Gas control device for myocardial cell hypoxia / reoxygenation experiments

Abstract

This invention provides a gas control device for experiments involving hypoxia / reoxygenation of cardiomyocytes. [Solution] The system comprises three parallel intake branch pipes 4 integrating a precision needle-type throttling structure, a multi-stage mixing chamber 6 connected thereto, a gradient switching mechanism, and a pressure balancing device. The gradient switching mechanism includes a fixed plate 9 and a movable plate, with multiple throttling holes of different diameters in the fixed plate and gas passage holes in the movable plate, allowing the airflow cross-sectional area to be switched in steps by rotating the movable plate. This solves the problem of pressure fluctuations during gas environment switching interfering with cellular responses, achieves highly accurate and smooth switching between hypoxia and reoxygenation, eliminates instantaneous pressure pulses, provides an accurate and uniform mixed gas for the hypoxia / reoxygenation (H / R) model, and is applicable to molecular mechanism studies of myocardial ischemia-reperfusion injury (MIRI).

Description

Technical Field

[0001] The present invention belongs to the technical field of gas control devices, and specifically relates to a gas control device for myocardial cell hypoxia / reoxygenation experiments.

Background Art

[0002] Myocardial ischemia-reperfusion injury (MIRI) is the main cause of poor prognosis in patients with acute myocardial infarction. In the treatment of acute myocardial infarction, rapid restoration of coronary perfusion by revascularization is the main means to rescue dying myocardium. However, after rapid restoration of blood perfusion, the injury of ischemic myocardium deteriorates instead, leading to an increase in myocardial infarction size and a decrease in cardiac function, that is, MIRI. Exploring effective intervention targets and elucidating their molecular protection mechanisms have important scientific value. In in vitro studies, usually, a hypoxia / reoxygenation (H / R) model is constructed using rat myocardial cells to simulate the MIRI process, and by precisely controlling the oxygen concentration environment, changes in cell apoptosis, release of inflammatory factors, and related signal transduction pathways are observed. The reproduction of this physiological process highly depends on a high-precision gas control device.

[0003] The development and onset mechanisms of MIRI are complex, involving a series of cellular processes including calcium overload, mitochondrial dysfunction, cellular inflammation, oxidative stress, and cellular apoptosis. Pyroptosis, a newly discovered mode of inflammatory programmed cell death, has been demonstrated to be triggered by activation of the NLRP3 / ASC / caspase-1 pathway and high levels of IL-1β expression, playing a crucial role in the development and onset of MIRI. At the forefront of current research, ubiquitin-specific protease 39 (USP39) has been identified as a negative regulator of the NF-κB inflammatory response, and the NF-κB pathway is a key regulatory system for myocardial ischemia-reperfusion. Simultaneously, nuclear factor E2-related factor 2 (Nrf2), as a key nuclear transcription factor that modulates antioxidant responses, exerts a protective effect on maintaining redox homeostasis during MIRI. Therefore, in order to deeply explore the specific mechanism by which USP39 controls the NF-κB-Nrf2 axis and mitigates MIRI cardiomyocyte damage, there is an extremely high demand for the accuracy of environmental simulations in in vitro H / R models.

[0004] Gas control systems play a central role in cardiomyocyte experiments, providing a dynamic metabolic environment that simulates ischemia and reperfusion for cells by adjusting the flow rates and ratios of gases such as oxygen, carbon dioxide, and nitrogen. Highly efficient gas control systems typically integrate precise gas pathways, mixing components, and corresponding monitoring sensors, maintaining the stability of gas components within the experimental chamber in real time and meeting the experimental precision requirements for detecting complex biological functions such as the expression of environmentally sensitive genes like USP39 and NF-κB pathway regulation.

[0005] However, existing gas control systems for cardiomyocyte experiments still have limitations in the precision of gas flow rate distribution and ratio adjustment during the rapid transition between hypoxic and reoxygenated environments. When experimental requirements include precise gas concentration gradient compensation and real-time adjustment of minute flow rates, the adjustment step sizes of existing systems often fail to accommodate the demands of minute airflow fluctuations, resulting in room for improvement in the real-time responsiveness of the gas switching process and the smoothness of the concentration curve. In particular, in experiments such as those studying the effects of USP39 expression changes on the NF-κB-Nrf2 axis, nonspecific pressure fluctuations and component stratification of the gas environment easily interfere with the true biochemical response of cells. Such control methods often make it difficult to ensure the accuracy of gas environment switching when supporting high-precision MIRI model simulation experiments. [Overview of the project] [Problems that the invention aims to solve]

