CN101390779A - Preparation method of rat chronic posterior-limb ischemia model - Google Patents
Preparation method of rat chronic posterior-limb ischemia model Download PDFInfo
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Abstract
The invention relates to a chronic hindlimb ischemia animal model construction method, belonging to medical biological technical field and experimental zoology field. The chronic hindlimb ischemia animal model construction method includes the steps of inserting line embolic material close to the knee joint in the femoral artery of the experimental animal and suturing the cut, which can lead to stenosis of the artery lumen and foreign body reaction. In this way, intimal hyperplasia gradually appears in the lumen, the bleeding of the hindlimb reduces gradually, and the chronic hindlimb ischemia animal model can be established. The chronic hindlimb ischemia animal model has the advantages of simple surgical principle, high survival rate of the animal after the surgery and good ischemia stability. The chronic hindlimb ischemia animal model is long in ischemia time and better preserves the structure of the hindlimb musculature, and ischemic states of different parts of the limb are different. The change of the vascular lumen is similar to human atherosclerotic plaque and is more similar to the symptoms of clinical patients. The chronic hindlimb ischemia animal model constructed through line embolism method is suitable for intervention experiment and study two weeks after the model is constructed.
Description
Technical field
The present invention relates to a kind of preparation method of chronic posterior-limb ischemia animal model, belong to medical biotechnology field and experimental zoology field.
Background technology
According to external statistics, have every year in the U.S. to surpass 150,000 people because the severe ischemic of lower limb and unable to walk is got involved or operative treatment, along with arteriosclerotic continuous progress, lower limb ischemia constantly increases the weight of.Last patient can only select amputation.In recent years along with the continuous development of regenerative medicine, constantly have new therapeutic angiogenesis means to occur as: various somatomedin are imported ishemic parts, to reach the purpose of angiogenesis (angiogenesis) or angiogenesis (arteriogenesis).Animal experiment find as: bone marrow stem cell, cytokine (VEGF, bBGF etc.) can effectively promote angiogenesis or angiogenesis to supply to improve lower limb blood, for clinical treatment does not have efferent tract, serious chronic lower limb ischemia patient provides new treatment thinking, effect but is difficult to lead the people satisfied but above treatment means is applied to clinical back, certainly, reason is many-sided, but undeniablely be, one of them important reasons is at present when research therapeutic angiogenesis means and mechanism, and the animal model that is utilized all is acute lower limb ischemia model, can not the chronic lower limb ischemia of reaction in all directions after the various pathophysiological change characteristics of body.
The used animal model of research limb ischemia all is based on acute ischemia both at home and abroad at present, preparation method mainly is by the ligation femoral artery, external iliac artery, the method for perhaps stripping off deep femoral artery causes the acute ischemia of lower limb, generally is to be considered to the chronic ischemia process in the later stage of acute ischemia.Bibliographical information acute ischemia takes place after, the local shearing force that produces; Inflammatory reaction; The part of endothelium chief cell is immersed, more than three big mechanism all participate in angiogenesis and generation at short notice.
But our chronic ischemia that on arteriosclerotic basis, causes lower limb often of facing clinically, secular chronic ischemia makes lower limb muscles adapt to change to ischemia by changing metabolic way and myofibrillar configuration, thereby avoided the necrosis that causes as acute lower limb ischemia, inflammatory reaction, rather than by the acute lower limb blood fortune improved after compensatory of local vascular.In sum, acute lower limb ischemia and clinical chronic lower limb ischemia have all many-sides there are differences, at present acute posterior-limb ischemia animal model of using and the needs that are not suitable for studying.
We know that animal model has significance to the generation of study of disease development and treatment, and animal model should have reliable and stablely accurately, and repeatability is strong, prepares characteristics such as simple.The limb ischemia model is used very extensive at home and abroad, but relevant limb ischemia model specialize in but one to rarely.Even the narration to this model in relevant document is also very few.
With regard to present list of references, ischemic state has the branch of temporary transient ischemia and chronic ischemia.The former is used among the research of ischemia pretreatment and ischemia-reperfusion more, and its production method has two kinds: the one, and rings such as external tourniquet haemostat or binder are tied in the limbs root, decontrol after a period of time again, reach the purpose of temporary transient ischemia with this; Another kind is to separate partial feeding artery, or closes blood flow with the temporary transient folder of bulldog clamp.
Acute ischemia is the main flow of limb ischemia model, is most widely used its manufacture method substantially with ligation and excise the feeding artery trunk of hind leg or it branches into the master.But in most animal model manufacturing process, if ligation is also cut off the near-end of a certain feeding artery, usually the situation that causes the necrosis of far-end limb ischemia is actually rare: it is reported that the lower limb ischemia model of dog needs 14 tremulous pulsies of ligation, comprise the pelvic cavity tremulous pulse, common iliac artery femoral artery,common and their branch.After 6 weeks, the trouble limb of these dogs can return to roughly normal blood flow level.Cut off the limb ischemia of the common iliac artery formation of rabbit and can only keep 17 days, after 30 days, rabbit does not show serious posterior-limb ischemia.
Also have researcher to adopt alternate manner to make the limb ischemia model: as Bian Jiefang etc. when making the dog posterior-limb ischemia model, cut an osculum in femoral artery wall, insert one of helical metal wire to far-end, sew up a pin fixing metal silk, sew up arteriotomy then tinsel is stayed in the femoral artery.Gale L.Tang after the rat femoral root adds ameroidconstrictor (a kind of absorb water shrink material), thereby make the femoral artery external diameter gradually constriction reach the purpose of the chronic lower limb ischemia model of preparation.But this class model also exists such as: lower limb ischemia time weak point of holding time; Physical property constriction blood vessel external diameter differs greatly in the clinical disease change; Shortcomings such as Ischemia Time is wayward are so its concrete effect of its ischemia model of producing is still to be verified.
Still the report that does not have other chronic lower limb ischemia models at present both at home and abroad.
Summary of the invention
The technical problem that the present invention will solve provides a kind of preparation method of chronic posterior-limb ischemia animal model.
For achieving the above object, the present invention takes following technical scheme:
A kind of preparation method of chronic posterior-limb ischemia animal model is inserted line bolt material near knee joint in the femoral artery of laboratory animal, sew up the incision; Cause the narrow and generation foreign body reaction of arterial lumen, make to produce neointimal hyperplasia in the tube chamber gradually, cause the minimizing of lower extremity blood flow gradually, set up the chronic posterior-limb ischemia animal model.
