CN105738585A - Drug screening kit - Google Patents
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- CN105738585A CN105738585A CN201410752047.3A CN201410752047A CN105738585A CN 105738585 A CN105738585 A CN 105738585A CN 201410752047 A CN201410752047 A CN 201410752047A CN 105738585 A CN105738585 A CN 105738585A
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Abstract
The invention discloses a drug screening kit and a use method thereof, and belongs to the technical field of biological detection. The kit comprises Caenorhabditis elegans, a Caenorhabditis elegans medium, Escherichia coli and a dye. Different dyeing effects of the dye 2',7'-dichlorodihydrofluorescein diethyl ester on a mutant and wild Caenorhabditis elegans are used to realize rapid, efficient and accurate simple-operation and high-flux screening of effective antitumor drugs, so the time and the cost of general research and development workers of antitumor drugs are saved.
Description
Technical field
The present invention relates to the using method of a kind of drug screening kit and this test kit, belong to raw
Thing pharmaceutical technology field.
Background technology
It is known that RAS signal approach is the signal path relevant with tumor generation.The mankind
In tumor, the dysfunction of RAS albumen generally exists.It is reported, the pulmonary carcinoma of about 50%
With there is ras mutant gene in intestinal cancer, in 95% cancer of pancreas.If RAS albumen is continuously in work
Change state, can be combined with the effect protein in downstream, pass the signal along to downstream signal element, can
The abnormality proliferation of cell can be caused, lead oncogenic generation.Therefore, the signal of RAS albumen leads to
Road becomes the target spot that antitumor medicine screening is new.
Tumor cell line, immortalized cells and primary cultured cell be research tumor aetiology and
Pathogenetic important models.But, many drug candidates activity in vivo and external activity
Dependency is very poor.Therefore, there is technological deficiency in the in-vitro screening of medicine in methodology.And medicine
The internal screening of thing is it can be avoided that this type of technological deficiency.Therefore, current people generally utilize mice
Screening cancer therapy drug as model organism, but but to there is the cycle long for the method, cost is high,
The big defect of workload, it is difficult to realize high flux screening.
Multiple studies have shown that, Caenorhabditis elegans can suppress ras proto-oncogene as screening
The model organism of the medicine of activity.But present stage uses Caenorhabditis elegans to yet suffer from necessarily
Difficulty, main cause be for common people from the point of view of, Caenorhabditis elegans modeling, cultivation,
Inspection etc. remains a need for technical training and the practice of system, just can complete drug screening, therefore, greatly
A kind of drug screening kit with Caenorhabditis elegans as model is badly in need of in family.
The present invention utilize Caenorhabditis elegans for model organism the most perfect utilize beautiful hidden
Rhabditida, as model organism, constructs Caenorhabditis elegans drug screening kit, this medicine
Screening reagent box has advantage simple to operate, quick, efficient, accurate, high-throughout.
Summary of the invention
It is an object of the invention to provide a kind of drug screening kit, this test kit can utilize show
Beautiful hidden rhabditida realizes the internal screening of antitumor drug.
It is a further object to provide the using method of said medicine screening reagent box, this
Invention utilizes dyeing different with Wild-type C. elegans to mutant for dyestuff H2DCF-DA
Effect reaches simple to operate, quick, efficient, accurate, high flux screening and goes out effective anti-swollen
The purpose of tumor medicine, the research staff for vast antitumor drug saves time and cost.
Drug screening kit disclosed by the invention includes following material:
1. Caenorhabditis elegans wild type N2；
2. Caenorhabditis elegans mutant MT2124；
3. Caenorhabditis elegans CB1275；
4. NGM culture medium；
5. escherichia coli OP50；
6. dyestuff: 2', 7'-dichlorofluorescin diethylester.
Wherein, described tumor is caused by ras proto-oncogene process LAN.Owing to ras believes
Number path is from nematicide to people's high conservative, and in Caenorhabditis elegans, let-60 gene code is protected
The RAS albumen kept, it has the homology of 83% compared with the RAS albumen of the mankind.If ras
Sudden change, can cause nematicide to produce many vaginal orifices in Caenorhabditis elegans, then cause in the mankind
The malignant proliferation of cell, leads oncogenic generation.Therefore, model organism Caenorhabditis elegans
(Caenorhabditis elegans) Ras/MAPK signal path excessive activation type mutant
It is widely used in screening suppression ras proto-oncogene path excessive activation as tumor model
Antitumor drug.
