CN105833295B

CN105833295B - A kind of construction method of Caenorhabditis elegans high lithemia model and the screening technique of application and the construction method

Info

Publication number: CN105833295B
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: 2018-11-20
Anticipated expiration: 2036-05-13
Also published as: CN105833295A

Abstract

The present invention relates to a kind of construction methods of Caenorhabditis elegans high lithemia model, and steps are as follows：(1) the intracorporal uric acid content of high effective liquid chromatography for measuring Caenorhabditis elegans is utilized；(2) optimal administration training method is determined；(3) determine optimal to medicinal substances, dosage；(4) the Best administration time of xanthine is determined；(5) serial experiment evaluation is carried out to construction method high lithemia Caenorhabditis elegans model obtained, obtain low damaging, efficient, stable, reliable Caenorhabditis elegans high lithemia model.This method, which is utilized, has high conservative with human gene, and uric acid metabolism approach and the much like model organism-Caenorhabditis elegans of the mankind establish hyperuricemia animal model, the model has typical pathology characterization, stablizes, is reliable, and every quantitative index has statistical significance, extensive, high-flux medicaments sifting for gout and high lithemia disease provide strong tool.

Description

A kind of construction method of Caenorhabditis elegans high lithemia model and application and the building side The screening technique of method

Technical field

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

Background technique

With the improvement of people's living standard and the change of dietary structure, in recent years, the disease incidence of hyperuricemia is alive Trend is significantly risen within the scope of boundary.Hyperuricemia itself not only carrys out very big threat to the health care belt of the mankind, can also produce Raw various complication, such as cardiovascular and cerebrovascular disease and atherosclerosis.Therefore reinforce the prevention and control and correlation to hyperuricemia The research and development of drug have become a urgent task.But the deficiency of existing hyperuricemia animal model has become hyperuricemia disease The major obstacle of reason research and new drug development.Therefore, establish science, rationally, similitude it is high, reproducible, highly reliable and can Easy hyperuricemia animal model is controlled to have important practical significance.

Hyperuricemia animal model is established, is the premise and basis for screening safe and efficient anti-trioxypurine drug.Currently, The animal for 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, operation, raising are all more convenient, small with non-human species' difference.But then, the uric acid of mouse, rat Metabolic pathway varied considerably with the mankind, there are uricases, and the mankind do not have so that modeling have certain uncertainty and by Limitation.Chicken and quail, uric acid synthesis and metabolism are very much like with the mankind, in vivo without uricase, cannot aoxidize uric acid, but and people Class does not belong to, and common lab raising is also inconvenient.

In addition, different administration modes can also have a huge impact when establishing hyperuricemia model.It establishes at present There are mainly five types of in the administration mode of model：Feeding, stomach-filling, intraperitoneal injection, subcutaneous injection and gene knockout.Birds with feed to Before medicine, rat and mouse four kinds of modes can modeling, gene knockout research be only limitted to mouse.Different administration modes respectively has excellent lack Point：The unevenness of feeding animals food-intake can make internal serum uric acid level unstable, but the duration is longer；Stomach-filling can be to avoid blood Uric acid level is unstable；Drug administration by injection is easy to operate, but high lithemia state maintenance time is short, is unfit to do grinding for pathogenesis Study carefully；Often effect is preferable for several ways administering drug combinations, and the hyperuricemia model of construction has the characteristics that duration, stability； The model mice time-to-live that gene knockout is established is short, cost is high.

Ideal high lithemia model should have：(1) uric acid metabolism is consistent with the mankind, no uricase；(2) blood uric acid increase compared with Fastly, modeling is stablized；(3) high lithemia state can maintain time enough；(4) not damaged to other organs such as kidney.It is comprehensive next It sees, there is no a kind of efficient, stable, reliable hyperuricemia animal model at present.

Although 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 animals, there is a various neurotransmitters, and regulation Caenorhabditis elegans is to being not suitable for temperature, mechanical stimulus etc. The adaptation of poor environment.In addition, Caenorhabditis elegans shares 19717 genes, 6 chromosomes, including 5 pairs of autosomes With l to sex chromosome.Introne is less in gene, and genome is small, only 97Mb, is the smallest multi-celled eukaryotes of gene. Caenorhabditis elegans gene and human gene have high conservative, so that Caenorhabditis elegans becomes the ideal of research human diseases Model.

Currently, Caenorhabditis elegans is widely used for grinding in terms of infective pathogen bacterium model, detection toxicity and disease model Study carefully.Studies have shown that current Caenorhabditis elegans simulation 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.

Although the intracorporal uric acid metabolism approach (such as Fig. 1) of Caenorhabditis elegans and the uric acid metabolism approach of the mankind are slightly different (such as Fig. 2), but it is substantially similar.Caenorhabditis elegans is also the first base from small-molecule substance synthesizing ribonucleotide, is then carried out again The circulation of nucleotide, the guanine unlike the mankind cannot directly be metabolized as xanthine, and adenine cannot directly be metabolized as secondary Xanthine is converted into xanthine in turn, so the precursor substance of uric acid only has hypoxanthine and Huang fast in Caenorhabditis elegans body Purine, and in human body there are four types of the precursor substances of uric acid, respectively adenine, guanine, hypoxanthine and xanthine.But it is beautiful hidden All without uricase, the final product of purine metabolism is excreted in the form of uric acid by rhabditida and the mankind, this is utilization Caenorhabditis elegans establishes the high lithemia animal model similar with mankind's uric acid metabolism approach and provides possibility.

By retrieval, there is not yet establishing the relevant report of hyperuricemia model using Caenorhabditis elegans.

Summary of the invention

It is an object of the invention to provide a kind of Caenorhabditis elegans high lithemia model in place of overcome the deficiencies in the prior art Construction method and application and the construction method screening technique, which utilizes classical mode biology-beautiful hidden bar line Worm, and the model that this method obtains is with typical pathology characterization, stable, reliable, science, reasonable, similitude is high, repeated Good, highly reliable and controllable advantages such as easy, and every quantitative index has statistical significance, not only gout and high lithemia disease Extensive, high-flux medicaments sifting provide strong tool.

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

A kind of construction method of Caenorhabditis elegans high lithemia model, steps are as follows：

Using Caenorhabditis elegans, using precursor substance xanthine, final concentration of 0.25-0.45mg/mL, fluid administration, 18-32h is cultivated under the conditions of 20-25 DEG C, that is, obtains low damaging, efficient, stable, reliable Caenorhabditis elegans high lithemia mould Type.

