CN108998496A - A kind of evaluation method of artificial light source visual system injury - Google Patents
A kind of evaluation method of artificial light source visual system injury Download PDFInfo
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- CN108998496A CN108998496A CN201810945868.7A CN201810945868A CN108998496A CN 108998496 A CN108998496 A CN 108998496A CN 201810945868 A CN201810945868 A CN 201810945868A CN 108998496 A CN108998496 A CN 108998496A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
Abstract
The invention particularly discloses a kind of evaluation methods of artificial light source visual system injury, step includes the 10min that thaws in the water-bath for be put into the cryopreservation tube for being stored with human retina cell 37 DEG C, it is charged with the sugared culture solution of DMEM high, it is placed in incubator and carries out recovery culture, then secondary culture is carried out to the cell after recovery, artificial light source is placed in into the incubator of the 3rd generation or the 4th generation cell, 12h is chosen respectively, the passage cell of 18h and for 24 hours illumination cultivation carries out subsequent experimental, finally using the passage cell of above-mentioned illumination cultivation as material extraction RNA, and cDNA is synthesized by template reverse transcription of mRNA, again using cDNA as template, inflammatory factor design special primer is caused to carry out quantitative fluorescent PCR in human retina cell, human retina is judged according to the expression quantity of inflammatory factor The degree of impairment of cell, the present invention can detecte various artificial light sources to the degree of impairment of vision system, efficient, sensitive.
Description
Technical field
The present invention relates to a kind of evaluation method of visual system injury, in particular to a kind of artificial light source visual system injury
Evaluation method.
Background technique
Contain melanin granule in Human RPE Cells in Vitro (RPE cell), is largely passed through into intraocular light
This layer absorbs.Under different refractive status, due to the reason of hue difference, the optical density for the different wave length coloured light that RPE absorbs is different
Sample, this may growth, form, function etc. on RPE have certain influence, to affect vision.Cell biology and molecule are raw
Object research confirms high color temperature LED light source to eye cornea epithelial cell, Lens Epithelial Cells, the primary retinal pigment epithelium of people
Cell photo-damage effect；Studies have found that high color temperature LED has damage Mouse Retina photoreceptor cell nuclei, and to cornea and crystalline substance
Shape body has no damage.With the development of science and technology, the electronic equipments such as mobile phone, notebook become increasingly popular, people are exposed to electronic equipment
Time in the environment of illumination is increasingly longer, but whether these illumination impact human eye retina and fail to cause people enough
Attention.
Therefore, it is necessary to which the safety spectrum and secure threshold to various novel artificial light sources are further studied, one is established
The method of kind novel artificial light source visual system injury evaluation.
Summary of the invention
In order to overcome the above-mentioned deficiencies of the prior art, the invention proposes a kind of evaluations of artificial light source visual system injury
Method, can detecte various artificial light sources to the degree of impairment of vision system, efficient, sensitive.
To achieve the goals above, the technical scheme adopted by the invention is that:
A kind of evaluation method of artificial light source visual system injury, comprising the following steps:
S1, the cryopreservation tube for being stored with human retina cell is put into rapidly in 37 DEG C of water-bath to the 10min that thaws；
S2, the human retina cell after defrosting is transferred in centrifuge tube, 3- is centrifuged with the revolving speed of 10000-12000rpm
5min, reject supernatant；
S3, the sugared culture solution of 3-5mLDMEM high is added into centrifuge tube, is inoculated in culture bottle after mixing gently, then sets
Recovery culture, the culture solution of replacement in every two days are carried out in incubator；
S4, after cell covers with 90% or more in culture bottle, by culture solution reject, sterilizing tire ox is added into culture bottle
Serum cleans 3 times；
S5,2mL digestive juice, 37 DEG C of digestion 30min are added into culture bottle；
S6,2mL complete culture solution is added into the culture bottle containing digestive juice, terminates the digestion of digestive juice；
S7,1.5mL 0.1%EDTA solution is added into the culture bottle after cleaning, gently shakes the cell in bottom of bottle is molten
Solution, makes cell completely fall off into cell suspension；
S8, cell suspension is transferred in centrifuge tube, is placed in centrifuge and is centrifuged with the revolving speed of 10000-12000rpm
5min, reject supernatant；
S9, the sugared culture solution of appropriate DMEM high is added into centrifuge tube, transfers it in multiple new culture bottles, juxtaposition
It is cultivated in incubator；
S10, secondary culture is carried out by the method for S4-S9, is placed in artificial light into the incubator of the 3rd generation or the 4th generation cell
Source, chooses 12h, 18h respectively and the passage cell of illumination cultivation carries out subsequent experimental for 24 hours；
S11, using the passage cell of above-mentioned illumination cultivation as material extraction RNA, and using mRNA as template reverse transcription synthesize
CDNA causes inflammatory factor design special primer to carry out quantitative fluorescent PCR in human retina cell then using cDNA as template,
The degree of impairment of human retina cell is judged according to the expression quantity of inflammatory factor.