Near described line bolt material to the method the knee joint of inserting in the femoral artery of laboratory animal is: the skin of longitudinal incision lower limb center, from groin to knee joint; In the groin root portion from femoral artery and femoral vein; 0.5cm divides other places blocking-up femoral artery femoral artery nearly far-end more under inguinal ligament, and ligation femoral artery,superficial and other branches, cuts femoral artery, and direct-view is inserted line bolt line of material down to femoral artery knee joint place; After the complete open femoral artery of art is seen no active hemorrhage, sew up the incision.
Described laboratory animal is Mus, rabbit, dog, sheep or pig.
Described laboratory animal is the Lewis rat.
Described line bolt material is 4-Oprolene line, hair or catgut, preferred 4-Oprolene line.The 4-Oprolene line is that proper line is fastened the method material at present, and histocompatibility is good, and is easy to operate.Tremulous pulse pathological change and arteriosclerosis that line is fastened are close.
The present invention is by inserting the 4-Oprolene line near knee joint in the Lewis rat femoral, cause the narrow and generation foreign body reaction of arterial lumen, make to produce neointimal hyperplasia in the tube chamber gradually, the minimizing that causes lower extremity blood flow gradually is to set up rat chronic posterior-limb ischemia model.By observing clinical manifestation, the blood pressure determination of noinvasive blood flow, angiography, histological examination, means evaluation such as immunohistochemistry and acute lower limb ischemia model comprehensively contrast.
Compare research from dynamic evolution, ischemic tissue intramuscular microenvironment changes of cytokine aspect and the acute lower limb ischemia model of ischemia performance, Histological change, blood flow:
(1) clinical observation result analysis: we carry out the preparation of acute and chronic lower limb ischemia respectively to rat, and its clinical observation result of carrying out 42 days by a definite date shown: acute lower limb ischemia model group animal have clinical manifestation that 8 rats show lower limb ischemia as: cyanosis appears in postoperative lower limb immediately, phenomenons such as walking lamely appears in viewing duration, pale.After other 1 rats underwent left side iliac artery excision+left femoral artery excision, necrosis, animal dead appearred in the hind leg short time.This phenomenon shows, the pathological condition of acute ischemia model is with the chronic difference that truly has, and laboratory animal must experience and just can enter so-called chronic ischemia state after ischemia adapts to, and shows the incompatibility of acute posterior-limb ischemia model with the clinical patients performance in sum; And chronic lower limb ischemia model has 4 the lower limb ischemia clinical manifestation to occur, lower limbs necrosis and animal dead do not occur.But observing the later stage atrophy phenomenon of hindlimb muscle again, this and clinical findings are similar.Therefore, have reason to believe the pathophysiological change that our animal model can truer reflection chronic ischemia.
(2) Mus hind leg blood flow and blood pressure dynamic evolution observed result are analyzed: this test adopt the laser Doppler flowmetry that generally adopts both at home and abroad and laser-Doppler blood flow imaging instrument by to hind leg blood flow and blood pressure after surgery different time points measure the blood flow and the blood pressure trend of analysis and more different model ischemia hind legs.We adopt LDF instrument and probe in process of the test; Immobile phase with the mensuration position, under constant relatively room temperature, operate, in conjunction with the method for fixing light cable to reduce test error to greatest extent.
1. laser-Doppler blood flow analysis: change and show by measuring the microcirculation of suffering from limb: chronic lower limb ischemia model occurs that the lower limb ischemia state is slow, evening time.But the speed that blood flow descends is but faster than the acute ischemia model, and the posterior-limb ischemia state is held time, and (the 42nd day after surgery still is 70% of strong side to length; Acute 42 days after surgery, ischemic state was recovered substantially).Reason is thought of as after femoral artery inserts the 4-Oprolene line, caused the interior physical property of blood vessel narrow in a short time, this moment, lower extremity blood flow was not cut off yet, according to the Hemodynamics Study result, lumen diameter is narrow〉50% or area narrow 75%, tangible hemodynamics can take place change, clinical demarcation is the ischemic state that needs surgery to get involved.Above result is with the result in 1 week is consistent after surgery through the analysis of laser-Doppler imager.After producing foreign body reaction in the lumen of vessels, inner membrance begins hypertrophy, causes tube chamber sharply narrow subsequently, and final inaccessible back lower limb are lasting ischemic state.
2. laser-Doppler analysis of blood pressure: postoperative shows suffering from limb and strong side limbs different time points continuous blood pressure measurement result: chronic lower limb ischemia model early than acute lower limb ischemia model after surgery 1 all blood pressure drops to minimum point subsequently blood pressure recover gradually.In conjunction with angiographic results, 7 days after surgery thigh portion collatoral vessels of chronic ischemia model are not seen obvious opening, and acute posterior-limb ischemia model 7 days after surgery, just there is a large amount of side Zhi Xunhuan open, this has also caused different in thigh portion blood pressure trend of acute posterior-limb ischemia model and chronic posterior-limb ischemia model, and the analysis of causes is: on the one hand, and under the normal physiological situation, tremulous pulse is unobstructed, collatoral vessel far-end and near-end do not have barometric gradient basically.When the blood flow of tremulous pulse stopped up, the pressure of the near-end of collatoral vessel rose, and far-end is because of no blood, and pressure sharply descends, and collatoral vessel has formed tangible barometric gradient, causes the blood flow shearing force sharply to rise.More than making most sides prop up systemic vascular can open at short notice, with compensatory rapid ischemic state, and the chronic ischemia model is owing to be the incomplete blocking-up of artery of lower extremity, the vascular scissors shear force that is produced is less than acute lower limb ischemia model, there is not the rising of tangible blood flow shearing force, so side Zhi Xunhuan is not exclusively open.The result has caused the difference of lower limb ischemia state between different models.On the other hand, because the characteristics of the hind leg blood vessel of rodent own, and there is not arteriosclerosis in laboratory animal, pathological changes such as diabetes, therefore to set up speed quite fast for rat hindlimb side Zhi Xunhuan, after showing as acute ischemia, muscular tissue is rare large stretch of downright bad, and collatoral vessel is open at short notice.
