CN105403593A - Method for detecting neurotoxicity of magnesium or magnesium alloy - Google Patents
Method for detecting neurotoxicity of magnesium or magnesium alloy Download PDFInfo
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- CN105403593A CN105403593A CN201510851921.3A CN201510851921A CN105403593A CN 105403593 A CN105403593 A CN 105403593A CN 201510851921 A CN201510851921 A CN 201510851921A CN 105403593 A CN105403593 A CN 105403593A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention belongs to the field of metal ion toxicity detection and discloses a method for detecting neurotoxicity of magnesium or magnesium alloy. The method comprises the steps of using a lactate dehydrogenase (LDH) method to detect cell viability; and using a chick embryo forebrain neuron-microelectrode array biosensor to detect neural network electrophysiological signals. Experimental results according to the detection method are stable and reliable, and the method is simple and convenient to operate and high in efficiency and is a high-throughput detection method.
Description
Technical field
The invention belongs to Metal detection field, specifically a kind of detection magnesium or the neurovirulent method of magnesium alloy.
Background technology
In recent years, Mg-based hydrogen storage is swift and violent as the research and development of implantable medical material, has become one of focus of novel biomaterial research in world wide.Magnesium alloy has good mechanical property, close with human bone mineral density; Wholly-degradable in vivo, can reduce the misery that second operation brings patient; The magnesium ion that degraded produces is one of kation of needed by human, has extraordinary application prospect, also expand to the fields such as surgical repair material, dental prosthetic material recently as orthopedic implanting material, vascular stent material etc.But it is too fast that magnesium alloy also also exists degradation speed, the problem that corrosion resistance is on the low side.The subject matter brought thus is whether the corrosion product that its fast degradation produces has good biocompatibility.Current biocompatibility in vitro research is mainly concentrated into osteocyte, fibroblast, vascular endothelial cell and blood compatibility, but for the more responsive neurocyte of toxicity, does not but almost report.
Although magnesium ion is the fourth-largest ion in human body, in body can tolerable concentration very high.But after magnesium alloy implants, degradation speed is very fast, local magnesium ion and alloy ion concentration thereof around implant will be caused to raise suddenly, genotoxic potential will be existed to peripheral nerve.And by blood circulation through blood-brain barrier, also potential hazard can be there is to central nervous system in a large amount of magnesium ion and alloy ion thereof.And its common alloying element aluminium in magnesium alloy, zinc, lithium, manganese etc. are all known neurotoxic substances.Before Mg-based hydrogen storage material enters clinical practice in a large number, study it and impact of neurocyte is necessary very much.Moreover, someone proposes magnesium alloy materials to be used as CO2 laser weld timbering material, and Preliminary Animal Experiment demonstrates its feasibility, therefore before Magnesium and magnesium alloys material enters clinical practice in a large number, be necessary very much to study its potential impact to neurocyte.
According to ISO10993 and GB/T16886 regulation, the detection method of biological material cell toxicity has contact method and extraction.Magnesium alloy materials can produce galvanic corrosion after implanting, and have violent chemical reaction and raise along with pH value and hydrogen generation, research shows the inapplicable contact method of magnesium alloy materials, and needs to adopt extraction.
Mtt assay is the cytoactive research method be most widely used at present, but mtt assay disturbing factor is more, easily causes experimental result stable not.
The neurocyte of in vitro culture can be spontaneous formation neural network and produce stable electricity physiological signal.Microelectrode array (MEA) be latest developments get up can Noninvasive, the technology of real-time, long-term detection Electrophysiology signal.The neural network of in vitro culture and microelectrode array are combined the neurovirulent detection of electro physiology that the biology sensor formed is widely used in medicine and chemical combination material in recent years.Because MEA biology sensor can realize high flux, the high detection exported, and its testing result obtained has good consistance with in vivo studies, is referred to as " the neurovirulent detection platform of 21st century physiology ".Although the neuro-physiology that MEA has been widely used in medicine detects, the detection for biomaterial have not been reported.
