CN102692493A - Method for rapidly screening plant extract with cell aberration resistance - Google Patents
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Abstract
The invention relates to a method for rapidly screening a plant extract with cell aberration resistance. Rapid preliminary screening and repeated screening of caenorhadits elegans are carried out based on an SOS/Umu testing system, particularly, the method comprises the following steps of: protecting salmonella typhimurium TA1535/pSK1002 by using the plant extract to be screened, treating the salmonella typhimurium TA1535/pSK1002 by taking mitomycin C as a mutagen, detecting the activity condition of beta-galactosidase, preliminarily screening seven extracts with cell aberration resistance, such as acanthopanax, garlic, green tea, soybean, aloe, radix sophorae flavescentis and grapestone; and feeding model organism caenorhabditis elegans by using the seven extracts which are obtained by preliminarily screening, treating the model organism caenorhabditis elegans by using MMC (Mitomycin C), detecting indexes, such as the over half lethal days, the service life, the generation cycle and the spawning conditions, and further screening six extracts with obvious aberration resistance, such as the acanthopanax, garlic, green tea, soybean, grapestone and aloe. The research proves that a white mouse is subjected to gavage treatment by using the six extracts, the blood, urine, biochemical and organ tissue structure of the white mouse are not influenced, and any toxic effect is not discovered. Through changes of the survival rate, micronucleus rate, thymus index and other parameters of the mouse, the six screened extracts of the acanthopanax, garlic, green tea, soybean, grapestone and aloe which are used by the white moused have obvious cell aberration resistance.
Description
Technical field
The present invention relates to a kind of rapid screening method, be specifically related to the application in anti-cell distortion drug screening of a kind of SOS/Umu test system and beautiful nematode with anti-cell distortion plant extracts.
Background technology
Chemotherapy and radiation is routine, the effective method of treatment and assisted surgery treatment cancer, but the toxic and side effect of chemicotherapy has been brought great misery to the cancer patient.Be that radiotherapy or chemotherapy all can be damaged normal tissues and cell.Radioactive exposure causes the decomposition of water, and the generation active oxygen radical (reactive oxygen species, ROS).Thereby ROS starts chemical peroxidating process destroys DNA and the big molecule of other biological, and chemotherapy is as a kind of important method of treatment cancer, widespread use clinically.But great majority effectively cancer therapy drug all have some toxicity, and a lot of cancer therapy drug shows mutagenesis, teratogenesis and carcinogenic effect in some experimental systems.So, seek anti-chemotherapeutics efficient, low toxicity and just become to prevent cellular damage, an important channel of prevention cell mutation.Research at present confirms that some Chinese herbal medicines can play a protective role to the distortion cell; And have the advantages that the medicine source is wide, toxicity is low; But mostly the screening technique of traditional anti-cell distortion medicine is to be based upon on the animal experiment, exists human and material resources consumption big, the shortcoming that the cycle is long.Therefore set up the rapid screening system and become the gordian technique that people seek anti-cell distortion Chinese herbal medicine.
Summary of the invention
The object of the present invention is to provide a kind of rapid screening method with anti-cell distortion plant extracts.
To achieve these goals, technical scheme of the present invention has adopted a kind of rapid screening method with anti-cell distortion plant extracts, is to screen with the method that quick primary dcreening operation of SOS/Umu test system and beautiful nematode are sieved again.
Specifically may further comprise the steps: salmonella typhimurium TA1535/pSK1002 is protected with plant extracts to be screened; Use mutagenic agent that this bacterium is handled; Detect β-gala glycosides enzymatic activity; Calculate inductivity R value and inhibiting rate, preliminary screening goes out garlic, wilsonii, green tea, soybean, grape pip, kuh-seng and seven kinds of extracts of aloe; Seven kinds of anti-cell distortion Chinese herbal medicine extracts that obtain with primary dcreening operation are again fed the beautiful stealthy nematode of model organism; And utilize chemotherapeutics MMC that it is handled; Through the detection of life-span length, generation cycle, the situation of laying eggs index, further screen wilsonii, garlic, green tea, soybean, grape pip and aloe and have the active plant extracts of strong anti-distortion for six kinds.
