Model for evaluating biological action of cigarette smoke aerosol as well as construction method and application of model
By designing a slice model and a gas-liquid interface exposure system, the shortcomings in evaluating the biological role of cigarette smoke aerosols in the prior art are solved, and the flue gas absorption process in vitro is more realistically simulated in vitro and more accurate evaluation of biological functions is provided.
Patent Information
- Application Number
- CN202510744637.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-05
- Publication Date
- 2025-09-02
AI Technical Summary
It is difficult to develop effective models for the evaluation of the biological role of cigarette smoke aerosols on the nervous system, there are differences between in vitro cell models and animal models and humans, and it is difficult for laboratory conditions to simulate the real smoking process, resulting in bias in the research results.
A new section model construction scheme was designed to expose experimental animal tissue sections through gas-liquid interface contact, accurately control section culture and aerosol preparation, simulate the in vivo state, and use vitrocell gas-liquid exposure system for exposure, and evaluate it in combination with immunofluorescence staining, LDH detection and other methods.
It realizes efficient culturing of cells in vitro, maintains the internal structure of tissues, truly simulates the flue gas absorption process in vivo, and provides more accurate biological function evaluation.
Smart Images
Figure CN120574777A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology and relates to a model for evaluating the biological effects of cigarette smoke aerosols and a construction method and application thereof. Background Art
[0002] Cigarette smoke aerosol contains over 7,000 chemical components, including nicotine, tar, polycyclic aromatic hydrocarbons, heavy metals, and free radicals, which can cross the blood-brain barrier and directly act on the central nervous system. Epidemiological studies have shown that long-term smoking is significantly associated with neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. At the neural circuit level, nicotine can activate the mesolimbic dopamine system and mediate reward effects through α4β2 nicotinic acetylcholine receptors. However, the deeper impacts of complex aerosols on synaptic plasticity, neural network synchronization, and other aspects require systematic research.
[0003] Currently, research on the harmful effects of cigarette smoke aerosol involves constructing cell models or animal models. Immortalized neuronal cell lines, such as PC12 cells, are commonly used to study the effects of cigarette smoke aerosol on neurons. PC12 cells possess neuronal-like properties, simulating the physiological and pathological processes of neurons. Studies have found that harmful components in cigarette smoke aerosol can cause oxidative stress damage, inflammation, and apoptosis in PC12 cells. Primary neural cells isolated and cultured from animal brains, such as primary cortical neurons, can more realistically reflect the physiological state of neurons. Studies have shown that cigarette smoke aerosol extracts can affect neurite outgrowth and neurotransmitter release in primary cortical neurons, thereby affecting the formation and function of neural circuits. Mice and rats are commonly used animal models for studying the effects of cigarette smoke aerosol on the nervous system. Their physiological and genetic backgrounds are somewhat similar to those of humans, and they are easy to maintain and manipulate. For example, studies have exposed mice to cigarette smoke aerosol to observe the effects on neurobehavior and neural cells. Through behavioral experiments, such as anxiety and depression-related tests, it was found that mice exposed to cigarette smoke aerosols exhibited anxiety- and depression-like behaviors, which may be related to changes in the function of nerve cells.
[0004] However, in vitro cell models are less likely to reflect the real situation in vivo, and animal models differ from humans in physiology and genetics, so the results of animal experiments cannot fully represent the human situation. For example, some neurotoxic effects observed in animal models may not be obvious in humans, and vice versa. In addition, the exposure system and conditions in the laboratory are difficult to fully simulate the complex conditions in the real smoking process, such as the frequency, depth, and duration of smoking, which may lead to certain deviations between the research results and the actual situation. Therefore, how to develop effective models and evaluation methods to evaluate the biological effects of cigarette smoke aerosols remains one of the problems that need to be solved urgently. Summary of the Invention
[0005] In response to the deficiencies of existing technologies and actual needs, the present invention provides a model for evaluating the biological effects of cigarette smoke aerosols, a construction method, and an application thereof, and designs a new model construction scheme to provide new tools and new ideas for the study of the biological effects of cigarette smoke aerosols.
