CN105928913A - Cell phenotype based high-content multi-index renal toxicity detection method and applications thereof - Google Patents
Cell phenotype based high-content multi-index renal toxicity detection method and applications thereof Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
Abstract
The invention discloses a cell phenotype based high-content multi-index renal toxicity detection method and applications thereof. According to the detection method, A fluorescent molecular probe that can change the functions of nucleus or mitochondrial and carry out specific detection is adopted to label cells; a high content screening system is taken as the detection means, based on the high content screening system, live cells, which are injected with drugs to be detected, are subjected to imaging analysis so as to determine at least one influence of the drugs on cell number, nucleus and mitochondrial, and thus the influence of the drugs on the cells can be determined. The invention also discloses applications of the detection method. The provided detection method has the advantages that multiple indexes can be detected, while the cell structure and functional integrity are maintained, and the detection method has the characteristics of rapidness, high flux, low cost, and high accuracy.
Description
Technical field
The present invention relates to drug toxicity detection field, be specifically related to a kind of high intension based on cell phenotype many
Index nephrotoxicity detection method and application thereof.
Background technology
Chinese herbal medicine is applied to the history of clinical existing more than 2000 year in China, but the research of its toxic and side effects is
At the early-stage, drug toxicity is the untoward reaction often occurred during development of Chinese herb products and Clinical practice.
Owing to people recognize Shortcomings to Chinese medicine safety issue, in voluntarily, for a long time, in a large number, blindly taking
The traditional Chinese medicine medicinal herbs resource damage that medical herbs causes increases in the recent period year by year, and kidney can be with unique blood because of its sigong
Circulation feature is the organ that untoward reaction easily injures.For the research of medicine in early days nephrotoxicity mainly with dynamic
The kidney cell of thing or people and animal is experimental model.But traditional animal toxicology experiment exist time length,
Cost is high, flux is low, animal usage amount is big, animal individual difference is big, lacked by Experimentation restriction etc.
Point.Conventional nephrotoxicity cell model detection method has the detection of mtt assay, cell cycle, mitochondrial membrane electricity
Position detection, apoptosis detections etc., these method indexs are single, and sensitivity is low, it is difficult to adapt to Chinese medicine and become
Divide complicated feature.High intension detection technique can be on the premise of keeping cellularity and functional completeness
Multiple indexs are detected simultaneously, there is quick, the advantage of high flux, low cost, high accuracy,
And it has been successfully applied to liver toxicity, cardiac toxicity, neurotoxicity and Genotoxic Assessment, to early discovery
Drug toxicity, reduces medicament research and development cost and has value greatly.
At present, there is no and high intension detection technique is applied to the pertinent literature report that Toxicity of Kidney is evaluated, pin
To with this, high intension detection means is applied to the detection of nephrotoxic drugs, evaluates whether medicine has kidney
Toxicity, and attempt being used for the method Literature Consult to the multiple Chinese medicine monomer with potential nephrotoxicity become
Go-on-go is surveyed, and with sensitivity and the accuracy of verification method, thus is evaluation the quickest, cost-effective
Chinese native medicine security provides more may.
Summary of the invention
Based on above-mentioned problems of the prior art, the present invention has designed and developed a kind of based on cell phenotype
The detection method of high intension multi objective nephrotoxicity, it is therefore an objective to be applied to kidney poison by high intension detection technique
Property evaluation, keep cellularity and fully functional on the premise of multiple indexs are detected simultaneously,
There is quick, the feature of high flux, low cost, high accuracy.
The present invention has also designed and developed the detection side of a kind of high intension multi objective nephrotoxicity based on cell phenotype
The application of method, thus provide more for the quickest, accurate, economy, effectively evaluating Chinese native medicine security
Possibility.
The technical scheme that the present invention provides is:
A kind of high intension multi objective nephrotoxicity detection method based on cell phenotype, including: using can be right
Nucleus or mitochondrial function change carry out the fluorescent molecular probe of specific detection and are marked cell,
And using High content screening system as detection means, based on High content screening system to having been added to medicine to be measured
The living cells of thing carries out imaging analysis, show that described medicine to be measured is to cell quantity, nucleus and mitochondrion
At least one impact, thus judge the impact on cell.
