CN107746873A - A kind of cutaneous safety evaluation method based on HaCaT cells to nano titanium oxide - Google Patents
A kind of cutaneous safety evaluation method based on HaCaT cells to nano titanium oxide Download PDFInfo
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Abstract
The invention provides a kind of cutaneous safety evaluation method based on HaCaT cells to nano titanium oxide, belong to external application material tests field.Methods described includes successively passing through HaCaT cells into the nanometer titanium dioxide titanium solution and UV treatment of various concentrations, determines cell survival rate；The concentration that selection makes HaCaT cell survival rates reach in 50%~90% nanometer titanium dioxide titanium solution is used as potential danger concentration, with reference to UV treatment, causes sialic acid expression in cell to occur unanimously to change with the horizontal of ROS in cell；Change finally by the expression of detection sialic acid judges influence of the potential danger concentration nano titanium oxide to cellular damage degree, so as to obtain the cutaneous safety evaluation method of standard set nano titanium oxide.The present invention provides reliable scientific basis for the safe handling of nano titanium oxide, also provides reference for the safety evaluatio of other nano materials.
Description
Technical field
The invention belongs to external application material tests field, and in particular to it is a kind of based on HaCaT cells to nano titanium oxide
Cutaneous safety evaluation method.
Background technology
Nano-TiO2(nano-TiO2) be a kind of widely used nano material, be mainly used in cosmetics, medicine, environmental protection,
The fields such as coating.Nano material is used widely because its unique advantage such as specific surface area is big, particle diameter is small in all trades and professions,
Especially in cosmetic industry, because nano titanium oxide has the characteristic for absorbing scattering ultraviolet light, it is widely used
To among suncream.The whole world is used for the nano-TiO of cosmetics every year at present2Thousands of tons of is reached.Exactly because also it these
Characteristic, may bring bigger harm, and nano titanium oxide is constantly subjected to scientific research personnel's height to the safety research of skin
Pay attention to.
Nano-TiO2Harm essentially consist in it there is photocatalytic, especially under ultraviolet light, it can produce substantial amounts of oxygen
Free radical (ROS), including HO, O2·-With1O2.Under normal condition, ROS passes to maintaining cell normal function in intracellular, intercellular
Delivery signal plays an important role；But when oxygen free radicals rise, considerably beyond itself normal level, cellular damage can be given, is broken
Bad normal configuration, influence function.Essentially consisted at present for research of the nano titanium oxide in cellular level, ROS can be caused carefully
After birth integrity violations, lipid peroxidation is caused, DNA break, the integrality of mitochondria is destroyed, causes cytoskeleton to damage, from
And influence the normal function of cell.But as the carbohydrate of one of cell main component, but rarely people studies it in nano material
What kind of change occurs in toxicity.Sugar forms glycoprotein, proteoglycans and sugar with covalent bond and protein or lipid binding
Fat, they participate in cell recognition and differentiation, cell adherence, and migration etc. plays a role.Such as Yin et al. was just sent out in 2012
Existing nano-TiO2Caused by phototoxicity damage with ultraviolet light (UV) dosage and nano material dosage increase and increase；Grain
Damaged caused by footpath is smaller bigger.It can produce substantial amounts of ROS under w light, cause lipid protein matter peroxidating inducing cell to wither
Die.
At present, the skin safe evaluation method for nano titanium oxide of report, is related to systems biology aspect, passes through
Metabolism network is established, observes the change of different metabolic thing in cell metabolism network, finding titania nanoparticles influences cell
The metabolic markers and action target spot of cutaneous safety, the inherent generation of comprehensive analysis titania nanoparticles inducing cell damage
Thank to path.Such as the patent that patent publication No. is CN102373261A discloses the cell safety evaluation of nano titanium oxide, builds
Formwork erection type, according to the change of difference metabolin in cell, builds metabolism network, and then inquiring into titanium dioxide granule influences cell
Metabolic mechanism, the relation between overall merit titania nanoparticles and cell cutaneous safety.Methods described can quickly,
Search out exactly and induce the disorderly related key metabolites of metabolism network to titania nanoparticles, but methods described needs
The change and statistical analysis of multiple metabolic pathways are analyzed, method is complicated, it is impossible to directly reflects security evaluation result.
The content of the invention
In view of this, it is an object of the invention to provide a kind of skin peace based on HaCaT cells to nano titanium oxide
Full property evaluation method, step is simple, can directly reflect the result of security evaluation.
In order to realize foregoing invention purpose, the present invention provides following technical scheme：
The invention provides a kind of cutaneous safety evaluation method based on HaCaT cells to nano titanium oxide, including
Following steps：
1) HaCaT cells are seeded in cell culture medium and cultivated, treat that the HaCaT cell growths to logarithmic phase is disappeared
Change, postdigestive HaCaT cell suspensions carry out adhere-wall culture 24h, obtain the HaCaT cells of adherent growth；
2) nano titanium oxide is dispersed in PBS, ultrasound, obtains nanometer titanium dioxide titanium solution；
3) the nanometer titanium dioxide titanium solution in the step 2) is diluted to some concentration, is added separately to the step 1)
In the HaCaT cells of obtained adherent growth, 21~22h is cultivated, then ultraviolet irradiates 30~60min；
4) the HaCaT cells after the step 3) middle-ultraviolet lamp is irradiated are cultivated, and the HaCaT cells after culture are existed
Continue to cultivate in MTT solution；HaCaT cells after culture under 492nm wavelength are determined into light absorption value in DMSO solution, obtained
Test results；
Zeroing group, the suction that the zeroing group result measures for the solution only successively comprising DMEM, MTT solution and DMSO are set
Luminosity；
Experimental comparison group is set, and the experimental comparison group result is successively through DMEM, MTT solution, DMSO by HaCaT cells
The absorbance measured after solution processing；
The test results, zeroing group result and experimental comparison group result are substituted into formula shown in Formulas I to be calculated carefully
Born of the same parents' survival rate；
The cell survival rate=(the ODexp-ODneg)/Formulas I of (ODcon-ODneg) × 100%
Wherein ODexp represents experimental group absorbance, and ODneg represents zeroing group absorbance, and ODcon represents experiment contrast
Group absorbance；
According to cell survival rate in experimental group 50%~90% nanometer titanium dioxide titanium solution concentration as potential danger
Dangerous concentration；
5) handled with the nanometer titanium dioxide titanium solution of the potential danger concentration obtained in the step 4) in the step 1)
Postdigestive logarithmic phase HaCaT cells 21 hours, the HaCaT cells after obtained processing carry out successively UV treatment 30~
60min, continue to cultivate 2h, the first cleaning, fixed, the addition FITC mark agglutinin solution into the HaCaT cells after fixation, according to
It is secondary be incubated, dyed, second cleaning, measure cleaning after HaCaT cells fluorescence intensity, obtain the saliva of HaCaT cells
Sour expression；
Zeroing group and experimental comparison group are set；The zeroing group result is only successively comprising nanometer titanium dioxide titanium solution, purple
External exposure and fluorescent staining, it is incubated, the fluorescence intensity that the solution after cleaning measures；The experimental comparison group result is by HaCaT
Cell is successively through nanometer titanium dioxide titanium solution, ultraviolet irradiation and fluorescent staining, the fluorescence intensity for being incubated, being measured after cleaning；Will be real
Test in a group result, zeroing group result and experimental comparison group result substitution formula II and calculate, obtain relative intensity of fluorescence；
Relative intensity of fluorescence=(the MFexp-MFneg)/formula of (MFcon-MFneg) × 100% II
Wherein MFexp represents experimental group fluorescence intensity, and MFneg represents zeroing group fluorescence intensity, and MFcon represents experiment contrast
Group fluorescence intensity；
Pass through saliva in the HaCaT cells of the nanometer titanium dioxide titanium solution under various concentrations and treatment with ultraviolet light by analyzing
Whether there is significant difference between sour expression and control group, to judge the degree of injury of cell；
There is no the limitation of time sequencing between the step 1) and step 2).
