vessel infused with the sample to be tested and the pipe diameter value after incubation of the sample to be tested of the isolated blood vessel infused with the sample to be tested.
The DMT120CP measuring system is schematically shown in FIG. 5, wherein a bath unit is a core component, and two ends of an isolated blood vessel are respectively fixed on a glass tube by nylon ropes and are placed in the bath. To fully simulate physiological conditions, PSS buffer solution was added to the bath, and gas (95% O) was continuously introduced 2 +5%CO 2 ) The bath temperature was set at 37 ℃. In-vitro blood vessels are PSS buffer solution, and normal flow of the buffer solution is realized by adjusting the pressure difference between the P1 end and the P2 end. The in vitro blood vessel is PSS solution, and the bath tank is small molecular medicine to be detected; according to literature reports, DMT120CP system is generally used to monitor the effect of small molecule drugs on the structure and function of isolated blood vessels, and because small molecule drugs are easy to permeate blood vessels to play a role, the monitoring can be performed by changing the solution outside the isolated blood vessels, namely, directly adding small molecule drugs with corresponding concentration into the bath.
In this example, the DMT120CP system was used to monitor the effect of HBOCs on isolated microvasculature, and by monitoring the diameter change, HBOCs-induced vascular activity was characterized. Based on the physical and chemical properties of HBOCs samples, the original DMT120CP system using method is not suitable for detecting the vascular activity, and the main reason is that the particle size of HBOCs is usually larger than 5nm and is difficult to penetrate the wall of a micro blood vessel, so that the operation method of the DMT120CP system needs to be improved, the HBOCs samples are added from the inside of the blood vessel, and the change of the external diameter of the blood vessel is monitored. Before the HBOCs sample is introduced into the micro-blood vessel, PSS buffer solution is required to be introduced for balancing, bubbles are easily generated during liquid exchange, and the measurement of the diameter of the blood vessel is affected, so that a three-way valve device is added at the liquid inlet of the DMT120CP system, the whole flow channel is filled with the HBOCs sample before the PSS buffer solution and the HBOCs sample are exchanged and poured, so that air is ensured to be removed, and then the three-way valve is regulated to connect the flow channel of the HBOCs to the inlet of the micro-blood vessel. And (3) injecting a sample by adopting a micro-injection pump, and controlling the pressure at two ends of the blood vessel by changing the pump speed of the micro-injection pump. As shown in fig. 1, the in-vitro blood vessel is the red blood to be measured and represents an oxygen carrier, the bath tank is the PSS solution, the P2 end is connected with the control pressure of the host, and the P1 end controls the pressure by adjusting the pump speed of the injection pump; capturing the outer diameter edge of the isolated blood vessel through high-resolution microscope and computer video imaging, and monitoring the diameter of the isolated blood vessel to generate diameter data. At this time, the isolated blood vessel is the hemoglobin oxygen carrier to be detected, and the bath is the PSS solution. The vascular diameter detection device also comprises a differential regulating ruler, wherein the differential regulating ruler is arranged at the upper end of the bath wall of the bath; the differential adjusting ruler is mainly used for adjusting the horizontal direction and the vertical direction of the two fixed pipes and the distance between the two fixed pipes, namely the distance which can be detected in the middle of the isolated blood vessel.
Fig. 2 is a schematic flowchart of a method for detecting a change in a vessel diameter according to a second aspect of the present invention, specifically, the following detection method is performed by using the detection device according to the first aspect, and the method includes the following steps:
101: determining a sample to be tested;
in one embodiment, the sample to be tested comprises any one or more of the following: bHb solution, malPEG-bHb solution. Wherein bHb (bovine hemoglobin) is bovine hemoglobin, and bHb is purified from bovine whole blood according to existing methods. The steps are briefly described as follows: firstly, centrifuging collected bovine blood, removing supernatant, and washing with 1.6% NaCl and 0.9% NaCl in sequence to obtain precipitated red blood cells; then, adding hypotonic dissolved blood into the precipitated red blood cells, stirring under the condition of nitrogen and 4 ℃, and releasing hemoglobin from the red blood cells; finally, subjecting the hemoglobin-dissolved blood to microfiltration (0.22 μm), chromatography (Q anion chromatographic column) and ultrafiltration (30 kDa) respectively to obtain purified bHb, and freezing in a refrigerator at-80deg.C for use. The preparation method of mPEG-Mal modification bHb (MalPEG-bHb) is as follows: bHb, 2-IT and mPEG-Mal were weighed in a molar ratio of 1:10:20, dissolved and dispersed in PBS buffer (pH 7.4), and the reaction system was left to stir slowly at 4 ℃. After 14 hours, the reaction was completed, and the reaction system was purified by using an ultrafiltration centrifuge tube having a molecular weight cut-off of 50kDa to remove mPEG-Mal and bHb which did not participate in the reaction, and the centrifugation condition was 3000g for 30 minutes.
