CN110108622B - Isolated microvascular endothelial barrier function detection method and device - Google Patents
Isolated microvascular endothelial barrier function detection method and device Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N15/0806—Details, e.g. sample holders, mounting samples for testing
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/08—Investigating permeability, pore-volume, or surface area of porous materials
- G01N15/082—Investigating permeability by forcing a fluid through a sample
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Abstract
An in vitro microvascular endothelial barrier function detection method and device belong to the field of basic medical experimental technology and experimental equipment. In particular to an experimental method and equipment for detecting the barrier function of the isolated microvascular endothelium of an experimental animal or human body. The invention provides an in vitro microvascular endothelial barrier function detection method and device without using virulent or fluorescent marker proteins. The invention relates to an isolated microvascular endothelial barrier function detection device, which comprises a constant temperature device and is characterized in that: the constant temperature device is internally provided with an external vascular environment chamber, a semipermeable membrane chamber is arranged in the external vascular environment chamber, two ends of the semipermeable membrane chamber are provided with a vascular irrigation inflow pipe and a vascular irrigation outflow pipe which are communicated with the outside, and the external vascular environment chamber is provided with an external vascular irrigation inflow pipe, an external vascular irrigation outflow pipe and an oxygen supply pipe.
Description
Technical Field
The invention belongs to the field of basic medical experimental technology and experimental equipment. In particular to an experimental method and equipment for detecting the barrier function of the isolated microvascular endothelium of an experimental animal or human body.
Background
In recent years, the research hot spot of circularity gradually shifts from the level of heart-large blood vessels to micro blood vessels, wherein the barrier function of the endothelium of micro blood vessels is an important research direction.
Under normal conditions, the microvascular endothelium is connected between cells by tight junction proteins and adhesion junction proteins, so that small molecular substances such as water, oxygen, carbon dioxide, glucose and the like can freely diffuse through cell membranes, and macromolecular substances such as proteins in blood plasma and tissue fluid are isolated. Under pathological conditions such as inflammation, ischemia-reperfusion and the like, tight connection between microvascular endothelium is ineffective, and substances such as protein in plasma leak into tissues through endothelium, namely microvascular endothelial barrier dysfunction.
At present, the research mode of microvascular endothelial dysfunction mainly comprises experimental techniques at both the whole animal level and the cellular level.
The experiment of the whole animal level mainly comprises the steps of injecting fluorescent marker proteins or large molecular substances such as Yi Venturi blue into an animal circulatory system, and detecting whether endothelial barrier dysfunction occurs in blood vessels by detecting the fluorescent intensity, tissue homogenate absorbance value or frozen section fluorescence and the like under a microscope.
Although the whole animal experiment is the most main research method for the barrier function of the microvascular endothelium, the whole animal experiment has more defects: because of the complex metabolic process in vivo, the physiological and pathological mechanisms of the barrier function of the microvascular endothelium are difficult to deeply study by using antagonists, activators, medicaments and the like; because of individual differences among animals, repeated experiments with more cases are needed by the whole animal method, so that the research expense and the labor capacity are increased; for experiments of real-time monitoring, only qualitative description of leakage occurrence can not provide quantitative leakage degree indexes, and for a tissue homogenate method of quantitative detection, the time-action relationship can not be known because only the endpoint condition can be detected. Meanwhile, in the two most widely used intravascular markers, the fluorescence-marked plasma protein is expensive, and the dosage of the fluorescence-marked plasma protein is huge according to the whole administration mode of the body weight, so that the experimental budget is greatly improved; while the Iwinian blue has stronger toxicity and weaker fluorescence intensity, and has adverse effects on experimental results while harming the health of experimental persons.
In addition, at the cellular level, the vascular endothelial barrier function is characterized by culturing various primary vascular endothelial cells or endothelial cell lines (strains) and examining the cell-cell tight junctions by trans-cell membrane impedance or cell-connexin fluorescent staining after formation of a continuous monolayer.
However, in the course of culture passage, the differentiation characteristics of cells gradually deteriorate, and the growth environment lacks cell polarity due to lack of induction of subendothelial matrix layer, resulting in a large difference in physiological characteristics of the cultured monolayer endothelial cells and vascular endothelium. This index only suggests the possibility of endothelial barrier dysfunction laterally, with a weaker evidence effect.
The isolated organ tissue level experiment is an important component of the functional experiment, and the isolated tissue level can well solve the defects of the whole and cell level research method while eliminating the interference of organism nerves, body fluid factors and metabolic environment and maintaining the specific structure and part of functions of the tissue. However, no technology for detecting the level of isolated tissues is available for the barrier function of the capillary endothelium.
Disclosure of Invention
The present invention is directed to the above-mentioned problems, and provides a method and apparatus for detecting the barrier function of the endothelial cells of isolated microvasculature without using highly toxic or fluorescent marker proteins.