Summary of the invention
The object of this invention is to provide the stable detection magnesium of a kind of result or the neurovirulent method of magnesium alloy.
For achieving the above object, the present invention by the following technical solutions:
A kind of detection magnesium or the neurovirulent method of magnesium alloy, described method comprises: detect cytoactive by LDH method; And, detect neural network electricity physiological signal with chicken proembryo brain neuron-microelectrode array biology sensor.
Further, described LDH method detection cytoactive comprises the following steps:
S1, the leaching liquor prepared magnesium ion solution, prepare magnesium or magnesium alloy;
S2, use Neurobasal nutrient solution cultivate chicken proembryo brain neuron;
S3, past chicken proembryo brain neuron add the leaching liquor of magnesium or magnesium alloy, detect cytoactive with LDH.
Further, described S1 is specially:
S11, the sample of magnesium or magnesium alloy is placed in the centrifuge tube of sealing, adds the lixiviate of Neurobasal nutrient solution;
Take out sample after S12,24h, after leaching liquor is centrifugal, get supernatant;
S13, the pH value detecting leaching liquor and magnesium ion concentration;
S14, leaching liquor save backup after membrane filtration, dilution.
Further, described S2 is specially:
96 orifice plates are spread polyethyleneimine by S21, kind day before cell;
S22, acquisition chicken proembryo brain tissue, use EDTA trypsinization, then blow and beat into single cell suspension, then centrifugal supernatant discarded, add M199 nutrient solution and make cell suspension, and add 96 orifice plates, be placed in incubator;
M199 nutrient solution is all changed into Neurobasal nutrient solution after S23,24h;
S24, within the 3rd day, add cytarabine.
Further, described acquisition chicken proembryo brain tissue is specially:
S221, get the fertilized eggs of 7th ~ 9 days, sterilization, breaks into pieces egg head with tweezers, shells, push shell membrane aside, takes out chicken embryo and is put into the double dish that HBSS is housed;
S222, to block with the head of tweezers by chicken embryo, put into the double dish that another has HBSS;
S223, the head back side adding chicken embryo are put upward, push down midbrain position, and the meninx of forebrain is opened, and take out two white tissues, are put in the double dish of HBSS, remove, obtaining chicken proembryo brain tissue by being attached to meninx remaining outside white tissues.
Further, described S3 is specially: discard whole supernatant when chicken proembryo brain neuron is cultured to the 5th day, add magnesium ion solution or leaching liquor, carries out LDH detection respectively at the 6th, 8,10 day.
Further, described LDH detects and is specially: from 96 orifice plate Aspirate supernatant to new 96 orifice plates, every hole adds reaction buffer, 30min is placed in room temperature dark place, then add stop solution mixing, be measure wavelength with 490nm, 680nm is reference wavelength, utilize microplate reader to measure the optical density value in each hole, calculate the relative release rate of LDH.
Further, describedly detect neural network electricity physiological signal with chicken proembryo brain neuron-microelectrode array biology sensor and comprise the following steps:
S5, prepare the leaching liquor of magnesium or magnesium alloy;
S6, Neurobasal nutrient solution is added microelectrode array, microelectrode array is placed in incubator, tracer signal;
S7, when electric signal reaches stable state, continuous three days, electric signal 30min when measuring balance every day;
S8, the 3rd day time, change the Neurobasal nutrient solution of 1/4th into leaching liquor, then continuous coverage three sferic signal, every day measures 30min;
S9, the 6th day time, entirely change nutrient solution into Nostoc commune Vanch liquid, continuous coverage three sferic signal, every day 30min.
The present invention has following beneficial effect:
The present invention is based on LDH method and chicken proembryo cranial nerve network-microelectrode array biology sensor, and establish and detect magnesium or the neurovirulent method of magnesium alloy, the method experimental result is reliable and stable, easy and simple to handle, and efficiency is high, is a kind of high-throughout detection method.