In one embodiment, bHb and MalPEG-bHb are characterized as follows: size exclusion chromatography: samples were analyzed using a chromatographic column Superdex 200, using a protein purification system. The column was equilibrated thoroughly with 10mM phosphate buffer solution (pH 7.0), and after loading, the sample was eluted at a flow rate of 0.05 mL/min, the detection wavelength was 280nm, and the elution profile was recorded.
Oxygen dissociation curve determination: the sample was dispersed into P50 buffer (4 mL) at a bHb concentration of 0.75 mg/mL. Sample P50 was measured using a blood-2018 erythrocyte oxygen carrying/releasing analyzer. Calculating the corresponding volume of 3 mg hemoglobin according to the concentration of the sample, mixing the sample with 4mL of P50 buffer solution, adding the mixture into a sample cell, introducing air to saturate the hemoglobin with oxygen, introducing nitrogen at 37 ℃ to start drawing and recording an oxygen dissociation curve, and obtaining the P50 value from the oxygen dissociation curve.
Colloid osmotic pressure measurement: the sample colloid osmotic pressure value was measured by colloid osmotic pressure meter OSMOMAT 050. 200 mu L of 40mg/mL sample is injected into the measuring cell through the plug by using a special injector, the indicator light is on, the instrument starts automatic measurement, the result is recorded, and the measurement is repeated three times.
And (3) measuring viscosity: the viscosity of the sample was measured using a rheometer. Setting the temperature to 37 ℃ and adjusting the shearing rate range to 75 s -1 The bHb concentration of the sample to be measured is adjusted to 40mg/mL, the experimental result is recorded, and the measurement is repeated three times.
Molecular particle size determination: the particle hydration diameter in the sample was measured by a particle size analyzer. And (3) adjusting the concentration of the sample bHb to be measured to be 0.4mg/mL, adding the sample into a sample cell, automatically measuring by an instrument, repeatedly measuring for three times, and recording the result.
In one embodiment, experimental data are expressed as mean ± SEM. All data were analyzed using a normalization test and a variance alignment test. Statistical analysis was performed using IBM SPSS Statistics system 26. The differences in different concentrations of NE were analyzed using analysis of variance (ANOVA). The comparison between each group adopts single factor independent analysis of variance. * P <0.05, < P <0.01 is statistically significant.
102: the sample to be detected is infused into the isolated blood vessel based on the blood vessel diameter detection equipment, and an initial pipe diameter value corresponding to the isolated blood vessel infused with the sample to be detected and a pipe diameter value after incubation of the sample to be detected of the isolated blood vessel infused with the sample to be detected are respectively obtained;
in one embodiment, the step of obtaining the initial pipe diameter value comprises:
after the sample to be detected is incubated in the isolated blood vessel for 0 min, determining the uppermost edge line and the lowermost edge line of the target position of the isolated blood vessel, and determining the initial horizontal reference lines of the two isolated blood vessels;
Determining the initial vessel diameter perpendicular to the initial horizontal direction reference line at the target position of the isolated vessel, namely the initial vessel diameter value;
optionally, the step of obtaining the tube diameter value of the sample to be tested after incubation includes:
after the sample to be detected is incubated in the isolated blood vessel for N min, adding a vasoconstrictor solution to the periphery of the isolated blood vessel based on the blood vessel diameter detection equipment, and changing the concentration of the vasoconstrictor solution in the peripheral solution of the isolated blood vessel to a target concentration; determining the uppermost and lowermost two edge lines of the target position of the isolated blood vessel based on the target concentration of the peripheral solution of the isolated blood vessel, and determining two horizontal reference lines after the treatment of the isolated blood vessel; determining the diameter of the blood vessel after incubation of the sample to be detected, which is vertical to the treated horizontal direction reference line, at the target position of the isolated blood vessel, namely the diameter value of the blood vessel after incubation of the sample to be detected;
optionally, the vasoconstrictor solution comprises: NE; optionally, the targetThe concentration range includes: 1X 10 -6 -3×10 -6 M。
In one embodiment, the target locations are the same, i.e., only the diameter of the vessel at a fixed location is selected each time, and the diameter at the fixed location is monitored during the vasoactivity measurement; each experiment only has one blood vessel, only has one initial diameter, and the initial diameters of each experiment can be different, but the blood vessels of the same position and the same grade are ensured to be taken each time. The diameter of the isolated blood vessel is all the diameter data generated automatically by capturing the outer diameter edge of the blood vessel by a high resolution microscope.
