CN1698906A - Duplication method of rat acute or persistent hyperuricemia model - Google Patents

Abstract

The invention discloses a duplication method of rat acute or persistent hyperuricemia model which comprises, (1) selecting male, female healthy rats with the specie of SD and Wistar and 150-450g of body weight, simultaneously administering purines by 100-2000 mg/kg and uric acid depressant by 5-1000mg/kg through stomach filling, hypodermic injection or abdominal cavity injection, (2) after 5-24 hours, administering the rats by the same mode and medicament dose, (3) administering the rats daily continually according to the step (1) and (2), continuing administration for one to two weeks.

Description

The clone method of rat acute or persistence hyperuricemia model

One, technical field

The present invention relates to the clone method of animal model, specifically relate to the clone method of rat acute or persistence hyperuricemia model.

Two, background technology

Gout be purine metabolic disturbance or (with) urate excretion reduces caused one group of different substantiality disease.Be principal character clinically with the hyperuricemia, hyperuricemia is the biochemical basis of gout, and gout is inevitable with hyperuricemia.In essence, there is no strict boundary between hyperuricemia and the gout.The pathogenesis and the Drug therapy thereof of Recent study gout become one of focus of medical circle, still lack the mature animal model of relevant gout at present both at home and abroad, and making up the hyperuricemia animal disease model then is the prerequisite of gout research.Also have a lot of incomplete places in the existing model, thereby brought great inconvenience for the research of gout.Below some representational rat hyperuricemia animal models are done and briefly introduced.

1.Wistar rat is male, body weight (200 ± 10) g, adenine 100mgkg ^-1+ ethambutol 250mgkg ^-1Gastric infusion, every day 1 time, continuous 7～21 days, the rat blood serum uric acid obviously raise.(Xiong Xiangming, Qu Qiuzhu. the rat hyperuricemia model is set up. Tianjin College of Traditional Chinese Medicine journal, 2001; 20 (4): 28～29)

2.SD rat or Wistar rat, male and female half and half, 110～150g is with adenine 300mgkg ^-1Irritate stomach, continuous 21 days, the rat serum uric acid obviously raise and chronic renal failure occurs.(Zhou Xiaozhou, Zhang Shengguang, Yang Xiao etc. the mechanism research of rat chronic renal failure due to the adenine. preclinical medicine and clinical, 1997; 17 (1): 54～57)

3.SD rat, body weight (200 ± 20) g evenly admixes yeast dry powder, adenine in the rat pellet of pulverizing, and content is respectively 10% and 01%, again pelletizing of raw material.Give rat high purine feedstuff, the food-intake of yeast, adenine is respectively 10gkg ^-1, 100mgkg ^-1The rat serum uric acid slightly raise in the 7th day, rat blood serum uric acid, creatinine, urea nitrogen content obviously raise in the 12 day, the 19 day, still continued behind the high purine feedstuff of stopping using to rise, and just began during second fortnight to fall after rise, in the time of the 52 day, compare with matched group and to still have significant difference.(Xi Jiuyi, Zhao Zhaolin, Lu Peiji etc. the experimental animal model research of hyperuricemic nephropathy. journal of shanghai Chinese medicine, 2001; (10): 10～12)

4.SD rat, 150～180g feeds rat with yeast, adenine, sardine and a small amount of commercially available powder feedstuff back that stirs, modeling after the 21st day rat serum uric acid, creatinine, blood urea nitrogen all raise.(He Liqun, Nie Yonghong, Zou Shilin. the foundation of novel urate nephropathy animal model. Shanghai laboratory animal science, 2001; 21 (1): 22～25)

5.SD rat or Wistar rat are male, body weight 250 ± 10g, adenine 2,4gkg ^-1Gastric infusion, the 3rd day rat serum uric acid level begins to raise, and obviously raises in the 7th day, reduces, and occurs renal function injury simultaneously in the tenth day.(Zhang Chao, Cao Keguang, Yang Chongqing etc. the foundation and the application of hyperuricemia and urate nephropathy animal model. laboratory animal science and management, 1999; 16 (4): 18～21)

