CN110699256A - Gas replacement device for constructing cell or tissue pathological model and construction method thereof - Google Patents
Gas replacement device for constructing cell or tissue pathological model and construction method thereof Download PDFInfo
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M21/00—Bioreactors or fermenters specially adapted for specific uses
- C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/26—Conditioning fluids entering or exiting the reaction vessel
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12M37/00—Means for sterilizing, maintaining sterile conditions or avoiding chemical or biological contamination
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- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C12N5/0693—Tumour cells; Cancer cells
Abstract
The invention discloses a gas replacement device for constructing a cell or tissue pathological model and a construction method thereof. The invention firstly provides a gas replacement device for constructing a cell or tissue pathological model, which comprises a gas replacement part and a gas supply part; the gas replacement part comprises a culture box, a gas inlet assembly and a gas outlet assembly; the gas supply component is connected with the gas inlet assembly through a gas conduit; the culture box consists of a box body and a box cover matched with the box body; the air inlet assembly consists of an air inlet pipe penetrating through the box cover and a first valve; the air outlet assembly consists of an air outlet pipe penetrating through the box cover and a second valve; the length of the pipe body of the air inlet pipe extending into the box body is shorter than that of the pipe body of the air outlet pipe extending into the box body. The device can realize gas replacement in the process of constructing a cell or tissue pathological model, and has wide popularization and application prospects.
Description
Technical Field
The invention belongs to the technical field of biomedicine. And more particularly, to a gas replacement device for constructing a cell or tissue pathology model and a construction method thereof.
Background
The method is a common method for simulating pathological environments such as oxygen deficiency in laboratories by placing experimental cells or tissues and the like in oxygen/carbon dioxide/nitrogen environments with different contents for culture. Currently, three-gas cell incubators (oxygen, carbon dioxide and nitrogen) are typically used for gas exchange. However, the cell culture box is bulky, and therefore, the cell culture box needs to be constantly filled with gas to maintain a stable gas environment, and space resources of the whole culture box need to be occupied during the modeling of experimental cells or tissues, which is relatively wasteful. Therefore, the development of the cell or tissue culture box with simple structure, small volume and flexible combination has important significance for researching and providing a method for simulating pathological environments such as oxygen deficiency, which is simple and convenient to operate, economic and environment-friendly.
Currently, the existing devices such as the combined cell hypoxia culture tank (stem cell Technologies, USA) imported from abroad have disadvantages and shortcomings of high price, long order cycle, troublesome part renewal, and incapability of flexibly customizing the size, although having a relatively small volume. Further, patent CN201520525373.0 discloses a culture apparatus for hypoxic cell embryos, etc., which employs a hypoxic culture air bag for gas replacement and inflation, and then seals the air bag opening with a heat sealing machine. The device can simulate the culture environment of hypoxic cell embryos and the like, is harmless to people and has low cost; however, the low-oxygen culture air bag adopted by the device cannot be reused, the air bag is troublesome to seal, and the shape of the low-oxygen culture air bag cannot be fixed, so that various culture vessels cannot be placed conveniently. Therefore, there is a need to develop a device that has a simple structure, a small size, a flexible combination, a simple operation, economy, environmental protection, a high gas replacement efficiency, and can be used for constructing a cell or tissue pathological model.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings of the conventional device capable of simulating culture environments such as hypoxic cells and the like, and provides a gas replacement device for constructing a cell or tissue pathological model and a construction method thereof.
It is an object of the present invention to provide a gas replacement device for use in constructing a model of a cellular or histopathology.
It is another object of the invention to provide the use of said device for constructing a model of cell or tissue pathology.
It is a further object of the present invention to provide a method for constructing a model of cellular or histopathology.
The above purpose of the invention is realized by the following technical scheme:
the invention firstly provides a gas replacement device for constructing a cell or tissue pathological model, which comprises a gas replacement part and a gas supply part; the gas replacement part comprises a culture box, a gas inlet assembly and a gas outlet assembly; the gas supply component is connected with the gas inlet assembly through a gas conduit; the culture box consists of a box body and a box cover matched with the box body; the air inlet assembly consists of an air inlet pipe penetrating through the box cover and a first valve; the air outlet assembly consists of an air outlet pipe penetrating through the box cover and a second valve; the length of the pipe body of the air inlet pipe extending into the box body is shorter than that of the pipe body of the air outlet pipe extending into the box body.
