CN105833295A

CN105833295A - Construction method and application of caenorhabditis elegans hyperuricemia model and screening method of construction method

Info

Publication number: CN105833295A
Application number: CN201610318064.5A
Authority: CN
Inventors: 李贞景; 王昌禄; 李志�; 熊春福; 陈勉华; 李风娟; 武淑芬
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2016-05-13
Filing date: 2016-05-13
Publication date: 2016-08-10
Anticipated expiration: 2036-05-13
Also published as: CN105833295B

Abstract

The invention relates to a construction method of a caenorhabditis elegans hyperuricemia model. The method comprises the following steps that 1, the uric acid content in caenorhabditis elegans is measured through a high performance liquid chromatography method; 2, an optimal drug delivery and culture mode is determined; 3, an optical drug delivery substance and drug delivery dosage are determined; 4, the optimal drug delivery time of xanthine is determined; 5, series experiment evaluation is conducted on the caenorhabditis elegans hyperuricemia model obtained through the construction method, and the caenorhabditis elegans hyperuricemia model which is low in damage, efficient, stable and reliable is obtained. According to the construction method, the hyperuricemia animal model is constructed by means of the model organism caenorhabditis elegans which has the high conservative property to human genes and has the similar uric acid metabolic pathway with human beings, the model has typical pathological characterization and is stable and reliable, various quantity indexes have the statistical significance, and a powerful tool is provided for large-scale and high-throughput drug screening of gout and the hyperuricemia symptom.

Description

The construction method of a kind of Caenorhabditis elegans high lithemia model and application and this structure side The screening technique of method

Technical field

The invention belongs to human chronic diseases's animal model technical field, relate to a kind of human chronic diseases's animal model Method for building up, the construction method of a kind of Caenorhabditis elegans high lithemia model and application and the screening side of this construction method Method.

Background technology

Along with improvement and the change of dietary structure of people's living standard, in recent years, the incidence of disease of hyperuricemia is alive Notable ascendant trend is had in the range of boundary.Hyperuricemia self brings the biggest threat not only to the health of the mankind, also can produce Raw various complication, such as cardiovascular and cerebrovascular disease and atherosclerotic etc..Therefore strengthen the prevention and control to hyperuricemia and be correlated with The research and development of medicine have become a urgent task.But it is sick that the deficiency of existing hyperuricemia animal model has become hyperuricemia Reason research and the major obstacle of new drug development.Therefore, set up science, rationally, similitude high, reproducible, highly reliable and can Control easy hyperuricemia animal model to have important practical significance.

Set up hyperuricemia animal model, be premise and the basis screening safe and efficient anti-trioxypurine medicine.At present, The animal of the hyperuricemia model having built up mainly has mouse, rat, chicken, quail etc..On the one hand rat, mouse are experiments The conventional animal of room, operate, raise the most more convenient, little with non-human species's difference.But then, the uric acid of mouse, rat Metabolic pathway vary considerably with the mankind, there is uricase, and the mankind do not have so that modeling has certain uncertainty to be subject to Limit.Chicken and quail, uric acid synthesis and metabolism are very much like with the mankind, internal without uricase, it is impossible to aoxidized by uric acid, but and people Class does not belongs to together, and common lab is raised also inconvenient.

It addition, when setting up hyperuricemia model, different administering modes also can have a huge impact.Set up at present Mainly have five kinds on the administering mode of model: feed, gavage, lumbar injection, hypodermic injection and gene knockout.Bird with feed to The front four kinds of modes of medicine, rat and mouse all can modeling, gene knockout research be only limitted to mouse.Different administering modes is respectively arranged with excellent lacking Point: the inequality of feeding animals food-intake can make internal serum uric acid level unstable, but the duration is longer；Gavage can avoid blood Uric acid level is unstable；Drug administration by injection is easy and simple to handle, but high lithemia state maintenance time is short, is unfit to do pathogenetic grinding Study carefully；Several ways administering drug combinations often effect is preferable, and the hyperuricemia model of structure has the feature such as continuation, stability； The model mice time-to-live that gene knockout is set up is short, cost is high.

Preferably high lithemia model should possess: (1) uric acid metabolism is consistent with the mankind, without uricase；(2) blood uric acid raises relatively Hurry up, modeling is stable；(3) high lithemia state can maintain time enough；(4) to other organ such as kidney not damaged.Comprehensive next See, there is no a kind of efficient, stable, reliable hyperuricemia animal model at present.

Though the adult of Caenorhabditis elegans only 1-1.5mm, have integumentary system, pharynx food system, nervous system, The complete structures such as defecation system, immunological regulation system and reproductive system.Although the neuronal cell of Caenorhabditis elegans is simple, But its function is as other animal, having various neurotransmitter, regulation and control Caenorhabditis elegans is to being not suitable for temperature, mechanical stimulus etc. The adaptation of poor environment.Additionally, Caenorhabditis elegans has 19717 genes, 6 chromosomes, including 5 pairs of autosomes With l to sex chromosome.In gene, introne is less, and genome is little, only 97Mb, is the multi-celled eukaryotes of gene minimum. Caenorhabditis elegans gene and human gene have high conservative so that Caenorhabditis elegans becomes the ideal of research human diseases Model.

At present, Caenorhabditis elegans be widely used for infective pathogen bacterium model, detection toxicity and disease model aspect grind Study carefully.Research shows, current Caenorhabditis elegans simulating human disease model is broadly divided into four classes, the show of metabolic syndrome disease Beautiful hidden rhabditida model, depressed Caenorhabditis elegans model and the nervous system disease model and amyotrophy model.

Uric acid metabolism approach (such as Fig. 1) in Caenorhabditis elegans body is although being slightly different with the uric acid metabolism approach of the mankind (such as Fig. 2), but basic simlarity.Caenorhabditis elegans is also first from the base of small-molecule substance synthesizing ribonucleotide, carries out the most again The circulation of nucleotides, guanine can not metabolism be directly xanthine unlike the mankind, and adenine can not metabolism be directly secondary Xanthine and then be converted into xanthine, so the precursor substance of uric acid only has hypoxanthine and Huang fast in Caenorhabditis elegans body Purine, and in human body, the precursor substance of uric acid has four kinds, respectively adenine, guanine, hypoxanthine and xanthine.But it is beautiful hidden Rhabditida and the mankind do not have uricase, and the end product of purine metabolism is all to excrete with the form of uric acid, and this is for utilizing Caenorhabditis elegans is set up the high lithemia animal model similar with human urine acid metabolic approach and is provided possibility.

By retrieval, there is not yet the relevant report utilizing Caenorhabditis elegans to set up hyperuricemia model.

Summary of the invention

In place of it is an object of the invention to overcome the deficiencies in the prior art, it is provided that a kind of Caenorhabditis elegans high lithemia model Construction method and application and the screening technique of this construction method, this construction method utilizes classical mode biology-beautiful hidden bar line Worm, and the model that obtains of the method have typical pathology characterize, stable, reliable, science, rationally, similitude high, repeated Good, the highly reliable and controlled advantage such as easy, and every quantitative index has statistical significance, the most only gout and high lithemia disease Extensive, high-flux medicaments sifting provide strong instrument.

The technical solution adopted for the present invention to solve the technical problems is as follows:

A kind of construction method of Caenorhabditis elegans high lithemia model, step is as follows:

Utilize Caenorhabditis elegans, use precursor substance xanthine, final concentration of 0.25-0.45mg/mL, fluid administration, Cultivate 18-32h under the conditions of 20-25 DEG C, i.e. obtain low Caenorhabditis elegans high lithemia mould damaging, efficient, stable, reliable Type.

And, step is as follows:

Utilize Caenorhabditis elegans, use precursor substance xanthine, final concentration of 0.25mg/mL, fluid administration, 20 DEG C of bars Cultivate 18h under part, i.e. obtain low Caenorhabditis elegans high lithemia model damaging, efficient, stable, reliable.

The application in anti-trioxypurine drug screening of the construction method of Caenorhabditis elegans high lithemia model as above.

The Caenorhabditis elegans high lithemia constructed by construction method of Caenorhabditis elegans high lithemia model as above Model application in anti-trioxypurine drug screening.