Described mutagenic agent is that methopterin is or/and mitomycin C.
The prescreening method of described SOS/Umu test system is following:
(1) gets the freezing Salmonella typhimuriumTA1535/PSK1002 of 80ul bacterium liquid in the LB of 20ml nutrient culture media, 37 ℃ of vibration cultivation 12h overnight; With 10～50 times of TGA nutrient solution dilutions, behind shaken cultivation 1.5～2h, use the LB nutrient culture media that nutrient solution light absorption value (600nm) is transferred to 0.2-0.4 again under 37 ℃ of conditions culture bacteria liquid next day;
(2) getting culture bacteria liquid 990uL and add in the 1.5mLEppendorf pipe, add the various Chinese herbal medicine solutions vibrations that 10ul prepares again and shake up, is solvent control with DMSO, 37 ℃ of shaken cultivation 2h; Culture plate is measured the absorbance of bacterium liquid down in the 600nm wavelength;
(3) get 200ul reaction bacterium liquid in addition and add 800uL damping fluid, 50ul 0.1%SDS, add the 10ul methenyl choloride again behind the mixing 5min, vibration shakes up 10min; The NaH that adds the PBS 0.1M that contains ONPG then
2PO
4And KH
2PO
4100ul reacts 25min down, the Na of adding 1000ul1M in 37 ℃ in above-mentioned solution
2CO
3The solution cessation reaction is drawn supernatant 200ul in 96 hole ELISA Plates, measures absorbance down respectively at 420nm and 550nm wavelength.
Described LB nutrient culture media consists of: 10g tryptone+5g sodium chloride+5g yeast extract is dissolved in the 1000ml ultrapure water.
Described TGA nutrient solution consists of: 10g tryptone+5g sodium chloride is dissolved in the 980ml ultrapure water, and it is that 10% glucose solution is preserved subsequent use in 4 ℃ of refrigerators that the sterilization back adds 20ml concentration.
The oscillation frequency of step (3) is 175rmin
-1
Described damping fluid consists of: Z-Buffer solution, 21.48g Na
2HPO
4.12H2O+6.24gNaH
2PO
4.2H2O+0.745g KCl+0.12gMgSO
4+ 3.9g beta-mercaptoethanol is dissolved in the 1000ml ultrapure water, and the sterilization back is preserved subsequent use in 4 ℃ of refrigerators.
Described beautiful stealthy nematode is sieved experimental technique again and may further comprise the steps:
(1) the nematode synchronization is cultivated
The beautiful nematode hermaphroditic adult in the egg-laying season is just washed the centrifugal 1min of 1500rpm, supernatant discarded from 90mm NGM culture plate with M9 buffer.The M9buffer solution that will contain beautiful nematode after as above method is cleaned once with M9 buffer again is settled to 500 μ l in 1.5ml EP pipe.Add lysate 500 μ l, the centrifugal 3min of 1500rpm behind the mixing 3～5min that turns upside down, supernatant discarded is collected bottom settlings; Deposition is used aseptic S-Medium instead and is cleaned 2～3 times repeatedly to the pH to 7.0, and this moment, beautiful nematode polypide was by cracking and worm's ovum is still intact; The aseptic S-Medium solution that will contain worm's ovum is put in 20 ℃ and cultivates 8～12h; Owing to there is not food, the ovum hatching can all stop at the L1 phase later, and the centrifugal 3min of 1500rpm collects larva, forwards in the NGM culture plate of E.coli OP50 to continue to cultivate;
(2) mensuration in nematode life-span
Beautiful nematode adopts liquid cultivating method to cultivate, and gets 96 orifice plates, and every hole adds nutrient solution S-Medium 70 μ l, E.coli OP50 to about the OD600=0.2, DhHP-6 to 50 μ M, 200 μ M, 500 μ M variable concentrations; Cultivate beautiful nematode the same period to the L4 phase, the single hermaphroditic nematode of picking is to the bottom, U type hole of 96 orifice plates, 20 ℃ of cultivations; In the egg-laying season, need every day with the nematode picking in the new nutrient solution to prevent that larva from too much influencing the growth of adult; Egg-laying season can change per 2～3 days into later and change liquid once; With initial 0 day of nematode life of line eggs hatching beginning to be designated as, to stop with getting the terminal light reactionless nematode life that is designated as of polypide of visiting of worm device.The nematode lifetime is designated as nematode from the initial time that stops to life of life; The nematode of losing, should from statistics, get rid of because of climbing to double dish wall dead nematode and " worm bag " (worm bag);
