CN103479680A - Method and kit for establishing experimental model of diabetic foot ulcer big mouse infected by staphylococcus aureus - Google Patents
Method and kit for establishing experimental model of diabetic foot ulcer big mouse infected by staphylococcus aureus Download PDFInfo
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- CN103479680A CN103479680A CN201310424824.7A CN201310424824A CN103479680A CN 103479680 A CN103479680 A CN 103479680A CN 201310424824 A CN201310424824 A CN 201310424824A CN 103479680 A CN103479680 A CN 103479680A
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Abstract
The invention discloses a method for establishing an experimental model of a diabetic foot ulcer big mouse infected by staphylococcus aureus. The method includes the following steps that after the big mouse is fed on high-fat diet for eight weeks, streptozotocin is injected, and a diabetic model is manufactured. After the diabetic model is stabilized, II-degree scalding is performed on the skin of the big mouse, and after three days, subcutaneous injection of suspension liquid with staphylococcus aureus as the bacterial strain is performed in each ulcer position. In addition, the invention further discloses a kit for establishing the experimental model of the diabetic foot ulcer big mouse infected by the staphylococcus aureus, wherein the kit comprises the staphylococcus aureus suspension liquid. The diabetic foot ulcer experimental animal model detects variation of biochemical indexes after pharmacological intervention, and a result shows that the model has the infection features of diabetic foot ulcer and concurrent gram-positive bacteria. The model has the advantages of being short in ulcer wound surface healing time, sensitive to drug reaction and the like.
Description
Technical field
The invention belongs to the experimental animal model field, be specifically related to a kind of method and test kit of setting up the diabetic foot ulcer rat experiment model of infection of staphylococcus aureus.
Background technology
Along with the onset diabetes rate of China sharply raises, diabetic ulcer patient's quantity also increases thereupon, set up the disease model of real simulation diabetic ulcer in the integral animal level, be the essential condition of inquiring into the medical researches such as diabetic ulcer pathogenesis and preclinical pharmacodynamics of San evaluation, there is very important scientific meaning and clinical meaning.
Animal model of diabetic foot ulcer mostly is bringing out property animal model at present.The literature research result shows, the single pathogenic factors of animal model analog of existing diabetic foot ulcer is (as the peripheral arterial pathological changes, neuropathy or infection), the animal model gap larger with the research purpose existence with the realistic simulation similarity degree simultaneously, and factor compound pathogenic more general clinically more than two kinds in fact.Therefore, multifactor compound animal model of diabetic foot ulcer more can be simulated the incidence of clinical practice.
Therefore, according to the pathogeneticing characteristic of clinical practice and pathological change, set up animal model that simulation degree is higher for the pathogenetic research of diabetic ulcer and relevant operation, the therapeutic evaluation of the interventions such as medicine has important practical significance.
Summary of the invention
It is high that one of the technical problem to be solved in the present invention is to provide a kind of repeatability, stable, and the disease simulation degree is high, is convenient to the method for building up of the diabetic foot ulcer rat laboratory animal model of the infection of staphylococcus aureus estimated.
Two of the technical problem to be solved in the present invention is to provide a kind of test kit for the diabetic foot ulcer rat experiment model of setting up infection of staphylococcus aureus.
Three of the technical problem to be solved in the present invention is to provide the application of a kind of mentioned reagent box in setting up the diabetic foot ulcer rat experiment model of infection of staphylococcus aureus.
In one aspect of the invention, a kind of method of setting up the diabetic foot ulcer rat experiment model of infection of staphylococcus aureus is provided, comprise the steps: that rat is after high lipid food is fed 8 weeks, the injection streptozotocin, make diabetes model, make deep II degree burn wound after stable on rat skin, after three days, each ulcer place subcutaneous injection bacterial strain is the staphylococcus aureus suspension.
As preferred technical scheme, described staphylococcus aureus suspension comprises staphylococcus aureus-ATCC25923 and diluent, and the concentration of this staphylococcus aureus suspension is 1 * 10
8ufcml
-1, this diluent is PBS liquid.
As preferred technical scheme, after described injection staphylococcus aureus suspension infects, 24 as a child went wound surface to do pathogenic microorganism and susceptibility detection, for determining the bacteria planting situation.
As preferred technical scheme, described injection streptozotocin is specially: 1.5% streptozotocin is dissolved in the citric acid-sodium citrate buffer of pH value 4.5 and injects at the rat left lower quadrant.