Preferably, described medicine to be measured includes: aristolochic acid A, ciclosporin A and cisplatin, calculates inspection
Surveying concentration is to cell quantity, nucleus and at least one shadow mitochondrial in the range of 0.01-100 μm ol/L
Ring.
Preferably, described pharmaceutical samples MEM basal medium dissolved dilution to be measured is extremely
0.01 μm ol/L, is configured in centrifuge tube, and aluminium foil lucifuge-20 DEG C seals and preserves.
Preferably, described cell is HEK293 cell, and it is with containing 10% hyclone, 1% blue or green strepto-
The MEM culture medium culturing of element, cell is at 37 DEG C, 5%CO2Incubator is cultivated, and within every three days, passes on once.
Preferably, described fluorescent molecular probe be Hoechst 33342, Mitotracker Deep Red FM
Or Rhodamine123.
Preferably, when described imaging analysis, select Hoechst 33342, Mitotracker Deep Red
FM and/or Rhodamine 123 passage carries out living cells imaging.
Preferably, described medicine is detected on when affecting of described cell, calculating cell quantity, nucleus
Area, nucleus circularity, mitochondrial mass and mitochondrial membrane potential index, investigate the medicine shadow to cell
Ring.
Preferably, described High content screening system includes as detection means, concrete operations: cell connects
Plant after orifice plate, collect exponential phase cell, with 1 × 104Individual/mL concentration adds culture plate,
100 μ L/ holes, 37 DEG C, 5%CO2Add after incubator is cultivated 24h and dilute with MEM basal medium
Medicine described to be measured, 37 DEG C of lucifuges, 5%CO2After cultivating 24 hours, add containing Hoechst 33342
With the MEM basal medium 50 μ L/ hole of Mitotracker Deep Red FM, 37 DEG C of lucifuges, 5%CO2
Cultivate 30min, add the MEM basal medium 100 μ L/ hole containing Rhodamine123, keep away for 37 DEG C
Light, 5%CO2Cultivate 30min, select Hoechst 33342, Mitotracker Deep Red FM and/
Or Rhodamine 123 passage carries out living cells imaging, image is acquired, calculating cell quantity,
Nuclear area, nucleus circularity, mitochondrial mass, the IC of 5 indexs of mitochondrial membrane potential50Value inspection
Survey the impact on cell.
The application of a kind of high intension multi objective nephrotoxicity detection method based on cell phenotype, is used for having kidney
The screening of toxic traditional Chinese medicine monomer.
Preferably, the impact of cell is detected selection cell quantity, nucleus face by described Chinese medicine monomer
Long-pending, nucleus circularity, mitochondrial mass and the IC of 5 indexs of mitochondrial membrane potential50Value.
The present invention is had the advantages that compared with prior art
In invention during foundation by high intension multi objective nephrotoxicity detection method, have selected respectively
Three kinds of mechanism of aristolochic acid A, ciclosporin A and cisplatin are different and clinic has different purposes but all serious with it
Toxicity of Kidney and use limited medicine and by this method 2 kinds are had nephrotoxicity report but mechanism still
Indefinite Chinese medicine monomer detects, and the positive drug median lethal dose(LD 50) measured by the present invention is reported with document
Close, and monomer Selection detectable concentration is low, and effect is obvious, it was demonstrated that set up based on multi objective high intension
Nephrotoxicity detection method compare other traditional methods for, have sensitiveer, the result of detection more accurately,
Evaluation index more comprehensively and the advantage of more convenient operation, and is keeping cellularity and fully functional
On the premise of property, can nucleus and multiple indexs such as mitochondrial form and function be carried out accurately simultaneously
Detection and analysis, by different pharmaceutical, the different damaging mechanisms of Toxicity of Kidney are not limited, for Chinese medicine
And the research of monomer component nephrotoxicity makes preliminary judgement, Chinese medicine monomer composition and Chinese medicine can be successfully applied to
The toxicity assessment of new drug and screening.
Accompanying drawing explanation
Fig. 1 is the high intension representative image of 3 kinds of positive medicines to be measured of the present invention.
