CN114532288A - Method for constructing diabetic pig chronic skin ulcer model - Google Patents
Method for constructing diabetic pig chronic skin ulcer model Download PDFInfo
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- CN114532288A CN114532288A CN202111396014.6A CN202111396014A CN114532288A CN 114532288 A CN114532288 A CN 114532288A CN 202111396014 A CN202111396014 A CN 202111396014A CN 114532288 A CN114532288 A CN 114532288A
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Images
Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/02—Breeding vertebrates
Abstract
The invention discloses a method for constructing a chronic skin ulcer model of a diabetic pig, and relates to the technical field of skin ulcer. The method comprises the following specific steps: constructing a diabetes model of the Bama miniature pig, and constructing the diabetes model in the Bama miniature pig; 3D printing a foreign matter filling design, creating a wound on the pig skin and filling foreign matters into the wound; constructing a diabetic chronic skin ulcer model, and constructing a chronic skin ulcer model in a pig wound. The invention is suitable for large animal models with thicker skin tissues, particularly pigs; and the model formation is evaluated from multiple angles, and the formation of the diabetic chronic skin ulcer is better simulated from pathogenic factors, pathological changes and mechanism changes.
Description
Technical Field
The invention belongs to the technical field of skin ulcer, and particularly relates to a method for constructing a chronic skin ulcer model of a diabetic pig.
Background
The pathogenesis of the chronic skin ulcer of the diabetes is complex, and the problems of difficult animal model construction and poor clinical treatment effect exist;
the existing construction mode of the chronic skin ulcer model of the diabetic pig mainly comprises the following steps: local radiotherapy, local pressure application, ischemic flap creation or artery ligation and other methods; wherein the chronic wound caused by radiotherapy does not conform to the pathogenic mechanism of the chronic wound related to diabetes and complications thereof; the ischemic skin flap is large, so that the ischemic skin flap is not suitable for being created at multiple places on the back of a single pig, the number of required experimental animals is large, and the experimental cost is high; the wound is created after artery ligation, is suitable for wound models at the far ends of four limbs, and has the problems of limited number of wounds created by a single animal, higher requirement on surgical skills for experimenters, high technical difficulty and high experimental cost.
Disclosure of Invention
The invention aims to develop an economic, practical and easy-to-produce pig chronic skin ulcer construction model suitable for thick skin; meanwhile, a plurality of angle evaluation standards of the formation time, pathological changes and mechanism changes of the chronic wound surface are provided, and the modeling benefit is tested.
In order to solve the technical problems, the invention is realized by the following technical scheme: a method for constructing a chronic skin ulcer model of a diabetic pig comprises the following specific steps:
constructing a diabetes model of the Bama miniature pig, and constructing the diabetes model in the Bama miniature pig;
3D printing a foreign matter filling design, creating a wound on the pig skin and filling foreign matters into the wound;
constructing a diabetic chronic skin ulcer model, and constructing a chronic skin ulcer model in a pig wound.
Further, the construction of the diabetes model of the Bama miniature pig comprises the following specific steps:
grouping, after adaptive feeding of male non-castrated Bama minipigs for 5 weeks, randomly dividing into two groups according to a non-equilibrium design in a complete random design, 6 experimental groups (D1-D6) and 2 control groups (C1, C2);
establishing a diabetes model of the Bama miniature pigs, feeding two groups by different schemes, and carrying out an experiment group: high-fat high-sugar feed feeding, control group: feeding with a control feed;
feeding conditions, namely controlling the temperature to be 18-22 ℃, feeding in cages and freely drinking water, wherein the illumination and darkness are 12 hours/day respectively;
STZ injection, in vivo STZ injection into experimental groups;
monitoring blood sugar and insulin, and detecting blood sugar and insulin data in the experimental group.
Further, the specific steps of the 3D printed foreign matter stuffing design are as follows:
the design of filling foreign matters, namely, cylinders with different lengths are designed according to the depths of wound surfaces of different parts, and the cylinders have the contact and compression effects on local foreign matters after the wound surfaces are filled;
the specific design scheme is that the 3D printing foreign matter consumable material design software is as follows: solid works, the consumable production is carried out by adopting an Allect 334 machine through an FDM hot melt stacking production process;
performing wound creation, namely, after general anesthesia, opening 3cm beside the two sides of the spine of the miniature pig, and cutting 5-6 skin full-layer wound surfaces with the diameter of 3cm by using a scalpel, wherein the wound surfaces are spaced by 3-4 cm;
chronic wound definition and evaluation indexes thereof;
HE dyeing;
immunofluorescence detecting macrophage markers;
and (3) detecting the dynamic change of the mRNA of the inflammatory factor by real-time fluorescence quantitative PCR.