Preferably, the gas supply part comprises a connecting pipe, a gas sterilizing filter, a gas flowmeter, a gas pressure reducing valve and a mixed gas bottle which are connected in sequence.
The gas sterilization filter is used for filtering and sterilizing gas, and the sterile state of cells or tissues in the culture process is ensured.
The gas flowmeter is used for monitoring the flow of input gas and controlling the flow of the gas.
Preferably, the gas conduit is sleeved on the connecting pipe to prevent gas from overflowing from the connection between the connecting pipe and the gas conduit.
In order to ensure the air tightness of the culture box, a sealing component is preferably arranged between the box body and the box cover; the sealing component is a sealing ring and a sealing buckle device which are arranged between the box body and the box cover.
More preferably, the sealing buckle device comprises a buckle fixing strip arranged at the edge of the box cover and a buckle protruding structure which is arranged at the upper edge of the box body and is matched with the buckle fixing strip.
Preferably, the sealing ring is arranged along the periphery of the box cover so as to ensure the air tightness of the culture box.
More preferably, the sealing ring is a rectangular sealing ring.
In order to prevent the gas from overflowing from the joints of the first valve and the second valve with the box cover and ensure the smooth circulation of the gas in the culture box, preferably, the first valve and the second valve respectively comprise an extension interface, a valve switch, an O-shaped sealing ring and a nut which are fixedly and hermetically connected in sequence; the air inlet pipe and the air outlet pipe are both composed of an inner box pipe and an outer box pipe, the outer box pipe is connected with the extension connector, and the inner box pipe is connected with the nut; the O-shaped sealing ring is connected with the box cover.
The O-shaped sealing ring is connected with the box cover to prevent the possibility of gas leakage between the gas inlet assembly and the box cover and between the gas outlet assembly and the box cover.
More preferably, the O-ring is coupled to a lower surface of the cap.
And a sealed gas passage is formed among the box outer tube, the extension interface, the valve switch, the O-shaped sealing ring, the nut and the box inner tube.
Preferably, the outer wall of the inner tube of the box is provided with a thread structure matched with the nut.
Preferably, the connection tube is connected to the cartridge outer tube in the inlet tube by a gas conduit.
In order to further ensure the air tightness of the culture box on the basis of the first valve and the second valve, the gas conduit is preferably provided with a switch device for controlling the gas to enter and exit.
More preferably, the switching means for controlling the ingress and egress of gas is a clamp.
For convenience of operation, the outer cartridge tube is preferably a tube of soft material.
In order to facilitate experimental observation and perform alcohol disinfection and ultraviolet disinfection on the culture box to prevent the cells and tissues from being polluted, preferably, the box body and the box cover are made of transparent plastic materials.
In order to facilitate the operation and further increase the air tightness of the device, the gas conduit, the sealing ring and the O-ring are preferably made of rubber.
The use of the device for constructing a model of a cellular or histopathology is also intended to be within the scope of the present invention.
In particular, the device can be used for culturing cells or tissues under different oxygen/carbon dioxide/nitrogen concentration conditions, constructing a low-oxygen or high-oxygen pathological model and detecting hypoxia endurance.
In addition, the invention also provides a method for constructing a cell or tissue pathological model, which comprises the steps of putting cells or tissues into the box body, and sealing the box body by using the box cover; connecting a gas conduit to a connecting pipe, opening a gas pressure reducing valve, loosening a switching device arranged on the gas conduit, opening a first valve and a second valve for ventilation, and adjusting a gas flowmeter to control the gas flow; after the ventilation is finished, simultaneously closing the first valve and the second valve, sequentially closing the gas flowmeter and the gas pressure reducing valve, closing a switching device arranged on the gas guide pipe, and loosening the connection between the gas guide pipe and the connecting pipe; and putting the culture box into a cell tissue culture box for culture, then taking out the culture box, sequentially opening the second valve and the box cover, and taking out the cells or tissues to obtain the cell or tissue pathological model.
