CN105969732B - A kind of neurocyte protection method - Google Patents

A kind of neurocyte protection method Download PDF

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Publication number
CN105969732B
CN105969732B CN201511009739.XA CN201511009739A CN105969732B CN 105969732 B CN105969732 B CN 105969732B CN 201511009739 A CN201511009739 A CN 201511009739A CN 105969732 B CN105969732 B CN 105969732B
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cell
plasma
nerve cell
nerve
power supply
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CN105969732A (en
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闫旭
欧阳吉庭
袁芳
乔雅隽
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Beijing Beizhuo Medical Technology Development Co.,Ltd.
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Beijing University of Technology
Beijing Neurosurgical Institute
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients

Abstract

The invention discloses a kind of neurocyte protection methods, belong to biomedical engineering field.A kind of neurocyte protection method, by the nerve cell of corona treatment culture, research finds that effective cytoprotection can be played using after low dose of short time processing cell.Present invention firstly provides " cytoprotection of plasma " concepts, and H can be effectively protected in the corona treatment for demonstrating low dosage for the first time in vitro2O2The nerve cell of damage, it is not only that neural cell injury reparation and the prevention and treatment of degenerative disease research provide new idea and method, and its price is more cheap, it operates more simple, carry out animal in the meaning of body research and clinical research with further, while there is extensive potential applicability in clinical practice.