Moreover, steps are as follows：

Using Caenorhabditis elegans, using precursor substance xanthine, final concentration of 0.25mg/mL, fluid administration, 20 DEG C of items 18h is cultivated under part, that is, obtains low damaging, efficient, stable, reliable Caenorhabditis elegans high lithemia model.

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

Caenorhabditis elegans high lithemia constructed by the construction method of Caenorhabditis elegans high lithemia model as described above Application of the model in anti-trioxypurine drug screening.

The screening technique of the construction method of Caenorhabditis elegans high lithemia model as described above, steps are as follows：

(1) the intracorporal uric acid content of high effective liquid chromatography for measuring Caenorhabditis elegans is utilized；

(2) solid form delivery is respectively adopted to the Caenorhabditis elegans of step (1) and fluid administration two ways is cultivated, led to The influence for comparing two kinds of administration modes to uric acid content in Caenorhabditis elegans body is crossed, determines optimal administration training method；

To step (2) determined by best administration mode give Caenorhabditis elegans hypoxanthine, uric acid, yellow fast respectively Four kinds of different concentration of drug of purine and adenine and different action time are measured beautiful hidden as unit of Caenorhabditis elegans The variation of uric acid content in rhabditida body determines optimal to medicinal substances, dosage；

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

Step (3) determined by most preferably give medicinal substances and dosage under the conditions of, measurement most preferably to medicinal substances in difference The variation of uric acid content, determines in influence and combination Caenorhabditis elegans body of the administration time to Caenorhabditis elegans average life span The best Best administration time for giving medicinal substances；

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

(5) Caenorhabditis elegans high lithemia model building method is established, to beautiful by construction method high lithemia obtained Hidden rhabditida model carries out serial experiment evaluation, and the serial experiment evaluation includes that the variation of Caenorhabditis elegans locomitivity is commented Valence, Caenorhabditis elegans egg laying amount Assessment of Changes, model stability evaluation, allopurinol drug evaluation, xanthine oxidase are living Property evaluation, determine the stability of high lithemia Caenorhabditis elegans model building method, reliability and to Caenorhabditis elegans It is damaging, it determines optimum condition, that is, obtains low damaging, efficient, stable, reliable Caenorhabditis elegans high lithemia model.

Moreover, steps are as follows：

(1) the intracorporal uric acid content of 400-1000 Caenorhabditis elegans of high effective liquid chromatography for measuring is utilized；

(2) solid form delivery is respectively adopted to the Caenorhabditis elegans of step (1) and fluid administration two ways is cultivated, led to The influence for comparing two kinds of administration modes to uric acid content in Caenorhabditis elegans body is crossed, optimal administration training method is determined, is Fluid administration；

To step (2) determined by best administration mode give Caenorhabditis elegans hypoxanthine, uric acid, yellow fast respectively Four kinds of different concentration of drug of purine and adenine and different action time, as unit of 400-1000 Caenorhabditis elegans, The variation for measuring uric acid content in Caenorhabditis elegans body, determines optimal to medicinal substances, dosage, is xanthine final concentration 0.25mg/mL；

Step (3) determined by most preferably give medicinal substances and dosage under the conditions of, measure xanthine in different dosing Between influence to Caenorhabditis elegans average life span and combine the variation of uric acid content in Caenorhabditis elegans body, determine xanthine Best administration time, be 18h；

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

(5) Caenorhabditis elegans high lithemia model building method is established, to beautiful by construction method high lithemia obtained Hidden rhabditida model carries out serial experiment evaluation, and the serial experiment evaluation includes that the variation of Caenorhabditis elegans locomitivity is commented Valence, Caenorhabditis elegans egg laying amount Assessment of Changes, model stability evaluation, allopurinol drug evaluation, xanthine oxidase are living Property evaluation, determine the stability of high lithemia Caenorhabditis elegans model building method, reliability and to Caenorhabditis elegans It is damaging, it determines optimum condition, that is, uses precursor substance xanthine, final concentration of 0.25-0.45mg/mL, fluid administration, 20- 25 DEG C, 18-32h obtain low damaging, efficient, stable, reliable Caenorhabditis elegans high lithemia model.

Moreover, (1) the step is handled before middle Caenorhabditis elegans measures through acetonitrile removing protein, and the additional amount of acetonitrile is： Acetonitrile 5.0mL is added in 500 Caenorhabditis elegans；

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

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

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

Detector：UV detector；

Column temperature：25℃；

Flow velocity：1mL/min；

Sample volume：30μL；

Specification Curve of Increasing：

4.0mg uric acid standard items are accurately weighed, 10mL is settled to the ammonium hydroxide ultrasonic dissolution that mass percent is 25%, obtains The uric acid standard mother liquor for being 400 μ g/mL to concentration is configured to the uric acid that concentration is 0,12.5,30,60,90,120,150 μ g/mL It is spare to cross film for standard solution；30 μ L of accurate sample introduction, according to peak area, the corresponding relationship directly proportional to standard solution sample introduction concentration is drawn Antidiuresis acidity scale directrix curve, acquires regression equation, related coefficient and its range of linearity.

Moreover, the step (2) in solid form delivery culture it is specific as follows：

Standard medium used in Caenorhabditis elegans is NGM culture medium, and configuration proportion and program are 3g NaCl, 17g fine jade Rouge, 2.5g peptone, 1L distilled water, after high pressure steam sterilization, the concentration that 1mL cholesterol is aseptically added 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 CaCl through high pressure steam sterilization₂The 1mol/L of solution and 25mL, the phosphoric acid through high pressure steam sterilization, pH=6.0 Potassium buffer, is sufficiently mixed, and pours into the culture dish that diameter is 9cm, cooling stand-by；

Wherein, the preparation process of the cholesterol ethanol solution is：Cholesterol using dehydrated alcohol carry out dissolution and Through 0.45 μm of membrane filtration degerming；

The larva SM liquid medium 1500-2000r/min hatched centrifugation 2-5min is collected children by nematode synchronization Worm goes on the NGM culture medium flat plate for being coated with Escherichia coli OP50, and plate is placed in 20 DEG C of incubators and continues to cultivate, culture It is stand-by after to 32h；

Drug hypoxanthine, uric acid, xanthine and the adenine of various concentration 0,0.05,0.15,0.25,0.35mg/mL, After culture medium is ready, 5mL drug is drawn, until in a sterilized 50mL centrifuge tube, then NGM culture medium is toppled over to volume 22.5mL, then plate is poured into, next step experiment is carried out after its solidification；Each concentration three parallel, this process of each drug It needs quickly, in case culture medium solidifies；5mL M9 buffer is added in blank plate；