[0006] This invention provides a gas control device for myocardial cell hypoxia / reoxygenation experiments, which enables highly accurate adjustment of gas component ratios and switching to a calm environmental state during the process of myocardial cell hypoxia and reoxygenation experiments. [Means for solving the problem]

[0007] The technical means employed in this invention are as follows:

[0008] A gas control device for myocardial cell hypoxia / reoxygenation experiments, A support base, on which a protective frame is attached, Three intake branch pipes arranged in parallel, mounted within the protective frame, with a precision needle-type throttling structure integrated within the piping of each intake branch pipe, and the outlet ends of the three intake branch pipes being joined by a three-way branch pipe, A multi-stage mixing chamber is installed within the protective frame, the top inlet of which communicates with the outlet of the three-way branch pipe, the inner wall of which is provided with a spiral groove, the edge of a spiral guide plate is fitted into the groove, a disturbance axis is provided vertically at the central axis of the multi-stage mixing chamber, and spherical protrusions are distributed on the surface of the disturbance axis. A gradient conversion mechanism provided between the multi-stage mixing chamber and the experimental chamber, comprising a fixed platen and a movable platen, wherein the inlet of the fixed platen communicates with the outlet end of the multi-stage mixing chamber, the platen surface has a plurality of throttling holes of different diameters, the movable platen has a single gas passage hole, the movable platen is rotatable relative to the fixed platen, and the gradient conversion mechanism is capable of directing the gas passage hole to communicate with any one of the throttling holes, A pressure balancing device, the inlet of which communicates with the gas passage hole of the movable platen, and the outlet which is used to connect to the experimental chamber, the pressure balancing device comprising a buffer bulb housing, and a pressure balancing device including an elastic pressure-sensitive membrane piece and a reset compression spring provided inside the buffer bulb housing, It is characterized by being equipped with [the following features].

[0009] Specifically, the precision needle-type diaphragm structure includes a valve body having an internal thread, a scale adjustment rod that screws into the internal thread, and a conical diaphragm core located inside the valve body, the conical angle range of the conical diaphragm core being 15° to 25°.

[0010] An impeller is attached to the bottom of the flow disturbance shaft, and the impeller is used to drive the rotation of the flow disturbance shaft under the action of the airflow. A gas collecting plate is provided at the bottom of the multi-stage mixing chamber, and the inside of the gas collecting plate is filled with a porous packing layer made of sintered stainless steel with a void ratio of 30% to 50%.

[0011] An operating lever is provided on the edge of the movable plate, and the operating lever extends outward through a long slit on the side of the support base, and a concentration grade scale is marked on the edge of the long slit. [Effects of the Invention]

[0012] Compared to conventional technology, this invention has the following beneficial effects.

[0013] This invention reduces the minimum step size for gas mixture adjustment by utilizing a micrometer-level stroke combination of a scale adjustment rod and a conical throttling core, through a precision needle-type throttling structure integrated on the intake branch tube. This provides a precise initial concentration base for nitrogen, oxygen, and carbon dioxide for experiments involving highly environmentally sensitive molecular mechanisms, such as the USP39 controlling the NF-κB-Nrf2 axis to mitigate MIRI cardiomyocyte damage. This solves the problem that the adjustment step sizes of existing devices are insufficient to accommodate minute airflow fluctuations.

[0014] This invention employs a structure in which a spiral guide plate and a disturbance shaft having spherical protrusions are combined inside a multi-stage mixing chamber, and the disturbance shaft is rotated by an impeller driven by airflow. This increases the residence time and collision probability of each component gas in the mixing chamber, effectively eliminating the stratification phenomenon that occurs during the gas mixing process and ensuring the uniformity of the gas components output to the experimental chamber. This avoids interference between gas component stratification and the detection results of biochemical responses such as cell apoptosis and the release of inflammatory factors in cardiomyocyte hypoxia / reoxygenation models.

[0015] This invention achieves stepwise switching of gas flow rate using a physical mechanical method by aligning multiple throttling holes of different diameters on a fixed plate and gas passage holes on a movable plate in a gradient switching mechanism. This avoids the influence of electromagnetic interference on the physiological state of cardiomyocytes and improves the real-time response speed in the transition between hypoxia and reoxygenation. At the same time, the pressure balancing device utilizes the elastic deformation of an elastic pressure-sensitive membrane and a reset compression spring to effectively absorb instantaneous pressure pulses generated during the switching process, smoothing the pressure curve of the output airflow and reducing fluctuations. This avoids activation of the mechanically sensitive signaling pathway of cardiomyocytes due to non-physiological physical shocks, ensuring that evaluation indicators such as pyroptosis and changes in inflammatory factors observed in the hypoxia / reoxygenation model truly reflect the preset biochemical stimuli. [Brief explanation of the drawing]