Description

A kind of neurocyte protection method
Technical field
The present invention relates to a kind of neurocyte protection methods, belong to biomedical engineering field.
Background technique
Cranial vascular disease be endanger human life and health common disease and frequently-occurring disease, be current China first it is lethal and cause Disabled sick, patient 50%-70% leaves the handicaps such as paralysis, aphasia in survivor, brings heavy bear to society and family Load.More and more data prove that irreversible regression occurs for intracerebral privileged site neuron and loss is to cause nervous system disease The major reason of disease.Its inducement mechanism is there are many hypothesis, mitochondria dysfunction theory, emerging such as the unbalance theory of neurotransmitter Nerve toxicity of putting forth energy theory, paraprotein build up theory, gene mutation, inflammatory process, dysimmunity and Apoptosis theory etc.. In these hypothesis, oxidative stress and active oxygen (reactive oxygen species, ROS) are generated and are removed in the cell It is abnormal, direct or indirect relationship is suffered from other several mechanism, important regulating and controlling effect is played to the damage of neuron.When The generation of ROS, which increases or removes, to be reduced, or when reducing to the macromolecular reparation of oxidative modification, oxidative stress will occur.ROS exists Key player is played in cellular signal transduction, participates in a variety of physiology and pathologic process.
Hydrogen peroxide (H2O2) it is the product being metabolized in vivo, while being also a kind of main source ROS, in Induction of neuronal Key effect has been played in the damage or even death of cell.H2O2Caused by cell oxidative damage have become Studies On Neuronal oxidation One of important method of damage.SH-SY5Y cell is derived from the transfer osteoma for the neuroblastoma patient that 1970 establish Subbreed (SK-N-SH → SH-SY → SH-SY5 → SH-SY5Y) of the stove cell SK-N-SH after three time clonings.During the cell is shown Deng horizontal dopamine-beta-hydroxy enzymatic activity, cell differentiation is lower, cellular morphology, physiology and biochemical function and normal mind It is similar through cell, and there is apparent aixs cylinder.SH-SY5Y cell is to H2O2Induced oxidation stress have high susceptibility, H2O2It lures Lead the research that SH-SY5Y cell oxidative damage model is widely used in the neuron degeneration apoptosis of oxidative stress induction.Institute Use SY5Y cell line SH-SY5Y cell as ex vivo nerve meta-model using this research.
In recent years, lower temperature plasma technology becomes an emerging research hotspot in the application of field of biomedicine.Deng Gas ions are the ionized gas that is excited, by a large amount of free electrons and ion and a small amount of unionized gas molecule and atom group At, and show as being similar to electroneutral on the whole.In the lab, plasma is generated by high voltagehigh frequency ionized gas, Its temperature is controlled to room temperature (low temperature), contains a large amount of O2-、NO-And N3-Isoreactivity ion, application are concentrated mainly on swollen In terms of apoptosis of tumor and wound repair.Protective effect about plasma to nerve cell, does not have been reported that so far.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of nerve cell of using plasma technical treatment cell guarantors Maintaining method.
To achieve the above object, the invention adopts the following technical scheme:
A kind of neurocyte protection method handle within 0.5-5 seconds to nerve cell, using pure when processing with plasma He gas, power supply 10-20kV, 20-50kHz, sine ac power supply, the nozzle distance tissue culture plate bottom surface of plasma Distance is 20-60mm.
The neurocyte protection method preferably handle within 0.5-5 seconds to nerve cell with plasma, and when processing makes With pure He gas, power supply 13-15kV, 20-40kHz, sine ac power supply, the nozzle distance cell culture board bottom of plasma The distance in face is 30-50mm.
The neurocyte protection method, optimal selection plasma carries out processing in 0.5-5 second to nerve cell, when processing Using pure He gas, power supply 13.4-13.6kV, 30kHz, alternating voltage voltage signal, the nozzle distance cell of plasma The distance of culture plate bottom surface is 40mm.
By the nerve cell of corona treatment culture, research finds to handle cell using the low dose of short time present invention Afterwards, effective cytoprotection can be played.The dosage of processing is depending on processing time, plasma intensity and nozzle and carefully The distance between born of the same parents.These conditions can be adjusted in a certain range according to practical operation situation, for example plasma is strong When degree increases, the processing time can slightly shorten；When Distance Shortened of the nozzle away from cell, plasma intensity can be reduced.
The nerve cell includes but is not limited to SH-SY5Y cell, HT-22 cell, PC-12 cell strain and primary nerve First cell.
The plasma is low temperature plasma.Plasma is considered as of the substance in addition to solid-state, liquid, gaseous state Four states, it by after atom or molecular ionization non-bound state electronics and particle buildup form, macroscopically be in electroneutral.In universe 90% or more substance is all existing for the state with plasma, such as the ionosphere around the earth, the sun, nebula, aurora, Common fluorescent lamp, plasma television, the waterproof coating on baby' diaper surface etc. in people's daily life.In plasma In, the temperature of plasma depends on ion temperature Ti.Plasma presses temperature classifications: referred to as high-temperature plasma when Ti >=104K Body is then known as low temperature plasma when Ti≤104K.And low temperature plasma is divided into hot plasma (Ti >=103K) and cold Plasma (Ti≤103K), the former electron temperature and ion temperature difference very little and close to neutral particle temperature, also referred to as Equilibrium plasma, such as arc-plasma；The latter's electron temperature is more much higher than ion temperature, also referred to as non-equilibrium or non-thermal etc. Gas ions, such as glow discharge, corona discharge and dielectric barrier discharge plasma.Plasma is generally in the non-thermodynamics of height Equilibrium state.
The low temperature plasma that present invention research uses is provided by Beijing Institute of Technology, is coaxial coplanar DBD electric discharge device, Article " the Electrical characteristics and formation delivered referring to Lijuan Liu et al. people in 2014 The mechanism of atmospheric pressure plasma jet (electrology characteristic and shape of atmosphere pressure plasma jet flow At mechanism) ", Applied Physics Letters 104,244108 (2014)；Doi:10.1063/1.4884939, article It is linked as http://dx.doi.org/10.1063/1.4884939.The size of medium tube is changed to internal diameter in the present invention For 1mm, outer diameter 2mm, two electrodes are entwined by the metal aluminum foil that width is 3mm, and the electrode on the left side is power electrode, driving Power supply is the ac signal that frequency is f=30kHz, and the electrode on the right is grounding electrode, and high-pure helium is from medium tube left end with solid Constant flow flows into.In addition to this, this may be implemented in other low-temperature plasma devices for being capable of providing Parameter Conditions of the present invention Goal of the invention.
Neurocyte protection of the present invention refers to that neural cell injury caused by mitigating oxidative stress and activity reduce.
The neural cell injury is by H2O2Caused by cellular damage caused by cell oxidative damage and activity reduce.
The research of the invention finds that low temperature plasma is used for the purposes of neurocyte protection.Espespecially by low temperature plasma For protecting the purposes of neural cell injury caused by oxidative stress.
Present invention firstly discovers that protective effect of the plasma to nerve cell, the discovery in the prior art with etc. from Daughter processing cell is completely opposite to the application for causing Apoptosis.The study find that having for neurocyte protection research Special Significance provides new idea and method for neural cell injury reparation and the prevention and treatment of degenerative disease research.
The invention has the advantages that present invention firstly provides " cytoprotection of plasma " concepts, and in vitro for the first time H can be effectively protected in the corona treatment for demonstrating low dosage2O2The nerve cell of damage, not only neural cell injury It repairs and the prevention and treatment of degenerative disease research provides new idea and method, and its price is more cheap, operation It is more simple, have and further carry out animal in the meaning of body research and clinical research, while before there is extensive clinical application Scape.
The present invention will be further described with reference to the accompanying drawings and detailed description, not limitation of the present invention, all According to this field equivalent replacement that the disclosure of invention carries out, the scope of the present invention is belonged to.
Detailed description of the invention
Fig. 1 is the cell survival rate figure after the corona treatment nerve cell of embodiment 1.
Fig. 2 is the cellular morphology figure after the corona treatment nerve cell of embodiment 1.
Specific embodiment
Embodiment 1: the neurocyte protection experiment of plasma
One, experimental material
1, SH-SY5Y cell is purchased from Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences's cell resource center.
2,1640 culture mediums and FBS (Fetal Bovine Serum) are purchased from Gibco company.
3, plasma device: Beijing Institute of Technology provides, and for coaxial coplanar DBD electric discharge device, medium bore is 1mm, outer diameter 2mm, two electrodes are entwined by the metal aluminum foil that width is 3mm, and the electrode on the left side is power electrode, driving electricity Source is the ac signal that frequency is f=30kHz, and the electrode on the right is grounding electrode, and high-pure helium is from medium tube left end with fixation Flow flows into.
4, CCK-8 cell Proliferation and activity detection kit: it is purchased from colleague's chemistry institute (CK04).
Two, experimental method
1, the preparation of experimental cell:
(1) SH-SY5Y cell culture, condition of culture: 1640 culture mediums, 15%FBS (Fetal Bovine Serum), 5%CO237 DEG C of incubator cultures.
(2) logarithmic phase SH-SY5Y cell is taken, adjusts concentration of cell suspension most 2*10 after pancreatin digestion5A/ml's or so It is added in 96 well culture plates, every 100 μ l of hole, 37 DEG C, 5%CO2Condition continues to cultivate.
2, corona treatment:
(1) use pure He gas, experimental power supply 13.6Kv, 30KHz, sine ac power supply, the nozzle of plasma away from The distance of 96 porocyte plates bottom surfaces is 40mm.
(2) 96 orifice plates are subjected to plasma treatment, temporally gradient divides 0.5S, 1S, 2S, 5S, 10S, each time gradient Handle 3 hole cells.Taking 1 group of cell simultaneously is control group, only carries out helium gas jet processing.
(3) culture solution in 96 orifice plates is sucked out after handling, cell replacement fresh culture continues to cultivate.
3、H2O2Damage and CCK-8 detection:
(1) after cultivating 18 hours, the culture medium in each hole cell of 96 porocyte plates is changed to containing 200 μM of final concentration H2O2Culture medium, handle 1h cell.
(2) the CCK-8 reagent of 10 μ l is added in every hole.
(3) 37 DEG C, 5%CO2Incubator is incubated for 2-4h.
(4) the OD value at microplate reader measurement 450nm.
(5) cell survival rate in each hole is calculated.
4, cell picture shooting
The cell to each grouping in 18 hours will be cultivated, is clapped with Cai Si Axio Observer A1 inverted microscope It takes the photograph, amplification factor is 100 times.
Three, experimental result and analysis
Shown in referring to Figures 1 and 2, with the growth of plasma treatment time, 0.5-5 seconds stages, H2O2That damages is thin Born of the same parents' survival rate gradually rises, and cell state is significantly improved.Show that corona treatment alleviates H2O2Caused by cell oxygen Changing cellular damage caused by damaging and activity reduces, and has significant protective effect to cell.
When the processing time reaches 10s, cell may receive damage, to exacerbate H2O2Degree of injury.