(3) rat hindlimb muscle histology analyzes: but the scholar thinks process at the later stage of acute ischemia chronic ischemia at present, so according to more than carried out the experimentation of a large amount of angiogenesiss: as Bone Marrow Stem Cells Transplantation etc., we are by after model prepares successfully, carry out muscular tissue in different time points and draw materials, carry out the conventional H .E dyeing and the VIII factor, a-actin immunohistochemical staining.
Structure to the ischemic muscle tissue, tissue morphology, reach capillary density and small artery density and carried out comprehensive comparative analysis, wherein the VIII factor is that endothelial cell membrane is specific expressed, be mainly used in research to the density of blood capillary, a-actin mainly is the research that is used at small artery density, experimental result is found: chronic lower limb ischemia model has well been preserved the integrity of lower limb muscles structure on the one hand, but also can find the meat fiber spotty necrosis, not have the phenomenon of large stretch of muscular death.Can find significantly muscle fiber atrophy in the later stage of observing.On the other hand, the infiltration of inflammatory cell shows comparatively obviously at chronic lower limb ischemia hindlimb muscle, especially in 7 days gastrocnemius of chronic lower limb ischemia model postoperative.
Chronic lower limb ischemia model the 7th day after surgery, capillary density obviously is less than acute lower limb ischemia model, arteriolar opening also obviously is less than the acute ischemia model, 14 days after surgery, chronic posterior-limb ischemia model is suffered from the limb capillary density apparently higher than the acute ischemia model, and this point can be confirmed by angiography.The above analysis of causes as a result appears in this experiment: though the Hemodynamics Study in early stage can see that blood flow appears in the chronic ischemia model, blood pressure drops is later than the chronic ischemia model, but ischemia, the anoxia longer duration, the less gradually trouble limb that caused of blood flow is in lasting low filling anaerobic condition for a long time, ischemia, anoxia is again by the HIF-1 signal transducting system: i.e. the anoxia of cell, suppress the HIF-1a transcription factor, cause many increases that cause various inflammatory cell chemokine secretions, the histology shows as the infiltration of inflammatory cell.All be various cytokines such as VEGF as mononuclear cell and macrophage in the inflammatory cell, the important source of MCP-1 etc., above cytokine has stronger chemotactic endotheliocyte, regulates the effect of angiogenesis.Because the secretion of a large amount of cytokine makes the rising of chronic posterior-limb ischemia model intramuscular blood capillary quantity, but early stage at ischemia, acute posterior-limb ischemia model is because the rapid rising of the interior shearing force of blood vessel makes small artery open in the short time, show as the 7th day after surgery, small artery density is higher than chronic posterior-limb ischemia model.
It is to be noted, chronic posterior-limb ischemia model after surgery side to prop up be that side is propped up the circulation opening or the new life of blood capillary is slower than acute posterior-limb ischemia model, these characteristics are less with clinical patient acute ischemia side Zhi Jianli, and chronic ischemia is set up more clinical manifestation and contradicted.Entangling its reason may be that prosperity of rodent primitive vessel or collateral blood vessels exist but unopened reason, if can directly causing limb necrosis at human acute ischemia, the chronic ischemia state can't occur.
(4) selection of line tying material: in the preliminary experiment part of this experiment, we once adopted common silk thread No. 0, catgut is dissatisfied as the material result: common silk thread is because the not enough relatively difficulty of inserting on the one hand of hardness and toughness, even another negative side is inserted in the blood vessel, the very fast and blood vessel wall adhesion of silk thread can't be carried to far-end.Catgut is to be the wire work of raw material with the biomaterial, though hardness and toughness can be inserted in the blood vessel smoothly, preliminary experiment is found, very fast and blood vessel wall generation inflammatory reaction, and all left and right sides blood vessel entirely shuts after surgery.Human hair too carefully can't impact the hematodinamics of blood vessel, and the Prolene line has good hardness and toughness makes convenient experimental operation, histocompatibility is better, in blood vessel, at first caused the luminal stenosis of physical property, the prolene line stimulates the intensive hypertrophy of inner membrance by foreign body reaction on this basis, further make gradually narrow of tube chamber, more than develop to such an extent that morphology is more similar with arteriosclerosis plaque.Can imitate the process that human atherosclerotic causes the lower limb chronic ischemia to a certain extent.
(5) acute lower limb ischemia model and chronic lower limb ischemia model hind leg VEGF, Flk-1, Ang-2, different and the analysis of Flk-1 and SDF-1 mRNA: VEGF (vascular endothelialgrowth factor, VEGF) and the family member can promote endothelial cell division, propagation and migration specifically, in the new vessels forming process, play an important role.Angiogenesis hormone (angioprotien, Ang) be the unique angiogenesis factor family of containing receptor stimulating agent and acceptor inhibitor that finds at present, be a class and vascularization proteins associated matter molecule, comprise angiogenesis hormone-1 (ang 1), angiogenesis hormone 2 (ang 1), angiogenesis hormone 3 (ang 1) and angiogenesis hormone 4 (ang 1), mainly play a role 12 by combining with endothelial cell specific tryrosinase receptor Tie 1], wherein Ang 1 has expression in tissues such as fetal livers blood vessel, adult ovary, Placenta Hominis and uterus.VEGF (vEGF), fibroblast growth factor FGF) and anoxia can increase the expression of Ang 1.Stroma cell derivative factor (stromal cell-derived factor-1 α, SDF-1 α) is a member of chemotaxis cytokine (chemotaxis) CXC subtribe, can promote the endothelial cell proliferation migration, and can promote endotheliocyte to express VEGF (VEGF), thereby promote blood vessel to take place, show that SDF-1 α has participated in the process that capillary endothelium forms new vessels, and have bone marrow stem cell had intensive chemotaxis.
We utilize Real-time PCR to detect acute posterior-limb ischemia model and 2 weeks of chronic posterior-limb ischemia model postoperative respectively, the quadriceps femoris and the gastrocnemius VEGF of strong side and Ipsilateral, the expression of Ang-2 and SDF mRNA, found that chronic lower limb ischemia model 2 weeks after surgery, suffer from the limb muscle especially that above-mentioned cytokine is the high expressed state in the gastrocnemius.Further confirm that the hypoxic-ischemic of muscular tissue can be induced the secretion of a large amount of angiogenesis promoting blood vessel factors under the chronic ischemia state, also increased the weight of simultaneously the local inflammation cell infiltration this conform to chronic lower limb histology result.