In one embodiment, the ex vivo blood vessel is a satisfactory ex vivo blood vessel prepared by:
fixing the isolated blood vessel at the fixing tube sides at the two ends in the bath, and pouring a PSS buffer solution into the bath and the isolated blood vessel; saline buffer (PSS): naCl 129.99 mM, KCl 4.69 mM, KH 2 PO4 1.18 mM, MgSO 4 ·7H 2 O 1.18 mM, NaHCO 3 14.88 mM, Glucose 5.55 mM, EDTA 0.029 mM, CaCl 2 1.60 mM 。
Adjusting the pressure values of the liquid inlet and the liquid outlet at two ends of the bath until reaching a target pressure value;
10ml KPSS solution is added into the bath tank to induce vasoconstriction, the stimulation is continued for M min, and the pipe diameter value of the isolated blood vessel and the percentage of the pipe diameter value relative to the initial pipe diameter value of the vascular ring are detected; repeating the operation, and if the percentage difference value of the pipe diameter values of the two adjacent isolated blood vessels is smaller than a third threshold value, obtaining the isolated blood vessels meeting the requirements; otherwise, the result that the isolated blood vessel is not in accordance with the requirement is obtained. The range of the third threshold value is 0-10%.
In one embodiment, the mesenteric artery is a typical resistance vessel, and the rat mesenteric artery diameter distribution ranges from 140.+ -.60. Mu.m, and the study is intended to use the rat mesenteric artery as a subject. The isolated blood vessel is an isolated mesenteric artery vascular ring, and the specific preparation method comprises the following steps: male SD rats were euthanized, and after laparotomy, intestinal tissue was rapidly removed, placed in a PSS solution at 4deg.C, and purged with 95% O 2 And 5% CO 2 And (3) mixing the gases. Cutting a section of intestinal tissue, fixing the intestinal tissue in a black culture dish by using a pin, peeling connective tissue and adipose tissue around the mesenteric artery under a split microscope, selecting a 2-3-level mesenteric artery, and cutting a complete vascular ring of about 2mm without branches and cracks for later use. The experiment adopts SPF grade SD rats with the age of 3-8 weeks and the weight of 220-260 g, which are purchased from Viton Lihua (Beijing) biotechnology Co. Most of the blood vessels that have an influence on the flow resistance of peripheral blood include arterioles, and arteriole branch vessels are collectively called resistance vessels. Blood flowing in the vascular system can cause contraction of smooth muscle of the vessel wall, change of blood vessel pressure, and thus cause resistance change due to external influence, which occurs mostly in arterioles, particularly arterioles. Arterioles and arterioles contract and relax, which can significantly affect blood flow in organs and tissues. The maintenance of normal blood pressure depends to some extent on the resistance of the peripheral vascular arterioles and arterioles to blood flow, i.e. peripheral resistance. Also known as the pre-capillary resistance vessel, because they are located before the capillaries. The mesenteric artery (mesenteric artery) of the vertebrate runs from the dorsal aorta in the mesentery, distributed in the arteries of the digestive tract. The superior mesenteric artery and the inferior mesenteric artery are divided. The former is mainly distributed on the left side of the small intestine, and the latter is distributed on the colon, large intestine, cloaca, etc.
Before an experiment, calibrating a tension sensor, a pressure sensor and a microscope of a DMT120CP system; both ends of the vascular ring were fixed with nylon ropes on glass tubes at the liquid inlet end (P1) and the liquid outlet end (P2) in the bath of the system, both ends were slowly pressurized at the same time, and 10ml of PSS solution was put into the bath and slowly heated to 37 ℃. The computer software was turned on and the recording was started, giving the vessel a certain initial perfusion pressure (10 mmHg), and then raising the perfusion pressure at both ends of the vessel to the target value (60 mmHg) at a rate of 10mmHg every 5 min. 95% O is required to be introduced into the whole experiment bath 2 And 5% CO 2 。
The internal and external solutions of the microvessels are PSS buffer solutions, the initial pressure values of the P1 end and the P2 end are respectively adjusted to be 10mmHg, the pressure at the two ends is adjusted by adopting a speed gradient of increasing 10mmHg every 5min until the target pressure value is reached, the balance is carried out for 60min, and the target pressure is 60mmHg for the mesenteric artery of the rat; as shown in fig. 6.
After the perfusion pressure rises to the target value required by the experiment, the PSS solution is replaced, 10ml KPSS is added to induce the vasoconstriction, the vasoconstriction is continuously stimulated for 10min, the operation is repeated, obvious constriction is carried out twice, the difference of the contraction amplitude is smaller than 10%, and the vascular ring tissue structure is considered to be complete, and the contraction function is normal; as shown in fig. 7.