6. the people Mus is raised the oxonicacid with 2%, and gives low salt diet, continues for 7 weeks, slight hyperuricemia can occur, and hypertension simultaneously, afferent glomerular arteriole thicken.(Mazzali?M，Kanellis?J，Han，etal.Hyperuricemia?induces?a?primary?renal?arteriolopathy?in?rats?by?a?bloodpressure?independent?mechanism.Am?J?Physiol?Renal?Physiol，2002；282(6)：F991～997)

7. give the Wistar rat oxonic acid 0.4gd that feeds ^-1With uric acid 0.6gd ^-1, after 3～4 weeks, rat serum uric acid persistence raises.(Bluestone?R，Waisman?J，Klinenberg?JR.Chronic?experimentalhyperuricemic?nephropathy.Lab?Invest，1975；33(3)：273～279)

Uric acid can not be utilized by human body or decompose in vivo, in the environment of normal person's body fluid pH7.4, be the uric acid na form, dissolubility 417 μ mol/L (7mg/dl) (the Yin Weihe River. hyperuricemia and gout [J]. the doctor studies magazine, 1998,21 (11): 573), just can be deposited in the tissue so uric acid concentration must be higher than this value.The clone method of the existing bibliographical information rat hyperuricemia medicines that give heavy dose of purine substance, suppress urate excretion that adopt more. thing or uricase inhibitor, promptly by increasing the incoming road of uric acid in the body, suppressing uric acid excretes, or inhibition uric acid decomposition in vivo, blood uric acid content is increased, cause the hyperuricemia model of rat.This kind model approaches the mechanism of production of human hyperuricemia and gout, provides feasible research method for studying gout and hyperuricemia and assessing medicine for treatment.But there are 2 deficiencies in above method: one, all need the long period just can finish replication of Model; Two, duplicated hyperuricemia model uric acid level is lower than uric acid more and produces crystalline saturated concentration.Above deficiency will be brought more difficulty for studying treatment hyperuricemia and gout class medicine: replicative cycle is long; The existing model of utilization only can be observed the therapeutical effect of medicine to slight metabolic arthritis; The metabolic arthritis level is lower than uric acid and produces sedimentary saturated concentration, and this will be unfavorable for gout replication of Model or the like.

Three, summary of the invention

1. goal of the invention

The objective of the invention is to overcome weak point of the prior art, provide a kind of and more approach formed rat acute of human purine metabolism obstacle or persistence hyperuricemia model, and serum uric acid level is higher than the method for saturated concentration.

2. technical scheme

2.1 animal: rat.Kind: SD, Wistar.Sex: female, male.Body weight: do not limit.

2.2 modeling medicine

Title: purine class: hypoxanthine, xanthine, uric acid; Uricase inhibitor: Oxonic acid, potassiumsalt (OA), allantoxanamide.

Route of administration: irritate stomach, subcutaneous injection, lumbar injection.

Dosage: purine class: 100～2000mgkg ^-1

Uricase inhibitor: 5～1000mgkg ^-1

Solvent: normal saline, liquid paraffin, CMC-Na, oil, soluble starch, glycerol, thickening agent.

Preparation requires: the preparation of suspensoid is must concentration even, will shake up during use.

Modeling blanking time: 5～24 hours.

2.3 the clone method of rat acute hyperuricemia model, its step is as follows:

(1) choose female, the robust health rat that kind is SD, Wistar, body weight 150～450g, fasting, non-fasting all can, under room temperature, give the purine class 100～2000mgkg simultaneously with the mode of irritating stomach, subcutaneous injection or lumbar injection to rat ^-1With uricase inhibitor 5～1000mgkg ^-1

(2) or through 5～12 hours, the above-mentioned same mode of reuse gave the purine class 100～2000mgkg once more ^-1And/or uricase inhibitor 5～1000mgkg ^-1But, ad lib, drinking-water after the modeling.

Above-mentioned purine class is hypoxanthine, xanthine, uric acid; Above-mentioned uricase inhibitor is 0A, allantoxanamide.

The optimum amount of above-mentioned uricase inhibitor is 100mgkg ^-1/ day/1 time or 200mgkg ^-1/ day/2 times, cooperate purine class 500mgkg ^-1/ day/1 time or 1000mgkg ^-1/ day/2 times.