Further, the creating method comprises the following specific steps:
preoperative preparation, namely weighing and preparing skin of a miniature pig and marking a part to be wounded one day before an operation;
preparing instruments and consumables, sterilizing a scalpel, scissors (big and small), tweezers (with teeth and without teeth), gauze, normal saline, an aseptic operating coat, an operating sheet, a right-angle crochet hook, No. 18 and No. 10 veterinary suture lines, 3D filling foreign matters and the like, and preparing adrenaline and nikkemin injection for first aid and standby;
anaesthesia, performing 15min before operation, subcutaneously injecting atropine sulfate (0.05mg/Kg) behind the ear, and then injecting Shutai 50(0.08mL/Kg, namely 4mg/Kg), inducing anaesthesia;
after the wound surface is thoroughly disinfected by iodophor for 3 times, the skin is incised along the skin marking area by using a scalpel until reaching a fat layer, and the whole layer tissue of the skin is removed until reaching a deep fascia, so that a whole layer skin defect wound surface is formed;
photographing and tracing the wound surface, wiping the skin after finishing the wound suturing, photographing the wound surface, and respectively tracing along the edges of the wound surface at two sides for area calculation;
the foreign matter is filled and fixed, the ring is fixed on the surface of the skin by pressing close to the skin of the back of the pig, and the end of the cylindrical fixing circular hole needs to be vertical to the spine, so that the cylindrical fixing is facilitated;
improving postoperative pain, injecting tramadol hydrochloride injection 1-2 mL/injection per day within 3 days after operation; subsequently adjusting according to the individual state;
dressing exchange after wound creation, injecting tramadol 30min before dressing change for analgesia, removing fixed suture every 6 days after wound creation, removing necrotic tissue in the wound surface after the cylinder is pulled out, continuously filling the cylinder after cleaning and disinfection, and fixing again.
Further, the specific steps of the definition of the chronic wound and the evaluation index thereof are as follows:
the time evaluation standard meets any one of the conditions that the area of the wound surface can not be reduced by 10-15% every week or the area of the wound surface can not be reduced by more than 50% every month or the wound surface is not healed for at least 8 weeks, and the establishment of the chronic wound model is defined to be successful;
HE pathological evaluation index, which is used for evaluating the inflammatory state of the wound surface tissue;
and (3) measuring the thickness of the HE new skin, wherein the measurement range is the boundary area of the new granulation and the skin, and the whole layer thickness of the mastoid area is avoided.
Further, the HE staining method comprises the following specific steps:
s1: selecting slices, and dewaxing and hydrating by adopting an automatic bleaching and drying instrument;
s2: after hematoxylin staining is carried out for 10-20 min, washing with tap water for 1-3 min;
s3: after the hydrochloric acid alcohol is differentiated for 5-10 s, washing for 1-3 min by tap water;
s4: putting into warm water of 50 deg.C or weakly alkaline aqueous solution to turn blue until blue color appears;
s5: washing with tap water for 1-3 min;
s6: putting the mixture into 85% alcohol for 3-5 min;
s7: carrying out eosin dyeing for 3-5 min, and then washing for 3-5 s;
s8: gradient alcohol dehydration
S9: after the xylene is transparent, the gel is sealed by neutral gum.
Further, the specific steps of the immunofluorescence detection of the macrophage marker are as follows:
s1: dropping pancreatin repair liquid into the dewaxed slices, and incubating for 40min at 37 ℃ in an incubator;
s2: washing with PBS for 5min for 3 times;
s3: 10% serum confining liquid is dripped, and the temperature is 30 minutes;
s4: primary antibody (CD68, concentration: 1:100) was added dropwise over night at 4 ℃;
s5: PBS wash same as item S2;
s6: dripping secondary antibody, and controlling the temperature at 37 ℃ for 30 minutes;
s7: dripping DAPI, and incubating at room temperature for 10 min;
s8: PBS wash same as item S2;
s9: sealing the wafer by using an anti-fluorescence attenuation sealing agent;
s10: and adopting a fluorescence scanning microscope camera system to collect images of the slices.
Further, the construction of the diabetic chronic skin ulcer model comprises the following specific steps:
constructing a diabetes model of the Bama miniature pig, and constructing the diabetes model of the pig by combining with STZ injection after feeding the Bama miniature pig for 14 weeks through high-fat high-sugar diet;
the chronic skin ulcer model is constructed by successfully filling the foreign body, and the chronic skin ulcer model is constructed by adopting a mode of combining surgical wound creation and local foreign body filling compression.
The invention has the following beneficial effects:
the invention is suitable for large animal models with thicker skin tissues, particularly pigs; and the model formation is evaluated from multiple angles, and the formation of the diabetic chronic skin ulcer is better simulated from pathogenic factors, pathological changes and mechanism changes.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a view showing the design of the packing of the present invention;
FIG. 2 is a view showing an application of the foreign object stuffing of the present invention;
FIG. 3 is a view of the present invention showing the sewing of a stationary ring;
FIG. 4 is a schematic drawing of a chronic wound specimen according to the present invention;
FIG. 5 is a schematic view of the creation of a diabetic chronic skin ulcer of the present invention;
FIG. 6 is a comparison of the thickness of the new epidermis of the wound surface at day 18 after two groups of two wounding according to the present invention;
FIG. 7 is a histopathological view of a diabetic chronic skin ulcer of the present invention over time;
FIG. 8 is a graph of the pathological scores of wound surfaces of two groups of miniature pigs during the construction of the DCU model of the present invention;
FIG. 9 is a graph comparing the fluorescence intensity of the two sets of macrophage markers CD68 according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to fig. 1-9, the present invention relates to a method for constructing a chronic skin ulcer model of a diabetic pig, comprising the following steps:
constructing a diabetes model of the Bama miniature pig, and constructing the diabetes model in the Bama miniature pig;
the construction method of the diabetes model of the Bama miniature pig comprises the following specific steps:
grouping, after adaptive feeding of male non-castrated Bama minipigs for 5 weeks, randomly dividing into two groups according to a non-equilibrium design in a complete random design, 6 experimental groups (D1-D6) and 2 control groups (C1, C2);
establishing a diabetes model of the Bama miniature pigs, feeding two groups by different schemes, and carrying out an experiment group: high-fat high-sugar feed feeding, control group: feeding with a control feed;
feeding conditions, namely controlling the temperature to be 18-22 ℃, feeding in cages and freely drinking water, wherein the illumination and darkness are 12 hours/day respectively;
STZ injection, in vivo STZ injection into experimental groups;
STZ injection included preparation of citrate buffer, preparation, hydration, STZ pre-injection, post-injection observation, and molding criteria.