Preferably, the human retina cell is Human RPE Cells in Vitro or retinal glial cells.
Preferably, the human retina cell is stored in cryopreservation tube, and then cryopreservation tube is placed in liquid nitrogen and is saved.
Preferably, the fetal calf serum containing 10% volume in the sugared culture solution of the DMEM high.
Preferably, the condition of culture of the incubator is 37 DEG C, 5% carbon dioxide.
Preferably, the complete culture solution includes 20% fetal calf serum, 1% mycillin and Hams-F12 culture solution.
Preferably, the digestive juice is 0.5% trypsase.
Preferably, the artificial light source includes LED light, mobile phone screen, notebook screens.
Preferably, control group is provided in S10 step, control group is protected from light culture with masking foil package.
Preferably, the A260/280 ratio of RNA described in S11 step is 1.9-2.0.
Compared with prior art, beneficial effects of the present invention:
(1) evaluation method of a kind of artificial light source visual system injury provided by the invention, can detecte various artificial lights
Source is efficient, sensitive to the degree of impairment of vision system；
(2) using evaluation method detection LED light, mobile phone screen and the notes of artificial light source visual system injury of the invention
Degree of impairment of the three kinds of artificial light sources of this screen to Human RPE Cells in Vitro, testing result discovery LED light, mobile phone screen
Under notebook screens light environment, a degree of damage, and light application time can occur for Human RPE Cells in Vitro
It is longer, damaged condition is bigger；
(3) using evaluation method detection LED light, mobile phone screen and the notes of artificial light source visual system injury of the invention
Three kinds of artificial light sources of this screen to the degree of impairment of retinal glial cells, testing result find LED light, mobile phone screen and
Under notebook screens light environment, a degree of damage can occur for retinal glial cells, and light application time more
Long, damaged condition is bigger.
Specific embodiment
Specific embodiments of the present invention will be further explained below.It should be noted that for these implementations
The explanation of mode is used to help understand the present invention, but and does not constitute a limitation of the invention.In addition, invention described below
Technical characteristic involved in each embodiment can be combined with each other as long as they do not conflict with each other.
Embodiment 1
A kind of evaluation method of artificial light source visual system injury, comprising the following steps:
S1, the cryopreservation tube for being stored with Human RPE Cells in Vitro is put into rapidly in 37 DEG C of water-bath to the 10min that thaws；
S2, the Human RPE Cells in Vitro after defrosting is transferred in centrifuge tube, is centrifuged with the revolving speed of 10000rpm
3min, reject supernatant；
S3, the sugared culture solution of 3mLDMEM high is added into centrifuge tube, is inoculated in culture bottle, is subsequently placed in after mixing gently
37 DEG C, recovery culture, the culture solution of replacement in every two days are carried out in the incubator of 5% carbon dioxide；
S4, after cell covers with 90% or more in culture bottle, by culture solution reject, sterilizing tire ox is added into culture bottle
Serum cleans 3 times；
S5,2mL digestive juice, 37 DEG C of digestion 30min are added into culture bottle；
S6,2mL complete culture solution is added into the culture bottle containing digestive juice, terminates the digestion of digestive juice；
S7,1.5mL 0.1%EDTA solution is added into the culture bottle after cleaning, gently shakes the cell in bottom of bottle is molten
Solution, makes cell completely fall off into cell suspension；
S8, cell suspension is transferred in centrifuge tube, is placed in centrifuge and 5min is centrifuged with the revolving speed of 10000rpm, abandoned
Except supernatant；
S9, the sugared culture solution of appropriate DMEM high is added into centrifuge tube, transfers it in multiple new culture bottles, juxtaposition
It is cultivated in 37 DEG C, the incubator of 5% carbon dioxide；
S10, carry out secondary culture by the method for S4-S9, be placed in LED light into the incubator of the 3rd generation cell, control group with
Masking foil package is protected from light culture, chooses 12h, 18h respectively and the passage cell of illumination cultivation carries out subsequent experimental for 24 hours；
