CN106568902A - Surgical smoke biological toxicity evaluation method - Google Patents
Surgical smoke biological toxicity evaluation method Download PDFInfo
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Abstract
The invention discloses a surgical smoke biological toxicity evaluation method. The method comprises the following steps: A, evaluating the in-vitro biological toxicity of luminescent bacteria; B, evaluating the in-vivo biological toxicity of ApoE gene-deficient mice exposed to surgical smoke; and C, evaluating the genetic toxicity of surgical smoke through micronucleus test. The method of the invention has the advantages of quickness and efficiency, accuracy and quantification, realness and reliability, multi-level evaluation, and the like, and has very high practical value and a broad market prospect. The method gets away from the embarrassing situation in which the biological toxicity of surgical smoke cannot be tested fully, and is expected to become an important way of evaluation and test for controlling surgical smoke pollution.
Description
Technical field
The present invention relates to a kind of operation smoke bio-toxicity evaluation methodology.
Background technology
With continuous development of the Minimally Invasive Surgery in modern medicine, the operation smoke produced in operation process has to health
Very big harm, can cause the acute and chronic poisoning of human body, operation smoke to directly result in operating room healthcare givers and patient
There are the symptoms such as headache, eye nose larynx pain inflammation.Pathogen is also possible to be propagated by operation smoke diffusion, causes operating room
Healthcare givers is infected.Operation smoke can also produce genetoxic, and numerous studies prove that operation smoke has potential mutagenicity
With carcinogenecity, genetoxic can be produced.But, these years since, due to the restriction and the cognition of level of condition, for operation
The research of smog is few, and particularly the country does not almost have, the approach cognitive so as to reduce people.Plus due to cigarette of performing the operation
Mist does not typically result in the acute and chronic poisoning with manifest symptom, thus is easily ignored by people.But operating room is cured
Treatment personnel are exposed to containing including due to long-time:Imperfect combustion tissue fragment, active pathogen and virus, cancer
Become the handss of the harmful substances such as cell, toxic and harmful substance, CO, acrylonitrile, formaldehyde, benzene homologues (particularly toluene), phenols
In art smog, chronicity, repeatedly contact cause poisonous effect to accumulate, so should be taken seriously, and the measure that properly protects.
At present, it is of common occurrence for the chemical analysis method of single toxic compounds in operation smoke, bio-toxicity evaluation
Data are also no lack of report.However, lacking for complicated component, the joint-biotoxicity of the miscellaneous operation smoke of noxious substance
Method for quantitatively evaluating invention and creation, future is expected to become the ecological risk assessment of operation smoke and ecological pollution control
Technical tool.
The content of the invention
To solve the shortcoming and defect part of prior art, it is an object of the invention to provide a kind of biology of operation smoke
Toxicity assessment method.The method is a kind of comprehensive bio-toxicity evaluation methodology of employing in vitro and in vivo, for operation smoke pair
The evaluation areas of human health damage and ecological risk, can provide for the ecological risk of operation smoke and to human health damage
Technology evaluation meanses.
The object of the invention is achieved through the following technical solutions:
A kind of operation smoke bio-toxicity evaluation methodology, comprises the following steps:A, photobacteria external biological toxicity assessment;
B, ApoE deficient mice bio-toxicity evaluation in vivo under operation smoke exposure, vivo biodistribution toxicity assessment result makes up body
The deficiency of outer biological toxicity assessment result;C, micronucleus test evaluate operation smoke genetoxic.
It is well known that operation smoke has the features such as chemical toxicity is relatively low, bio-toxicity is relatively low, and photobacteria and other
Antibacterial is compared, with sensitivity it is high the characteristics of, and response speed is fast, low cost, simple to operate, is suitable for operation smoke poison
The detection of property.In the culture fluid incubation containing dissolving operation smoke solvent, photobacteria is by harmful substance in operation smoke
Poison, bacterial respiratory is reduced with physiological process activity, so as to bacterial luminescence intensity changes, by determining photobacteria
The bio-toxicity of luminous intensity quantitatively characterizing operation smoke.
Preferably, described photobacteria is Vibrio-qinghaiensis sp. Q67 (Vibrio qinghaiensis sp.-Q67).