Fig. 2 a is 3 kinds of positive drug amount effect relation curves to cell survival rate situation of change, and tables of data is shown as:
Mean ± SEM, n=4.
Fig. 2 b is 3 kinds of positive drug amount effect relation curves to mitochondrial mass situation of change, and tables of data is shown as:
Mean ± SEM, n=4.
Fig. 2 c is 3 kinds of positive drug amount effect relation curves to mitochondrial membrane potential situation of change, and data represent
For: mean ± SEM, n=4.
Fig. 2 d is 3 kinds of positive drug amount effect relation curves to nuclear area situation of change, and tables of data is shown as:
Mean ± SEM, n=4.
Fig. 2 e is 3 kinds of positive drug amount effect relation curves to nucleus circularity situation of change, and tables of data is shown as:
Mean ± SEM, n=4.
Fig. 3 is the high intension representative image of triptolide and cantharidin.
Fig. 4 a is triptolide and the cantharidin amount effect relation curve to survival rate situation of change, tables of data
It is shown as: mean ± SEM, n=3.
Fig. 4 b is triptolide and the cantharidin amount effect relation curve to mitochondrial mass situation of change, number
According to being expressed as: mean ± SEM, n=3.
Fig. 4 c is triptolide and the amount effect relation curve of cantharidin mitochondrial membrane potential situation of change, number
According to being expressed as: mean ± SEM, n=3.
Fig. 4 d is triptolide and the amount effect relation curve of cantharidin nuclear area situation of change, data
It is expressed as: mean ± SEM, n=3.
Fig. 4 e is triptolide and the amount effect relation curve of cantharidin nucleus circularity situation of change, data
It is expressed as: mean ± SEM, n=3.
Detailed description of the invention
The present invention is described in further detail below in conjunction with the accompanying drawings, to make those skilled in the art's reference
Description word can be implemented according to this.
The invention provides a kind of high intension multi objective nephrotoxicity detection method based on cell phenotype, including:
Nucleus or mitochondrial function can be changed and carry out the fluorescent molecular probe of specific detection to cell by employing
It is marked, and using High content screening system as detection means, based on High content screening system to
The living cells adding medicine to be measured carries out imaging analysis, show that described medicine to be measured is to cell quantity, cell
Core and at least one impact mitochondrial, thus judge the impact on cell.
In another kind of embodiment, described medicine to be measured includes: aristolochic acid A, ciclosporin A and cisplatin,
Calculate detectable concentration be in the range of 0.01-100 μm ol/L to cell quantity, nucleus and mitochondrial at least
A kind of impact.
In another kind of embodiment, described pharmaceutical samples MEM basal medium dissolved dilution to be measured is extremely
0.01 μm ol/L, is configured in centrifuge tube, and aluminium foil lucifuge-20 DEG C seals and preserves.
In another kind of embodiment, described cell is HEK293 cell, its with containing 10% hyclone,
The MEM culture medium culturing of 1% mycillin, cell is at 37 DEG C, 5%CO2Incubator is cultivated, and every three
It passes on once.
In another kind of embodiment, described fluorescent molecular probe is Hoechst 33342, Mitotracker
Deep Red FM or Rhodamine123.
In another kind of embodiment, when described imaging analysis, select Hoechst 33342, Mitotracker
Deep Red FM and/or Rhodamine 123 passage carries out living cells imaging.
In another kind of embodiment, detect described medicine to when affecting of described cell, calculate cell quantity,
Nuclear area, nucleus circularity, mitochondrial mass and mitochondrial membrane potential index, investigate medicine to carefully
The impact of born of the same parents.
