CN116042763A - Method for amplifying vascular activity, product and application thereof - Google Patents
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Abstract
The invention discloses a method for amplifying vasoactivity, a product and application thereof, in particular relates to the effect of norepinephrine on amplifying vasoactivity induced by vasoactive drugs.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a method for amplifying vascular activity, a product and application thereof.
Background
Blood vessels are conduits for transporting blood biologically, and can be divided into arteries (arteries), veins (veins) and capillaries (Capillary) depending on the direction of transport. Arteries carry blood from the heart to body tissue, veins carry blood from tissue to heart, and microvasculature connects arteries to veins, which are the primary sites for mass exchange between blood and tissue. The vascular patterns possessed by various organisms vary. Vascular activity refers to the ability of blood vessels to contract and relax, and is affected by a variety of factors, playing a very important role in various vital activities in the living body.
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. Hypertension is one of the side effects of Hemoglobin-based oxygen carrier (HBOCs) blood substitutes in clinical studies, affecting their clinical use.
Disclosure of Invention
In order to make up the defects of the prior art, the invention aims to provide a method for amplifying the vascular activity, a product and application thereof.
To achieve our object, a first aspect of the present invention provides a method of amplifying vasoactive drug-induced vasoactivity, the method comprising the step of using norepinephrine.
Further, the blood vessel comprises an ex vivo blood vessel.
The sources of ex vivo blood vessels in the present invention include, but are not limited to, human and non-human animals. Non-human animals include all vertebrates, for example, mammals, such as non-human primates (particularly higher primates), sheep, dogs, rodents (such as mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and any domestic animals or pets; and non-mammals such as amphibians, reptiles, and the like. In a preferred embodiment, the ex vivo blood vessels used in the experiments were derived from SD rats.
Further, the isolated blood vessel comprises an isolated arterial blood vessel, an isolated venous blood vessel and an isolated capillary blood vessel.
Further, the isolated blood vessel comprises an isolated mesenteric artery vessel.
In some preferred embodiments, the isolated blood vessel is a resistance blood vessel. In a specific embodiment, the isolated blood vessel is an isolated mesenteric artery blood vessel, preferably a grade 2-3 mesenteric artery, which requires more than 2 mm intact blood vessels without branches and tears.
The term "resistance vessel" and "arterial vessel" as used herein are used interchangeably to refer to a conduit between a ventricle and a capillary vessel, where the arterial vessel is near the ventricle, has a large vessel diameter, and has a relatively thick vessel wall. Through repeated branching, the pipe diameter is gradually reduced, the pipe wall is thinned, and finally, a capillary anterior arteriole similar to the capillary structure is formed and connected with the capillary. The size of the artery diameter and the thickness of the artery wall are quite different, but the structures have the same common point. Generally consists of 3 membranes, the innermost layer called the intima (tunica intema), consisting of connective tissue lining the endothelium and the wales; the middle layer, called the mesomembrane (tu-nica media), is made up of a circular array of tissues; the outermost layer, called the adventitia (tunica adventitia), is made up of connective tissue arranged in wales.
Further, the vascular activity includes a change in vessel diameter.
Further, the concentration of norepinephrine is 1×10 -12 -1×10 -4 M。
Further, the norepinephrine concentration is 1×10 -6 -4×10 -6 M。
In some specific embodiments, the norepinephrine concentration is 1×10 -6 -4×10 -6 The vascular contraction rate at M is stepped down。
Further, the norepinephrine concentration is 1×10 -6 -3×10 -6 M。
Further, the method comprises the following steps:
1) Continuously introducing a buffer solution into the isolated blood vessel;
2) Adding a vasoactive drug;
3) Adding norepinephrine and adjusting to 1×10 -12 -1×10 -4 M。
Further, the vasoactive drugs include antiplatelet aggregation drugs, vasodilators, vasoconstrictors, lipid-modulating drugs, plaque-stabilizing beta-blockers or Chinese patent drugs.