The Vibrio-qinghaiensis sp. Q67 (Vibrio qinghaiensis sp.-Q67) of the present invention belongs to well known in the prior art
Bacterial strain, it is all on the books in many books or periodical literature, books such as written by red legend outstanding person (red legend is outstanding, Zheng Tianling, Li Weimin. send out
Photobacteria is detected with environmental toxicity【M】. Beijing:China Light Industry Press, 2009.175~225), the document report of MAMEI etc.
(MAMEI, virgin China, Wang Zijian, Yang Ducai, red legend is outstanding. new luminescent bacterium (Vibrio qinghaiensis sp.-Q67)
It is applied to the preliminary study of environmental sample toxotest.《ACTA Scientiae Circumstantiae》, 01 phase in 1998).Vibrio-qinghaiensis sp. Q67 is a kind of
Rare fresh water photobacteria, is characterized in compared with other Marine Luminous Bacterias, need not add during culture larger amount of
NaCl (up to 3%), so as to avoid the possibility thus brought from affecting.After appropriate CMC model, Vibrio-qinghaiensis sp. Q67 can send
Macroscopic blue green light.And Vibrio-qinghaiensis sp. Q67 is adapted to middle isosmoticity, adaptive temperature scope close with general cell
Larger (10~30 DEG C), have wider adaptive faculty (adapting to pH=5.5~9.0) to pH, so preferably using Qinghai in the present invention
Vibrio Q67.
Preferably, the bio-toxicity evaluation methodology of the operation smoke specifically includes following step:
A () carries out external biological toxicity quantitative assessment to operation smoke by detecting Vibrio-qinghaiensis sp. Q67 luminous intensity, obtain
Bio-toxicity evaluation quantifies testing result;
B () is exposed to median lethal concentration operation smoke with 6 monthly age ApoE deficient mices as biological subject material
In, blood fat and histopathology situation are checked, compensate for operation smoke external biological toxicity method for quantitatively evaluating can not detect not
The deficiency of insoluble gas, obtains the true detection by quantitative result of operation smoke bio-toxicity;
C () makes cell smear with the bone marrow of step (b) mice, basis of microscopic observation contains the thermophilic of micronucleus more and incarnadines carefully
Born of the same parents, according to the micronuclear rateses of polychromatic erythrocyte, evaluate the genetoxic of operation smoke.
It is furthermore preferred that select ethanol to be GAS ABSORPTION liquid in step (a), it is dense in different pollutant from Vibrio-qinghaiensis sp. Q67
Certain hour is acted in the GAS ABSORPTION liquid of degree, index is turned to the change of its luminous intensity, it is possible to achieve be quick, efficient, accurate
Quantitatively evaluating operation smoke bio-toxicity.
Ethanol can dissolve most organic exhaust gas, and the impact of its bio-toxicity to photobacteria is less, institute
To be suitable as the absorbing liquid of operation smoke.
It is furthermore preferred that the inspection blood fat and histopathology situation described in step (b), specifically refers to:Check mice blood
Lipid level and aorta tangent plane Lipid Plaque situation of change.
The photobacteria luminous intensity external biological toxicity method for quantitatively evaluating mentioned in step (a) is present can not be detected not
The deficiency of insoluble gas toxicity, needs, by checking ApoE deficient mices blood fat and histopathology situation in (b), to enter one
Step is modified the result in external biological toxicity assessment method, reduces true bio-toxicity evaluation result.
For the operation smoke of complicated component, Luminous bacteria can not detect the gas insoluble in ethanol, such as CO, CO2, because
This, it is comprehensive to adopt representative mammal, ApoE deficient mices to receive as vivo biodistribution toxicity assessment method
Examination is biological, more sensitive to the endocrine disruption toxicity of operation smoke, can be with accurate simulation operation smoke contaminated air after, human body
Bio-toxicity effect in intake, metabolic process, more closing to reality pollution condition and poisonous effect.It is simultaneously biological to contamination
Medullary cell carry out micronucleus test, detect its genetoxic, acquired results can correct Luminous bacteria evaluation result not
Foot so that the bio-toxicity evaluation result of operation smoke is more comprehensively and true and reliable.
Step (c) takes mouse bone marrow cells in step (b) and makes cell smear, examines under a microscope polychromatic erythrocyte, counts
Calculate micronuclear rateses;Micronuclear rateses refer to the polychromatic erythrocyte number containing micronucleus, and with permillage it is represented;According to micronuclear rateses to operation smoke
Genetoxic evaluated.