Sequentially and cumulatively adding NE (norepinephrine) into the bath tank to ensure that the concentration of the NE in the bath tank is 1×10 -6 M、2*10 -6 M and 3 x 10 -6 M, dripping for 3min each time, recording the diameters of blood vessels, changing the solution in the bath to be PSS solution, and starting the subsequent experiment. This step is to determine the appropriate NE concentration; as shown in fig. 8.
103: determining the diameter change value of the isolated blood vessel according to the initial pipe diameter value and the pipe diameter value of the sample to be detected after incubation;
optionally, the method for determining the diameter variation value of the isolated blood vessel comprises the following steps: and subtracting the pipe diameter value of the sample to be detected after incubation from the initial pipe diameter value. The scheme is used for constructing a vascular activity detection device based on isolated rat mesenteric resistance arteries, can be used for in-vitro evaluation of the vascular activity of HOBCs blood substitutes, and can be used for calculating the vascular contraction percentage and reflecting the size of the vascular activity by measuring the change of the vascular diameter through experimental verification. The in vitro detection method is more beneficial to the deep research and discussion of the mechanism and elimination mechanism of the vascular activity by controlling a single variable due to the fact that the in vivo complex physiological environment is separated.
In one embodiment, the method further comprises: calculating a percent shrinkage of the isolated blood vessel based on the diameter change value; obtaining the activity of the isolated blood vessel infused with the sample to be tested based on the contraction percentage; wherein the shrinkage percentage is larger than or equal to a first threshold value, and a result of large activity of the isolated blood vessel infused with the sample to be detected is obtained; the shrinkage percentage is less than or equal to a first threshold value, and the node with small activity of the isolated blood vessel infused with the sample to be tested is obtained And (5) fruits. Notably, the most ideal case for blood substitutes is the absence of vascular activity, i.e. vasoconstriction is the same as in the negative control group (PSS); therefore, the first threshold is not limited to be fixed, and when the first threshold is 0, the in vitro vascular activity is small, that is, no vascular activity exists, and the effect is the best, that is, the aim is to construct the HBOCs blood substitute without vascular activity, and when the first threshold is 0, the most ideal is the case. However, due to the specificity of the isolated blood vessel itself, the isolated blood vessel was not substantially contracted to 0, and as shown in FIG. 12A, when NE was added as a vasoconstrictor to the bath, the negative control group (PSS) also contracted the isolated blood vessel, and the blood vessel contraction was not 0. Thus, the first threshold will typically be greater than a certain value of 0. The method for detecting the vascular activity comprises the following steps: regulating the flow rate of the three-way valve connector and the microinjection pump, maintaining the pressure of the inlet end of the microvasculature between 60 and 65mmHg, starting to introduce the sample to be detected (MalPEG-bHb and bHb), starting to time when the sample is full of the whole blood vessel, regulating the position of the three-way valve connector, closing the microinjection pump, stopping introducing the sample to be detected, incubating the sample to be detected and the microvasculature for 15min, gradually adding the NE solution into the bath tank to ensure that the concentration gradient of the NE solution rises (1 multiplied by 10) -6 ~3×10 -6 M), the change in microvascular diameter was recorded.
After the experiment is finished, verifying the reliability of the experimental data, eluting the solution in the bath, dripping 10ml of KPSS solution, observing whether the blood vessel is contracted or not, and if the difference between the contraction amplitude and the KPSS first induction is less than 10%, proving that the experimental data are reliable and effective. High potassium physiological saline buffer (KPSS): naCl 74.76. 74.76 mM, KCl 59.95. 59.95 mM, KH 2 PO 4 1.18 mM, MgSO 4 ·7H 2 O 1.18 mM, NaHCO 3 14.88 mM, Glucose 5.55 mM, EDTA 0.029 mM, CaCl 2 1.60 mM. Optionally, the calculation mode of the shrinkage percentage of the isolated blood vessel is as follows: (initial pipe diameter value-pipe diameter value after incubation of sample to be tested)/initial pipe diameter value is 100%.
In one embodiment, the method further comprises: obtaining a quality classification result of the sample to be detected based on the shrinkage percentage; wherein the shrinkage percentage is larger than or equal to a second threshold value, and a result of bad quality of the sample to be detected is obtained; and the shrinkage percentage is smaller than or equal to a second threshold value, and a result with good quality of the sample to be detected is obtained. Notably, the most ideal case for blood substitutes is the absence of vascular activity, i.e. vasoconstriction is the same as in the negative control group (PSS); therefore, the second threshold is not limited in this case, and when the second threshold is 0, the shrinkage percentage is 0, and the quality of the sample to be measured is the best, that is, the objective is to construct an HBOCs blood substitute without vascular activity, and when the second threshold is 0, the best condition is obtained. As shown in fig. 12A, when NE is added to the bath as a vasoconstrictor, the negative control group (PSS) also constricts the isolated blood vessel, and the vasoconstrictor is not 0, because of the specificity of the isolated blood vessel itself, in which case the isolated blood vessel constriction percentage does not substantially reach 0, as the principle of determining the magnitude of the isolated blood vessel activity based on the constriction percentage. Therefore, the second threshold will typically be greater than a certain value of 0.