S11, RNA is extracted respectively using the passage cell of above-mentioned illumination cultivation and cellular control unit as material, and detect RNA's
The A260/280 ratio of purity, RNA should be between 1.9-2.0, then using mRNA as template reverse transcription synthesis cDNA, then with
CDNA is template, causes inflammatory factor design special primer to carry out quantitative fluorescent PCR Human RPE Cells in Vitro, due to
The inflammatory factor for causing human retina cellular damage includes NF- κ B, MCP-1 and IL-8, and for human retina when lesion occurs, this three
Kind of inflammatory factor expression quantity will increase, therefore can be sentenced according to the expression quantity of NF- κ B, MCP-1 and IL-8 these three inflammatory factors
The degree of impairment of disconnected Human RPE Cells in Vitro.For the accuracy for guaranteeing experiment, this experiment is repeated three times.Wherein,
Primer used by these three inflammatory factors of NF- κ B, MCP-1 and IL-8 is as follows:
After quantitative fluorescent PCR, by each control group 12h, 18h and for 24 hours human retinal pigment after LED light illumination cultivation
Epithelial cell NF- κ B, MCP-1 and IL-8 these three inflammatory factors expression quantity be labeled as (1 ± 0.00), and record each experimental group
The ratio of three kinds of inflammatory factor expression quantity and three kinds of inflammatory factor expression quantity of control group, as a result, it has been found that, LED lamplight shines 12h, 18h
The expression quantity of Human RPE Cells in Vitro NF- κ Factor B is control group (1 ± 0.00) (1.43 after illumination cultivation for 24 hours
± 0.14), (1.52 ± 0.15) and (1.61 ± 0.34) times；The expression quantity of the MCP-1 factor is control group (1 ± 0.00)
(1.38 ± 0.23), (1.47 ± 0.34) and (1.52 ± 0.51) times；The expression quantity of the IL-8 factor is control group (1 ± 0.00)
(1.49 ± 0.35), (1.61 ± 0.61) and (1.73 ± 0.41) times.It can be seen that under LED light light environment, human retina color
A degree of damage can occur for plain epithelial cell, and light application time is longer, and damaged condition is bigger.
Embodiment 2
A kind of evaluation method of artificial light source visual system injury, comprising the following steps:
S1, the cryopreservation tube for being stored with Human RPE Cells in Vitro is put into rapidly in 37 DEG C of water-bath to the 10min that thaws；
S2, the Human RPE Cells in Vitro after defrosting is transferred in centrifuge tube, is centrifuged with the revolving speed of 11000rpm
4min, reject supernatant；
S3, the sugared culture solution of 4mLDMEM high is added into centrifuge tube, is inoculated in culture bottle, is subsequently placed in after mixing gently
37 DEG C, recovery culture, the culture solution of replacement in every two days are carried out in the incubator of 5% carbon dioxide；
S4, after cell covers with 90% or more in culture bottle, by culture solution reject, sterilizing tire ox is added into culture bottle
Serum cleans 3 times；
S5,2mL digestive juice, 37 DEG C of digestion 30min are added into culture bottle；
S6,2mL complete culture solution is added into the culture bottle containing digestive juice, terminates the digestion of digestive juice；
S7,1.5mL 0.1%EDTA solution is added into the culture bottle after cleaning, gently shakes the cell in bottom of bottle is molten
Solution, makes cell completely fall off into cell suspension；
S8, cell suspension is transferred in centrifuge tube, is placed in centrifuge and 5min is centrifuged with the revolving speed of 11000rpm, abandoned
Except supernatant；
S9, the sugared culture solution of appropriate DMEM high is added into centrifuge tube, transfers it in multiple new culture bottles, juxtaposition
It is cultivated in 37 DEG C, the incubator of 5% carbon dioxide；
S10, secondary culture is carried out by the method for S4-S9, mobile phone screen light source is placed in into the incubator of the 3rd generation cell,
Control group is protected from light culture with masking foil package, chooses 12h, 18h respectively and the passage cell of illumination cultivation carries out subsequent reality for 24 hours
It tests；
S11, RNA is extracted respectively using the passage cell of above-mentioned illumination cultivation and cellular control unit as material, and detect RNA's
The A260/280 ratio of purity, RNA should be between 1.9-2.0, then using mRNA as template reverse transcription synthesis cDNA, then with