In plate after culture medium solidification, the plate for selecting bubble-free, surface smooth draws 100 μ L large intestine bars with liquid-transfering gun Bacterium OP50 is smeared uniformly on culture medium, and bacterium solution is dried stand-by apart from ware wall 20mm when smearing；

It will cultivate after the nematode after 32h rinsed 2-3 time with M9 buffer, constant volume, then with liquid-transfering gun absorption 0.2- 2mL is added dropwise in the NGM plate center for being coated with OP50, plate is sealed with sealed membrane, then 20 DEG C are put into after being wrapped with preservative film It is cultivated for 24 hours in incubator；

Nematode is collected, is cleaned 2-3 times repeatedly with M9 buffer, guarantees that all nematodes are all collected, is settled to 1mL, is put into In refrigerator or carry out liquid phase pre-treatment；

Alternatively, the step (2) in fluid administration culture it is specific as follows：

After synchronization, the larva SM liquid medium 1500-2000r/min hatched centrifugation 2-5min is collected into larva, It goes on the NGM plate for being coated with Escherichia coli OP50, plate is placed in 20 DEG C of incubators and continues to cultivate, cultivate 32h；

Prepared SM liquid medium is aseptically distributed into 6 well culture plates, every hole 3.6mL is added Drug hypoxanthine, uric acid, xanthine and the adenine of prepared various concentration 0,0.05,0.15,0.25,0.35mg/mL, Every hole 1.2mL, each concentration three of each drug are parallel；

By micro- sem observation, selects the identical plate of growing way to be rinsed well with M9 buffer, goes in 15mL centrifuge tube, 1500-2000r/min is centrifuged 2-5min, draws supernatant, and shake up after constant volume and be distributed into 6 well culture plates, every hole is made to contain polypide 500 ± 5,20 DEG C of incubators are put in after sealing with sealing film and are cultivated respectively to 0,6,10,18,24,32h, liquid relief is then used Rifle sucks in 15mL centrifuge tube, then 1500-2000r/min centrifugation is settled to 1mL, is put into refrigerator and is saved or carried out liquid Phase pre-treatment.

Moreover, (4) specific step is as follows for the step：

Synchronization Caenorhabditis elegans is taken, solid culture 32h is swept away with M9, cleaned, and collects nematode；

Take six orifice plates, Liquid Culture Caenorhabditis elegans, then with 0.25mg/mL xanthine it is administered respectively 18h, for 24 hours, 32h, control group are M9 solution；Control group and the caenorhabditis elegan number of each experimental group are 500 ± 5；

The caenorhabditis elegan of control group and each experimental group be all respectively transferred to be coated with OP50 containing 5 FU 5 fluorouracil On NGM culture medium, the mortality and number of missing of daily accurate recording caenorhabditis elegan, until all tested beautiful hidden bar lines Worm is all dead, judges that the standard of nematode death is not replied outside stimulus, dead on wall if any climbing to, not counting at it It is interior；

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

Moreover, (5) specific step is as follows for middle Caenorhabditis elegans locomitivity Assessment of Changes for the step：

1. taking synchronization Caenorhabditis elegans；

2. nematode solid culture 32h, is swept away with M9, cleaned, nematode is collected；

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

4. picking Caenorhabditis elegans after 2min, is observed, on the NGM culture medium of no OP50 with nematode under the microscope Head pendulum in the past swing back again be it is primary, be recorded in the head oscillation number of Caenorhabditis elegans in 1min, in total picking 10 To observe；Sinusoidal motion with one wavelength of nematode be it is primary, be recorded in Caenorhabditis elegans in 20s body bending time Number, picking 10 is observed altogether；

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

Alternatively, (5) specific step is as follows for middle Caenorhabditis elegans egg laying amount Assessment of Changes for the step：

1. taking synchronization Caenorhabditis elegans；

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

4. 15 Caenorhabditis elegans of each picking are on 15 NGM culture mediums without OP50 respectively, daily beautiful hidden bar Nematode is transferred on the NGM culture medium of new no OP50, counts worm's ovum number under the microscope, until oviposition terminates；

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

Moreover, (5) specific step is as follows for middle model stability evaluation for the step：

The Caenorhabditis elegans of blank group and experimental group is subjected to solid culture at 20 DEG C, and guarantees sufficient food, Respectively culture 6,12,18,24, after 30h, rinse caenorhabditis elegan with M9, collect, carry out liquid phase pre-treatment, measure respectively beautiful hidden The content of uric acid in rhabditida body；

Wherein, blank group is unmodeled preceding caenorhabditis elegan, and experimental group is the beautiful hidden bar of culture different time after modeling Nematode；

At least in triplicate, experimental result carries out statistical disposition with SPSS software for parallel test；

Alternatively, (5) specific step is as follows for middle allopurinol evaluation for the step：

Give the Caenorhabditis elegans established before and after model to allopurinol final concentration 0.05,0.15,0.5mg/mL respectively, After cultivating 12h on NGM culture medium, Caenorhabditis elegans is collected, high effective liquid chromatography for measuring Caenorhabditis elegans is intracorporal Uric acid content；Wherein, blank group is unmodeled preceding Caenorhabditis elegans, and model group is the Caenorhabditis elegans after modeling, is put down At least in triplicate, experimental result carries out statistical disposition with SPSS software for row test.

Moreover, (5) specific step is as follows for middle xanthine oxidase activity evaluation for the step：

1. taking synchronization Caenorhabditis elegans；

2. L1 phase larva equivalent is transferred to after cultivating 32h in solid medium, keep the nematode population on each plate equal, And quantity is 500 ± 5；

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

By micro- sem observation, selects the identical plate of growing way to be rinsed well with M9 buffer, goes in 15mL centrifuge tube, 1500-2000r/min is centrifuged 2-5min, draws supernatant, and shake up after constant volume and be distributed into 6 well culture plates, every hole is made to contain polypide 500 ± 5,20 DEG C of incubator culture 18h modelings, nematode after must modeling are put in after sealing with sealing film；

4. nematode after modeling is sucked out from orifice plate, M9 is cleaned three times, under condition of ice bath, is sufficiently homogenized, homogenate is made, Refrigerated centrifuge 3000-5000r/min is centrifuged 5-15min, supernatant is taken to be measured；

5. building up the behaviour of the operating instruction in the xanthine oxidase kit specification of Bioengineering Research Institute according to Nanjing Make.