Accompanying drawing explanation
Fig. 1 is the concentration effect curve of magnesium ion to neural cell activity;
Fig. 2 is the testing result of 100% leaching liquor to neural cell activity;
Fig. 3 is the testing result of 50% leaching liquor to neural cell activity;
Fig. 4 is the testing result of 25% leaching liquor to neural cell activity;
Fig. 5 be continuous three days effect 25% leaching liquors or 3mM magnesium ion on the impact of neural network spike potential frequency;
Fig. 6 is that continuous three days effect 25% leaching liquors or 3mM magnesium ion break out the impact of frequency to neural network;
Fig. 7 is that continuous three days effect 25% leaching liquors or 3mM magnesium ion account for the impact of outburst number percent to neural network spike potential;
Fig. 8 is that continuous three days effect 25% leaching liquors or 3mM magnesium ion break out the impact of length to neural network;
Fig. 9 is the impact of spike potential frequency in continuous three days effect 25% leaching liquors or 3mM magnesium ion break out neural network;
Figure 10 be continuous three days effect 25% magnesium alloy leaching liquors or 3mM magnesium ion on the impact of neural network spike potential amplitude.
Embodiment
Below in conjunction with specific embodiment, the present invention is described further:
embodiment 1
Preparation magnesium ion solution
Take 1.015gMgCl
26H
2o(M2670, Sigma), be dissolved in 50mL chicken proembryo brain tissue neuronal cell cultures base (serum-free), obtain the magnesium ion solution of 100mM, dilute the solution that different multiples obtains 30mM, 10mM, 3mM, 1mM, 0.3mM, 0.1mM magnesium ion respectively, 4 DEG C of refrigerator and cooled are frozen stand-by.
Prepare the leaching liquor of magnesium or magnesium alloy
(1) by polishing to the pure magnesium of as cast condition of 2000# and WE43(Hunan rare earth metal research institute, 1.5cm × 1.5cm × 1cm), ultrasonic cleaning 20 minutes in ethanol (analyzing pure), drying at room temperature in super-clean bench.
(2) being placed in by sample in the centrifuge tube that can seal, is 3cm by leaching liquor and surface area ratio
2the ratio of/mL adds chicken proembryo brain neuron nutrient solution (Neurobasal, 2%B27,1%GluMax, 0.25% is dual anti-), is placed in the incubator lixiviate 24h of 37 DEG C.
Take out after (3) 24 hours, and with the centrifugation 5min of 2000rpm, take out supernatant.
(4) detect the pH value of leaching liquor with pH instrument, supernatant uses ICP-AES (ICP-AES, Optima5300DV, PerkinElmer, USA) to detect magnesium ion concentration after diluting 10 times.
(5) leaching liquor for cell chulture adopts 0.22 μm of membrane filtration, is diluted to 50%, 25% with ordinary cells nutrient solution, and be placed in 4 DEG C of refrigerators and save backup, the time is no more than 1 week.
Cultivate chicken proembryo brain neuron
(1) plant day before cell, 96 orifice plates (Corning) are spread 50 μ L0.05%w/v polyethyleneimine (PEI, Sigma), is placed in 37 DEG C of incubators and spends the night, within second day, suck supernatant, and wash three times with deionized water, dry up stand-by in super-clean bench.
(2) get the white Leghorn fertilized eggs of the 7th day to the 9th day, spray 70% alcohol disinfecting, all dissection apparatus are put into dissection super-clean bench.
(3) with blunt medium size tweezers, egg head (major part) is broken into pieces, and removes eggshell carefully, push shell membrane aside, take out chicken embryo be put into ice-cold HBSS(Gibco is housed) 35mm double dish in.
(4) with No. 5 tweezers, the head of chicken embryo is blocked, put into another new having in the 35mm double dish of HBSS.