Preparing a citric acid buffer solution: 1.9212g citric acid (molecular weight 192.12) is added into 100mL sterile water to prepare A solution, 2.5807g trisodium citrate (molecular weight 258.07) is added into 100mL sterile water to prepare B solution, and A, B solution is mixed according to the weight ratio of 1: mixing at a ratio of 1, measuring pH, and adjusting to about 4.5. Filtering with 0.22 μm filter, sterilizing at high temperature and high pressure, and keeping.
Preparation: after fasting for 12-18 hours, the weight and fasting eartip blood sugar of the miniature pig are measured; an ear vein and a hind limb vein indwelling needle are arranged in advance, the ear vein is used for injecting medicines, glucose and the like, and the hind limb vein is used for collecting blood and monitoring blood sugar and insulin changes.
Hydration: about 500mL of physiological saline is instilled into each pig, and after an indwelling needle is set, hydration is carried out immediately and is carried out for 1 hour at the speed of 10 mL/kg/h.
Prior to STZ injection: fasting venous blood is collected for testing fasting blood glucose and insulin. Intramuscular injection of 0.3mg/kg of mildazolam for sedation 10-30 min before injection can be divided into two injections, wherein 2/3 is injected first, and whether the rest 1/3 is added is determined after observing the situation.
And (3) injection: the dose of the first STZ injection is 125mg/kg, the weighed STZ is added into prepared citric acid buffer solution (1g of STZ:10mL of buffer solution), the mixture is shielded from light, cooled and mixed uniformly, the STZ is injected through an auricular vein indwelling needle for 2-5 minutes, and then the tube is flushed by 10mL of physiological saline. Immediately intramuscular injection of metoclopramide 0.25mg/kg for antiemetic effect;
post-injection observation: closely observing the state of each miniature pig, measuring the blood sugar of the ear tip every 2h, collecting venous blood, and adjusting the blood sugar measuring frequency according to the change of the blood sugar of the ear tip. Treatment for hypoglycemia: if the blood sugar appears less than 3mmol/L during 24h of STZ injection, the pigs are fed freely, and if the pigs are refused to feed, 50% glucose solution is injected into the marginal veins of the ears; different individuals react to STZ differently due to individual differences, and in order to avoid hypoglycemia, the pigs are allowed to eat freely in the first three days, and hyperglycemia is treated: when diabetic ketosis occurs or blood glucose is >27.8mmol/L, insulin treatment is initiated; the target is as follows: controlling blood glucose to be less than 27.8 mmol/L; and (4) recommending a scheme: insulin glargine was injected subcutaneously, 4U initiated once a day, and dose was adjusted according to real-time blood glucose monitoring. ② 30R subcutaneous injection of norgestimate, 4U starting, twice a day, dose adjustment according to real-time blood sugar monitoring. After one month, if a stable hyperglycemic state can be formed, the STZ is not injected, otherwise, the dose of the STZ for secondary injection is determined according to the blood sugar condition.
And (3) modeling evaluation standard: in the molding process, if the miniature pig has no obvious symptoms of polydipsia, polyphagia, polyuria, weight reduction and the like, the fasting blood sugar is more than or equal to 7.0mmol/L and the random blood sugar is more than or equal to 11.1 mmol/L; if the fasting blood sugar is more than or equal to 7.0mmol/L or the random blood sugar is more than or equal to 11.1mmol/L, the diagnosis of diabetes mellitus can be confirmed only if the fasting blood sugar is more than or equal to 7.0mmol/L or the random blood sugar is more than or equal to 11.1mmol/L and the fasting blood sugar still reaches the standard after repeated at another time. If necessary, one month after the model was established, an Oral Glucose Tolerance Test (OGTT) was performed to determine whether the diabetes model was stable.