[0016] [Figure 1] Figure 1 is a schematic diagram showing the overall structure of the present invention. [Figure 2] Figure 2 is a cross-sectional view of the multi-stage mixing chamber. [Figure 3] Figure 3 is a schematic diagram showing the structure of the gradient conversion mechanism. [Modes for carrying out the invention]

[0017] The embodiments of the present invention will be described below with reference to the drawings. These embodiments are provided for the purpose of clearly and completely illustrating the present invention and do not limit its scope.

[0018] Referring to Figures 1 to 3, the present invention provides a gas control device for myocardial cell hypoxia / reoxygenation experiments. This device is primarily applied in situations where precise intervention is required regarding the component ratio and flow state of the ambient gas during in vitro experiments on myocardial cells. The physical structure of the device allows for adjustment of the mixing ratio of nitrogen, oxygen, and carbon dioxide at different concentrations, while maintaining pressure stability during the gas transport process.

[0019] This invention structurally includes a support base 1, an intake air regulation assembly, a multi-stage mixing chamber 6, a gradient conversion mechanism, and a pressure balance device. Among these, The support base 1 includes a support plate 2, support columns 3, and swivel casters with locking mechanisms. The support columns 3 are provided at the four corners of the support plate 2, and the swivel casters are provided at the bottom of the support plate 2 to achieve the movement and fixation of the entire device. The surface of the support plate 2 is coated with anti-corrosion Teflon (registered trademark), which has hydrophobicity and chemical inertness. A protective frame is provided above the support plate 2, and a wire mesh is stretched inside the protective frame, and its physical state constitutes a physical shield for the internal precision adjustment components.

[0020] The intake air regulation assembly is provided inside the protective frame and includes three intake air branch pipes 4 provided in parallel. The three intake air branch pipes are respectively used for nitrogen input, oxygen input, and carbon dioxide input. A precision needle-type throttle structure is integrated in the middle part of the pipeline of the intake air branch pipe 4. The precision needle-type throttle structure includes a valve body with internal threads, a scale adjustment rod that engages with the internal threads, and a conical throttle core located inside the valve body. A knurled knob 5 is fixedly connected to the top end of the scale adjustment rod. Annular equally divided scale lines are engraved on the surface of the scale adjustment rod. By rotating the knurled knob 5, the scale adjustment rod generates a spiral displacement inside the valve body. The conical angle range of the conical throttle core is from 15° to 25°. When the conical throttle core is axially displaced inside the internal flow path of the intake air branch pipe 4, the internal fluid passage cross-sectional area of the intake air branch pipe 4 changes correspondingly. A fluororubber sealing ring is inserted at the rear position of the conical throttle core inside the valve body. After being pressurized, the sealing ring closely contacts the outer wall of the scale adjustment rod and constitutes a physical block for the path of gas leaking to the outside along the thread gap.

[0021] The multi-stage mixing chamber 6 is mounted in the central region within the protective frame. The multi-stage mixing chamber 6 is a cylindrical outer shell, and the outlet ends of the three intake branch pipes are converged by a three-way branch pipe before passing through to the top inlet of the multi-stage mixing chamber 6. A spiral groove is machined into the inner wall of the outer shell, and the edge of the spiral guide plate 7 is fitted into the groove, forming a geometric path through which the gas descends in a spiral. Multiple ventilation micropores are provided on the surface of the spiral guide plate 7, distributed in a plum blossom pattern, and the diameter of the ventilation micropores decreases from the starting point to the end point of the spiral path, and this pore diameter gradient constitutes the stepwise induction of the local pressure of the airflow. A disturbance axis 8 is provided vertically at the central axis of the multi-stage mixing chamber 6. One end of the disturbance axis 8 is connected to the top of the multi-stage mixing chamber 6. Multiple spherical protrusions are distributed alternately on the surface of the disturbance axis 8. An impeller is attached to the bottom of the disturbance shaft 8. After the airflow flows into the multi-stage mixing chamber 6, the kinetic energy of the airflow acts on the impeller, driving the disturbance shaft 8 to rotate around its axis. During the rotation process, the spherical protrusions generate shear and collision effects on the swirling gas, increasing the probability of random collisions between gas molecules. A gas collecting plate is provided at the bottom of the multi-stage mixing chamber 6, and a honeycomb-shaped porous packing layer is provided inside the gas collecting plate. The material of this porous packing layer is sintered stainless steel, and its porosity is between 30% and 50%.