CDNA is template, causes inflammatory factor design special primer to carry out quantitative fluorescent PCR Human RPE Cells in Vitro, due to
The inflammatory factor for causing Human RPE Cells in Vitro to damage includes NF- κ B, MCP-1 and IL-8, thus according to NF- κ B,
The expression quantity of these three inflammatory factors of MCP-1 and IL-8 may determine that the degree of impairment of Human RPE Cells in Vitro.To protect
Confirm the accuracy tested, this experiment is repeated three times.Wherein, these three inflammatory factors of NF- κ B, MCP-1 and IL-8 are used
Primer it is as follows:
After quantitative fluorescent PCR, by each control group 12h, 18h and mobile phone screen illumination cultivation descendant retinal color for 24 hours
Plain epithelial cell NF- κ B, MCP-1 and IL-8 these three inflammatory factors expression quantity be labeled as (1 ± 0.00), and record each experiment
The ratio of group three kinds of inflammatory factor expression quantity and three kinds of inflammatory factor expression quantity of control group, as a result, it has been found that, mobile phone screen illumination
The expression quantity of Human RPE Cells in Vitro NF- κ Factor B is control group (1 ± 0.00) after 12h, 18h and for 24 hours illumination cultivation
(1.22 ± 0.31), (1.31 ± 0.43) and (1.44 ± 0.33) times；The expression quantity of the MCP-1 factor be control group (1 ±
0.00) (1.28 ± 0.12), (1.36 ± 0.35) and (1.43 ± 0.27) times；The expression quantity of the IL-8 factor be control group (1 ±
0.00) (1.27 ± 0.25), (1.39 ± 0.52) and (1.48 ± 0.11) times.It can be seen that under mobile phone screen light environment,
A degree of damage can occur for Human RPE Cells in Vitro, and light application time is longer, and damaged condition is bigger.
Embodiment 3
A kind of evaluation method of artificial light source visual system injury, comprising the following steps:
S1, the cryopreservation tube for being stored with Human RPE Cells in Vitro is put into rapidly in 37 DEG C of water-bath to the 10min that thaws；
S2, the Human RPE Cells in Vitro after defrosting is transferred in centrifuge tube, is centrifuged with the revolving speed of 12000rpm
5min, reject supernatant；
S3, the sugared culture solution of 5mLDMEM high is added into centrifuge tube, is inoculated in culture bottle, is subsequently placed in after mixing gently
37 DEG C, recovery culture, the culture solution of replacement in every two days are carried out in the incubator of 5% carbon dioxide；
S4, after cell covers with 90% or more in culture bottle, by culture solution reject, sterilizing tire ox is added into culture bottle
Serum cleans 3 times；
S5,2mL digestive juice, 37 DEG C of digestion 30min are added into culture bottle；
S6,2mL complete culture solution is added into the culture bottle containing digestive juice, terminates the digestion of digestive juice；
S7,1.5mL 0.1%EDTA solution is added into the culture bottle after cleaning, gently shakes the cell in bottom of bottle is molten
Solution, makes cell completely fall off into cell suspension；
S8, cell suspension is transferred in centrifuge tube, is placed in centrifuge and 5min is centrifuged with the revolving speed of 12000rpm, abandoned
Except supernatant；
S9, the sugared culture solution of appropriate DMEM high is added into centrifuge tube, transfers it in multiple new culture bottles, juxtaposition
It is cultivated in 37 DEG C, the incubator of 5% carbon dioxide；
S10, secondary culture is carried out by the method for S4-S9, notebook screens light is placed in into the incubator of the 4th generation cell
Source, control group with masking foil package be protected from light culture, respectively choose 12h, 18h and for 24 hours illumination cultivation passage cell progress it is subsequent
Experiment；
S11, RNA is extracted respectively using the passage cell of above-mentioned illumination cultivation and cellular control unit as material, and detect RNA's
The A260/280 ratio of purity, RNA should be between 1.9-2.0, then using mRNA as template reverse transcription synthesis cDNA, then with
CDNA is template, causes inflammatory factor design special primer to carry out quantitative fluorescent PCR Human RPE Cells in Vitro, due to
The inflammatory factor for causing Human RPE Cells in Vitro to damage includes NF- κ B, MCP-1 and IL-8, thus according to NF- κ B,
The expression quantity of these three inflammatory factors of MCP-1 and IL-8 may determine that the degree of impairment of Human RPE Cells in Vitro.To protect
Confirm the accuracy tested, this experiment is repeated three times.Wherein, these three inflammatory factors of NF- κ B, MCP-1 and IL-8 are used