Preferably, the step 3) and the power of step 5) middle-ultraviolet lamp irradiation are 6~10W；The wavelength of the ultraviolet
For 365nm, distance is 9~11cm between the light source and HaCaT cells of the ultraviolet.
Preferably, FITC marks the synthetism lignin solution that agglutinin solution is FITC marks in the step 5)；It is described
The mass concentration of the synthetism lignin solution of FITC marks is 8~12 μ g/mL.
Preferably, the first time being incubated was 50~70min in the step 5).
Preferably, the first cleaning and the second cleaning with solution are PBS in the step 5).
Preferably, it is paraformaldehyde solution that mass concentration is 4% to be fixed in the step 5) with solution；The fixation
Time is 12~17min.
Preferably, it is characterised in that the excitation wave of the fluorescence intensity of the HaCaT cells in the step 5) after measure cleaning
A length of 488nm.
Preferably, the cell concentration of postdigestive HaCaT cell suspensions is 1x10 in the step 1)5/mL；The step
3) gradient concentration of nanometer titanium dioxide titanium solution is 10~1000 μ g/mL in；The addition volume of the nanometer titanium dioxide titanium solution
It is identical with the volume of cell culture medium during HaCaT cell attachment cultures.
Preferably, cell culture medium is the culture medium based on DMEM complete mediums in the step 1), includes matter
Measure percentage composition be 1% penicillin, weight/mass percentage composition be 1% streptomysin and weight/mass percentage composition be 10% tire ox blood
Clearly.
Preferably, the granularity of nano titanium oxide is 20~800nm in the step 2)；The quality of nano titanium oxide is dense
Degree is in 10~1000 μ g/mL.
The invention provides a kind of cutaneous safety evaluation method based on HaCaT cells to nano titanium oxide, compare
In traditional nano titanium oxide biological evaluation method mainly for inhereditary material, protein, the change and life of lipid
The influence of thing function, the biological evaluation method of nano titanium oxide of the invention is in ultraviolet light based on nano titanium oxide
Under the conditions of the change of polysaccharose substance (sialic acid belongs to polysaccharose substance) that damages of inducing cell, the reason for analyzing its change,
Help to obtain the means and method of effectively evaluating nano titanium oxide cell biological.Can by the experimental result of the present invention
To find out, nanometer titanic oxide material can not only have an impact to DNA, protein, lipid etc., can also influence polysaccharide structure and
Function, this contributes to influence of the more deep understanding nano titanium oxide to cell, also can more objective, in-depth study it
Mechanism；The present invention also provides reliable scientific basis for the safe handling of nano titanium oxide simultaneously, is also other nano materials
Safety evaluatio provide reference.
Brief description of the drawings
Fig. 1 is the influence of nano titanium oxide and HaCaT Cells cell survival rate in embodiment 1；
Fig. 2 is α -2,6 saliva of the HaCaT cells after nano titanium oxide and ultraviolet processing on cell in embodiment 2
The expression change of acid；
Fig. 3 is that sialic acid of the HaCaT cells after nano titanium oxide and ultraviolet processing on cell is relative in embodiment 2
Fluorescence intensity；
Fig. 4 is HaCaT cells intracellular caused ROS after nano titanium oxide and UV treatment in embodiment 2
The change of amount；
Fig. 5 is expression change and the ROS relation of α -2,6 sialic acids on HaCaT cells in embodiment 3；
Fig. 6 is the change for the D-MANNOSE expression for carrying out quantitative cell in embodiment 4 with sepectrophotofluorometer；
Fig. 7 is nm TiO 2-base in embodiment 5 because of toxicity detection experimental result；
Fig. 8 is that nano titanium oxide influences experiment knot to the hepatocuprein (SOD) of HaCaT cells in embodiment 6
Fruit；
Fig. 9 is that nano titanium oxide influences experiment knot to the hepatocuprein (SOD) of HaCaT cells in embodiment 7
Fruit；
Lipid peroxidation testing results of the Figure 10 for nano titanium oxide in embodiment 8 to HaCaT cells.