The term "vasoactive drug" as used in the present invention refers to a drug that achieves the objective of shock resistance by modulating vasomotor status, altering vascular function and improving microcirculation blood perfusion. Including vasoconstrictors and vasodilators.
Further, the vasoactive drug is HBOCs.
The term "HBOCs" used in the present invention refers to a hemoglobin-based oxygen carrier, which is a red blood cell substitute with oxygen carrying function obtained by modifying hemoglobin-based by crosslinking, microencapsulation, chemical modification and the like. HBOCs are mainly based on human or animal hemoglobin and are obtained by polymerization, modification or encapsulation. The bovine hemoglobin has wide sources and stable and controllable quality, and the homology with human hemoglobin is more than 85%, so the bovine hemoglobin is the first choice raw material for preparing HBOCs.
In a second aspect the invention provides a product for amplifying vascular activity, the product comprising an agent for use in the method as hereinbefore described.
Further, the products include kits, devices, apparatuses.
Further, the kit includes an agent capable of amplifying the vascular activity.
Further, the product is a product that amplifies vasoactive drug-induced vasoactivity.
The term "kit" as used in the present invention refers to a collection of the above components, preferably provided separately or in a single container. The container also preferably contains instructions for practicing the methods of the present disclosure. Examples of these components of the kit and methods of use thereof have been given in this specification. Preferably, the kit may additionally comprise instructions, for example, a user manual for adjusting the components (e.g., concentration of the detection agent) and for interpreting the results of any assays regarding the diagnosis provided by the methods of the present disclosure. The user manual may be provided in paper or electronic form (e.g., stored on a CD or CD ROM). The disclosure also relates to the use of the kit in any method according to the disclosure.
In some specific embodiments, the device comprises a DMT120CP detection system and modifications thereof, and the device further comprises reagents for use in the methods described above.
The term "apparatus" as used in the present invention may refer to either a device or a module that is configured based on hardware or a module that is configured based on software.
The term "apparatus" as used in the present invention relates to a device system comprising at least the above-mentioned devices which are operatively interconnected to allow diagnosis to be made. How the devices are operatively connected will depend on the type of device included in the apparatus. Preferred detection devices are disclosed above in connection with embodiments relating to the methods of the present disclosure. In this case, the device is operatively connected such that the user of the system, due to the description and explanation given in the manual, combines the determination of the measurement with its diagnostic value. In such embodiments the device may be presented as a separate apparatus and preferably packaged together as a kit. Those skilled in the art will appreciate how to contact the device without further inventive skill. Preferred devices are those that can be applied without the specific knowledge of a skilled clinician, such as test strips or electronic devices that need only to be loaded with a sample.
The term "amplification" as used in the present invention means amplification of an image, sound, function, effect, etc., and the effect of amplification is independent of the quality of the object to be amplified, i.e., whether beneficial or detrimental effects are amplified. In some embodiments, "amplifying" is the amplification of the effect of vascular activity, particularly where the effect is a functional effect, particularly where the amplification of vascular activity requires the addition of a vasoactive drug.
A third aspect of the invention provides a system for amplifying vascular activity using the method described above.
Further, the system comprises a detection unit, a data processing unit or a result determination unit.
Further, the detection unit is used for detecting blood vessel related information to obtain a detection result.
Further, the data processing unit is used for analyzing and processing the detection result of the detection unit.
Further, the result determination unit is configured to compare the detected value of the vascular activity with a detected average value of the vascular activity without drug addition in a control.
Further, the system is a system that amplifies vasoactive drug-induced vasoactivity.
In a fourth aspect, the present invention provides a method of screening for a vasoactive agent for non-therapeutic purposes, the method comprising the method as described hereinbefore, the method of screening for a vasoactive agent comprising the steps of:
1) Continuously introducing a buffer solution into the isolated blood vessel;
2) Adding the medicine to be screened;
3) Adding norepinephrine and adjusting to 1×10 -12 -1×10 -4 M。
Further, the method control experiment steps are as follows:
1) Continuously introducing a buffer solution into the isolated blood vessel;
2) Norepinephrine is added and adjusted to the same concentration as norepinephrine in the previously described method.