In order to achieve the above purpose, the invention adopts the following technical scheme that the isolated microvascular endothelial barrier function detection device comprises a constant temperature device and is characterized in that: the constant temperature device is internally provided with an external vascular environment chamber, a semipermeable membrane chamber is arranged in the external vascular environment chamber, two ends of the semipermeable membrane chamber are provided with a vascular irrigation inflow pipe and a vascular irrigation outflow pipe which are communicated with the outside, and the external vascular environment chamber is provided with an external vascular irrigation inflow pipe, an external vascular irrigation outflow pipe and an oxygen supply pipe.
As a preferable scheme of the invention, the vascular perfusion tube is connected with an intravascular perfusion fluid tank with a constant temperature device through a pressure regulating buffer pump; the blood vessel perfusion outlet pipe is connected with the perfusate collecting vessel through a pressure regulating buffer pump.
As another preferable scheme of the invention, the outer chamber perfusion inflow pipe and the outer chamber perfusion outflow pipe are respectively connected with the outer chamber perfusion liquid tank and the outer chamber liquid collecting dish through the double-channel peristaltic pump.
As a third preferred scheme of the invention, the semi-permeable membrane chamber is provided with a small chamber liquid eduction tube and a pressure stabilizing tube which are connected with the outside, and the pressure stabilizing tube is connected with a small chamber internal pressure stabilizer.
The invention relates to an in-vitro microvascular endothelial barrier function detection method, which is characterized by comprising the following steps of: separating microvessels of tissue beside an animal or human body, connecting two ends of the microvessels with a vascular perfusion tube and a vascular perfusion tube respectively, establishing an intraductal flow path, and perfusing physiological perfusion liquid containing measurable high molecular substances in the microvessels; the space outside the capillary section is sealed by a semipermeable membrane chamber containing a semipermeable membrane wall, and the semipermeable membrane chamber can allow small molecular substances such as ions, glucose, dissolved oxygen in water, water molecules and the like to pass freely, but isolate high molecular substances; an open extravascular environment chamber is arranged outside the semipermeable membrane chamber, the extravascular environment chamber is filled with isotonic physiological solution containing glucose, oxygenation is carried out at the same time, and the vascular endothelial barrier function is represented by detecting the content of high molecular substances in the semipermeable membrane chamber at each time point and the high molecular substances penetrating through the wall of the micro-blood vessel.
As a preferred embodiment, the micro-intravascular perfusion uses serum or serum containing serum proteins instead of the solution, the semi-permeable membrane chamber and the extravascular environment chamber use common physiological solution without protein, and the protein content in the semi-shielded chamber is detected by protein quantification.
Further, the normal physiological solution without protein is either wilt's solution or artificial cerebrospinal fluid.
The invention has the beneficial effects that: 1. the invention adopts an in-situ in-vitro microvascular segment, and can embody the in-situ microvascular function specificity of each organ.
2. The perfusion adopts a pressure regulating buffer pump, and under the condition of experimental design requirement, the influence of different pressure conditions on the barrier function of vascular endothelium can be detected.
3. The isolated intravascular flow path and the intravascular pool environment allow the organ in-situ environment to be different from the intravascular environment under different physiological and pathological conditions, and have good verification effect.
4. The isolated blood vessel is incubated by a small amount of the antagonist, so that the channel blocking type research can be developed, conditions are provided for deep research of physiological and pathological mechanisms, and expenditure is reduced.
Drawings
Fig. 1 is a schematic structural view of the present invention.
In the drawings, a small chamber internal pressure stabilizer, a small chamber liquid eduction tube, a blood vessel external environment chamber, a semipermeable membrane chamber, a constant temperature device, an intravascular perfusion liquid tank, a pressure regulating buffer pump, a blood vessel perfusion tube, a micro blood vessel, an external chamber perfusion tube, an oxygen bottle, an oxygen supply tube, an external chamber perfusion liquid tank, a double-channel peristaltic pump, an external chamber liquid collection vessel, an external chamber perfusion outflow tube, a constant pressure tube, a perfusion liquid collection vessel and a blood vessel perfusion outflow tube are respectively arranged in the figures 1, 2, 3, 16, 17, 18, and 19.
Detailed Description
The invention relates to an isolated microvascular endothelial barrier function detection device, which comprises a constant temperature device 5 and is characterized in that: an extravascular environment chamber 3 is arranged in the constant temperature device 5, a semipermeable membrane chamber 4 is arranged in the extravascular environment chamber 3, a vascular irrigation inflow pipe 8 and a vascular irrigation outflow pipe 19 which are communicated with the outside are arranged at two ends of the semipermeable membrane chamber 4, and an extravascular irrigation inflow pipe 10, an extravascular irrigation outflow pipe 16 and an oxygen supply pipe 12 are arranged on the extravascular environment chamber 3.
As a preferable scheme of the invention, the vascular perfusion tube 8 is connected with the vascular perfusion fluid tank 6 with the constant temperature device 5 through the pressure regulating buffer pump 7; the vascular perfusion outflow pipe 19 is connected with a perfusion fluid collecting dish 18 through a pressure regulating buffer pump 7.
As another preferred embodiment of the present invention, the outer chamber perfusion tube 10 and the outer chamber perfusion tube 16 are connected to the outer chamber perfusion liquid tank 13 and the outer chamber liquid collection dish 15 respectively through a dual-channel peristaltic pump 14.