The screening technique of the construction method of Caenorhabditis elegans high lithemia model as above, step is as follows:

(1) utilize the uric acid content in high effective liquid chromatography for measuring Caenorhabditis elegans body；

(2) step Caenorhabditis elegans (1) is respectively adopted solid form delivery and fluid administration two ways is cultivated, logical Cross and compare two kinds of administering modes to the impact of uric acid content in Caenorhabditis elegans body, determine optimal administration training method；

To step (2) determined by optimal administering mode give Caenorhabditis elegans hypoxanthine, uric acid, yellow fast respectively Concentration that four kinds of medicines of purine and adenine are different and different action time, in units of Caenorhabditis elegans, measure beautiful hidden The change of uric acid content in rhabditida body, determines optimal administration material, dosage；

Described different concentration is 0,0.05,0.15,0.25,0.50,0.65mg/mL；Described different action time is 0,6,10,18,24,32,48h；

Step (3) determined by optimal be administered material and dosage under the conditions of, measure the optimal material that is administered in difference Administration time, on the impact of Caenorhabditis elegans average life span the change of uric acid content in combining Caenorhabditis elegans body, determines The optimal Best administration time being administered material；

The described different dosing time is 0,18,24,32,48h；

(5) establish Caenorhabditis elegans high lithemia model building method, beautiful to the high lithemia obtained by this construction method Hidden rhabditida model carries out serial experiment evaluation, and described serial experiment evaluation includes that the change of Caenorhabditis elegans locomitivity is commented Valency, Caenorhabditis elegans egg laying amount Assessment of Changes, model stability evaluation, allopurinol drug evaluation, xanthine oxidase are lived Property evaluate, determine the stability of high lithemia Caenorhabditis elegans model building method, reliability and to Caenorhabditis elegans Damaging, determine optimum condition, i.e. obtain low Caenorhabditis elegans high lithemia model damaging, efficient, stable, reliable.

And, step is as follows:

(1) utilize the uric acid content in high effective liquid chromatography for measuring 400-1000 bar Caenorhabditis elegans body；

(2) step Caenorhabditis elegans (1) is respectively adopted solid form delivery and fluid administration two ways is cultivated, logical Cross and compare two kinds of administering modes to the impact of uric acid content in Caenorhabditis elegans body, determine optimal administration training method, for Fluid administration；

To step (2) determined by optimal administering mode give Caenorhabditis elegans hypoxanthine, uric acid, yellow fast respectively Concentration that four kinds of medicines of purine and adenine are different and different action time, in units of 400-1000 bar Caenorhabditis elegans, Measure the change of uric acid content in Caenorhabditis elegans body, determine optimal administration material, dosage, for xanthine final concentration 0.25mg/mL；

Step (3) determined by optimal be administered material and dosage under the conditions of, measure xanthine when different dosing Between on the impact of Caenorhabditis elegans average life span the change of uric acid content in combining Caenorhabditis elegans body, determine xanthine Best administration time, for 18h；

The described different dosing time is 0,18,24,32,48h；

(5) establish Caenorhabditis elegans high lithemia model building method, beautiful to the high lithemia obtained by this construction method Hidden rhabditida model carries out serial experiment evaluation, and described serial experiment evaluation includes that the change of Caenorhabditis elegans locomitivity is commented Valency, Caenorhabditis elegans egg laying amount Assessment of Changes, model stability evaluation, allopurinol drug evaluation, xanthine oxidase are lived Property evaluate, determine the stability of high lithemia Caenorhabditis elegans model building method, reliability and to Caenorhabditis elegans Damaging, determine optimum condition, i.e. use precursor substance xanthine, final concentration of 0.25-0.45mg/mL, fluid administration, 20- 25 DEG C, 18-32h, i.e. obtain low Caenorhabditis elegans high lithemia model damaging, efficient, stable, reliable.

And, described step (1) middle Caenorhabditis elegans processes through acetonitrile removing protein before measuring, and the addition of acetonitrile is: Article 500, Caenorhabditis elegans adds acetonitrile 5.0mL；

Or, the condition of described Urine by HPLC acid content is as follows:

Chromatographic column: Intertsil ODS-SP chromatographic column, 5 μm, 4.6mm × 250mm；

The ammonium acetate of flowing phase: 10mmol/L；

Detector: UV-detector；

Column temperature: 25 DEG C；

Flow velocity: 1mL/min；

Sample size: 30 μ L；

Specification Curve of Increasing:

Accurately weigh 4.0mg uric acid standard items, be settled to 10mL with the ammoniacal liquor ultrasonic dissolution that mass percent is 25%, To the uric acid standard mother liquor that concentration is 400 μ g/mL, it is configured to the uric acid that concentration is 0,12.5,30,60,90,120,150 μ g/mL Standard liquid, crosses film standby；Accurately sample introduction 30 μ L, paints according to the corresponding relation that peak area is directly proportional to standard liquid sample introduction concentration Antidiuresis acidity scale directrix curve, tries to achieve regression equation, coefficient correlation and the range of linearity thereof.

And, the cultivation of described step (2) middle solid form delivery is specific as follows:

Standard medium used by Caenorhabditis elegans is NGM culture medium, and its configuration proportion and program are 3g NaCl, 17g fine jade Fat, 2.5g peptone, 1L distilled water, after high pressure steam sterilization, the concentration aseptically adding 1mL cholesterol is 5mg/mL Cholesterol ethanol solution, 1mL concentration be 1mol/L and the MgSO through high pressure steam sterilization₄Solution, 1mL concentration are 1mol/L and through the CaCl of high pressure steam sterilization₂Solution and the 1mol/L of 25mL, through high pressure steam sterilization, the phosphoric acid of pH=6.0 Potassium buffer solution, is fully mixed, and pours the culture dish of a diameter of 9cm into, cools down stand-by；

Wherein, the preparation process of described cholesterol ethanol solution is: cholesterol use absolute ethyl alcohol to carry out dissolving and Degerming through 0.45 μm membrane filtration；

Nematode synchronization, is centrifuged the larva SM liquid medium 1500-2000r/min hatched 2-5min and collects children Worm, forwards to scribble on the NGM culture medium flat plate of Escherichia coli OP50, flat board is placed in 20 DEG C of incubators continuation and cultivates, cultivate To 32h stand-by；

Variable concentrations 0,0.05, medicine hypoxanthine, uric acid, xanthine and the adenine of 0.15,0.25,0.35mg/mL, After culture medium is ready, draws 5mL medicine, to a sterilized 50mL centrifuge tube, then topple over NGM culture medium to volume 22.5mL, then pour flat board into, after it solidifies, carry out next step experiment；Each concentration of each medicine three is parallel, this process Need quickly, in order to avoid culture medium solidifying；Blank plate adds 5mL M9 buffer solution；

In flat board after culture medium solidifying, select bubble-free, ganoid flat board, draw 100 μ L large intestine bars with liquid-transfering gun Bacterium OP50 is on culture medium, and smears uniformly, and when smearing, bacterium solution distance ware wall 20mm, dries stand-by；

After having cultivated nematode M9 wash buffer after 32h 2-3 time, constant volume, then draw 0.2-with liquid-transfering gun 2mL dropping is scribbling the NGM flat board central authorities of OP50, is sealed by flat board sealed membrane, then puts into 20 DEG C after wrapping with preservative film Incubator is cultivated 24h；

Collect nematode, repeatedly clean 2-3 time with M9 buffer solution, it is ensured that all nematodes are all collected, and are settled to 1mL, put into In refrigerator or carry out liquid phase pre-treatment；

Or, the cultivation of described step (2) middle fluid administration is specific as follows:

After synchronization, the larva SM liquid medium 1500-2000r/min hatched is centrifuged 2-5min and collects larva, Forward to scribble on the NGM flat board of Escherichia coli OP50, flat board is placed in 20 DEG C of incubators continuation and cultivates, cultivate 32h；

The SM liquid medium that will prepare, is aseptically distributed in 6 well culture plates, and every hole 3.6mL adds Medicine hypoxanthine, uric acid, xanthine and the adenine of the variable concentrations 0,0.05,0.15,0.25,0.35mg/mL prepared, Every hole 1.2mL, each concentration of each medicine three is parallel；

Observed by microscope, select the flat board M9 wash buffer that growing way is identical clean, go in 15mL centrifuge tube, 1500-2000r/min is centrifuged 2-5min, shakes up and is distributed into 6 well culture plates, make every hole contain polypide after drawing supernatant, and constant volume Article 500 ± 5, seal with sealed membrane and be put in 20 DEG C of incubators afterwards and cultivate respectively to 0,6,10,18,24,32h, then with moving liquid Rifle sucks in 15mL centrifuge tube, and 1500-2000r/min is centrifuged, and is then settled to 1mL, puts into refrigerator and carry out preserving or carrying out liquid Phase pre-treatment.

