CN108553473A - A kind of method for building up of liver-kidney deficiency card animal model - Google Patents
A kind of method for building up of liver-kidney deficiency card animal model Download PDFInfo
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Abstract
The present invention relates to the method for building up that a kind of liver-kidney deficiency demonstrate,proves animal model, include the following steps：It chooses appropriate rat and carries out power daily and exhaust swimming, rest 1.5h after swimming suspends 2h in midair.Modeling 6 days weekly.In modeling the 22nd day, the 25th day rat skin lower injection acetylphenylhydrazine 20mg/kg, intraperitoneal injection of cyclophosphamide 10mg/kg from the 25th day, continuous 4 days.During modeling, by observing the general status of rat, the present invention is respectively adopted and formulates rat macro-indicators grade form and the progress model evaluation of four methods of diagnosis quantizating index, the results showed that after above-mentioned modeling method modeling 4 weeks, liver-kidney deficiency card animal model can be successfully established.It is high, reproducible that the present invention provides a kind of stability, facilitates the liver-kidney deficiency of popularization to demonstrate,prove rat model, compensates for the vacancy of tcm syndrome model, the animal model for meeting theory of traditional Chinese medical science is provided for the class traditional Chinese medicine research that tonifies the liver and kidney.
Description
Technical field
The present invention relates to traditional Chinese medical theory studying technological domains, and in particular to a kind of foundation of liver-kidney deficiency card animal model
Method.
Background technology
Tcm theory thinks, liver and kidney it is in close relations, be mainly manifested in " The liver and the kidney have a common source ", " essence and blood sharing the same origin ", " rush down Tibetan
Mutually with " and " negative and positive are mutually made " etc..Under pathological state, deficiency of liver-blood influences each other more with deficiency of kidney-essence, to head occur
The card that the livers kidney essense blood two such as dizzy dizzy, Hiccough and deaf, soreness and weakness of waist and knees lose, abbreviation liver-kidney deficiency.The card mostly strains because of prolonged illness, Nian Gao
Weak or damage of kidney-essence causes deficiency of liver-blood or deficiency of liver-blood to cause caused by deficiency of kidney-essence.
According to the theoretical point view of Chinese medicine " The liver and the kidney have a common source ", " kidney storing essence ", " blood stored in the liver ", the mutual metaplasia of essence and blood, deficiency of kidney-essence
Then deficiency of liver-blood, the liver failing to be nourished, liver-blood deficiency then lead to kidney essence deficiency；Liver blood deficiency and the existing causality of kidney-jing deficiency, and mutually
It influences, is eventually exhibited as liver-kidney deficiency.Will lose inadequate natural endowment, the day after tomorrow support, year height physically weak, prolonged illness wound just, feelings will internal injury, work and rest immoderation
Deng can cause liver-kidney deficiency.Pain in the back knee is soft, deficient in energy, dizziness and tinnitus, insomnia forgetfulness, pink tongue, tongue is thin, arteries and veins is thin etc. to distinguish
Demonstrate,prove main points.
The accompanying symptoms that liver-kidney deficiency system deficiency of liver-blood and deficiency of kidney-essence two are demonstrate,proved.Liver-kidney deficiency card never adds kidney equal to syndrome of deficiency of liver blood
The simple addition of insufficiency of essence card, liver blood deficiency and the existing causality of kidney essence deficiency, and influence each other.Chinese medicine thinks, blood stored in the liver, kidney
Store essential substances, essence and blood alternate.《The General Treatise on the Cause and Symptoms of Diseases》It says：" smart person, the institute of blood at." blood energy production of sperm；《Zhang Shi doctors are logical》Cloud：" (kidney) essence
It does not let out, returns and be skillful in liver and change and purify the blood." essence again can metaplasia blood.In addition, kidney energy store essential substances gives birth to marrow, marrow includes marrow.Modern medicine is recognized
It is important blood forming organ for marrow, this is very consistent with traditional Chinese medicine essence and blood alternate theory.Essence in kidney fills Sheng, then liver has been
It supports, blood is filled；The store blood amount of liver is full, then kidney is hidden, and essence is provided.In addition, kidney water nourishes edema with the liver involved, so that irritability catharsis
Item reaches；The normal catharsis of irritability can also promote the regeneration and storage of kidney essense.Conversely, then kidney essence deficiency can cause liver mistake to support, liver blood
Virtual loss；Deficiency of liver-blood can lead to damage of kidney-essence, be eventually exhibited as liver-kidney deficiency.
