CN104523348A - Upper-jiao fluid retention stagnating in the interior rat model and construction method thereof - Google Patents
Upper-jiao fluid retention stagnating in the interior rat model and construction method thereof Download PDFInfo
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Abstract
The invention discloses an upper-jiao fluid retention stagnating in the interior rat model construction method. The method includes the steps of (1) placing an SPF Wistar rat in a purification cage with separate air supply and air exhaust, feeding the rat with common food and enabling the rat to eat food and drink water freely; (2) subcutaneously injecting the Wistar rat of step (1) with isoproterenol of 20 mg/kg on the first day, 10 mg/kg on the second day, 5 mg/kg on the third day and 3 mg/kg/d from the fourth day for 20 days and observing for two weeks; (3) arranging a trachea built-in catheter in the trachea of the Wistar rat; (4) placing the Wistar rat with the trachea built-in catheter in a freezer at the temperature of 0 DEG C for 4 hours every day to accept cold stimulation for seven consecutive days to obtain a upper-jiao fluid retention stagnating in the interior rat model. The upper-jiao fluid retaining rat model construction method solves the problem of no upper-jiao fluid retention stagnating in the interior rat model currently, provides a new method for normal, standard and scientific evaluation of traditional Chinese medicine and has the advantages of being simple in model construction, easy to operate and popularize, similar to clinic and the like.
Description
Technical field
The present invention relates to a kind of part of the body cavity above the diaphragm housing the heart and lungs fluid-retention stagnating in the interior rat model and construction method thereof, belong to medical care evaluation, detection technique field.
Background technology
The traditional Chinese medical science thinks that lung occupy the part of the body cavity above the diaphragm housing the heart and lungs, therefore claims " canopy " to be referred to as again in " on water source ".The main Physiological Function of lung is main gas, department breathes, main Xuan Fasu falls, regulating fluid apssage.Operation compress and a surname of lung of body fluid fall move relevant." spleen-QI transporting the essence to the whole body, is above attributed to lung, " lung by a surname outbreak with by body fluid compress to all keys of women's head-ornaments, fur space between skin and muscles.And through its respectful effect of falling, body fluid being delivered to other internal organs, bet bladder, so then crystal four cloth, the Five Classics walks abreast, and internal organs and whole body all obtain nutrition.The lung being the upper source of fluids, dereliction of duty falls in lung qi a surname, and crystal must not four cloth, then in prolonged fluid-retention and lung for expectorant is for drink.Phlegm retention is accumulate lung and lung qi can be caused again strongly fragrant and not declare or inverse and do not fall, i.e. so-called " expectorant becomes because of gas, and gas becomes because of expectorant ".
Part of the body cavity above the diaphragm housing the heart and lungs fluid-retention stagnating in the interior is clinical common pattern of syndrome, and many chronic diseases are all demonstrate,proved with part of the body cavity above the diaphragm housing the heart and lungs fluid-retention stagnating in the interior.The reasons such as it is mainly due to airway obstruction, affection of exogenous wind-cold, congestion resistance lung cause impairment of dispersing and descending function of the lung, and logical tune is neglected one's duty, and polywater is phlegm retention." through twelve interior organs's shape scape seal in Thorough Knowledge of Medicine ": " be lung under larynx, two leaves are lustrous and transparent in vain, the canopy of meaning, all dirty to cover, empty as Nidus Vespae, the saturating key of lower unit, therefore suction is then full, and that exhales is then empty, and one exhales a suction, and originally active, without also finite, is pure and impure shipping, bag a unit of measure used in ancient China of the person ".Lung is bag a unit of measure used in ancient China of breathings, is that clearing heat in QI system sucks, the product institute that foul smell is discharged, and this is pure and impure ships is that lung declares the concrete manifestation sending out respectful to fall.Airway obstruction, be then an impediment to the coordination mutual aid that lung a surname sends out respectful to fall, and causes water to wet and stop gathering.Jing-Yue Complete Works cloud: " expectorant of wind and cold with heresy from fur, invasion and attack and lung, the unclear and even raw expectorant of lung qi ".Visible affection by exopathogenic cold-wind, inside gives up in lung, affects the gasification of lung, failure of the lung to distribute body fluid to all parts of the body, can coagulate for phlegm retention.Teacher Guan Youbo also proposes " expectorant and blood belong to the moon together, are easy to cementing and solidify, and QI and blood smoothness then body fluid walks abreast, and without expectorant with life, qi depression to blood stasis expectorant is tied " is that the silt of speech blood hinders and raw expectorant." gold is deficient " edema with the heart involved person, its body weak breath, must not crouch, restlessness and irritability, its people's swelling of the vulva.These descriptions are similar to heart failure clinical symptoms.Visible the heart controlling blood circulation, blood is unfavorable, and congestion, once be formed, also can affect impairment of dispersing and descending function of the lung, water is wet and stops consor phlegm retention.