And, described step specifically comprising the following steps that (4)

Take synchronization Caenorhabditis elegans, solid culture 32h, sweep away with M9, clean, collect nematode；

Take six orifice plates, Liquid Culture Caenorhabditis elegans, then with 0.25mg/mL xanthine it is administered respectively 18h, 24h, 32h, control group is M9 solution；The C. Elegans Automatic Screening number of control group and each experimental group is 500 ± 5；

The C. Elegans Automatic Screening of control group and each experimental group be transferred to the most respectively to scribble OP50 containing 5 FU 5 fluorouracil On NGM culture medium, every day accurate recording C. Elegans Automatic Screening mortality and number of missing, until all tested beautiful hidden bar lines Worm is the most dead, it is judged that the standard of nematode death is not to reply outside stimulus, if any climbing to death on wall, not very at it In；

In triplicate, experimental result SPSS software carries out statistical disposition in parallel test.

And, specifically comprising the following steps that of described step (5) middle Caenorhabditis elegans locomitivity Assessment of Changes

1. synchronization Caenorhabditis elegans is taken；

2. nematode solid culture 32h, sweeps away with M9, cleans, and collects nematode；

3. taking six orifice plates, Liquid Culture nematode, with 0.25mg/mL xanthine, it is administered 18h respectively, control group is that M9 is molten Liquid；The Caenorhabditis elegans number of control group and each experimental group is 500 ± 5；

4. picking Caenorhabditis elegans is in without on the NGM culture medium of OP50, after 2min, examines under a microscope, with nematode Head pendulum swings back in the past as once again, records the head oscillation number of times of Caenorhabditis elegans, altogether picking 10 in 1min Observe；With the sinusoidal motion of one wavelength of nematode for once, the health bending time of record Caenorhabditis elegans in 20s Number, picking 10 is observed altogether；

5. parallel test is in triplicate, and experimental result SPSS software carries out statistical disposition；

Or, specifically comprising the following steps that of described step (5) middle Caenorhabditis elegans egg laying amount Assessment of Changes

1. synchronization Caenorhabditis elegans is taken；

3. taking six orifice plates, Liquid Culture nematode, with 0.25mg/mL xanthine, it is administered 18h respectively, control group is that M9 is molten Liquid；The C. Elegans Automatic Screening number of control group and each experimental group is 500 ± 5；

4. distinguishing 15 Caenorhabditis elegans of each picking in 15 without on the NGM culture medium of OP50, every day is beautiful hidden bar Nematode is transferred to, on the new NGM culture medium without OP50, count worm's ovum number under the microscope, until end of laying eggs；

5. parallel test is repeated twice, and experimental result SPSS software carries out statistical disposition.

And, specifically comprising the following steps that of described step (5) middle model estimation of stability

The Caenorhabditis elegans of blank group and experimental group is carried out at 20 DEG C solid culture, and ensures sufficient food, Cultivate 6 respectively, 12,18,24, after 30h, rinse C. Elegans Automatic Screening with M9, collect, carry out liquid phase pre-treatment, measure beautiful hidden respectively The content of uric acid in rhabditida body；

Wherein, blank group is the C. Elegans Automatic Screening before not modeling, and experimental group is to cultivate the beautiful hidden bar of different time after modeling Nematode；

The most in triplicate, experimental result SPSS software carries out statistical disposition in parallel test；

Or, specifically comprising the following steps that of described step (5) middle allopurinol evaluation

By set up the Caenorhabditis elegans before and after model give respectively allopurinol final concentration 0.05,0.15,0.5mg/mL, After NGM culture medium cultivates 12h, collect Caenorhabditis elegans, in high effective liquid chromatography for measuring Caenorhabditis elegans body Uric acid content；Wherein, blank group is the Caenorhabditis elegans before not modeling, and model group is the Caenorhabditis elegans after modeling, flat The most in triplicate, experimental result SPSS software carries out statistical disposition in row test.

And, specifically comprising the following steps that of described step (5) middle xanthine oxidase activity evaluation

1. synchronization Caenorhabditis elegans is taken；

2., after proceeding to L1 phase larva equivalent solid medium cultivates 32h, make the nematode population on each flat board equal, And quantity is 500 ± 5；

3. taking six orifice plates, every hole adds the xanthine of 1.2mL, 0.25mg/mL, carries out Liquid Culture, the SM that will prepare Liquid medium, is aseptically distributed in 6 well culture plates, every hole 3.6mL, and three parallel；

Observed by microscope, select the flat board M9 wash buffer that growing way is identical clean, go in 15mL centrifuge tube, 1500-2000r/min is centrifuged 2-5min, shakes up and is distributed into 6 well culture plates, make every hole contain polypide after drawing supernatant, and constant volume Article 500 ± 5, seal with sealed membrane and be put in 20 DEG C of incubators cultivation 18h modelings afterwards, obtain nematode after modeling；

4. nematode sucking-off from orifice plate after modeling, M9 cleans three times, under condition of ice bath, is fully homogenized, makes homogenate, Refrigerated centrifuge 3000-5000r/min, centrifugal 5-15min, take supernatant and be measured；

5. the operational procedure behaviour on the xanthine oxidase kit specification of Bioengineering Research Institute is built up according to Nanjing Make.

The present invention obtain advantage and good effect be:

1. the inventive method utilizes and has high conservative, and the mould that uric acid metabolism approach is much like with the mankind with human gene Formula biology Caenorhabditis elegans establishes hyperuricemia animal model, this model have typical pathology characterize, stable, can Lean on, science, rationally, high, reproducible, the highly reliable and controlled advantage such as easy of similitude, and every quantitative index has system Meter is learned extensive, the high-flux medicaments sifting of meaning, the most only gout and high lithemia disease and is provided strong instrument, Er Qiewei The pathomechanism research of hyperuricemia and ventilation provides the experimental model of good medicine, is also other metabolic disease animal models Foundation provide beneficial reference, the hyperuricemia animal model of foundation has important practical significance.

2. this method utilizes the Caenorhabditis elegans similar to human urine acid metabolic as model organism, not only increases show Beautiful hidden rhabditida is in the value of field of medicaments, and modeling indifference is different in nature, and the high lithemia disease model of foundation is the big of high lithemia disease Scale, high-flux medicaments sifting provide instrument accurately, to find more safe and efficient medicine, can be the mankind's Health brings glad tidings.

3. this method finally determines that employing precursor substance xanthine can be set up low damaging, efficient, stable, reliable and practical , and the Caenorhabditis elegans high lithemia model of mankind's high lithemia essence can be reacted.

The construction method of Caenorhabditis elegans high lithemia model the most of the present invention can be applied in anti-trioxypurine drug screening, with Time, the Caenorhabditis elegans high lithemia model constructed by construction method of Caenorhabditis elegans high lithemia model of the present invention can also Apply in anti-trioxypurine drug screening.

Accompanying drawing explanation

Fig. 1 is the uric acid metabolism figure of Caenorhabditis elegans in the present invention；

Fig. 2 is the uric acid metabolism figure of human body；

Fig. 3 is the liquid chromatogram of high performance liquid chromatography detection uric acid mark product in the present invention；

Fig. 4 is uric acid calibration curve in the present invention；

Fig. 5 is the HPLC chromatogram of uric acid sample in the present invention；

Fig. 6 is the comparison diagram of different modes of administration in the present invention；

Fig. 7 is that the hypoxanthine of variable concentrations in the present invention, uric acid, adenine, four kinds of modeling agent of xanthine are to beautiful hidden In rhabditida body, uric acid content affects figure；

(± SD) (* p < 0.05, * * p < 0.01, * * * p < 0.001vs. blank group)；

(±SD)(*p<0.05,**p<0.01,***p<0.001vs.0group)；

Fig. 8 is that hypoxanthine in the present invention, uric acid, xanthine, four kinds of modeling agent difference modeling times of adenine are to beautiful In hidden rhabditida body, uric acid content affects figure；

(± SD) (* p < 0.05, * * p < 0.01, * * * p < 0.001vs. blank group)；

(±SD)(*p<0.05,**p<0.01,***p<0.001vs.0group)；

Fig. 9 is that in the present invention, xanthic administration time affects figure to the survival rate of Caenorhabditis elegans；

(± SD) (* p < 0.05, * * p < 0.01, * * * p < 0.001vs. blank group)；

(±SD)(*p<0.05,**p<0.01,***p<0.001vs.0group)；

Figure 10 is that administration time that in the present invention, xanthine is different affects figure to Caenorhabditis elegans average life span；

(± SD) (* p < 0.05, * * p < 0.01, * * * p < 0.001vs. blank group)；

(±SD)(*p<0.05,**p<0.01,***p<0.001vs.0group)；

Figure 11 be in the present invention before and after modeling in Caenorhabditis elegans body uric acid content scheme over time；

(± SD) (* p < 0.05vs.0 group, * * * p < 0.001vs.-18h group)；

(± SD) (* p < 0.05vs.0group, * * * p < 0.001vs.-18group)；

Figure 12 be in the present invention allopurinol of variable concentrations on the impact of uric acid content in model Caenorhabditis elegans body Figure；

(± SD) (* * p < 0.01vs. model group, * * * p < 0.001vs. blank group)；(±SD)(**p<0.01vs.model Group, * * * p < 0.001vs.normal control group)；

Figure 13 is the variation diagram of Caenorhabditis elegans survival rate before and after modeling in the present invention；

Figure 14 is before and after modeling Caenorhabditis elegans head oscillation to be affected figure (± SD) in the present invention；

Figure 15 is to affect figure (± SD) to what Caenorhabditis elegans health bent before and after modeling in the present invention；

Figure 16 is the variation diagram (± S.D) of Caenorhabditis elegans egg laying amount before and after modeling in the present invention.