According to Chinese medicine Viscera-State Doctrine and theory of seven emotions, the present invention replicates liver-kidney deficiency using complex factors and demonstrate,proves animal model.
Specific modeling method is as follows：Acetylphenylhydrazine is strong oxidizer, can destroy erythrocyte membrane；Cyclophosphamide has very strong bone marrow suppression poison
Side effect can be such that the generation of hematopoietic cell reduces.According to《The official of Chinese and Western Correspondent ancient Chinese medical classics essential ideas internal organs》" then marrow is sufficient for essence foot ",
《The big opinion of Plain Questions YIN YANG classification of natural phenomena》" kidney producing bone marrow, the kidney promoting the liver " is theoretical, and liver blood deficiency syndrome is replicated using composite algorithm.Basis again《Element
Ask that act pain is discussed》" terror impariring kidney ",《Ling Shu Miraculous Pivot or Divine Axis perverse trend cacosplanchnia shape》" overstrain damage kidney essense ", exhausts trip using suspension stress composite force
Swimming method replicates kidney essence deficiency syndrome.The liver and the kidney have a common source, and kidney deficiency can influence and liver, and essence, which is lost, can cause essence not green blood, liver to lose and support.Using
Complex factors modeling, to replicate " liver-kidney deficiency " animal syndrome model.The present invention is based on Basic Theories of Chinese Medicine, establish liver kidney essense
The rat model of anemia void, compensates for the vacancy of tcm syndrome model, is provided for the class traditional Chinese medicine research that tonifies the liver and kidney and meets middle medical knowledge
The animal model of opinion.
Invention content
The object of the present invention is to provide the method for building up that a kind of liver-kidney deficiency demonstrate,proves animal model, can be obtained using this method
Animal model with liver kidney essense anemia virtual state.It is the clone method stability, reproducible, it is consistent with theory of traditional Chinese medical science.
The present invention is achieved by the following technical solutions：A kind of method for building up of liver-kidney deficiency card animal model, it is special
Sign is：Appropriate rat is chosen, the daily power that carries out exhausts swimming, and rest 1.5h after swimming suspends 2h in midair, weekly modeling 6 days.In modeling
22 days, the 25th day rat skin lower injection acetylphenylhydrazine 20mg/kg, intraperitoneal injection of cyclophosphamide 10mg/kg from the 25th day, continuous 4
It.By modeling in 28 days, liver-kidney deficiency card animal model can be obtained.
The method for building up of above-mentioned liver-kidney deficiency card animal model, specifically includes following steps：
(1) rat adaptability is raised 1 week, during adaptability is raised, carries out an adaptability trip daily since the 5th day
Swimming.
(2) during modeling, rat carries out power and exhausts swimming daily, and swimming time is no less than 2h；Modeling 6 days weekly.
(3) rest 1.5h after swimming, by rat hindlimb immobilization with adhesive tape (tail can be freely movable), later again by rat head
2h is suspended in midair upward towards lower tail；Modeling 6 days weekly.
(4) intraperitoneal injection ring phosphinylidyne is played in 22d, 25d rat skin lower injection acetylphenylhydrazine 20mg/kg, 25d respectively
Amine 10mg/kg, continuous 4 days.
The specific method that the power exhausts swimming is：Rat is put in the drum went swimming of high 630mm, diameter 500mm, water temperature 32
± 2 DEG C, depth of water 500mm prevents rat from touching bottom of cylinder, and entering water swimming with rat reaches after power exhausts standard, pulls out immediately
Rat, and its fur is dried up.
The standard that the power exhausts swimming is：1. it is more than 10s that rat nose, which sinks under the water and can not return to the water surface, and is placed on plane
It can not complete righting reflex；2. the rat coordinated movement of various economic factors disappears, run helter-skelter in no direction in water, though not up to 10s is also set to power
It exhausts；3. in the rat that relative short time internal force exhausts, after pulling rest 5min out, it is further continued for carrying out swimming exercise, makes its run duration
No less than 2h.
The modeling time is 28 days.
The animal is the rat (SD, Wistar) of 150~250g of weight.