[0006] To achieve this object, the present invention adopts the following technical solutions:
[0007] In a first aspect, the present invention provides a method for constructing a model for evaluating the biological effects of cigarette smoke aerosols, the method comprising:
[0008] Tissue slices from experimental animals are cultured, and cigarette smoke aerosols are exposed to the tissue slices via a gas-liquid interface. The cigarette smoke aerosol preparation method comprises: taking cigarettes and preparing the cigarette smoke aerosol using a smoking machine, wherein the smoking mode of the smoking machine is: a frequency of 1 to 2 puffs per minute, a puff duration of 1.6 to 4 seconds, and a puff volume of 33 to 37 mL per puff.
[0009] The present invention designs a new scheme for constructing a slice model stimulated by cigarette smoke aerosols, designs and precisely controls the slice culture, aerosol preparation, and gas-liquid interface exposure processes, and realizes efficient model preparation through overall coordination. This allows for the in vitro culture of developing cells while maintaining the in vivo structure of the tissue, effectively simulating the in vivo state. In addition, aerosol exposure based on the gas-liquid interface is gas-liquid exposure that is closer to the in vivo environment, and can be closer to the process of cigarette absorption when the human body smokes cigarettes, thereby more realistically reflecting the biological functions of smoke aerosols.
[0010] Preferably, the experimental animals include rats or mice.
[0011] Preferably, the experimental animals include rats, and their age is within 7 days after birth, such as 12 hours, 24 hours, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days, etc., preferably within 24 hours, and more preferably within 12 hours.
[0012] In the present invention, the processing operation conditions of the experimental animal tissue before slicing are: culturing in a pre-cooled culture medium at 0-4°C for 20-40 minutes.
[0013] Preferably, the tissue slice comprises a brain tissue slice.
[0014] In the present invention, the processing operation conditions of the brain tissue before slicing are: culturing in a pre-cooled culture medium at 0-4°C for 20-40 minutes.
[0015] In the present invention, a model is constructed using brain tissue slices, which can be applied to the study of nerve cells and neural circuits.
[0016] Preferably, the thickness of the tissue slice is 200-400 μm, for example, it can be 210, 220, 230, 240, 250, 260, 270, 280, 300, 320, 350, 360, 370, 380 or 390 μm, and the slicing direction is coronal or sagittal section.
[0017] Preferably, the culturing method comprises: inoculating and culturing in a container having a permeable membrane at the bottom;
[0018] Preferably, the container comprises a Transwell chamber.
[0019] Preferably, the culture medium comprises DMEM / F12 medium containing 5% to 15% FBS (for example, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13% or 14%, etc.) and 0.5% to 1.5% penicillin (for example, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3% or 1.4%), with a pH of 7.3 to 7.5.
[0020] Preferably, the culture medium further contains 0.5-1.5 mL of B27 (for example, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3 or 1.4 mL, etc.), 15-25 ng of EGF (for example, 16, 17, 18, 19, 20, 21, 22, 23 or 24 ng, etc.) and 5-15 ng of bFGF (for example, 6, 7, 8, 9, 10, 11, 12, 13 or 14 ng, etc.) per 100 mL.
[0021] In the present invention, the Transwell chamber is shaped like a small cup that can be placed in a well plate. The chamber is placed in a culture plate, and the inner part of the chamber is called the upper chamber, and the inner part of the culture plate is called the lower chamber. The upper and lower culture fluids are separated by a polycarbonate membrane.
[0022] Preferably, the culturing time is at least 7 days.
[0023] Preferably, the step of equilibrating the cigarettes is further included before preparing the cigarette smoke aerosol. The equilibrating conditions include: a temperature of 20-23° C., a humidity of 25%-62% (for example, 26%, 27%, 28%, 29%, 30%, 35%, 38%, 40%, 45%, 50%, 55%, 58%, 59%, 60%, or 61%, etc.), and a time of 36-72 hours (for example, 37, 38, 39, 40, 45, 50, 55, 60, 65, 68, 70, or 71 hours, etc.).
[0024] Preferably, the number of cigarettes smoked is 1-20, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 15, 16, 17, 18 or 19 cigarettes.
[0025] Preferably, the exposure method comprises using a vitrocell gas-liquid exposure system for exposure.
[0026] Preferably, the exposure time is 1 h to 144 h, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 h, etc.
[0027] Preferably, the exposure amount is an amount of aerosol prepared from 2 to 5 cigarettes.
[0028] In a second aspect, the present invention provides a model for evaluating the biological effects of cigarette smoke aerosols. The model is constructed by the method for constructing a model for evaluating the biological effects of cigarette smoke aerosols described in the first aspect.