Detailed description of the invention

Below in conjunction with embodiment, the present invention is further described；Following embodiment is illustrative, is not determinate, Protection scope of the present invention can not be limited with following embodiment.

Raw material used in the present invention, if no special instructions, is the commercially available prod of routine；Used in the present invention Method, if no special instructions, is the conventional method of this area.

The culture of heretofore described Caenorhabditis elegans is uracil-deficient type Escherichia coli (OP50).

In the present invention, Caenorhabditis elegans used medium is standard NGM culture medium.

Associated materials used in 1 present invention can be such that

1.1 materials: Caenorhabditis elegans.

1.2 solution preparations

Uric acid: accurately weigh 15.0mg uric acid, adds the DMSO dissolution of trace (50-300 μ L), takes 15mL distilled water, super Sound water-bath is dissolved, then is diluted to desired concn respectively.

Hypoxanthine: accurately weigh hypoxanthine 15.0mg, adds 10mL distilled water, ultrasonic 30min, then is diluted to respectively Desired concn.

Xanthine: accurately weigh 15.0mg xanthine, adds the ammoniacal liquor dissolution of trace (50-300 μ L), then takes 15mL and steams Distilled water ultrasonic water bath is dissolved, then is diluted to desired concn respectively, adjusts pH to about 7.5.

Adenine: accurately weigh adenine 15.0mg, takes 15mL distilled water ultrasonic dissolution, then is diluted to required dense respectively Degree.

Uric acid standard reserving solution: accurately weigh uric acid mark product 4.0mg, takes the ammoniacal liquor that 10mL mass percent is 25%, molten Solve.

Allopurinol: take 20.0mg allopurinol, the hot bath that adds water (37-60 DEG C) is dissolved, is settled to 10mL, is diluted to institute Need concentration.

1.3 kit

Xanthine oxidase kit: Bioengineering Research Institute is built up in Nanjing.

Total number born kit: Bioengineering Research Institute is built up in Nanjing.

1.4 instrument and equipment

U.S. high speed freezing centrifuge CR3i, Thermo company；U.S. ultra low temperature freezer Forma-700, Themo；The U.S. High performance liquid chromatograph Agilent 1200, Anjelen Sci. & Tech. Inc；Japan biomicroscope CX41, OLYMPUS company； Japan's fluorescence inverted microscope, Nikon.

More preferably, step is as follows:

The screening technique of the construction method of above-mentioned Caenorhabditis elegans high lithemia model, step is as follows:

The described different dosing time is 0,18,24,32,48h；

Specifically, the screening technique of the construction method of above-mentioned Caenorhabditis elegans high lithemia model, step is as follows:

The described different dosing time is 0,18,24,32,48h；

1. Urine by HPLC acid content

High performance liquid chromatography condition

Chromatographic column: Intertsil ODS-SP chromatographic column (5 μm, 4.6mm × 250mm)；

The ammonium acetate of flowing phase: 10mmol/L

Detector: UV-detector

Column temperature: 25 DEG C；

Flow velocity: 1mL/min；

Sample size: 30 μ L.

Specification Curve of Increasing

Accurately weigh 4.0mg uric acid standard items, be settled to 10mL with the ammoniacal liquor ultrasonic dissolution that mass percent is 25%, To the uric acid standard mother liquor that concentration is 400 μ g/mL, it is configured to the uric acid that concentration is 0,12.5,30,60,90,120,150 μ g/mL Standard liquid, crosses film standby.Accurately sample introduction 30 μ L, paints according to the corresponding relation that peak area is directly proportional to standard liquid sample introduction concentration Antidiuresis acidity scale directrix curve, tries to achieve regression equation, coefficient correlation and the range of linearity thereof.

Sample treatment

Liquid-transfering gun draws M9 solution, is all swept away by the nematode on pretreatment flat board, and 1500-2000r/min is centrifuged 2- 5min, abandoning supernatant.

Repeatedly clean 3 times as above-mentioned, retain bottom precipitation nematode.

Nematode is ground broken, by slurry hydraulic control system at 3-4mL, in 15mL centrifuge tube through mill.

In slurry liquid, add 6mL ammoniacal liquor carry out ultrasonic wave process more than 30min, add 4mL acetonitrile, vortex oscillation 8min, stands 20min, 3000-5000r/min and is centrifuged 5-10min.

Discard precipitation, take supernatant and carry out rotary evaporation, redissolve with ammoniacal liquor after rotary evaporation, and ultrasonic promotion uric acid is molten Solving, be finally settled to 2mL ,-20 DEG C preserve or direct injected mensuration.

2. the solid culture of Caenorhabditis elegans

Solid culture Caenorhabditis elegans is typically used in laboratory, and several the nematodes that picking upgrowth situation is good are put into and scribble greatly On the NGM culture medium of enterobacteria OP50,20 DEG C of cultivations.During Selective agar medium flat board, bubble-free to be selected, ganoid flat Plate, if there being gap, nematode will crawl into culture medium, the accuracy of impact experiment.

General solid culture mode is improved by the present invention, cultivates and is administered and carries out simultaneously, specifically comprises the following steps that

Nematode synchronization, is centrifuged the larva SM liquid medium 1500r/min hatched 2min and collects larva, and forwards to be coated with Have on the NGM flat board of Escherichia coli OP50, flat board is placed in 20 DEG C of incubators continuation and cultivates, cultivate to 32h stand-by.

Aseptically, by NGM culture medium good for sterilizing, (in the present invention, standard medium used by Caenorhabditis elegans is NGM culture medium, its configuration proportion and program are 3g NaCl, 17g agar, 2.5g peptone, 1L distilled water, high pressure steam sterilization Afterwards) aseptically add the cholesterol ethanol solution that concentration is 5mg/mL of 1mL cholesterol, 1mL concentration is 1mol/ L and through the MgSO of high pressure steam sterilization₄Solution, 1mL concentration are 1mol/L and the CaCl through high pressure steam sterilization₂Solution and 25mL 1mol/L, through high pressure steam sterilization, the kaliumphosphate buffer of pH=6.0, be fully mixed, pour the culture dish of a diameter of 9cm into, Cool down stand-by；

Wherein, the preparation process of described cholesterol ethanol solution is: cholesterol use absolute ethyl alcohol to carry out dissolving and Degerming through 0.45 μm membrane filtration.

Variable concentrations 0,0.05, medicine hypoxanthine, uric acid, xanthine and the adenine of 0.15,0.25,0.35mg/mL, After culture medium is ready, draws 5mL drug solution, to a sterilized 50mL centrifuge tube, then topple over NGM culture medium to body Long-pending about 22.5mL, then pour flat board into, after it solidifies, carry out next step experiment.Each concentration of each medicine three is parallel, This process needs quickly, in order to avoid culture medium solidifying.Blank plate adds 5mL M9 buffer solution.

In flat board after culture medium solidifying, draw 100 μ L Escherichia coli OP50 on culture medium with liquid-transfering gun, and smear uniformly (bacterium solution distance ware wall 20mm when smearing), dries stand-by.

After having cultivated nematode M9 wash buffer after 32h 2-3 time, it is settled to 1mL, then draws with liquid-transfering gun Proper volume (0.2-2mL) dropping is scribbling the NGM flat board central authorities of OP50, is sealed by flat board sealed membrane, then uses preservative film bag Wrap rear (in case microbiological contamination) and put into cultivation 24h in 20 DEG C of incubators.

Collect nematode, repeatedly clean 2-3 time with M9 buffer solution, it is ensured that all nematodes are all collected.It is settled to 1mL, puts into In refrigerator or carry out liquid phase pre-treatment.