(5) put upward at the head back side of chicken embryo, midbrain position pushed down by left hand No. 5 tweezers, and the meninx of forebrain opened by the right hand No. 5 tweezers, then two white tissue holders gone out.
(6) two white organizing being transferred to one newer has in the 35mm double dish of HBSS, removes, the white tissues of these two crescent shapes and forebrain tissue by being attached to meninx remaining outside white tissues.
(7) with 0.25%EDTA pancreatin (Trypsin, Sigma) at 37 DEG C of digestion chicken proembryo brain tissue 3min, then single cell suspension is blown and beaten into 1mL rifle head, the then centrifugal 5min of 1000rpm, abandoning supernatant, adding M199 nutrient solution (Sigma) (containing 2%B27), to be prepared into density be 6.4 × 10
5the cell suspension of/mL; 100 μ L cell suspensions are added bag by 96 good orifice plates, be placed in 37 DEG C, 5%CO
2in incubator.
(8) M199 nutrient solution is all changed into NeuroBasal nutrient solution (Gibco) after 24h.
Within (9) the 3rd days, add 50 μ L3 μM cytarabine (AraC, Sigma), be used for suppressing glia proliferation.
Within (10) the 5th days, discard whole supernatant, add 150 μ L containing the nutrient solution of magnesium ion of variable concentrations or different dilution magnesium or magnesium alloy leaching liquor, normal incubation medium is blank negative control, and often group establishes 5 Duplicate Samples; LDH detection is carried out respectively at the 6th, 8,10 day (after dosing the 1st, 3,5 day).
(11) LDH detects: from each hole, draw 50 μ L supernatants to new 96 orifice plates, LDH according to Pierce company detects suit, every hole adds the reaction buffer that 50 μ L prepare, and places 30min, then add 50 μ L stop solutions and slightly mix in room temperature dark place.Be measure wavelength with 490nm, 680nm is reference wavelength, utilizes microplate reader (BioTek, Synergy
tM4) optical density (OD) value in each hole is measured, the relative release rate according to the LDH of following formulae discovery cell: (test group OD value)/(negative control OD value) × 100%.
All results are all represent with mean value ± standard error (SEM).LDH result all represents with the percent relative to negative control.Use SPSS20.0 software, multiple-group analysis single factor test ANOVA and Turkey compares between two, and two groups are compared and do statistical test by the t method of inspection.With GraphpadPrism5.0 software matching amount effect curve, and estimate EC
50(medium effective concentration) and hill coefficient.P<0.01 indicates significant difference (* p<0.01, * * p<0.001).
To the concentration effect curve of neural cell activity as described in Figure 1, magnesium ion is to the EC of cytoactive for magnesium ion
50value (mM) and nH value as shown in table 1.When magnesium ion concentration is increased to 10mM from 0.01mM time, cell mortality does not all significantly increase.1st day, the 3rd day, the EC of the 5th day
50value is respectively 38.59mM, 36.37mM, 23.02mM.Along with magnesium ion increases action time, the pharmacodynamic profiles of magnesium ion is from the pattern of advancing by leaps and bounds (nH>6), to transition type pattern (2<nH<6), arrive incremental model (nH<2) again, show that the tolerance of cell magnesium ion reduces gradually.T inspection shows, when magnesium ion concentration the 1st day, the 3rd day, within the 5th day, to reach 30mM(115.50% respectively), 30mM(156.71%), 30mM(171.94%) and the increase of cell lethality has significant difference.
100% magnesium leaching liquor and 100%WE43 leaching liquor to the result of neural cell activity as shown in Figure 2, the LDH burst size of two kinds of leaching liquors far above control group, and along with the increase of time, the trend obviously increased.Adding of WE43 leaching liquor makes cell at the 1st day, and the 3rd day, the LDH burst size of the 5th day reached 182.22%, 168.96%, 175.30% of control group respectively, and pure magnesium leaching liquor is then 169.98%, 158.40%, 179.49% respectively.Obvious significant difference is not had between two groups.