Claims (7)

1. a kind of external guard method of nerve cell, it is characterised in that: carried out 0.5-5 seconds with low temperature plasma to nerve cell Processing, uses pure He gas when processing, power supply 10-20kV, 20-50kHz, sine ac power supply, the nozzle of plasma away from It is 20-60mm with a distance from tissue culture plate bottom surface；The nerve cell is SH-SY5Y cell.
2. the external guard method of a kind of nerve cell according to claim 1, it is characterised in that: with low temperature plasma pair Nerve cell handle within 0.5-5 second, using pure He gas when processing, power supply 13-15kV, 20-40kHz, and sinusoidal ac Source, the distance of the nozzle distance tissue culture plate bottom surface of plasma are 30-50mm.
3. the external guard method of a kind of nerve cell according to claim 2, it is characterised in that: with low temperature plasma pair Nerve cell handle within 0.5-5 second, using pure He gas when processing, power supply 13.6kV, 30kHz, and sine ac power supply, etc. The distance of the nozzle distance tissue culture plate bottom surface of gas ions is 40mm.
4. according to claim 1 to the external guard method of a kind of nerve cell described in any one of 3, it is characterised in that: institute It states neurocyte protection and refers to that neural cell injury caused by mitigating oxidative stress and activity reduce.
5. the external guard method of a kind of nerve cell according to claim 4, it is characterised in that: the neural cell injury And it is by H that activity, which reduces,2O2Caused by cellular damage caused by cell oxidative damage and activity reduce.
6. low temperature plasma described in claims 1 to 3 is used for the purposes that nerve cell is protected in vitro, the cell is SH- SY5Y cell.
7. low temperature plasma described in claims 1 to 3 is for mitigating the external of neural cell injury caused by oxidative stress Purposes, the cell are SH-SY5Y cell.
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CN101702866A (en) * 2009-11-27 2010-05-05 北京理工大学 Controllable plate-like plasma generating device

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US20130197023A1 (en) * 2012-01-27 2013-08-01 California Institute Of Technology Neuroprotection by pharmacological chaperoning of nicotinic acetylcholine receptors

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Publication number Priority date Publication date Assignee Title
CN101702866A (en) * 2009-11-27 2010-05-05 北京理工大学 Controllable plate-like plasma generating device

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