3. fluid administration cultivates Caenorhabditis elegans

After synchronization, the larva SM liquid medium 1500-2000r/min hatched is centrifuged 2-5min and collects larva, Forward to scribble on the NGM flat board of Escherichia coli OP50, flat board is placed in 20 DEG C of incubators continuation and cultivates, cultivate 32h.

The SM nutrient solution that will prepare, is aseptically distributed in 6 well culture plates, and every hole 3.6mL adds preparation Good variable concentrations 0, the every hole of medicine hypoxanthine, uric acid, xanthine and adenine of 0.05,0.15,0.25,0.35mg/mL 1.2mL, each concentration of each medicine three is parallel.

Observed by microscope, select the flat board M9 wash buffer that growing way is identical clean, go in 15mL centrifuge tube, 1500-2000r/min is centrifuged 2-5min, draws supernatant, and is settled to 1mL and shakes up and be distributed into 6 well culture plates, makes every hole contain worm Body 500 ± 5, seals with sealed membrane and is put in 20 DEG C of incubators afterwards and cultivates 0,6,10,18,24,32h and then suck with liquid-transfering gun In 15mL centrifuge tube, centrifugal 1500-2000r/min is centrifuged 2-5min, is then settled to 1mL, puts into refrigerator and preserves or enter Row liquid phase pre-treatment.

4. Caenorhabditis elegans life experiment

Synchronization Caenorhabditis elegans.

Solid culture, through 32h, sweeps away with M9, cleans, and collects nematode.

Take six orifice plates, Liquid Culture Caenorhabditis elegans, then with 0.25mg/mL xanthine it is administered respectively 18h, 24h, 32h, control group is M9 solution.The C. Elegans Automatic Screening number of control group and each experimental group is 500 ± 5.

The C. Elegans Automatic Screening of control group and each experimental group be transferred to the most respectively to scribble OP50 containing 5 FU 5 fluorouracil On NGM culture medium, the mortality of C. Elegans Automatic Screening and number of missing under every day accurate recording, until all tested beautiful hidden bars Nematode is the most dead.Judge that the dead standard of nematode is not to reply outside stimulus.If any climbing to death on wall, not very exist In it.

5. the motion conditions of Caenorhabditis elegans

(1) synchronization Caenorhabditis elegans.

(2) solid culture is through 32h, sweeps away with M9, cleans, and collects nematode.

(3) taking six orifice plates, Liquid Culture nematode, with 0.25mg/mL xanthine, it is administered 18h respectively, control group is M9 Solution.The Caenorhabditis elegans number of control group and each experimental group is 500 ± 5.

(4) picking Caenorhabditis elegans is in without on the NGM culture medium of OP50, after 2min, examines under a microscope, with nematode Head pendulum swings back in the past as once again, records the head oscillation number of times of Caenorhabditis elegans, altogether picking 10 in 1min Observe；With the sinusoidal motion of one wavelength of nematode for once, the health bending time of record Caenorhabditis elegans in 20s Number, picking 10 is observed altogether.

(5) parallel test is in triplicate, and experimental result SPSS software carries out statistical disposition.

6. the egg laying amount of Caenorhabditis elegans

(1) synchronization Caenorhabditis elegans.

(3) taking six orifice plates, Liquid Culture nematode, with 0.25mg/mL xanthine, it is administered 18h respectively, control group is M9 Solution.The C. Elegans Automatic Screening number of control group and each experimental group is 500 ± 5.

(4) 15 Caenorhabditis elegans of each picking are in 15 without on the NGM culture medium of OP50 respectively, and every day is beautiful hidden bar Nematode is transferred to, on the new NGM culture medium without OP50, count worm's ovum number under the microscope, until end of laying eggs.

(5) parallel test is repeated twice, and experimental result SPSS software carries out statistical disposition.

7. model stability evaluation

The most in triplicate, experimental result SPSS software carries out statistical disposition in parallel test.

8. allopurinol evaluation

Specifically comprise the following steps that

By set up the Caenorhabditis elegans before and after model give respectively allopurinol final concentration 0.05,0.15,0.5mg/mL, After solid culture 12h, collect Caenorhabditis elegans, the uric acid content in high effective liquid chromatography for measuring Caenorhabditis elegans body； Wherein, blank group is the Caenorhabditis elegans before not modeling, and model group is the Caenorhabditis elegans after modeling, and parallel test is at least In triplicate, experimental result SPSS software carries out statistical disposition.

9. the mensuration of xanthine oxidase activity (XOD)

Experiment use Nanjing build up biotechnology research xanthine oxidase kit measurement, principle is that xanthine can Catalysis hypoxanthine generates xanthine, and superoxide anion can generate aubergine with developer effect, can be according to absorbance Change calculations xanthine oxidase vigor.Concrete operation step is as follows:

(1) synchronization Caenorhabditis elegans

(2), after proceeding to L1 phase larva equivalent solid medium cultivates 32h, make the nematode population on each flat board as far as possible Equal, and quantity is suitable (about 500).

Taking six orifice plates, every hole adds 1.2mL, 0.25mg/mL xanthine, carries out Liquid Culture, the SM liquid that will prepare Nutrient solution, is aseptically distributed in 6 well culture plates, every hole 3.6mL, and three parallel；

Observed by microscope, select the flat board M9 wash buffer that growing way is identical clean, go in 15mL centrifuge tube, 1500-2000r/min is centrifuged 2-5min, shakes up and is distributed into 6 well culture plates, make every hole contain polypide after drawing supernatant, and constant volume Article 500 ± 5, seal with sealed membrane and be put in 20 DEG C of incubators cultivation 18h modelings afterwards, obtain nematode after modeling.

(3) nematode sucking-off from orifice plate after modeling, M9 cleans three times, under condition of ice bath, is fully homogenized, makes homogenate Liquid.Refrigerated centrifuge 3000-5000r/min, centrifugal 5-15min, take supernatant and be measured.

(4) operation on xanthine oxidase (XOD) the kit specification of Bioengineering Research Institute is built up according to Nanjing Guidelines.

(5) in tissue, the activity of xanthine oxidase is the per gram of tissue albumen substrate 37 DEG C of conversion 1 μm ol per minute Required enzyme amount is an enzyme activity unit.

Computing formula:

In formula: U represents xanthine oxidase vigor (U/gprot), A₂It is to measure pipe OD value, A₁Being blank tube OD value, m is Colour generation thing molar extinction coefficient: 12.6*10^-3, V is the cumulative volume (mL) of reactant liquor, V₁For sampling amount (mL), d represents optical path, t Representing the reaction time (min), c represents homogenate proteins concentration (gprot/L).

The related test results of the present invention:

1. the mensuration of uric acid content in Caenorhabditis elegans body

Fig. 3 is high performance liquid chromatography detection method uric acid mark product liquid chromatogram, and uric acid retention time is 2.941min.Figure 4 is uric acid calibration curve, calibration curve try to achieve peak area (y) and to the equation of linear regression of uric acid concentration (x) be: y= 37.88x-401.54, coefficient R²=0.9982, the equation has significant correlation.

Fig. 5 is the HPLC chromatogram of uric acid sample, utilizes uric acid in high effective liquid chromatography for measuring Caenorhabditis elegans body Content, utilize acetonitrile removing protein to carry out pre-treatment.In mensuration Caenorhabditis elegans body, uric acid level is 5.58 μ g/100 bars.

2. the determination of Caenorhabditis elegans administering mode

After Caenorhabditis elegans synchronization, the larva SM liquid medium 1500r/min hatched is centrifuged 2min and collects Larva, forwards to scribble on the NGM flat board of Escherichia coli OP50, flat board is placed in 20 DEG C of incubators continuation and cultivates, cultivate 32h. Being allocated into by equal medicine in SM fluid nutrient medium, NGM solid medium respectively, each concentration of each medicine three is parallel, and point Do not access Escherichia coli.After having cultivated Caenorhabditis elegans M9 wash buffer after 32h 2-3 time, it is settled to certain Add in culture medium (about 500) after volume, put into cultivation 24h in 20 DEG C of incubators.

Fig. 6 is the comparison of different modes of administration, Caenorhabditis elegans solid form delivery system and the blank of fluid administration mode Control group is without significant difference (p > 0.05).Caenorhabditis elegans when Liquid Culture mode is administered, more blank group of liter of uric acid content High 5.39 μ g/500 bars, and when solid culture is administered, more blank group of uric acid content only raises 2.08 μ g/500 bars, so, this reality Test and use the mode of Liquid Culture to set up Caenorhabditis elegans high lithemia model.