The high expressed of cytokine in the chronic posterior-limb ischemia model gastrocnemius, the ischemia model gastrocnemius ischemic state that indication wire is fastened the method preparation is more serious than quadriceps femoris, above interpretation of result, it is relevant can to obtain a part of blood flow compensation from pelvic cavity or median sacral artery with the rat muscle of thigh, and because femoral artery,superficial, popliteal arterial occlusion mainly influences Calf muscle, the pathological changes of above blood vessel is directly reflected as the obvious ischemia of Calf muscle, this and human atherosclerotic obliterans, the diseased region of patient with diabetic feet is close.But above each cytokine obviously is not the high expressed state in the acute ischemia model, do not meet with theory.But comprehensive integral animal ischemic state is seen, because the side Zhi Kaifang of acute ischemia model significantly better than chronic ischemia, therefore may not produce the mechanism of directly inducing inflammatory factor to produce.
Advantage of the present invention is: the method by line bolt femoral artery can successfully prepare the lower limb ischemia model, and the operation principle is simple, animal postoperative survival rate height, ischemia good stability.By comparing with acute lower limb ischemia model, the chronic lower limb ischemia model ischemia time is long, has preserved the lower limb muscles organizational structure better, and there is position difference in trouble limb ischemic state.Membrane change is close with the human atherosclerotic speckle in the lumen of vessels, more approaching patient performance clinically.There is significant difference in chronic lower limb ischemia model in the posterior-limb ischemia distributions: the gastrocnemius ischemic state is the most serious, and is consistent with the diseased region of human atherosclerotic obliterans and patient with diabetic feet, is more suitable for the observation of curative effect.The chronic lower limb ischemia model that line is fastened the method preparation 2 weeks of modeling postoperative relatively is being fit to carry out intervention experiment research.
The invention will be further described below in conjunction with the specific embodiment; be not the qualification to invention, according to prior art well known in the art, embodiments of the present invention are not limited to this; therefore all this areas of having done according to present disclosure be equal to replacement, all belong to protection scope of the present invention.
Description of drawings
Fig. 1 is the imaging of animal ischemia model laser-Doppler blood perfusion: Figure 1A is one week of acute postoperative; Figure 1B is chronic one week of postoperative.Found that early stage after surgery acute posterior-limb ischemia model suffers from that the limb blood flow significantly reduces and the chronic posterior-limb ischemia model blood flow is minimizing trend.
Fig. 2 A is the blood flow analysis of chronic lower limb ischemia animal model; Fig. 2 B is the blood flow analysis of acute lower limb ischemia animal model; Fig. 2 C is the analysis of blood pressure of chronic lower limb ischemia animal model; Fig. 2 D is the analysis of blood pressure of acute lower limb ischemia animal model.
The specific embodiment
Embodiment 1: the modeling experiment
One. laboratory animal and grouping:
1.SPF level Lewis rat, male, body weight 250～300g is provided by Beijing Vital River Experimental Animals Technology Co., Ltd., and credit number is SCXK (capital) 2007-0001.Totally 24.The raising condition: 23～25 ℃ of room temperatures, to put in the rat Rotating Stainless Steel Cage tool and raise, 5 in every cage is freely taken the photograph water and is ingested.
2.20 only laboratory animal adopts the method for random packet to be divided into two groups, acute lower limb ischemia group and chronic lower limb ischemia group, 10 every group.In addition 3 are carried out line and fasten thing and select test, 1 the left iliac artery excision+femoral artery of row excision.
Two. modelling:
1. acute lower limb ischemia model:
Chloral hydrate solution 4ul/kg intraperitoneal injection of anesthesia, it is fixing to lie on the back, the conventional preserved skin sterilization of left lower extremity, the skin of longitudinal incision left lower extremity center, from groin to knee joint.Sharp property is isolated femoral artery under groin, scope from inguinal ligament until knee joint.The ligation femoral artery is in all branches of thigh portion respectively.Femoral artery is cut off under inguinal ligament, and dual ligation near-end is peeled off to knee joint vascular bifurcation place to the sharp property of far-end subsequently, and knot bundle popliteal tremulous pulse and peroneal artery, and art finishes, and sews up the incision.
2. chronic lower limb ischemia Preparation of model and line are fastened the selection of thing:
Anesthesia is the same, the conventional preserved skin sterilization of left lower extremity, the skin of longitudinal incision left lower extremity center, from groin to knee joint.In the groin root portion from femoral artery and femoral vein.0.5cm divides other places blocking-up femoral artery femoral artery nearly far-end more under inguinal ligament, and ligation femoral artery,superficial and other branches, cuts femoral artery, and direct-view is inserted the thread line to femoral artery knee joint place down.After the complete open femoral artery of art is seen no active hemorrhage, sew up the incision.
The blood vessel line is fastened the selection of thing: in limited scope, select suitable line to fasten thing, observe the reaction of lumen of vessels to it, we select the 4-0prolene line respectively for use, catgut, human hair carries out Preparation of model, and gets institute's vascular embolization on the 14th day in postoperative and carry out conventional H .E dyeing.
The research of embodiment 2. laboratory animal lower limb ischemia states
One. observation index and method
1. general state is observed:
The conventional feed water inlet of embodiment 1 postoperative animal, 5 one cages are raised.Observe the animal activity situation and suffer from limb blood fortune, animal carried out ischemic state and assesses evaluation index: (0=is normal, and it is pale that 1+=suffers from limb, the score of 2+=blood pressure, the downright bad or intermittent limping of 3+=) in respectively at postoperative the 3rd, 7 in 14,21,28,42 days
2. laser-Doppler blood flow imaging and laser-Doppler laser blood flowmeter (Sweden lark prestige Medical Instruments China company) detect:
Every treated animal is got 2 (n=2) week row laser-Doppler blood flow imaging inspection after surgery respectively.And in postoperative the 3rd, 7,14,28,42 days, every group get 2 (n=2) conventional anesthesia after, row laser-Doppler hind leg blood flowmeter detects.