The described Caenorhabditis elegans used by test kit is the nematicide after synchronization, and nematicide synchronizes
The step changed is:
1. select in the ripe Caenorhabditis elegans centrifuge tube extremely equipped with 1ml M9 liquid, 4000
Rpm is centrifuged 5 minutes, removes supernatant；
2. adding 500 μ L 1M sodium hydroxide in centrifuge tube, 500 μ L6.4% (V/V) are secondary
Sodium chlorate, concussion cracking；
3. remove supernatant, to precipitation in add 1.5 μ L M9 liquid, shake up, 4000rpm from
The heart 5 minutes, repeats this step 3 time；
4. in clean ovum, add 500 μ L M9 liquid, within 24 hours, hatch into L1 for 18 DEG C
Phase Caenorhabditis elegans.
Wherein, the concentration of described Caenorhabditis elegans is 100/20 μ l.
The using method of the above-mentioned drug screening kit that the present invention provides, described test kit
Using method be:
(1) dyeing of Caenorhabditis elegans and detection
L1 phase Caenorhabditis elegans MT2124 after synchronization is mixed, digit's liquid bulk
In long-pending, the number of Caenorhabditis elegans, standby；96 orifice plates take 2 holes, each Kong Zhongxian
Add the S liquid of 140 μ L, be subsequently adding 20 μ L coli somatic solution, each Kong Zhongjia
Entering the Caenorhabditis elegans MT2124 of about 90, first hole is negative control, adds 20
μ L distilled water, another hole adds for reagent thing；The liquid volume finally making each hole is
200μL；Then by liquid blending, 20 DEG C of shaken cultivation about 6 days, treat that it is grown for becoming
Worm, after i.e. producing false vaginal orifice, sweeps away from flat board with M liquid, transfers to, in centrifuge tube, treat show
Beautiful hidden rhabditida natural sedimentation, removes supernatant, leaves and takes Caenorhabditis elegans；Add containing 0.05%
(V/V) the M9 liquid of polysorbas20, treats Caenorhabditis elegans natural sedimentation, removes supernatant,
Leave and take Caenorhabditis elegans；Again with pure M9 liquid washing to clarification, shift beautiful hidden bar line
Worm is on other 96 new orifice plates, and load position is corresponding with original 96 plates, and each hole does three
Individual parallel control, adds dyestuff, dyes respectively, and the Caenorhabditis elegans after dyeing shows at fluorescence
Micro-Microscopic observation, takes pictures；And utilize fluorescence microplate reader to detect fluorescent value, excitation wavelength
485nm, wavelength of transmitted light 535nm；
(2) experimental data is processed, measure the nematicide fluorescence intensity after dyeing.
Wherein, during described Caenorhabditis elegans dyeing, dye concentrations is
50 μ g/mL, dyeing time is 1.5 hours, dyeing temperature 20 DEG C.
The invention have the benefit that
1. the present invention is to utilize dyestuff H2Caenorhabditis elegans vacation vaginal orifice is contaminated by DCF-DA specificity
Color, then detects the fluorescence intensity of Caenorhabditis elegans colony with fluorescence microplate reader, it is possible to realize
High flux screening, has simple to operate, the advantage that data are objective and accurate.
2. very poor due to many drug candidates activity in vivo and external activity dependency, because of
This, there is technological deficiency in the external high flux screening of medicine in methodology.The present invention uses show
Beautiful hidden rhabditida, as the model organism of internal screening of medicaments, solves the fraud of in-vitro screening medicine
End, and screen cancer therapy drug compared to mice as model organism, the cycle is short, low cost,
Simple to operate, effective antitumor medicine can be gone out, for grinding of vast antitumor drug by high flux screening
The personnel of sending out save time and cost.
Accompanying drawing explanation
Fig. 1 is the fluorescence photo of Caenorhabditis elegans mutant MT2124 dyeing 1.5h；
Fig. 2 is the fluorescence photo of Caenorhabditis elegans wild type N2 dyeing 1.5h；
Fig. 3 is the fluorescent value of the different nematode population dyeing of WT ratios；
Fig. 4 is the result of Sorafenib checking drug screening kit；
Fig. 5 is the Sorafenib impact on Ras/MAPK path many vaginal orifices mutant.
