CN111856025B - Neuronal synaptobrevin protein SYN sensitive to terahertz-wave radiation - Google Patents
Neuronal synaptobrevin protein SYN sensitive to terahertz-wave radiation Download PDFInfo
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Abstract
The invention provides application of SYN protein in detecting neuron state under the action of terahertz wave radiation. The inventor finds that SYN protein has sensitivity to terahertz wave radiation, and the expression quantity of the SYN protein is reduced under the action of the terahertz wave radiation, so that abnormal neuron state is caused, and nerve information transmission is blocked. Therefore, by detecting the variation of the expression level of SYN protein in the neuron under the action of terahertz wave radiation, whether the state of the neuron is abnormal or not can be effectively judged.
Description
Technical Field
The present invention relates to the field of biology. In particular, the invention relates to neuronal synaptobrevin proteins SYN sensitive to terahertz wave radiation. More specifically, the invention relates to application of SYN protein in detecting neuron state under the action of terahertz wave radiation, application of a reagent for detecting SYN protein in preparing a kit, a method for detecting neuron state under the action of terahertz wave radiation and a method for regulating SYN protein expression quantity in neurons.
Background
Terahertz (THz) waves are electromagnetic waves with frequencies between 100GHz and 10 THz. Along with the progress of science and technology, the THz wave technology is mature, and the application prospect is huge. Along with the continuous popularization of the THz wave technology in the aspects of security check, nondestructive detection, imaging, communication and the like, the biological effect and the biological safety of the THz wave technology are increasingly concerned. Studies have shown that THz waves can cause resonance and rotational energy level transitions of biological macromolecules, potentially producing unique effects on biological tissues. However, so far, little research on nerve information transmission by THz wave, especially presynaptic vesicle release change is not reported, and a detection method for nerve information transmission abnormality is not yet known. Therefore, if the neuronal synapse vesicle protein sensitive to the terahertz wave radiation can be found, the neuronal synapse vesicle protein can be used for detecting whether the terahertz wave radiation causes abnormal transmission of the neuronal information.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems existing in the prior art to at least some extent.
The present invention has been completed based on the following findings by the inventors:
6h after the terahertz wave irradiates the primary hippocampal neurons, the immunoblotting result shows that compared with a control group, SYN expression of the experimental group is down-regulated (P is less than 0.05), vesicle accumulation and release disorder are shown, and the neuron information transmission is abnormal.
For this purpose, in one aspect of the invention, the invention proposes the use of SYN proteins for detecting neuronal states under the action of terahertz wave radiation.
Complexes of presynaptic proteins such as SYN, rSec6, SNAP and VAMP play a key role in neurotransmitter release. Neuronal Synaptobrevin (SYN) has the function of modulating the release of transmitters in neuronal synaptovesicles and can be involved in modulating the number of synaptosomes released by exocytosis. rSec6 is an exocytosis secreted protein that regulates the exocytosis release process of the transmitter vesicles. SNAP is a synaptosomal nerve-related protein that is closely related to neurotransmitter exocytosis. VAMP is a synaptic particle-related protein that is involved in adhesion and fusion of synaptic particles and plays an important role in synaptic particle exocytosis.
When the inventor researches the presynaptic proteins, only SYN proteins have sensitivity to terahertz wave radiation, and the expression quantity of the SYN proteins can be reduced under the action of the terahertz wave radiation, so that abnormal neuron states are caused, and the transmission of nerve information is blocked. Therefore, by detecting the change of the expression level of SYN protein in the neuron under the action of terahertz wave radiation, whether the state of the neuron is abnormal or not can be effectively judged.
According to an embodiment of the invention, the neuronal status is abnormal when the SYN protein expression is down-regulated. The inventor finds that under the action of terahertz wave radiation, SYN protein expression quantity in neurons is down-regulated, and the synapse vesicles of the neurons are released from disorder and abnormal in state.
According to an embodiment of the present invention, the terahertz wave radiation has an intensity of 30 to 60 milliwatts and a frequency of 0.1 to 0.3THz. Under the irradiation condition, SYN protein expression is down-regulated, so that abnormal neuron state occurs and nerve information transmission is blocked.
According to the embodiment of the invention, the time of the terahertz wave irradiation is 50-70 minutes. Under the irradiation condition, SYN protein expression is down-regulated, so that abnormal neuron state occurs and nerve information transmission is blocked.
In another aspect of the invention, the invention proposes the use of a reagent for detecting SYN protein in the preparation of a kit. According to an embodiment of the invention, the kit is used for detecting the state of neurons under the action of terahertz wave radiation. Under the action of terahertz wave radiation, the expression quantity of SYN protein is reduced, so that abnormal neuron information transmission is caused. Therefore, the expression quantity of SYN protein is detected by utilizing the reagent for specifically detecting SYN protein, so that whether the neuron state is abnormal or not can be effectively judged under the action of terahertz wave radiation.