Experimental procedure: the conventional row of Lewis rat intraperitoneal injection chloral hydrate anesthesia.Animal is got dorsal position, and two upper limb are fixed, two lower limb free positions.After keeping 28 degrees centigrade of room temperatures constant relatively,, place the laser-Doppler probe in forefoot position, the vola of Lewis rat.Continuous measurement is also write down the lower extremity blood flow curve, treats behind the blood flow value stabilization test constantly 3 minutes, record bilateral blood flow basic value.Ratio to two groups of operation sides and non-operation side limbs basis blood perfusion values (perfusion unit PU) compares research.
3. laser-Doppler lower limb thigh portion blood pressure determination:
In postoperative the 3rd, 7,14,28,42 days, every group get 2 (n=2) conventional anesthesia after, the blood pressure determination of row laser-Doppler lower limb.
Experimental procedure: the conventional row of Lewis rat intraperitoneal injection chloral hydrate anesthesia.Animal is got dorsal position, and two upper limb are fixed, and two lower limb free positions are widened the 1.2cm cuff outward respectively at two lower limb thigh roots and placed the general probe of reining in of laser in the vola simultaneously.After cuff was forced into 200mmhg, decompression gradually saw that corresponding blood flow measurement curve begins to descend this moment.The blood flow situation of change is write down in lasting decompression simultaneously, the pairing pressure value of starting point that begins to recover with blood flow is this side limbs systolic pressure, continuous measurement is averaged for three times and is Ipsilateral and strong side pressure value, and the ratio of two groups of operations sides and non-operation side blood pressures is compared research.
4. lower extremities ct angiography:
Laboratory animal is in postoperative the 3rd day, and the 7th day, the 14th day, the 28th day, 42 days, get two (n=2) respectively for every group, carry out two lower extremities ct angiography arts through the abdominal cavity.
Experimental procedure: behind abdominal cavity chloral hydrate injecting anesthetic, open abdomen, cut off posterior peritoneum, sharp property separation infrarenal abdominal aorta, postcava, near-end blocking-up ventral aorta.After blood vessel remaining needle punctures with infrarenal abdominal aorta far away in blocking-up, sees the activeness blood back, injecting heparin normal saline 10ml.Animal is got dorsal position, and two lower limb are fixed in and stretch the position.Adjust contrast machine pipe ball in two lower limb scopes.Speed injection of contrast medium (cardiografin) with 2ml/s is total to 3-5ml continuously, simultaneously two lower limb is taken pictures continuously, handles the radiography result with the method for Digital Subtraction.
Interpretation of result: taking the photograph sheet when developing back 1.5s with the ventral aorta crotch is foundation, calculates the number of blood vessel by femur middle point vertical line, the open degree of expression collateral blood vessels.(two independently observer to vertically counting) by the collateral blood vessels of femur mid point
5. histological examination
(1) HE dyeing: visualization is finished, and adopts the method that strengthens anaesthesia dosage to put to death rat.Get the two lower limb quadriceps femoris of rat respectively, gastrocnemius stage casing fixed position and fixed volume muscular tissue cut the femoral artery of line bolt, more than after formaldehyde fixed, use paraffin embedding, make the 10um slab, every section is dyeed with hematoxylin and Yihong.
After drawing materials, 10% neutral buffered formalin is fixed 24-48h; Take out the tissue after fixing, tap water fully soaks, through 70%, 80%, 90%, 95%, 100% each 2h of ethanol, to slough the moisture in the piece of tissue.Piece of tissue after the dehydration is soaked into transparent through dimethylbenzene, be put in the paraffin of thawing, makes wax immerse tissue and dimethylbenzene is replaced out; Carry out embedding behind the waxdip.Slice thickness 4 μ m.
Hematoxylin--Yihong staining: section is gone into dimethylbenzene 2 times, and each 5-10min is to slough paraffin.Go into the ethanol that concentration is successively decreased, about 5min of per step.Add distilled water, add Harris hematoxylin dye liquor dyeing 1.5min,, add 1% hydrochloride alcohol color separation with the unnecessary dye liquor of tap water flush away.Painted darker to nucleus, be advisable when other structures are colourless.The tap water oil blackeite, the distillation washing.Add 1% eosin stain dyeing 8min.Through the dehydration of alcohol that concentration increases progressively, dimethylbenzene is transparent, the neutral gum mounting.
(2) VIII factor immunohistochemical staining (test kit is purchased in Wuhan doctor's moral gon company): acute ischemia group and chronic ischemia treated animal be respectively at postoperative 7 days (n=2), 14 days (n=2), visualization after a large amount of chloral hydrate lumbar injection put to death.Cut bilateral lower limb muscles tissue, row VIII factor immunohistochemical staining after the formalin fixed.Be aided with Lecia micro image analysis system (brown particle number in the unit visual field) under the light microscopic.With positive cell number in the unit visual field/muscle fiber number, the expression capillary density.
The section routine dewaxes to water.PBS flushing, 3min * 3 time.3% hydrogen peroxide is eliminated endogenous peroxidase activity, incubated at room 10min.Distillation washing, PBS flushing, 3min * 3 time.Hot antigen retrieval, PBS flushing, 3min * 3 time.Drip one of suitably dilution and resist, (Von Willibrand Factor, 1:200), 4 ℃ are spent the night.PBS flushing, 3min * 3 time.Drip goat anti-rabbit igg antibody-HRP polymer, incubated at room 40min.PBS flushing, 5min * 4 time.The DAB colour developing.Distilled water flushing, (haematoxylin redyeing), the conventional dehydration of section, dimethylbenzene is transparent, gummy mounting.
(3) α-actin immunohistochemical staining: acute ischemia group and chronic ischemia treated animal are respectively at postoperative 7 days (n=2), and visualization is after a large amount of chloral hydrate lumbar injection execution.Cut bilateral lower limb muscles tissue, be aided with Lecia micro image analysis system (brown particle number in the unit visual field) under row α-actin immunohistochemical staining light microscopic after the formalin fixed.With positive cell number in the unit visual field/muscle fiber number, expression small artery density.
The section routine dewaxes to water.PBS flushing, 3min * 3 time.3% hydrogen peroxide is eliminated endogenous peroxidase activity, incubated at room 10min.Distillation washing, PBS flushing, 3min * 3 time.Hot antigen retrieval, PBS flushing, 3min * 3 time.Drip one of suitably dilution and resist, (α-actin 1:1500), 4 ℃ are spent the night.PBS flushing, 3min * 3 time.Drip goat anti-rabbit igg antibody-HRP polymer, incubated at room 40min.PBS flushing, 5min * 4 time.The DAB colour developing.Distilled water flushing, (haematoxylin redyeing), the conventional dehydration of section, dimethylbenzene is transparent, gummy mounting.