Detailed description of the invention
Specific embodiment presented below is not to realize drug screening kit of the present invention, but not
It is limited to these embodiments.
1. reagent
(1) M9 buffer (M9buffer): every liter buffer contains: 12gNa2HPO4·
12H2O (or 6g Na2HPO4), 3g KH2PO4, 5g NaCl, 0.25g MgSO4·7H2O,
Preferably matching while using.
(2) S buffer (S buffer): 0.1mol/L NaC1,0.05mmol/L
K2HPO4-KH2PO4Buffer (pH=6.0).
(3) NGM (Nematode Growth Medium) culture medium: 1.21g NaCl,
6.8g potassium dihydrogen phosphate, 0.923g dipotassium hydrogen phosphate, 1.08g peptone, 6.86g agar
Add water to 400mL, after sterilizing, add the 1mol/L Adlerika of 400 μ L, 400 μ L
1mol/L calcium chloride solution, the cholesterol solution of 400 μ L 5mg/mL, be down flat plate.
(4) LB culture medium: peptone 10g, sodium chloride 10g, yeast extract 5g, add water
To 1000mL, adjust pH to 7.0.
(5) dyestuff
Dyestuff: 2', 7'-dichlorofluorescin diethylester (dichlorofluorescein
Diacetate, H2DCF-DA), purchased from Sigma company.
(6) Sorafenib, is mainly used in renal carcinoma, primary hepatocarcinoma, and colon cancer is the most right
Pulmonary carcinoma, cancer of pancreas etc. has certain effect.The antitumor action that Mutiple Targets is dual, Sorafenib can
Lived by serine and the threonine kinase of suppression Raf-1, the BRAf of wild type and V599E sudden change
Property, block RAS/RAF/MEK/ERK signal path, thus block cell cycle, induced tumor
Apoptosis, directly suppresses tumor cell proliferation.
2. biomaterial
(1) Caenorhabditis elegans wild type N2, purchased from CGC (Caenorhabditis Genetics
Center), this strain nematicide does not has false vaginal orifice.
(2) Caenorhabditis elegans mutant MT2124, purchased from CGC (Caenorhabditis
Genetics Center), this strain nematicide has false vaginal orifice, and can be along with anti-ras excessive activation
The performance of medicine effect and transfer wild type to, false vaginal orifice also can disappear therewith.
Ras is a kind of small molecular protein, and participate in during regulation and control animal development is the most important
Link.Ras signal path from nematicide to people's high conservative, let-60 in Caenorhabditis elegans
The RAS albumen that gene code is conservative, it have compared with the RAS albumen of the mankind 83% same
Source property.If ras suddenlys change, nematicide can be caused in Caenorhabditis elegans to produce many vaginal orifices,
The mankind are then the malignant proliferations causing cell, lead oncogenic generation.Therefore, beautiful hidden bar
Nematicide (Caenorhabditis elegans) Ras/MAPK signal path excessive activation type suddenlys change
Body is widely used in screening suppression proto-oncogene ras path excessive activation as tumor model
Antitumor drug.
MT2124 is the Caenorhabditis elegans of let-60/ras excessive activation, can produce many vaginal orifices
Phenotype.Medicine is acted on Caenorhabditis elegans MT2124, if it is possible to suppress tested line
The ratio that the many vaginal orifices generation of worm colony is individual, and the wild type nematicide individuality ratio of only one of which vaginal orifice
Example rises, then explanation medicine acts on ras path, has lowered the excessive activation of this path, tool
There is anti-tumor activity.In colony, the ratio of wild type nematicide is the highest, then drug effectiveness is the most aobvious
Write.As represented then contrary with mutant ratio, wherein, WT ratios (%)=100%-dashes forward
Variant ratio (%)
(3) experiment material: Caenorhabditis elegans CB1275, genotype: lin-1 (e1275) IV
Purchased from CGC (Caenorhabditis Genetics Center).
(4) escherichia coli OP50 (uracil leaky mutant), purchased from CGC (Caenorhabditis
Genetics Center), for Caenorhabditis elegans food.