The advantages of present invention obtains and good effect are：

1. the method for the present invention, which is utilized, has high conservative, and uric acid metabolism approach and the much like mould of the mankind with human gene Formula biology-Caenorhabditis elegans establishes hyperuricemia animal model, the model have typical pathology characterization, stablize, can Lean on, science, rationally, similitude is high, reproducible, highly reliable and controllable advantages such as easy, and every quantitative index has and unites Meter learns meaning, and extensive, the high-flux medicaments sifting of only gout and high lithemia disease do not provide strong tool, Er Qiewei Hyperuricemia and the research of the pathomechanism of ventilation provide the experimental model of good medicine, are also other metabolic disease animal models Foundation provide beneficial reference, the hyperuricemia animal model of foundation has important practical significance.

2. this method not only increases show using Caenorhabditis elegans similar with mankind's uric acid metabolism as model organism Beautiful hidden rhabditida is in the value of field of medicaments, and modeling indifference is anisotropic, and the high lithemia disease model of foundation is the big of high lithemia disease Scale, high-flux medicaments sifting provide accurate tool, can be for the mankind's to find more safe and efficient therapeutic agents Health brings glad tidings.

3. this method finally determine using precursor substance xanthine can establish it is low damaging, efficient, stable, reliable and practical , and the Caenorhabditis elegans high lithemia model of mankind's high lithemia essence can be reacted.

4. the construction method of Caenorhabditis elegans high lithemia model of the present invention can be applied in anti-trioxypurine drug screening, together When, Caenorhabditis elegans high lithemia model constructed by the construction method of Caenorhabditis elegans high lithemia model of the present invention can also be with It applies in anti-trioxypurine drug screening.

Detailed description of the invention

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 that high performance liquid chromatography detects uric acid mark product in the present invention；

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

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

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

Fig. 7 is four kinds of modeling agent of hypoxanthine, uric acid, adenine, xanthine of various concentration in the present invention to beautiful hidden The influence diagram of uric acid content in rhabditida body；

(±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 hypoxanthine, uric acid, xanthine, four kinds of modeling agent difference modeling times of adenine in the present invention to beautiful The influence diagram of uric acid content in hidden rhabditida body；

(±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 influence diagram of the administration time to the survival rate of Caenorhabditis elegans of xanthine in the present invention；

(±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 the different administration time of xanthine in the present invention to the influence diagram of 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 is that uric acid content changes with time figure in Caenorhabditis elegans body before and after modeling in the present invention；

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

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

Figure 12 is influence of the allopurinol of various concentration in the present invention to 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 modeling front and back in the present invention to the influence diagram (± SD) of Caenorhabditis elegans head oscillation；

Figure 15 is modeling front and back in the present invention to the curved influence diagram of Caenorhabditis elegans body (± SD)；

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

Specific embodiment

Below with reference to embodiment, the present invention is further described；Following embodiments be it is illustrative, be not restrictive, It cannot be limited the scope of protection of the present invention with following embodiments.

Raw material used in the present invention is unless otherwise specified conventional commercial product；Used in the present invention Method is unless otherwise specified the conventional method of this field.

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

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

Associated materials used in 1 present invention can be as follows：

1.1 material：Caenorhabditis elegans.

1.2 solution are prepared

Uric acid：15.0mg uric acid is accurately weighed, the DMSO dissolution of micro (50-300 μ L) is added, takes 15mL distilled water, is surpassed Sound water-bath dissolution, then it is diluted to required concentration respectively.

Hypoxanthine：Hypoxanthine 15.0mg is accurately weighed, 10mL distilled water, ultrasonic 30min is added, then be diluted to respectively Required concentration.

Xanthine：15.0mg xanthine is accurately weighed, the ammonium hydroxide dissolution of micro (50-300 μ L) is added, then 15mL is taken to steam The dissolution of distilled water ultrasonic water bath, then it is diluted to required concentration respectively, adjust pH to 7.5 or so.

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

Uric acid standard reserving solution：Uric acid mark product 4.0mg is accurately weighed, taking 10mL mass percent is 25% ammonium hydroxide, molten Solution.

Allopurinol：20.0mg allopurinol is taken, adds (37-60 DEG C) of hydro-thermal water-bath dissolution, 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；Japanese biomicroscope CX41, OLYMPUS company； Japanese fluorescence inverted microscope, Nikon.

More preferably, steps are as follows：

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

The 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, steps are as follows：

The 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)；

Mobile phase：The ammonium acetate of 10mmol/L

Detector：UV detector

Column temperature：25℃；

Flow velocity：1mL/min；

Sample volume：30μL.

Specification Curve of Increasing

4.0mg uric acid standard items are accurately weighed, 10mL is settled to the ammonium hydroxide ultrasonic dissolution that mass percent is 25%, obtains The uric acid standard mother liquor for being 400 μ g/mL to concentration is configured to the uric acid that concentration is 0,12.5,30,60,90,120,150 μ g/mL It is spare to cross film for standard solution.30 μ L of accurate sample introduction, according to peak area, the corresponding relationship directly proportional to standard solution sample introduction concentration is drawn Antidiuresis acidity scale directrix curve, acquires regression equation, related coefficient and its range of linearity.

Sample treatment

Liquid-transfering gun draws M9 solution, and the nematode pre-processed on plate is all swept away, and 1500-2000r/min is centrifuged 2- 5min discards supernatant liquid.

It is cleaned 3 times repeatedly as above-mentioned, retains bottom precipitation nematode.

Nematode is broken by dismembyator grinding, by slurry hydraulic control system in 3-4mL, in 15mL centrifuge tube.

6mL ammonium hydroxide is added into slurry liquid and carries out ultrasonication 30min or more, 4mL acetonitrile, vortex oscillation is added 8min, stands 20min, and 3000-5000r/min is centrifuged 5-10min.

Precipitating is discarded, takes supernatant to carry out rotary evaporation, is redissolved after rotary evaporation with ammonium hydroxide, and ultrasound promotes uric acid molten Solution is finally settled to 2mL, -20 DEG C of preservations or direct injected measurement.

2. the solid culture of Caenorhabditis elegans

Solid culture Caenorhabditis elegans is generally used in laboratory, and the good several nematodes of picking upgrowth situation, which are put into, to be coated with greatly On the NGM culture medium of enterobacteria OP50,20 DEG C of cultures.When Selective agar medium plate, to select bubble-free, surface are smooth to put down Plate, if had the gap, nematode will crawl into culture medium, influence the accuracy of experiment.

The present invention improves general solid culture mode, cultivates and is administered while carrying out, specific step is as follows：

The larva SM liquid medium 1500r/min hatched centrifugation 2min is collected larva, goes to painting by nematode synchronization Have on the NGM plate of Escherichia coli OP50, plate is placed in 20 DEG C of incubators and continues to cultivate, it is stand-by after culture to 32h.