In another kind of embodiment, described High content screening system includes as detection means, concrete operations:
After cell is inoculated in orifice plate, collect exponential phase cell, with 1 × 104Individual/mL concentration adds cultivates
Plate, 100 μ L/ holes, 37 DEG C, 5%CO2Add after incubator cultivates 24h and use MEM basal medium
The medicine described to be measured of dilution, 37 DEG C of lucifuges, 5%CO2After cultivating 24 hours, add containing Hoechst
The MEM basal medium 50 μ L/ hole of 33342 and Mitotracker Deep Red FM, 37 DEG C of lucifuges,
5%CO2Cultivate 30min, add the MEM basal medium 100 μ L/ hole containing Rhodamine123,
37 DEG C of lucifuges, 5%CO2Cultivate 30min, select Hoechst 33342, Mitotracker Deep Red FM
And/or Rhodamine 123 passage carries out living cells imaging, image is acquired, calculating cell quantity,
Nuclear area, nucleus circularity, mitochondrial mass, the IC of 5 indexs of mitochondrial membrane potential50Value inspection
Survey the impact on cell.
Present invention also offers answering of a kind of high intension multi objective nephrotoxicity detection method based on cell phenotype
With, for having the screening of nephrotoxicity Chinese medicine monomer.
In another kind of embodiment, the impact of cell is detected by described Chinese medicine monomer select cell quantity,
Nuclear area, nucleus circularity, mitochondrial mass and the IC of 5 indexs of mitochondrial membrane potential50Value.
Embodiment
1 materials and methods
1.1 experiment materials and instrument
Table 1. experiment material
Table 2. experimental apparatus
1.2 experimental technique
1.2.1HEK293 cell is cultivated
Cell culture condition is recommended according to American Type Culture Collection committee of Chinese Academy of Sciences Shanghai cell bank,
HEK293 cell with containing 10%FBS, the MEM culture medium culturing of 1% mycillin, cell at 37 DEG C,
5%CO2Incubator is cultivated, and within every three days, passes on once.
1.2.2 passage
Cell passes on every other day, discards former training base when passing on, and 4mL FBS rinses 2 times, adds 3mL pancreas
Protease, 37 DEG C of digestion 1min；Pat a bottle wall and make cell detachment, add the full culture medium of 3mL and terminate digestion；
Pouring sterile centrifugation tube into, 1000r is centrifuged 3min；Abandon supernatant, add the full culture medium of 3mL resuspended uniformly
After, take 1mL cell suspension and add 75cm2Culture bottle, adds the full culture medium of 10mL, puts into 37 DEG C,
5%CO2Incubator is cultivated.
1.2.3 sample configuration
Aristolochic acid A (AA), ciclosporin A (CsA), cisplatin (CDDP) are respectively with containing MEM base
Basal culture medium dissolves and stepwise dilution is to testing desired concn；Root according to the literature, AA, CsA, CDDP
EC50It is respectively 20 μ g/ml, 5～50 μm ol/L, 10-50 μm ol/L, therefore, selects according to medicine EC50
Concentration selects 0.01-100 μm ol/L as positive drug experimental concentration；According to Literature Consult, selected cantharidin
And triptolide is related to the Chinese medicine monomer composition of nephrotoxicity report and carries out toxicity screening, all samples are used
MEM basal medium dissolved dilution, to 0.01 μm ol/L, is configured in EP pipe, and aluminium foil lucifuge-20 DEG C is close
Envelope preserves stand-by.
The foundation of the highest intension multi objective nephrotoxicity detection method and optimization
In 96 hole black reveal the exact details Tissue Culture Plate, every hole adds 0.1mg mL-1D-type poly-D-lysine is molten
Liquid 50 μ L；Being coated sucking-off remaining liq after overnight, aseptic FBS solution rinses 3 times；Collect logarithmic growth
Phase cell, with 1 × 104Individual/mL concentration adds culture plate, 100 μ L/ holes；37 DEG C, 5%CO2Incubator
Middle cultivation 24h；Discard culture medium, be separately added into the medicine to be measured with the dilution of MEM basal medium
100 μ L/ holes, 37 DEG C of lucifuges, 5%CO2Cultivate 24h；Adding containing concentration is 1.67 μ g mL-1Hoechst
The MEM basis of 33342 and Mitotracker Deep Red FM that concentration is 0.08 μM of ol/L is cultivated
Base 50 μ L/ hole, 37 DEG C of lucifuges, 5%CO2Cultivate 30min；Discard culture medium, add containing 1.26 μ g mL-1
The MEM basal medium 100 μ L/ hole of Rhodamine123,37 DEG C of lucifuges, 5%CO2Cultivate
30min；Discarding culture medium, MEM culture medium 100 μ L/ hole rinses 1 time；High content screening Systematic selection
Hoechst 33342, Mitotracker 123 3 passages of Deep Red FM and Rhodamine carry out living carefully
Born of the same parents' imaging.