During modeling, macro-indicators scoring is carried out to rat, four methods of diagnosis quantizating index dynamic analysis modeling method is to rat
It influences, using peripheral red blood cells counting, content of hemoglobin measurement, organ coefficient and bone marrow smear pathologic finding, it was demonstrated that make
Mould rat belongs to liver-kidney deficiency card.There is macro-indicators 13~15 points of score of scoring in modeling rat, and peripheral red blood cells quantity is small
In 5.5 × 1012/ L, content of hemoglobin are less than 5.5g/L, and anxiety degree is more than+50, and mobility is less than -30, can determine whether animal mould
Type is successfully established.
The present invention solves the deficiency for lacking liver-kidney deficiency syndrome animal model at present, for specification, standard, scientifically evaluates
Chinese medicine provides a kind of new method, and popularization simple, reproducible, easily operated with modeling method, close to tcm clinical practice
Feature.
Description of the drawings
Fig. 1 is the normally comparison of group and model group rats weight before and after modeling, compared with normal group,*P ＜ 0.05,**P ＜
0.01。
Fig. 2 is the normally comparison of group and the scoring of model group rats macro-indicators before and after modeling.
Fig. 3 is the comparison of 5 weeks normal groups and model group rats dietary amount of modeling.
Fig. 4 is the comparison of 5 weeks normal groups and model group rats amount of drinking water of modeling.
Fig. 5 is the comparison of 5 weeks normal groups and model group rats body weight growth rate of modeling, compared with normal group,**P ＜
0.01,**P ＜ 0.001.
Fig. 6 is 5 weeks model group rats anxiety degree of modeling and mobility situation of change.
Fig. 7 is the comparison of 5 weeks normal groups and model group rats auscultation and olfaction W values of modeling.
Fig. 8 is the comparison of normal group and model group rats bone marrow smear.
Specific implementation mode
With reference to specific embodiment, the present invention is further illustrated.
Embodiment 1 establishes liver-kidney deficiency card animal model
1 material requested
1.1 experimental animal
SPF grades of male SD rats 14,180~220g of weight are provided by Zhejiang Province's Experimental Animal Center, and animal productiong is permitted
It can the number of card SCXK (Zhejiang) 2014-0001.Give the periodicity of illumination (illumination in 12 hours, 12 hour night) of standard, indoor temperature (25
± 2) DEG C, humidity (50 ± 10) %, adaptability are raised 1 week, are tested whole process and are given fed standard chow.
1.2 drug and reagent
Syklofosfamid ampoule (lot number：16021725) Jiangsu Sheng Di Pharmaceuticals Ltds, are purchased from；Acetylphenylhydrazine (lot number：
20160407), it is purchased from Sinopharm Chemical Reagent Co., Ltd.；Erythrocyte diluting fluid (lot number：20160405), hemoglobin is dilute
Release liquid (lot number：20160405) it, is purchased from Nanjing and builds up Bioengineering Research Institute.
1.3 key instrument
Multiskan GO microplate reader (Thermo companies)；752N types ultraviolet specrophotometer (Shanghai precision scientific instrument
Co., Ltd).
2 methods
2.1 liver-kidney deficiency demonstrate,prove modeling
2.1.1 adaptability swimming and animal packet
Animal adaptability is raised 1 week, carries out an adaptability swimming daily since the 5th day.Rat is put into plastic barrel
In (height 630mm, diameter 500mm), 5 every barrel, depth of water 500mm, water temperature maintains (32 ± 2) DEG C, the 5th day trip 20min,
6th day trip 30min, the 7th day trip 40min.It is layered grouping at random by weight, normal group 4, model group 10.
2.1.2 modeling method
2.1.2.1 power exhausts swimming
Rat is put into plastic barrel, and 5 every barrel, depth of water 500mm, water temperature maintains (32 ± 2) DEG C, carries out one daily
Secondary power exhausts swimming.The swimming state for examining rat pulls rat out immediately after reaching power and exhausting standard, and by its fur
Drying, prevents animals got sick.
Power exhausts criterion：1. it is more than 10s that rat nose, which sinks under the water and can not return to the water surface, and is placed on plane and can not complete
Righting reflex；2. the rat coordinated movement of various economic factors disappears, run helter-skelter in no direction in water, though not up to 10s is also set to power and exhausts.Opposite
The rat that short time internal force exhausts after pulling rest 5min out, is further continued for carrying out swimming exercise, its run duration is made to be no less than 2h.Just
Normal rat is placed in during model group processing in cage, fasting for solids and liquids.