Aseptically, by the NGM culture medium to have sterilized, (standard medium used in Caenorhabditis elegans is in the present invention NGM culture medium, configuration proportion and program are 3g NaCl, 17g agar, 2.5g peptone, 1L distilled water, high pressure steam sterilization The cholesterol ethanol solution that the concentration of 1mL cholesterol is 5mg/mL is aseptically added afterwards), 1mL concentration is 1mol/ L and MgSO through high pressure steam sterilization₄Solution, 1mL concentration are 1mol/L and the CaCl through high pressure steam sterilization₂Solution and 25mL 1mol/L, the kaliumphosphate buffer through high pressure steam sterilization, pH=6.0, be sufficiently mixed, pour into diameter be 9cm culture dish, It is cooling stand-by；

Wherein, the preparation process of the cholesterol ethanol solution is：Cholesterol using dehydrated alcohol carry out dissolution and Through 0.45 μm of membrane filtration degerming.

Drug hypoxanthine, uric acid, xanthine and the adenine of various concentration 0,0.05,0.15,0.25,0.35mg/mL, After culture medium is ready, 5mL drug solution is drawn, until in a sterilized 50mL centrifuge tube, then NGM culture medium is toppled over to body Product 22.5mL or so, then plate is poured into, next step experiment is carried out after its solidification.Each concentration three of each drug are parallel, This process needs quickly, in case culture medium solidifies.5mL M9 buffer is added in blank plate.

In plate after culture medium solidification, 100 μ L Escherichia coli OP50 are drawn on culture medium with liquid-transfering gun, and smear uniform (bacterium solution is apart from ware wall 20mm when smearing) dries stand-by.

It will cultivate after the nematode after 32h rinsed 2-3 times with M9 buffer, be settled to 1mL, then drawn with liquid-transfering gun Proper volume (0.2-2mL) is added dropwise in the NGM plate center for being coated with OP50, and plate is sealed with sealed membrane, then with preservative film packet It is put into 20 DEG C of incubators and cultivates for 24 hours (to prevent microbiological contamination) after wrapping.

Nematode is collected, is cleaned 2-3 times repeatedly with M9 buffer, guarantees that all nematodes are all collected.It is settled to 1mL, is put into In refrigerator or carry out liquid phase pre-treatment.

3. fluid administration culture Caenorhabditis elegans

After synchronization, the larva SM liquid medium 1500-2000r/min hatched centrifugation 2-5min is collected into larva, It goes on the NGM plate for being coated with Escherichia coli OP50, plate is placed in 20 DEG C of incubators and continues to cultivate, cultivate 32h.

Prepared SM culture solution is aseptically distributed into 6 well culture plates, every hole 3.6mL adds preparation Good various concentration 0,0.05,0.15,0.25,0.35mg/mL drug hypoxanthine, uric acid, xanthine and the every hole of adenine 1.2mL, each concentration three of each drug are parallel.

By micro- sem observation, selects the identical plate of growing way to be rinsed well with M9 buffer, goes 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, and every hole is made to contain worm Body 500 ± 5, it is put in 20 DEG C of incubator cultures 0,6,10,18,24,32h after sealing with sealing film and then is sucked with liquid-transfering gun In 15mL centrifuge tube, centrifugation 1500-2000r/min be centrifuged 2-5min, be then settled to 1mL, be put into refrigerator carry out save or into Row liquid phase pre-treatment.

4. Caenorhabditis elegans life experiment

Synchronization Caenorhabditis elegans.

Solid culture passes through 32h, is swept away, is cleaned with M9, collects nematode.

Take six orifice plates, Liquid Culture Caenorhabditis elegans, then with 0.25mg/mL xanthine it is administered respectively 18h, for 24 hours, 32h, control group are M9 solution.Control group and the caenorhabditis elegan number of each experimental group are 500 ± 5.

The caenorhabditis elegan of control group and each experimental group be all respectively transferred to be coated with OP50 containing 5 FU 5 fluorouracil On NGM culture medium, the mortality and number of missing of caenorhabditis elegan under daily accurate recording, until all tested beautiful hidden bars Nematode is all dead.The standard for judging nematode death is not replied outside stimulus.It is dead on wall if any climbing to, not counting In it.

5. the motion conditions of Caenorhabditis elegans

(1) synchronization Caenorhabditis elegans.

(2) solid culture passes through 32h, is swept away, is cleaned with M9, collects nematode.

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

(4) picking Caenorhabditis elegans after 2min, is observed, on the NGM culture medium of no OP50 with nematode under the microscope Head pendulum in the past swing back again be it is primary, be recorded in the head oscillation number of Caenorhabditis elegans in 1min, in total picking 10 To observe；Sinusoidal motion with one wavelength of nematode be it is primary, be recorded in Caenorhabditis elegans in 20s body bending time Number, picking 10 is observed altogether.

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

6. the egg laying amount of Caenorhabditis elegans

(1) synchronization Caenorhabditis elegans.

(3) six orifice plates are taken, Liquid Culture nematode it is administered respectively 18h, control group M9 with 0.25mg/mL xanthine Solution.Control group and the caenorhabditis elegan number of each experimental group are 500 ± 5.

(4) each 15 Caenorhabditis elegans of picking are on 15 NGM culture mediums without OP50 respectively, daily beautiful hidden bar Nematode is transferred on the NGM culture medium of new no OP50, counts worm's ovum number under the microscope, until oviposition terminates.

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

7. model stability is evaluated

At least in triplicate, experimental result carries out statistical disposition with SPSS software for parallel test.

8. allopurinol is evaluated

Specific step is as follows：

Give the Caenorhabditis elegans established before and after model to allopurinol final concentration 0.05,0.15,0.5mg/mL respectively, After solid culture 12h, Caenorhabditis elegans, the intracorporal uric acid content of high effective liquid chromatography for measuring Caenorhabditis elegans are collected； Wherein, blank group is unmodeled preceding Caenorhabditis elegans, and model group is the Caenorhabditis elegans after modeling, and parallel test is at least In triplicate, experimental result carries out statistical disposition with SPSS software.

9. the measurement of xanthine oxidase activity (XOD)

The xanthine oxidase kit measurement of biotechnology research institute is built up in experiment using Nanjing, and principle is that xanthine can It is catalyzed hypoxanthine and generates xanthine, superoxide anion can act on color developing agent and generate aubergine, can be according to absorbance Variation calculates xanthine oxidase vigor.Concrete operation step is as follows：

(1) synchronization Caenorhabditis elegans

(2) L1 phase larva equivalent is transferred to after cultivating 32h in solid medium, makes the nematode population on each plate as far as possible It is equal, and quantity is suitable for (500 or so).