Select to calculate cell quantity, nuclear area, nucleus circularity, mitochondrial mass, mitochondrial membrane
5 indexs of current potential, investigate the medicine impact on cell.
1.2.5 statistical method
Preferred as one, high intension shooting image Harmony 3.0 computed in software is quantized data,
Use SPSS 17.0 Software of Data Statistics, data are carried out test of normality, compare employing between many groups single
Analysis of variance, analysis method is LSD, with P ＜ 0.05 for having significant difference, with P ＜ 0.01
For having pole significant difference.
2 experimental results
2.1 positive drug high intension experimental results
2.1.1 positive drug high intension image taking results
3 kinds of positive drug high intension image taking results are as shown in Figure 1；Wherein, figure a, e, i, m is empty
Organizing cell image in vain, figure b, f, j, n are aristolochic acid A group cell image, and figure c, g, k, o are
Ciclosporin A group cell image, d, h, l, p are cisplatin group cell image, can be observed three from picture
The cell quantity of individual positive drug (figure bcd) is considerably less than blank group (figure a), and from the n amplified,
It can be seen that the obvious change of karyomorphism in o, p figure, wherein o schemes visible significantly cell shrinkage.
2.1.2 positive drug is to surveyed each index dose-effect relationship result
To the amount effect relation curve of each index as in figure 2 it is shown, wherein, figure a is cell survival for 3 kinds of positive drug
Rate situation of change, figure b is mitochondrial mass situation of change, and figure c is mitochondrial membrane potential situation of change,
Figure d is nuclear area situation of change, and figure e is nucleus circularity situation of change；Can from Fig. 2 a～2e
Find out, each positive drug IC to cell survival rate50It is respectively aristolochic acid A 6.474 μMs, ciclosporin A
61.90 μMs, cisplatin 8.288 μMs (Fig. 2 a)；Cause the IC that mitochondrial mass increases50Value is aristolochic acid A
1.420 μMs, ciclosporin A 33.68 μMs, cisplatin 4.029 μMs (Fig. 2 b)；Mitochondrial membrane potential is caused to decline
IC50Value is aristolochic acid A 8.861 μMs, ciclosporin A 12.03 μMs, cisplatin 25.51 μMs (Fig. 2 c)；
Cause the IC that nuclear area declines50Value for aristolochic acid A 145.8 μMs, ciclosporin A 30.17 μMs,
Cisplatin 3.447 × 106μM (Fig. 2 d)；Cause the IC that nucleus circularity changes50Value is respectively aristolochic acid A
7.326 μM, ciclosporin A 13.81 μMs, cisplatin 5.132 μMs (Fig. 2 e)；Wherein, except AA and CDDP
Nuclear area IC50Beyond value is relatively big compared with other desired value corresponding, remaining each index IC50Value
All it is closer to, and with the medicine of document report causes apoptotic EC50It is worth close.
2.2 Chinese medicine monomer composition high intension nephrotoxicity the selection result
2.2.1 Chinese medicine monomer composition high intension image taking results
Preferred as one, the triptolide (TR) clearer and more definite with nephrotoxicity and cantharidin (CA)
Image taking results is as representative, as it is shown on figure 3, triptolide and cantharidin monomer component are all with 10-8
The less concentration of Mol/L is screened, and figure a, d, g, j be blank group cell image, scheme b, e, h,
K is cantharidin group cell image, and figure c, f, i, l are triptolide group cell image, can from picture
Observe in b, c figure that cell quantity is considerably less than a figure, and it can be seen that thin from j, k, l figure amplified
The obvious change of karyon form, wherein the visible significantly cell shrinkage of k figure, can be seen that nucleus in l figure
Substantially become big.
