CN103463135A - Method and reagent kit for establishing experimental model of diabetic foot ulcer rat with mixed infection of Gram positive bacteria and negative bacteria - Google Patents
Method and reagent kit for establishing experimental model of diabetic foot ulcer rat with mixed infection of Gram positive bacteria and negative bacteria Download PDFInfo
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- CN103463135A CN103463135A CN2013104232944A CN201310423294A CN103463135A CN 103463135 A CN103463135 A CN 103463135A CN 2013104232944 A CN2013104232944 A CN 2013104232944A CN 201310423294 A CN201310423294 A CN 201310423294A CN 103463135 A CN103463135 A CN 103463135A
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Abstract
The invention discloses a method and a reagent kit for establishing an experimental model of a diabetic foot ulcer rat with mixed infection of Gram positive bacteria and Gram negative bacteria. The method comprises the following steps: after the rat is fed by a high fat feed for eight weeks, injecting streptozotocin to generate a diabetic model, after the diabetic model is stable, generating a scald with the depth of II degrees in the skin of the diabetic rat, and three days later, injecting mixed suspension of the Gram positive bacteria and the Gram negative bacteria at the scald. In addition, the invention also discloses the reagent kit for establishing the experimental model of the diabetic foot ulcer rat with the mixed infection of the Gram positive bacteria and the Gram negative bacteria. The reagent kit comprises the mixed suspension of the Gram positive bacteria and the Gram negative bacteria. According to the diabetic ulcer experimental animal model disclosed by the invention, by detecting the changes of biochemical indicators after drug intervention, results show that the model has the characteristic of being capable of generating the diabetic foot ulcer with various bacteria colony infections, and the characteristics of long healing time of an ulcer wound surface, sensitivity 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 gram positive bacteria and negative bacterium mixed infection.
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 gram positive bacteria estimated and negative bacterium mixed infection.
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 gram positive bacteria and negative bacterium mixed infection.
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 the diabetic foot ulcer rat experiment model of setting up gram positive bacteria and negative bacterium mixed infection.
In one aspect of the invention, a kind of method of setting up the diabetic foot ulcer rat experiment model of gram positive bacteria and negative bacterium mixed infection 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 3 days, scald place injection gram positive bacteria and gram negative bacteria suspension.
As preferred technical scheme, described gram positive bacteria and gram negative bacteria bacterium suspension are: concentration is 1 * 10
8ufcml
-1staphylococcus aureus ATCC25923 and escherichia coli ATCC285922 equal proportion mixed liquor.
As preferred technical scheme, described injection gram positive bacteria and gram negative bacteria suspension are got the wound surface pus and are done pathogenic microorganism and susceptibility detection after 24 hours, for determining the wound surface bacteria planting.
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 gram positive bacteria and negative bacterium mixed infection, this test kit comprises gram positive bacteria and gram negative bacteria suspension.
As preferred technical scheme, described gram positive bacteria and gram negative bacteria suspension are that concentration is 1 * 10
8ufcml
-1staphylococcus aureus ATCC25923 and escherichia coli ATCC25922 equal proportion mixed liquor.
As preferred technical scheme, described test kit also comprises 1.5% streptozotocin, the citric acid-sodium citrate buffer of pH value 4.5, PBS liquid, syringe, sterile swab, sterile gloves, An Er iodine liquid (for abandoning the deactivation of bacterium liquid).
In addition, in another aspect of this invention, provide the application of mentioned reagent box in the diabetic foot ulcer rat experiment model of setting up gram positive bacteria and negative bacterium mixed infection.
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 that diabetic foot ulcer accompanies many floras to infect, and traumatic infection pus is obviously rotten, the ulcer wound surface healing time is longer, to characteristics such as drug reaction sensitivities.
The accompanying drawing explanation
Fig. 1 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, adopting subcutaneous injection Quality Control bacterium staphylococcus aureus-ATCC25923 (Staphylococcus aureus) and escherichia coli-ATCC25922(Escherichia coli, E.coli) the strain equivalent mixed liquor makes 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), Quality Control bacterial strain escherichia coli-ATCC25922(Escherichia coli, E.coli), 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: 48 rats are divided into normal ulcer group (N) (12) at random, and diabetic ulcer group (DU) (12) infects matched group (IU-C) (12), treatment of infection group (IU-T) (12).Normal ulcer group normal diet is fed; Diabetic ulcer group and infect the matched group fed with high 8 weeks, on an empty stomach after 24 hours, lumbar injection 1.5% streptozotocin 35mgkg
-1, tail venous blood sampling measuring blood after 3 days>and 16.7mmolL
-1, make diabetes model; Diabetic ulcer group and infection matched group blood glucose>16.7mmolL
-13% pentobarbital sodium 2mlKg after one week
-1scald apparatus scald under the dosage intraperitoneal injection of anesthesia (1000g, 90 ℃, 10S), after three days, diabetic ulcer group (DU), infect matched group (IU-C), and treatment of infection group (IU-T) is the PBS liquid of lumbar injection 0.1ml respectively, 10 of 0.1ml
8ufcml
-1the equivalent mixed liquor of staphylococcus aureus and escherichia coli.Respectively organize sterile swab after 48 hours and get pathogenic microorganism cultivation and susceptibility check, the fixedly standby inspection of bark fetching skin tissue 10% formaldehyde.