[0029] In a third aspect, the present invention provides a method for evaluating the biological effects of cigarette smoke aerosol. The method for constructing a model for evaluating the biological effects of cigarette smoke aerosol described in the first aspect is used to construct a model, and biological tests are performed on the model to evaluate the biological effects of cigarette smoke aerosol. The biological tests include immunofluorescence staining of tissue sections, LDH detection, BrdU / Edu staining of tissue section cells, nuclear staining (such as PI or DAPI), or ATP detection. The biological behaviors of the tissue sections, such as growth status, differentiation direction, and migration trend, are evaluated.
[0030] Compared with the prior art, the present invention has at least the following beneficial effects:
[0031] The present invention designs a new scheme for constructing a slice model stimulated by cigarette smoke aerosols, designs and precisely controls the slice culture, aerosol preparation, and gas-liquid interface exposure processes, and realizes efficient model preparation through overall coordination. This allows for the in vitro culture of developing cells while maintaining the in vivo structure of the tissue, effectively simulating the in vivo state. In addition, aerosol exposure based on the gas-liquid interface is gas-liquid exposure that is closer to the in vivo environment, and can be closer to the process of cigarette absorption when the human body smokes cigarettes, thereby more realistically reflecting the biological functions of smoke aerosols. BRIEF DESCRIPTION OF THE DRAWINGS
[0032] Figure 1 Figure 2 shows the effect of dissection culture medium temperature on the survival of organotypic brain tissue based on air-liquid interface exposure.
[0033] Figure 2The figure shows the age-dependent organotypic brain tissue survival changes in newborn mice based on air-liquid interface exposure.
[0034] Figure 3 This figure shows the effects of cigarette smoke on the survival of organotypic brain tissue in an air-liquid interface exposure system. DETAILED DESCRIPTION
[0035] The technical solution of the present invention will be further described below with reference to the accompanying drawings and through specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.
[0036] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.
[0037] LDH is an enzyme that exists stably in the cell cytoplasm. Under normal conditions, it is only distributed inside the cell. When the cell is stimulated to die, the plasma membrane ruptures and LDH is rapidly released outside the cell. Therefore, it is one of the most widely used markers in cytotoxicity research.
[0038] Example 1
[0039] This example constructs a model for evaluating the biological effects of cigarette smoke aerosol.
[0040] 1. Select newborn rats 12 hours after birth, remove the intact brain, and culture in 4°C pre-cooled culture medium (HBSS, 26% inactivated horse serum, 1% penicillin-streptomycin) after removing the meninges for 30 minutes.
[0041] 2. Place the brain, after removing the meninges, on the stage of an oscillating microtome and slice to a thickness of 300 μm.
[0042] 3. Transfer brain tissue sections to a Transwell microporous membrane. Add 1 mL of culture medium (DMEM / F12 medium containing 10% FBS and 1% penicillin, with 1 mL of B27, 20 ng of EGF, and 10 ng of bFGF per 100 mL, pH 7.4) to the bottom of the membrane. Incubate the sections in a sterile incubator at 37°C and 5% CO2, changing the medium by half every 3 days.
[0043] 4. Equilibrate the cigarettes at a temperature of 22°C and a humidity of 60% for 48 h. Then, prepare cigarette smoke aerosol using a single-channel smoking machine with a puff rate of 1 puff / min, a puff duration of 2 s, and a puff volume of 35 mL / puff.
[0044] 5. After one week of culture, the organotypic brain tissue slices were transferred to the in vitrocell air-liquid exposure system for culture and tobacco smoke aerosol treatment. Each Transwell chamber was stimulated with 5 cigarettes. The control group (Ctrl) was stimulated with clean air at the same flow rate.
[0045] 6. After 48 hours of air-liquid exposure, remove the Transwell chamber and use a kit to detect the amount of LDH released in the culture medium of the lower chamber.
[0046] 7. Meanwhile, the exposed organotypic brain tissue sections were fixed and immunofluorescence stained.
[0047] Example 2
[0048] This example constructs a model for evaluating the biological effects of cigarette smoke aerosol.
[0049] 1. Select newborn rats 12 hours after birth, remove the intact brain, and culture in 4°C pre-cooled culture medium (HBSS, 26% inactivated horse serum, 1% penicillin-streptomycin) after removing the meninges for 30 minutes.
[0050] 2. Place the brain, after removing the meninges, on the stage of an oscillating microtome and slice to a thickness of 300 μm.