(3) mensuration of egg laying amount
With being cultured to the U type hole bottom of hermaphroditic beautiful nematode picking of L4 phase to 96 orifice plates that contain 50 μ l nutrient solutions the single same period, every separated 24h changes the hole once, finishes until the egg-laying season.The nutrient solution that product is had an ovum continues to cultivate in 20 ℃ and grew to L3～L4 phase to larva in 2～3 days, and the porose interior filial generation larva number of record institute is egg laying amount; The time interval when egg-laying season finished to finish to laying eggs for the L4 phase; The equal METHOD FOR CONTINUOUS DETERMINATION of all data beautiful nematode more than 10 is averaged;
(4) mensuration of generation cycle length
The some NGM of preparation are dull and stereotyped earlier, move into adult of 5 pregnancies above each is dull and stereotyped, behind 20 ℃ of cultivation 2h, collect worm's ovum; In 96 microwell plates, add siberian Ginseng P.E+OP50 bacterium liquid+MMC+k nutrient culture media mixed liquor, move into 20 fresh worm's ovums, 20 ℃ of constant temperature culture in bottom, U type hole; Clock and be 0h this moment, and every the taking-up at a distance from 3h observed after the 48h, until there being ovum to occur.Record the time that second generation ovum occurs.
The present invention has set up a kind of method of sieving again based on the quick primary dcreening operation of SOS/Umu test system and beautiful nematode; SOS/Umu test is the short-term shaker test of testing environment paramorphogen, and it is based on the dna damage thing and induces the SOS reaction and express and set up on the basis of umuC gene.Compare with the anti-distortion medicaments sifting model of setting up based on the chemotherapy sensitive cells, this method has fast, advantage such as sensitive, accurate.The present invention protects salmonella typhimurium TA1535/pSK1002 with Chinese herbal medicine extract to be screened; Use methopterin and mitomycin C (mitomycin C; MMC) as mutagenic agent this bacterium is handled, thereby and detect its β-gala glycosides enzymatic activity situation and filter out Chinese herbal medicine extract with anti-cell distortion function.Adopt this method from nearly 20 kind of plant extracts rapid screening to seven kinds of extracts such as soybean, green tea.But salmonella typhimurium is a kind of prokaryotes after all, and its genetic background and the mankind differ greatly, and Caenorhabditis elegans (Caenorhabditis elegans) is a kind of very effective model organism on toxicology and human Physiological Mechanism.Therefore; On the basis of unicellular organism screening system; Anti-cell distortion Chinese herbal medicine extract so that primary dcreening operation obtains is fed the beautiful stealthy nematode of model organism; And utilize chemotherapeutics MMC that it is handled, through the detection of life-span length, generation cycle, the indexs such as situation of laying eggs, further filter out wilsonii, garlic, green tea, soybean, grape pip and six kinds of significant Chinese herbal medicine extracts of anti-distortion effect of aloe.At last, to screening to such an extent that six kind of plant extracts carry out acute toxicity test, research shows: with six kinds of extracts mice lavage is handled, big white mouse blood, urine, biochemistry and organs and tissues structure are not all had influence, do not find any toxic action.The extract of having verified six kinds of screenings such as wilsonii, green tea, soybean, grape pip through the isoparametric variation of mouse survival rate, micronuclear rates and thymus index has remarkable anti-cell distortion effect after mouse is used.