Six orifice plates are taken, 1.2mL is added in every hole, and 0.25mg/mL xanthine carries out Liquid Culture, by prepared SM liquid Culture solution is aseptically distributed into 6 well culture plates, every hole 3.6mL, and three parallel；

By micro- sem observation, selects the identical plate of growing way to be rinsed well with M9 buffer, goes in 15mL centrifuge tube, 1500-2000r/min is centrifuged 2-5min, draws supernatant, and shake up after constant volume and be distributed into 6 well culture plates, every hole is made to contain polypide 500 ± 5,20 DEG C of incubator culture 18h modelings, nematode after must modeling are put in after sealing with sealing film.

(3) nematode after modeling is sucked out from orifice plate, M9 is cleaned three times, under condition of ice bath, is sufficiently homogenized, homogenate is made Liquid.Refrigerated centrifuge 3000-5000r/min is centrifuged 5-15min, supernatant is taken to be measured.

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

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

Calculation formula：

In formula：U represents xanthine oxidase vigor (U/gprot), A₂It is measurement pipe OD value, A₁It is blank tube OD value, m is Colour generation object molar extinction coefficient：12.6*10^-3, V is the total volume (mL) of reaction solution, V₁For sampling amount (mL), d indicates optical path, t It indicates reaction time (min), c indicates homogenate proteins concentration (gprot/L).

Related test results of the invention：

1. the measurement 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 be uric acid standard curve, and acquire peak area (y) by standard curve is to the equation of linear regression of uric acid concentration (x)：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 carry out pre-treatment.Measuring uric acid level in Caenorhabditis elegans body is 5.58 μ g/100 items.

2. the determination of Caenorhabditis elegans administration mode

After Caenorhabditis elegans synchronization, the larva SM liquid medium 1500r/min hatched centrifugation 2min is collected Larva goes on the NGM plate for being coated with Escherichia coli OP50, and plate is placed in 20 DEG C of incubators and continues to cultivate, and cultivates 32h. Respectively by same drug supplying SM fluid nutrient medium, NGM solid medium, each concentration three of each drug is parallel, and point It Jie Ru not Escherichia coli.It will cultivate after the Caenorhabditis elegans after 32h rinses 2-3 time with M9 buffer, be settled to centainly It is added in culture medium (500 or so) after volume, is put into 20 DEG C of incubators and cultivates for 24 hours.

Fig. 6 is the comparison of different modes of administration, the blank of Caenorhabditis elegans solid form delivery system and fluid administration mode Control group is without significant difference (p>0.05).For Caenorhabditis elegans when Liquid Culture mode is administered, uric acid content is compared with blank group liter High 5.39 μ g/500 item, and when solid culture administration, uric acid content only increases 2.08 μ g/500 items compared with blank group, so, this reality It tests by the way of Liquid Culture and establishes Caenorhabditis elegans high lithemia model.

3. most suitable modeling drug, concentration and the determination of time

According to solid form delivery system research various concentration (0.05,0.15,0.25mg/mL) and be administered 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 Influence, as unit of 500 Caenorhabditis elegans.

Fig. 7 is four kinds of modeling agent of hypoxanthine, uric acid, adenine, xanthine of various concentration to Caenorhabditis elegans body The influence of interior uric acid content.Fig. 8 is hypoxanthine, uric acid, xanthine, four kinds of modeling agent difference modeling times of adenine to beautiful The influence of uric acid content in hidden rhabditida body.

By Fig. 7 and Fig. 8, compare four kinds to medicinal substances, three kinds of hypoxanthine, uric acid, xanthine drugs give beautiful hidden bar After nematode, intracorporal uric acid content has a degree of raising, and adenine drug is substantially unchanged, statistically without significant Difference (p>0.05).And group, administration 32h group are more empty for 24 hours for xanthine concentration 0.25mg/mL group and xanthine administration 18h group, administration White group increases the intracorporal uric acid content of Caenorhabditis elegans than other materials in the case where various concentration and administration different time It is all more, and have extremely significant difference (p with blank group<0.001).

Hypoxanthine, uric acid, xanthine are when concentration is 0.25mg/mL it can be seen from Fig. 7, Fig. 8, beautiful hidden bar line The intracorporal uric acid of worm increases obviously, increases 10.02 μ g, 11.42 μ g, 19.20 μ g respectively compared with blank group, statisticallys analyze, and secondary Huang is fast Purine and uric acid (0.25mg/mL) group are compared with blank group significant difference (p<0.05), xanthine group (0.25mg/mL) group is poor compared with blank group Heteropolar significant (p<0.001).

As can be seen from the results, give Caenorhabditis elegans hypoxanthine, uric acid, xanthine optium concentration be 0.25mg/ ML, still, 0.25mg/mL xanthine keeps uric acid raising in Caenorhabditis elegans body the most obvious in these three substances, after institute It is continuous to test 0.25mg/mL xanthine to establish Caenorhabditis elegans high lithemia model.

It may not be that action time is longer in view of the influence that drug itself grows Caenorhabditis elegans, uric acid liter It is high The more the better, it needs to select a suitable administration time, Caenorhabditis elegans can neither be caused compared with major injury, and can be with Caenorhabditis elegans is set to reach high lithemia state.Life experiment is the Classic Experiments for evaluating drug toxicity.

Fig. 9 is influence of the administration time to the survival rate of Caenorhabditis elegans of xanthine.Figure 10 is that xanthine is different Influence of the administration time to Caenorhabditis elegans average life span.

As shown in Figure 9, blank group MaLS 39d, administration 18h, for 24 hours, the MaLS of 32h reduces respectively compared with blank group 1d, 3d and 7d.As it can be seen that xanthine has a degree of injury to Caenorhabditis elegans.Blank group 21d, administration 18h group 15d, 13d, 32h group 10d are organized for 24 hours, and the survival rate of Caenorhabditis elegans is 50% or more.

As shown in Figure 10, the equal decrease to some degree of each group average life span, administration 18h group reduce 1.68d compared with blank group, Administration group average life span for 24 hours reduces 4.85d compared with blank group, and administration 32h group reduces 8.69d compared with blank group average life span.Statistical 18h is administered compared with blank group in analysis, and average life span is without significant difference (p>0.05) it, is administered to have with administration 32h for 24 hours and extremely show Write difference (p<0.01).So administration can cause compared with major injury Caenorhabditis elegans with 32h for 24 hours, though and 18h group is administered So there is certain injury to Caenorhabditis elegans, but is also not up to the level of signifiance.

The result shows that xanthine drug has a certain impact to Caenorhabditis elegans, with the extension of administration time, service life shadow Ring aggravation.But high lithemia state is had arrived in 18h, Caenorhabditis elegans body, there is extremely significant difference with blank group, to keep away Large effect may be caused to Caenorhabditis elegans by exempting from drug itself, and increase uric acid significantly, so the most suitable time is selected Select administration 18h.