The Color replication experiment of embodiment 1 Caenorhabditis elegans
(1) synchronization of Caenorhabditis elegans
1. select in the ripe Caenorhabditis elegans 2ml centrifuge tube extremely equipped with 1ml M9 liquid,
4000rpm is centrifuged 5 minutes, removes supernatant；
2. in centrifuge tube, add 500 μ L 1M sodium hydroxide, 500 μ L6.4% (V/V)
Sodium hypochlorite, concussion cracking；
3. remove supernatant, to precipitation in add 1.5 μ L M9 liquid, shake up, 4000rpm from
The heart 5 minutes, repeats this step 3 time；
4. in clean ovum, add 500 μ L M9 liquid, within 24 hours, hatch into L1 for 18 DEG C
Phase Caenorhabditis elegans.
(2) dyeing of Caenorhabditis elegans and detection method
1. the dyeing of Caenorhabditis elegans and detection
L1 phase Caenorhabditis elegans MT2124 after above-mentioned synchronization is mixed, digit's liquid
In body volume, the number of Caenorhabditis elegans, standby；96 orifice plates take 2 holes, Mei Gekong
In be initially charged the S liquid of 140 μ L, be subsequently adding 20 μ L coli somatic solution, each
Adding the Caenorhabditis elegans MT2124 of about 90 in hole, first hole is negative control,
Adding 20 μ L distilled water, another hole adds for reagent thing；Finally make the liquid in each hole
Volume is all 200 μ L；Then by liquid blending, 20 DEG C of shaken cultivation about 6 days, treat
After it is grown for the false vaginal orifice of adult, i.e. generation, sweep away from flat board with M liquid, transfer to be centrifuged
Guan Zhong, treats Caenorhabditis elegans natural sedimentation, removes supernatant, leaves and takes Caenorhabditis elegans；
Add the M9 liquid of the polysorbas20 containing 0.05% (V/V), treat Caenorhabditis elegans natural sedimentation,
Remove supernatant, leave and take Caenorhabditis elegans；Again with pure M9 liquid washing to clarification, turn
Moving Caenorhabditis elegans on other 96 new orifice plates, load position is corresponding with original 96 plates,
Three parallel controls are done in each hole, add dyestuff, dye respectively, the beautiful hidden bar line after dyeing
Worm, at fluorescence microscopy Microscopic observation, is taken pictures；And utilize fluorescence microplate reader to detect fluorescent value, swash
Emission wavelength 485nm, wavelength of transmitted light 535nm；
2. experimental data is processed, measure the nematicide fluorescence intensity after dyeing.
(3) the dyeing effect of Caenorhabditis elegans
By above-mentioned (1), the step process nematicide of (2), from Fig. 1, Fig. 2 Caenorhabditis elegans
Color figure see to learn, (1.5 is little to utilize the dyeing suitable time of suitable concentration
Time), Caenorhabditis elegans mutant MT2124 can be made to dye, it is possible to specific make show
The false vaginal orifice position dyeing of beautiful hidden rhabditida mutant MT2124, and Wild-type C. elegans
Then cannot colour.
(4) fluorescent value detection after the nematode population dyeing that WT ratios is different
By the present embodiment (1) described method by Caenorhabditis elegans wild type N2With beautiful hidden bar
Nematicide mutant MT2124 synchronization respectively, then by respective synchronized Caenorhabditis elegans
Mixing, is cultivating containing on colibacillary NGM culture medium flat plate respectively, and temperature is 20 DEG C,
After it is grown for adult, sweep away from flat board with M liquid, transfer in centrifuge tube, treat beautiful
Hidden rhabditida natural sedimentation, removes supernatant, leaves and takes Caenorhabditis elegans；Add containing 0.05%
(V/V) the M9 liquid of polysorbas20, treats Caenorhabditis elegans natural sedimentation, removes supernatant,
Leave and take Caenorhabditis elegans；Again with pure M9 liquid washing to clarification, respective digit liquid
In body volume, the number of Caenorhabditis elegans, standby.96 orifice plates take 11 holes, every hole
Add the Caenorhabditis elegans colony of different mutants ratio, Caenorhabditis elegans C. in each hole
Elegans sum is at 70, and WT ratios is followed successively by 0%, and 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90%, 100%, it is subsequently adding dyestuff so that it is final concentration of 50
μ g/mL, 20 DEG C of dyeing 1.5h, survey each hole fluorescent value.Three parallel controls are done in each hole.