The sterilized water dissolving is mainly adopted in the preparation of the plant extracts solution that the present invention relates to, and extracting section thing solute effect is bad, can add 0.1% DMSO and heating hydrotropy.
The method of the quick primary dcreening operation of SOS/Umu test system that the present invention relates to is:
1. (10g tryptone+5g sodium chloride+5g yeast extract is dissolved in the 1000ml ultrapure water in the LB of 20ml nutrient culture media to get the freezing Salmonella typhimuriumTA1535/PSK1002 of 80ul bacterium liquid; Preserve subsequent use in 4 ℃ of refrigerators behind the autoclaving) in, 37 ℃ of vibration cultivation 12h overnight.Next day, (10g tryptone+5g sodium chloride was dissolved in the 980ml ultrapure water with the TGA nutrient solution with culture bacteria liquid; Adding the 20ml10% glucose solution behind the autoclaving preserves subsequent use in 4 ℃ of refrigerators) dilute 10～50 times; After 1.5～2h is cultivated in vibration (175rmin-1) under 37 ℃ of conditions, use the LB nutrient culture media that nutrient solution light absorption value (600nm) is transferred to about 0.3 again.
2. getting culture bacteria liquid 990uL and add in the 1.5mLEppendorf pipe, add 10ul again and shaken up by the various Chinese herbal medicine solution vibrations that instance 1 prepares, is solvent control with DMSO, 37 ℃ of shaken cultivation 2h.Culture plate is measured the absorbance of bacterium liquid down in the 600nm wavelength.
3. get 200ul reaction bacterium liquid in addition and add 800uL damping fluid (Z-Buffer solution, 21.48g Na
2HPO
4.12H2O+6.24gNaH
2PO
4.2H2O+0.745g KCl+0.12gMgSO
4+ 3.9g beta-mercaptoethanol is dissolved in the 1000ml ultrapure water, preserves subsequent use in 4 ℃ of refrigerators behind the autoclaving), 50ul 0.1%SDS, add the 10ul methenyl choloride again after mixing 5min, vibration shakes up 10min.The NaH that adds the PBS 0.1M that contains ONPG
2PO
4And KH
2PO
4) 100ul in above-mentioned solution, in 37 ℃ of following reaction 25min, add the Na of 1000ul 1M
2CO
3The solution cessation reaction is drawn supernatant 200ul in 96 hole ELISA Plates, measures absorbance down respectively at 420nm and 550nm wavelength.
According to computes B2 galactosidase induced activity:
β-Galactosidase?Activity(IU)＝1000*(A420-1.75*A550)/(t*v*A600)
Wherein, t is the reaction time behind the adding ONPG, and v is 0.2 in this research for the volume of reaction bacterium liquid.
A600 is final nutrient solution bacteria content;
A420 is the content of enzyme-to-substrate reaction back product;
A550 represents and has or not the graininess chaff interference in the reactive system, usually near 0 "
Calculate the ratio of inducing according to the IU value through dilution back sample
(R) value: induce ratio (R)=developmental tube IU value/control tube IU value (2)
Numerical value is with means standard deviation (standard deviation, SD) expression.Carry out t-check analysis conspicuousness with SPSS 13.0 for Windows softwares, the p value reaches the level of signifiance less than 0.05.
Embodiment
Embodiment one: according to the consumption of GB regulation, with waiting that sieving first plant extracts is mixed with following working concentration.
Table 1 Chinese herbal medicine extract and active constituent content
Embodiment two: through of the protection of said herbal medicine extract, carry out SOS/umu test screening experiment then to salmonella typhimurium TA1535/pSK1002, be provided with simultaneously do not add any plant extracts as control group.Calculate R value and inhibiting rate, find that anti-distortion activity has garlic, wilsonii, green tea, soybean, grape pip, kuh-seng and seven kinds of extracts of aloe significantly, the result sees the highly significant in the table 2.