In yet another aspect, the present invention provides a method of detecting a state of a neuron under the action of terahertz wave radiation. According to an embodiment of the invention, the method comprises: applying terahertz wave radiation to neurons, and measuring the expression quantity of SYN protein in the neurons before and after the radiation, wherein the expression quantity of SYN protein is down-regulated and is an indication of abnormal state of the neurons. Thus, by using the method according to the embodiment of the invention, whether the state of the neuron is abnormal under the action of terahertz wave radiation can be effectively detected.
According to an embodiment of the present invention, the terahertz wave radiation has an intensity of 30 to 60 milliwatts and a frequency of 0.1 to 0.3THz. Under the irradiation condition, SYN protein expression is down-regulated, so that abnormal neuron state occurs and nerve information transmission is blocked.
According to the embodiment of the invention, the time of the terahertz wave irradiation is 50-70 minutes. Under the irradiation condition, SYN protein expression is down-regulated, so that abnormal neuron state occurs and nerve information transmission is blocked.
In yet another aspect, the invention features a method of regulating expression of a SYN protein in a neuron. According to an embodiment of the invention, the method comprises: causing terahertz waves to radiate the neurons. The inventor finds that SYN protein has sensitivity to terahertz wave radiation, and the expression amount of the SYN protein can be changed under the action of the terahertz wave radiation.
According to an embodiment of the invention, the SYN protein expression is down-regulated. Under the action of terahertz wave radiation, SYN protein expression quantity is down-regulated.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 shows a change in rat brain slice and hippocampal neuronal synaptic structure after terahertz wave irradiation of 0.16THz/50mW, wherein A is a pseudo-irradiation group hippocampal neuronal synaptic structure, in accordance with one embodiment of the present invention; b is the synapse ultrastructure of the terahertz wave radiation group neuron;
FIG. 2 shows changes in hippocampal pre-synaptic vesicle associated protein following terahertz wave irradiation of 0.16THz/50mW, wherein A is SYN, rSec6, SNAP, and VAMP western blotting strips, according to one embodiment of the invention; b is a SYN, rSec6, SNAP and VAMP protein expression profile showing P <0.05 compared to group C;
FIG. 3 shows the change in expression of hippocampal neurons SYN after irradiation with terahertz waves of 0.16THz/50mW, wherein A is the result of pseudo-irradiation group hippocampal neurons SYN immunofluorescence, according to one embodiment of the present invention; b is a result of SYN immunofluorescence of the terahertz wave radiation group hippocampal neurons; c is SYN immunofluorescence result statistical plot showing P <0.05 compared to group C.
Detailed Description
The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1
1 Experimental method
1.1 preparation of rat hippocampal brain slice and primary hippocampal neuron culture method
Rat hippocampal brain slice preparation: after the rat was sacrificed, the brain was quickly craniotomized and placed in the low temperature artificial cerebrospinal fluid for incubation and continuous perfusion with 95% oxygen and 5% carbon dioxide gas. Rat brain was divided into two hemispheres from sagittal plane using a pathology cutter, rat brain hemispheres were fixed on a shake microtome using 502 glue, and low temperature artificial cerebrospinal fluid was incubated and continuously perfused with 95% oxygen and 5% carbon dioxide gas. The rat brain was corrected by setting a slice thickness of 200 μm to expose the hippocampus, setting a slice thickness of 50 μm, preparing a rat brain slice with a hippocampal structure, continuously preparing a plurality of brain slice slices, and incubating in artificial cerebrospinal fluid for 30min at 37 ℃ with a perfusion of 95% oxygen and 5% carbon dioxide gas using a brain slice incubation box. After the rat was sacrificed, the brain was rapidly opened and placed in low-temperature artificial cerebrospinal fluid for incubation, 95% oxygen and 5% carbon dioxide gas were used for continuous perfusion, 50 μm thick rat brain slices containing hippocampal structures were prepared with an oscillation microtome, and incubated in artificial cerebrospinal fluid for 30min for experiments.
Primary hippocampal neuron culture: taking a Wistar milk mouse within 12h of new born, and soaking and sterilizing the Wistar milk mouse in 75% alcohol. Breaking the end under aseptic condition, and taking brain. Under dissecting microscope, removing Hippocampus with ophthalmic forceps, digesting and dispersing, diluting cell suspension to 5×10 with planting solution 5 The density per ml was inoculated into polylysine-coated 35mm dishes and incubated in a carbon dioxide incubator. After the cells are attached, the culture medium is replaced, all the planting liquid is sucked, 2ml of feed liquid is added, cytarabine with the final concentration of 3-5 mug/ml is added into the culture liquid on the 3 rd day of culture, after 24 hours, half liquid replacement is performed, and then half liquid replacement is performed 2 times per week.
1.2 rat brain slice and primary hippocampal neuron terahertz wave radiation method
The rat brain slice and the primary hippocampal neuron cells are randomly divided into a pseudo-radiation group (group C) and a terahertz wave radiation group (group R) 2, wherein the group C is placed in an incubator and is not irradiated, the group R uses QS2-180THz wave emission sources, and the power range is 1-65 mW. The output power was 50mW when irradiated, the corresponding frequency was 0.16THz, and the irradiation time was 60min.