Further, the method comprises comparing the vascular activity of the control group to that of the experimental group.
The term "non-therapeutic purpose" as used herein is synonymous with "non-therapeutic", "non-therapeutic" and refers to the methods of the present invention and/or the methods of diagnosis and treatment employing exclusion of diseases prescribed in clause 25 of the chinese patent law.
In certain embodiments, the addition of the test drug provides a significant distinction between the control and test groups, indicating that the drug is the drug to be screened. In these specific protocols, the effect of norepinephrine amplification is used on drugs that are otherwise weak in vasoactivity, allowing drugs that are regarded as errors or are not found to be screened for vasoactivity.
In some embodiments, the function of norepinephrine to amplify vascular activity is also used to screen currently used drugs, which are specific to drugs whose vascular activity is adverse, and in these embodiments, drugs whose vascular activity is so weak that it is not conventionally detected or detected but is considered to be an error are screened for clinical risk by the amplifying vascular activity of norepinephrine. In some cases, the secretion of autologous norepinephrine may lead to an amplification of the vasoactive adverse effects of the above drugs, with adverse consequences.
The term "drug" as used herein refers to a substance composed of at least one active ingredient with/without the addition of other chemical ingredients including carriers, stabilizers, diluents, adjuvants, dispersants, suspending agents, thickening agents and/or excipients. Modes of administration of the "drug" include, but are not limited to, intravenous, oral, aerosol, parenteral, intraocular, pulmonary, and topical administration.
In a fifth aspect the invention provides the use of norepinephrine in the manufacture of a product for potentiating vasoactivity induced by a vasoactive drug.
The term "norepinephrine" as used in the present invention is the same as "NE" and the generic name 1- (3, 4-dihydroxyphenyl) -2-aminoethanol, which is a substance formed by removing N-methyl groups from epinephrine, and also belongs to catecholamines in chemical structure. In particular embodiments of the invention "norepinephrine" is used as a vasoconstrictor or vasoconstrictor.
Drawings
FIG. 1 is an original schematic diagram and an improved schematic diagram of a DMT120CP system device, wherein A is an original schematic diagram of a DMT120CP measurement system device, and B is an improved schematic diagram of the DMT120CP system device;
FIG. 2 is a graph of the distribution of the diameters of blood vessels before and after the equilibrium of the mesenteric artery, wherein A is the distribution of the diameter of the unbalanced mesenteric artery, B is the distribution of the diameter of the mesenteric artery after the completion of the equilibrium, and C is a four-dimensional graph of the distribution of the diameters of the blood vessels before and after the equilibrium;
FIG. 3 is a graph of KPSS solution testing vascular activity;
FIG. 4 is a graph of molecular particle size distribution, wherein A is a graph of molecular particle size of bHb measured by dynamic light scattering, and B is a graph of molecular particle size of MalPEG-bHb measured by dynamic light scattering;
FIG. 5 is a graph of molecular exclusion chromatography and oxygen saturation dissociation, wherein A is bHb and MalPEG-bHb molecular exclusion chromatography, and B is bHb and MalPEG-bHb oxygen carrying-releasing capacity;
FIG. 6 is a graph showing the change in vessel diameter after adding a vasoconstrictor to a bath, wherein A is a graph showing the percentage of change in vessel diameter relative to angiotensin II at different concentrations, B is a graph showing the percentage of change in vessel diameter relative to norepinephrine at different concentrations, and C is 1X 10 -6 ~4*10 -6 A graph of percent change in vessel diameter relative to concentration of norepinephrine;
FIG. 7 is a graph showing the change of the blood vessel diameter with time after the blood vessel is perfused with different solutions, wherein A is a graph showing the change of the blood vessel diameter with time before and after the perfusion of the PSS solution, B is a graph showing the change of the blood vessel diameter with time before and after the perfusion of bHb, and C is a graph showing the change of the blood vessel diameter with time before and after the perfusion of MalPEG-bHb;
FIG. 8 is a graph showing the trend of the blood vessel diameter with the concentration of norepinephrine, wherein A is a graph showing the trend of the blood vessel diameter with the concentration of NE after the blood vessel is perfused with PSS, bHb and MalPEG-bHb, and B is a graph showing the blood vessel taken during the course of the blood vessel activity detection experiment; and (3) injection: significant differences in percent of bHbvs PSS vessel diameter shrinkage: * P < 0.05, < 0.01,bHb vs MalPEG-bHb percent of vessel diameter contractionSignificant differences in ratios: & P＜0.05, && P＜0.01。
Detailed Description
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are complete or merely performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, stability, etc.), but some experimental errors and deviations should be accounted for.