Primer it is as follows:
After quantitative fluorescent PCR, by each control group 12h, 18h and for 24 hours human retina after notebook screens illumination cultivation
Pigment epithelial cell NF- κ B, MCP-1 and IL-8 these three inflammatory factors expression quantity be labeled as (1 ± 0.00), and record each reality
The ratio of three kinds of inflammatory factor expression quantity and three kinds of inflammatory factor expression quantity of control group of group is tested, as a result, it has been found that, notebook screens light
According to 12h, 18h and for 24 hours after illumination cultivation the expression quantity of Human RPE Cells in Vitro NF- κ Factor B be control group (1 ±
0.00) (1.25 ± 0.26), (1.37 ± 0.52) and (1.46 ± 0.13) times；The expression quantity of the MCP-1 factor is control group (1
± 0.00) (1.30 ± 0.32), (1.39 ± 0.31) and (1.46 ± 0.54) times；The expression quantity of the IL-8 factor is control group (1
± 0.00) (1.29 ± 0.17), (1.42 ± 0.76) and (1.53 ± 0.37) times.It can be seen that notebook screens illumination ring
Under border, a degree of damage can occur for Human RPE Cells in Vitro, and light application time is longer, and damaged condition is got over
Greatly.
Embodiment 4
A kind of evaluation method of artificial light source visual system injury, comprising the following steps:
S1, the cryopreservation tube for being stored with retinal glial cells is put into rapidly in 37 DEG C of water-bath to the 10min that thaws；
S2, the retinal glial cells after defrosting are transferred in centrifuge tube, are centrifuged with the revolving speed of 10000rpm
3min, reject supernatant；
S3, the sugared culture solution of 3mLDMEM high is added into centrifuge tube, is inoculated in culture bottle, is subsequently placed in after mixing gently
37 DEG C, recovery culture, the culture solution of replacement in every two days are carried out in the incubator of 5% carbon dioxide；
S4, after cell covers with 90% or more in culture bottle, by culture solution reject, sterilizing tire ox is added into culture bottle
Serum cleans 3 times；
S5,2mL digestive juice, 37 DEG C of digestion 30min are added into culture bottle；
S6,2mL complete culture solution is added into the culture bottle containing digestive juice, terminates the digestion of digestive juice；
S7,1.5mL 0.1%EDTA solution is added into the culture bottle after cleaning, gently shakes the cell in bottom of bottle is molten
Solution, makes cell completely fall off into cell suspension；
S8, cell suspension is transferred in centrifuge tube, is placed in centrifuge and 5min is centrifuged with the revolving speed of 10000rpm, abandoned
Except supernatant；
S9, the sugared culture solution of appropriate DMEM high is added into centrifuge tube, transfers it in multiple new culture bottles, juxtaposition
It is cultivated in 37 DEG C, the incubator of 5% carbon dioxide；
S10, carry out secondary culture by the method for S4-S9, be placed in LED light into the incubator of the 3rd generation cell, control group with
Masking foil package is protected from light culture, chooses 12h, 18h respectively and the passage cell of illumination cultivation carries out subsequent experimental for 24 hours；
S11, RNA is extracted respectively using the passage cell of above-mentioned illumination cultivation and cellular control unit as material, and detect RNA's
The A260/280 ratio of purity, RNA should be between 1.9-2.0, then using mRNA as template reverse transcription synthesis cDNA, then with
CDNA is template, carries out quantitative fluorescent PCR to the cytokine design special primer of retinal glial cells secretion, due to
The cell factor of retinal glial cells secretion includes VEGF and TGF-β, and for human retina when lesion occurs, both are thin
Intracellular cytokine expression quantity will increase, therefore may determine that retina mind according to the expression quantity of VEGF and TGF-β both cell factors
Lesion situation through spongiocyte.For the accuracy for guaranteeing experiment, this experiment is repeated three times.Wherein, VEGF and TGF-β this
Primer used by two kinds of cell factors is as follows:
After quantitative fluorescent PCR, by each control group 12h, 18h and for 24 hours retina neuroglia after LED light illumination cultivation
The expression quantity of both cell factors of cell plastid VEGF and TGF-β is labeled as (1 ± 0.00), and records two kinds of cells of each experimental group