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 1, normal ulcer group (N), diabetic ulcer group (DU), infect matched group (IU-C), treatment of infection group (IU-T) rat skin disorganization reaches skin corium, hair follicle, and the appendages of skin such as sweat gland destroy, inflammatory cell infiltration, meet deep II degree burn wound.Stablize after 48 hours and enter the next stage experiment.
3. model infects preparatory phase
Method: select microorganism detection Quality Control strain, staphylococcus aureus-ATCC25923, escherichia coli-ATCC25922, set up bacteria suspension concentration: 1 * 10
8ufc/ml, equal proportion is mixed rear each wound surface subcutaneous injection dosage 0.1m.(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).
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: normal ulcer group (N), diabetic ulcer group (DU), infect matched group (IU-C) normal saline every day and change dressings, and treatment of infection group (IU-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 | 2.330±0.194 |
| The DU group | 6 | 4.392±0.563 |
| The IU-C group | 6 | 5.581±0.3219 |
| The IU-T group | 6 | 3.255±0.263 |
Table 1 explanation: diabetic ulcer group (DU), infect matched group (IU-C), treatment of infection group (IU-T) compares with normal ulcer group (N), and the P value all is less than 0.05, has significant difference.The IU-C group, the IU-T group is P=0.000<=0.05 relatively, has significant difference.
Table 1 result shows: the DU group, and the IU-C group, in IU-T group rat body, the NO level raises than the N group, illustrates that in body, oxidation resistance weakens, and there is the Oxidation-antioxidation system imbalance in body.Bfgf-ESSEX can reduce the content of serum NO level to the intervention of infected group model.
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 IU-C group | 6 | 45.327±3.340 |
| The IU-T group | 6 | 32.348±1.888 |
Table 2 explanation: diabetic ulcer group (DU), infect matched group (IU-C), treatment of infection group (IU-T) compares with normal ulcer group (N), and the P value all is less than 0.05, has significant difference.The IU-C group, the IU-T group is P=0.000<=0.05 relatively, has significant difference.
Table 2 result shows: the DU group, and the IU-C group, in IU-T group rat body, the NO level raises than the N group, illustrates that TNF-α plays an important role in the generation of diabetes and evolution.
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 IU-C group | 6 | 20.075±1.248 |
| The IU-T group | 6 | 15.677±1.451 |
Table 3 explanation: diabetic ulcer group (DU), infect matched group (IU-C), treatment of infection group (IU-T) compares with normal ulcer group (N), and the P value all is less than 0.05, has significant difference.The IU-C group, the IU-T group is P=0.000<=0.05 relatively, has significant difference.
Table 3 result shows: the U group, and the IU-C group, in IU-T group rat body, the IL1 level raises than the N group, illustrates that vivo immuning system is in the imbalance state.The IU-T group is lower than IU-C group IL1 level, illustrates after Bfgf-ESSEX is treated and can reduce inflammatory reaction.
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 IU-C group | 6 | 32.3±1.88 |
| The IU-T group | 6 | 26.5±1.35 |
Table 4 explanation: diabetic ulcer group (DU) and infection matched group (IU-C), relatively, the P value all is less than 0.05 to treatment of infection group (IU-T), and result has significant difference.
Table 4 result shows: infection matched group and treatment of infection group sugar ulcer are long than the healing time of diabetic ulcer group ulcer, more meet the feature of diabetic foot chronic ulcer.
Claims (9)
1. a method of setting up the diabetic foot ulcer rat experiment model of gram positive bacteria and negative bacterium mixed infection, it is characterized in that, 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 3 days, scald place injection gram positive bacteria and gram negative bacteria suspension.
2. by method claimed in claim 1, it is characterized in that, described gram positive bacteria and gram negative bacteria bacterium suspension are: concentration is 1 * 10
8ufcml
-1staphylococcus aureus ATCC25923 and escherichia coli ATCC25922 equal proportion mixed liquor.
3. by method claimed in claim 1, it is characterized in that, described injection gram positive bacteria and gram negative bacteria suspension are got the wound surface pus and are done pathogenic microorganism and susceptibility detection after 24 hours, for determining the wound surface bacteria planting.
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 gram positive bacteria and negative bacterium mixed infection, is characterized in that, this test kit comprises gram positive bacteria and gram negative bacteria suspension.
7. test kit as claimed in claim 6, is characterized in that, described gram positive bacteria and gram negative bacteria suspension are that concentration is 1 * 10
8ufcml
-1staphylococcus aureus ATCC25923 and escherichia coli ATCC25922 equal proportion mixed liquor.
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, PBS liquid, 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 the diabetic foot ulcer rat experiment model of setting up gram positive bacteria and negative bacterium mixed infection.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
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| CN101347624A (en) * | 2008-06-13 | 2009-01-21 | 上海师范大学 | Construction method of animal model with diabetes skin ulcer |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
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Application publication date: 20131225 |