To the result of neural cell activity as shown in Figure 3, although 50% leaching liquor makes cell LDH burst size low compared with 100%, comparing control group still has obviously significant difference for 50% magnesium leaching liquor and 50%WE43 leaching liquor.Adding of 50%WE43 leaching liquor makes cell at the 1st day, and the 3rd day, the LDH burst size of the 5th day reached 138.81%, 130.24%, 122.30% of control group respectively, and pure magnesium leaching liquor is then 125.04%, 123.68%, 123.95% respectively.Leaching liquor causes cell burst size the highest at first day, along with the time increases, has decline trend.Obvious significant difference is not had between WE43 and two groups, pure magnesium.
25% magnesium leaching liquor and 25%WE43 leaching liquor are to the result of neural cell activity as shown in Figure 4, can find out, when first day, WE43 and pure magnesium leaching liquor make cell LDH discharge and are respectively 121.03% of control group, 113.76%, compare obvious significant difference with control group, during to the 3rd day and the 5th day, although comparatively control group also increases to some extent, there is no obvious significant difference.And the LDH amount that the leaching liquor of WE43 discharges than pure magnesium leaching liquor is slightly high, but does not more also have significant difference between two.In summary, along with dilution increase, the neurotoxicity of magnesium alloy leaching liquor reduces gradually, and LDH burst size is the highest relative to control group when first day.
embodiment 2
The preparation of the leaching liquor of magnesium or magnesium alloy is identical with embodiment 1.
The outer microelectrode array technology of born of the same parents refers on glass or silicon base; with microelectronic processing technique, the metals such as Au, Ir or Pt are deposited formation electrode and lead-in wire on it; adopt passivation layer protection lead-in wire; electrode exposes and cells contacting region, transmits and record the isoparametric cell sensor of action potentials of cells frequency, amplitude, waveform and speed.It providing the method for a kind of long-term non-destructive monitoring cellular electrophysiologicalsensor activity, by high flux passage, transmitting out from having the data obtained in electroactive extracellular change in electric.Due to its make simple, good biocompatibility, can reuse, the advantage such as available conventional microscope observation, be widely applied in neural, cardiac muscle cell field.According to different designs, MEA both can also can electricity physiological signal outside detection bodies in detection bodies.The neural network of in vitro culture is formed biology sensor in conjunction with MEA and is used for nervous physiology toxicity detection by the present invention.MEA can realize detection rapidly and efficiently, and can obtain the result parameter of many terminals in conjunction with Computer Analysis technology simultaneously.
At present, MEA technology is very ripe, and commercialization.Now main commercial company has seven: the AlphaMEDSciences of Japan, AxionBiosystems, Plexon of the U.S., Inc. and Tucker-DavisTechnologies, AyandaBiosystems and 3-Brain of Switzerland, the MultiChannelSystems of Germany.What the present embodiment adopted is the standard microelectrode array chip that German MCS company produces.
An advantage of MEA is exactly multiple supplemental characteristics that simultaneously can provide Time and place, and the result of this high information quantity provides the neurovirulent platform of multi-angular analysis.Example introduction is trained for below with spike potential, spike potential training just refers to that the signal (spike potential and outburst) of MEA identifies and extraction according to certain standard by the software of application signal transacting, and extracts more parameter on this basis to analyze multichannel signal mutual relationship over time and space.Spike potential training is the most basic data analysing method of MEA, and has developed some commercialization analysis software at present, as Neuroexplorer(NexTechologies, U.S.), more laboratory applications be the analysis software of independent research.
The most direct signal that MEA obtains is exactly spike potential, and therefore spike potential frequency is most widely used parameter.The drug concentration of spike potential frequency and variable concentrations simulates semilog dosage beneficial effect curve, and obtains the pharmacodynamic parameters about this medicine thus, as EC
50(medium effective concentration) EC
10, EC
90, hill coefficient etc.After the training of application spike potential, the correlation parameter of more neural network can be obtained, the influencing mechanism of chemical substance to bioelectrical activity can be understood further.