As seen from Figure 3 when different Caenorhabditis elegans colonies is carried out dyeing,
Dye the identical time time, the mutant Caenorhabditis elegans institute accounting in Caenorhabditis elegans colony
Example becomes positive correlation with the fluorescence intensity detected.
Utilize dyestuff can Caenorhabditis elegans be dyeed, make mutant Caenorhabditis elegans
False vaginal orifice specificity coloring, due to the ratio of mutant and luciferase in Caenorhabditis elegans colony
The fluorescence intensity that mark instrument detects becomes positive correlation, and the dyeing time of Caenorhabditis elegans colony is with glimmering
The fluorescence intensity that light microplate reader detects becomes positive correlation, is therefore detected beautiful by fluorescence microplate reader
The intensity of the fluorescent value that hidden rhabditida colony sends, may determine that Caenorhabditis elegans colony accordingly
WT ratios.And then judge whether the process of medicine can lower excessively swashing of ras gene
Live.
Embodiment 2 antitumor drug candidate is to Caenorhabditis elegans CB1275 (lin-1 deletion mutation)
Effect
Lin-1 is a gene in ras downstream, is can be by the nuclear transcription factor of MAPK phosphorylation
Son, is the negative growth factor of vaginal orifice growth, can regulate the letter that the vaginal orifice in let-60 downstream is grown
Number path.Lin-1 Yu lin-31 forms complex, lin-1 rock code ETS family member, and lin-31
Coding winged helix transcription factor, during ras signal un-activation, lin-1 Yu lin-31 interacts
Composition complex, this complex is combined with the actuating section of oopod functional gene and stops it
Expression.After lin-1 deletion mutation, complex just cannot be formed, and complex can not be formed,
Cannot be combined with the actuating section of oopod functional gene, oopod gene expression, and then produce
False vaginal orifice is grown and is formed many vaginal orifices phenotype.
If the result of embodiment 1 shows many vaginal orifices phenotype of Drug inhibition nematicide, but this
The site that one result only points out medicine significantly to lower let-60/ras excessive activation be let-60 or its
Downstream, then whether medicine has lin-1 Inactivating mutations many vaginal orifices phenotype in let-60 downstream
Inhibitory action？Therefore the present embodiment can suppress MT2124 (let-60 sudden change) many
The medicine of vaginal orifice phenotype processes Caenorhabditis elegans CB1275 equally, if them can not be suppressed many
Vaginal orifice phenotype, finds the constant rate of mutant, illustrates that medicine action site exists after i.e. measuring
Lin-1 upstream.Additionally, due to lin-1 is nuclear factor, interacts with lin-31 and form
Complex, regulates the cytocerastic process of vaginal orifice.If it is the most cloudy that this gene inactivation is suddenlyd change by medicine
Door phenotype has significant inhibitory action, then explanation medicine can directly suppress sending out of vaginal orifice precursor
Educate, stop it to form many vaginal orifices.
The result of the present embodiment is if many vaginal orifices phenotype of CB1275 strain nematicide is not affected by
The impact of antitumor drug candidate, indicates that medicine acts on ras signal path and can suppress ras
The false vaginal orifice that excessive activation is formed, it is impossible to the false vaginal orifice phenotype that suppression lin-1 deletion mutation is formed,
Illustrate that antitumor drug candidate acts on ras and do not acts on lin-1, it was demonstrated that antitumor candidate's medicine
Thing has been lowered the excessive activation of ras rather than has directly inhibited the proliferation and differentiation of cell to work
, i.e. antitumor drug candidate works is that ras pathways relies on ground, rather than the thinnest
Cytotoxicity.
The assembling of embodiment 3 drug screening kit
Drug screening kit of the present invention, this test kit includes following reagent:
1. Caenorhabditis elegans wild type N2；
2. Caenorhabditis elegans mutant MT2124；
3. Caenorhabditis elegans CB1275；
4. NGM culture medium；
5. escherichia coli OP50；
6. dyestuff: 2', 7'-dichlorofluorescin diethylester.
Wherein, the concentration of described Caenorhabditis elegans is 100/20 μ l, escherichia coli OP50
Concentration be 10mg/ml, the final concentration of 50 μ g/mL of dyestuff.