The suitableeest modeling medicine, concentration and the determination of time

According to solid form delivery system research variable concentrations (0.05,0.15,0.25mg/mL) and administration different time (6,12, 18,24,30h) four kinds of modeling agent hypoxanthine, uric acid, xanthine and adenine to uric acid content in Caenorhabditis elegans body Impact, in units of 500 Caenorhabditis elegans.

Fig. 7 is that the hypoxanthine of variable concentrations, uric acid, adenine, four kinds of modeling agent of xanthine are to Caenorhabditis elegans body The impact of interior uric acid content.Fig. 8 is that hypoxanthine, uric acid, xanthine, four kinds of modeling agent difference modeling times of adenine are to beautiful The impact of uric acid content in hidden rhabditida body.

By Fig. 7 and Fig. 8, comparing four kinds and be administered material, hypoxanthine, uric acid, three kinds of medicines of xanthine give beautiful hidden bar After nematode, internal uric acid content has a certain degree of rising, and adenine medicine is the most unchanged, statistically without notable Difference (p > 0.05).And xanthine concentration 0.25mg/mL group and xanthine be administered 18h group, be administered 24h group, to be administered 32h group more empty White group raises the uric acid content in Caenorhabditis elegans body than other material in the case of variable concentrations and administration different time The most, and have pole significant difference (p < 0.001) with blank group.

By Fig. 7, Fig. 8 it can be seen that hypoxanthine, uric acid, xanthine are when concentration is 0.25mg/mL, beautiful hidden bar line Uric acid in polypide raises substantially, and relatively blank elements does not raise 10.02 μ g, 11.42 μ g, 19.20 μ g, and statistical analysis, secondary Huang is fast Purine and uric acid (0.25mg/mL) more blank group of significant difference (p < 0.05) of group, xanthine group (0.25mg/mL) organizes more blank group of difference Heteropole is notable (p < 0.001).

From result, give Caenorhabditis elegans hypoxanthine, uric acid, xanthic optium concentration are 0.25mg/ ML, but, in these three material, 0.25mg/mL xanthine makes Caenorhabditis elegans body, uric acid raises the most obvious, after institute Caenorhabditis elegans high lithemia model set up by continuous experiment 0.25mg/mL xanthine.

The impact grown Caenorhabditis elegans in view of medicine itself, may not be that action time is the longest, uric acid liter High The more the better, need to select a suitable administration time, Caenorhabditis elegans can not be caused relatively major injury, the most permissible Caenorhabditis elegans is made to reach high lithemia state.Life experiment is the Classic Experiments evaluating drug toxicity.

Fig. 9 is the impact on the survival rate of Caenorhabditis elegans of the xanthic administration time.Figure 10 is that xanthine is different The administration time impact on Caenorhabditis elegans average life span.

As shown in Figure 9, blank group MaLS 39d, the MaLS relatively blank elements being administered 18h, 24h, 32h does not reduce 1d, 3d and 7d.Visible, xanthine has a certain degree of injury to Caenorhabditis elegans.Blank group 21d, be administered 18h group 15d, 24h group 13d, 32h group 10d, the survival rate of Caenorhabditis elegans is more than 50%.

As shown in Figure 10, the equal decrease to some degree of average life span of each group, it is administered more blank group of 18h group and reduces 1.68d, It is administered more blank group of 24h group average life span and reduces 4.85d, be administered the more blank group of average life span of 32h group and reduce 8.69d.Statistical Analysis, is administered 18h and compares with blank group, and average life span is without significant difference (p > 0.05), and is administered 24h and administration 32h and all has the most aobvious Write difference (p < 0.01).So, it is administered 24h and 32h and all Caenorhabditis elegans can be caused relatively major injury, though and being administered 18h group So Caenorhabditis elegans is had certain injury, but is also not up to the level of signifiance.

Result shows, Caenorhabditis elegans is had a certain impact by xanthine medicine, with the prolongation of administration time, life-span shadow Ring aggravation.But in 18h, Caenorhabditis elegans body, have arrived at high lithemia state, have pole significant difference, for keeping away with blank group Exempt from medicine itself and Caenorhabditis elegans may be caused large effect, make again uric acid significantly raise, so the choosing of most suitable time Select administration 18h.

4. model persistence is evaluated

After research modeling, in Caenorhabditis elegans body, uric acid content over time, measures beautiful hidden bar line before and after modeling In polypide, uric acid content changes over situation, and its result is as shown in figure 11.

-18h represents and utilizes xanthine 0.25mg/mL to begin setting up Caenorhabditis elegans high lithemia model, 0h representative model Group, as seen from Figure 11, in 0h Caenorhabditis elegans body, uric acid content significantly raises (raising about 52.48%), statistical analysis Difference is extremely notable (p < 0.001).Model group uric acid content in 12h, model Caenorhabditis elegans body remains stable substantially High lithemia state, after 12h, the uric acid content in Caenorhabditis elegans body is on a declining curve.Uric acid in 24h Caenorhabditis elegans body More blank group of content becomes that there were significant differences (p < 0.05), although showing that high lithemia has declined, but beautiful hidden with non-administration Still there were significant differences for uric acid content in rhabditida body.So, Caenorhabditis elegans high lithemia model can maintain 12h.

Some model is held time the shortest, the mouse high lithemia model set up such as lumbar injection Oteracil Potassium, is only capable of dimension Hold 5h, be not provided that time enough for screening and treatment hyperuricemia.The Caenorhabditis elegans high lithemia that this experiment is set up Model is basicly stable, can maintain about 12h, and is drug screening and study mechanism provides possibility.

5. the allopurinol of variable concentrations is on the impact of uric acid content in model Caenorhabditis elegans body

Set up the Caenorhabditis elegans after model give respectively allopurinol final concentration 0.05,0.15,0.5mg/mL, solid After cultivating 12h, collecting Caenorhabditis elegans, HPLC measures the uric acid content in Caenorhabditis elegans body.Blank group is not for model Front Caenorhabditis elegans, model group is the Caenorhabditis elegans after modeling, and the most in triplicate, experimental result is used in parallel test SPSS software carries out statistical disposition.

Its result is as shown in figure 12.In model group Caenorhabditis elegans body, more blank group of uric acid content significantly raises, about 52.4%, statistics has extremely notable meaning (p < 0.001).After giving allopurinol 0.05,0.15mg/mL, Caenorhabditis elegans More blank group of internal uric acid content has pole significant difference (p < 0.001).After giving allopurinol 0.25mg/mL, beautiful hidden bar line In polypide, uric acid content relatively model group is decreased significantly, and about 15.0%, there were significant differences for statistics (p < 0.01), and allopurinol (0.25mg/mL) there were significant differences (p < 0.05) for more blank group of Caenorhabditis elegans of group, illustrates with 0.25mg/mL allopurinol pair After Caenorhabditis elegans is treated, in Caenorhabditis elegans body, uric acid is decreased significantly, but is also not up to normal level.

6. Caenorhabditis elegans survival rate and the investigation of average life span before and after modeling

It is transferred to the Caenorhabditis elegans of control group and each experimental group the most respectively scribble urinating containing 5-fluorine of OP50 On the NGM culture medium of pyrimidine, the mortality of Caenorhabditis elegans and number of missing under every day accurate recording, until all tested Caenorhabditis elegans the most dead.Judge that the dead standard of Caenorhabditis elegans is not to reply outside stimulus.If any climbing Death on wall, the most within it.In triplicate, before and after research modeling, Caenorhabditis elegans survival rate is with average in parallel test The situation of change in life-span, experimental result SPSS software carries out statistical disposition.Result is as shown in table 1 and Figure 13.

The average life span table (± SD) of Caenorhabditis elegans before and after table 1 modeling

As shown in Table 1, the average life span of model group Caenorhabditis elegans and blank group are without significant difference (p > 0.05).Result Show, set up modeling agent xanthine (0.25mg/mL, 18h) of Caenorhabditis elegans high lithemia model use not to beautiful hidden Rhabditida causes notable injury.

As shown in Figure 13, the survival rate of model group Caenorhabditis elegans and blank group are without significant difference (p > 0.05).Result Show, set up modeling agent xanthine (0.25mg/mL, 18h) of Caenorhabditis elegans high lithemia model use not to beautiful hidden Rhabditida causes notable injury.

7. the investigation of Caenorhabditis elegans motion conditions before and after modeling

Picking Caenorhabditis elegans, in without on the NGM culture medium of OP50, after 2min, is examined under a microscope, with beautiful hidden Rhabditida head pendulum swings back in the past as once again, records the head oscillation number of times of Caenorhabditis elegans in 1min, altogether chooses Take 10 to observe；With the sinusoidal motion of one wavelength of Caenorhabditis elegans for once, record beautiful hidden bar line in 20s The health number of bends of worm, picking 10 is observed altogether.In triplicate, experimental result SPSS software is added up in parallel test Process.