Embodiment
The invention provides a kind of cutaneous safety evaluation method based on HaCaT cells to nano titanium oxide, including
Following steps：
1) HaCaT cells are seeded in cell culture medium and cultivated, treat that the HaCaT cell growths to logarithmic phase is disappeared
Change, postdigestive HaCaT cells are subjected to adhere-wall culture 24h, obtain the HaCaT cells of adherent growth；
2) nano titanium oxide is dispersed in PBS, ultrasound, obtains nanometer titanium dioxide titanium solution；
3) the nanometer titanium dioxide titanium solution in the step 2) is diluted to some concentration, is added separately to the step 1)
In obtained adherent growth HaCaT cells, 21~22h is cultivated, ultraviolet irradiates 30~60min；
4) the HaCaT cells that the step 3) middle-ultraviolet lamp irradiates are cultivated, the HaCaT cells after culture is existed
Continue to cultivate in MTT solution；HaCaT cells after culture are determined into light absorption value as test results in DMSO solution；
Withered group is set, the extinction that the zeroing group measures for the mixed solution only comprising DMEM, MTT solution and DMSO
Degree；
Experimental comparison group is set, and the experimental comparison group is successively through DMEM, MTT solution, DMSO processing by HaCaT cells
The absorbance measured afterwards；
The test results, zeroing group result and experimental comparison group result are substituted into Formulas I and calculated, obtains cell survival
Rate；The cell survival rate=(the ODexp-ODneg)/Formulas I of (ODcon-ODneg) × 100%
Wherein ODexp represents experimental group, and ODneg represents zeroing group, and ODcon represents experimental comparison group；
Using cell survival rate in experimental group 50%~90% nanometer titanium dioxide titanium solution concentration as potential danger
Concentration；
5) handled in the step 1) and digested with the nano titanium oxide of the potential danger concentration obtained in the step 4)
Logarithmic phase HaCaT cells afterwards 21 hours, the HaCaT cells after obtained processing carry out 30~60min of UV treatment successively,
Continue to cultivate, the first cleaning is fixed, and FITC mark agglutinin solution is added into the HaCaT cells after fixation, is incubated successively
Educate, dye, the second cleaning, the absorbance of the HaCaT cells after measure cleaning, obtain the sialic acid expression of HaCaT cells；
Zeroing group and experimental comparison group are set；The zeroing group result is only successively comprising nanometer titanium dioxide titanium solution, purple
External exposure and fluorescent staining, it is incubated, the fluorescence intensity that the solution after cleaning measures；The experimental comparison group result is by HaCaT
Cell is successively through nanometer titanium dioxide titanium solution, ultraviolet irradiation and fluorescent staining, the fluorescence intensity for being incubated, being measured after cleaning；Will be real
Test in a group result, zeroing group result and experimental comparison group result substitution formula II and calculate, obtain relative intensity of fluorescence.
Relative intensity of fluorescence=(the MFexp-MFneg)/formula of (MFcon-MFneg) × 100% II
Wherein MFexp represents experimental group fluorescence intensity, and MFneg represents zeroing group fluorescence intensity, and MFcon represents experiment contrast
Group fluorescence intensity；
Pass through saliva in the HaCaT cells of the nanometer titanium dioxide titanium solution under various concentrations and treatment with ultraviolet light by analyzing
Whether there is significant difference between sour expression and control group, to judge the degree of injury of cell；
There is no the limitation of time sequencing between the step 1) and step 2).
HaCaT cells are seeded in cell culture medium in the present invention and cultivated, treat the HaCaT cell growths to logarithmic phase
Digested, postdigestive HaCaT cells are subjected to adhere-wall culture 24h, obtain the HaCaT cells of adherent growth.
In the present invention, the source of the HaCaT cells is not particularly limited, and use is well-known to those skilled in the art
HaCaT cells.In the embodiment of the present invention, the HaCaT cells are provided by Chinese cellular resources storehouse.
The present invention does not have particular/special requirement to the method for inoculation, is using inoculation method well-known to those skilled in the art
Can.The inoculum concentration of HaCaT cells is preferably per hole 6000~12000, more preferably per 10000, hole cell.The culture is excellent
It is selected in the progress of 96 holes.
In the present invention, the cell culture medium is the culture medium based on DMEM complete mediums, includes quality percentage
The streptomysin and weight/mass percentage composition that penicillin that content is 1%, weight/mass percentage composition are 1% are 10% hyclone.
In the present invention, the form of the cell of the logarithmic phase is middle wide fusiformis pointed at both ends, is born to surrounding and touches foot.
In the present invention, the digestion is preferably carried out using pancreatin.The mass concentration of the pancreatin is preferably 0.2~0.3%,
More preferably 0.25%.The digestion time of the pancreatin is preferably 3~5min, more preferably 4min.
In the present invention, the conditional sampling for continuing culture after the culture and digestion is：The temperature of culture is preferably 36.5~
37.5 DEG C, more preferably 37 DEG C.The CO2Volumetric concentration be preferably 4%~6%, more preferably 5%.
In the present invention, the HaCaT cell suspensions that preferred pair obtains after digestion culture are diluted.After the dilution
The concentration of HaCaT cell suspensions is preferably 0.8~1.5x105/ mL, more preferably 1x105/mL。
Nano titanium oxide is dispersed in PBS by the present invention, ultrasound, obtains nanometer titanium dioxide titanium solution.
In the present invention, the granularity of the nano titanium oxide is preferably 10~800nm, and more preferably 10~100nm is optimal
Elect 25nm as.The mass concentration of nanometer titanium dioxide titanium solution is in 10~1000 μ g/mL.The present invention is to the nano titanium oxide
Source is not particularly limited, using nano titanium oxide well-known to those skilled in the art.In the embodiment of the present invention, institute
State nano titanium oxide and be purchased from German goldschmidt chemical corporation.
In the present invention, the PBS for be 0.067M comprising molar concentration phosphate buffer solution；The PBS delays
The pH value of fliud flushing is preferably 6.8~7.4, and more preferably 7.2.
In the present invention, the ultrasonic frequency is preferably 20~40KHz, the ultrasonic power be preferably 25KHz~
30KHzW, the ultrasonic time is preferably 4~6min, more preferably 5min.The present invention does not have to the ultrasound with instrument and equipment
Have it is specifically limited, using ultrasonic instrument equipment well-known to those skilled in the art.
It is of the invention by the nanometer after obtaining nanometer titanium dioxide titanium solution and postdigestive logarithmic phase HaCaT cell suspensions
Titania solution is diluted to some concentration, is added separately in the postdigestive logarithmic phase HaCaT cell suspensions, culture 21
~22h, ultraviolet irradiate 30~60h.
In the present invention, dilution nanometer titanium dioxide titanium solution is preferably DMEM culture mediums with diluent.The nanometer titanium dioxide
Some concentration of titanium solution are 10~1000 μ g/mL, more preferably 10,50,100,500,1000 μ g/mL.