[0051] 3. Transfer the brain tissue slices to the microporous membrane of Transwell, add 1 mL of culture medium to the bottom, and culture in a sterile incubator at 37°C and 5% CO2, changing half the medium every 3 days.
[0052] 4. Equilibrate the cigarettes at a temperature of 23°C and a humidity of 50% for 72 h. Then, prepare cigarette smoke aerosol using a single-channel smoking machine with a puff rate of 2 puffs / min, a puff duration of 1.6 s, and a puff volume of 37 mL / puff.
[0053] 5. After one week of culture, the organotypic brain tissue slices were transferred to the in vitrocell air-liquid exposure system for culture and smoke aerosol treatment. The stimulation dose per Transwell chamber was 2 cigarettes. The control group was stimulated with clean air at the same flow rate.
[0054] 6. After 24 hours of air-liquid exposure, remove the Transwell chamber and use a kit to detect the amount of LDH released in the culture medium of the lower chamber.
[0055] 7. Meanwhile, the exposed organotypic brain tissue sections were fixed and immunofluorescence stained.
[0056] Example 3
[0057] This example constructs a model for evaluating the biological effects of cigarette smoke aerosol.
[0058] 1. Select newborn rats 12 hours after birth, remove the intact brain, and culture in 4°C pre-cooled culture medium (HBSS, 26% inactivated horse serum, 1% penicillin-streptomycin) after removing the meninges for 30 minutes.
[0059] 2. Place the brain, after removing the meninges, on the stage of an oscillating microtome and slice to a thickness of 300 μm.
[0060] 3. Transfer the brain tissue slices to the microporous membrane of Transwell, add 1 mL of culture medium to the bottom, and culture in a sterile incubator at 37°C and 5% CO2, changing half the medium every 3 days.
[0061] 4. Cigarettes were equilibrated at 20°C and 25% humidity for 36 hours, and then cigarette smoke aerosols were generated using a single-channel smoking machine with a puff rate of 1 puff / min, a puff duration of 4 seconds, and a puff volume of 33 mL / puff. 5. After one week of culture, organotypic brain tissue slices were transferred to a Vitrocell air-liquid exposure system for incubation and cigarette smoke aerosol exposure. Each Transwell chamber was stimulated with two cigarettes, while a control group was stimulated with clean air at the same flow rate.
[0062] 6. After 24 hours of air-liquid exposure, remove the Transwell chamber and use a kit to detect the amount of LDH released in the culture medium of the lower chamber.
[0063] 7. Meanwhile, the exposed organotypic brain tissue sections were fixed and immunofluorescence stained.
[0064] Example 4
[0065] Compared with Example 1, the only difference is that the mouse brain tissue is dissected in a culture medium at room temperature (22° C.).
[0066] like Figure 1 As shown in the figure, the LDH release experiment (detection wavelength 490nm) quantitatively showed that in the air-liquid interface exposure system, the cytotoxicity level of the organotypic brain tissue slices in the low temperature (4℃) group (0.261±0.012) was significantly lower than that in the room temperature group (0.299±0.024), and the difference was statistically significant (*p<0.05, mean±SD), indicating that low temperature culture medium is more conducive to the survival of organotypic brain tissue.
[0067] Example 5
[0068] Compared with Example 1, the only difference is that the brain tissues of mice 3 and 7 days after birth were selected as research objects.
[0069] like Figure 2 As shown, LDH release experiments (detection wavelength 490 nm) quantitatively demonstrated that, in an air-liquid interface exposure system, the cytotoxicity level of brain organoids in the 12-hour neonatal group (0.261±0.012) was significantly lower than that in the 3-day (0.309±0.011) and 7-day (0.405±0.0219) groups (p<0.01), showing a clear age-dependent effect (ANOVA test: 87.27, ****p<0.0001). These results indicate that the viability of organotypic brain tissue shows a progressive decline over the developmental period of neonatal mice (**p<0.01, mean±SD).
[0070] Test Case
[0071] This test example tested the model constructed in Example 1. 12-hour-old mice were cultured in pre-cooled medium for 30 minutes, and then stimulated with air and smoke from 5 cigarettes for 48 hours before LDH detection.
[0072] Detection method: Use LDH detection kit for detection, and detect at 490nm after the reaction is completed according to the instructions.