Effectively Chinese medicine is treated, the necessary research carried out part of the body cavity above the diaphragm housing the heart and lungs fluid-retention stagnating in the interior rat model and copy for further further investigated part of the body cavity above the diaphragm housing the heart and lungs fluid-retention stagnating in the interior is essential and seek it.The biological characteristics of animal need be considered during model copy, select similar to human physiology, metabolism, disease feature, the animal of Pass Test object as far as possible.Due to the complexity that animal symptom, sign gather, therefore the animal model of rationality is not only required on symptom and sign close with people or identical, more needs the objective indicator with quantizing to carry out accurate evaluation.
At present, yet there are no part of the body cavity above the diaphragm housing the heart and lungs fluid-retention stagnating in the interior rat model.Rarely seen expectorant is turbid accumulates lung, and cold fluid-retention accumulates lung, waits the report of rat model, adopts the method modeling of fumigation, tracheal instillation lipopolysaccharide, additional cold stimulation more.But the modeling that the cause of disease, performance etc. meet the guidance of theory of Chinese medical science is actually rare, the animal model meeting tcm symptom feature is needed in the research of tcm syndrome badly.
Summary of the invention
For above-mentioned prior art, the object of this invention is to provide a kind of part of the body cavity above the diaphragm housing the heart and lungs fluid-retention stagnating in the interior rat model and construction method thereof, with impairment of dispersing and descending function of the lung fluid-retention stagnating in the interior Pathogenesis Theory for instructing, adopt rat trachea intubate, in conjunction with abdominal part hypodermic isoproterenol, additional cold stimulation simulation impairment of dispersing and descending function of the lung, and design special sequential, induce 40 days with rat, set up a kind of part of the body cavity above the diaphragm housing the heart and lungs fluid-retention stagnating in the interior rat model.And mainly wetting from water that this evaluates on the one hand, result shows that the method successfully can copy part of the body cavity above the diaphragm housing the heart and lungs fluid-retention stagnating in the interior animal model, and this clone method stability, reproducible, simple, objective, meets the guidance of theory of Chinese medical science.
For achieving the above object, the present invention adopts following technical proposals:
A construction method for part of the body cavity above the diaphragm housing the heart and lungs fluid-retention stagnating in the interior rat model, comprises the following steps:
(1) your (Wistar) rat of no-special pathogen (SPF) level Vist is placed in independent air-blowing-returning purification cage (IVC), raises with normal diet, ad lib, drinking-water;
(2) give isoproterenol abdominal part hypodermic to Wistar rat, first day 20mg/kg, second day 10mg/kg, the 3rd day 5mg/kg, 3mg/kg.d from the 4th day, continuous 20 days, observe two weeks;
(3) circulation of qi promoting pipe built-in pipe: the 34th day, uses tracheal inserting device (comprise trachea cannula outer sleeve, interior pipe, built-in pipe is placed on Inner pipe top) to carry out inserting of trachea built-in pipe.Rat lies on the back fixing, and air flue, above esophagus, gag is chosen, visible gas port, glottis is the folding of fish mouth shape, by intubate radian upward, get hold of rats breathing rhythm, take advantage of glottis to open moment and rapid ready Intubaction device is inserted rat trachea to labelling position, 1cm under glottis, first extracts out Inner pipe, by trocar sheath, the trachea built-in pipe on top stops that rear disconnection is connected with device, extract trocar sheath out, built-in pipe is just placed in animal trachea, retains 7 days;
(4) the Wistar rat after circulation of qi promoting pipe built-in pipe is placed in the refrigerator-freezer 4 hours that temperature is 0 DEG C every day, accepts cold stimulation, continuous 7 days.Obtain part of the body cavity above the diaphragm housing the heart and lungs fluid-retention stagnating in the interior rat model.