Fig. 3 is a schematic flow chart of a detection system for vascular diameter variation according to a third aspect of the present invention, including:
an acquisition unit 301 for determining a sample to be measured;
the first processing unit 302 infuses the sample to be tested into the isolated blood vessel based on the blood vessel diameter detection device of the first aspect, and respectively obtains an initial tube diameter value corresponding to the isolated blood vessel infused with the sample to be tested and a tube diameter value after incubation of the sample to be tested of the isolated blood vessel infused with the sample to be tested;
the second processing unit 303 is configured to determine a diameter variation value of the isolated blood vessel according to the initial tube diameter value and the tube diameter value of the sample to be tested after incubation;
a system for detecting a change in vessel diameter, comprising a computer program which, when executed by a processor, implements the method for detecting a change in vessel diameter according to the second aspect.
Fig. 4 is a schematic diagram of an apparatus for detecting a change in vessel diameter according to a fourth aspect of the present invention, where the apparatus includes: a memory and a processor; the memory is used for storing program instructions; the processor is used for calling program instructions, and when the program instructions are executed, the processor is used for executing the vascular caliber detection device.
FIG. 6 is a graph showing the distribution of vessel diameters before and after equilibrium of mesenteric artery provided by the example of the present invention; wherein, FIG. 6A is a freshly intercepted unbalanced mesenteric artery vessel diameter distribution in the range of 110-160 μm; FIG. 6B is a graph showing that after balancing is completed, the pressure at two ends of a blood vessel is regulated to be 60mmHg, and the distribution of the diameters of mesenteric artery blood vessels under the in-vivo physiological condition is simulated, wherein the range is 150-200 mu m; FIG. 6B is a quarter-turn plot of the vessel diameter distribution with corresponding minima, 25%, median, 75%, maximum and mean values of 112.4 μm, 134.1 μm, 145.0 μm, 156.5 μm, 168.4 μm and 143.8 μm, respectively, before balancing, 150.7 μm, 170.9 μm, 184.2 μm, 197.8 μm, 220.0 μm and 184.9 μm, respectively, after balancing.
FIG. 7 is a schematic representation of KPSS-stimulated vasoconstrictor restorative activity provided by an embodiment of the present invention; after the mesenteric artery blood vessel is balanced, the KPSS solution is replaced to stimulate the blood vessel to shrink, the activity of the blood vessel to shrink is recovered, and the integrity of endothelial cells is verified. As shown in FIG. 7, after KPSS is added into the bath, the blood vessel is contracted from 185 μm to about 100 μm, after KPSS is replaced, the diameter of the blood vessel is restored to an equilibrium state, the operation is repeated twice, the difference between the diameters of the blood vessel contracted by two times is not more than 10%, and after KPSS is replaced, the diameter of the blood vessel is restored to an initial state, so that the blood vessel is proved to have perfect function and can be used for subsequent experiments.
FIG. 8 is a schematic representation of the enhancement of vasoconstriction by varying concentrations of NE provided by an embodiment of the present invention; wherein, fig. 8A is a graph showing the change of the vessel diameter with time after PSS solution and bHb solution are respectively introduced into the mesenteric artery vessel at the time of the blood vessel activity detection. When the PSS solution perfuses the vessel, the vessel diameter remains unchanged, and after bHb perfusion, the contraction amplitude remains within 3%. FIG. 8B shows the change in the percent shrinkage of vascular diameter with NE concentration after infusion of the PSS solution into the mesenteric artery vessel by addition of NE to the bath. When the concentration of NE ranges from 1 x 10 -6 M~1*10 -4 At M, the blood vessel appears to shrink obviously. On the basis of whichThe NE concentration was further explored above. As shown in fig. 8C, when the NE concentration reaches 3×10 -6 At M, the percent of vessel diameter contraction and NE concentration were 1 x 10 -4 M was close, indicating that the blood vessels were at a concentration of NE of 3 x 10 -6 M reaches the shrink threshold. Thus, 1 x 10 is selected -6 M~3*10 -6 The concentration gradient of NE in the M range was increased to amplify the vasoconstrictor effect.