The ratio of factor expression amount and two kinds of cytokine-expressing amounts of control group, as a result, it has been found that, LED lamplight shines 12h, 18h and illumination for 24 hours
After culture the expression quantity of retinal glial cells VEGF cell factor be control group (1 ± 0.00) (1.21 ± 0.34),
(1.25 ± 0.41) and (1.27 ± 0.37) times；The expression quantity of TGF-β cell factor be control group (1 ± 0.00) (1.24 ±
0.13), (1.27 ± 0.35) and (1.29 ± 0.10) times.It can be seen that retinal neuroglia is thin under LED light light environment
A degree of damage can occur for born of the same parents, and light application time is longer, and damaged condition is bigger.
Embodiment 5
A kind of evaluation method of artificial light source visual system injury, comprising the following steps:
S1, the cryopreservation tube for being stored with retinal glial cells is put into rapidly in 37 DEG C of water-bath to the 10min that thaws；
S2, the retinal glial cells after defrosting are transferred in centrifuge tube, are centrifuged with the revolving speed of 11000rpm
4min, reject supernatant；
S3, the sugared culture solution of 4mLDMEM high is added into centrifuge tube, is inoculated in culture bottle, is subsequently placed in after mixing gently
37 DEG C, recovery culture, the culture solution of replacement in every two days are carried out in the incubator of 5% carbon dioxide；
S4, after cell covers with 90% or more in culture bottle, by culture solution reject, sterilizing tire ox is added into culture bottle
Serum cleans 3 times；
S5,2mL digestive juice, 37 DEG C of digestion 30min are added into culture bottle；
S6,2mL complete culture solution is added into the culture bottle containing digestive juice, terminates the digestion of digestive juice；
S7,1.5mL 0.1%EDTA solution is added into the culture bottle after cleaning, gently shakes the cell in bottom of bottle is molten
Solution, makes cell completely fall off into cell suspension；
S8, cell suspension is transferred in centrifuge tube, is placed in centrifuge and 5min is centrifuged with the revolving speed of 11000rpm, abandoned
Except supernatant；
S9, the sugared culture solution of appropriate DMEM high is added into centrifuge tube, transfers it in multiple new culture bottles, juxtaposition
It is cultivated in 37 DEG C, the incubator of 5% carbon dioxide；
S10, secondary culture is carried out by the method for S4-S9, mobile phone screen light source is placed in into the incubator of the 3rd generation cell,
Control group is protected from light culture with masking foil package, chooses 12h, 18h respectively and the passage cell of illumination cultivation carries out subsequent reality for 24 hours
It tests；
S11, RNA is extracted respectively using the passage cell of above-mentioned illumination cultivation and cellular control unit as material, and detect RNA's
The A260/280 ratio of purity, RNA should be between 1.9-2.0, then using mRNA as template reverse transcription synthesis cDNA, then with
CDNA is template, carries out quantitative fluorescent PCR to the cytokine design special primer of retinal glial cells secretion, due to
The cell factor of retinal glial cells secretion includes VEGF and TGF-β, and for human retina when lesion occurs, both are thin
Intracellular cytokine expression quantity will increase, therefore may determine that retina mind according to the expression quantity of VEGF and TGF-β both cell factors
Lesion situation through spongiocyte.For the accuracy for guaranteeing experiment, this experiment is repeated three times.Wherein, VEGF and TGF-β this
Primer used by two kinds of cell factors is as follows:
After quantitative fluorescent PCR, by each control group 12h, 18h and for 24 hours retina neural after mobile phone screen illumination cultivation
The expression quantity of both cell factors of spongiocyte VEGF and TGF-β is labeled as (1 ± 0.00), and records two kinds of each experimental group thin
The ratio of intracellular cytokine expression quantity and two kinds of cytokine-expressing amounts of control group, as a result, it has been found that, LED lamplight light according to 12h, 18h and for 24 hours
Expression quantity according to retinal glial cells VEGF cell factor after culture be control group (1 ± 0.00) (1.23 ±
0.23), (1.26 ± 0.53) and (1.30 ± 0.38) times；The expression quantity of TGF-β cell factor is control group (1 ± 0.00)
(1.25 ± 0.26), (1.28 ± 0.43) and (1.31 ± 0.23) times.It can be seen that under mobile phone screen light environment, retina
A degree of damage can occur for Deiter's cells, and light application time is longer, and damaged condition is bigger.