In modeling process, observe rat general status.After model is set up and is terminated, objective indicator using cardiopulmonary histopathology as evaluation of tissue degree of injury, carrys out the success or not of overall merit part of the body cavity above the diaphragm housing the heart and lungs fluid-retention stagnating in the interior Establishment of Rat Model in conjunction with other Heart and lungs injury indexs (lung permeation index, bronchoalveolar lavage fluid yield, lung wet dry weight express than W/D, cardiopulmonary organ index, creatine kinase CK).
The present invention mainly wets from water, and this evaluates part of the body cavity above the diaphragm housing the heart and lungs fluid-retention stagnating in the interior rat model on the one hand, and concrete evaluation index is as follows:
1. general status is observed: model group rats occurs that dry skin and hair is dark and gloomy, rapid breathing, with sound of stridulating, and the symptoms such as words mucosa is purple dark, and nose has a small amount of courageous and upright secretions, activity difference;
2. lung permeation index, bronchoalveolar lavage fluid yield: statistical result shows that model group rats lung permeation index obviously raises (P<0.05) bronchoalveolar lavage fluid yield and obviously raises (P<0.05);
3. Rats Organs and Tissues index: statistical result shows that part of the body cavity above the diaphragm housing the heart and lungs fluid-retention stagnating in the interior model group rats cardiac weight and heart organ index significantly raise (P<0.01), the lung dry weight ratio that wets is increased significantly (P<0.05), and lung weight, lung organ index are on the rise;
4. creatine kinase CK: statistical result shows that in part of the body cavity above the diaphragm housing the heart and lungs fluid-retention stagnating in the interior model group rats serum, creatine kinase CK obviously raises (P<0.05);
5. lung tissue disease's Neo-Confucianism observation part of the body cavity above the diaphragm housing the heart and lungs fluid-retention stagnating in the interior model group rats lung tissue alveolar septum edema thickens, and inflammatory cell infiltration, is full of pink edematous fluid in alveolar space, in homogenizing shape, have pulmonary edema to exist;
6. heart tissue's pathological observation part of the body cavity above the diaphragm housing the heart and lungs fluid-retention stagnating in the interior model group rats cardiac myocyte hypertrophy, part myocardial cell has acidophilia's degeneration and (or) necrosis.
Beneficial effect of the present invention:
(1) the invention solves the deficiency not having part of the body cavity above the diaphragm housing the heart and lungs fluid-retention stagnating in the interior rat model at present, for the evaluation Chinese medicine of specification, standard, science provides a kind of new method, have modeling method simple, be easy to operation and promote, the advantages such as approach clinic.
(2) modeling method stability of the present invention, reproducible, simple, objective, meets the guidance of theory of Chinese medical science.
(3) the present invention is using Wistar rat as the experimental animal of modeling, that zoopery rat kind is commonly used the most and uses the kind that history is the longest in biomedical research, be widely used in the experiment in biomedical each field, close with people on its symptom and sign, modeling more rationality, objective.
Accompanying drawing explanation
Fig. 1 lung tissue of rats pathology optical microscope figure;
In figure, 1 is blank group lung tissue of rats hematoxylin-eosin stains × 40,2 is model group rats lung hematoxylin-eosin stains tissue × 40, and 3 is blank group lung tissue of rats hematoxylin-eosin stains × 100, and 4 is model group rats lung tissue hematoxylin-eosin staining procedures × 100;
Fig. 2 rat heart tissue pathology optical microscope figure;
In figure, 1 is blank group rat heart tissue hematoxylin-eosin stains × 200,2 is model group rats heart tissue hematoxylin-eosin stains × 200, and 3 is blank group rat heart tissue hematoxylin-eosin stains × 400, and 4 is model group rats heart tissue hematoxylin-eosin stains × 400.