[0022] The gradient conversion mechanism is provided at the connection part between the multi-stage mixing chamber 6 and the experimental chamber. The gradient conversion mechanism includes a fixed plate 9 and a movable plate 10. A self-lubricating spacer made of polytetrafluoroethylene is sandwiched between the fixed plate 9 and the movable plate 10. The central position of the fixed plate 9 is connected to the outlet end of the multi-stage mixing chamber 6. Four throttle holes with diameters increasing along the circumferential direction are opened on the plate surface of the fixed plate 9. An annular sealing groove is opened at the circumferential edge position of each throttle hole on the back surface of the fixed plate 9, and a silicone rubber sealing ring is fitted into the sealing groove. One gas passage hole adapted to the throttle hole is opened on the movable plate 10. An operation lever 11 is provided at the edge of the movable plate 10, and an anti-slip rubber sleeve is mounted on the surface of the operation lever 11. The operation lever 11 extends to the outside through a long slit on the side surface of the support base 1. Digital scales representing different concentration levels are marked at the edge position of the long slit on the side surface of the support base 1. By operating the operation lever 11 to rotationally drive the movable plate 10, the gas passage hole is sequentially aligned with throttle holes of different diameters on the fixed plate 9, constituting a stepped switching of the total cross-sectional area of the air flow path.

[0023] The pressure balancing device is connected to the output end of the gradient conversion mechanism. The pressure balancing device includes a buffer ball chamber 12, an elastic pressure-sensitive diaphragm, and a reset compression spring. The internal chamber volume of the buffer ball chamber 12 is larger than the inner diameter volume of the intake branch pipe 4. The edge of the elastic pressure-sensitive diaphragm is fixed to the inner wall flange of the buffer ball chamber 12 by a pressing ring. The elastic pressure-sensitive diaphragm is horizontally spanned across the middle of the buffer ball chamber 12, partitioning the inner cavity of the ball chamber into an air flow chamber and a compensation chamber. The reset compression spring is provided in the compensation chamber and abuts against the back surface of the elastic pressure-sensitive diaphragm. At the moment of air flow switching, the pressure fluctuation acts on the elastic pressure-sensitive diaphragm, and through the deformation of the diaphragm and the feedback of the elastic force of the reset compression spring, a physical absorption of the pressure pulse is constituted. The bottom of the buffer ball chamber 12 is connected to the total outlet pipe through a flange, and a pressure gauge head is attached to the pipe wall of the total outlet pipe.

[0024] The operation flow of the present invention is as follows.

[0025] First, the operator connects the nitrogen, oxygen, and carbon dioxide gas source pipes to the intake fittings of the three intake branch pipes, respectively, and moves the scale adjustment rod by rotating the knurled knob 5 on each branch pipe. The scale adjustment rod is positioned based on the annular, equally spaced scale lines on its surface, changing the position of the conical throttling core in the flow path, thereby adjusting the initial inflow rates of the three gases. After the three component gases are preliminarily merged at the three branch pipes, they enter the multi-stage mixing chamber 6. The airflow descends in a spiral motion along the spiral guide plate 7, passing through the plum blossom-shaped distribution of micro-vents. Simultaneously, the airflow strikes the impeller at the bottom of the disturbance shaft 8, driving the disturbance shaft 8 to rotate. The spherical protrusions on the surface of the disturbance shaft 8 create continuous mechanical disturbance to the airflow. The mixed gas enters the gas collecting plate, undergoes secondary separation and filtration through a porous packed bed of sintered stainless steel, and then enters the gradient conversion mechanism.

[0026] Subsequently, the operator holds the operating lever 11 by hand and slides it along the elongated slit, according to the requirements of the experimental stage. The operating lever 11 drives the movable platen 10 to rotate relative to the fixed platen 9, connecting the gas passage holes on the movable platen 10 to a throttling hole of a specific diameter on the fixed platen 9. When switching from a hypoxia experiment to a re-oxygenation experiment, the operating lever 11 is operated to switch to a larger diameter throttling hole, increasing the instantaneous flow rate of the mixed gas. Pressure fluctuations that occur during this process are physically absorbed by the elastic pressure-sensitive film and reset compression spring in the buffer bulb chamber 12, and finally a calm airflow enters the experimental chamber through the exhaust pipe, satisfying the subsequent detection requirements.