2.4 the clone method of rat persistence hyperuricemia model, its step is as follows:

(1) choose female, the robust health rat that kind is SD, Wistar, body weight 150-450g, fasting, non-fasting all can, under room temperature, give the purine class 100～2000mgkg simultaneously with irritating stomach, subcutaneous injection or lumbar injection to rat ^-1And uricase inhibitor, 5～1000mgkg ^-1

(2),, give the purine class 100～2000mgkg once more simultaneously with above-mentioned same mode through 5～24 hours ^-1With uricase inhibitor 5～1000mgkg ^-1But, ad lib, drinking-water after the modeling;

(3) press continuously the method modeling of (1), (2) step, one thoughtful two weeks of successive administration every day.

Above-mentioned purine class is hypoxanthine, xanthine, uric acid; Above-mentioned uricase inhibitor is OA, allantoxanamide.

The optimum amount of above-mentioned uricase inhibitor is 100mgkg ^-1/ day/1 time or 200mgkg ^-1The optimum amount 500mgkg of/day 2 cooperation purine classes ^-1/ day/1 time or 1000mgkg ^-1/ sky 2 times.

3. beneficial effect

In this experiment, we use uricase inhibitor to cooperate the purine class, can obtain stable acute or persistence rat hyperuricemia model.The serum uric acid level of animal can improve significantly, and the metabolic arthritis level still is higher than uric acid and produces crystalline saturated concentration 417 μ molL behind the 12h ^-1, the acute hyperuricemia model replicative cycle is short, and matches with the mechanism of hyperuricemia.If every day, modeling twice, uric acid level is apparently higher than saturated concentration, and uric acid level can constantly accumulate rising, forms persistence rat hyperuricemia model, wherein is also shown in the gout rat model of some.The mechanism of this and hyperuricemia and gout is coincide, increase the interior purine class of animal body to increase the raw material that generates uric acid, use the decomposition of uricase inhibitor inhibition simultaneously to uric acid, reduce the outlet of uric acid with this, both are collaborative, thereby obviously improve serum uric acid level, and the metabolic arthritis level is held time and obviously prolonged, and uric acid continues to be in the over-saturation level in animal body, will deposit to the joint of animal chamber and form the gout model.As seen, purine class and uricase inhibitor are used, and are the desirable combination that copies the persistence hyperuricemia model.For the percentage rate that further improves the generation of gout model provides valuable reference.

Four, the specific embodiment

The clone method of embodiment 1. rat acute hyperuricemia models, its method is as follows:

Choose the Wistar rat of male and healthy, body weight 250g ± 10g at room temperature, pours into xanthine 500mgkg with irritating the stomach mode ^-1, inject OA 200mgkg with the lumbar injection mode ^-1

The clone method of embodiment 2. rat acute hyperuricemia models, its method is as follows:

1. choose the Wistar rat of male and healthy, body weight 250g ± 10g at room temperature, pours into xanthine 500mgkg with irritating the stomach mode ^-1, inject OA 100mgkg with the lumbar injection mode ^-1

2.5h after, lumbar injection OA 100mgkg once more ^-1

The clone method of embodiment 3. rat acute hyperuricemia models, its method step is as follows:

1. choose the Wistar rat of male and healthy, body weight 250g ± 10g at room temperature, pours into xanthine 500mgkg with irritating the stomach mode ^-1, inject OA 200mgkg with the lumbar injection mode ^-1

2.5h after, irritate stomach once more and give xanthine 500mgkg ^-1

The clone method of embodiment 4. rat acute hyperuricemia models, its method step is as follows:

1. choose the Wistar rat of male and healthy, body weight 250g ± 10g at room temperature, pours into xanthine 500mgkg with irritating the stomach mode ^-1, inject OA 100mgkg with the lumbar injection mode ^-1

2.5h after, with above-mentioned same mode, give the xanthine and the OA of same amount once more simultaneously.