As preferred technical scheme, the component that described high lipid food comprises following percentage by weight: normal feedstuff 73%, Adeps Sus domestica 20%, milk powder 2%, cholesterol 1%, sucrose 4%; Described normal feedstuff is comprised of the component of following percentage by weight: Fructus Hordei Vulgaris 15%, Semen Tritici aestivi 31%, Semen Maydis 15%, wheat bran 15%, fish flour 6%, bean cake, oil and salt totally 18%.
In another aspect of this invention, provide a kind of test kit for the diabetic foot ulcer rat experiment model of setting up infection of staphylococcus aureus, this test kit comprises the staphylococcus aureus suspension.
As preferred technical scheme, described staphylococcus aureus suspension comprises staphylococcus aureus-ATCC25923 and diluent, and the concentration of this staphylococcus aureus suspension is 1 * 10
8ufcml
-1, this diluent is PBS liquid.
As preferred technical scheme, described test kit also comprises 1.5% streptozotocin, the citric acid-sodium citrate buffer of pH value 4.5, syringe, sterile swab, sterile gloves, An Er iodine liquid.
In another aspect of this invention, also provide the application of mentioned reagent box in setting up the diabetic foot ulcer rat experiment model of infection of staphylococcus aureus.
Experimental animal model of the present invention belongs to the compound diabetic foot ulcer rat model for preparing, the model preparation is divided into three phases, and its concrete preparation method and evaluation methodology is respectively arranged, and specifically is divided into the diabetes model preparatory phase, the Ulcer Models preparatory phase, MOI model preparatory phase.
Diabetic ulcer experimental animal model of the present invention is by the detection of the variation to biochemical indicator after pharmaceutical intervention, result shows, this model has the feature of 2 property diabetic foot chronic ulcers, the healing time of the more common ulcer of diabetic foot ulcer healing time of infection of staphylococcus aureus is longer, to characteristics such as drug reaction sensitivities.
The accompanying drawing explanation
Fig. 1 is diabetic foot ulcer rat animal model manufacture method flow chart in the embodiment of the present invention 1.
Fig. 2 is the dyeing of the pathological section HE after burned rats schematic diagram in the embodiment of the present invention 1.
The specific embodiment
Model of the present invention preparation and be verified following experiment and confirm:
embodiment 1
The rats with type 2 diabetes that uses high lipid food to coordinate streptozotocin to induce, use scald apparatus to make dark II degree scald and induce skin ulcer, adopt subcutaneous injection Quality Control bacterium staphylococcus aureus-ATCC25923 (Staphylococcus aureus) strain to make diabetic foot ulcer infected rats model.
1 materials and methods
1.1 animal: the SD male rat, the SPF level, body weight (180 ± 10) g, provided by Shanghai Univ. of Traditional Chinese Medicine's Experimental Animal Center.
1.2 main agents and medicine: streptozotocin (Streptozotocin, STZ), purchased from Alexis company, high lipid food (composition: normal feedstuff 73%, Adeps Sus domestica 20%, milk powder 2%, cholesterol 1%, sucrose 4%, normal feedstuff formula wherein: Fructus Hordei Vulgaris 15%, Semen Tritici aestivi 31%, Semen Maydis 15%, wheat bran 15%, fish flour 6%, bean cake, oil and salt totally 18%, this normal feedstuff is purchased from People's Liberation Army Navy Medical Research Institute), the preparation method of high lipid food is: at first, by Fructus Hordei Vulgaris 15%, Semen Tritici aestivi 31%, Semen Maydis 15%, wheat bran 15%, after fish flour 6% is uniformly mixed in proportion, add bean cake, oil, salt mixes rear extrusion forming and makes normal feedstuff, then, by normal feedstuff 73%, Adeps Sus domestica 20%, milk powder 2%, cholesterol 1%, sucrose 4% is uniformly mixed in proportion rear extrusion forming and makes high lipid food, finished product is the cylinder bulk, Quality Control strain golden color staphylococcus-ATCC25923 (Staphylococcus aureus), by SHUGUANG HOSPITAL pathogenic microorganism chamber, provided, primary source: purchased from US mode culture collection warehousing (American type culture collection), secondary source is: the international experimental work of Shanghai Disease Prevention and Control Centre station, Recombinant Bovine Basic Fibroblast Growth Factor for External Use (trade name: Bfgf-ESSEX, Recombinant Bovine Basic Fibroblast Growth Factor For External Use, rb-bFGF), Yisheng Biological Pharmaceutical Co., Ltd., Shuhai, the accurate word S10980077 of traditional Chinese medicines.An Er iodine liquid (abandoning the deactivation of bacterium liquid uses); The 1ml syringe, sterile swab, sterile gloves.