2.1.2.2 suspension stress
By rat hindlimb immobilization with adhesive tape (tail can be freely movable), by rat, tail suspends 2h in midair upward upside down again later,
Period is fasting for solids and liquids.
2.1.2.3 cyclophosphamide+acetylphenylhydrazine hematopoiesis is empty
Acetylphenylhydrazine 20mg/kg, intraperitoneal injection of cyclophosphamide from the 25th day is subcutaneously injected in experiment the 22nd day, the 25th day
10mg/kg, continuous 4 days.
The Indexs measure of 2.2 normal groups and model group rats
2.2.1 weight
The weight of daily measurement experiment animal in experimental period.
2.2.2 ordinary circumstance and Macroscopic score
In experimental period, daily observe rat general status index, including spirit, activity with react, fur, ear, tail,
Onyx, binocular, muffle, two just etc., and macro-indicators scoring (being shown in Table 1) is carried out to rat according to macro-indicators score by rules.It calculates
Overall score, score value is higher, indicates that liver-kidney deficiency sign is more apparent.
1 rat macro-indicators score by rules of table
2.2.3 peripheral red blood cells count and content of hemoglobin measures
Each group rat the 5th, 6 weekend eye sockets after modeling take blood, and anticoagulant heparin is added.10 μ L of anticoagulation are taken, red blood cell is used
200 times of mixings of diluted, take 10 μ L to be added on red blood cell count(RBC) plate, red blood cell count(RBC) are carried out under microscope low power lens.
It takes anticoagulation to measure content of hemoglobin according to kit specification method, is counted after result of calculation.
2.3 statistical method
Experimental result carries out statistical analysis using 19.0 statistical softwares of SPSS, with 5 softwares of Graphpad Prism
It maps.Testing index group difference compares when modeling 5,6 weeks carries out statistical procedures using independent samples t test.P ＜
0.05 thinks that significant difference, P ＜ 0.01 think that difference is extremely notable.As a result with means standard deviationMode is shown.
3 results
3.1 weight
It will be seen from figure 1 that there was no significant difference for each group rat body weight before modeling.Modeling 1 week, model group rats weight with
Compared to being decreased obviously, body weight increase is significantly inhibited (P ＜ 0.01) normal group.With the extension of modeling time, body weight increase
Inhibit bigger.
3.2 ordinary circumstances and macro-indicators scoring
The rat state of mind is good before modeling, and the eyes are bright and piercing, autonomic activities are more, and reaction is quick, and fur is bright and clean neat.Modeling
1 week, model group rats fur was gradually crude loose.Modeling 2 weeks, model group rats dry skin and hair lose China, and autonomic activities are reduced, ear
The light red Shaoze of tail, onyx, binocular, muffle, the relatively normal group of model group macro-indicators scoring is significantly raised, there is pole significant difference (P
＜ 0.01).To modeling 4 weeks, the rat state of mind was gradually dispirited, slow in reacting, and whole body fur is withered to be dredged without pool, ear tail, onyx, double
Mesh, muffle bleach, and irritability, slightly shout, and bait.The score modeling 1 week of rat macro-indicators is i.e. on the rise, until modeling 2 weeks
Occurs significant difference compared with normal group, modeling macro-indicators scoring in 3 weeks is lasting to be increased, and modeling macro-indicators scoring in 4 weeks becomes
In stabilization, modeling starts for 5 weeks, and macro-indicators scoring is stablized, and group difference reduces (result is shown in Fig. 2).
The influence of 3.3 human peripheral blood red blood cell count(RBC)s and content of hemoglobin
Modeling 5,6 weeks, taking whole blood to carry out, peripheral red blood cells count and content of hemoglobin measures.The results show that modeling
5,6 weeks, model group erythrocyte number, content of hemoglobin were remarkably decreased (P ＜ 0.01) pole compared with normal group, used in prompt
The modeling of complex factors modeling method can cause the rat model deficiency of blood (liver blood deficiency) model for 5 weeks.(the results are shown in Table 2)
2 rat peripheral red blood cell count of table and content of hemoglobin situation of change
Note：Compared with normal group,**P ＜ 0.01.