The clone method of embodiment 5. rat acute hyperuricemia models, its method is as follows:

Choose the SD rat of female Sexual health, body weight 250g ± 10g at room temperature, with irritating the stomach mode, irritates to rat hypoxanthine 500mgkg ^-1, with the mode of lumbar injection, injection OA 50mgkg ^-1

The clone method of embodiment 6. rat acute hyperuricemia models, its method is as follows:

Choose the SD rat of female Sexual health, body weight 250g ± 10g at room temperature, with irritating the stomach mode, irritates to rat hypoxanthine 500mgkg ^-1, with the mode of lumbar injection, injection OA 10mgkg ^-1

The clone method of embodiment 7. rat acute hyperuricemia models, its method is as follows:

Choose the SD rat of female Sexual health, body weight 250g ± 10g at room temperature, with irritating the stomach mode, irritates to rat hypoxanthine 250mgkg ^-1, with the mode of lumbar injection, injection OA 50mgkg ^-1

The clone method of embodiment 8. rat acute hyperuricemia models, its method is as follows:

Choose the SD rat of female Sexual health, body weight 250g ± 10g at room temperature, with irritating the stomach mode, irritates to rat hypoxanthine 250mgkg ^-1, with the mode of lumbar injection, injection OA 10mgkg ^-1

The clone method of embodiment 9. rat acute hyperuricemia models, its method is as follows:

Choose the SD rat of male and healthy, body weight 250g ± 10g at room temperature, pours into hypoxanthine 500mgkg with irritating the stomach mode ^-1, use the lumbar injection mode, injection OA 50mgkg ^-1

The clone method of embodiment 10. rat acute hyperuricemia models, its method is as follows:

Choose the SD rat of male and healthy, body weight 250g ± 10g at room temperature, pours into hypoxanthine 500mgkg with irritating the stomach mode ^-1, use the subcutaneous injection mode, injection OA 50mgkg ^-1

The clone method of embodiment 11. rat acute hyperuricemia models, its method step is as follows:

1. choose the SD rat of male and healthy, body weight 300g ± 20g at room temperature, with irritating the stomach mode, irritates to rat hypoxanthine 500mgkg ^-1With OA 100mgkg ^-1

2. through 12h, repeat above step.

The clone method of embodiment 12. rat persistence hyperuricemia models, its method step is as follows:

1. choose the SD rat of male and healthy, body weight 300g ± 20g at room temperature, with irritating the stomach mode, irritates to rat hypoxanthine 500mgkg ^-1With OA 100mgkg ^-1

2. through 12h, repeat above step.

3. every day is according to step 1,2 continuous modelings, one week of successive administration.

Duplicating of embodiment 13. rat acutes or persistence hyperuricemia model

1. experiment material

1.1 animal: SD, Wistar rat, male, 180～320g.Experimental animal feeding credit number: SYXK (Soviet Union) 2002-0002 is provided by Nanjing University of Traditional Chinese Medicine's Experimental Animal Center.

1.2 medicine: Oxonic acid, potassium salt (OA), sigma-Aldrich company, Sigma company.With normal saline or oil preparation, add a little solvent during preparation earlier, after putting into mortar and grinding well, residual solvent is being added mixing.Xanthine (Xanthine, X) sigma-Aldrich company, Sigma company.Hypoxanthine (Hypoxanthine, HX), chemical plant, the northeast part of China, Tianjin.By soluble starch preparation, preparation 3% soluble starch boils earlier, adds earlier after the cooling to grind well to the mortar that fills purine on a small quantity, will remain the soluble starch adding again, mixing.The uric acid reagent box is Sysmex company product.

1.3 instrument: Hitachi's 7020 automatic clinical chemistry analyzers.

2. method and result

2.1X the influence of share the rat serum uric acid level with OA is divided into 5 groups at random with rat, normal control group (C), X1+OA1 group, X1+OA2 group, X2+OA1 group, X2+OA2 group, and 6 every group, single cage is raised.X1+OA1 group lumbar injection OA200mgkg ^-1, irritate stomach behind the 1h and give X500mgkg ^-1X1+OA2 group lumbar injection OA100mgkg ^-1, irritate stomach behind the 1h and give X500mgkg ^-1, lumbar injection OA100mgkg once more behind the 5h ^-1X2+OA1 group lumbar injection OA200mgkg ^-1, irritate stomach behind the 1h and give X500mgkg ^-1, irritate stomach behind the 5h once more and give X500mgkg ^-1X2+OA2 group lumbar injection OA100mgkg ^-1, irritate stomach behind the 1h and give X500mgkg ^-1, lumbar injection OA100mgkg once more behind the 5h ^-1, irritate stomach and give X500mgkg ^-1C group lumbar injection equal-volume normal saline and filling stomach give the equal-volume distilled water.Give every rat extracting blood respectively at 2h, 6h after irritating for the first time X, centrifuging and taking serum is surveyed the hematuria acid number.The results are shown in Table 1.