The culture box can be designed with different capacities, sizes and shapes according to experiment requirements, and can be flexibly matched with a cell or tissue culture box for culture. For example, when the volume of the culture box is 1L, the gas flow rate is set to 1LPM, and the aeration is carried out for 5min, so that the ideal gas replacement effect can be achieved.
Cell culture bottles, cell culture dishes or cell culture plates with different specifications can be placed in the culture box according to experiment needs.
The invention has the following beneficial effects:
the invention provides a gas replacement device for constructing a cell or tissue pathological model and a construction method thereof. The invention provides a gas replacement device for constructing a cell or tissue pathological model, which can be used for culturing cells or tissues under the conditions of different oxygen/carbon dioxide/nitrogen concentrations, constructing a low-oxygen or high-oxygen pathological model and detecting the anoxia endurance, is simple and convenient to operate, can be flexibly matched with culture boxes with different capacities according to experimental requirements, can be flexibly matched with cell culture bottles or culture dishes with different specifications, effectively simulates the pathological environments of low oxygen, high oxygen and the like, successfully constructs the cell or tissue pathological model, effectively avoids pollution, saves experimental space and reduces gas and material consumption; in addition, the device is small in size, flexible in combination, resistant to disinfection and sterilization, free of expensive parts and low in cost, all the parts can be reused, and the device has wide popularization and application prospects.
Drawings
FIG. 1 is a schematic diagram of a gas replacement device for use in constructing a model of a cellular or histopathology; reference numerals: 100-culture box; 200-a first valve; 300-a second valve; 400-air inlet pipe; 500-air outlet pipe; 600-a gas conduit; 700-connecting pipe; 800-gas sterilizing filter; 900-gas flow meter; 110-gas pressure reducing valve; 120-mixed gas bottle; 130-O type seal ring; 140-a clip; 201-extension interface.
FIG. 2 is a schematic view of the structure of the culture cassette; reference numerals: 102-a box cover; 103-sealing ring; 104-snap fastener strips.
FIG. 3 is a schematic view of the structure of the culture cassette; reference numerals: 101-box body.
FIG. 4 is a schematic illustration of the structure of the intake valve; reference numerals: 201-an extension interface; 202-a nut; 203-valve switch; 130-O-ring.
FIG. 5 is a diagram showing the result of the morphological changes of neurone cell Neuron-2a and vascular endothelial cell EA.hy926 after sugar deficiency and hypoxia modeling; wherein, A) is a diagram of the result of the morphological change of Neuron-2a cells; B) the figure is a graph of the morphological change result of vascular endothelial cell EA.hy926.
Fig. 6 is a graph showing the result of mitochondrial damage state of vascular endothelial cell ea.hy926 after sugar-deficient hypoxia modeling.
FIG. 7 is a graph showing the result of apoptosis of vascular endothelial cell EA.hy926 after sugar-deficient hypoxia modeling.
FIG. 8 is a graph showing the results of the activation of lysosomal signals by vascular endothelial cells EA.hy926 after sugar-deficient hypoxia modelling; wherein, A) is the activation of cathepsins B and ubiquitin ligase E6 AP; B) the figure is a) the Z-axis superimposed colocalization map of the figure.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
EXAMPLE 1A gas replacement device for constructing a model of cell or tissue pathology
As shown in fig. 1, the present embodiment provides a gas replacement device for constructing a cell or tissue pathology model, including a gas replacement part and a gas supply part; the gas replacement part comprises a culture box 100, a gas inlet component and a gas outlet component; the gas supply component is connected with the gas inlet component through a gas conduit 600; the culture box 100 consists of a box body 101 and a box cover 102 matched with the box body; the air inlet assembly consists of an air inlet pipe 400 penetrating through the box cover 102 and a first valve 200; the air outlet assembly consists of an air outlet pipe 500 penetrating through the box cover 102 and a second valve 300; the length of the pipe body of the air inlet pipe 400 extending into the box body 101 is shorter than the length of the pipe body of the air outlet pipe 500 extending into the box body 101.