4 experiment conclusions
Experimental result shows that modeling rat macro-indicators scoring in 1 week is i.e. on the rise, until modeling organizes phase in 2 weeks with normal
Than there is significant difference, modeling macro-indicators scoring in 3 weeks is lasting to be increased, and modeling macro-indicators scoring in 4 weeks tends towards stability, modeling
Macro-indicators scoring in 5~6 weeks is stablized, and group difference reduces, and prompts the standards of grading more objective, reasonable.Refer to according to animal macroscopic view
It marks appraisal result to judge, after modeling 4 weeks, macro-indicators scoring is scored at 13~15 points, can reach liver-kidney deficiency syndrome state.It adopts
Combine suspension stress with compound deficiency of blood method and power exhausts swimming reproducible liver-kidney deficiency syndrome model from etiology and pathogenesis.Experimental result
The scoring of rat macro-indicators increases after showing modeling；There is apathetic, delay of response in animal, and more anger are baited, lip, ear, pawl, tail
Color is light, the sings and symptoms such as weight loss；Peripheral red blood cells quantity and content of hemoglobin decline.Therefore, which meets
Chinese medicine liver-kidney deficiency syndrome standard.
Embodiment 2 demonstrate,proves liver-kidney deficiency based on the Chinese medicine four methods of diagnosis method evaluation study of Establishment of Rat Model
1 material requested
1.1 experimental animal
SPF grades of male SD rats 16,180~220g of weight are provided by Zhejiang Province's Experimental Animal Center, and animal productiong is permitted
It can the number of card SCXK (Zhejiang) 2014-0001.Give the periodicity of illumination (illumination in 12 hours, 12 hour night) of standard, indoor temperature (25
± 2) DEG C, humidity (50 ± 10) %, adaptability are raised 1 week, are tested whole process and are given fed standard chow.
1.2 drug and reagent
Syklofosfamid ampoule (lot number：7F72A), hundred special (China) limited investment public companies are purchased from；Acetylphenylhydrazine (batch
Number：20160407), it is purchased from Sinopharm Chemical Reagent Co., Ltd.；Erythrocyte diluting fluid (lot number：20180509), blood red egg
White dilution (lot number：20180509), quick Wright's stain (lot number：20180315) it, is purchased from Nanjing and builds up bioengineering and grind
Study carefully institute.
1.3 key instrument
Multiskan GO microplate reader (Thermo), Micro CL 21R refrigerated centrifuges (Thermo), ultraviolet point of N4S types
Light photometer (Shanghai INESA Analytical Instrument Co., Ltd.), electronic thermometer MC-347 types (Omron), DSC-TX100 number phases
Machine (SONY).
2 methods
2.1 liver-kidney deficiency demonstrate,prove modeling
After Animal adaptability is swum, it is layered grouping at random by weight, normal group 6, model group 10.Model group is big
Mouse carries out modeling according to 2.1.2 the methods in embodiment 1.
The acquisition and detection of 2.2 each indexs of liver-kidney deficiency model of a syndrome
2.2.1 the acquisition of four methods of diagnosis information
2.2.1.1 interrogation
Interrogation is that the disease event of other side is grasped by way of dialogue, due to that can not be carried out truly to rat
Interrogation originally determines the following interrogation letter of acquisition according to the way of inquisition of the experimental animal of the interrogation and document report of Chinese medicine routine
Breath：1. ordinary circumstance；2. living environment；3. the cause of disease influences；4. two just situations etc..
2.2.1.2 observation
(1) body is hoped
Using modeling the previous day weight as original body mass, weight is measured daily, calculates body weight growth rate.Body weight growth rate=
(per daily weight-original body mass) ÷ original body mass × 100%.
(2) observation of mental state and facial expression, activity and react
Modeling the 1st, 2,3,4,5 weekends, observation each group rat spirit, activity and response situation, the specific method is as follows：
Selection specification is (40 × 25 × 25) cm3Wooden case, with (25 × 25) cm2Face be place when bottom surface, the bottom of
Face diagonal line intersection point cuts out the round wood chip that radius is 1.5cm as the center of circle.Under quiet environment, hole downward horizontal
After fixation, 1 rat is placed per case, every nest rat records every rat probe number in 10min with batch observation.With " anxiety
Degree " the evaluation rat state of mind, anxiety degree=(model group is popped one's head in, and number mean value-is normal to organize probe number mean value)/normal group are visited
Head number mean value × 100%.