Above reagent is sub-packed in suitable container, is placed in outsourcing test kit, wherein
M9 buffer, S buffer, LB culture medium are recorded in operation instructions in detail.
The checking of embodiment 4 drug screening kit reliability
As described in embodiment 1, method is by nematicide MT2124 synchronization, by hatching after synchronization
Caenorhabditis elegans MT2124 mixes, the number of Caenorhabditis elegans in digit's liquid volume
Mesh, standby.96 orifice plates take 2 holes, each hole are initially charged the S liquid of 140 μ L,
Being subsequently adding 20 μ L coli somatic solution, in each hole, addition about 90 is beautiful
Hidden rhabditida MT2124, first hole is negative control, adds 20 μ L distilled water, another
Individual hole adds the Sorafenib that 20 μ L concentration are 60 μMs.Finally make the liquid bulk in each hole
Long-pending is all 200 μ L.Then by liquid blending, 20 DEG C of shaken cultivation about 6 days, treat it
After growing for the false vaginal orifice of adult, i.e. generation, sweep away from flat board with M liquid, transfer to centrifuge tube
In, treat Caenorhabditis elegans natural sedimentation, remove supernatant, leave and take Caenorhabditis elegans；Add
Enter the M9 liquid of the polysorbas20 containing 0.05%, treat Caenorhabditis elegans natural sedimentation, remove supernatant
Liquid, leaves and takes Caenorhabditis elegans；Again with pure M9 liquid washing to clarification, shift beautiful hidden
Rhabditida is on other 96 new orifice plates, and position is corresponding with original 96 plates, is subsequently adding dye
Material so that it is final concentration of 50 μ g/mL, 20 DEG C of dyeing 1.5h, survey each hole fluorescent value.Often
Three parallel controls are done in individual hole.
By method described above process nematicide CB1275, add 1 μM, 2 μMs, 4 μMs, 6 μMs not
Sorafenib with concentration.
(1) Sorafenib checking drug screening kit verification the verifying results
As seen from Figure 4, blank group is to be added without Caenorhabditis elegans, only adds solution
Group, blank group fluorescence intensity is extremely low, and the group without Caenorhabditis elegans is nearly free from fluorescence.Cloudy
Property comparison be add Caenorhabditis elegans and be not added with the group of medicine, by comparison, add Suo Lafei
The experimental group of Buddhist nun, the fluorescence intensity that Caenorhabditis elegans colony sends substantially reduces, and reagent is described
Box is respond well, it is possible to the sensitive fluorescence detected by drug treating Caenorhabditis elegans colony
Strength difference.
And found that RAF targeted inhibition agent Sorafenib can suppress ras dose-dependant by Fig. 5
The activity of excessive activation, the most several to transcription factor lin-1 (lf) many vaginal orifices mutant downstream
Not impact.
In the present embodiment, we use have good result clinically to press down cancer drug Sorafenib, right
Drug screening kit of the present invention is verified, result shows: add the comparison of positive drug
Group, the fluorescence intensity that Caenorhabditis elegans colony sends substantially reduces, and shows drug selection agent
Box can reach expected effect.
In a word, the present invention utilizes dyestuff to dye mutant and Wild-type C. elegans not
Reach simple to operate, quick, efficient, accurate, high flux screening with effect and go out effective anti-
The purpose of tumour medicine, the research staff for vast antitumor drug saves time and cost.I
Utilize positive drug Sorafenib that drug screening kit is verified, it was demonstrated that its effect
Fruit is good, is a kind of quick, accurately, may be used for high-flux medicaments sifting test kit.
Claims (8)
1. a drug screening kit, described test kit includes following material:
1. Caenorhabditis elegans wild type N2；
2. Caenorhabditis elegans mutant MT2124；
3. Caenorhabditis elegans CB1275；
4. NGM culture medium；
5. escherichia coli OP50；
6. dyestuff: 2', 7'-dichlorofluorescin diethylester.
Drug screening kit the most according to claim 1, it is characterised in that described
Tumor is caused by ras proto-oncogene process LAN.