6. different parts muscular tissue realtime-PCR detects:
(1) primer sequence is synthetic: institute's primer sequence that adopts is held up Bioisystech Co., Ltd of section by Beijing and is synthesized all from document.
Table 1: the detection of the rat muscle inner cell factor, detect gene primer sequence and characteristics.
(2) tissue T rizol extracts total RNA
Behind the cell centrifugation, abandon supernatant.Add 1000ul Trizol, lash repeatedly, concuss.Placed room temperature 5 minutes.Get 200ul chloroform (chloroform), the concussion mixing left standstill 3 minutes.4 ℃ of centrifugal 15min of 13000r/min then.Draw the upper strata water and (note not contacting intermediary albumin layer) in accordingly new EP pipe, then, add the isopropyl alcohol of about equal volume, mixing leaves standstill 10min under the room temperature.4 ℃ of centrifugal 10min of 13000r/min.Remove supernatant, go again to get rid of, blot the liquid of the inside, clean the inside again after, add 75% ethanol of 1ml; Turn upside down several times 4 ℃ of centrifugal 5min of 13000r/min.Remove supernatant, go again to get rid of, blot the liquid of the inside, clean the inside again after, add 75% ethanol of 1ml; Turn upside down several times 4 ℃ of centrifugal 5min of 13000r/min.Outwell supernatant, get rid of, blot the liquid of clean the inside, according to precipitation size DEPC water dissolution.
(3) RNA detects:
A. the mensuration of concentration and purity: get the 1ul cell total rna, add 0.1%DEPC treating water 3ml.Make blank with the 0.1%DEPC treating water, read optical density value (OD value) under 260nm, 280nm and the 230nm wavelength respectively, change when wavelength is measured at every turn and all should readjust zero point at ultraviolet-uisible spectrophotometer.Calculate A260nm/A280nm respectively, the ratio of A260nm/A230nm.Calculate total rna concentration according to formula:
Total rna concentration (the ug/ul)=A260nm value of reading * 40 * extension rate/1000
B. total RNA integrity detection (denaturing formaldehyde agarose gel electrophoresis method): prepare 1% denaturing formaldehyde agarose gel.The total RNA of application of sample: 4.5ul (about 20-30ug), 2.0ul 5 * MOPS, 3.5ul formaldehyde, 10.0ul Methanamide are total to 20.0ul in the 0.5mlEppendorf pipe in the following order, and mixing is centrifugal.65 ℃ of water-baths 15 minutes, ice bath.Get above-mentioned mixed liquor 10.0ul, add 6 * RNA sample loading buffer 2ul, application of sample is in 1% denaturing formaldehyde agarose gel well behind the mixing.On 1% denaturing formaldehyde agarose gel, carry out horizontal strip electrophoresis, voltage 5V/cm, electrophoresis time 40 minutes.Under visible ultraviolet light detector 254nm ultraviolet light perspective, the result is directly observed.
(4) cDNA's is synthetic
A. reverse transcription: in sterile tube, add following sample:
Total RNA 1-10ul (1ng-2ng)
Oligo?Dt 2ul
Dntp 4ul
Add the nuclease free contaminant water to 16ul
B.70 ℃ heating is 5 minutes, of short duration centrifugal being placed on ice; Mix following sample:
Above-mentioned product 16ul (total RNA, Oligo Dt, Dntp, nuclease free contaminant water)
10 * RT reaction buffer 2ul
RNase inhibitor 1ul
M-MuLv reverse transcription 1ul
Cumulative volume 20ul
C.42 ℃ incubation is 1 hour; 95 ℃ made enzyme deactivation in 5 minutes; Survey its concentration with the SMA3000 quantitative instrument, and be diluted between the 50-100ng/ul, help the PCR reaction and carry out;
(5)Realtime?PCR
A. reaction system: 10 * Buffer 1ul
dNTP(10mM) 0.25ul
primer 2ul
Taq enzyme 0.1ul
SYBR?Green?I 0.5ul
Template cDNA 1ul
ddH20 5.15ul
Cumulative volume 10ul
B. response procedures: pre-95 ℃ of 2min of degeneration
94 ℃ of 30 circulation degeneration, 10sec
Anneal 60 ℃ ± 10 ℃ 10sec
Extend 68 ℃/72 ℃, 30sec
Read plate
50～95 ℃ of solubility curves
4 ℃ of cessation reaction coolings, ∞
(6) date processing: calculate
Folds＝2
-△△Ct
△△Ct＝(Ct1-Ct2)-(Ct3-Ct4)
Ct1: the Ct that handles the sample testing gene
Ct2: the Ct that handles the sample house-keeping gene
Ct3: the Ct of control sample testing gene
Ct4: the Ct of control sample house-keeping gene
If Folds is high expresseds greater than 2, if be low the expression less than 0.5.
Two. statistical analysis:
Adopt the SPSS11.5 statistical software that experimental result is analyzed, (X ± S) expression relatively adopts the t check of variance analysis or two independent sample means to each batch total amount data between group, the relatively employing x2 check of rate between each group with mean ± standard deviation.The check significance is got α=0.05, and there is significant difference P＜0.05.