The umu gene expression influence that table 2 Chinese herbal medicine extract causes MMC
Annotate: inhibiting rate (%)=100 * (R
Control-R
Sample)/R
Control *P＜0.05,
*P＜0.01.
Embodiment three: culture of nematodes adopts standard program; Experiment is all carried out toxicity test with the nematode that is in the L4 stage in larvae development later stage; Experiment is carried out in the double dish of culture of nematodes, and the active significant Chinese herbal medicine extract solution of seven kinds of anti-distortion that SOS/Umu test system primary dcreening operation is obtained are dissolved in the K-medium that contains E.coli OP50 respectively and (contain 53mM NaCl, 32mM KCl; 121 ℃ of autoclavings) in; And it is carried out chemotherapeutics MMC (50 μ g/ml) handle, 1 control group and 3 parallel-group are set simultaneously, control group does not add Chinese herbal medicine protection for only adding MMC.Then through detection to its half fatal rate, life-span length, generation cycle, the indexs such as situation of laying eggs.The result sees table 3, and it is active that promptly garlic, wilsonii, green tea, soybean and grape pip, aloe all have stronger anti-distortion.
Table 3 Chinese herbal medicine extract is to the influence of the anti-distortion effect of beautiful nematode
With the siberian Ginseng P.E is that example describes: MMC is 65 μ g/ml to the LC50 of beautiful stealthy nematode, and concentration is that the siberian Ginseng P.E of 60 μ g/ml can make the mortality ratio of nematode reduce to 20%.Under the MMC of 80 μ g/ml influence, average laying is reduced to 82.4 by 232, and under the siberian Ginseng P.E protection of 50 μ g/ml, its egg laying amount can return to normal level.The MMC of 80 μ g/ml can make the mean lifetime of beautiful stealthy nematode reduce to 11.5d by 21.8d, in concentration is can make the nematode mean lifetime return to 20d under the protection of siberian Ginseng P.E of 40 μ g/ml.Under 20 ℃ of conditions, the generation cycle average out to 82.4h of nematode, the toxic action of MMC can make it prolong, and are that its generation cycle can recover normally to reach 82.1h basically under the wilsonii protection of 60 μ g/ml in concentration.
Embodiment four: in order to verify the anti-distortion effect of the six kind of plant extracts that obtain through the present invention screening, be animal used as test with the mouse, male and female property half and half, select at random body weight close be divided into control group and experimental group.Mouse is irritated stomach, and wherein experimental group is got six kind of plant extract solutions respectively mouse is irritated stomach, and control group is got physiological saline and irritated stomach, irritates the stomach amount and calculates by weight, and every 10g irritates stomach 0.1ml, continuous irrigation stomach ten days.With MMC mouse was carried out lumbar injection in continuous seven days then, ID is calculated by weight, every 10g injection 0.1mlMMC.Through marrow micronuclear rates and the thymus index of test mouse, data statistic analysis postevaluation six kind of plant extracts are to the protective effect of mouse anti distortion.The result sees table 4, confirms that garlic, wilsonii, green tea, soybean and grape pip, aloe all have stronger anti-distortion active.
Table 4 Chinese herbal medicine extract is to the effect of mouse anti distortion
Claims (9)
1. the rapid screening method with anti-cell distortion plant extracts is characterized in that: be to screen with the method that quick primary dcreening operation of SOS/Umu test system and beautiful nematode are sieved again.