FIG. 9 is a graph showing the molecular size distribution of dynamic light scattering analysis roar for molecular sizes of bHb and MalPEG-bHb provided in the examples of the present invention; wherein, fig. 9A is a dynamic light scattering measurement bHb molecular particle size; FIG. 9B is a dynamic light scattering measurement of molecular particle size of MalPEG-bHb; as shown in fig. 9 and table 1, the molecular particle sizes of bHb and MalPEG-bHb exhibited a single peak, indicating that the purity of the samples was higher. The molecular particle size of bHb is 4.7+/-1. 1 nm, and after PEG modification, the molecular particle size of MalPEG-bHb is 15.5+/-1.4 nm, and the molecular particle size is obviously increased. Table 1 shows the physicochemical properties of bHb and MalPEG-bHb;
FIG. 10 is a graph of size exclusion chromatography and oxygen saturation dissociation provided by an embodiment of the present invention; as shown in FIG. 10A, the molecular exclusion chromatography was performed on bHb and MalPEG-bHb. bHb and MalPEG-bHb showed a single elution peak after elution, indicating higher sample purity. The peak positions of the two elution peaks are 1.78mL and 1.37mL respectively, and compared with bHb, the peak positions of the MalPEG-bHb elution peaks are obviously shifted to the left, which proves that the apparent relative molecular weight of bHb is obviously increased by PEG modification. The oxygen saturation curve can be used to measure the oxygen carrying-releasing function of hemoglobin, and the oxygen partial pressure (P) at 50% hemoglobin oxygen saturation can be obtained from the curve 50 ). As shown in fig. 10B, P of bHb 50 23.73mmHg, modified with PEG, P of MalPEG-bHb 50 The oxygen affinity was increased at 6.99 mmHg.
FIG. 11 is a graph showing the variation of vessel diameter with time when PSS, malPEG-bHb and bHb samples are infused into the vessel, respectively, in an in vitro vasoconstrictor activity assay provided by an embodiment of the present invention; which is a kind ofFIG. 11A shows the change of the vessel diameter with time before and after the infusion of the PSS solution; fig. 11B shows the change in vessel diameter with time before and after bHb infusion, and fig. 11C shows the change in vessel diameter with time before and after MalPEG-bHb infusion. Through the balancing stage, the diameter of the blood vessel of the three experiments slowly rises to reach a plateau value, and the process is mainly used for simulating the physiological condition of the isolated blood vessel in the rat body. The subsequent addition of KPSS stimulated vasoconstriction, restoring vasoconstrictor activity and simultaneously detecting vascular endothelial cell integrity. The vessel was then perfused with sample and added to the bath gradient 1 x 10 -6 ~4*10 -6 M NE amplifies vasoconstrictor active effects. As shown in FIG. 11, when the NE concentration in the bath is 1×10 -6 In the M process, the blood vessel contraction of the perfused PSS solution is not obvious, the contraction amplitude is about 2 mu M, the blood vessel of the perfused MalPEG-bHb solution is slightly contracted, the diameter is contracted from 200 mu M to 190 mu M, the contraction amplitude is 10 mu M, the blood vessel of the perfused bHb solution is obviously contracted, the diameter of the blood vessel is contracted from 200 mu M to 170 mu M, the contraction amplitude is 30 mu M, and compared with MalPEG-bHb, the blood vessel contraction rate is increased; when the concentration of NE in the bath is 2 x 10 -6 M is increased to 4 x 10 -6 In M, the blood vessels perfused with the three samples are obviously contracted, and the contraction amplitude is gradually increased along with the increase of the concentration gradient of the NE, when the concentration of the NE in the bath is 4 x 10 -6 At M, the vasoconstriction amplitude of the perfused three samples reached unity, about 70 μm, all reaching the constriction threshold.
FIG. 12 is a graph showing the variation of the diameter of an isolated blood vessel with the concentration of NE according to an embodiment of the present invention; wherein fig. 12A is the percentage of vessel diameter relative to the initial diameter after mesenteric artery vessels were perfused with PSS, malPEG-bHb and bHb samples, respectively. When the concentration of NE in the bath is 1 x 10 -6 In M, the percent diameter shrinkage of the perfused vessels of the three groups of PSS, malPEG-bHb and bHb, relative to the initial diameter of the vessels, was 0.5%,3% and 9%, respectively, and the magnitude of the vasoconstriction after bHb perfusion was significantly different from that of PSS and MalPEG-bHb (P <0.05 A) is provided; when the concentration of NE in the bath is 2 x 10 -6 At M, the percent diameter shrinkage of the perfused vessels was 8%,12% and 25% for the three groups of PSS, malPEG-bHb and bHb, respectively, and there was a significant difference between the bHb group and the PSS and MalPEG-bHb groups (P<0.05 A) is provided; when the concentration of NE in the bath is 3 x 10 -6 M, three blood vessels shrink by nearly the same magnitude, where the primary factor affecting vasoconstriction is from NE and not the constrictive effect of the sample on the blood vessel. Fig. 12B is a photograph of blood vessels taken during a blood vessel activity detection experiment.