Examples
1. Experimental materials and methods
1. Experimental materials
1) Experimental animal
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.
2) Experimental instrument
DMT120CP microvascular pressure-diameter perfusion detection system (DMT, denmark), OLYMPUS SZ61 integral microscope (OLYMPUS, japan), MS-H-S electronic balance (SCILOGEX Co., U.S.A.), milli-Q ultra-pure water instrument (Millipore, U.S.A.).
2. Experimental method
1) Working fluid arrangement
PSS buffer: naCl 129.99 mM, KCl 4.69 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.
KPSS buffer: 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.
2) Improvements in detection systems
The schematic diagram of the DMT120CP measurement system is shown in FIG. 1A, a bath unit is used as a core component, two ends of an isolated blood vessel are respectively fixed on a glass tube by nylon ropes, and the glass tube is placed in a bath. In order to adequately simulate the physiological state of a patient,adding PSS buffer solution into the bath, and continuously introducing air (95% O) 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. 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.
The DMT120CP system was used in this study to monitor the effect of HBOCs on isolated microvasculature, by monitoring diameter changes, to characterize HBOCs-induced vascular activity. 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 5 nm 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, so that air bubbles are generated in the process of changing liquid or the damage to the blood vessel is avoided due to discontinuous liquid circulation, a three-way valve device is added at the liquid inlet of the DMT120CP system, the sample is injected by adopting a micro-injection pump, and the pressure at two ends of the blood vessel is controlled by changing the pump speed of the micro-injection pump. A schematic diagram of the modified DMT120CP assay system is shown in FIG. 1B.
3) Preparation of isolated mesenteric arterial vascular ring
Male SD rats were euthanized, and after laparotomy, intestinal tissue was rapidly removed, placed in a PSS solution at 4deg.C, and 95% O was passed through 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, removing connective tissues and adipose tissue around the mesenteric artery under a split microscope, selecting a 2-3 grade mesenteric artery, and cutting out a complete vascular ring with about 2 mm and no branches or cracks for later use.
Before an experiment, calibrating a tension sensor, a pressure sensor and a microscope of a DMT120CP system; fixing both ends of the vascular ring with nylon ropeIs positioned on a glass tube at a liquid inlet end (P1) and a liquid outlet end (P2) in a bath of the system, the two ends are slowly pressurized at the same time, 10 ml of PSS solution is put in the bath, and the temperature is 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 10 mmHg 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 10 mmHg, the pressure at the two ends is adjusted by adopting a speed gradient of increasing 10 mmHg every 5 min until the target pressure value is reached, the balance is carried out for 60 min, and the target pressure is 60 mmHg for the mesenteric artery of the rat.
After the perfusion pressure rises to the target value required by the experiment, the PSS solution is replaced, 10 ml KPSS is added to induce vasoconstriction, the vasoconstriction is continuously stimulated for 10 min, 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.