Monitoring blood sugar and insulin, and detecting blood sugar and insulin data in an experimental group;
blood sugar monitoring: the rogowski glucometer monitors the fasting ear tip, every 4 weeks before the diabetes is modeled, and every week after the modeling. The venous blood sugar is measured by a redox method and is used for detecting the blood sugar level during STZ molding and OGTT. The insulin detection is determined by an iodine 125 radioimmunoassay;
3D printing a foreign matter filling design, creating a wound on the pig skin, and filling foreign matters into the wound;
the specific steps of the 3D printing foreign matter filling design are as follows:
the design of filling foreign matters, namely, cylinders with different lengths are designed according to the depths of wound surfaces of different parts, and the cylinders have the contact and compression effects on local foreign matters after the wound surfaces are filled;
filling foreign matters by adopting 3D printing combining polylactic acid (PLA) materials and thermoplastic polyurethane elastomer (TPU);
the using method comprises the following steps: cylinders with different lengths are designed according to the depths of wound surfaces at different positions, and the cylinders have the contact and compression effects on local foreign matters after the wound surfaces are filled; the ring fixes the edge of the wound surface, the fixed cylinder is mainly used, and the wound surface can be partially inhibited from shrinking towards the center; and the ring can be used to secure the dressing during the treatment phase;
the specific design scheme is that the 3D printing foreign matter consumable material design software is as follows: solid works, the consumable production is carried out by adopting an Allect 334 machine through an FDM hot melt stacking production process;
wherein the production material is polylactic acid (PLA), and the fixed ring production material is thermoplastic polyurethane elastomer (TPU);
performing wound creation, namely, after general anesthesia, opening 3cm beside the two sides of the spine of the miniature pig, and cutting 5-6 skin full-layer wound surfaces with the diameter of 3cm by using a scalpel, wherein the wound surfaces are spaced by 3-4 cm;
the specific steps of the wound are as follows:
preoperative preparation, weighing and preparing skin of the miniature pig and marking a planned wound part one day before operation, and no food or drink is taken 20 o' clock before operation until the day of operation;
preparing instruments and consumables, sterilizing a scalpel, scissors (large and small), forceps (toothed or toothless), gauze, normal saline, a sterile operating coat, an operating sheet, a right-angle crochet hook, No. 18 and No. 10 veterinary sutures, 3D filling foreign matters and the like, and preparing epinephrine and Nicotimam injection for first aid in advance;
performing anesthesia, performing preoperative 15min, performing subcutaneous injection of atropine sulfate (0.05mg/Kg) at the back of an ear, then performing injection of Shutai 50(0.08mL/Kg, namely 4mg/Kg), inducing anesthesia, and performing tracheal intubation after the miniature pig is completely anesthetized after 5-10 min. Tracheal intubation: using a No. 8 catheter, after successful intubation, setting the tidal volume to be 8-10 mL/Kg, fixing 1 person to monitor the pig anaesthesia state, and then using isoflurane to maintain anaesthesia;
after the wound surface and iodophor are thoroughly disinfected for 3 times, a scalpel is used for cutting the skin along the skin marking area until the fat layer, the whole layer of tissue of the skin is removed until the fascia is deep, a whole layer of skin defect wound surface is formed, then, a suture is sutured in the area 1cm away from the edge of the wound surface and used for fixing a circular ring, and at least 4 suture lines are arranged on each wound surface. Closely observing the anesthesia state of the piglet in the operation, and if the skin of the piglet is cut and restless and other reactions occur, adding the isoflurane dosage according to the situation;
photographing and tracing the wound surface, wiping the skin after the wound creating and sewing are finished, photographing the wound surface (photographing lenses are perpendicular to the wound surface and are spaced by 20cm), and respectively tracing along the edges of the wound surface at two sides by 2 fixed experimenters for area calculation by using sterile transparent films;
the foreign matter is filled and fixed, and the ring fixed mode is crucial to model application effect, and the best fixed mode in practice is: (1) completely sterilizing a right-angle crochet hook and a No. 18 veterinary suture before operation, tracing the edge of a wound surface by adopting a fixed ring, and determining the suture position; (2) tracing the position by a scalpel beyond 5mm of the edge of the wound surface according to the aperture, cutting 4 full-layer incisions with 2 incisions at each position, wherein each incision is about 2-3mm and is used for fixing sutures; (3) a right-angle crochet needle is adopted to enable the double-strand 18-gauge suture to penetrate through a full-layer incision from the inside of a wound surface, a dead knot is fixed on one side of the incision, and 2 longer double-strand wires are reserved; (4) after all incisions are sutured, the suture lines respectively penetrate through the small fixing holes in the circular rings and are close to the skin of the back of the pig, the circular rings are fixed on the surface of the skin, and the ends of the circular fixing holes of the cylinder body need to be perpendicular to the spine, so that the cylinder body is convenient to fix;
improving postoperative pain, injecting tramadol hydrochloride injection 1-2 mL/injection per day within 3 days after operation; subsequently adjusting according to the individual state;