Select (38 × 27 × 19) cm of 3 × 3 grid of bottom surface band3Rat cage tool as rat across lattice number measurement work
Tool.Rat is gently caught and is put in cage, waits for that its autonomic activities adapts to 15s, rat autonomic activities video recording 60s is recorded, calculates in 60s
Across the lattice total degrees of shoulder of rat.Activities in rats state, mobility=(across the lattice number mean values-of model group are evaluated with " mobility "
Across the lattice number mean values of normal group)/across lattice number mean value × 100% of normal group.
(3) pawl, tail are hoped
Respectively at modeling the 1st, 2,3,4,5 weekends using digital camera shooting pawl, the photo of tail.It is kept fixed ground when acquisition
The principle of point, same light source, same sample point.The Eyedropper tool of Adobe Photoshop CS5 softwares is used after Image Acquisition
R (red), G (green) at the reading pawl centre of the palm, tail point 1/3 and B (blue) component value, and then find out the r of the red degree of reflection
Value, r=R/ (R+G+B) × 100%.R values can reflect the red degree of pawl, tail skin, and r values are bigger, and red degree is bigger, liver blood
Empty degree is smaller.
(4) tongue inspection
Rat lingual surface is shot with 25% urethane anesthetized rat in the 5th weekend of modeling, image uses Adobe
The Eyedropper tool of Photoshop CS5 softwares reads rgb value, and calculates lingual surface r values.
(5) fur is hoped
In experimental period, rat fur state change is observed and recorded daily, including whether glossiness, perpendicular hair；In modeling
1st, 2,3,4, the observation scoring of 5 weekends, fur score by rules is shown in Table 3.
3 rat fur of table observes item and corresponding score value
2.2.1.3 auscultation and olfaction
Auscultation and olfaction predict the patient's condition by smelling and listening.During experiment, in modeling the 1st, 2,3,4,5 weekends, see
It examines and records bedding and padding smell in big mouse cage and change；It is daily to observe and record the ring of rat nose, breathing situation；Auscultation and olfaction score by rules is shown in Table
4.Auscultation and olfaction W values are calculated, is scored using W values as rat auscultation and olfaction, is compared with model group W values to normally organizing, reflect modeling method
Influence to auscultation and olfaction.
4 auscultation and olfaction of table acquire project and content
Note：W values=smelling × listening × 0.5 0.5+.
2.2.1.4 diagnosis
Tentatively judge that rat temperature, body whether there is lump by palpation；Rat anus temperature is detected using electronic thermometer；
The power of its resistance is observed and recorded when capturing rat.
2.2.2 peripheral red blood cells count and content of hemoglobin measures
With 2.2.3 in embodiment 1.
2.2.3 organ coefficient
Last day is tested, with 25% urethane anesthetized rat, arteria carotis communis takes blood.After taking blood, rat cervical dislocation is put to death,
Dissection, takes liver, spleen, thymus gland, Bilateral Renal and bilateral testes, is cleaned twice with physiological saline, weighed after blotting, and calculates internal organs
Coefficient.
2.2.4 bone marrow smear
After putting to death animal, rat femur is quickly removed, picks and takes suitable marrow, with 45 °~50 ° angles, Quick uniform push jack.
It is dyed according to Wright's stain specification after natural drying, the read tablet under microscope.
2.3 statistical method
Experimental result carries out statistical analysis using 22.0 statistical softwares of IBM SPSS Statistics, uses
6 softwares of Graphpad Prism are mapped.Normal distribution data uses independent samples t test；Non-Gaussian Distribution data uses
Non-parametric test；P ＜ 0.05 think that significant difference, P ＜ 0.01, P ＜ 0.001 think that difference is extremely notable.As a result with mean value ±
Standard deviationDisplaying.
3 results
3.1 four methods of diagnosis results
3.1.1 interrogation
3.1.1.2 ordinary circumstance
Two groups of rat ordinary circumstances, diet conditions of water drinking, living environment are consistent；Normal group is without apparent pathogenic factor, mould
Type group receives multiple factors method modeling, is specifically shown in Table 5.