In the present invention, the volume ratio of the HaCaT cell suspensions liquid and nanometer titanium dioxide titanium solution is preferably 1：1.
In the present invention, the condition that cell is cultivated in nanometer titanium dioxide titanium solution includes：The temperature of environment culture is excellent
Elect 36.5~37.5 DEG C, more preferably 37 DEG C as；CO2Volumetric concentration be preferably 4%~6%, more preferably 5%.
In the present invention, ultraviolet irradiation is carried out in PBS solution again after preferably HaCaT cells are cleaned after culture.It is described clear
The number washed is preferably 2~4 times, more preferably 3 times.Cleaning is PBS solution with solution.
In the present invention, the ultraviolet irradiation is preferably carried out using uviol lamp；The power of the uviol lamp is preferably 6~
10W, more preferably 8W；The wavelength of the ultraviolet is preferably 365nm, and distance is preferred between uviol lamp and the HaCaT cell
For 9~11cm, more preferably 10cm.
After ultraviolet irradiates, the present invention is cultivated the HaCaT cells that the ultraviolet irradiates, after culture
HaCaT cells continue to cultivate in MTT solution；HaCaT cells after being cultivated in the MTT solution are determined into suction in DMSO
Light value, obtain test results；Zeroing group is set, and the zeroing group is the mixed solution only comprising DMEM, MTT solution and DMSO
The absorbance measured；Experimental comparison group is set, and experimental comparison group is the MTT solution successively through DMEM by HaCaT cells, at DMSO
The absorbance measured after reason under 492nm wavelength；By the test results, zeroing group result and experimental comparison group result generation
Enter and cell survival rate is obtained in Formulas I；The cell survival rate=(ODexp-ODneg)/(formula of (ODcon-ODneg) × 100%
I), wherein ODexp represents experimental group absorbance, and ODneg represents zeroing group absorbance, and ODcon represents experimental comparison group absorbance.
In the present invention, the time that the HaCaT cells of the ultraviolet irradiation are cultivated is preferably 1h, so that cell recovers
Vigor.The HaCaT cells of the ultraviolet irradiation are preferably cultivated in the environment of DMEM culture mediums.
In the present invention, the time that the HaCaT cells continue culture in MTT solution is preferably 3.5~5h, more preferably
4h.The HaCaT cells are preferably cleaned before continuing culture in MTT solution using PBS solution.The number of the cleaning is preferably
2~4 times, more preferably 3 times.The mass concentration of the MTT solution is preferably 0.03~0.08mg/mL, more preferably 0.05mg/
mL.The purpose for continuing to cultivate HaCaT cells with MTT solution is whether detection cell damages, and judges the nanometer of various concentrations
The phototoxicity variation tendency of titanium dioxide.The present invention is not particularly limited to the condition cultivated in the MTT solution, using training
Support condition of culture in base.In the present invention, cleaned using the preferably above-mentioned PBS of use after MTT hydroponics.It is described clear
The number washed is preferably 2~3 times.
In the present invention, the wavelength of the measure light absorption value is 492nm.
In the present invention, by determine various concentrations nano titanium oxide and UV treatment to caused by HaCaT cells
Phototoxic influence, it can obtain having lethal to cell under certain ultraviolet radiation situation by calculating cell survival rate
The concentration of nanometer titanium dioxide titanium solution, so as to obtain not having the potential danger concentration of lethal to cell.It is different dense by calculating
The cell survival rate of the nanometer titanium dioxide titanium solution of degree, determines that cell survival rate is defined as nano titanium oxide 50%~90%
Molten potential danger concentration, it is easy to subsequently selected cell can be made to be in different apoptosis periods according to the potential danger concentration
The concentration of nanometer titanium dioxide titanium solution.In the present invention, the power of the uviol lamp is 6~10W, and the wavelength of ultraviolet is 365nm,
Distance is the molten potential danger concentration of nano titanium oxide under 9~11cm treatment conditions between uviol lamp and the HaCaT cell
Preferably 10~50 μ g/ml, more preferably 50 μ g/ml.
Potential danger concentration according to nanometer titanium dioxide titanium solution obtained above to HaCaT cells, select potential danger
The titanium nanometer titanium dioxide solution of concentration handles the postdigestive logarithmic phase HaCaT cells 21 hours, after obtained processing
HaCaT cells carry out 30~60min of UV treatment successively, continue to cultivate, the first cleaning, fixed, thin to the HaCaT after fixation
FITC mark agglutinin solution is added in born of the same parents, is incubated successively, fluorescent staining, second cleans, and the HaCaT after measure cleaning is thin
The fluorescence intensity of born of the same parents, obtain the sialic acid expression of HaCaT cells.
The condition of condition and UV treatment during titania solution processing is the same as the above-mentioned cell survival rate that obtains
Technical scheme.
In the present invention, the time of incubation is preferably 50~70min, more preferably 60min.The condition of the incubation is：Temperature
Degree is preferably 36.5~37.5 DEG C, more preferably 37 DEG C；CO2Concentration be preferably 4~6%, more preferably 5%.
In the present invention, first cleaning and the second cleaning are preferably PBS with solution.
In the present invention, the fixation is with the paraformaldehyde solution that solution is that mass concentration is preferably 4%；The fixation when
Between be preferably 12~17min, more preferably 15min.
In the present invention, the FITC marks agglutinin solution is preferably the synthetism lignin solution of FITC marks；The FITC
The mass concentration of the synthetism lignin solution of mark is preferably 8~12 μ g/mL, more preferably 10 μ g/mL.The agglutinin of FITC marks
The addition volume of solution is preferably 500~1000 μ L, more preferably 800 μ L.
The present invention is not particularly limited to the dyeing, using Staining Protocol well-known to those skilled in the art.
In the present invention, the excitation wavelength of the fluorescence intensity of the HaCaT cells after the measure cleaning is preferably 488nm.
In the present invention, measure HaCaT cells fluorescence intensity be test results after, preferably also include set zeroing group and
Experimental comparison group.The zeroing group result is incubated only successively to include nanometer titanium dioxide titanium solution, ultraviolet irradiation and fluorescent staining
Educate, clean after the fluorescence intensity that measures of solution；The experimental comparison group result is successively through nanometer titanium dioxide by HaCaT cells
Titanium solution, ultraviolet irradiation and fluorescent staining, the fluorescence intensity for being incubated, being measured after cleaning；By test results, zeroing group result and
Experimental comparison group result is substituted into formula II and calculated, and obtains relative intensity of fluorescence.