2. the rapid screening method with anti-cell distortion plant extracts according to claim 1; It is characterized in that: specifically may further comprise the steps: salmonella typhimurium TA1535/pSK1002 is protected with plant extracts to be screened; Use mutagenic agent that this bacterium is handled; Detect β-gala glycosides enzymatic activity, calculate inductivity R value and inhibiting rate, preliminary screening goes out garlic, wilsonii, green tea, soybean, grape pip, kuh-seng and seven kinds of extracts of aloe; Seven kinds of anti-cell distortion Chinese herbal medicine extracts that obtain with primary dcreening operation are again fed the beautiful stealthy nematode of model organism; And utilize chemotherapeutics MMC that it is handled; Through the detection of life-span length, generation cycle, the situation of laying eggs index, further screen wilsonii, garlic, green tea, soybean, grape pip and aloe and have the active plant extracts of strong anti-distortion for six kinds.
3. the rapid screening method with anti-cell distortion plant extracts according to claim 2, it is characterized in that: described mutagenic agent is that methopterin is or/and mitomycin C.
4. the rapid screening method with anti-cell distortion plant extracts according to claim 1, it is characterized in that: the prescreening method of described SOS/Umu test system is following:
(1) gets the freezing Salmonella typhimuriumTA1535/PSK1002 of 80ul bacterium liquid in the LB of 20ml nutrient culture media, 37 ℃ of vibration cultivation 12h overnight; With 10～50 times of TGA nutrient solution dilutions, behind shaken cultivation 1.5～2h, use the LB nutrient culture media that nutrient solution light absorption value (600nm) is transferred to 0.2-0.4 again under 37 ℃ of conditions culture bacteria liquid next day;
(2) getting culture bacteria liquid 990uL and add in the 1.5mLEppendorf pipe, add the various Chinese herbal medicine solutions vibrations that 10ul prepares again and shake up, is solvent control with DMSO, 37 ℃ of shaken cultivation 2h; Culture plate is measured the absorbance of bacterium liquid down in the 600nm wavelength;
(3) get 200ul reaction bacterium liquid in addition and add 800uL damping fluid, 50ul 0.1%SDS, add the 10ul methenyl choloride again behind the mixing 5min, vibration shakes up 10min; The NaH that adds the PBS 0.1M that contains ONPG then
2PO
4And KH
2PO
4100ul reacts 25min down, the Na of adding 1000ul 1M in 37 ℃ in above-mentioned solution
2CO
3The solution cessation reaction is drawn supernatant 200ul in 96 hole ELISA Plates, measures absorbance down respectively at 420nm and 550nm wavelength.
5. the rapid screening method with anti-cell distortion plant extracts according to claim 4, it is characterized in that: described LB nutrient culture media consists of: 10g tryptone+5g sodium chloride+5g yeast extract is dissolved in the 1000ml ultrapure water.
6. the rapid screening method with anti-cell distortion plant extracts according to claim 4; It is characterized in that: described TGA nutrient solution consists of: 10g tryptone+5g sodium chloride is dissolved in the 980ml ultrapure water, and it is that 10% glucose solution is preserved subsequent use in 4 ℃ of refrigerators that the sterilization back adds 20ml concentration.
7. the rapid screening method with anti-cell distortion plant extracts according to claim 4, it is characterized in that: the oscillation frequency of step (3) is 175rmin
-1
8. the rapid screening method with anti-cell distortion plant extracts according to claim 4, it is characterized in that: the damping fluid of said step (3) consists of: Z-Buffer solution, 21.48g Na
2HPO
4.12H2O+6.24gNaH
2PO
4.2H2O+0.745g KCl+0.12gMgSO
4+ 3.9g beta-mercaptoethanol is dissolved in the 1000ml ultrapure water, and the sterilization back is preserved subsequent use in 4 ℃ of refrigerators.