Detailed description of the invention
The present invention is further illustrated in conjunction with the embodiments, should be noted that following explanation is only to explain the present invention, not limiting its content.
Embodiment 1: the structure of part of the body cavity above the diaphragm housing the heart and lungs fluid-retention stagnating in the interior rat model and evaluation thereof
1. experiment material
The healthy Vist that of 1.1 laboratory animals (Wistar) rat 40, male and female half and half, body weight (200 ± 20) g, purchased from Lu Kang Pharmaceutical Group limited company Experimental Animal Center (licence numbering SCXK Shandong 20130001).No-special pathogen (SPF) level environment is fed.
1.2 experimental apparatus L-20A type completely automated high speed refrigerated centrifuges, Hunan centrifuge factory; The multi-functional full-automatic microplate reader of RT-2100C type, Shenzhen Lei Du company; Analytical balance, Shenyang Longteng Electronic Co., Ltd.: Nikon scientific research microscope (NikonECLIPSE Ci-L), NIKON.
1.3 experiment reagent BCA determination of protein concentration test kits, purchased from Beijing DingGuo ChangSheng Biology Technology Co., Ltd.Rat creatine kinase (CK) enzyme-linked immunologic detecting kit, purchased from Beijing equation biotech firm.Isoproterenol, purchased from upper Haidah Er Da biotech firm.
2. experimental technique
2.1 models are set up
Your (Wistar) rat of no-special pathogen (SPF) level Vist is placed in independent air-blowing-returning purification cage (IVC), raises with normal diet, ad lib, drinking-water.Model group rats gives isoproterenol abdominal part hypodermic, and first day 20mg/kg, second day 10mg/kg, the 3rd day 5mg/kg, 3mg/kg/d from the 4th day, continuous 20 days, observe 2 weeks, blank group abdominal part hypodermic normal saline.
34th day, after model group rats gives chloral hydrate (3ml/kg) intraperitoneal injection of anesthesia of 10%, tracheal cannula device (comprise trachea cannula outer sleeve, interior pipe, built-in pipe is placed on Inner pipe top) is used to carry out inserting of trachea built-in pipe.Lain on the back by rat fixing, air flue, above esophagus, gag is chosen, visible gas port, glottis is the folding of fish mouth shape, by intubate radian upward, get hold of rats breathing rhythm, take advantage of glottis to open moment and rapid ready Intubaction device is inserted rat trachea to labelling position, 1cm under glottis, first extracts out Inner pipe, by trocar sheath, the trachea built-in pipe on top stops that rear disconnection is connected with device, extract trocar sheath out, built-in pipe is just placed in animal trachea, retains 7 days.After circulation of qi promoting pipe built-in pipe, 2:00-6:00, was placed in the refrigerator-freezer 4 hours that temperature is 0 DEG C, continuous 7 days, obtained part of the body cavity above the diaphragm housing the heart and lungs fluid-retention stagnating in the interior rat model by rat every afternoon.
The evaluation of 2.2 part of the body cavity above the diaphragm housing the heart and lungs fluid-retention stagnating in the interior rat models
2.2.1 lung permeation index (LPI) mensuration is carried out the abundant lavation of left bronchus alveolar with 4 DEG C of normal saline and reclaims bronchoalveolar lavage fluid (BALF) (response rate > 80%), and adopts BCA determination of protein concentration kit measurement bronchoalveolar lavage fluid in rats total protein and total serum protein.
Lung permeation index measures total protein concentration/total protein concentration of serum in (LPI)=BALF.The results are shown in Table 1.
2.2.2 bronchoalveolar lavage fluid yield measures and opens thoracic cavity, ligation postcava, below trachea crotch, dissociate ligation after right principal bronchus, to dissociate escape pipe in cervical region again, under trachea laryngeal cartilages, an inverse-T-shaped mouth is cut at 0.5cm place, 10ml syringe (removing syringe needle) is placed in trachea intracavity, reach 1cm place, fix with No. 0 stitching thread, connect 2ml syringe (including 1ml normal saline), slow flushing for several times, every minor tick about 30s, continuous 4 times (resorption rate is more than 80%), reclaims bronchoalveolar lavage fluid (BALF) and weighs volume.The results are shown in Table 1.