Drug screening kit the most according to claim 1, it is characterised in that described
Caenorhabditis elegans used by test kit is the nematicide after synchronization, the synchronized step of nematicide
For:
1. select in the ripe Caenorhabditis elegans centrifuge tube extremely equipped with 1ml M9 liquid, 4000
Rpm is centrifuged 5 minutes, removes supernatant；
2. adding 500 μ L 1M sodium hydroxide in centrifuge tube, 500 μ L 6.4% (V/V) are secondary
Sodium chlorate, concussion cracking；
3. remove supernatant, to precipitation in add 1.5 μ L M9 liquid, shake up, 4000rpm from
The heart 5 minutes, repeats this step 3 time；
4. in clean ovum, add 500 μ L M9 liquid, within 24 hours, hatch into L1 for 18 DEG C
Phase Caenorhabditis elegans.
Drug screening kit the most according to claim 1, it is characterised in that described
The concentration of Caenorhabditis elegans is 100/20 μ l.
5. the using method of drug screening kit as claimed in claim 3, its feature exists
In, the using method of described test kit is:
(1) dyeing of Caenorhabditis elegans and detection
L1 phase Caenorhabditis elegans MT2124 after synchronization as claimed in claim 3 is mixed,
In digit's liquid volume, the number of Caenorhabditis elegans, standby；96 orifice plates take 2
Hole, is initially charged the S liquid of 140 μ L in each hole, be subsequently adding 20 μ L coli somatics molten
Liquid, adds the Caenorhabditis elegans MT2124 of about 90 in each hole, first hole is cloudy
Property comparison, add 20 μ L distilled water, another hole adds for reagent thing；Finally make each
The liquid volume in hole is all 200 μ L；Then by liquid blending, 20 DEG C of shaken cultivation 6 days
Left and right, treating that it is grown is adult, after i.e. producing false vaginal orifice, sweeps away with M liquid from flat board, turns
Move on in centrifuge tube, treat Caenorhabditis elegans natural sedimentation, remove supernatant, leave and take beautiful hidden
Rhabditida；Add the M9 liquid of the polysorbas20 containing 0.05% (V/V), treat that Caenorhabditis elegans is certainly
So precipitation, removes supernatant, leaves and takes Caenorhabditis elegans；Wash extremely with pure M9 liquid again
Clarification, on transfer Caenorhabditis elegans to 96 new orifice plates, load position and original 96 plates pair
Should, three parallel controls are done in each hole, add dyestuff, dye respectively, beautiful hidden after dyeing
Rhabditida, at fluorescence microscopy Microscopic observation, is taken pictures；And utilize fluorescence microplate reader to detect fluorescent value,
Excitation wavelength 485nm, wavelength of transmitted light 535nm；
(2) experimental data is processed, measure the nematicide fluorescence intensity after dyeing.
The using method of drug screening kit the most according to claim 5, its feature exists
In, during described Caenorhabditis elegans dyeing, dye concentrations is 50 μ g/mL.
The using method of drug screening kit the most according to claim 5, its feature exists
In, during described Caenorhabditis elegans dyeing, dyeing time is 1.5 hours.
The using method of drug screening kit the most according to claim 5, its feature exists
In, during described Caenorhabditis elegans dyeing, dyeing temperature 20 DEG C.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107875374A (en) * | 2017-10-13 | 2018-04-06 | 西安电子科技大学 | The method that Caenorhabditis elegans infection model is precisely quickly established based on orthogonal optimization |
| CN111398573A (en) * | 2019-01-02 | 2020-07-10 | 广东省第二人民医院 | Drug screening method based on anti-lung cancer drug resistance effect of caenorhabditis elegans |
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| WO2000073493A2 (en) * | 1999-05-27 | 2000-12-07 | Pharmacia & Upjohn | A nematode drug screen for modulators of mammalian disorders |
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| CN107875374A (en) * | 2017-10-13 | 2018-04-06 | 西安电子科技大学 | The method that Caenorhabditis elegans infection model is precisely quickly established based on orthogonal optimization |
| CN111398573A (en) * | 2019-01-02 | 2020-07-10 | 广东省第二人民医院 | Drug screening method based on anti-lung cancer drug resistance effect of caenorhabditis elegans |
| CN111398573B (en) * | 2019-01-02 | 2024-04-30 | 广东省第二人民医院 | Method for screening medicines with anti-lung cancer drug resistance based on caenorhabditis elegans |
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