Table 1X and OA share influence (X ± S) (n=6) to the rat serum uric acid

Group	X dosage/mgkg -1	OA dosage/mgkg -1	Hematuria acid number/umolL -1
					??2h	?6h
			Matched group X1+OA1 X1+OA2 X2+OA1 X2+OA2	???- ???500 ???500 ? ???500*2 ???500*2			??- ??200 ??100*2 ? ??200 ??100*2	??199.0±39.6 ??460.3±162.5* ??622.0±268.9* ? ??521.8±177.6* ??643.3±255.4*	?222.5±63.2 ?185.8±63.76 ?593.0±245.0* ?189.3±32.75 ?889.3± ?134.0**

Compare * P＜0.05, * * P＜0.01 with matched group.

The result shows that after giving X2h for the first time, each is organized serum uric acid level and all is higher than normal control group (P＜0.05); After irritating X6h for the first time, X1+OA2 group serum uric acid level and normal control group have certain difference (P＜0.05), and X2+OA2 group serum uric acid level obviously raises, and with the normal control group significant difference (P＜0.01) is arranged.The above results shows: though once give X and OA the rat blood serum uric acid level that can raise, it is shorter to hold time, administration after six hours serum uric acid level near normal.The total amount of OA and X is taked once or at twice to give in one day, or reduces by half and give, and different changes has appearred in the rat blood serum uric acid level.According to the visible OA of table 1 100mgkg no matter ^-1Or 200mgkg ^-1To the equal no significant difference of rising of the rat blood serum uric acid level due to the X that gives same dose, OA one daily amount 200mgkg ^-1Though once give the rat blood serum uric acid level that to raise, only keep less than six hours.And with OA one daily amount 200mgkg ^-1Give at twice, once give X and then can make the rat blood serum uric acid level continue to raise, and maintain more than six hours, the dosage that OA is described is at 100mgkg ^-1The time can bring into play inhibitory action to uricase, as increasing the dosage of X on this basis simultaneously, then not only can make the rat blood serum uric acid level continue to raise, the rat blood serum uric acid level is raise reach 800umolL ^-1More than.

2.2 various dose HX and OA share the influence of rat serum uric acid level one diurnal variation is divided into six groups of A, B, C, D, E, F at random with rat.The B group is irritated stomach and is given HX 500mgkg ^-1C group lumbar injection OA50mgkg ^-1+ irritate stomach to give HX 500mgkg ^-1D group lumbar injection OA 10mgkg ^-1+ irritate stomach to give HX 500mgkg ^-1E group lumbar injection OA 50mgkg ^-1+ irritate stomach to give HX 250makg ^-1F group lumbar injection OA10mgkg ^-1+ irritate stomach to give HX 250mgkg ^-1A group lumbar injection equal-volume normal saline+filling stomach gives the equal-volume distilled water; During respectively at 1h, 3h, 5h, 7h, 9h, 24h, get blood for each group, centrifuging and taking serum is surveyed blood uric acid concentration.The results are shown in Table 2.

Table 2 HX and OA share the influence of rat serum uric acid (X ± S) (n=5)

Group	HX dosage/mgkg 1	OA dosage/mgkg 1	????1h	??3h	??5h	??7h	??9h	??24h
									??A ??B ? ??C ? ??D ? ??E ? ??F	????-- ????500 ? ????500 ? ????500 ? ????250 ? ????250	????-- ????-- ? ????50 ? ????10 ? ????50 ? ????10	????80.2±12.4 ????77.8±34.9 ? ????334.6±58.1** ? ????285.2±60.1** ? ????270.0±167.4* ? ????371.0±170.4**	??71.8±21.7 ??74.4±18.9 ? ??506.0±148.5** ? ??99.2±38.4 ? ??246.2±152.4* ? ??217.8±164.1	?104.0±66.2 ?103.0±30.3 ?446.4± ? ??277.5* ?90.2±35.5 ? ?128.4±98.5 ? ?123.4±63.6	??94.8±31.7 ??90.4±28.3 ? ??429.4±343.6 ? ??82.6±24.6 ? ??101.6±37.1 ? ??114.4±48.2	??99.2±41.0 ??84.8±29.6 ? ??261.8±221.6 ? ??82.8±46.4 ? ??131.8±62.2 ? ??57.0±15.7	??90.2±68.3 ??80.0±16.5 ? ??79.2±21.3 ? ??61.8±16.0 ??111.0± ? ????37.1 ??74.0±60.3