2.2.3 organ index measures and adopts analytical balance to weigh the heart, lung weight, and rat body weight measured by electronic balance, organ index=organ weights/body weight × 100%.The results are shown in Table 3.
2.2.4 the lung dry weight that wets gets rat superior lobe of right lung than mensuration, and weigh rearmounted 80 DEG C of oven cooking cycle 48h, is dried to constant weight.Lung wets dry weight (W/D)=weight in wet base/dry weight frequently.The results are shown in Table 2.
2.2.5 serum cardiac enzymatic determination adopts creatine kinase (CK) enzyme-linked immunologic detecting kit to measure the content of creatine kinase CK in rat blood serum.The results are shown in Table 4.
2.2.6 pathological study middle lobe of right lung lung tissue fixes 24h in 10% paraformaldehyde solution configured with PBS, thick about 5um lung tissue is cut along sagittal plane from right lung door, dehydration, transparent, waxdip, paraffin embedding, section, the heart is observed in row HE dyeing Nikon scientific research microscope (Nikon ECLIPSE Ci-L), lung morphology changes.Result is shown in Fig. 1 and Fig. 2 respectively.
2.2.7 date processing experimental data SPSS17.0 software statistics is analyzed, and continuous data all represents with mean ± standard deviation, and adopting one factor analysis of variance, is that difference has statistical significance with P<0.05.
3. result
3.1 rat general status is observed
Compared with blank group, model group rats occurs that dry skin and hair is dark and gloomy, rapid breathing, and with sound of stridulating, the symptoms such as words mucosa is purple dark, and nose has a small amount of courageous and upright secretions, activity difference, match with traditional Chinese medical science part of the body cavity above the diaphragm housing the heart and lungs fluid-retention stagnating in the interior syndrome.
3.2 induced lung permeation indexs and alveolar wass yield
Table 1 lung permeation index, bronchoalveolar lavage fluid pumpback amount compare (n=20, mean ± standard deviation)
Note: # compares with blank group, P<0.05.
The change of lung permeation index and the change of pulmonary capillary permeability proportional, be commonly used to evaluate pulmonary vascular endothelial cell and alveolar epithelial cells degree of impairment.Table 1 shows, and model group compares with matched group, and induced lung permeation index obviously raises (P<0.05).Increasing of vascular permeability and pulmonary can cause body fluid in interstitial lung and alveolar inner accumulated.Model group rats bronchoalveolar lavage fluid yield obviously raises (P<0.05), may have the retention of water liquid in hints model group alveolar.
3.3 lungs wet dry weight ratio (W/D).
Table 2 lung wets dry weight ratio (n=20, mean ± standard deviation)
Note: * compared with blank group, P<0.01; # compared with blank group, P<0.05.
The 3.4 rat hearts, pulmonary organ coefficient
Table 3 organ weights and organ index (n=20, mean ± standard deviation)
Note: * compared with blank group, P<0.01.
Table 2, table 3 show, model group compares with matched group, induced lung wets dry weight than obviously raising (P<0.05), and cardiac weight and heart organ index significantly raise (P<0.01), and lung weight, lung organ index are on the rise.Lung W/D, lung organ index are the easy of the reflection pulmonary edema order of severity, accurately responsive index.Lung (W/D) obviously raises, and occurs water retention in lung organ index hints model group on the rise induced lung.Heart organ index significance increases, and hints model treated animal causes cardiac function to be damaged because of myocardial damage, thus causes heart congestion, and blood volume is many.
3.5 Serum fibrosis markers
Table 4 serum creatine kinase CK (n=20, mean ± standard deviation)
Note: * compared with blank group, P<0.01.