On the impact of concrete each index respectively as shown in Fig. 4 a～Fig. 4 e, with triptolide and cantharidin it is
Represent, its dose-effect relationship is investigated, 10-4-10-8In Mol/L concentration range, calculate gained medicine
IC to cell survival rate50It is respectively cantharidin 8.689 μMs, triptolide 0.09466 μM (Fig. 4 a)；
Cause the IC that mitochondrial mass increases50Value is cantharidin～5665 μMs, triptolide 29.88 μMs (figure
4b)；Cause the IC that mitochondrial membrane potential declines50Value is cantharidin 50.19 μMs, triptolide
0.1863 μM (Fig. 4 c)；And cantharidin causes the IC of nuclear area shrinkage50Value is 51.15 μMs, Thunder God
Rattan A prime causes the IC of long term voyage swelling50Value is 0.006012 μM (Fig. 4 d)；Two guiding drugs play nucleus circularity
The IC of change50Value is respectively cantharidin～8875 μMs, triptolide 0.0484 μM (Fig. 4 e)；Result is demonstrate,proved
Bright, two medicines all can cause the significant change of each index under low concentration, with the selection result in 10-8Mol/L
Cell quantity can be caused to decline for concentration and morphologic change is consistent, demonstrate that experiment set up based on cell
The accuracy of the high intension multiple determination method of phenotype.
3. brief summary
During this experiment high intension multi objective nephrotoxicity detection method is set up, we have selected horse respectively
Three kinds of mechanism of Semen Oroxyli acid A, ciclosporin A and cisplatin are different and clinic has different purposes but all serious with it
Toxicity of Kidney and use limited medicine and 2 kinds had nephrotoxicity report but the Chinese medicine that is still not clear of mechanism
Monomer detects.Experiment is demonstrated by these three medicine in difference accurately with HEK293 for cell model
Impact on Lymphocytic phenotype under concentration.From experimental result, under three kinds of positive drug effects, carefully
Born of the same parents' survival rate all rises with drug level and is gradually reduced；Mitochondrial mass is with drug level rising gradually
Raise；Mitochondrial membrane potential raises with drug level and reduces；Nuclear area and nucleus circularity also with
The concentration medicine raises the change that generation is certain.Show that these 3 positive drug are to nucleus and mitochondrion merit
Certain impact can be created, and then cause apoptosis, and with institute's method for building up to 2 kinds of Chinese medicine monomer
When composition detects, although detectable concentration is extremely low, from these results, 2 kinds of monomer components pair
Nucleus and mitochondrion cause damage in various degree.
Positive drug median lethal dose(LD 50) measured by this experiment is close with document report, and monomer Selection
Detectable concentration is relatively low, and effect is more apparent, it was demonstrated that the nephrotoxicity detection side set up based on multi objective high intension
For method compares other traditional methods, have sensitiveer, the result of detection more accurately, evaluation index more complete
Face and the advantage of more convenient operation, can carry out essence to nucleus and mitochondrial form and function simultaneously
True analysis, studies for Chinese medicine and monomer component nephrotoxicity thereof and makes preliminary judgement, in can being successfully applied to
Medicine monomer component and the toxicity assessment of new Chinese medicine and screening.
Although embodiment of the present invention are disclosed as above, but it is not restricted to description and embodiment party
Listed utilization in formula, it can be applied to various applicable the field of the invention completely, for being familiar with ability
For the personnel in territory, be easily achieved other amendment, therefore without departing substantially from claim and etc. homotype
Enclosing under limited general concept, the present invention is not limited to specific details and shown here as the figure with description
Example.
Claims (10)
1. a high intension multi objective nephrotoxicity detection method based on cell phenotype, it is characterised in that bag
Include: use and nucleus or mitochondrial function can be changed the fluorescent molecular probe pair carrying out specific detection
Cell is marked, and using High content screening system as detection means, based on High content screening system pair
The living cells having been added to medicine to be measured carries out imaging analysis, draw described medicine to be measured to cell quantity,
Nucleus and at least one impact mitochondrial, thus judge the impact on cell.
2. high intension multi objective nephrotoxicity detection method based on cell phenotype as claimed in claim 1,
It is characterized in that, described medicine to be measured includes: aristolochic acid A, ciclosporin A and cisplatin, calculates detection
Concentration is on cell quantity, nucleus and at least one impact mitochondrial in the range of 0.01-100 μm ol/L.