In this embodiment, the gas supply unit includes a connection pipe 700, a gas sterilizing filter 800, a gas flow meter 900, a gas pressure reducing valve 110, and a mixed gas bottle 120, which are connected in this order.
In this embodiment, the gas conduit 600 is sleeved on the connection tube 700 to prevent gas from overflowing from the connection position between the connection tube 700 and the gas conduit 600.
In order to ensure the air tightness of the culture box 100, in the embodiment, a sealing component is arranged between the box body 101 and the box cover 102; the sealing component is a sealing ring 103 and a sealing buckle device which are arranged between the box body 101 and the box cover 102; the sealing buckle device comprises a buckle fixing strip 104 arranged at the edge of the box cover 102 and a buckle protruding structure which is arranged at the upper edge of the box body 101 and is matched with the buckle fixing strip 104.
In this embodiment, the sealing ring 103 is disposed along the periphery of the box cover 102 to ensure the air tightness of the culture box 100.
In this embodiment, the sealing ring 103 is a rectangular sealing ring.
In order to prevent the gas from overflowing from the joints between the first valve 200 and the second valve 300 and the box cover 102 and ensure the smooth circulation of the gas in the culture box 100, in this embodiment, the first valve 200 and the second valve 300 each include an extension connector 201, a valve switch 203, an O-ring 130, and a nut 202, which are fixedly and hermetically connected in sequence; the air inlet pipe 400 and the air outlet pipe 500 are both composed of an inner box pipe and an outer box pipe, the outer box pipe is connected with the extension connector 201, and the inner box pipe is connected with the nut 202; the O-ring 130 is connected to the cap 102.
In this embodiment, the O-ring 130 is connected to the lower surface of the cover 102 to prevent the possibility of gas leakage between the cover 102 and the inlet and outlet assemblies.
In this embodiment, a sealed gas passage is formed between the outer cartridge tube, the extension port 201, the valve switch 203, the O-ring 130, the nut 202, and the inner cartridge tube.
In this embodiment, the outer wall of the inner tube of the box is provided with a thread structure matched with the nut 202.
In this embodiment, the connection tube 700 is connected to the cartridge outer tube in the inlet tube 400 through the gas conduit 600.
In order to further ensure the air tightness of the culture box 100 based on the first valve 200 and the second valve 300, in this embodiment, a clamp 140 for controlling the gas to enter and exit is provided on the gas conduit 600.
In order to facilitate the operation, in this embodiment, the outer tube is a soft material tube.
In order to facilitate experimental observation and perform alcohol disinfection and ultraviolet disinfection on the culture box 100 to prevent contamination of cells and tissues, in this embodiment, the box body 101 and the box cover 102 are made of transparent plastic materials.
In order to facilitate the operation and further increase the air tightness of the device, in this embodiment, the gas conduit 600, the sealing ring 103 and the O-ring 130 are made of rubber.
Example 2A method for constructing a model of cell or tissue pathology
A method for constructing cell or tissue pathological model comprises placing cell or tissue into a container 101, and sealing the container 101 with a sealing cover 102; sleeving the gas conduit 600 on the connecting pipe 700, opening the gas pressure reducing valve 110 and loosening the clamp 140, opening the gas inlet valve 200 and the gas outlet valve 300 for ventilation, and adjusting the gas flow meter 900 to control the gas flow; after ventilation is finished, the air inlet valve 200 and the air outlet valve 300 are closed at the same time, the gas flowmeter 900 and the gas reducing valve 110 are closed in sequence, the gas guide pipe 600 is clamped by the clamp 140, and the sleeving connection between the gas guide pipe 600 and the connecting pipe 700 is released; and (3) putting the culture box 100 into a cell tissue culture box for culture, then taking out the culture box 100, sequentially opening the air outlet valve 300 and the sealing cover 102, and taking out the cells or tissues to obtain the cell or tissue pathological model.
In this example, the volume of the culture cassette 100 was 1L, the gas flow rate was 1LPM, and the aeration was performed for 5 min.