The interrogation result of 5 each group rat of table
3.1.1.2 diet, amount of drinking water situation of change
Fig. 3,4 displays, compared with normal group, the diet amount of drinking water fluctuation of model group rats day is larger, and is significantly lower than just
Normal group.
3.1.2 observation
3.1.2.1 body is hoped
As shown in Figure 5, compared with normal group, during model group modeling, body weight increase is slow；Compared with normal group, from making
Mould starts on the 1st week, and model group body weight growth rate pole is remarkably decreased (P ＜ 0.001).The result shows that during modeling, modeling rat compared with
Normal rat weight is light, and body weight increase is slow, meets the syndrome characteristics of liver-kidney deficiency card " body is become thin ".
3.1.2.2 observation of mental state and facial expression, activity and react
As shown in Table 6, with the increase of modeling number of days, normal group probe number is without significant change, compared with normal group, mould
Type group probe number gradually increases, and has pole significant difference (P ＜ 0.01) since 3 weekend of modeling.Model group rats are being made
In mold process, autonomic activities show first to increase, the exception of rear reduction, and capture reaction fierceness, bait, roar；Modeling 1,2,3
Week, across the lattice number showed increaseds of rat；Modeling 4,5 weeks, across lattice numbers substantially reduce, and the results are shown in Table 7.Model group as shown in Figure 6
Rat modeling 3 weeks~5 weeks, anxiety degree significantly increases, and substantially remains in+60 or so；Model group rats mobility first increases to drop afterwards
Low, 3 weeks~5 weeks mobilities of modeling substantially remain between -30~-40.
6 modeling of table, 5 weeks rats probe number situation of change (it is secondary,)
Note：Compared with normal group,**P ＜ 0.01,***P ＜ 0.001.
Across the lattice number situations of change of 7 modeling of table, 5 weeks rats (it is secondary,)
Note：Compared with normal group,*P ＜ 0.05,**P ＜ 0.01,***P ＜ 0.001.
3.1.2.3 pawl, tail are hoped
By table 8,9 it is found that with modeling number of days increase, model group rats pawl, tail r values are bright compared with normal rats
It is aobvious to decline.Modeling the 4th week, compared with normal group, model group rats pawl, tail r values pole are remarkably decreased (P ＜ 0.001).
8 modeling of table, 5 weeks rat pawl r values situations of change (%,)
Note：Compared with normal group,*P ＜ 0.05,***P ＜ 0.001.
9 modeling of table, 5 weeks big rat-tail r values situation of change (%,)
Note：Compared with normal group,**P ＜ 0.01,***P ＜ 0.001.
3.1.2.4 the tongue inspection
As shown in Table 10, after modeling 5 weeks, compared with normal group, model group rats tongue r values are remarkably decreased (P ＜ 0.01).
10 modeling of table, 5 weeks Rat Tongue r values situations of change (%,)
Note：Compared with normal group,**P ＜ 0.01.
3.1.2.5 fur is hoped
Rat fur state is observed during experiment, and is scored according to from quasi- rat fur in modeling the 1st, 2,3,4,5 weekends
Detailed rules and regulations score.Compared with normal group, model group rats fur relaxation, hair owes gloss, easily dries up, falls off, and perpendicular hair is more,
Hair is not submissive, and sense of touch is poor；Individual rat furs are filthy.Appraisal result shows to increase with modeling number of days, model group rats skin
Hairy state is gradually deteriorated, and has pole significant difference compared with normal group, the results are shown in Table 11.
The situation of change of 11 rat fur of table
Note：Compared with normal group,**P ＜ 0.01,***P ＜ 0.001.
3.1.3 auscultation and olfaction
During experiment, normal rats smell and sound are normal, and it is different not occur foul defecation, nose ring, lung's rale etc.
Often occur as.And the accidental foul defecation of model group rats, nasal sound, rale.As shown in Figure 7, with the increase of modeling number of days, model group
Auscultation and olfaction scoring W values become larger, and illustrate that modeling method can make rat smell and cacophonia.
3.1.4 diagnosis
As shown in Table 12, since modeling the 2nd week, model group rats anus temperature more normally organizes apparent increase, and there were significant differences
(P ＜ 0.05), and with the increase of modeling number of days, model group rats anus temperature also gradually rises.