3. high intension multi objective nephrotoxicity detection side based on cell phenotype as claimed in claim 1 or 2
Method, it is characterised in that described pharmaceutical samples MEM basal medium dissolved dilution to be measured is extremely
0.01 μm ol/L, is configured in centrifuge tube, and aluminium foil lucifuge-20 DEG C seals and preserves.
4. high intension multi objective nephrotoxicity detection method based on cell phenotype as claimed in claim 3,
It is characterized in that, described cell is HEK293 cell, and it is with containing 10% hyclone, 1% mycillin
MEM culture medium culturing, cell is at 37 DEG C, 5%CO2Incubator is cultivated, and within every three days, passes on once.
5. high intension multi objective nephrotoxicity detection method based on cell phenotype as claimed in claim 4,
It is characterized in that, described fluorescent molecular probe is Hoechst 33342, Mitotracker Deep Red FM
Or Rhodamine123.
6. based on cell phenotype the high intension multi objective nephrotoxicity detection side as described in claim 4 or 5
Method, it is characterised in that when described imaging analysis, selects Hoechst 33342, Mitotracker Deep
Red FM and/or Rhodamine 123 passage carries out living cells imaging.
7. high intension multi objective nephrotoxicity detection method based on cell phenotype as claimed in claim 6,
It is characterized in that, detect described medicine to when affecting of described cell, calculating cell quantity, nucleus face
Long-pending, nucleus circularity, mitochondrial mass and mitochondrial membrane potential index, investigate the medicine impact on cell.
8. based on cell phenotype the high intension multi objective as according to any one of claim 1,2 and 7
Nephrotoxicity detection method, it is characterised in that described High content screening system, as detection means, is specifically grasped
Work includes: after cell is inoculated in orifice plate, collects exponential phase cell, with 1 × 104Individual/mL concentration
Addition culture plate, 100 μ L/ holes, 37 DEG C, 5%CO2Add after incubator cultivates 24h and use MEM base
The medicine described to be measured of basal culture medium dilution, 37 DEG C of lucifuges, 5%CO2After cultivating 24 hours, addition contains
The MEM basal medium 50 μ L/ hole of Hoechst 33342 and Mitotracker Deep Red FM, 37 DEG C
Lucifuge, 5%CO2Cultivate 30min, add the MEM basal medium 100 μ L/ containing Rhodamine123
Hole, 37 DEG C of lucifuges, 5%CO2Cultivate 30min, select Hoechst 33342, Mitotracker Deep Red
FM and/or Rhodamine 123 passage carries out living cells imaging, is acquired image, calculates cell
Quantity, nuclear area, nucleus circularity, mitochondrial mass, the IC of 5 indexs of mitochondrial membrane potential50
The value detection impact on cell.
9. an application for high intension multi objective nephrotoxicity detection method based on cell phenotype, its feature exists
In, for having the screening of nephrotoxicity Chinese medicine monomer.
10. high intension multi objective nephrotoxicity detection method based on cell phenotype as claimed in claim 9
Application, it is characterised in that the impact of cell is detected by described Chinese medicine monomer and selects cell quantity, thin
Karyon area, nucleus circularity, mitochondrial mass and the IC of 5 indexs of mitochondrial membrane potential50Value.
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Cited By (5)
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| CN107475346A (en) * | 2017-08-11 | 2017-12-15 | 北京汉典制药有限公司 | A kind of method of in-vitro screening medicine for treating diabetic nephropathy |
| CN109556999A (en) * | 2018-12-13 | 2019-04-02 | 中南大学湘雅医院 | A kind of application method based on high intension technology in poikilocyte detection |
| CN113960302A (en) * | 2021-09-29 | 2022-01-21 | 中国人民解放军军事科学院军事医学研究院 | Kidney toxicity detection method based on high content technology and application thereof |
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| CN116912825A (en) * | 2023-09-14 | 2023-10-20 | 生态环境部华南环境科学研究所(生态环境部生态环境应急研究所) | High content chemical pulmonary toxicity screening method by utilizing artificial intelligence and machine learning |
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