1.3 key instrument: scald apparatus SQL-5Q scald apparatus, the glad soft Information technology company limited in Shanghai.
1.4 method: 60 rats are divided into normal ulcer group (N) (12) at random, diabetes matched group (DM) (12), diabetic ulcer group (DU) (12), golden Portugal's bacterium matched group (SD-C) (12), golden Portugal's bacterium treatment group (SD-T) (12).Normal ulcer group normal diet is fed; Diabetes matched group and golden Portugal bacterium matched group, golden Portugal bacterium treatment group fed with high 8 weeks, on an empty stomach after 24 hours, lumbar injection 1.5%STZ35mgkg
-1, tail venous blood sampling measuring blood after 3 days>and 16.7mmolL
-1, make diabetes model; Model group blood glucose>16.7mmolL
-1after one week; Normal ulcer group (N), diabetic ulcer group (DU), golden Portugal's bacterium matched group (SD-C), golden Portugal's bacterium treatment group (SD-T) 3% pentobarbital sodium 2mlKg
-1scald apparatus scald under the dosage intraperitoneal injection of anesthesia (9.8N, 90 ℃, 10S), after three days, diabetic ulcer group (DU), golden Portugal's bacterium matched group (SD-C), gold Portugal's bacterium treatment group (SD-T) is the PBS liquid of ulcer spot injection 0.1ml respectively, the PBS liquid of 0.1ml, 10
8ufcml
-1staphylococcus aureus.Respectively organize sterile swab after 48 hours and get ulcer surface secretions and do that pathogenic microorganism is cultivated and the susceptibility check, bark fetching skin tissue 10% formaldehyde fixedly standby is examined.Flow chart is shown in Fig. 1.
As shown in Figure 1, preparation and the evaluation methodology of diabetic foot experimental animal model are as follows, specifically are divided into 3 experimental stages:
1. diabetes model preparatory phase
Method: after high lipid food is fed 8 weeks, 1.5% streptozotocin is dissolved in the dosage left lower quadrant injection of the citric acid-sodium citrate buffer of pH value 4.5 by 35mg/kg.
Result: inject blood glucose after 24 hours and be greater than 16.7mml/L, stablize and detect blood glucose after 3 days and still be greater than 16.7mml/L, the diabetes model success, after stablizing 2 weeks, enter the next stage experiment.
2. diabetic ulcer model preparatory phase
Method: use the SQL-5Q scald apparatus, 90 ℃ of temperature, time of contact 10S, pressure 9.8N, area 4cm
2, after anesthesia, in back part skin, scalded.
Result: observed and recorded scald place change of skin.Get junctional area skin after 24h after scalding and do pathological section, HE dyeing is observed and is reached degree of depth II scald, as shown in Figure 2, diabetes matched group (DM) skin histology, epidermis exists, visible a large amount of skin appendages, normal ulcer group (N), diabetes matched group (DM), diabetic ulcer group (DU), gold Portugal's bacterium matched group (SD-C), gold Portugal's bacterium treatment group (SD-T) skin histology destroys and reaches skin corium, hair follicle, and the appendages of skin such as sweat gland destroy, inflammatory cell infiltration, meet dark II degree and scald.Stablize after 48 hours and enter the next stage experiment.
3. MOI model (moist diabetic foot model) preparatory phase
Method: select microorganism detection Quality Control strain, staphylococcus aureus-ATCC25923, bacteria suspension concentration: 1 * 10
8ufc/ml.Operational approach: after 10 times of gradient dilutions of bacterial suspension, counting is used spectrophotometer, OD value 600, and analysing luminosity is 1, is 1*10
8ufc/ml.Each wound surface subcutaneous injection dosage 0.1ml.