4) Addition of vasoactive amplification agent and detection of vasoactivity
The PSS buffer solution is continuously introduced into the blood vessel, and the reagent (angiotensin II or norepinephrine) with the function of vasoconstriction is sequentially and cumulatively added into the bath, so that the concentration of the vasoconstrictor in the bath is gradually increased, and the instrument automatically records the change condition of the diameter of the blood vessel.
And (3) adjusting the flow rate of the three-way valve connecting port and the microinjection pump, maintaining the pressure of the inlet end of the microvascular at 60-65 mmHg, starting to introduce the sample to be detected (mPEG-bHb and bHb), starting to time when the sample is full of the whole blood vessel, adjusting the position of the three-way valve connecting port, closing the microinjection pump, stopping introducing the sample to be detected, incubating the sample to be detected and the microvascular for 15 min, gradually adding a reagent with a vasoconstriction function into the bath tank, and recording the diameter change condition of the microvascular. After the experiment is completed, eluting the solution in the bath, dripping 10 ml of KPSS solution, and observing whether the blood vessel is contracted or not, wherein the difference between the contraction amplitude and the first induction of KPSS is less than 10%, so that the experimental data is proved to be reliable and effective.
5) Purification of bovine hemoglobin (bovine hemoglobin, bHb)
According to the existing method, purified bHb is prepared from bovine whole blood. 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.
6) Preparation of mPEG-Mal modification bHb (MalPEG-bHb)
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 50 kDa to remove mPEG-Mal and bHb which did not participate in the reaction, and the centrifugation condition was 3000 g for 30 minutes.
7) Characterization of bHb and MalPEG-bHb
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 (Ph 7.0), and after loading, the column was eluted at a flow rate of 0.05 mL/min, the wavelength was measured at 280nm, and the elution profile was recorded.
Oxygen dissociation curve determination: 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 4 mL 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 an oxygen dissociation curve, and reading P50.
Colloid osmotic pressure measurement: the sample colloid osmotic pressure value was measured by colloid osmotic pressure meter OSMOMAT 050. 200 mu L of 4g/dL 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 to be 50-200 -s The experimental results were recorded and the measurements were 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 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.
8) Statistical analysis
Experimental data shows that the methods were all performed using x±sd, statistical analysis was performed using IBM SPSS Statistics system, and comparison between groups was performed using repeated measures of variance analysis, P <0,05, P <0,01, indicating statistical significance compared to the initial diameter.
2. Experimental results
1. Vascular activity detection
The distribution of the diameters of the mesenteric artery before and after the equilibrium is shown in figure 2, and the distribution of the diameters of the mesenteric artery without the equilibrium is shown in figure 2A, and the range of the diameters of the mesenteric artery is 110-160 mu m; after balancing is completed, regulating the pressure at two ends of the blood vessel to 60 mmHg, and simulating the distribution situation of the diameters of the mesenteric artery blood vessels under the physiological conditions in the body, wherein the distribution situation is shown in figure 2B; FIG. 2C is a quarter-turn plot of a vessel diameter distribution, wherein the corresponding minimum, 25%, median, 75%, maximum and average values are 112.4 μm, 145.5 μm, 166.7 μm, 183.9 μm, 220 μm and 164 μm, respectively.
After the mesenteric artery vessel is balanced, the KPSS solution is replaced to stimulate the vessel contraction, restore the activity of the vessel contraction and verify the integrity of endothelial cells. As shown in FIG. 3, 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.
2. Characterization of bHb and MalPEG-bHb
Dynamic light scattering analysis was performed on molecular particle sizes of bHb and MalPEG-bHb. As shown in FIG. 4 and Table 1, the molecular particle sizes of bHb and MalPEG-bHb exhibited a single peak, indicating that the purity of the samples was high. The molecular particle size of bHb shown in FIG. 4A is 4.7+ -1 nm; as shown in FIG. 4B, after PEG modification, the molecular particle size of MalPEG-bHb is 15.5+ -1.4 nm, and the molecular particle size is obviously increased.
bHb the molecular particle size is 4.5 nm, the colloid osmotic pressure is 10.0 mmHg, the relative molecular mass is obviously increased after PEG modification, the molecular particle size of MalPEG-bHb is 17.6 nm, and the colloid osmotic pressure is 64.1 mmHg. Viscosity analysis was performed on bHb and MalPEG-bHb, and the measurements were repeated three times at 37℃and a shear rate of 75/s, and after modification, the viscosity of MalPEG-bHb increased but was still lower than the blood viscosity.