Embodiment 6
A kind of evaluation method of artificial light source visual system injury, comprising the following steps:
S1, the cryopreservation tube for being stored with retinal glial cells is put into rapidly in 37 DEG C of water-bath to the 10min that thaws；
S2, the retinal glial cells after defrosting are transferred in centrifuge tube, are centrifuged with the revolving speed of 12000rpm
5min, reject supernatant；
S3, the sugared culture solution of 5mLDMEM high is added into centrifuge tube, is inoculated in culture bottle, is subsequently placed in after mixing gently
37 DEG C, recovery culture, the culture solution of replacement in every two days are carried out in the incubator of 5% carbon dioxide；
S4, after cell covers with 90% or more in culture bottle, by culture solution reject, sterilizing tire ox is added into culture bottle
Serum cleans 3 times；
S5,2mL digestive juice, 37 DEG C of digestion 30min are added into culture bottle；
S6,2mL complete culture solution is added into the culture bottle containing digestive juice, terminates the digestion of digestive juice；
S7,1.5mL 0.1%EDTA solution is added into the culture bottle after cleaning, gently shakes the cell in bottom of bottle is molten
Solution, makes cell completely fall off into cell suspension；
S8, cell suspension is transferred in centrifuge tube, is placed in centrifuge and 5min is centrifuged with the revolving speed of 12000rpm, abandoned
Except supernatant；
S9, the sugared culture solution of appropriate DMEM high is added into centrifuge tube, transfers it in multiple new culture bottles, juxtaposition
It is cultivated in 37 DEG C, the incubator of 5% carbon dioxide；
S10, secondary culture is carried out by the method for S4-S9, notebook screens light is placed in into the incubator of the 4th generation cell
Source, control group with masking foil package be protected from light culture, respectively choose 12h, 18h and for 24 hours illumination cultivation passage cell progress it is subsequent
Experiment；
S11, RNA is extracted respectively using the passage cell of above-mentioned illumination cultivation and cellular control unit as material, and detect RNA's
The A260/280 ratio of purity, RNA should be between 1.9-2.0, then using mRNA as template reverse transcription synthesis cDNA, then with
CDNA is template, carries out quantitative fluorescent PCR to the cytokine design special primer of retinal glial cells secretion, due to
The cell factor of retinal glial cells secretion includes VEGF and TGF-β, and for human retina when lesion occurs, both are thin
Intracellular cytokine expression quantity will increase, therefore may determine that retina mind according to the expression quantity of VEGF and TGF-β both cell factors
Lesion situation through spongiocyte.For the accuracy for guaranteeing experiment, this experiment is repeated three times.Wherein, VEGF and TGF-β this
Primer used by two kinds of cell factors is as follows:
After quantitative fluorescent PCR, by each control group 12h, 18h and for 24 hours after notebook screens illumination cultivation, retina is refreshing
Expression quantity through spongiocyte VEGF and TGF-β both cell factors is labeled as (1 ± 0.00), and records two kinds of each experimental group
The ratio of cytokine-expressing amount and two kinds of cytokine-expressing amounts of control group, as a result, it has been found that, notebook screens illumination 12h, 18h
The expression quantity of retinal glial cells VEGF cell factor is control group (1 ± 0.00) (1.30 after illumination cultivation for 24 hours
± 0.21), (1.34 ± 0.36) and (1.38 ± 0.24) times；The expression quantity of TGF-β cell factor is control group (1 ± 0.00)
(1.28 ± 0.62), (1.33 ± 0.22) and (1.37 ± 0.17) times.It can be seen that under notebook screens light environment, view
A degree of damage can occur for film Deiter's cells, and light application time is longer, and damaged condition is bigger.