The gas replacement device for constructing a cell or tissue pathology model obtained in example 1 can be used for culturing cells or tissues under different oxygen/carbon dioxide/nitrogen concentration conditions, constructing a hypoxia or hyperoxic pathology model and detecting hypoxia endurance. The specific experimental methods and experimental results are as follows:
application example 1 establishment of pathological models of glucose and oxygen deficiency of neuroma cells and vascular endothelial cells and morphological change of cells
1. Experimental methods
The preparation method comprises the following steps of respectively inoculating neurone-2 a and vascular endothelial cells EA.hy926 of neuroma cells into a T25 cell culture bottle, putting the bottle into a cell culture box for conventional culture and adherence for 24 hours, replacing a conventional high-sugar cell culture medium with a sugar-free culture medium, and performing sugar-deficiency hypoxia (OGD) molding, wherein the specific method comprises the following steps:
firstly, taking out a cell culture bottle, sucking out original culture solution in a super clean bench, and using sugar-free culture medium HBSS (NaCl8.00g/L, KCl 0.20g/L, CaCl)20.14g/L,MgSO4.7H2O 0.20g/L,Na2HPO4.H2O 0.06g/L,KH2PO40.06g/L,NaHCO30.35g/L) cells were washed 3 times, followed by addition of HBSS for culture under sugarless conditions; the cells were then placed in the culture chamber of the gas replacement device for constructing a model of cell or tissue pathology obtained in example 1, according to the procedure of example 2, using 5% CO2+95％N2The premixed gas is used for carrying out gas replacement of the culture box, so that the cells are in an anoxic environment, and sugar-deficient and anoxic cell molding can be carried out; among them, neurone cell neurone-2 a was OGD-treated for 3 hours (OGD 3h group), vascular endothelial cell EA.hy926 was OGD-treated for 6 hours (OGD 6h group), and neurone cell neurone-2 a and vascular endothelial cell EA.hy926, which were not subjected to the sugar-deficient hypoxia molding, were used as controls (Con group). After the sugar-deficiency and oxygen-deficiency modeling is finished, the morphological changes of neuroma cell Neuron-2a and vascular endothelial cell EA.hy926 are respectively observed under a microscope and photographed and recorded.
2. Results of the experiment
After sugar-deficiency and hypoxia modeling, the morphological change results of neuroma cell Neuron-2a and vascular endothelial cell EA.hy926 are shown in FIG. 5, wherein, A) is a morphological change result graph of neuroma cell Neuron-2a, compared with neuroma cell Neuron-2a in Con group which is spread and has full morphology, after neuroma cell Neuron-2a is subjected to OGD treatment for 3 hours, the cell body part of the cell is obviously shrunk and has stringiness pseudopodia; B) the figure shows the results of morphological changes of vascular endothelial cells ea.hy926, and it can be seen that the cell morphology became flat and pseudopodia was evident after 6 hours of OGD treatment of vascular endothelial cells ea.hy926, compared with the fusiform and stereoscopic satiated adherent spreading of vascular endothelial cells ea.hy926 in the Con group. The results show that after the gas replacement device for constructing the cell or tissue pathological model is applied to sugar-deficient hypoxia modeling, cells are all subjected to morphological damage, and the device can effectively simulate the closed hypoxia state.
Application example 2 establishment of pathological model of sugar deficiency and hypoxia of vascular endothelial cells and damage state of mitochondria of cells
The MTT method is an important means for detecting cell mitochondrial damage, and the working principle of the MTT method is that MTT is deposited and forms needle crystals under the action of cell mitochondrial succinate dehydrogenase under normal conditions; when the mitochondria of the cells are pathologically damaged, the needle-shaped crystals can not be effectively formed. Mitochondrial damage is an important link of cellular hypoxia and glucose deprivation molding damage, and therefore, in this embodiment, mitochondrial damage is used as one of the indexes of cellular hypoxia and glucose deprivation effect. The specific experimental methods and experimental results are as follows:
1. experimental methods
Vascular endothelial cells ea.hy926 were seeded in a 96-well plate, placed in a cell culture chamber for 24 hours after being subjected to conventional culture and adherence, and placed in the culture cassette of the gas replacement device for constructing a cell or tissue pathology model obtained in example 1, and the vascular endothelial cells ea.hy926 were subjected to OGD treatment for 6 hours (OGD 6h group) and 12 hours (OGD 12h group) according to the procedure of example 2, and vascular endothelial cells ea.hy926 that were not subjected to cell-sugar-deficiency hypoxia molding were used as a control (Con group). After the completion of the sugar-deficient and oxygen-deficient molding, MTT solution (5mg/mL stock solution, 20 uL/well) was added and incubated in a 37 ℃ cell incubator for 4 hours to observe "needle crystals" under a microscope.