As shown in fig. 5A, 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 1.78 mL and 1.37 mL, respectively, and compared with bHb, the peak position of the MalPEG-bHb elution peak is obviously shifted to the left, which indicates that the PEG modification significantly increases the apparent relative molecular weight of bHb. 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. 5B, P of bHb 50 23.73 mmHg, modified with PEG, P of MalPEG-bHb 50 The oxygen affinity was increased at 6.99 mmHg.
3. Selection of vasoactive amplifying agent
The isolated mesenteric artery is endovascularly filled with a PSS buffer solution, and a reagent with vasoconstrictor function is added into the bath as a vasoactive amplifier, wherein FIG. 6A is angiotensin II, and FIG. 6B and FIG. 6C are norepinephrine. As can be seen from FIG. 6A, the concentration of angiotensin II is 0 to 1×10 -4 M, the diameter of the blood vessel is not obviously changed; as can be seen from fig. 6B, as the norepinephrine concentration is increased from 1×10 -12 ~1*10 -4 M, significant shrinkage of vessel diameter occurred and according to further experimentsFound at 1 x 10 -6 ~4*10 -6 Within the M range (fig. 6C), gradient constriction of vessel diameter occurs, so norepinephrine may be used in vasoactivity experiments to amplify the change in vessel diameter induced by HBOCs samples.
4. Detection of vascular Activity of bHb and MalPEG-bHb
In vitro vasoconstrictor activity assay, the trace of change of vessel diameter over time when PSS, malPEG-bHb and bHb samples were infused into the vessel, respectively, is shown in FIG. 7. 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. 7, 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. 8A is a graph of the blood vessel diameter as a percentage of the initial diameter after the mesenteric artery blood vessel was perfused with PSS, malPEG-bHb and bHb samples, respectively, and fig. 8B is a photograph of the blood vessel taken during the course of the vasoactivity test. From FIG. 8A, it can be seen that the NE concentration in the bath is 1X 10 -6 In M, the diameter shrinkage percentage of the blood vessels perfused by the three groups of PSS, malPEG-bHb and bHb relative to the initial diameter of the blood vessels is 0.5%,3% and 9%, respectively, and the blood vessel shrinkage amplitude after bHb perfusion and the blood vessel after PSS perfusion are respectivelySignificant differences in tube shrinkage amplitude (×p)<0.01 bHb and MalPEG-bHb, respectively && P<0.01 A) is provided; when the concentration of NE in the bath is 2 x 10 -6 In M, the diameter shrinkage percentages of the blood vessels perfused by the three groups of PSS, malPEG-bHb and bHb are 8%,12% and 25%, respectively, and the blood vessel shrinkage amplitude after bHb perfusion is significantly different from that after PSS perfusion (P)<0.01 bHb and MalPEG-bHb, respectively && P<0.01 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.
The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.
Claims (10)
1. A method for amplifying vasoactivity induced by a vasoactive agent, comprising the steps of inducing vasoactivity in a blood vessel by adding a vasoactive agent and amplifying the vasoactivity with norepinephrine at a concentration of 1 x 10 for the vasoactivity induced by the vasoactive agent -6 -3×10 -6 M。
2. The method of claim 1, wherein the blood vessel comprises an ex vivo blood vessel.
3. The method of claim 2, wherein the isolated blood vessel comprises an isolated mesenteric artery vessel.
4. The method of claim 1, wherein the vascular activity comprises a change in vessel diameter.
5. The method according to claim 1, characterized in that it comprises the steps of:
1) Continuously introducing a buffer solution into the isolated blood vessel;
2) Adding a vasoactive drug;
3) Adding norepinephrine and adjusting to 1×10 -6 -3×10 -6 M。
6. The method of claim 1, wherein the vasoactive drug comprises an anti-platelet aggregation drug, a vasodilator drug, a vasoconstrictor drug, a lipid-modulating drug, a plaque-stabilizing drug beta-blocker, or a chinese patent drug.