Pure magnesium and WE43 leaching liquor are on the detection of the impact of electricity physiological signal:
The signal considering each neural network every day fluctuates in certain scope, therefore within continuous three days, measures electric signal and average to observe leaching liquor to the recovery situation after the impact of MEA signal and wash-out.
(1) Neurobasal nutrient solution is added microelectrode array, microelectrode array is placed in incubator, when the electric signal of MEA reaches more stable state, continuous three days, measures electric signal 30min when balancing every day.
When (2) the 3rd days, the nutrient solution of 1/4th is changed into the pure magnesium of 250 μ L or the magnesium ion nutrient solution of WE43 leaching liquor or 12mM, then continuous coverage three sferic signal, every day measures 30min.
When (4) the 6th days, nutrient solution is changed entirely into fresh Nostoc commune Vanch liquid, continuous coverage three sferic signal, every day 30min.
The result of embodiment 1 shows that 25% pure magnesium and WE43 magnesium alloy leaching liquor have no significant effect the activity of neurocyte after 3 days in effect, therefore selects the detection of the leaching liquor application point physiological signal of 25%.Again because the concentration of extra magnesium ion is about 2.43mM in 25% pure magnesium leaching liquor, in WE43, extra magnesium ion concentration is about 2.8mM, has therefore selected the magnesium ion of 3mM in contrast.Measure the electric signal 30min of equilibrium state every day, using the mean value of first three day of dosing as baseline, the mean value action effect in dosing period three sky, the mean value of three days after wash-out is that recovery is added up.Often group test obtains by three MEA result statistics.
Because the signal difference of each electrode of MEA is very large, in order to directly make comparisons, the value standardization (value divided by the baseline of correspondence) of each electrode is calculated later.All results are all represent with mean value ± standard error (SEM).Do statistical test with SPSS20.0 software, same MEA different time points compares with baseline and compares with single factor test ANOVA and Dunnet ' s afterwards, compares and compare between two with single factor test ANOVA and Turkey between different groups.Two groups are compared and check with t.With GraphpadPrism5.0 software matching amount effect curve, and estimate EC
50(medium effective concentration) and hill coefficient.P<0.01 indicates significant difference (* p<0.01, * * p<0.001).
On the impact of signal frequency
On the impact of neural network spike potential frequency as shown in Figure 5, when magnesium alloy leaching liquor or magnesium ion solution act on neural network, spike potential frequency is all obviously suppressed for continuous three days effect 25% leaching liquors or 3mM magnesium ion.After the effect of 25%WE43 leaching liquor, spike potential frequency only has 17.73% of baseline, after 25% pure magnesium leaching liquor effect, only have 20.42% of baseline, and by contrast, the effect of 3mM magnesium ion solution has only been suppressed to 50.15% of control group, far above leaching liquor group (p<0.001).After 25%WE43 leaching liquor wash-out, spike potential frequency retrieval, to 75.67%, after pure magnesium leaching liquor wash-out, returned to 73.52%, and after 3mM magnesium ion solution wash-out, spike potential frequency but drops to 20.33%, and leaching liquor group has notable difference.
On the impact of neural network outburst frequency as shown in Figure 6, after leaching liquor or magnesium ion solution act on neural network and wash-out, the change of outburst frequency is more consistent with the variation tendency of spike potential frequency for continuous three days effect 25% leaching liquors or 3mM magnesium ion.Application single factor test ANOVA compares to compare between two with Turkey and draws, 25%WE43 leaching liquor group and 25% pure magnesium leaching liquor group are not obviously distinguished, but all have significant difference between 3mM magnesium ion solution group and this two leaching liquor groups.