The results of the verification of the present verification embodiment show that assigning an inherent weight to an indication may moderately improve the performance of the present method relative to the default settings.
It will be clear to those skilled in the art that, for convenience and brevity of description, specific working procedures of the above-described systems, apparatuses and units may refer to corresponding procedures in the foregoing method embodiments, which are not repeated herein.
In the several embodiments provided in this application, it should be understood that the disclosed systems, apparatuses, and methods may be implemented in other ways. For example, the apparatus embodiments described above are merely illustrative, e.g., the division of the units is merely a logical function division, and there may be additional divisions when actually implemented, e.g., multiple units or components may be combined or integrated into another system, or some features may be omitted or not performed. Alternatively, the coupling or direct coupling or communication connection shown or discussed with each other may be an indirect coupling or communication connection via some interfaces, devices or units, which may be in electrical, mechanical or other form.
The units described as separate units may or may not be physically separate, and units shown as units may or may not be physical units, may be located in one place, or may be distributed on a plurality of network units. Some or all of the units may be selected according to actual needs to achieve the purpose of the solution of this embodiment.
In addition, each functional unit in the embodiments of the present invention may be integrated in one processing unit, or each unit may exist alone physically, or two or more units may be integrated in one unit. The integrated units may be implemented in hardware or in software functional units.
Those of ordinary skill in the art will appreciate that all or part of the steps in the various methods of the above embodiments may be implemented by a program to instruct related hardware, the program may be stored in a computer readable storage medium, and the storage medium may include: read Only Memory (ROM), random access Memory (RAM, random Access Memory), magnetic or optical disk, and the like.
Those of ordinary skill in the art will appreciate that all or a portion of the steps in implementing the methods of the above embodiments may be implemented by a program to instruct related hardware, where the program may be stored in a computer readable storage medium, where the storage medium may be a read only memory, a magnetic disk or optical disk, etc.
While the foregoing describes a computer device provided by the present invention in detail, those skilled in the art will appreciate that the foregoing description is not meant to limit the invention thereto, as long as the scope of the invention is defined by the claims appended hereto.
Claims (10)
1. The vascular diameter detection equipment is used for fixing the isolated blood vessel and monitoring the diameter of the blood vessel corresponding to the isolated blood vessel infused with the sample to be detected; the vascular caliber detection device comprises:
the isolated blood vessel incubation part is used for incubating the isolated blood vessel and the isolated blood vessel infused with the sample to be detected; the first end of the isolated blood vessel incubation part is provided with a liquid inlet; a liquid outlet is formed in the second end of the isolated blood vessel incubation part;
the first pressure adjusting part is used for adjusting the pressure value of the first end of the isolated blood vessel incubation part; the first pressure regulating part is connected with the liquid inlet;
The second pressure adjusting part is used for adjusting the pressure value of the second end of the isolated blood vessel incubation part; the second pressure regulating part is connected with the liquid outlet.
2. The vascular caliber detection device according to claim 1, wherein the ex vivo vascular incubation portion comprises: a bath and a fixed tube; the fixed pipe is positioned in the bath; the two ends of the isolated blood vessel are respectively fixed on the side of the fixed tube.
3. The vascular caliber detection device according to claim 1, wherein the first pressure regulating part comprises a liquid collecting unit connected with the liquid inlet, and the liquid collecting unit is connected with a host to control the pressure value of the first end of the isolated vascular incubation part;
optionally, the first pressure adjusting part includes: a syringe pump, an injection unit, and a connector; the first end of the connector is connected with the injection pump, the second end of the connector is connected with the liquid collecting unit filled with buffer solution, and the third end of the connector is connected with the liquid inlet; the injection unit is arranged on the injection pump, a sample to be detected is arranged in the injection unit, and the sample to be detected in the injection unit is infused into the isolated blood vessel through the connector and the liquid inlet sequentially by using the injection pump; controlling the pressure value of the first end of the isolated blood vessel incubation part by changing the pump speed of the injection pump; optionally, the second pressure adjusting part comprises a liquid collecting unit connected with the liquid outlet, and the liquid collecting unit is connected with the host to control the pressure value at the second end of the isolated blood vessel incubation part.
4. The vascular caliber detection device according to claim 1, wherein the blood vessel diameter corresponding to the isolated blood vessel into which the sample to be detected has been infused includes: the initial pipe diameter value corresponding to the isolated blood vessel infused with the sample to be tested and the pipe diameter value after incubation of the sample to be tested of the isolated blood vessel infused with the sample to be tested.