On the impact of Caenorhabditis elegans head oscillation, impact such as table 2, Figure 14, Tu15Suo of health bending before and after modeling Show.

The head oscillation frequency of Caenorhabditis elegans and health corner frequency table (± SD) before and after table 2 modeling

As seen from Figure 14, the head oscillation frequency of model group is organized without significant difference (p > 0.05) with blank, so, profit It is administered 18h with modeling agent xanthine the head oscillation of model Caenorhabditis elegans do not resulted in significantly affect.

As seen from Figure 15, the health corner frequency of model group group is organized without significant difference (p > 0.05) with blank, so, Utilize modeling agent xanthine administration 18h that the health bending of model Caenorhabditis elegans is not resulted in significantly affect.

Test result indicate that, utilize modeling agent xanthine (0.25mg/mL, the 18h) modeling can't be to Caenorhabditis elegans Cause notable injury, thus demonstrate the reliability of model.

8. the investigation of Caenorhabditis elegans egg laying amount before and after modeling

Take six orifice plates, Liquid Culture Caenorhabditis elegans, with 0.25mg/mL xanthine, it is administered respectively 18h, control group For M9 solution.The Caenorhabditis elegans number of control group and each experimental group is 500 ± 5.Each picking 15 is beautiful hidden respectively Rhabditida is in 15 without on the NGM culture medium of OP50, and every day transfers to the new NGM culture medium without OP50 Caenorhabditis elegans On, count worm's ovum number under the microscope, until end of laying eggs, parallel test is repeated twice, and experimental result SPSS software enters Row statistical disposition.

Before and after modeling, the result of variations of Caenorhabditis elegans egg laying amount is as shown in table 3, Figure 16.

Before and after table 3 modeling, Caenorhabditis elegans is laid eggs the change table (± S.D) of situation

Result shows, before and after modeling, the egg laying amount of Caenorhabditis elegans is without significantly changing (p > 0.05).So, utilize modeling The situation of laying eggs of Caenorhabditis elegans is not resulted in and significantly affects by agent xanthine (0.25mg/mL, 18h) modeling.

The change of xanthine oxidase activity in nematode body before and after research modeling, its result is as shown in table 4.

The change table (± S.D) of xanthine oxidase activity in nematode body before and after table 4 modeling

It is one that XOD vigor is defined as every gram of homogenate proteins enzyme amount required for the substrate of 37 DEG C of conversion 1 μm ol per minute Individual enzyme activity unit.As shown in Table 4, more blank group of model group xanthine oxidase vigor has significantly rising (p < 0.001).Knot Fruit shows, can set up the reason of nematode high lithemia model with modeling agent xanthine is to make the xanthine oxidase in nematode body live Property significantly improves.In purine metabolism, xanthine oxidase is the key enzyme of regulation and control speed and process.Xanthine oxidase Activity improves, and beneficially xanthine converts towards the direction of uric acid, and in making nematode body, uric acid content raises.On the other hand, yellow Purine converts towards uric acid direction and is also beneficial to hypoxanthine and is oxidized to xanthine, and then reoxidizes as uric acid, makes internal purine The speed of metabolism is accelerated, so that uric acid content can significantly raise in nematode body.

By testing result above it can be seen that use precursor substance xanthine (0.25mg/mL, 18h) that low damage can be set up Property, efficiently, stable, reliable Caenorhabditis elegans high lithemia model.This research is not only hyperuricemia pathological study with new Medicine research and development add new research tool, and the foundation for other metabolic disease animal models provides beneficial reference.

As can be seen here, the construction method of Caenorhabditis elegans high lithemia model of the present invention can be applied at anti-trioxypurine drug sieve Choose；The Caenorhabditis elegans high lithemia model constructed by construction method of the inventive method Caenorhabditis elegans high lithemia model Can also apply in anti-trioxypurine drug screening.

Claims

1. the construction method of a Caenorhabditis elegans high lithemia model, it is characterised in that: step is as follows:

Utilize Caenorhabditis elegans, use precursor substance xanthine, final concentration of 0.25-0.45mg/mL, fluid administration, 20-25 Cultivate 18-32h under the conditions of DEG C, i.e. obtain low Caenorhabditis elegans high lithemia model damaging, efficient, stable, reliable.

The construction method of Caenorhabditis elegans high lithemia model the most according to claim 1, it is characterised in that: step is such as Under:

Utilize Caenorhabditis elegans, use precursor substance xanthine, final concentration of 0.25mg/mL, fluid administration, under the conditions of 20 DEG C Cultivate 18h, i.e. obtain low Caenorhabditis elegans high lithemia model damaging, efficient, stable, reliable.

3. the construction method of Caenorhabditis elegans high lithemia model as claimed in claim 1 or 2 is in anti-trioxypurine drug screening Application.

4. the beautiful hidden bar line constructed by construction method of Caenorhabditis elegans high lithemia model as claimed in claim 1 or 2 The application in anti-trioxypurine drug screening of the worm high lithemia model.

5. the screening technique of the construction method of Caenorhabditis elegans high lithemia model as claimed in claim 1 or 2, its feature exists In: step is as follows:

(2) step Caenorhabditis elegans (1) is respectively adopted solid form delivery and fluid administration two ways is cultivated, by than Relatively two kinds of administering modes, on the impact of uric acid content in Caenorhabditis elegans body, determine optimal administration training method；

To step (2) determined by optimal administering mode give respectively Caenorhabditis elegans hypoxanthine, uric acid, xanthine and Concentration that four kinds of medicines of adenine are different and different action time, in units of Caenorhabditis elegans, measure beautiful hidden bar line The change of uric acid content in polypide, determines optimal administration material, dosage；

Described different concentration is 0,0.05,0.15,0.25,0.50,0.65mg/mL；Described different action time is 0,6, 10,18,24,32,48h；

Step (3) determined by optimal be administered material and dosage under the conditions of, measure the optimal material that is administered in different dosing Time, on the impact of Caenorhabditis elegans average life span the change of uric acid content in combining Caenorhabditis elegans body, determines optimal It is administered the Best administration time of material；

The described different dosing time is 0,18,24,32,48h；

(5) establish Caenorhabditis elegans high lithemia model building method, hidden bar beautiful to the high lithemia obtained by this construction method Nematode model carries out serial experiment evaluation, and described serial experiment evaluation includes Caenorhabditis elegans locomitivity Assessment of Changes, show Beautiful hidden rhabditida egg laying amount Assessment of Changes, model stability evaluation, allopurinol drug evaluation, xanthine oxidase activity are commented Valency, determines the stability of high lithemia Caenorhabditis elegans model building method, reliability and the damage to Caenorhabditis elegans Property, determine optimum condition, i.e. obtain low Caenorhabditis elegans high lithemia model damaging, efficient, stable, reliable.

The screening technique of the construction method of Caenorhabditis elegans high lithemia model the most according to claim 5, its feature exists In: step is as follows:

(2) step Caenorhabditis elegans (1) is respectively adopted solid form delivery and fluid administration two ways is cultivated, by than Relatively two kinds of administering modes, on the impact of uric acid content in Caenorhabditis elegans body, determine optimal administration training method, for liquid It is administered；

To step (2) determined by optimal administering mode give respectively Caenorhabditis elegans hypoxanthine, uric acid, xanthine and Concentration that four kinds of medicines of adenine are different and different action time, in units of 400-1000 bar Caenorhabditis elegans, measure The change of uric acid content in Caenorhabditis elegans body, determines optimal administration material, dosage, for xanthine final concentration 0.25mg/mL；

Step (3) determined by optimal be administered material and dosage under the conditions of, measure xanthine in the different dosing time pair The impact of Caenorhabditis elegans average life span the change of uric acid content in combining Caenorhabditis elegans body, determine xanthic Good administration time, for 18h；

The described different dosing time is 0,18,24,32,48h；

(5) establish Caenorhabditis elegans high lithemia model building method, hidden bar beautiful to the high lithemia obtained by this construction method Nematode model carries out serial experiment evaluation, and described serial experiment evaluation includes Caenorhabditis elegans locomitivity Assessment of Changes, show Beautiful hidden rhabditida egg laying amount Assessment of Changes, model stability evaluation, allopurinol drug evaluation, xanthine oxidase activity are commented Valency, determines the stability of high lithemia Caenorhabditis elegans model building method, reliability and the damage to Caenorhabditis elegans Property, determine optimum condition, i.e. use precursor substance xanthine, final concentration of 0.25-0.45mg/mL, fluid administration, 20-25 DEG C, 18-32h, i.e. obtains low Caenorhabditis elegans high lithemia model damaging, efficient, stable, reliable.