On the impact of outburst structure
Continuous three days effect 25% leaching liquors or 3mM magnesium ion account for the impact of outburst number percent as shown in Figure 7 to neural network spike potential, and when 3mM magnesium ion solution effect three days, the number percent that spike potential accounts for outburst slightly declined, and leaching liquor does not significantly affect.After wash-out, the number percent that these three groups of spike potentials account for outburst declines all to some extent, but magnesium ion solution group drops to 66.82%, is starkly lower than 25%WE43 leaching liquor group (88.03%) and 25% magnesium alloy leaching liquor group (86.19%).
On the impact of neural network outburst length as shown in Figure 8, between 25%WE43 leaching liquor action period, 125.14%, the 25% pure magnesium leaching liquor that outburst length increases baseline is that outburst length increases 133.48% for continuous three days effect 25% leaching liquors or 3mM magnesium ion.But 3mM magnesium ion solution makes outburst length decline slightly to some extent, only has 87.44% of baseline.After wash-out, two kinds of leaching liquors outburst length returned to baseline values, and 3mM magnesium ion solution group continue decline, only have 67.12% of baseline.
On the impact of spike potential frequency in neural network outburst as shown in Figure 9, can find out, this parameter is more consistent with the variation tendency of outburst length parameter for continuous three days effect 25% leaching liquors or 3mM magnesium ion.When between two kinds of leaching liquor action periods, in outburst, spike potential frequency increases all to some extent, and 25%WE43 leaching liquor is increased to 123.08% of baseline, and the pure magnesium leaching liquor of 25% is increased to 123.97% of baseline.Then after wash-out, all returned to baseline values (91.38% and 99.84%).And 3mM magnesium ion solution has no significant effect spike potential frequency in outburst between action period, but after wash-out, have decreased to 77.42% of baseline, be starkly lower than baseline group, also have significant difference (p<0.01) with leaching liquor group.
Can be found out with the parameter that outburst structure is relevant by these three, the impact of 3mM magnesium ion solution group on the outburst structure of neural network is greater than leaching liquor group.
On the impact of spike potential amplitude
Continuous three days effect 25% magnesium alloy leaching liquors or 3mM magnesium ion are on the impact of neural network spike potential amplitude as shown in Figure 10, two kinds of leaching liquors all significantly do not affect spike potential amplitude, but magnesium ion solution is in effect during three days, spike potential amplitude is 91.58% of baseline, be 91.33% of baseline after wash-out, all slightly decline.
Experimental result:
Between the pure magnesium 25% and WE43 lixiviate action period, signal frequency also has obvious suppression, and inhibiting rate is higher than magnesium ion group.Moreover, during drug effect, leaching liquor is also completely different to the influence mode of outburst structure with magnesium ion.Spike potential is accounted for the number percent of outburst, two groups of leaching liquors have no significant effect, and magnesium ion group decreases; To outburst length, two groups of leaching liquors obviously increase, and magnesium ion obviously reduces; To the frequency of spike potential in outburst, two groups of leaching liquors increase to some extent, and magnesium ion group has no significant effect.As can be seen here, leaching liquor and the influencing mechanism of magnesium ion to neurocyte electricity physiological signal are different.
From the recovery of electric signal after effect three days, leaching liquor is also completely different to the influence mode of outburst structure with magnesium ion.Two groups of leaching liquor signal frequency recover to some extent but do not arrive baseline values, and the signal frequency of magnesium ion group to be compared to the used time lower; The ratio of spike potential in outburst in outburst structural parameters, leaching liquor group decreases, but magnesium ion group is lower; Length and these two parameters of spike potential frequency in outburst of outburst, leaching liquor group returns to baseline values, and magnesium ion group all obviously reduces.Result shows, is no matter the structure from signal frequency or outburst, all will be worse than leaching liquor group after the effect of 3mM magnesium ion to the signal recuperation of neural network.