dressing exchange after wound creation, intramuscular injection of tramadol 30min before dressing change for analgesia, removing fixed sutures every 6 days after wound creation, pulling out the cylinder, cleaning necrotic tissues in the wound surface, continuously filling the cylinder after cleaning and disinfection, fixing again, determining whether to remove a fixed ring and dig granulation tissues according to the line knot inflammatory reaction at the fixed part of the periphery of the wound surface and the size of the wound surface, taking tissues at the edge of the wound surface every 6 days for detection, wherein 1/2 specimens are fixed by 4% paraformaldehyde, and storing at-80 ℃ after 1/2 liquid nitrogen treatment;
chronic wound definition and evaluation indexes thereof;
the specific steps of the definition of the chronic wound surface and the evaluation index thereof are as follows:
the time evaluation standard meets any one of the conditions that the area of the wound surface can not be reduced by 10-15% every week or the area of the wound surface can not be reduced by more than 50% every month or the wound surface is not healed for at least 8 weeks, and the establishment of the chronic wound model is defined to be successful;
HE pathology assessment index, for the assessment of inflammatory state of wound tissue, the assessment scale is shown in tables 1-1 and 1-2, wherein tables 1-2 are neutrophilic granulocyte and eosinophilic granulocyte supplements in tables 1-1;
TABLE 1-1 pathological Scoring for Chronic wound inflammation status
TABLE 1-2 neutrophil, eosinophil pathology scores
Measuring the thickness of HE new epidermis, wherein the measuring range is the boundary area of new granulation and epidermis, the whole thickness of mastoid area is avoided, a Pannoramic 250 digital slice scanner is adopted to collect images of the slice after HE dyeing, 1 picture of 20 times is collected for each sample to be measured, 3 groups of data are measured for each sample, and the Liner measurement reporting in Annotations is used for measuring;
HE staining;
the HE staining method comprises the following specific steps:
s1: selecting slices, and dewaxing and hydrating by adopting an automatic bleaching and drying instrument;
s2: after hematoxylin staining is carried out for 10-20 min, washing with tap water for 1-3 min;
s3: after the hydrochloric acid alcohol is differentiated for 5-10 s, washing for 1-3 min by tap water;
s4: putting into warm water of 50 deg.C or weakly alkaline aqueous solution to turn blue until blue color appears;
s5: washing with tap water for 1-3 min;
s6: putting into 85% alcohol for 3-5 min;
s7: carrying out eosin dyeing for 3-5 min, and then washing for 3-5 s;
s8: gradient alcohol dehydration
S9: after the xylene is transparent, the gel is sealed by neutral gum.
Immunofluorescence detecting macrophage markers;
the specific steps of the immunofluorescence detection macrophage marker are as follows:
s1: dropping pancreatin repair liquid into the dewaxed slices, and incubating for 40min at 37 ℃ in an incubator;
s2: washing with PBS for 5min for 3 times;
s3: 10% serum confining liquid is dripped, and the temperature is 30 minutes;
s4: primary antibody (CD68, concentration: 1:100) was added dropwise over night at 4 ℃;
s5: PBS wash same as item S2;
s6: dripping secondary antibody, and controlling the temperature at 37 ℃ for 30 minutes;
s7: adding DAPI dropwise, and incubating at room temperature for 10 min;
s8: PBS wash with item S2;
s9: sealing the wafer by using an anti-fluorescence attenuation sealing agent;
s10: and adopting a fluorescence scanning microscope camera system to collect images of the slices.
Detecting dynamic change of inflammatory factor mRNA by real-time fluorescence quantitative PCR;
the real-time fluorescence quantitative PCR detection of the dynamic change of the inflammatory factor mRNA comprises tissue grinding, tissue total RNA extraction, reverse transcription and real-time fluorescence quantitative PCR;
grinding the tissues;
precooling magnetic beads, a 1.5mL EP tube and a centrifugal plate;
transferring a proper amount of tissue specimen in the frozen tube to a precooled centrifuge tube with the volume of 1.5mL, and adding 4 magnetic beads and 1mL Trizol into the centrifuge tube;
setting a grinding mode: 60HZ, continuing for 30s, interrupting for 20s, continuing for 2-4 times according to the tissue grinding effect until the tissue is broken, and operating at low temperature.
Extracting total RNA of tissues;
adding 200 mu L of chloroform into an enzyme-free EP tube containing Trizol, violently shaking and uniformly mixing for 15s, standing for 5min at room temperature, and centrifuging for 15min at 4 ℃ under 12000 prm;
secondly, after centrifugation, carefully absorbing 350-400 mu L of supernatant into an enzyme-free EP tube, adding isopropanol with the same volume into the enzyme-free EP tube, uniformly mixing, standing for 10min at room temperature, and centrifuging for 10min at 4 ℃ under 12000 prm;
thirdly, abandoning the supernatant, adding 1mL of 75% ethanol into an EP tube, repeatedly beating and washing thoroughly, centrifuging for 5min under the conditions of 4 ℃ and 7500prm, and repeatedly washing for 1 time. After the supernatant is poured off, idling for 1 time again, sucking and removing the redundant supernatant, and airing the supernatant on an ultra-clean bench for about 5 min;
preheating 30-50 mu L DEPC water at 55 ℃ for dissolving RNA, sucking 5 mu L of the DEPC water, measuring the RNA concentration in 95 mu L DEPC water, and placing the rest on ice to be subjected to reverse transcription;
detecting the concentration: taking an RNA sample diluted by 20 times to determine the RNA concentration on an enzyme-labeling instrument, wherein the ratio of OD 260/OD 280 is more than 1.8; from the measured RNA concentration, the volume required for 1. mu.g of cDNA was calculated.
Reverse transcription;
(gDNA) reaction for removing genomic DNA: the reference reverse transcription Kit PrimeScript RT reagent Kit with gDNA Eraser was performed at room temperature for 10min in the reaction system shown in tables 1-3.