Compare * P＜0.05, * * P＜0.01 with matched group.

The result shows, OA 50mgkg ^-1With HX 500mgkg ^-1Dosage 1h after administration, the hematuria acid number promptly has significant rising, in 1h, 3h, 5h, 7h place and blank group significant difference (P＜0.01) is arranged promptly, at 9h place and blank group also variant (P＜0.05).OA 100mgkg ^-1With HX 500mgkg ^-1Dosage 1h after administration, hematuria acid number and blank group have significant difference (P＜0.05), at 3h place and blank group also variant (P＜0.05).OA 50mgkg ^-1With HX 250mgkg ^-1Dosage in 1h, 3h place and blank group significant difference (P＜0.01) is arranged promptly, at 24h place and blank group also variant (P＜0.05).OA 10mgkg ^-1With HX 250mgkg ^-1Dosage in 1h, 3h place and blank group significant difference (P＜0.01) is arranged promptly.

2.3 different way of administration is divided into matched group, lumbar injection group and subcutaneous injection group to the influence of rat serum uric acid at random with rat.To abdominal cavity group rats by intraperitoneal injection OA (normal saline preparation) 50mgkg ^-1+ irritate stomach to give HX (distilled water preparation) 500mgkg ^-1, give subcutaneous group of rat skin lower injection OA 50mgkg ^-1+ irritate stomach to give HX 500mgkg ^-11h, 3h, 5h, 7h, 9h, 11h, 24h give rat extracting blood after modeling, and be centrifugal, gets serum, surveys the hematuria acid number.The results are shown in Table 3.

Table 3 different way of administration is to influence (X ± S) (n=5) of rat serum uric acid

Group	Dosage/mgkg -1		Uric acid level/mgkg -1
										????0A	????HX	????1h	??3h	??5h	??7h	??9h	??11h	????24h
	Subcutaneous group of matched group abdominal cavity group	? ????-- ? ? ????50 ? ? ????50	? ????-- ? ? ????500 ? ? ????500	????141.3± ? ????????34.6 ????376.3± ? ?????????175.9 ????426.5± ? ?????????140.8*	??175.0± ? ?????26.6 ??560.0± ? ????308.3 ??703.0± ? ????115.7**	??138.3± ? ????11.1 ??576.0± ? ?????291.2 ??587.0± ? ????147.5**	??164.0± ? ????19.3 ??470.7± ? ????301.6 ??471.5± ? ????128.6*	??212.0± ? ????61.5 ??335.0± ? ????196.0 ??336.8± ? ????116.1	??152.3± ? ????18.2 ??273.7± ? ????122.9 ??264.5± ? ????144.1										????132.0± ? ????????22.3 ????185.3± ? ????????38.0 ????133.8± ? ????????26.2

The result shows, all variant (P＜0.01 of subcutaneous injection group rat 1h, 3h, 5h, 7h and matched group after modeling, P＜0.05), serum uric acid level after two groups of modelings, all be higher than the abdominal cavity group in the subcutaneous group of rat serum uric acid 9h, blood uric acid drops to uric acid and produces sedimentary saturated concentration place during 7h, and 7h bleeding from anus uric acid level returns to normally gradually.By comparing between group, we select subcutaneous injection OA and irritate stomach to give HX route of administration as modeling.

In above experimentation, we find the OA for preparing with normal saline and all do not dissolve with the HX of distilled water preparation, very fast meeting layering, the uniformity to drug solution has very big influence, therefore the dispersion of duplicated animal model uric acid level is bigger, still consider that following experiment makes suspensoid with medicine, improve the uniformity of medicine dissolution.