7. according to the screening technique of the construction method of the Caenorhabditis elegans high lithemia model described in claim 5 or 6, its feature It is: described step (1) middle Caenorhabditis elegans processes through acetonitrile removing protein before measuring, and the addition of acetonitrile is: 500 shows Beautiful hidden rhabditida adds acetonitrile 5.0mL；

Or, the condition of described Urine by HPLC acid content is as follows:

The ammonium acetate of flowing phase: 10mmol/L；

Detector: UV-detector；

Column temperature: 25 DEG C；

Flow velocity: 1mL/min；

Sample size: 30 μ L；

Specification Curve of Increasing: accurately weigh 4.0mg uric acid standard items, with the ammoniacal liquor ultrasonic dissolution constant volume that mass percent is 25% To 10mL, obtaining the uric acid standard mother liquor that concentration is 400 μ g/mL, being configured to concentration is 0,12.5,30,60,90,120,150 μ The uric acid standard liquid of g/mL, crosses film standby；Accurately sample introduction 30 μ L, is directly proportional to standard liquid sample introduction concentration according to peak area Corresponding relation draws uric acid calibration curve, tries to achieve regression equation, coefficient correlation and the range of linearity thereof；

Or, the cultivation of described step (2) middle solid form delivery is specific as follows:

Standard medium used by Caenorhabditis elegans is NGM culture medium, its configuration proportion and program be 3g NaCl, 17g agar, 2.5g peptone, 1L distilled water, after high pressure steam sterilization, the concentration aseptically adding 1mL cholesterol is 5mg/mL's Cholesterol ethanol solution, 1mL concentration are 1mol/L and the MgSO through high pressure steam sterilization₄Solution, 1mL concentration are 1mol/L And through the CaCl of high pressure steam sterilization₂Solution and the 1mol/L of 25mL, buffer through the potassium phosphate of high pressure steam sterilization, pH=6.0 Liquid, is fully mixed, and pours the culture dish of a diameter of 9cm into, cools down stand-by；

Wherein, the preparation process of described cholesterol ethanol solution is: cholesterol uses absolute ethyl alcohol to carry out dissolving and warp 0.45 μm membrane filtration is degerming；

Nematode synchronization, is centrifuged the larva SM liquid medium 1500-2000r/min hatched 2-5min and collects larva, turn On the NGM culture medium flat plate scribbling Escherichia coli OP50, flat board is placed in 20 DEG C of incubators continuation and cultivates, cultivate to 32h The most stand-by；

Variable concentrations 0,0.05, medicine hypoxanthine, uric acid, xanthine and the adenine of 0.15,0.25,0.35mg/mL, cultivation After benchmark is standby, draws 5mL medicine, to a sterilized 50mL centrifuge tube, then topple over NGM culture medium to volume 22.5mL, then pour flat board into, after it solidifies, carry out next step experiment；Each concentration of each medicine three is parallel, this process Need quickly, in order to avoid culture medium solidifying；Blank plate adds 5mL M9 buffer solution；

In flat board after culture medium solidifying, select bubble-free, ganoid flat board, draw 100 μ L Escherichia coli with liquid-transfering gun OP50 is on culture medium, and smears uniformly, and when smearing, bacterium solution distance ware wall 20mm, dries stand-by；

After having cultivated nematode M9 wash buffer after 32h 2-3 time, constant volume, then draw 0.2-2mL with liquid-transfering gun and drip It is added in the NGM flat board central authorities scribbling OP50, flat board sealed membrane is sealed, then puts into 20 DEG C of incubators after wrapping with preservative film Middle cultivation 24h；

Collect nematode, repeatedly clean 2-3 time with M9 buffer solution, it is ensured that all nematodes are all collected, and are settled to 1mL, put into refrigerator In or carry out liquid phase pre-treatment；

The SM liquid medium that will prepare, is aseptically distributed in 6 well culture plates, and every hole 3.6mL adds preparation Good variable concentrations 0, medicine hypoxanthine, uric acid, xanthine and the adenine of 0.05,0.15,0.25,0.35mg/mL, every hole 1.2mL, each concentration of each medicine three is parallel；

8. according to the screening technique of the construction method of the Caenorhabditis elegans high lithemia model described in claim 5 or 6, its feature It is: described step specifically comprising the following steps that (4)

Take six orifice plates, Liquid Culture Caenorhabditis elegans, then with 0.25mg/mL xanthine, it is administered respectively 18h, 24h, 32h, Control group is M9 solution；The C. Elegans Automatic Screening number of control group and each experimental group is 500 ± 5；

The C. Elegans Automatic Screening of control group and each experimental group is transferred to scribble the training of the NGM containing 5 FU 5 fluorouracil of OP50 the most respectively Support on base, every day accurate recording C. Elegans Automatic Screening mortality and number of missing, until all tested Caenorhabditis elegans are complete Portion is dead, it is judged that the standard of nematode death is not to reply outside stimulus, if any climbing to death on wall, the most within it；

In triplicate, experimental result SPSS software carries out statistical disposition in parallel test；

Or, specifically comprising the following steps that of described step (5) middle Caenorhabditis elegans locomitivity Assessment of Changes

1. synchronization Caenorhabditis elegans is taken；

3. taking six orifice plates, Liquid Culture nematode, with 0.25mg/mL xanthine, it is administered 18h respectively, control group is M9 solution； The Caenorhabditis elegans number of control group and each experimental group is 500 ± 5；

4. picking Caenorhabditis elegans is in without on the NGM culture medium of OP50, after 2min, examines under a microscope, with nematode head Pendulum swings back in the past as once again, and record head oscillation number of times of Caenorhabditis elegans in 1min, picking 10 is seen altogether Examine；With the sinusoidal motion of one wavelength of nematode for once, record health number of bends of Caenorhabditis elegans in 20s, Picking 10 is observed altogether；

1. synchronization Caenorhabditis elegans is taken；

3. taking six orifice plates, Liquid Culture nematode, with 0.25mg/mL xanthine, it is administered 18h respectively, control group is M9 solution； The C. Elegans Automatic Screening number of control group and each experimental group is 500 ± 5；

4. distinguishing 15 Caenorhabditis elegans of each picking in 15 without on the NGM culture medium of OP50, every day is Caenorhabditis elegans Transfer to, on the new NGM culture medium without OP50, count worm's ovum number under the microscope, until end of laying eggs；

9. according to the screening technique of the construction method of the Caenorhabditis elegans high lithemia model described in claim 5 or 6, its feature It is: specifically comprising the following steps that of described step (5) middle model estimation of stability

The Caenorhabditis elegans of blank group and experimental group is carried out at 20 DEG C solid culture, and ensures sufficient food, respectively Cultivate 6,12,18,24, after 30h, rinse C. Elegans Automatic Screening with M9, collect, carry out liquid phase pre-treatment, measure beautiful hidden bar line respectively The content of uric acid in polypide；

Wherein, blank group is the C. Elegans Automatic Screening before not modeling, and experimental group is to cultivate the Caenorhabditis elegans of different time after modeling；

By set up the Caenorhabditis elegans before and after model give respectively allopurinol final concentration 0.05,0.15,0.5mg/mL, After cultivating 12h on NGM culture medium, collect Caenorhabditis elegans, the urine in high effective liquid chromatography for measuring Caenorhabditis elegans body Acid content；Wherein, blank group is the Caenorhabditis elegans before not modeling, and model group is the Caenorhabditis elegans after modeling, parallel The most in triplicate, experimental result SPSS software carries out statistical disposition in test.

10., according to the screening technique of the construction method of the Caenorhabditis elegans high lithemia model described in claim 5 or 6, it is special Levy and be: specifically comprising the following steps that of described step (5) middle xanthine oxidase activity evaluation

1. synchronization Caenorhabditis elegans is taken；

2., after proceeding to L1 phase larva equivalent solid medium cultivates 32h, make the nematode population on each flat board equal, and number Amount is 500 ± 5；

3. taking six orifice plates, every hole adds the xanthine of 1.2mL, 0.25mg/mL, carries out Liquid Culture, the SM liquid that will prepare Nutrient solution, is aseptically distributed in 6 well culture plates, every hole 3.6mL, and three parallel；

4. nematode sucking-off from orifice plate after modeling, M9 cleans three times, under condition of ice bath, is fully homogenized, makes homogenate, freezing Centrifuge 3000-5000r/min, centrifugal 5-15min, take supernatant and be measured；

5. the operational procedure operation on the xanthine oxidase kit specification of Bioengineering Research Institute is built up according to Nanjing.