TABLE 1-3 genomic DNA removal reaction configuration
Reverse transcription to synthesize cDNA: the reaction conditions were 37 ℃, 15min, 85 ℃, 5s, 4 ℃, holing (on ice operation). The reaction systems are shown in tables 1-4.
Tables 1-4 reverse transcription reaction configuration
And storing the cDNA sample at-20 deg.c for further use.
A. Real-time fluorescent quantitative PCR
Firstly, primer design: designing species-specific primers and carrying out detection; the sequence results are shown in tables 1-5;
② referring to QPCR kit TB Green Premix Ex Taq II, the experiment is carried out in a LightCycler 96 system (the operation of adding reagents on ice is needed). The reaction system is shown in tables 1-6, and the reaction conditions are shown in tables 1-7;
③ after the reaction, GAPDH was used as an internal reference for the relative quantification of mRNA. The measurement was repeated three times for each sample, and the relative expression was calculated using the statistical method of 2^ Delta Ct.
TABLE 1-5 Bama minipig QPCR primer sequences
Tables 1-6 QPCR reaction configurations
TABLE 1-7 QPCR reaction conditions
Constructing a diabetes chronic skin ulcer model, namely constructing a chronic skin ulcer model in a pig wound;
the construction method of the diabetic chronic skin ulcer model comprises the following specific steps:
constructing a diabetes model of the Bama miniature pig, and constructing the diabetes model of the pig by combining with STZ injection after feeding the Bama miniature pig for 14 weeks through high-fat high-sugar diet; only D2 has obvious symptoms of polydipsia, diuresis and weight loss, the fasting blood sugar is up to more than 20mmol/L, and the diabetes needs to be treated by insulin, other experimental group miniature pigs only diagnose the diabetes for slight blood sugar rise or OGTT, but the blood sugar is reduced to a certain degree along with the time prolongation, all experimental group miniature pigs successfully construct stable diabetes models within 48 weeks after multiple times of supplementation within 13 weeks, and the STZ injection scheme is as shown in tables 1-8. Wherein D1, D2 and D4 need long-term insulin subcutaneous injection treatment, and fasting blood glucose is controlled below 8-20 mmol/L for a long time; d3 and D5 maintained stable mild blood sugar rise with fasting fluctuations of 7-11.3 mmol/L. During induction, D6 showed no significant change in blood glucose at 1 st additional STZ, and increased blood glucose at 2 nd additional STZ, but died after 1 week due to pancreatitis. Therefore, the actual bama minipigs diabetic models were finally 5 and used for subsequent experiments.
Constructing a DCU model starting at the age of 76 weeks of piglets, at which time the experimental group of piglets has formed a stable diabetes model for 29 weeks;
TABLE 1-8 STZ injection protocol
Successfully filling foreign matters to construct a chronic skin ulcer model, and constructing the chronic skin ulcer model by adopting a mode of combining surgical trauma and local foreign matter filling compression;
the specific creation scheme in the experimental process is as follows: for the first time, after surgical wound is caused, the foreign body is filled and the fixed circular ring around the wound is sutured, and because the surface of the skin suture position is shallow, the inflammatory reaction at the fixed knot is serious, the foreign body can be removed only by pressing for 6 days; secondly, on the 18 th day after the wound, the anesthesia risk and the recovery situation of the inflammation around the wound surface are integrated, the wound surface is only surgically created (secondarily created with the diameter of 3cm), and the wound surface is only padded by gauze after the operation without being padded by foreign matters and fixed around the wound; thirdly, on the 43 th day after the wound, on the basis of the first scheme, after the skin tissues around the wound are sutured in a full layer, the inflammatory reaction of the fixed part of the wound line knot is obviously improved, and the foreign body fixing effect achieves the expected effect; in order to facilitate the experiment in the treatment stage, the fixed column is pulled out when the column is pressed to the 53 th day after the wound, and the wound circumference ring is reserved for dressing fixation;
before the creation and during the model construction, periodically detecting indexes such as blood sugar, body weight, blood routine, albumin, total protein and the like of the miniature pig; the results show that the hemoglobin level and the albumin/total protein ratio of two groups of pigs are reduced to a certain extent during the wound-creating period;
comparing the thickness of the new epidermis at the edge of the wound surface 18 days after the first wound and the second wound, wherein the thickness of the new epidermis at the edge of the wound surface 18 days after the first wound is 130.550 +/-33.646 microns in a control group, and the thickness of the new epidermis at the edge of the wound surface is 110.114 +/-16.767 microns in an experimental group; on day 18 after the second wound, the control group was 212.150. + -. 77.359 μm, and the experimental group was 167.333. + -. 38.628 μm (FIGS. 1-9). The results show that the epidermis thickness of the two groups of miniature pigs is 18 days after the second wound and is more obvious than the epidermis thickness of the two groups of miniature pigs 18 days after the first wound, and the differences are more obvious in the experimental group (p is less than 0.001); however, the neoepidermal thickness in the experimental group was significantly lower than that in the normal control group (p ═ 0.002). The results suggest that diabetes combined with foreign body tamponade (local pressure) can reduce neonatal skin thickness in piglets;
the chronic inflammation state evaluation is carried out on the two groups of minipigs on the 6 th day, the 24 th day and the 56 th day before and after the wound respectively, and the results show that:
before wound, the epidermis structure of the skin tissue of the control group is complete and clear, the hornification layer is obvious, the cells are arranged neatly and tightly, and the collagen fibers in the dermis layer are arranged in a staggered way and are more compact; the epidermal cells in the experimental group are sparsely arranged, and the collagen fibers in the dermis layer are arranged in parallel. No obvious inflammatory cell infiltration phenomenon is seen in normal skin tissues between two groups, and the pre-traumatic inflammation score is 0;