2.4 various dose, different volumes OA are divided into matched group, big concentration (C) small size (V) group, the big V group of middle C, the little V group of middle C, the big V group of little C to the influence of rat serum uric acid level at random with rat.OA (solvent is an edible oil) subcutaneous injection administration, HX (solvent is a soluble starch) gastric infusion, dosage and volume see Table 4.Behind the 12h, repeat above step.5h, 12h, 24h give rat extracting blood after the modeling first time, and be centrifugal, gets serum, surveys the hematuria acid number.The results are shown in Table 4.

Table 4 various dose, different volumes OA are to influence (X ± S) (n=6) of rat serum uric acid level

Group	Dosage/mgkg -1		Concentration/mgml -1		Volume/mlkg -1		Uric acid level/mgkg -1
										?HX	?OA	????HX	????OA	????HX	????OA	??5h	?12h	??24h
	The little V of C organized the big V group of little C during the big V of C organized in the little V group of the big C of matched group	?-- ?500*2 ?500*2 ?500*2 ?500*2	?-- ?100*2 ?100*2 ?50*2 ?50*2	????-- ????50 ????50 ????50 ????50	????-- ????80 ????40 ????40 ????20	????-- ????10 ????10 ????10 ????10	????-- ????1.25 ????2.5 ????1.25 ????2.5	??127.7±26.95 ??924.2±184.12** ??648.5±185.80** ??573.0±305.51* ??519.0±256.08*	?113.3±6.66 ?524.3±427.51 ?257.7±131.4 ?240.3±173.71 ?199.7±74.56										??125.7±16.17 ??928.2±747.89 ??496.2±210.61* ??525.2±515.57 ??350.8±274.69

Compare * P＜0.05, * * P＜0.01 with matched group.

The result shows that each model group 5h after the modeling first time all is higher than matched group (P＜0.01, P＜0.05).12h after the modeling first time, the little V group of only big C hematuria acid number is higher than uric acid and produces crystalline saturated concentration, and all the other are respectively organized the hematuria acid number and have been lower than the uric acid saturated concentration.24h after the modeling for the first time (i.e. 12h after the modeling for the second time), remove the big V group of little C, the animal blood uric acid of its excess-three group all exceeds the uric acid saturated concentration, but the big V group of C has certain difference (P＜0.05) with matched group only, the little V group of big C 24h uric acid level is higher than all the other each model group, but performance statistics meaning not, this is main relevant greatly with the data discrete degree, the factor that influences dispersion mainly comprises the uniformity of modeling medicine and the individual variation of animal etc., prompting will overcome above influence factor when model copy from now on, can be by enlarging the experiment sample number, the uniformity of strict control pharmaceutical suspension etc.Find that from obduction subcutaneous injection small size group rat back skin injury is little in addition, basic noresidue solvent to animal.And the residual big gauging of subcutaneous injection large volume group rat back can't be absorbed, so be unfavorable for the carrying out of long-term experiment.According to above experimental result, the consumption of final definite employing OA is 200mgkg ^-1/ day/2 times, 80mg/ml, 1.25ml/kg cooperates HX 1000mgkg ^-1/ day/2 times (OA adopts the oil preparation, and HX adopts the soluble starch preparation) duplicates the rat acute hyperuricemia model.

2.5 duplicating of rat persistence hyperuricemia model is divided into matched group, thin oil (solvent is an edible oil) model group, viscous crude (solvent is an edible oil, 10% glycerol, poloxamer 1%) model group at random with rat.Model group subcutaneous injection OA 1.25ml/kg (80mg/ml) irritates stomach and gives HX 10ml/kg (50mg/ml), and matched group gives equal-volume edible oil and soluble starch; Behind the 12h, repeat above step.Continuous three days.12h gives rat extracting blood after each modeling, and is centrifugal, gets serum, surveys the hematuria acid number.The results are shown in Table 5.After three days rat is put to death, observe the skin of back degree of injury.