Creatine kinase CK in Serum fibrosis markers is one of important diagnostic mark of myocardial ischemia.Table 4 shows, and model group and matched group compare, and in model group rats serum, creatine kinase CK obviously raises (P<0.05), hints model group rat myocardium from injury.
3.6 lungs, heart tissue's pathological observation
Show under lung tissue disease's light microscopic of science: Fig. 1 blank group lung tissue of rats clear in structure, alveolar wall structure is normal, alveolar size and form normal, interstitial lung has no hypertrophy, and alveolar septum thickness is normal, without the pulmonary collapse and vessel wall thickening, in bronchial lumen and alveolar space, have no inflammatory exudate; Model group rats lung tissue, alveolar wall telangiectasis is congested, and alveolar septum inflammatory cell infiltration, alveolar septum is widened, and is full of containing pink edematous fluid in alveolar, part visible red cell, leukocyte and the epithelial cell come off.Lung pathology observes directly prompting the existence of pulmonary edema.
Show under heart tissue's pathology light microscopic: Fig. 2 blank group visible cell form myofilament marshalling, muscle segment light and shade band is clear, mitochondrion along myofilament major axis marshalling, nuclear membrane complete display, chromatin is comparatively uniformly distributed; Model group heart tissue pathological observation cardiac myocyte hypertrophy, part myocardial cell has acidophilia's degeneration and (or) necrosis.
Thicken by the part of the body cavity above the diaphragm housing the heart and lungs fluid-retention stagnating in the interior rat model pathological section lung tissue alveolar septum edema of above-mentioned method establishment, inflammatory cell infiltration, is full of pink edematous fluid in most alveolar space, in homogenizing shape; Bronchoalveolar lavage fluid yield obviously raises, and dry weight that heart organ index significantly raises, lung wets is more on the rise than obviously rising, lung organ index, the success of prompting part of the body cavity above the diaphragm housing the heart and lungs fluid-retention stagnating in the interior rat model.
Claims (4)
1. a construction method for part of the body cavity above the diaphragm housing the heart and lungs fluid-retention stagnating in the interior rat model, is characterized in that, comprises the following steps:
(1) SPF level Wistar rat is placed in independent air-blowing-returning purification cage, raises with normal diet, ad lib, drinking-water;
(2) give isoproterenol abdominal part hypodermic to the Wistar rat of step (1), first day 20mg/kg, second day 10mg/kg, the 3rd day 5mg/kg, 3mg/kg.d from the 4th day, continuous 20 days, observe two weeks;
(3) circulation of qi promoting pipe built-in pipe, inserts in Wistar rat trachea by built-in pipe, retain 7 days;
(4) the Wistar rat after circulation of qi promoting pipe built-in pipe is placed in the refrigerator-freezer 4 hours that temperature is 0 DEG C every day, accepts cold stimulation, continuous 7 days, obtain part of the body cavity above the diaphragm housing the heart and lungs fluid-retention stagnating in the interior rat model.
2. the construction method of a kind of part of the body cavity above the diaphragm housing the heart and lungs fluid-retention stagnating in the interior rat model as claimed in claim 1, it is characterized in that, in step (3), the method for posting of described built-in pipe is: use tracheal inserting device to carry out inserting of built-in pipe, rat is lain on the back fixing, choose with on gag, visible gas port, glottis is the folding of fish mouth shape, by intubate radian upward, take advantage of glottis open moment rapid rat trachea that tracheal inserting device is inserted to labelling position, 1cm under glottis, first Inner pipe is extracted out, by trocar sheath, the built-in pipe on top stops that rear disconnection is connected with device, extract trocar sheath out, built-in pipe is just in implanted animal trachea.
3. the construction method of a kind of part of the body cavity above the diaphragm housing the heart and lungs fluid-retention stagnating in the interior rat model as claimed in claim 2, it is characterized in that, described tracheal inserting device, comprises trocar sheath, and interior pipe is positioned at described trocar sheath, and the top of described interior pipe is provided with built-in pipe.
4. the part of the body cavity above the diaphragm housing the heart and lungs fluid-retention stagnating in the interior rat model that the construction method described in any one of claims 1 to 3 obtains.
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Application publication date: 20150422 |