[0073] Test results such as Figure 3 As shown in the figure, the results of the LDH release experiment (detection wavelength 490nm) showed that in the air-liquid interface exposure system, the cytotoxicity of the organotypic brain tissue in the cigarette smoke treatment group (119.2±3.626%) was significantly higher than that in the clean air control group (100.0±3.480%). The difference was statistically significant, indicating that cigarette smoke stimulation would reduce the survival of the organotypic brain tissue, *p<0.05, mean±SD.
[0074] In summary, the present invention designs a new scheme for constructing a slice model stimulated by cigarette smoke aerosols, designs and precisely controls the slice culture, aerosol preparation, and gas-liquid interface exposure processes, and realizes efficient model preparation through overall coordination. It can allow developing cells to be cultured in vitro while maintaining the in vivo structure of the tissue, effectively simulating the in vivo state. In addition, aerosol exposure based on the gas-liquid interface is gas-liquid exposure that is closer to the in vivo environment, and can be closer to the process of cigarette absorption when the human body smokes cigarettes, thereby more realistically reflecting the biological functions of smoke aerosols.
[0075] The applicant declares that the above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the scope of protection and disclosure of the present invention.
Claims
1. A method for constructing a model for evaluating the biological effects of cigarette smoke aerosol, characterized in that: The construction method comprises: Tissue slices from experimental animals are cultured, and cigarette smoke aerosols are exposed to the tissue slices via a gas-liquid interface. The cigarette smoke aerosol preparation method comprises: taking cigarettes and preparing the cigarette smoke aerosol using a smoking machine, wherein the smoking mode of the smoking machine is: a frequency of 1 to 2 puffs per minute, a puff duration of 1.6 to 4 seconds, and a puff volume of 33 to 37 mL per puff.
2. The method for constructing a model for evaluating the biological effects of cigarette smoke aerosol according to claim 1, characterized in that: The experimental animals include rats or mice; Preferably, the experimental animals include rats within 7 days after birth.
3. The method for constructing a model for evaluating the biological effects of cigarette smoke aerosol according to claim 1 or 2, characterized in that: The tissue slices include brain tissue slices; Preferably, the thickness of the tissue slice is 200-400 μm, and the slicing direction is coronal or sagittal section.
4. The method for constructing a model for evaluating the biological effects of cigarette smoke aerosol according to any one of claims 1 to 3, characterized in that: The culture method comprises: inoculating and culturing in a container having a permeable membrane at the bottom; Preferably, the container comprises a Transwell chamber; Preferably, the culture medium comprises a DMEM / F12 culture medium containing 5% to 15% FBS and 0.5% to 1.5% penicillin, with a pH of 7.3 to 7.5; Preferably, the culture medium further contains 0.5-1.5 mL B27, 15-25 ng EGF and 5-15 ng bFGF per 100 mL.
5. The method for constructing a model for evaluating the biological effects of cigarette smoke aerosol according to any one of claims 1 to 4, characterized in that: The culturing time is at least 7 days.
6. The method for constructing a model for evaluating the biological effects of cigarette smoke aerosol according to any one of claims 1 to 5, characterized in that: Before preparing the cigarette smoke aerosol, the step of balancing the cigarette is also included. The conditions of the balancing cigarette include: temperature of 20-23° C., humidity of 25%-62%, and time of 36-72 hours.
7. The method for constructing a model for evaluating the biological effects of cigarette smoke aerosol according to any one of claims 1 to 6, characterized in that: The number of cigarettes smoked is 1-20.
8. The method for constructing a model for evaluating the biological effects of cigarette smoke aerosol according to any one of claims 1 to 7, characterized in that: The exposure method includes using a vitrocell gas-liquid exposure system for exposure; Preferably, the exposure time is 1 hour to 144 hours.
9. A model for evaluating the biological effects of cigarette smoke aerosol, characterized in that: The model is constructed by the method for constructing a model for evaluating the biological effects of cigarette smoke aerosols according to any one of claims 1 to 8.
10. A method for evaluating the biological effects of cigarette smoke aerosol, characterized in that: Constructing a model using the method for constructing a model for evaluating the biological effects of cigarette smoke aerosols according to any one of claims 1 to 8, and performing biological testing on the model to evaluate the biological effects of cigarette smoke aerosols; The biological detection includes at least one of immunofluorescence staining of tissue sections, BrdU / EdU staining of tissue section cells, cell nucleus staining, LDH detection or ATP detection.