2. Results of the experiment
After sugar-deficiency and hypoxia modeling, the result of the mitochondrial damage state of the vascular endothelial cell EA.hy926 is shown in fig. 6, and it can be seen that compared with the Con group vascular endothelial cell EA.hy926, obvious needle-shaped crystals are formed around the cell body, and after the vascular endothelial cell EA.hy926 is subjected to OGD treatment for 6 hours, the needle-shaped crystals are not formed and only granular purple precipitates are formed; the purple pellet was further reduced after 12 hours of OGD treatment of vascular endothelial cells ea.hy926. The results show that after the gas replacement device for constructing the cell or tissue pathological model is applied to sugar-deficient hypoxia modeling, the mitochondria of the vascular endothelial cells are damaged, and the mitochondria of the vascular endothelial cells are time-dependent along with the prolongation of the OGD treatment time.
Application example 3 establishment of pathological model of sugar deficiency and hypoxia of vascular endothelial cells and apoptosis condition of vascular endothelial cells
1. Experimental methods
Vascular endothelial cells EA.hy926 were inoculated into a T25 flask, and after growing to 90% confluency, the cells were placed in the culture cassette of the gas replacement device for constructing a cell or tissue pathology model obtained in example 1, and the vascular endothelial cells EA.hy926 were subjected to hypoxia or sugar deficiency treatment for 6 hours (4 groups in total: normal group, sugar deficiency group, hypoxic group, sugar deficiency hypoxic group) according to the procedure of example 2. After the treatment is finished, collecting a sample of vascular endothelial cells EA.hy926 to extract protein for immunoblot analysis, and inspecting the expression changes of endoplasmic reticulum damage marker protein (Calnexin protein) and apoptosis damage marker protein (PARP-1 protein).
2. Results of the experiment
The apoptosis result of vascular endothelial cells EA.hy926 after sugar-deficient hypoxia modeling is shown in figure 7, and the expression of Calnexin protein of cells in a hypoxia group and a sugar-deficient hypoxia group is reduced compared with that in a normal group; the sugar-deficient group, the oxygen-deficient group and the sugar-deficient oxygen-deficient group all cause the cleavage of cell PARP-1 protein to generate a small molecular weight band, and indicate that the vascular endothelial cell EA.hy926 generates apoptosis damage. The above results show that the cells are apoptotic after sugar-deficient hypoxia modeling by applying the gas replacement device for constructing cell or tissue pathological models of the present invention.
Application example 4 establishment of pathological model of sugar deficiency and hypoxia of vascular endothelial cells and activation condition of lysosome signal of pathological model
1. Experimental methods
The vascular endothelial cells ea.hy926 were seeded on the polylysine-treated slide glass, and after it had grown to 80% confluency, they were put into the culture box of the gas replacement device for constructing a cell or tissue pathology model obtained in example 1, and the vascular endothelial cells ea.hy926 were OGD-treated for 6 hours according to the procedure of example 2. The vascular endothelial cells ea.hy926 samples were subsequently fixed with 4% PFA solution for cellular immunofluorescence staining. The vascular endothelial cell EA.hy926 sample is subjected to serum blocking, primary antibody incubation (autophagy-lysosome activation marker protein Cathepsin B antibody and ubiquitin ligase E6AP antibody), fluorescent secondary antibody incubation, DAPI staining and mounting treatment, and then is subjected to imaging photographing by using a fluorescence microscope.