Relative intensity of fluorescence=(the MFexp-MFneg)/formula of (MFcon-MFneg) × 100% II
Wherein MFexp represents experimental group fluorescence intensity, and MFneg represents zeroing group fluorescence intensity, and MFcon represents experiment contrast
Group fluorescence intensity.
In the present invention, after nano titanium oxide and treatment with ultraviolet light, intracellular ROS contents can increase, ROS water
The flat α -2 that can make cell, 6 sialic acid expressions increase, therefore, can obtain α -2, the expression of 6 sialic acids can react
The degree of cellular damage, therefore, by detecting α -2, the expression of 6 sialic acids can reflect the damage feelings of existing cell
Condition, so as to the growth conditions of cell survived.
By analysis pass through between nano material and the cell sialic acid expression and control group for the treatment of with ultraviolet light whether
There is significant difference, to judge the degree of injury of cell.
In the present invention, cell is handled to verify its safety using the nanometer titanium dioxide titanium solution of obtained potential danger concentration
Property, the results showed that：D-MANNOSE, hepatocuprein, the base that nano titanium oxide can be given in HaCaT cells under ultraviolet light
Cause, glutathione (GSH), lipid occur peroxidating and bring very macrolesion and destruction.This illustrates that method provided by the invention can have
Effect obtains the method for the concentration safe to use of nanometer titanium dioxide titanium solution.
With reference to embodiment to a kind of skin safe based on HaCaT cells to nano titanium oxide provided by the invention
Property evaluation method is described in detail, but they can not be interpreted as limiting the scope of the present invention.
Material
Cell line
Human keratinocytes HaCaTATCC
Main agents
Embodiment 1
Cell culture：HaCaT cell is complete containing 10% hyclone, 1% penicillin and the DMEM of streptomysin
Cultivated in culture medium, culture environment is 5% CO2With 37 DEG C of constant incubators.Liquid is changed daily, the cell in growth period of taking the logarithm enters
Row experiment.
The preparation of nano titanium oxide suspension：It is accurate to weigh 5mg nano titanium oxides, after adding 1mLPBS mixing, ultrasound
5min, it is standby to be diluted to 10,50,100,500,1000 μ g/mL with DMEM culture mediums.
Nano titanium oxide phototoxicity experiments：Take the logarithm the HaCaT cells in growth period, add the digestion of 0.25% pancreatin, carefully
Born of the same parents' suspension 1x105/ mL, add in 96 orifice plates per the μ L of hole 100, cultivated 24 hours in incubator.Nano titanium oxide is used
DMEM dilution various concentrations 10,50,100,500,1000 μ g/mL add 100 μ L per hole and sequentially added, while set and be free of nanometer
The DMEM of titanium dioxide cultivates cell as a control group, 6 multiple holes of every group of setting.After culture 21 hours, suction is abandoned containing nanometer two
The supernatant of titanium oxide, cleaned 2~3 times with PBS, 100 μ LPBS are added per hole, 365nm is used under 8W small uviol lamp
Ultraviolet light 1 hour, lamp is apart from cell 10cm；With DMEM substituting PBS after the completion of irradiation, to continue to be placed in incubator culture 2 small
When.Supernatant is discarded afterwards, and PBS is washed 2-3 times, adds 0.05mg/mL MTT solution, continues culture 4 hours.Supernatant is discarded, is added
Enter 150 μ LDMSO, after fully dissolving purple crystal thing, determine light absorption value, while zeroing group is set in ELIASA 492nm：Only
Add DMEM and MTT solution, DMSO；Control group：HaCaT cells, DMEM, MTT solution, DMSO.Using origin7.0 softwares to reality
Test result to be analyzed, p<0.01 is significant difference.Cell survival rate=(ODexp-ODneg)/(ODcon-ODneg)
X100%, wherein ODexp represent experimental group, and ODneg represents zeroing group, and ODcon represents experimental comparison group.
As a result：HaCaT cells are after the nano titanium oxide processing of various concentrations, and cytoactive is with nano-silica
Change the rise of titanium concentration and reduce.Such as Fig. 1 under low dosage level (10 μ g/mL, 50 μ g/mL), nano titanium oxide is to cell
Survival rate compare and have little to no effect with control group, cell survival rate is more than 90%；But under middle high dose (100,
500,1000 μ g/mL) under, cytotoxicity substantially increases, and cell survival rate is below 70%.After adding ultraviolet light, hence it is evident that
Increasing the toxicity of nano titanium oxide, under low dosage, cell survival rate reaches 90% for independent nano titanium oxide processing, but
To add ultraviolet light, under the processing of 50 μ g/mL nano titanium oxides, cell survival rate 50% or so and afterwards 100,
500th, 1000 μ g/mL, on the basis of the survival rate reduced originally, continue to reduce.This explanation, ultraviolet light and nano titanium oxide are total to
Same-action can produce bigger cytotoxicity.
Embodiment 2
α -2 of cell, the change of 6 sialic acid expressions：Take the logarithm the HaCaT cells in growth period, with every hole 3x106It is individual
Cell is added in 6 orifice plates of the cover glass containing sterilizing, cultivates 24h.Add the nano titanium oxide that concentration is 0,50 μ g/mL
DMEM medium treatment HaCaT cells 21 hours.Wash away supernatant, after adding PBS, after 365nm treatment with ultraviolet light 1 hour,
PBS is replaced with DMEM culture mediums, is then incubated again in incubator 2 hours.Supernatant is discarded afterwards, is washed 2-3 times with PBS, is used
4% paraformaldehyde fixes cell 15 minutes, and the SNA for then adding 10 μ g/mL FITC marks is incubated 1 hour, is washed away with PBS more
Remaining dyestuff.Add DAPI to dye 15 minutes, excess dyestuff is washed away with PBS.Cover glass is transferred on slide, cell with
Anti- fluorescence quenching is added dropwise between slide, then with resinene mounting.Band resinene air-dries, you can with burnt aobvious with copolymerization
Micro mirror detects.Nucleus is in blueness when taking pictures, and α -2,6 sialic acids are in green.Carry out that quantitative (result is shown in sepectrophotofluorometer
Fig. 3).