Compared with prior art, the present invention has advantages below and beneficial effect:
(1) high sensitivity of photobacteria, it is all very sensitive to many toxicants, more adapt to cigarette of performing the operation compared with other antibacterials
The low concentration of mist, hypotoxicity.Using Vibrio-qinghaiensis sp. Q67, it is not necessary to add substantial amounts of NaCl, osmotic pressure medium, adaptive temperature and pH
Scope is larger, can more accurately realize the bio-toxicity of quantitative assessment operation smoke, and operation smoke composition is solved well
Complicated, external biological evaluation carrier selects difficult, it is impossible to detects the pressing problem of joint-biotoxicity, is expected to become government and ring
Protect the tool method that monitoring department detects operation smoke pollution situation.
(2) present invention adopts ApoE deficient mice vivo biodistribution toxicity assessment methods, more truly simulates sky
After gas is polluted by operation smoke, bio-toxicity effect of the human body in intake, metabolic process more truly reflects operation smoke
The poisonous effect of actual pollution condition and human body.The biological genetoxic of contamination is detected simultaneously, can make up external biological toxicity
The deficiency of evaluation methodology so that the bio-toxicity evaluation result of operation smoke is more true and reliable.
To sum up, the method for the invention have rapidly and efficiently, precise quantification, true and reliable, mutiple-stage model etc. it is many excellent
Point, with very high practical value and wide market prospect, is expected to become the important evaluation detection of control operation smoke pollution
Means.
Specific embodiment
With reference to embodiment, the present invention is described in further detail, but embodiments of the present invention not limited to this.
Embodiment 1
Step one:Vibrio-qinghaiensis sp. Q67 recovery bacterium solution is prepared, 10ml operation smokes are gathered in 5ml ethanol with gas flowmeter
In absorbing liquid, the photobacteria suspension of 0.1mL is added to respectively the operation smoke sample of warmed-up (or cold) to 20~25 DEG C
In stock solution, 1.25 times of dilution, 2.5 times of dilution, 5 times of dilution, the 1ml experiment water samples of 10 times of dilution (3 Duplicate Samples of each concentration),
Accurately after 15~20min of effect, its luminous intensity is surveyed successively.Record sample liquid concentration and its luminous intensity values, and with testing tube
In luminous intensity as control light intensity (the interior sample liquid for fresh air), calculate relative suppression light rate.It is with sample concentration
Abscissa, relative suppression light rate are vertical coordinate mapping, and the relative corresponding sample liquid concentration in the place of light rate 50% that presses down is water sample
EC50 values, that is, luminous intensity be maximum emission intensity half when exhaust gas concentration value.
Experimental result shows, operation smoke sample stock solution, 1.25 times of dilution, 2.5 times of dilution, 5 times of dilution, 10 times of dilution
The relative suppression light rate of Vibrio-qinghaiensis sp. Q67 is 70%, 60%, 40%, 30%, 20% in experiment water sample, and the relative light rate that presses down is with operation
Linear relationship is good between the concentration of smog, calculates EC50 for 1.22ml/ml.Drawn according to the percent grade that Bulich is proposed
Minute mark is accurate, and it is micro- poison that conclusion is the bio-toxicity that operation smoke has, although will not cause injury immediately to human body, but for a long time
Suction accumulation in situations in the surgery room can have certain infringement to human body.