Table 5 rat persistence hyperuricemia model duplicate result (X ± S) (n=6)

Group	Dosage/mgkg -1		Uric acid level/mgkg -1
									??HX	??OA	?12h	?24h	??36h	?48h	????60h	??72h
	Matched group thin oil model group viscous crude model group	??-- ? ??500*2 ? ??500*2	??-- ? ??100*2 ? ??100*2	?209.7± ??71.9 ?636.3± ??232.65* ?285.8± ??152.17	?229.7± ??79.15 ?906.0± ??445.37* ?454.2± ??259.38	??152.0± ????21.66 ??1149.5 ??±794.8 ??560.3± ????277.00*	?191.3± ??73.70 ?970.8± ??631.54 ?541.5± ??270.12	????187.0± ????????91.28 ????1107.3± ????????757.79 ????733.0± ????????553.04									??169.0± ????39.74 ??876.7± ????571.69 ??870.5± ????824.78

Compare * P＜0.05, * * P＜0.01 with matched group.

The result shows, can reach the saturated concentration of uric acid sodian deposition after the modeling of thin oil group immediately, modeling three days, and serum uric acid level is high, and first day hematuria acid number and matched group comparison, and certain difference (P＜0.05) is arranged.12h after the modeling for the first time in first day of viscous crude group, still concentration immediately can not reach capacity, concentration can reach capacity behind the 12h, 36h place hematuria acid number and matched group have certain difference (P＜0.05), but the metabolic arthritis level that the viscous crude group reaches animal all is lower than the thin oil group at each point, again from the state of two treated animals, after the execution, dissect skin of back and observe the damage of modeling animal, the viscous crude group is bigger to the back part of animal damage, and the substrate majority remains in the back and is not absorbed, and produces inflammation, the back part of animal adhesion, it is downright bad to suppurate.Still think and select the solvent of edible oil for use as OA, soluble starch is as the solvent of HX, adopting the consumption of OA is 200mgkg ^-1/ day/2 times, 80mg/ml, 1.25ml/kg cooperates HX 1000mgkg ^-1/ day/2 times, continuously modeling reproduciblely goes out stable rat persistence hyperuricemia model.

In sum, select suitable modeling medicine, solvent, make appropriate dosage, administration time, administering mode, copy and more approach the formed rat persistence of human purine metabolism obstacle hyperuricemia model, will make us deeply observe the influence that animal pattern kidney uric acid is discharged by the reagent thing, become possibility in the influence of articular cavity process, thereby provide necessary and animal model reliably for the research of the screening of anti-gout drugs and the mechanism of action to the effect link of purine metabolism and to urate deposition.

Claims (5)

1. the clone method of rat acute or persistence hyperuricemia model, its method step is as follows:

(1) choose female, the robust health rat that kind is SD, Wistar, body weight 150-450g, fasting or non-fasting, under room temperature, with a jar stomach, subcutaneous injection or lumbar injection mode give purine and uricase inhibitor simultaneously to rat;

(2) or through 5-12 hour again to rat with above-mentioned same mode and same drug dose modeling, but ad lib, drinking-water after the modeling.

2. the clone method of a rat persistence hyperuricemia model, its method step is as follows:

(1) choose female, the robust health rat that kind is SD, Wistar, body weight 150-450g, fasting or non-fasting, under room temperature, with a jar stomach, subcutaneous injection or lumbar injection mode give purine and uricase inhibitor simultaneously to rat;

(2) through 5-24 hour, with above-mentioned same mode and same drug dose once more to the rat modeling, but ad lib, drinking-water after the modeling;

(3) continue medication thoughtful two weeks by the method modeling of above-mentioned (1), (2) step every day.

3. clone method according to claim 1 and 2 is characterized in that the purine class described in the step (1) is hypoxanthine, xanthine, uric acid; Described uricase inhibitor is Potassium salt (OA), allantoxanamide.

4. clone method according to claim 1 and 2 is characterized in that the optimum amount of purine class in the step (1) is 100-2000mgkg ^-1With the consumption of uricase inhibitor be 5-1000mgkg ^-1

5. clone method according to claim 4, the optimum amount that it is characterized in that uricase inhibitor is 100mgkg ^-1/ day/1 time or 200mgkg ^-1/ sky 2 times, 800mg/ml, it is 500mgkg that 1.25mg/ml cooperates the optimum amount of purine class ^-1/ day/1 time or 1000mgkg ^-1/ sky 2 times.