As a result：HaCaT cells are after nano titanium oxide and treatment with ultraviolet light, α -2 on its cell, 6 sialic acids
Significant change occurs for expression.As shown in Fig. 2 compared with control group, at single nano titanium oxide and ultraviolet light
Reason, α -2 of cell surface, 6 sialic acid expressions have certain increase.But after being jointly processed by both passing through, its expression quantity
It is increased more obvious, increase about 6 times (Fig. 4).So these results show that nano titanium oxide and treatment with ultraviolet light
The increase of α -2,6 sialic acids expression can be caused after HaCaT cells.
Embodiment 3
The change of the sialic acid expression of cell under by ultraviolet light ROS caused by nano titanium oxide influenceed：Cell is used
After 0,50 μ g/mL nano titanium oxide and treatment with ultraviolet light, 10 μm of ol/L DCFH-DA is added, continues to be incubated 30min, uses
PBS is washed 2-3 times, can use fluorescence microscope and flow cytomery intracellular ROS level, excitation wavelength 488nm, transmitting
Wavelength 530nm.ROSup is a kind of ROS positive reagents, and substantial amounts of ROS is produced after handling cell, adds 50 μ g/mL ROSup examinations
After agent, and cell incubation 24h, dyed with the SNA of FITC marks, observe result.Vitamin C (VC) is that a kind of antioxidant can be with
ROS is removed, after addition 1mM vitamin C is incubated jointly while nano titanium oxide and ultraviolet processing cell is added, is passed through
Agglutinin SNA is dyed, and detects HaCaT
Pass through the result of fluorescence microscope, it can be seen that after nano titanium oxide or treatment with ultraviolet light, cell
Interior ROS contents have a little increase；And after nano titanium oxide and ultraviolet light are jointly processed by, the intracellular ROS of HaCaT substantially increase
Add.According to streaming quantitative result, it can be seen that after cell is jointly processed by by nano titanium oxide and ultraviolet light, into the cell
ROS compare and add 10 times (Fig. 5) with control group.And intracellular ROS result and the cell that becomes of α -2,6 sialic acid expressions
The change of sialic acid expression to be influenceed gesture consistent by ROS caused by nano titanium oxide under by ultraviolet light, in order to verify the two
Between relation, using ROSup reagents as positive controls, it can make cell produce substantial amounts of ROS；Vitamin C can be with
Remove ROS.Test result indicates that：The HaCaT cells that ROSup is incubated 24h, its α -2 are added, 6 sialic acid expressions substantially increase
Add；And, add vitamin C, α -2 of HaCaT cells, 6 sialic acid tables. in ultraviolet light and nano titanium oxide processing cell simultaneously
Compared up to horizontal with ultraviolet light with cell after nano titanium oxide processing, hence it is evident that reduce.These results it may be said that clear-cells α-
2,6 sialic acid expressions are influenceed by ROS.
Embodiment 4
The change of the D-MANNOSE expression of cell：Take the logarithm the HaCaT cells in growth period, with every hole 3x106It is individual thin
Born of the same parents are added in 6 orifice plates of the cover glass containing sterilizing, cultivate 24h.Add the nano titanium oxide that concentration is 0,50 μ g/mL
DMEM medium treatment HaCaT cells 21 hours.Wash away supernatant, after adding PBS, after 365nm treatment with ultraviolet light 1 hour,
PBS is replaced with DMEM culture mediums, is then incubated again in incubator 2 hours.Supernatant is discarded afterwards, is washed 2-3 times with PBS, is used
4% paraformaldehyde fixes cell 15 minutes, and the PSA for then adding 10 μ g/mL FITC marks is incubated 1 hour, is washed away with PBS more
Remaining dyestuff.Add DAPI to dye 15 minutes, excess dyestuff is washed away with PBS.Cover glass is transferred on slide, cell with
Anti- fluorescence quenching is added dropwise between slide, then with resinene mounting.Treat that resinene air-dries, you can with burnt aobvious with copolymerization
Micro mirror detects.Nucleus is in blueness when taking pictures, and PSA is in green.Carried out quantitative (result is shown in Fig. 6) with sepectrophotofluorometer.
As a result：HaCaT cells are after nano titanium oxide and treatment with ultraviolet light, the D-MANNOSE expression on its cell
Significant change occurs for level.As shown in fig. 6, compared with control group, by single nano titanium oxide and treatment with ultraviolet light, carefully
The D-MANNOSE expression of cellular surface has certain increase.But after being jointly processed by both passing through, its expression quantity is increased more
Add substantially, increased about 8 times (Fig. 6).So these results show that nano titanium oxide and treatment with ultraviolet light HaCaT cells
The increase of D mannoses expression can be caused afterwards.
Embodiment 5
Nm TiO 2-base is tested because of toxicity detection：
1st, take the logarithm the HaCaT cells in growth period, add the digestion of 0.25% pancreatin, cell suspension 1x106/mL, per hole 1mL
Add in 12 orifice plates, cultivated 24 hours in incubator.
2nd, nano titanium oxide is diluted into the every hole addition 1mL of 50 μ g/mL with DMEM to sequentially add, while sets and be free of nanometer
The DMEM of titanium dioxide cultivates cell as a control group, 3 multiple holes of every group of setting.After culture 21 hours, suction is abandoned containing nanometer two
The supernatant of titanium oxide, cleaned 2~3 times with PBS, 1mLPBS is added per hole, it is purple with 365nm under 8W small uviol lamp
Outer light irradiation 1 hour, lamp is apart from cell 10cm；With DMEM substituting PBS after the completion of irradiation, to continue to be placed in incubator culture 2 small
When.
3rd, supernatant is discarded afterwards, is washed 2-3 times with cryostat PBS, 0.5mL cell pyrolysis liquids is then added per hole, on ice
Cell lysis about 15min.Then pyrolysis product is centrifuged into 15min on 12000g centrifuge, takes supernatant as production to be detected
Product.
4 and then carry out single cell gel electrophoresis, by sample on agarose gel electrophoresis with 25V, under conditions of 300mA
30min electrophoresis is run, then takes out slide, with Tri-HCl and gel 15min, finally uses ethidium bromide staining.Using ultraviolet
Imaging system, result is obtained, then analysis of accounts is carried out to result with CASP image processing softwares.
DNA damage=total comet amount of fluorescence the x100% of DNA afterbodys amount of fluorescence/DNA
It may determine that the power of its genotoxicity of the cell of different disposal.