4. model persistence is evaluated

Uric acid content changes with time in Caenorhabditis elegans body after research modeling, beautiful hidden bar line before and after measurement modeling Uric acid content changes over time situation in polypide, and result is as shown in figure 11.

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

Some models hold time it is especially short, such as be injected intraperitoneally Oteracil Potassium establish mouse high lithemia model, be only capable of tieing up 5h is held, fails to provide time enough to screen and treating hyperuricemia.The Caenorhabditis elegans high lithemia that this experiment is established Model is basicly stable, can maintain 12h or so, and provides possibility for drug screening and mechanism study.

5. influence of the allopurinol of various concentration to uric acid content in model Caenorhabditis elegans body

Caenorhabditis elegans after establishing model gives allopurinol final concentration 0.05,0.15,0.5mg/mL, solid respectively After cultivating 12h, Caenorhabditis elegans is collected, HPLC measures the intracorporal uric acid content of Caenorhabditis elegans.Blank group is unmodeled Preceding Caenorhabditis elegans, model group are the Caenorhabditis elegans after modeling, and at least in triplicate, experimental result is used for parallel test SPSS software carries out statistical disposition.

Its result is as shown in figure 12.Uric acid content is significantly increased compared with blank group in model group Caenorhabditis elegans body, about 52.4%, statistics has extremely significant meaning (p<0.001).After giving allopurinol 0.05,0.15mg/mL, Caenorhabditis elegans Internal uric acid content has extremely significant difference (p compared with blank group<0.001).After giving allopurinol 0.25mg/mL, beautiful hidden bar line Uric acid content is decreased significantly compared with model group in polypide, about 15.0%, statistics there were significant differences (p<0.01), allopurinol (0.25mg/mL) organizes Caenorhabditis elegans compared with blank group there were significant differences (p<0.05), illustrate with 0.25mg/mL allopurinol pair After Caenorhabditis elegans is treated, uric acid is decreased significantly in Caenorhabditis elegans body, but is also not up to normal level.

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

Control group and the Caenorhabditis elegans of each experimental group are all transferred to respectively and are coated with urinating containing 5- fluorine for OP50 On the NGM culture medium of pyrimidine, the mortality and number of missing of Caenorhabditis elegans under daily accurate recording, until all tested Caenorhabditis elegans it is all dead.The standard for judging Caenorhabditis elegans death is not replied outside stimulus.If any climbing It is dead on to wall, not counting in it.Parallel test in triplicate, studies modeling front and back Caenorhabditis elegans survival rate and is averaged The situation of change in service life, experimental result carry out statistical disposition with SPSS software.As a result as shown in table 1 and Figure 13.

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

As shown in Table 1, the average life span of model group Caenorhabditis elegans and blank group are without significant difference (p>0.05).As a result Show to establish modeling agent xanthine (0.25mg/mL, 18h) that Caenorhabditis elegans high lithemia model uses not to beautiful hidden Rhabditida causes significantly to injure.

As shown in Figure 13, the survival rate and blank group of model group Caenorhabditis elegans are without significant difference (p>0.05).As a result Show to establish modeling agent xanthine (0.25mg/mL, 18h) that Caenorhabditis elegans high lithemia model uses not to beautiful hidden Rhabditida causes significantly to injure.

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

Picking Caenorhabditis elegans after 2min, is observed, on the NGM culture medium of no OP50 with beautiful hidden under the microscope Rhabditida head pendulum in the past swing back again be it is primary, be recorded in the head oscillation number of Caenorhabditis elegans in 1min, choose in total 10 are taken to observe；Sinusoidal motion with one wavelength of Caenorhabditis elegans be it is primary, be recorded in beautiful hidden bar line in 20s The body number of bends of worm, picking 10 is observed altogether.In triplicate, experimental result is counted with SPSS software for parallel test Processing.

Influence, body curved influence such as table 2, Figure 14, Tu15Suo before and after modeling to Caenorhabditis elegans head oscillation Show.

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

As seen from Figure 14, the head oscillation frequency of model group and blank group are without significant difference (p>0.05), so, benefit The head oscillation of model Caenorhabditis elegans will not be significantly affected with modeling agent xanthine administration 18h.

As seen from Figure 15, the body corner frequency of model group group and blank group are without significant difference (p>0.05), so, The body bending of model Caenorhabditis elegans will not be significantly affected using modeling agent xanthine administration 18h.

The experimental results showed that can't be to Caenorhabditis elegans using modeling agent xanthine (0.25mg/mL, 18h) modeling It causes significantly to injure, to demonstrate the reliability of model.

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

Six orifice plates are taken, Liquid Culture Caenorhabditis elegans it is administered respectively 18h, control group with 0.25mg/mL xanthine For M9 solution.Control group and the Caenorhabditis elegans number of each experimental group are 500 ± 5.Difference each picking 15 beautiful hidden Rhabditida is daily transferred to Caenorhabditis elegans the NGM culture medium of new no OP50 on 15 NGM culture mediums without OP50 On, under the microscope count worm's ovum numbers, until oviposition terminate until, parallel test is repeated twice, experimental result with SPSS software into Row statistical disposition.

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

The variation table (± S.D) of Caenorhabditis elegans oviposition situation before and after 3 modeling of table

The result shows that the egg laying amount of Caenorhabditis elegans is without significantly changing (p before and after modeling>0.05).So utilizing modeling (0.25mg/mL, the 18h) modeling of agent xanthine will not significantly affect the oviposition situation of Caenorhabditis elegans.

The variation of xanthine oxidase activity in the nematode body of modeling front and back is studied, the results are shown in Table 4.

The variation table (± S.D) of xanthine oxidase activity in the nematode body of 4 modeling of table front and back

It is one that XOD vigor, which is defined as every gram of homogenate proteins enzyme amount required for 37 DEG C per minute substrate of 1 μm of ol of conversion, A enzyme activity unit.As shown in Table 4, model group xanthine oxidase vigor has extremely significant raising (p compared with blank group<0.001).Knot Fruit shows that the reason of can establish nematode high lithemia model with modeling agent xanthine is to keep the intracorporal xanthine oxidase of nematode living Property significantly improves.In purine metabolism, xanthine oxidase is the key enzyme for regulating and controlling speed and process.Xanthine oxidase Activity improves, and is conducive to xanthine and converts towards the direction of uric acid, increases uric acid content in nematode body.On the other hand, yellow Purine is also beneficial to hypoxanthine and is oxidized to xanthine towards the conversion of uric acid direction, and then reoxidizes as uric acid, makes internal purine The speed of metabolism is accelerated, to make uric acid content in nematode body that can significantly increase.