[0027] For example, in detecting complex biological functions such as altered USP39 gene expression, activation of the NF-κB pathway, and Nrf2-mediated antioxidant reactions, the relevant experimental procedures are as follows:

[0028] H9c2 cardiomyocytes are collected and divided into groups such as a control group, H / R group, H / R+USP39 knockdown group, H / R+USP39 overexpression group, and H / R+USP39 knockdown + NF-κB inhibitor (JSH-23) co-treatment group. The operator first adjusts the flow ratios of nitrogen, oxygen, and carbon dioxide through precision needle-type throttling structures on three inspiratory branch tubes, precisely controlling the oxygen concentration in the mixed gas to less than 1%, and then introduces it into the experimental chamber to establish a hypoxic environment. After the hypoxic treatment is complete, the operator rapidly switches to a re-oxygenated environment where the oxygen concentration returns to 21% by operating the lever of the gradient switching mechanism, and the buffer bulb chamber automatically absorbs the pressure fluctuations during the switch.

[0029] After collecting cells from each group, cell proliferation ability is detected by the CCK-8 method, apoptosis rate is detected by flow cytometry, the content of inflammatory cytokines IL-1β, IL-6, and TNF-α is measured by ELISA, mitochondrial membrane potential is evaluated by JC-1 staining, lactate dehydrogenase release is measured by colorimetric method, and further interactions between USP39 and NF-κB, as well as changes in the expression of pathway genes such as NF-κB, Nrf2, Keap1, and HO-1, are analyzed by immunoprecipitation (Co-IP) and qPCR, respectively.

[0030] All of the above-mentioned detections can be completed in a stable, pressure-free gas environment provided by this invention, thereby enabling the acquisition of experimental data that accurately reflects how USP39 controls cardiomyocyte pyroptosis and inflammation via the NF-κB-Nrf2 axis. [Explanation of Symbols]

[0031] 1. Support base 2 Support plate 3 Support pillar 4. Intake branch pipe 5 knurled knobs 6 Multi-stage mixing chamber 7. Spiral Guide Board 8 Flow axis 9 Fixed plate 10 Movable plate 11. Operating lever 12 Buffer Ball Chambers

Claims

1. A gas control device for myocardial cell hypoxia / reoxygenation experiments, A support base, on which a protective frame is attached, Three intake branch pipes arranged in parallel, mounted within the protective frame, with a precision needle-type throttling structure integrated within the piping of each intake branch pipe, and the outlet ends of the three intake branch pipes being joined by a three-way branch pipe, A multi-stage mixing chamber is installed within the protective frame, the top inlet of which communicates with the outlet of the three-way branch pipe, the inner wall of which is provided with a spiral groove, the edge of a spiral guide plate is fitted into the groove, a disturbance axis is provided vertically at the central axis of the multi-stage mixing chamber, and spherical protrusions are distributed on the surface of the disturbance axis. A gradient conversion mechanism provided between the multi-stage mixing chamber and the experimental chamber, comprising a fixed platen and a movable platen, wherein the inlet of the fixed platen communicates with the outlet end of the multi-stage mixing chamber, the platen surface has a plurality of throttling holes of different diameters, the movable platen has a single gas passage hole, the movable platen is rotatable relative to the fixed platen, and the gradient conversion mechanism is capable of directing the gas passage hole to communicate with any one of the throttling holes, A pressure balancing device, the inlet of which communicates with the gas passage hole of the movable platen, and the outlet which is used to connect to the experimental chamber, the pressure balancing device comprising a buffer bulb housing, and a pressure balancing device including an elastic pressure-sensitive membrane piece and a reset compression spring provided inside the buffer bulb housing, A gas control device for myocardial cell hypoxia / reoxygenation experiments, characterized by comprising the following features.

2. The precision needle-type throttling structure includes a valve body having an internal thread, a scale adjustment rod that screws into the internal thread, and a conical throttling core located inside the valve body, wherein the conical angle range of the conical throttling core is from 15° to 25°, as described in claim 1, for use as a gas control device for myocardial cell hypoxia / reoxygenation experiments.

3. The gas control device for myocardial cell hypoxia / reoxygenation experiments according to claim 1, characterized in that an impeller is attached to the bottom of the disturbance shaft, the impeller is used to drive the rotation of the disturbance shaft under the action of airflow, a gas collecting plate is provided at the bottom of the multi-stage mixing chamber, and the inside of the gas collecting plate is filled with a porous packed layer made of sintered stainless steel with a porosity of 30% to 50%.

4. The gas control device for myocardial cell hypoxia / reoxygenation experiments according to claim 1, characterized in that an operating lever is provided on the edge of the movable plate, the operating lever extends to the outside through a long slit on the side of the support base, and a concentration grade scale is marked on the edge of the long slit.

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