1.3 observation of synaptic ultrastructural view of rat hippocampal brain slice neurons
When THz wave radiation is carried out, the cell culture dish is coated by polylysine, artificial cerebrospinal fluid is infused, 95% oxygen and 5% carbon dioxide gas are continuously infused, brain slices are put in the cell culture dish, and the brain slices can be used for THz wave radiation after being completely stretched and attached by means of gravity. Immediately after THz wave irradiation, a rat brain slice model was prepared as a transmission electron microscope sample, and an image was observed and collected using an H7650 transmission electron microscope.
1.4 detection of primary hippocampal neuronal presynaptic vesicle-associated proteins SYN, rSec6, SNAP and VAMP
Collecting the primary hippocampal neurons of the groups C and R, treating the primary hippocampal neurons with RIPA lysate containing 1% protease inhibitor 6h after terahertz wave irradiation, performing ice lysis for 10min, adding the cell lysate into an EP tube, and adding loading buffer for boiling for 10min. Presynaptic vesicle-associated proteins SYN, rSec6, SNAP, and VAMP expression were detected using Western blot, incubated with the corresponding primary antibodies: rabbit anti-rSec 6 monoclonal antibody (1:1000), rabbit anti-SNAP monoclonal antibody (1:1000), rabbit anti-SYN monoclonal antibody (1:5000), rabbit anti-VAMP monoclonal antibody (1:5000) and rabbit anti-GAPDH polyclonal antibody (1:10000), overnight at 4 ℃. Adding corresponding secondary antibody (HRP-labeled rabbit or mouse-derived secondary antibody), incubating at normal temperature for 1h, washing with TBS-T buffer solution for 3 times, each time for 15min, dripping ECL+ luminescent solution onto the strip, and collecting image with FluorChem FC2 chemiluminescent imaging system.
1.5 verification of primary hippocampal neuronal SYN expression
The cell climbing tablet of the primary hippocampal neuron is prepared, the cell climbing tablet is fixed for 10min by using a mixed solution of methanol and acetone in a ratio of 1:1 after terahertz wave radiation for 6h, and the cell climbing tablet is preserved for standby at the temperature of minus 20 ℃ after air drying. The method for detecting the synapse-related protein by adopting the immunofluorescence method comprises the following specific steps: after washing the cell slide with PBS-T buffer solution, adding penetrating fluid (0.2% Triton-PBS), standing at room temperature for 10-15 min, adding blocking fluid (0.1% Tween-20,3% BSA PBS), and incubating at 37deg.C for 30min. The blocking solution was blotted off, SYN antibody diluted with 1:50PBS-T, added dropwise to the slide, and placed in a humidity box overnight at 4 ℃. After rewarming, the climbing slices are washed 3 times by PBS-T buffer solution for 5min each time. Under the dark condition, adding FITC fluorescent secondary antibody prepared by PBS-T buffer solution in a ratio of 1:200, and incubating for 45min at room temperature in dark condition. The cells were washed 3 times with PBS-T buffer for 5min each. In the dark, DAPI-containing caplets were used. And observing by a laser confocal microscope, randomly selecting 5 visual fields for photographing on each sample, taking FITC-labeled target protein as green fluorescence under a microscope, taking DAPI-labeled cell nuclei as blue fluorescence, and analyzing the IOD of the protein fluorescence by using Image-Proplus software.
2 results
2.1 disorder of release of synapse vesicles in hippocampal neurons due to terahertz wave radiation
The THz wave is immediately irradiated to prepare a sample, and the sample is observed under a transmission electron microscope, so that a synaptic structure is formed among brain slice and hippocampal neurons of the group C rats, vesicles are gathered in a presynaptic membrane, a synaptic gap is clearly visible, and a postsynaptic membrane thickens to form a postsynaptic compact substance, which is shown in A of figure 1. The number of presynaptic vesicles seen in group R was significantly greater than in group C, see B of fig. 1, suggesting a disorder in vesicle accumulation and release.
2.2 terahertz wave radiation can cause the SYN expression of the synaptobrevin of the hippocampal neurons to be down-regulated, but has no obvious influence on the expression of other vesicle proteins rSec6, SNAP and VAMP.
6h after the terahertz wave irradiates the primary hippocampal neurons, immunoblotting results show that compared with group C, the expression of SYN in group R is down-regulated (P < 0.05), while the expression of rSec6, SNAP and VAMP is not obviously changed (P > 0.05), as shown in figure 2; immunofluorescence results further demonstrated significant downregulation of SYN expression (P < 0.05), see fig. 3.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (1)
1. A method of modulating the downregulation of SYN protein expression in murine neurons, comprising: causing terahertz waves to radiate the murine neurons;
the intensity of the terahertz wave radiation is 30-60 milliwatts, and the frequency is 0.1-0.3 THz;
the time of the terahertz wave radiation is 50-70 minutes;
the murine neurons are rat primary hippocampal neuronal cells.
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