Interpretation of result：As shown in Figure 7, after single nano titanium oxide processing cell, its genotoxicity is not strong,
Simply increase slightly than control group.But after 50 μ g/mL and ultraviolet light are jointly processed by, the genotoxicity of cell substantially increases
Add, increase to 20% or so from original 5% or so.These explanation nano titanium oxides can be brought seriously to cell under ultraviolet light
Gene damage.
Embodiment 6
Nano titanium oxide influences experiment to the hepatocuprein (SOD) of HaCaT cells：
1st, take the logarithm the HaCaT cells in growth period, add the digestion of 0.25% pancreatin, cell suspension 1x106/ mL, per hole 1mL
Add in 12 orifice plates, cultivated 24 hours in incubator.
2nd, nano titanium oxide is diluted into the every hole addition 1mL of various concentrations 0 μ g/mL, 50 μ g/mL with DMEM to sequentially add,
DMEM cultures cell without nano titanium oxide is set simultaneously as a control group, 3 multiple holes of every group of setting.Culture 21 hours
Afterwards, inhale and abandon the supernatant containing nano titanium oxide, cleaned 2~3 times with PBS, 1mLPBS is added per hole, in the small of 8W
365nm ultraviolet lights are used under uviol lamp 1 hour, lamp is apart from cell 10cm；PBS is substituted after the completion of irradiation with DMEM to continue to be placed on
Cultivated 2 hours in incubator.
3rd, supernatant is discarded afterwards, is washed 2-3 times with cryostat PBS, 0.5mL cell pyrolysis liquids is then added per hole, on ice
Cell lysis about 15min.Then pyrolysis product is centrifuged into 15min on 12000g centrifuge, takes supernatant as production to be detected
Product.
4 then using xanthine oxidase measure superoxide dismutase vigor.SOD kits, according to wherein saying
Bright book, different reagents are sequentially added, after reacting 5min, add developer, examined under the ultraviolet specrophotometer at 550nm
Survey.
5th, SOD vigor (U/ml)=(OD measure-OD blank)/(OD standard-OD blank)/50x extension rates/protein matter
Measure concentration (mg/mL).
Interpretation of result：As shown in Figure 8, after simple nano titanium oxide processing cell, the SOD activity level phases of cell
It is only a little to reduce for more unprocessed group of cell.But after 50 μ g/mL and ultraviolet light are jointly processed by, cell
SOD values substantially reduce, be reduced to 1 or so from original 3.5 or so.These explanation nano titanium oxides can be given under ultraviolet light
The hepatocuprein of HaCaT cells brings very havoc.
Embodiment 7
Nano titanium oxide influences experiment to the glutathione (GSH) of HaCaT cells：
1st, take the logarithm the HaCaT cells in growth period, add the digestion of 0.25% pancreatin, cell suspension 1x106/ mL, per hole 1mL
Add in 12 orifice plates, cultivated 24 hours in incubator.
2nd, nano titanium oxide is diluted into the every hole addition 1mL of the μ g/mL of various concentrations 0,50 with DMEM to sequentially add, set simultaneously
Put the DMEM cultures cell without nano titanium oxide as a control group, 3 multiple holes of every group of setting.After culture 21 hours, suction is abandoned
Supernatant containing nano titanium oxide, cleaned 2~3 times with PBS, 1mLPBS is added per hole, in 8W small uviol lamp
Lower to use 365nm ultraviolet lights 1 hour, lamp is apart from cell 10cm；PBS is substituted after the completion of irradiation with DMEM to continue to be placed on incubator
Middle culture 2 hours.
3rd, supernatant is discarded afterwards, is washed 2-3 times with cryostat PBS, 0.5mL cell pyrolysis liquids is then added per hole, on ice
Cell lysis about 15min.Then pyrolysis product is centrifuged into 15min on 12000g centrifuge, takes supernatant as production to be detected
Product.
4 and then GSH kits are used, according to wherein specification, sequentially add different reagents, after reacting 5min, add
Developer, detected under the ultraviolet specrophotometer at 412nm.
5th, GSH concentration (μm ol/L)=(OD measure-OD blank)/(OD standard-OD blank) x standard concentrations x dilutions times
Number/protein quality concentration (mg/mL).
Interpretation of result：As shown in Figure 9, after simple nano titanium oxide processing cell, the GSH activity level phases of cell
It is only a little to reduce for more unprocessed group of cell.But after 50 μ g/mL and ultraviolet light are jointly processed by, cell
GSH values substantially reduce, be reduced to 1.9 or so from original 4.5 or so.These explanation nano titanium oxides can be given under ultraviolet light
The glutathione of HaCaT cells brings very havoc.
Embodiment 8
Nano titanium oxide detects to the lipid peroxidation of HaCaT cells：
1. the HaCaT cells in growth period of taking the logarithm, add the digestion of 0.25% pancreatin, cell suspension 1x106/mL, per hole 1mL
Add in 12 orifice plates, cultivated 24 hours in incubator.
2. nano titanium oxide is diluted into the every hole addition 1mL of various concentrations 0 μ g/mL, 50 μ g/mL with DMEM to sequentially add,
DMEM cultures cell without nano titanium oxide is set simultaneously as a control group, 3 multiple holes of every group of setting.Culture 21 hours
Afterwards, inhale and abandon the supernatant containing nano titanium oxide, cleaned 2~3 times with PBS, 1mLPBS is added per hole, in the small of 8W
365nm ultraviolet lights are used under uviol lamp 1 hour, lamp is apart from cell 10cm；PBS is substituted after the completion of irradiation with DMEM to continue to be placed on
Cultivated 2 hours in incubator.
3. discarding supernatant afterwards, washed 2-3 times with cryostat PBS, 0.5mL cell pyrolysis liquids are then added per hole, on ice
Cell lysis about 15min.Then pyrolysis product is centrifuged into 15min on 12000g centrifuge, takes supernatant as production to be detected
Product.
4. and then use and use TBA experimental evaluations nano titanium oxide under action of ultraviolet light to cytolipin peroxidating shadow
Ring.Using TBAR kits, according to wherein specification, different reagents are sequentially added, after reacting 5min, add developer,
Detected under ultraviolet specrophotometer at 535nm.