Three. experimental result
1. lower limb ischemia clinical observation:
(1) acute lower limb ischemia group:
Acute lower limb ischemia model after surgery 3 lower limb foot pale (n=3) promptly appears, the limping of lower limb (n=2) appears at 48 days viewing duration by a definite date, cyanosis (n=2), pale (n=3), the atrophy of muscle (n=1). be divided into 2+ in two weeks after surgery, be divided into 1+ at 48 days, we are to a Lewis rats underwent left side iliac artery excision+left lower extremity femoral artery excision in addition, the ischemic necrosis of lower limb promptly appearred on the 1st in postoperative, death during radiography after three days.(as table 2)
(2) chronic lower limb ischemia model group:
Chronic lower limb ischemia model after surgery more shows as the property crossed a trouble limb cyanosis, but spontaneous remission in several minutes subsequently.Only have 1 (n=1) to show as lower limb in viewing duration and walk lamely, but minority has the phenomenon (n=2) of the atrophy of muscle, but find no the appearance of lower limbs necrosis and ulcer, this is close with relevant list of references.(as table 2)
Table 2: acute lower limb ischemia and chronic lower limb ischemia model clinical observation table
*Be row left side iliac artery excision+left femoral artery excision
2. laser-Doppler blood perfusion imager check result:
We have the 7th day after surgery, acute group model and chronic group model have been carried out the inspection of laser-Doppler hind leg blood perfusion imager respectively, find the acute ischemia model in the week after surgery Ipsilateral hind leg blood flow with respect to the chronic ischemia model, blood flow significantly reduces, and chronic posterior-limb ischemia model hind leg blood flow is the part minimizing.(Figure 1A and Figure 1B)
3. laser-Doppler hind leg blood flow detection result:
(1) acute ischemia rat model hind leg blood flow detection result: the blood flow of suffering from limb the decline of blood flow promptly occurs in art after one week, is 40% of normal side limbs to minimum point around the after surgery, returns to more than 90% of offside (normally limbs) after surgery 6 weeks.(Fig. 2 B).
(2) chronic lower limb ischemia rat model hind leg blood flow detection result: the decrease speed of suffering from the limb blood flow is slower than acute lower limb ischemia model, and the plateau of a period of time occurs.It is 50% of normal side LBF that but minimum point second week appears in blood flow after surgery, and the blood flow of suffering from limb then recovers gradually, returns to 80% of offside (normal limbs) again in the 6th week of postoperative, is lower than the acute ischemia group.(Fig. 2 A).
4. laser-Doppler hind leg blood pressure testing result:
(1) acute posterior-limb ischemia model: blood pressure obviously descended in second day after surgery, and second all blood pressure drops are to minimum point, after recover gradually, to the 6th week of postoperative recovering normal substantially.(Fig. 2 D)
(2) chronic posterior-limb ischemia model: after postoperative first week arrival blood pressure was minimum, longer recovery time with respect to the acute ischemia group, the 6th week still presented ischemic state after surgery.(Fig. 2 C)
5. animal model angiography result:
(1) in the acute ischemia model group: angiographic results shows that ischemia side far-end does not have blood vessel and develops, and suffers from limb the 7th day after surgery, and it is open that side Zhi Xunhuan promptly appears in thigh portion, shows as the little blood vessel quantity of Ipsilateral more than strong side.The 6th week peaked after surgery, and Ipsilateral blood vessel quantity is significantly higher than strong side.The 8th all both sides collatoral vessel quantity is basic identical after surgery.Row left side iliac artery+femoral artery excision animal blood vessels radiography shows: the trouble limb does not have the blood vessel development and does not also see that side Zhi Xunhuan sets up.
(2) chronic ischemia model group: angiography was presented at postoperative 7 days, and the blood vessel of institute's thromboembolism develops substantially, but had the filling defect of part, and the 14th day after surgery, still have the part blood vessel to develop, the contrast agent color is thin out, and far-end develops not good.Side is propped up the systemic vascular number and analyzed discovery by statistics: do not see tangible side Zhi Kaifang in the 7th day after surgery, collatoral vessel increased in the 14th day, but femoral artery attenuates.And 42 days after surgery, operation side limbs collatoral vessel number still is lower than strong side.
6. ischemia model muscular tissue H.E coloration result:
(1) acute posterior-limb ischemia model: the Lewis rat lower limb muscles portion of tissue HE discovery of dyeing: most of muscular tissue is not found significantly downright bad performance.Minority (n=4) shows as: the necrosis of branch point-like partly appears in the early stage ischemia meat of postoperative, show as the endochylema uniformity, to mid-term occurring the destruction of myoarchitecture, during massive inflammatory cells infiltrated, atrophy appears in the muscle of later stage lower limb, and showing as spatium intermusculare significantly increases.
(2) chronic posterior-limb lacks model: dyeing is found through H.E: Lewis rat ischemia position muscular tissue structure is intact, do not find muscular death, but the especially interior visible significantly infiltration of inflammatory cell of gastrocnemius of part rat hindlimb muscle is arranged, occur atrophic in the later stage part muscular tissue and change (n=2), showing as the muscle bundle gap increases, but does not find that tangible fibrosis changes.
(3) to inserting 4-Oprolene line and catgut, the blood vessel HE dyeing of hair is found:
The 4-Oprolene line: the histological structure of blood vessel is intact, and the hypertrophy of tunica intima has promptly appearred in 2 weeks after surgery, does not see the formation of thrombosis.To a large amount of hypertrophy of later stage, almost caused blood vessel inaccessible completely owing to tunica intima.
Hair: in tube chamber, insert hair and also can cause certain neointimal hyperplasia, but a large amount of inflammatory cell infiltration of the omnidistance appearance of blood vessel wall does not cause tangible physical property narrow to tube chamber.
Catgut: do not find the hypertrophy of inner membrance for inserting the visible a large amount of inflammatory cell infiltration of catgut blood vessel wall in the tube chamber, destroying appears in inner membrance, and tube chamber is almost occupied by catgut.
7. ischemic muscle VIII factor immunohistochemical staining result:
We to the animal of lower limb ischemia model respectively at postoperative 7 days, 14 days.Strong side and Ipsilateral lower limb muscles carry out VIII factor immunohistochemical staining result respectively and show: positive findings is positioned the endotheliocyte of blood vessel, the blood capillary stained positive be the dark-brown spot distribution between muscle fiber, what have is tangible tubular structure.
(1) acute ischemia and chronic ischemia VIII factor positive cell number comparative result: early stage at acute ischemia, VIII factor positive cell number significantly reduces in ischemic tissue.Chronic lower limb ischemia model, trouble limb muscle is early stage after surgery, suffers from limb intramuscular VIII factor positive cell number and does not see obvious decline.