As a third preferred aspect of the present invention, the semipermeable membrane chamber 4 is provided with a cell liquid outlet pipe 2 and a pressure stabilizing pipe 17 connected to the outside, and the pressure stabilizing pipe 17 is connected to the cell internal pressure stabilizer 1.
The invention relates to an in-vitro microvascular endothelial barrier function detection method, which is characterized by comprising the following steps of: separating the tissue microvessels 9 beside the tumor of the animal or human body, respectively connecting two ends of the microvessels 9 with a vascular perfusion tube 8 and a vascular perfusion tube 19, establishing an intraductal flow path, and perfusing physiological perfusion liquid containing measurable high molecular substances in the microvessels 9; the outer space of the section 9 of the capillary is sealed by a semipermeable membrane chamber 4 containing a semipermeable membrane wall, and the semipermeable membrane chamber 4 can allow small molecular substances such as ions, glucose, dissolved oxygen in water, water molecules and the like to pass freely, but isolate high molecular substances; an extravascular environment chamber 3 which is opened is arranged outside the semipermeable membrane chamber 4, the extravascular environment chamber 3 is filled with isotonic physiological solution containing glucose, oxygenation is carried out at the same time, and the vascular endothelial barrier function is represented by detecting the content of high molecular substances in the semipermeable membrane chamber 4 at each time point and the quality of the high molecular substances penetrating through the wall of the microvascular 9.
As a preferred option, the micro-blood vessels 9 are perfused with serum or serum containing serum proteins instead of solution, the semi-permeable membrane chamber 4 and the extravascular environment chamber 3 are used with normal physiological solution without protein, and protein content in the semi-shielded cells is detected by protein quantification.
Further, the normal physiological solution without protein is either wilt's solution or artificial cerebrospinal fluid.
The oxygen supply pipe 12 is connected with an oxygen bottle 11.
Examples: animal tissue is taken, placed in a physiological solution saturated with pre-oxygen, microvessels 9 with diameters of 100-400 microns and no branches are separated under a microscope, surrounding connective tissues are carefully peeled off, the blood vessels are separated, one end of each blood vessel is ligated to an exhausted vascular perfusion tube 8 by a microsurgery line, blood in each blood vessel is carefully flushed out, the other end of each blood vessel is ligated to a vascular perfusion tube 19, and vascular perfusion liquid is injected under constant pressure. After the solution is updated, the semipermeable membrane chamber 4 is closed, the ventilation and liquid updating of the extravascular environment chamber 3 are kept, a gradient vascular flushing activation program is operated, after the completion, the solution in the semipermeable membrane chamber 4 is updated, and after the corresponding stimulation is given, the time and the leakage rate of endothelial dysfunction of the microvasculature 9 are calculated by extracting and detecting substances in the solution in the inner chamber.
It should be understood that the foregoing detailed description of the present invention is provided for illustration only and is not limited to the technical solutions described in the embodiments of the present invention, and those skilled in the art should understand that the present invention may be modified or substituted for the same technical effects; as long as the use requirement is met, the invention is within the protection scope of the invention.
Claims (1)
1. An isolated microvascular endothelial barrier function detection device comprising a thermostat (5), characterized in that: an external vascular environment chamber (3) is arranged in the constant temperature device (5), a semipermeable membrane chamber (4) is arranged in the external vascular environment chamber (3), a vascular inflow pipe (8) and a vascular outflow pipe (19) which are communicated with the outside are arranged at two ends of the semipermeable membrane chamber (4), and an external vascular inflow pipe (10), an external vascular outflow pipe (16) and an oxygen supply pipe (12) are arranged on the external vascular environment chamber (3); the vascular perfusion tube (8) is connected with an intravascular perfusion tank (6) with a constant temperature device (5) through a pressure regulating buffer pump (7); the vascular perfusion outflow pipe (19) is connected with a perfusate collecting vessel (18) through a pressure regulating buffer pump (7); the outer chamber perfusion tube (10) and the outer chamber perfusion tube (16) are respectively connected with the outer chamber perfusion liquid tank (13) and the outer chamber liquid collecting dish (15) through a double-channel peristaltic pump (14); the semi-permeable membrane chamber (4) is provided with a small chamber liquid eduction tube (2) and a pressure stabilizing tube (17) which are connected with the outside, and the pressure stabilizing tube (17) is connected with the small chamber internal pressure stabilizer (1).
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| CN210090269U (en) * | 2019-06-17 | 2020-02-18 | 辽宁中医药大学 | In vitro microvascular endothelial barrier function detection equipment |
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| US7622298B2 (en) * | 2006-03-24 | 2009-11-24 | Norits, Inc. | Method for creating perfusable microvessel systems |
| EP3110490B1 (en) * | 2013-12-12 | 2021-05-12 | Michael S. Mirizzi | Multiple chamber, expandable therapeutic agent delivery device |
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| WO2008005998A2 (en) * | 2006-07-07 | 2008-01-10 | Georgia Tech Research Corporation | Centimeter-scale, integrated diagnostics incubator for biological culturing |
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