Low damage can be established using precursor substance xanthine (0.25mg/mL, 18h) it can be seen from testing result above Property, efficient, stable, reliable Caenorhabditis elegans high lithemia model.This research is not only hyperuricemia pathological study and new Medicine research and development increase new research tool, and the foundation for other metabolic disease animal models provides beneficial reference.

It can be seen that the construction method of Caenorhabditis elegans high lithemia model of the present invention can be applied in anti-trioxypurine drug sieve It chooses；Caenorhabditis elegans high lithemia model constructed by the construction method of the method for the present invention Caenorhabditis elegans high lithemia model It can also apply in anti-trioxypurine drug screening.

Claims

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

Using Caenorhabditis elegans, using precursor substance xanthine, final concentration of 0.25-0.45mg/mL, fluid administration, 20-25 18-32h is cultivated under the conditions of DEG C, i.e. acquisition Caenorhabditis elegans high lithemia model；

The screening technique of the construction method of the Caenorhabditis elegans high lithemia model, steps are as follows：

(2) solid form delivery is respectively adopted to the Caenorhabditis elegans of step (1) and fluid administration two ways is cultivated, pass through ratio Influence compared with two kinds of administration modes to uric acid content in Caenorhabditis elegans body determines optimal administration training method；

To step (2) determined by best administration mode give respectively Caenorhabditis elegans hypoxanthine, uric acid, xanthine and Four kinds of different concentration of drug of adenine and different action time measure beautiful hidden bar line as unit of Caenorhabditis elegans The variation of uric acid content in polypide determines optimal to medicinal substances, dosage；

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

Step (3) determined by most preferably give medicinal substances and dosage under the conditions of, measurement most preferably to medicinal substances in different dosing The variation of uric acid content, determines best in influence and combination Caenorhabditis elegans body of the time to Caenorhabditis elegans average life span To the Best administration time of medicinal substances；

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

(5) Caenorhabditis elegans high lithemia model building method is established, to by the beautiful hidden bar of construction method high lithemia obtained Nematode model carries out serial experiment evaluation, and the 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 Valence determines the stability, reliability and the damage to Caenorhabditis elegans of high lithemia Caenorhabditis elegans model building method Property, determine optimum condition, i.e. acquisition Caenorhabditis elegans high lithemia model；

(1) the step is handled before middle Caenorhabditis elegans measures through acetonitrile removing protein, and the additional amount of acetonitrile is：500 beautiful Acetonitrile 5.0mL is added in hidden rhabditida；

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

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

Detector：UV detector；

Column temperature：25℃；

Flow velocity：1mL/min；

Sample volume：30μL；

Specification Curve of Increasing：4.0mg uric acid standard items are accurately weighed, the ammonium hydroxide ultrasonic dissolution constant volume for being 25% with mass percent To 10mL, the uric acid standard mother liquor that concentration is 400 μ g/mL is obtained, being configured to concentration is 0,12.5,30,60,90,120,150 μ It is spare to cross film for the uric acid standard solution of g/mL；Accurately 30 μ L of sample introduction, it is directly proportional to standard solution sample introduction concentration according to peak area Corresponding relationship draws uric acid standard curve, acquires regression equation, related coefficient and its range of linearity；

Alternatively, the step (2) in solid form delivery culture it is specific as follows：

Standard medium used in Caenorhabditis elegans is NGM culture medium, configuration proportion and program be 3g NaCl, 17g agar, 2.5g peptone, 1L distilled water, after high pressure steam sterilization, the concentration that 1mL cholesterol is aseptically added 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 the CaCl through high pressure steam sterilization₂The 1mol/L of solution and 25mL, the potassium phosphate buffering through high pressure steam sterilization, pH=6.0 Liquid is sufficiently mixed, and pours into the culture dish that diameter is 9cm, cooling stand-by；

Wherein, the preparation process of the cholesterol ethanol solution is：Cholesterol is dissolved and is passed through using dehydrated alcohol 0.45 μm of membrane filtration degerming；

The larva SM liquid medium 1500-2000r/min hatched centrifugation 2-5min is collected larva, turned by nematode synchronization Onto the NGM culture medium flat plate for being coated with Escherichia coli OP50, plate is placed in 20 DEG C of incubators and continues to cultivate, culture to 32h It is stand-by afterwards；

Drug hypoxanthine, uric acid, xanthine and the adenine of various concentration 0,0.05,0.15,0.25,0.35mg/mL, culture After benchmark is standby, 5mL drug is drawn, until in a sterilized 50mL centrifuge tube, then NGM culture medium is toppled over to volume 22.5mL, then plate is poured into, next step experiment is carried out after its solidification；Each concentration three parallel, this process of each drug It needs quickly, in case culture medium solidifies；5mLM9 buffer is added in blank plate；

In plate after culture medium solidification, the plate for selecting bubble-free, surface smooth draws 100 μ L Escherichia coli with liquid-transfering gun OP50 is smeared uniformly on culture medium, and bacterium solution is dried stand-by apart from ware wall 20mm when smearing；

It will cultivate after the nematode after 32h rinsed 2-3 time with M9 buffer, constant volume, then with liquid-transfering gun absorption 0.2-2mL drop It is added in the NGM plate center for being coated with OP50, plate is sealed with sealed membrane, then be put into 20 DEG C of incubators after being wrapped with preservative film Middle culture is for 24 hours；

Nematode is collected, is cleaned 2-3 times repeatedly with M9 buffer, guarantees that all nematodes are all collected, is settled to 1mL, is put into refrigerator In or carry out liquid phase pre-treatment；

After synchronization, the larva SM liquid medium 1500-2000r/min hatched centrifugation 2-5min is collected into larva, is gone to It is coated on the NGM plate of Escherichia coli OP50, plate is placed in 20 DEG C of incubators and continues to cultivate, cultivate 32h；

Prepared SM liquid medium is aseptically distributed into 6 well culture plates, every hole 3.6mL adds preparation Good various concentration 0,0.05,0.15,0.25,0.35mg/mL drug hypoxanthine, uric acid, xanthine and adenine, every hole 1.2mL, each concentration three of each drug are parallel；

2. the construction method of Caenorhabditis elegans high lithemia model according to claim 1, it is characterised in that：Step is such as Under：

Using Caenorhabditis elegans, using precursor substance xanthine, final concentration of 0.25mg/mL, fluid administration, under the conditions of 20 DEG C 18h is cultivated, i.e. acquisition Caenorhabditis elegans high lithemia model.