Result: inject and get pus cultivation detection in latter 48 hours: use Phoenix-100-BD, do Bacteria Identification and susceptibility.Open wound after 24 hours, carry out the pharmaceutical intervention evaluation.
embodiment 2western medicine-Recombinant Bovine Basic Fibroblast Growth Factor for External Use (trade name: Bfgf-ESSEX) intervene result of the test
1. method: diabetes matched group (DM) non-processor; Normal ulcer group (N), diabetic ulcer group (DU), golden Portugal's bacterium matched group (SD-C), every day, normal saline was changed dressings; Gold Portugal's bacterium treatment group (SD-T), Bfgf-ESSEX 800iu wound is changed dressings every day.The measurement rear 0d that changes dressings, 3d, 10d, wound surface area after healing, after healing, 3% pentobarbital sodium is pressed 2mlKg
-1the dosage intraperitoneal injection of anesthesia, ventral aorta blood sampling, centrifugalize serum, the standby inspection of bark fetching skin tissue.
2. date processing: all adopt SPSS16.0 to carry out statistical analysis, measurement data is used
mean, meet the data one factor analysis of variance of normality and homogeneity of variance, assembly adopts paired t-test, does not meet the data non parametric tests of normality or homogeneity of variance, with P<0.05, statistical significance is arranged.
Table 1 respectively organize after serum NO level treatment relatively (
μ mol/l)
| Group | n | After medication |
| The N group | 6 | 18.921±2.189 |
| The DM group | 6 | 25.045±2.9105 |
| The DU group | 6 | 22.303±1.618 |
| The SD-C group | 6 | 22.374±2.982 |
| The SD-T group | 6 | 23.177±1.191 |
Table 1 explanation: diabetic ulcer group (DU), golden Portugal's bacterium mould matched group (SD-C), golden Portugal's bacterium treatment group (SD-T) compares with normal ulcer group (N), and the P value all is less than 0.05, and result has significant difference.The SD-C group, the SD-T group is P=0.561>=0.05 relatively, no difference of science of statistics.
Table 1 result shows: the DM group, and the DU group, the SD-C group, in SD-T group rat body, the horizontal compared with normal group of NO raises, and illustrates that in body, oxidation resistance weakens, and there is the Oxidation-antioxidation system imbalance in body.
Table 2 respectively organize after Serum Level of Tumor Necrosis Factor-α (TNF-α) treatment relatively (
μ mol/l)
| Group | n | After medication |
| The N group | 6 | 27.273±2.609 |
| The DM group | 6 | 38.191±3.128 |
| The DU group | 6 | 45.327±3.340 |
| The SD-C group | 6 | 32.348±1.888 |
| The SD-T group | 6 | 41.82±4.700 |
Table 2 explanation: diabetic ulcer group (DU), golden Portugal's bacterium matched group (SD-C), golden Portugal's bacterium treatment group (SD-T) compares with normal ulcer group (N), and the P value all is less than 0.05, and result has significant difference.The SD-C group, the SD-T group is P=0.003<=0.05 relatively, has significant difference.
Table 2 result shows: the DM group, and the DU group, the SD-C group, in SD-T group rat body, TNF-alpha levels compared with normal group raises, and illustrates that TNF-α plays an important role in the generation of diabetes and evolution; Bfgf-ESSEX can make the TNF-α level raise to the bacterium model intervention of golden Portugal.
Table 3 respectively organize after serum IL 1 treatment relatively (
ng/l)
| Group | n | After medication |
| The N group | 6 | 9.094±1.010 |
| The DM group | 6 | 16.263±11.575 |
| The DU group | 6 | 18.281±0.942 |
| The SD-C group | 6 | 11.575±0.685 |
| The SD-T group | 6 | 15.074±1.3520 |
Table 3 explanation: diabetic ulcer group (DU), golden Portugal's bacterium matched group (SD-C), golden Portugal's bacterium treatment group (SD-T) compares with normal ulcer group (N), and the P value all is less than 0.05, and result has significant difference.The SD-C group, the SD-T group is P=0.000<=0.05 relatively, has significant difference.
Table 3 result shows: the DM group, and the DU group, the SD-C group, in SD-T group rat body, the IL1 level raises than the N group, illustrates that vivo immuning system is in the imbalance state.The SD-T group is higher than SD-C group IL1 level, illustrates that the rear immunoreation of Bfgf-ESSEX treatment is comparatively active.
Table 4 respectively organize the diabetic foot the average healing relatively (
my god)
| Group | n | Time |
| The DU group | 6 | 15.41±1.25 |
| The SD-C group | 6 | 33.3±1.68 |
| The SD-T group | 6 | 26.7±2.45 |
Table 4 explanation: diabetic ulcer group (DU) and golden Portugal's bacterium matched group (SD-C), relatively, the P value all is less than 0.05 to golden Portugal's bacterium treatment group (SD-T), and result has significant difference.