(2) acute ischemia and chronic ischemia blood capillary/muscle fiber ratios (capillary density): prepared successfully back 7 days at ischemia model, the chronic ischemia model is suffered from the limb capillary density and is significantly less than acute ischemia model trouble limb, 14 days after surgery, chronic posterior-limb ischemia model was suffered from the limb capillary density apparently higher than the acute ischemia model.(table 3)
Table 3: the early stage after surgery capillary density of acute ischemia and chronic ischemia relatively
1: anxious strong; 2: anxious suffering from; 3: strong slowly; 4: suffer from slowly
Anxious being good for-strong slowly: P=0.034;
Anxious being good for-suffer from slowly: P=0.006;
Anxious strong 1w-2w:t=-13.931, P=0.004
Anxious 1w-2w:t=-7.936, the P=0.015 of suffering from
All the other respectively organize indifference.
8. ischemic muscle a-actin immunohistochemical staining result
Acute ischemia group and chronic ischemia treated animal be respectively at postoperative 7 days, row a-actin immunohistochemical staining after the formalin fixed, and the result shows: positive findings is positioned in the endochylema of arteriolar smooth muscle cell; Small artery dyeing for dark-brown in tubular construction, be distributed in spatium intermusculare.
(1) acute posterior-limb ischemia model and chronic posterior-limb ischemia model a-actin positive cell number comparative result: at acute ischemia early stage (postoperative 7 days), the a-actin positive cell number is apparently higher than strong pleural muscle meat in the acute ischemia tissue, chronic ischemia models show different parts a-actin is positive, and the quantity difference is not obvious, but obviously is less than acute posterior-limb ischemia model.(as Fig. 8-1)
(2) acute posterior-limb ischemia model and chronic posterior-limb ischemia model muscle small artery/muscle fiber ratios (small artery density) coloration result: the 7th day after surgery small artery density of chronic posterior-limb ischemia model quadriceps femoris is significantly less than acute posterior-limb ischemia model quadriceps femoris (table 4); Chronic posterior-limb ischemia model is in the limbs of the same side, and small artery density is but apparently higher than quadriceps femoris in the gastrocnemius.(table 5)
Table 4: acute and chronic small artery density data (n=3)
LSD check: ★: compare P=0.000 with anxious strong group; Compare P=0.007 with strong group slowly; Compare P=0.000 with the group of trouble slowly; ▲: compare P=0.000 with the group of trouble slowly.
Table 5: chronic posterior-limb ischemia model hind leg different parts small artery density data (n=3)
LSD check: ★: compare P=0.011 with strong burst of group; Compare P=0.017 with strong calf group; Compare P=0.006 with trouble calf group.
9. ischemic muscle VEGF, Ang-2, SDF-1, Flk-1mRNA RT-PCR result:
The intramuscular a-actin internal reference crt gene of the strong side limbs of acute ischemia model is adopted in this test, and destination gene expression and its ratio (fold) value are as a result of in other position muscular tissue: Folds is high expresseds greater than 2, if be to hang down expression less than 0.5.
(1) acute posterior-limb ischemia model: compare with strong pleural muscle meat tissue, acute posterior-limb ischemia model was suffered from the limb intramuscular the 14th day after surgery, VEGF, and SDF-1, the expression of ANG-2 and FLK-1 mRNA does not have remarkable increase.
(2) chronic posterior-limb ischemia model: compare with strong pleural muscle meat tissue, the chronic ischemia model is at Ipsilateral VEGF, and SDF-1, ANG-2 and FLK-1mRNA obviously are the high expressed state in gastrocnemius, expresses and be low in the Ipsilateral quadriceps femoris.
Sequence table
＜110〉Xuanwu Hospital of Capital University of Medical Science
＜120〉a kind of preparation method of rat chronic posterior-limb ischemia model
<130>
<160> 8
<170> PatentIn?version?3.5
<210> 1
<211> 21
<212> DNA
＜213〉synthetic VEGF forward primer
<400> 1
<210> 2
<211> 21
<212> DNA
＜213〉synthetic VEGF downstream primer
<400> 2
<210> 3
<211> 21
<212> DNA
＜213〉synthetic Ang-2 forward primer
<400> 3
<210> 4
<211> 21
<212> DNA
＜213〉synthetic Ang-2 downstream primer
<400> 4
<210> 5
<211> 20
<212> DNA
＜213〉synthetic SDF-1 forward primer
<400> 5
<210> 6
<211> 21
<212> DNA
＜213〉synthetic SDF-1 downstream primer
<400> 6
<210> 7
<211> 18
<212> DNA
＜213〉synthetic β-Actin forward primer
<400> 7
<210> 8
<211> 18
<212> DNA
＜213〉synthetic β-Actin downstream primer
<400> 8
Claims (6)
1. the preparation method of a rat chronic posterior-limb ischemia model is characterized in that: insert line bolt material in the femoral artery of laboratory animal near knee joint, sew up the incision; Cause the narrow and generation foreign body reaction of arterial lumen, make to produce neointimal hyperplasia in the tube chamber gradually, cause the minimizing of lower extremity blood flow gradually, set up the chronic posterior-limb ischemia animal model.
2. the preparation method of a kind of rat chronic posterior-limb ischemia model according to claim 1, it is characterized in that: near described line bolt material to the method the knee joint of inserting in the femoral artery of laboratory animal is: the skin of longitudinal incision lower limb center, from groin to knee joint; In the groin root portion from femoral artery and femoral vein; 0.5cm divides other places blocking-up femoral artery femoral artery nearly far-end more under inguinal ligament, and ligation femoral artery,superficial and other branches, cuts femoral artery, and direct-view is inserted line bolt line of material down to femoral artery knee joint place; After the complete open femoral artery of art is seen no active hemorrhage, sew up the incision.
3. the preparation method of a kind of rat chronic posterior-limb ischemia model according to claim 1 and 2, it is characterized in that: described laboratory animal can also be rabbit, dog, sheep or pig.
4. the preparation method of a kind of rat chronic posterior-limb ischemia model according to claim 1 and 2, it is characterized in that: described laboratory animal is the Lewis rat.
5. the preparation method of a kind of rat chronic posterior-limb ischemia model according to claim 1 and 2, it is characterized in that: described line bolt material is 4-0prolene line, hair or catgut.
6. the preparation method of a kind of rat chronic posterior-limb ischemia model according to claim 5, it is characterized in that: described line bolt material is the 4-0prolene line.
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| CN103750800A (en) * | 2011-12-30 | 2014-04-30 | 广州宝胆医疗器械科技有限公司 | Capsule enteroscope system with electronic CCD (charge coupled device) camera system and Doppler laser system |
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