two groups of similar manifestations after the wound are: necrosis of collagen fibers in the dermis layer continues to 56 days after the wound, and the experimental group is more obvious; the necrotic area and the subcutaneous fat layer are infiltrated by lymphocytes, neutrophils and eosinophils, and inflammatory cells are more common as lymphocytes are more visible with the time; from day 6 after the wound, both groups had a certain fibroblast proliferation and neovascularization, and increased significantly at day 56 after the wound; the difference between the two groups after the creation is that: the control group has the phenomenon of thickening epidermis at the 6 th day after the wound, while the experimental group has obvious appearance at the 24 th day, which is consistent with the result that the thickness of the new epidermis of the control group is obviously higher than that of the experimental group at the 18 th day after the wound twice; at each time point after the creation of the wound, the inflammation score fluctuates between 0 and 3 points and is mostly 1 to 2 points. Analysis of the data showed that the two groups differed only statistically in time (tables 1-9). The results indicate that the skin of the diabetes mellitus 'normal' before the DCU model is constructed has the phenomenon that epidermal cells become thin and the parallel arrangement of dermal collagen fibers is changed; during the construction of the DCU model, two groups of wound surfaces continuously have an inflammation state mainly caused by lymphocyte immersion, and in addition, the epidermal neogenesis capacity of the diabetic miniature pigs is remarkably lower than that of control miniature pigs;
table 1-9 two-factor repeated measures analysis of variance for pathology scores
Two groups of mini pigs were immunofluorescent stained for the macrophage marker CD68 (green fluorescence) on skin tissue before and 56 days post-wound, and the results showed: the CD68 expression in the experimental group before the wound was significantly higher than that in the control group (p ═ 0.043), while the difference between the two groups at the 56 th day after the wound had no statistical significance; compared with the control group before the wound, the CD68 expression is remarkably increased (p is 0.001) at the 56 th day after the wound of the control group, and the difference of the experimental group has no statistical significance; the results suggest that macrophage infiltration in normal skin tissue is more than normal control in the bama minidiabetic pig model, and macrophage infiltration after wound is similar to normal skin before wound; in contrast, in the control group, the macrophage infiltration after the wound is more than that of the normal skin before the wound, which is similar to the experimental group, and the inflammatory reaction exists in the diabetic pig model even if the skin is normal, so that the invisible damage exists in the normal skin of the diabetic pig.
In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The preferred embodiments of the invention disclosed above are intended to be illustrative only. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise embodiments disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best utilize the invention. The invention is limited only by the claims and their full scope and equivalents.
Claims (8)
1. A method for constructing a chronic skin ulcer model of a diabetic pig is characterized by comprising the following steps: the method comprises the following specific steps:
constructing a diabetes model of the Bama miniature pig, and constructing the diabetes model in the Bama miniature pig;
3D printing a foreign matter filling design, creating a wound on the pig skin and filling foreign matters into the wound;
constructing a diabetic chronic skin ulcer model, and constructing a chronic skin ulcer model in a pig wound.
2. The method for constructing the model of chronic skin ulcer of diabetic pigs according to claim 1, which is characterized by comprising the following steps: the construction method of the diabetes model of the Bama miniature pig comprises the following specific steps:
grouping, after adaptive feeding of male non-castrated Bama minipigs for 5 weeks, randomly dividing into two groups according to a non-equilibrium design in a complete random design, 6 experimental groups (D1-D6) and 2 control groups (C1, C2);
establishing a diabetes model of the Bama miniature pigs, feeding two groups by different schemes, and carrying out an experiment group: high-fat high-sugar feed feeding, control group: feeding with a control feed;
feeding conditions, namely controlling the temperature to be 18-22 ℃, feeding in cages and freely drinking water, wherein the illumination and darkness are 12 hours/day respectively;
STZ injection, in vivo STZ injection into experimental groups;
monitoring blood sugar and insulin, and detecting blood sugar and insulin data in the experimental group.
3. The method for constructing the model of chronic skin ulcer of diabetic pigs according to claim 1, which is characterized by comprising the following steps: the specific steps of the 3D printing foreign matter filling design are as follows:
the design of filling foreign matters, namely, cylinders with different lengths are designed according to the depths of wound surfaces of different parts, and the cylinders have the contact and compression effects on local foreign matters after the wound surfaces are filled;
the specific design scheme is that the 3D printing foreign matter consumable material design software is as follows: solidworks, consumable production is carried out by adopting an Allect 334 machine through an FDM hot melt stacking production process;
performing wound creation, namely, after general anesthesia, opening 3cm beside the two sides of the spine of the miniature pig, and cutting 5-6 skin full-layer wound surfaces with the diameter of 3cm by using a scalpel, wherein the wound surfaces are spaced by 3-4 cm;
chronic wound definition and evaluation indexes thereof;
HE dyeing;
immunofluorescence detecting macrophage markers;
and (3) detecting the dynamic change of the mRNA of the inflammatory factor by real-time fluorescence quantitative PCR.