The above; be only the specific embodiment of the present invention, but protection scope of the present invention is not limited thereto, anyly belongs to those skilled in the art in the technical scope that the present invention discloses; the change that can expect easily or replacement, all should be encompassed within protection scope of the present invention.Therefore, protection scope of the present invention should be as the criterion with the protection domain of claim.
Claims (8)
1. detect magnesium or the neurovirulent method of magnesium alloy, it is characterized in that, described method comprises: detect cytoactive by LDH method; And, detect neural network electricity physiological signal with chicken proembryo brain neuron-microelectrode array biology sensor.
2. method according to claim 1, is characterized in that, described LDH method detects cytoactive and comprises the following steps:
S1, the leaching liquor prepared magnesium ion solution, prepare magnesium or magnesium alloy;
S2, use Neurobasal nutrient solution cultivate chicken proembryo brain neuron;
S3, past chicken proembryo brain neuron add the leaching liquor of magnesium or magnesium alloy, detect cytoactive with LDH.
3. method according to claim 2, is characterized in that, described S1 is specially:
S11, the sample of magnesium or magnesium alloy is placed in the centrifuge tube of sealing, adds the lixiviate of Neurobasal nutrient solution;
Take out sample after S12,24h, after leaching liquor is centrifugal, get supernatant;
S13, the pH value detecting leaching liquor and magnesium ion concentration;
S14, leaching liquor save backup after membrane filtration, dilution.
4. method according to claim 2, is characterized in that, described S2 is specially:
96 orifice plates are spread polyethyleneimine by S21, kind day before cell;
S22, acquisition chicken proembryo brain tissue, use EDTA trypsinization, then blow and beat into single cell suspension, then centrifugal supernatant discarded, add M199 nutrient solution and make cell suspension, and add 96 orifice plates, be placed in incubator;
M199 nutrient solution is all changed into Neurobasal nutrient solution after S23,24h;
S24, within the 3rd day, add cytarabine.
5. method according to claim 4, is characterized in that, described acquisition chicken proembryo brain tissue is specially:
S221, get the fertilized eggs of 7th ~ 9 days, sterilization, breaks into pieces egg head with tweezers, shells, push shell membrane aside, takes out chicken embryo and is put into the double dish that HBSS is housed;
S222, to block with the head of tweezers by chicken embryo, put into the double dish that another has HBSS;
S223, the head back side adding chicken embryo are put upward, push down midbrain position, and the meninx of forebrain is opened, and take out two white tissues, are put in the double dish of HBSS, remove, obtaining chicken proembryo brain tissue by being attached to meninx remaining outside white tissues.
6. method according to claim 2, is characterized in that, described S3 is specially: discard whole supernatant when chicken proembryo brain neuron is cultured to the 5th day, add magnesium ion solution or leaching liquor, carries out LDH detection respectively at the 6th, 8,10 day.
7. method according to claim 6, it is characterized in that, described LDH detects and is specially: from 96 orifice plate Aspirate supernatant to new 96 orifice plates, every hole adds reaction buffer, places 30min in room temperature dark place, then adds stop solution mixing, be measure wavelength with 490nm, 680nm is reference wavelength, utilizes microplate reader to measure the optical density value in each hole, calculates the relative release rate of LDH.
8. method according to claim 1, is characterized in that, describedly detects neural network electricity physiological signal with chicken proembryo brain neuron-microelectrode array biology sensor and comprises the following steps:
S5, prepare the leaching liquor of magnesium or magnesium alloy;
S6, Neurobasal nutrient solution is added microelectrode array, microelectrode array is placed in incubator, tracer signal;
S7, when electric signal reaches stable state, continuous three days, electric signal 30min when measuring balance every day;
S8, the 3rd day time, change the Neurobasal nutrient solution of 1/4th into leaching liquor, then continuous coverage three sferic signal, every day measures 30min;
S9, the 6th day time, entirely change nutrient solution into Nostoc commune Vanch liquid, continuous coverage three sferic signal, every day 30min.
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