Table 4 result shows: the healing time of the more common ulcer of diabetic foot ulcer of infection of staphylococcus aureus is long, has the feature of diabetic foot chronic ulcer.
Claims (9)
1. a method of setting up the diabetic foot ulcer rat experiment model of infection of staphylococcus aureus, it is characterized in that comprising the steps: that rat is after high lipid food is fed 8 weeks, the injection streptozotocin, make diabetes model, make deep II degree burn wound after stable on rat skin, after three days, each ulcer place subcutaneous injection bacterial strain is the staphylococcus aureus suspension.
2. by method claimed in claim 1, it is characterized in that, described staphylococcus aureus suspension comprises staphylococcus aureus-ATCC25923 and diluent, and the concentration of this staphylococcus aureus suspension is 1 * 10
8ufcml
-1, this diluent is PBS liquid.
3. by method claimed in claim 1, it is characterized in that, after described injection staphylococcus aureus suspension infects, 24 as a child went wound surface to do pathogenic microorganism and susceptibility detection, for determining the bacteria planting situation.
4. by method claimed in claim 1, it is characterized in that, described injection streptozotocin is specially: 1.5% streptozotocin is dissolved in the citric acid-sodium citrate buffer of pH value 4.5 and injects at the rat left lower quadrant.
5. by method claimed in claim 1, it is characterized in that the component that described high lipid food comprises following percentage by weight: normal feedstuff 73%, Adeps Sus domestica 20%, milk powder 2%, cholesterol 1%, sucrose 4%; Described normal feedstuff is comprised of the component of following percentage by weight: Fructus Hordei Vulgaris 15%, Semen Tritici aestivi 31%, Semen Maydis 15%, wheat bran 15%, fish flour 6%, bean cake, oil and salt totally 18%.
6. the test kit for the diabetic foot ulcer rat experiment model of setting up infection of staphylococcus aureus, is characterized in that, this test kit comprises the staphylococcus aureus suspension.
7. test kit as claimed in claim 6, is characterized in that, described staphylococcus aureus suspension comprises staphylococcus aureus-ATCC25923 and diluent, and the concentration of this staphylococcus aureus suspension is 1 * 10
8ufcml
-1, this diluent is PBS liquid.
8. test kit as described as claim 6 or 7, is characterized in that, described test kit also comprises 1.5% streptozotocin, the citric acid-sodium citrate buffer of pH value 4.5, syringe, sterile swab, sterile gloves, An Er iodine liquid.
9. as the application of the described test kit of claim 6-8 any one in setting up the diabetic foot ulcer rat experiment model of infection of staphylococcus aureus.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108125987A (en) * | 2018-01-25 | 2018-06-08 | 楚雄医药高等专科学校 | A kind of method for building staphylococcus aureus subcutaneous infection model |
| CN110150229A (en) * | 2019-06-20 | 2019-08-23 | 上海市中西医结合医院 | A kind of production method of diabetes tendon degeneration necrosis disease model rodent |
| CN111616106A (en) * | 2020-06-23 | 2020-09-04 | 成都欧林生物科技股份有限公司 | Method for establishing fracture-related staphylococcus aureus infected animal model |
| CN113575516A (en) * | 2021-08-27 | 2021-11-02 | 河北生命原点生物科技有限公司 | Method for establishing type II diabetes rat model |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108125987A (en) * | 2018-01-25 | 2018-06-08 | 楚雄医药高等专科学校 | A kind of method for building staphylococcus aureus subcutaneous infection model |
| CN110150229A (en) * | 2019-06-20 | 2019-08-23 | 上海市中西医结合医院 | A kind of production method of diabetes tendon degeneration necrosis disease model rodent |
| CN111616106A (en) * | 2020-06-23 | 2020-09-04 | 成都欧林生物科技股份有限公司 | Method for establishing fracture-related staphylococcus aureus infected animal model |
| CN113575516A (en) * | 2021-08-27 | 2021-11-02 | 河北生命原点生物科技有限公司 | Method for establishing type II diabetes rat model |
| CN113575516B (en) * | 2021-08-27 | 2023-03-07 | 河北生命原点生物科技有限公司 | Method for establishing type II diabetes rat model |
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Application publication date: 20140101 |