Above the embodiments of the present invention are described in detail, but the present invention is not limited to described embodiments.It is right
For those skilled in the art, in the case where not departing from the principle of the invention and spirit, these embodiments are carried out more
Kind change, modification, replacement and modification, still fall in protection scope of the present invention.
Claims (10)
1. a kind of evaluation method of artificial light source visual system injury, it is characterised in that: the following steps are included:
S1, the cryopreservation tube for being stored with human retina cell is put into rapidly in 37 DEG C of water-bath to the 10min that thaws；
S2, the human retina cell after defrosting is transferred in centrifuge tube, 3-5min is centrifuged with the revolving speed of 10000-12000rpm,
Reject supernatant；
S3, the sugared culture solution of 3-5mLDMEM high is added into centrifuge tube, is inoculated in culture bottle after mixing gently, is subsequently placed in training
It supports in case and carries out recovery culture, the culture solution of replacement in every two days；
S4, after cell covers with 90% or more in culture bottle, by culture solution reject, sterilizing fetal calf serum is added into culture bottle
Cleaning 3 times；
S5,2mL digestive juice, 37 DEG C of digestion 30min are added into culture bottle；
S6,2mL complete culture solution is added into the culture bottle containing digestive juice, terminates the digestion of digestive juice；
S7,1.5mL 0.1%EDTA solution is added into the culture bottle after cleaning, gently shakes the cell dissolution in bottom of bottle,
Cell is set to completely fall off into cell suspension；
S8, cell suspension is transferred in centrifuge tube, is placed in centrifuge and 5min is centrifuged with the revolving speed of 10000-12000rpm,
Reject supernatant；
S9, the sugared culture solution of appropriate DMEM high is added into centrifuge tube, transfers it in multiple new culture bottles, is placed in training
It supports and is cultivated in case；
S10, secondary culture is carried out by the method for S4-S9, is placed in artificial light source into the incubator of the 3rd generation or the 4th generation cell, point
Not Xuan Qu 12h, 18h and for 24 hours illumination cultivation passage cell carry out subsequent experimental；
S11, using the passage cell of above-mentioned illumination cultivation as material extraction RNA, and using mRNA as template reverse transcription synthesize cDNA, so
Afterwards using cDNA as template, inflammatory factor design special primer is caused to carry out quantitative fluorescent PCR in human retina cell, according to inflammatory
The expression quantity of the factor judges the degree of impairment of human retina cell.
2. a kind of evaluation method of artificial light source visual system injury according to claim 1, it is characterised in that: the people
Retina cell is Human RPE Cells in Vitro or retinal glial cells.
3. a kind of evaluation method of artificial light source visual system injury according to claim 1, it is characterised in that: the people
Retina cell is stored in cryopreservation tube, and then cryopreservation tube is placed in liquid nitrogen and is saved.
4. a kind of evaluation method of artificial light source visual system injury according to claim 1, it is characterised in that: described
Fetal calf serum containing 10% volume in DMEM high sugar culture solution.
5. a kind of evaluation method of artificial light source visual system injury according to claim 1, it is characterised in that: the training
The condition of culture for supporting case is 37 DEG C, 5% carbon dioxide.
6. a kind of evaluation method of artificial light source visual system injury according to claim 1, it is characterised in that: described complete
Full nutrient solution includes 20% fetal calf serum, 1% mycillin and Hams-F12 culture solution.
7. a kind of evaluation method of artificial light source visual system injury according to claim 1, it is characterised in that: described to disappear
Change liquid is 0.5% trypsase.
8. a kind of evaluation method of artificial light source visual system injury according to claim 1, it is characterised in that: the people
Work light source includes LED light, mobile phone screen, notebook screens.
9. a kind of evaluation method of artificial light source visual system injury according to claim 1, it is characterised in that: S10 step
Control group is provided in rapid, control group is protected from light culture with masking foil package.
10. a kind of evaluation method of artificial light source visual system injury according to claim 1, it is characterised in that: S11 step
The A260/280 ratio of RNA described in rapid is 1.9-2.0.
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