7. The method of claim 6, wherein the vasoactive agent is HBOCs.
8. A product for amplifying vascular activity, characterized in that it comprises an agent for use in the method according to any one of claims 1-7, or that it is a system using the method according to any one of claims 1-7.
9. A method for screening for a vasoactive agent for non-therapeutic purposes, comprising the steps of:
1) Continuously introducing a buffer solution into the isolated blood vessel;
2) Adding the medicine to be screened;
3) Adding norepinephrine and adjusting to 1×10 -6 -3×10 -6 M。
10. Use of norepinephrine in the preparation of a product for amplifying vasoactivity induced by a vasoactive drug.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030049250A1 (en) * | 2001-09-05 | 2003-03-13 | Matti Karvonen | Method for enhancing endothelial function in humans |
| US20040081642A1 (en) * | 1999-10-29 | 2004-04-29 | Joseph Loscalzo | Methods of treating vascular diseases characterized by nitric oxide insufficiency related applications |
| CN102238968A (en) * | 2008-10-21 | 2011-11-09 | 强生医药研究及开发有限责任公司 | Animal model for evaluating vasomotor response in vivo |
| CN104349778A (en) * | 2012-04-11 | 2015-02-11 | 简·布卢门施泰因 | Composition for prevention of vasoactivity in the treatment of blood loss and anemia |
| US20180028595A1 (en) * | 2015-02-13 | 2018-02-01 | University Of Virginia Patent Foundation | Compositions and methods for regulating blood pressure |
| CN115317595A (en) * | 2021-09-27 | 2022-11-11 | 中国人民解放军军事科学院军事医学研究院 | Pegylated bovine hemoglobin oxygen carrier and preparation method and application thereof |
-
2023
- 2023-03-07 CN CN202310207077.5A patent/CN116042763A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040081642A1 (en) * | 1999-10-29 | 2004-04-29 | Joseph Loscalzo | Methods of treating vascular diseases characterized by nitric oxide insufficiency related applications |
| US20030049250A1 (en) * | 2001-09-05 | 2003-03-13 | Matti Karvonen | Method for enhancing endothelial function in humans |
| CN102238968A (en) * | 2008-10-21 | 2011-11-09 | 强生医药研究及开发有限责任公司 | Animal model for evaluating vasomotor response in vivo |
| CN104349778A (en) * | 2012-04-11 | 2015-02-11 | 简·布卢门施泰因 | Composition for prevention of vasoactivity in the treatment of blood loss and anemia |
| US20180028595A1 (en) * | 2015-02-13 | 2018-02-01 | University Of Virginia Patent Foundation | Compositions and methods for regulating blood pressure |
| CN115317595A (en) * | 2021-09-27 | 2022-11-11 | 中国人民解放军军事科学院军事医学研究院 | Pegylated bovine hemoglobin oxygen carrier and preparation method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| H. W. KIM等: "Hemoglobin Mediated Vasoactivity in Isolated Vascular Rings", 《 ARTIFICIAL CELLS, BLOOD SUBSTITUTES, AND BIOTECHNOLOGY》, vol. 23, no. 3, pages 303 - 309 * |
| 刘强等: "葛根素对大鼠豚鼠主动脉肺动脉血管平滑肌的作用", 《中国医科大学学报》, vol. 31, no. 6, pages 401 - 403 * |
| 谢湘林等: "血管紧张素Ⅱ对家兔离体血管条去甲肾上腺素收缩作用的增敏效应", 《吉林大学学报(医学版)》, vol. 17, no. 5, pages 2 - 4 * |
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