5.MDA concentration (μm ol/L)=(OD measure-OD blank)/(OD standard-OD blank) x10/ protein quality concentration
(mg/mL)
Interpretation of result：As shown in Figure 10, after simple nano titanium oxide processing cell, the lipid oxidation of cell is horizontal
For cell compared to more unprocessed group, only a little increase.But after 50 μ g/mL and ultraviolet light are jointly processed by, carefully
The lipid oxidation of born of the same parents substantially increases, and 4 or so are reduced to from original 1 or so.These explanation nano titanium oxides under ultraviolet light can
Peroxidating occurs for the lipid allowed in HaCaT cells, brings damage.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of cutaneous safety evaluation method based on HaCaT cells to nano titanium oxide, comprise the following steps：
1) HaCaT cells are seeded in cell culture medium and cultivated, disappeared when the HaCaT cell growths are to logarithmic phase
Change, postdigestive HaCaT cell suspensions carry out adhere-wall culture 24h, obtain the HaCaT cells of adherent growth；
2) nano titanium oxide is dispersed in PBS, ultrasound, obtains nanometer titanium dioxide titanium solution；
3) the nano titanium oxide solution gradient in the step 2) is diluted to some concentration, is added separately to the step 1)
In the HaCaT cells of obtained adherent growth, after cultivating 21~22h, then ultraviolet irradiates 30~60min；
4) the HaCaT cells after the step 3) middle-ultraviolet lamp is irradiated are cultivated, by the HaCaT cells after culture in MTT
Continue to cultivate in solution；HaCaT cells after MTT hydroponics are determined into light absorption value in DMSO under 492nm wavelength, as reality
Test a group result；
Zeroing group is set, and the result of the zeroing group is the extinction that the solution only successively comprising DMEM, MTT solution and DMSO measures
Degree；
Experimental comparison group is set, and the result of the experimental comparison group is successively through DMEM, MTT solution and DMSO by HaCaT cells
The absorbance measured after solution processing；
Formula shown in the test results, zeroing group result and experimental comparison group result substitution Formulas I is calculated into cell to deposit
Motility rate：
The cell survival rate=(the ODexp-ODneg)/Formulas I of (ODcon-ODneg) × 100%；
Wherein ODexp represents experimental group absorbance, and ODneg represents zeroing group absorbance, and ODcon represents experimental comparison group suction
Shading value；
The concentration of nanometer titanium dioxide titanium solution is as nano-silica according to used in cell survival rate in experimental group 50%~90%
Change the potential danger concentration of titanium solution；
5) handled in the step 1) and digested with the nanometer titanium dioxide titanium solution of the potential danger concentration obtained in the step 4)
Logarithmic phase HaCaT cells afterwards 21 hours, the HaCaT cells after being handled carry out successively 30~60min of UV treatment, after
Continuous culture 2h, the first cleaning and fixed, the addition FITC mark agglutinin solution into the HaCaT cells after the fixation, then according to
It is secondary be incubated, fluorescent staining and second cleaning, measure cleaning after HaCaT cells fluorescence intensity, obtain HaCaT cells
Sialic acid expression；
Zeroing group and experimental comparison group are set；The zeroing group result is only successively comprising nanometer titanium dioxide titanium solution, ultraviolet photograph
Penetrate and fluorescent staining, be incubated, the fluorescence intensity that the solution after cleaning measures；The experimental comparison group result is by HaCaT cells
Successively through nanometer titanium dioxide titanium solution, ultraviolet irradiation and fluorescent staining, the fluorescence intensity for being incubated, being measured after cleaning；By experimental group
As a result, zeroing group result and experimental comparison group result, which are substituted into formula II, calculates, and obtains relative intensity of fluorescence；
Relative intensity of fluorescence=(the MFexp-MFneg)/formula of (MFcon-MFneg) × 100% II
Wherein MFexp represents experimental group fluorescence intensity, and MFneg represents zeroing group fluorescence intensity, and it is glimmering that MFcon represents experimental comparison group
Luminous intensity；
Pass through sialic acid table in the HaCaT cells of the nanometer titanium dioxide titanium solution under various concentrations and treatment with ultraviolet light by analyzing
Whether there is significant difference up between horizontal and control group, to judge the degree of injury of cell；
There is no the limitation of time sequencing between the step 1) and step 2).
2. cutaneous safety evaluation method according to claim 1, it is characterised in that purple in the step 3) and step 5)
The power of outside line irradiation is 6~10W；The wavelength of the ultraviolet is 365nm, and the light source and HaCaT of the ultraviolet irradiation are thin
Distance is 9~11cm between born of the same parents.
3. cutaneous safety evaluation method according to claim 1, it is characterised in that FITC marks are solidifying in the step 5)
Integrate the synthetism lignin solution that plain solution marks as FITC；The mass concentration of the synthetism lignin solution of the FITC marks is 8~12 μ
g/mL。
4. cutaneous safety evaluation method according to claim 1, it is characterised in that the time being incubated in the step 5)
50~70min.
5. cutaneous safety evaluation method according to claim 1, it is characterised in that in the step 5) first cleaning and
Second cleaning is PBS with solution.
6. cutaneous safety evaluation method according to claim 1, it is characterised in that fixation solution in the step 5)
The paraformaldehyde solution for being 4% for mass concentration；The regular time is 12~17min.
7. the cutaneous safety evaluation method according to any one in claim 1~6, it is characterised in that the step
5) excitation wavelength of the fluorescence intensity of the HaCaT cells in after measure cleaning is 488nm.
8. cutaneous safety evaluation method according to claim 1, it is characterised in that postdigestive in the step 1)
The cell concentration of HaCaT cell suspensions is 1x105/mL；The gradient concentration of nanometer titanium dioxide titanium solution is 10 in the step 3)
At least any two kinds of concentration in~1000 μ g/mL；The addition volume of the nanometer titanium dioxide titanium solution is trained with HaCaT cell attachments
The volume of cell culture medium is identical when supporting.
9. cutaneous safety evaluation method according to claim 1, it is characterised in that cell culture medium in the step 1)
It is the culture medium based on DMEM complete mediums, including penicillin, weight/mass percentage composition that weight/mass percentage composition is 1% is
1% streptomysin and weight/mass percentage composition is 10% hyclone.
10. the cutaneous safety evaluation method according to claim 1 or 9, it is characterised in that nanometer two in the step 2)
The granularity of titanium oxide is 20~800nm；The mass concentration of nanometer titanium dioxide titanium solution is in 10~1000 μ g/mL.
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