5. A method for detecting a change in vessel diameter, the following method being performed using the detection apparatus according to any one of claims 1 to 4:
determining a sample to be tested;
the sample to be detected is infused into the isolated blood vessel based on the blood vessel diameter detection equipment, and an initial pipe diameter value corresponding to the isolated blood vessel infused with the sample to be detected and a pipe diameter value after incubation of the sample to be detected of the isolated blood vessel infused with the sample to be detected are respectively obtained;
and determining the diameter change value of the isolated blood vessel according to the initial pipe diameter value and the pipe diameter value of the sample to be detected after incubation.
6. The method of claim 5, wherein the step of obtaining the initial tube diameter value comprises:
after the sample to be detected is incubated in the isolated blood vessel for 0 min, determining the uppermost edge line and the lowermost edge line of the target position of the isolated blood vessel, and determining the initial horizontal reference lines of the two isolated blood vessels;
Determining the initial vessel diameter perpendicular to the initial horizontal direction reference line at the target position of the isolated vessel, namely the initial vessel diameter value;
optionally, the step of obtaining the tube diameter value of the sample to be tested after incubation includes:
after the sample to be detected is incubated in the isolated blood vessel for N min, adding a vasoconstrictor solution to the periphery of the isolated blood vessel based on the blood vessel diameter detection equipment, and changing the concentration of the vasoconstrictor solution in the peripheral solution of the isolated blood vessel to a target concentration;
determining the uppermost and lowermost two edge lines of the target position of the isolated blood vessel based on the target concentration of the peripheral solution of the isolated blood vessel, and determining two horizontal reference lines after the treatment of the isolated blood vessel;
and determining the diameter of the blood vessel after incubation of the sample to be detected, which is vertical to the treated horizontal reference line, at the target position of the isolated blood vessel, and namely the diameter value of the blood vessel after incubation of the sample to be detected.
7. The method for detecting a change in vessel diameter according to claim 5, further comprising: calculating a percent shrinkage of the isolated blood vessel based on the diameter change value; obtaining the activity of the isolated blood vessel infused with the sample to be tested based on the contraction percentage; wherein the shrinkage percentage is larger than or equal to a first threshold value, and a result of large activity of the isolated blood vessel infused with the sample to be detected is obtained; the shrinkage percentage is smaller than or equal to a first threshold value, and a result of small activity of the isolated blood vessel infused with the sample to be detected is obtained;
Optionally, the method further comprises: obtaining a quality classification result of the sample to be detected based on the shrinkage percentage; wherein the shrinkage percentage is larger than or equal to a second threshold value, and a result of bad quality of the sample to be detected is obtained; and the shrinkage percentage is smaller than or equal to a second threshold value, and a result with good quality of the sample to be detected is obtained.
8. The method for detecting changes in vessel diameter according to any one of claims 5 to 7, wherein the isolated vessel is a satisfactory isolated vessel prepared by:
fixing the isolated blood vessel at the fixing tube sides at the two ends in the bath, and pouring a PSS buffer solution into the bath and the isolated blood vessel;
adjusting the pressure values of the liquid inlet and the liquid outlet at two ends of the bath until reaching a target pressure value;
10ml KPSS solution is added into the bath tank to induce vasoconstriction, the stimulation is continued for M min, and the pipe diameter value of the isolated blood vessel and the percentage of the pipe diameter value relative to the initial pipe diameter value of the vascular ring are detected; repeating the operation, and if the percentage difference value of the pipe diameter values of the two adjacent isolated blood vessels is smaller than a third threshold value, obtaining the isolated blood vessels meeting the requirements; otherwise, the result that the isolated blood vessel is not in accordance with the requirement is obtained.
9. A system for detecting a change in vessel diameter, comprising:
an acquisition unit for determining a sample to be measured;
a first processing unit for infusing the sample to be tested into the isolated blood vessel based on the blood vessel diameter detection equipment of any one of claims 1-4, and respectively obtaining an initial tube diameter value corresponding to the isolated blood vessel infused with the sample to be tested and a tube diameter value after incubation of the sample to be tested of the isolated blood vessel infused with the sample to be tested;
the second processing unit is used for determining the diameter change value of the isolated blood vessel according to the initial pipe diameter value and the pipe diameter value of the sample to be detected after incubation.
10. A device for detecting a change in vessel diameter, the device comprising: a memory and a processor;
the memory is used for storing program instructions; the processor is configured to invoke program instructions, which when executed, are configured to perform the method of detecting a change in vessel diameter according to any of claims 5-8.
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