Step 2:5 preliminary experiment groups, 10 per group 6 monthly age ApoE deficient mices (mice sources are set:And south
University life science and technology institute), wherein female, male mice is each 5.The mice being grouped is taken, is put in dynamic formula toxicant exposure cabinet, prohibit water
Fasting.Operation smoke concentration is 60.5ml/m in operating room3, according to design dosage and each toxicant exposure cabinet volume, calculating needs what is added
The volume of operation smoke, makes each toxicant exposure cabinet operation smoke concentration determine 5,10,20,40, the 80 of concentration close to operating room respectively
Times concentration.Then operation smoke is passed through in cabinet, timing 12h, and records contamination in-cabinet temperature, observation animal poisoning symptom and dead
Die the indexs such as number of individuals.Median lethal concentration LC is measured according to experimental result50For 2.42 × 103ml/m3。
5 experimental grouies and 1 matched group, 10 per group 6 monthly age ApoE deficient mices, male and female half and half are set.Experiment
Mice is put in dynamic formula toxicant exposure cabinet group, fasting for solids and liquids, and setting concentration of contamination is median lethal concentration LC50.During contamination, matched group
Mice is also removed water and removes food, and receives fresh air.The jejunitas 12h of mice, orbital venous plexus take blood, centrifugation, separate serum, determine little
Mus T-CHOL (TC), low-density lipoprotein cholesterol (LDL-C), HDL-C (HDL-C), triglyceride
(TG), apolipoprotein B.Compared with matched group, experimental group TC and LDL-C are significantly raised, and HDL-C is significantly reduced, apolipoprotein B water
Flat is 1.5~3 times of control group mice.At the disconnected marrow of mice after death, whole-heartedly frozen section is carried out after dirty specimen liquid nitrogen quenching, is cut
Piece oil red O (ORO) dyes the speckle formational situation of the different tangent planes of observation, and experimental group plaque area is the 1.5~2 of matched group
Times, comparing with external biological toxicity assessment result, external biological toxicity assessment conclusion is correct.
Meanwhile, the bone marrow smear of experimental mice is prepared, carry out Micronucleus test.Observation is used containing micronucleus
Polychromatic erythrocyte.Polychromatic erythrocyte (PCE) is in dusty blue, and acellular core, mature erythrocyte (NCE) is in light salmon pink.
Micronucleus great majority are in single circle, and the smooth of the edge is neat, and thermophilic color is consistent with caryoplasm, in aubergine or bluish violet.Per only moving
Thing at least counts 1000 polychromatic erythrocytes.Micronuclear rateses refer to the polychromatic erythrocyte number containing micronucleus, with permillage (‰) table
Show it.And calculate the ratio of PCE/NCE in 200 cells, normal PCE/NCE ratios are about 1 (normal 0.6~1.2), and real
The average PCE/NCE ratios of group mice are tested less than 0.5, is represented that PCE is formed and is suppressed by thing of contaminating.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention not by above-described embodiment
Limit, other any spirit without departing from the present invention and the change, modification, replacement made under principle, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (5)
1. a kind of operation smoke bio-toxicity evaluation methodology, it is characterised in that comprise the following steps:A, photobacteria external biological
Toxicity assessment;The bio-toxicity evaluation in vivo under operation smoke exposure of B, ApoE deficient mice;C, micronucleus test evaluate handss
Art smog genetoxic.
2. a kind of operation smoke bio-toxicity evaluation methodology according to claim 1, it is characterised in that described is luminous thin
Bacterium is Vibrio-qinghaiensis sp. Q67.
3. a kind of operation smoke bio-toxicity evaluation methodology according to claim 2, it is characterised in that specifically include following
Step:
A () carries out external biological toxicity quantitative assessment to operation smoke by detecting Vibrio-qinghaiensis sp. Q67 luminous intensity, obtain biological
Toxicity assessment quantifies testing result;
B () in being exposed to median lethal concentration operation smoke, is examined with 6 monthly age ApoE deficient mices as biological subject material
Have a blood test fat and histopathology situation, compensate for operation smoke external biological toxicity method for quantitatively evaluating can not detect insoluble gas
The deficiency of body, obtains the true detection by quantitative result of operation smoke bio-toxicity;
C () makes cell smear with the bone marrow of step (b) mice, basis of microscopic observation contains the polychromatic erythrocyte of micronucleus,
According to the micronuclear rateses of polychromatic erythrocyte, the genetoxic of operation smoke is evaluated.
4. a kind of operation smoke bio-toxicity evaluation methodology according to claim 3, it is characterised in that choosing in step (a)
It is GAS ABSORPTION liquid with ethanol, Vibrio-qinghaiensis sp. Q67 is acted on into certain hour in the GAS ABSORPTION liquid of different pollutant levels, with
The change of its luminous intensity turns to index, obtains bio-toxicity evaluation and quantifies testing result.
5. a kind of operation smoke bio-toxicity evaluation methodology according to claim 3, it is characterised in that institute in step (b)
The inspection blood fat stated and histopathology situation, specifically refer to:Check that lipid of mice level and aorta tangent plane Lipid Plaque become
Change situation.
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