2. Results of the experiment
After sugar deficiency and hypoxia modeling, the activation condition of the lysosome signal of the vascular endothelial cell EA.hy926 is shown in figure 8, wherein, A) is the activation condition of cell Cathepsin B and ubiquitin ligase E6AP, and it can be seen that the expression of the Cathepsin B antibody in vascular endothelial cell EA.hy926 plasma is obviously increased and the E6AP antibody is also obviously increased after OGD treatment; B) the graph is a Z-axis superimposed co-localization graph of the graph A), and it can be seen that the E6AP antibody and the Cathepsin B antibody have better co-localization expression and are involved in activation of lysosomal signals. The results show that when the gas replacement device for constructing the cell or tissue pathological model is used for sugar-deficient hypoxia modeling, the lysosome signal is activated after the vascular endothelial cells are induced by sugar-deficient hypoxia injury.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A gas replacement device for constructing a model of a cellular or histopathology, comprising a gas replacement component and a gas supply component; the gas replacement part comprises a culture box (100), a gas inlet component and a gas outlet component; the gas supply component is connected with the gas inlet component through a gas conduit (600); the culture box (100) consists of a box body (101) and a box cover (102) matched with the box body; the air inlet assembly consists of an air inlet pipe (400) penetrating through the box cover (102) and a first valve (200); the air outlet assembly consists of an air outlet pipe (500) penetrating through the box cover (102) and a second valve (300); the length of the pipe body of the air inlet pipe (400) extending into the box body (101) is shorter than that of the pipe body of the air outlet pipe (500) extending into the box body (101).
2. The apparatus according to claim 1, wherein the gas supply means comprises a connection pipe (700), a gas sterilizing filter (800), a gas flow meter (900), a gas pressure reducing valve (110), and a mixed gas bottle (120) which are connected in this order.
3. The device according to claim 2, wherein the gas conduit (600) is sleeved on the connecting tube (700).
4. The device according to claim 1, characterized in that a sealing component is arranged between the box body (101) and the box cover (102); the sealing component is a sealing ring (103) and a sealing buckle device which are arranged between the box body (101) and the box cover (102).
5. The device according to claim 4, characterized in that the sealing snap means comprise a snap fixing strip (104) arranged at the edge of the lid (102) and a snap protruding structure arranged at the upper edge of the body (101) adapted to the snap fixing strip (104).
6. The device according to claim 1, wherein the first valve (200) and the second valve (300) each comprise an extension port (201), a valve switch (203), an O-shaped sealing ring (130) and a nut (202) which are fixedly and hermetically connected in sequence; the air inlet pipe (400) and the air outlet pipe (500) are both composed of an inner box pipe and an outer box pipe, the outer box pipe is connected with the extension connector (201), and the inner box pipe is connected with the nut (202); the O-shaped sealing ring (130) is connected with the box cover (102).
7. The device according to claim 1, characterized in that the gas conduit (600) is provided with a switching device for controlling the gas in and out.
8. The device according to claim 1, wherein the box body (101) and the box cover (102) are made of transparent plastic.
9. Use of the device of any one of claims 1 to 8 for the construction of a model of cell or tissue pathology.
10. A method for constructing a cell or tissue pathological model is characterized in that cells or tissues are placed in a box body (101), and the box body (101) is sealed by a box cover (102); connecting the gas guide pipe (600) to the connecting pipe (700), opening the gas pressure reducing valve (110), loosening a switch device arranged on the gas guide pipe (600), opening the first valve (200) and the second valve (300) for ventilation, and adjusting the gas flow meter (900) to control the gas flow; after ventilation is finished, simultaneously closing the first valve (200) and the second valve (300), sequentially closing the gas flowmeter (900) and the gas reducing valve (110), closing a switching device arranged on the gas guide pipe (600), and loosening the connection between the gas guide pipe (600) and the connecting pipe (700); and (3) putting the culture box (100) into a cell tissue culture box for culture, then taking out the culture box (100), sequentially opening the second valve (300) and the box cover (102), and taking out cells or tissues to obtain the cell or tissue pathological model.
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