4. The method for constructing the model of chronic skin ulcer of diabetic pigs according to claim 3, which is characterized by comprising the following steps: the specific steps of the wound are as follows:
preoperative preparation, namely weighing and preparing skin of a miniature pig and marking a part to be wounded one day before an operation;
preparing instruments and consumables, sterilizing a scalpel, scissors (big and small), tweezers (with teeth and without teeth), gauze, normal saline, an aseptic operating coat, an operating sheet, a right-angle crochet hook, No. 18 and No. 10 veterinary suture lines, 3D filling foreign matters and the like, and preparing adrenaline and nikkemin injection for first aid and standby;
anaesthesia, performing 15min before operation, performing subcutaneous injection of atropine sulfate (0.05mg/Kg) and performing postinjection of Shutai 50(0.08mL/Kg, namely 4mg/Kg) to induce anaesthesia;
after the wound surface is thoroughly disinfected by iodophor for 3 times, a scalpel is used for incising the skin along the skin marking area until reaching the fat layer, and the whole layer of tissue of the skin is removed until reaching the deep fascia, so that a whole layer of skin defect wound surface is formed;
photographing and tracing the wound surface, wiping the skin after finishing the wound suturing, photographing the wound surface, and respectively tracing along the edges of the wound surface at two sides for area calculation;
the foreign matter is filled and fixed, the ring is fixed on the surface of the skin by pressing close to the skin of the back of the pig, and the end of the cylindrical fixing circular hole needs to be vertical to the spine, so that the cylindrical fixing is facilitated;
improving postoperative pain, injecting tramadol hydrochloride injection 1-2 mL/injection per day within 3 days after operation; subsequently adjusting according to the individual state;
dressing exchange after wound creation, injecting tramadol 30min before dressing change for analgesia, removing fixed suture every 6 days after wound creation, removing necrotic tissue in the wound surface after the cylinder is pulled out, continuously filling the cylinder after cleaning and disinfection, and fixing again.
5. The method for constructing the model of chronic skin ulcer of diabetic pigs according to claim 3, which is characterized by comprising the following steps: the specific steps of the definition of the chronic wound surface and the evaluation index thereof are as follows:
the time evaluation standard meets any one of the conditions that the area of the wound surface can not be reduced by 10-15% every week or the area of the wound surface can not be reduced by more than 50% every month or the wound surface is not healed for at least 8 weeks, and the establishment of the chronic wound model is defined to be successful;
HE pathological evaluation index, which is used for evaluating the inflammatory state of the wound surface tissue;
and (3) measuring the thickness of the HE new skin, wherein the measurement range is the boundary area of the new granulation and the skin, and the whole layer thickness of the mastoid area is avoided.
6. The method for constructing the model of chronic skin ulcer of diabetic pigs according to claim 3, which is characterized by comprising the following steps: the HE staining method comprises the following specific steps:
s1: selecting slices, and dewaxing and hydrating by adopting an automatic bleaching and drying instrument;
s2: after hematoxylin staining is carried out for 10-20 min, washing with tap water for 1-3 min;
s3: after the hydrochloric acid alcohol is differentiated for 5-10 s, washing for 1-3 min by tap water;
s4: putting into warm water of 50 deg.C or weakly alkaline aqueous solution to turn blue until blue color appears;
s5: washing with tap water for 1-3 min;
s6: putting the mixture into 85% alcohol for 3-5 min;
s7: carrying out eosin dyeing for 3-5 min, and then washing for 3-5 s;
s8: gradient alcohol dehydration
S9: after the xylene is transparent, the gel is sealed by neutral gum.
7. The method for constructing the model of chronic skin ulcer of diabetic pigs according to claim 3, which is characterized by comprising the following steps: the specific steps of the immunofluorescence detection macrophage marker are as follows:
s1: dropping pancreatin repair liquid into the dewaxed slices, and incubating for 40min at 37 ℃ in an incubator;
s2: washing with PBS for 5min for 3 times;
s3: 10% serum confining liquid is dripped, and the temperature is 30 minutes;
s4: primary antibody (CD68, concentration: 1:100) was added dropwise over night at 4 ℃;
s5: PBS wash same as item S2;
s6: dripping secondary antibody, and controlling the temperature at 37 ℃ for 30 minutes;
s7: adding DAPI dropwise, and incubating at room temperature for 10 min;
s8: PBS wash with item S2;
s9: sealing the wafer by using an anti-fluorescence attenuation sealing agent;
s10: and adopting a fluorescence scanning microscope camera system to collect images of the slices.
8. The method for constructing the model of chronic skin ulcer of diabetic pigs according to claim 1, which is characterized by comprising the following steps: the construction method of the diabetic chronic skin ulcer model comprises the following specific steps:
constructing a diabetes model of the Bama miniature pig, and constructing the diabetes model of the pig by combining with STZ injection after feeding the Bama miniature pig for 14 weeks through high-fat high-sugar diet;
the chronic skin ulcer model is constructed by successfully filling the foreign body, and the chronic skin ulcer model is constructed by adopting a mode of combining surgical wound creation and local foreign body filling compression.
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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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