9. the rapid screening method with anti-cell distortion plant extracts according to claim 1, it is characterized in that: described beautiful stealthy nematode is sieved experimental technique again and may further comprise the steps:
(1) the nematode synchronization is cultivated
The beautiful nematode hermaphroditic adult in the egg-laying season is just washed the centrifugal 1min of 1500rpm, supernatant discarded from 90mm NGM culture plate with M9 buffer.The M9buffer solution that will contain beautiful nematode after as above method is cleaned once with M9 buffer again is settled to 500 μ l in 1.5ml EP pipe.Add lysate 500 μ l, the centrifugal 3min of 1500rpm behind the mixing 3～5min that turns upside down, supernatant discarded is collected bottom settlings; Deposition is used aseptic S-Medium instead and is cleaned 2～3 times repeatedly to the pH to 7.0, and this moment, beautiful nematode polypide was by cracking and worm's ovum is still intact; The aseptic S-Medium solution that will contain worm's ovum is put in 20 ℃ and cultivates 8～12h; Owing to there is not food, the ovum hatching can all stop at the L1 phase later, and the centrifugal 3min of 1500rpm collects larva, forwards in the NGM culture plate of E.coli OP50 to continue to cultivate;
(2) mensuration in nematode life-span
Beautiful nematode adopts liquid cultivating method to cultivate, and gets 96 orifice plates, and every hole adds nutrient solution S-Medium 70 μ l, E.coli OP50 to about the OD600=02, DhHP-6 to 50 μ M, 200 μ M, 500 μ M variable concentrations; Cultivate beautiful nematode the same period to the L4 phase, the single hermaphroditic nematode of picking is to the bottom, U type hole of 96 orifice plates, 20 ℃ of cultivations; In the egg-laying season, need every day with the nematode picking in the new nutrient solution to prevent that larva from too much influencing the growth of adult; Egg-laying season can change per 2～3 days into later and change liquid once; With initial 0 day of nematode life of line eggs hatching beginning to be designated as, to stop with getting the terminal light reactionless nematode life that is designated as of polypide of visiting of worm device; The nematode lifetime is designated as nematode from the initial time that stops to life of life; The nematode of losing, should from statistics, get rid of because of climbing to double dish wall dead nematode and " worm bag " (worm bag);
(3) mensuration of egg laying amount
With being cultured to the U type hole bottom of hermaphroditic beautiful nematode picking of L4 phase to 96 orifice plates that contain 50 μ l nutrient solutions the single same period, every separated 24h changes the hole once, finishes until the egg-laying season.The nutrient solution that product is had an ovum continues to cultivate in 20 ℃ and grew to L3～L4 phase to larva in 2～3 days, and the porose interior filial generation larva number of record institute is egg laying amount; The time interval when egg-laying season finished to finish to laying eggs for the L4 phase; The equal METHOD FOR CONTINUOUS DETERMINATION of all data beautiful nematode more than 10 is averaged;
(4) mensuration of generation cycle length
The some NGM of preparation are dull and stereotyped earlier, move into adult of 5 pregnancies above each is dull and stereotyped, behind 20 ℃ of cultivation 2h, collect worm's ovum; In 96 microwell plates, add siberian Ginseng P.E+OP50 bacterium liquid+MMC+k nutrient culture media mixed liquor, move into 20 fresh worm's ovums, 20 ℃ of constant temperature culture in bottom, U type hole; Clock and be 0h this moment, and every the taking-up at a distance from 3h observed after the 48h, until there being ovum to occur; Record the time that second generation ovum occurs.
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| CN111411082A (en) * | 2020-03-26 | 2020-07-14 | 中山大学孙逸仙纪念医院 | Culture medium for culturing CD90posi cells and culture method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105911238A (en) * | 2016-04-13 | 2016-08-31 | 中国科学院东北地理与农业生态研究所 | Method for detecting titanium dioxide by using Caenorhabditis elegans mutant RB2612 |
| CN111411082A (en) * | 2020-03-26 | 2020-07-14 | 中山大学孙逸仙纪念医院 | Culture medium for culturing CD90posi cells and culture method thereof |
| CN112843068A (en) * | 2021-01-07 | 2021-05-28 | 西藏自治区高原生物研究所 | Application of polyporus albus polyphenol in preparation of product for prolonging service life of nematodes |
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