12 rat anus temperature situation of change of table (DEG C,)
Note：Compared with normal group,*P ＜ 0.05,**P ＜ 0.01,***P ＜ 0.001.
3.2 Microscopic Indexes testing results
As shown in Table 13, after modeling 5 weeks, model group erythrocyte number, content of hemoglobin are with normal group compared to extremely showing
It writes and declines (P ＜ 0.01), the result is consistent with embodiment 1, illustrates that the modeling method stability of the present invention is good, repeats.
The situation of change of table 13 rat peripheral red blood cell count and content of hemoglobin
Note：Compared with normal group,**P ＜ 0.01.
3.3 Rats Organs and Tissues coefficients
As shown in Table 14, the 5th weekend of modeling, compared with normal group, the organ coefficient of model group rats liver, kidney and spleen is equal
No conspicuousness variation；Illustrate modeling method to rats'liver, kidney, the not damaged effect of spleen.Compared with normal group, testis, thymus gland are dirty
Device coefficient is substantially reduced, and has conspicuousness (P ＜ 0.01, P ＜ 0.001), prompts modeling 5 weeks rat testicles, atrophy of thymus gland, at this time
There are kidney essence deficiencies for rat.
14 Rats Organs and Tissues coefficient of table situation of change (‰,)
Note：Compared with normal group,**P ＜ 0.01,***P ＜ 0.001.
3.4 the influence to bone marrow smear
As shown in figure 5, normally red system, myeloid cell are more in group bone marrow cell, accidental fat drop.Compared with normal group,
Red system, myeloid cell are less in model group bone marrow smear, and fat drop is more；Illustrate that modeling rat hematopoiesis function is low, exists
Slight bone marrow suppression.
4 experiment conclusions
Normal rats body is solid, and the state of mind is excellent, is quick on the draw, hair luster, and eye, ear, pawl, tail color are micro- red,
The tactile warm of skin, pawl, tail, cry is without exception, and bedding and padding sawdust is relatively dry in cage, and two is just normal, and stink is lighter in cage.Model group
During modeling, fur state is gradually deteriorated rat；Cerebration and abnormal reaction, autonomic activities show first to increase, after
It reduces, and captures reaction fierceness, bait, roar, modeling the 4th week~5 weeks, model group rats anxiety degree is more than+50, and mobility is small
In -30, model group rats lesion is located in " liver " at this time for prompt.Body weight increase is slow during model group rats modeling, and body is thin
It is thin, and rat reaction is fierce when crawl, involuntary emission easily occurs, this is kidney deficiency, the cardinal symptom of deficiency of kidney-essence, hints model group rat
Lesion is located in " kidney ".Model group rats pawl, tail, tongue r values are lower than normally organizing, and r values can reflect deficiency of blood degree, the smaller deficiency of blood of r values
Degree is heavier, illustrates that model group rats meet the feature of tcm clinical practice " liver blood deficiency "：Colourless is simple and unadorned, and onyx, lip color are light, tongue fur
It is light.In anus temperature, model group rats anus temperature significantly increases, and body temperature can be used as one of dialectical index of negative and positive asthenic symptoms, in conjunction with big
Mouse anus temperature and cerebration and the variation reacted can initial guess model group rats be yin deficiency syndrome.
After modeling 35 days, model group rats peripheral red blood cells quantity is less than 5.5 × 1012/ L, content of hemoglobin are less than
5.5g/L illustrates that rat is in deficiency of blood state at this time, and cyclophosphamide combines the success of hematopoiesis virtual model with acetylphenylhydrazine；" Shen controls bone
Raw marrow, marrow includes marrow ", bone marrow smear result can reflect that modeling rat hemopoietic system function, results of animal show this
Modeling method can cause model of blood dificiency.Significant change does not occur for modeling rats'liver kidney organ coefficient, and modeling method does not produce liver kidney
It is raw to influence；And there are atrophy phenomenons for modeling rat testicle and thymus gland, there is involuntary emission in modeling rat during modeling, illustrates kidney essence deficiency
Demonstrate,prove modeling success.
Comprehensive animal four methods of diagnosis information analysis, modeling method are to the blood count of modeling rat peripheral, content of hemoglobin, again
Want the influence of organ coefficient and bone marrow smear of internal organs etc., it may be determined that this experiment can be successfully, reproduced liver kidney using complex factors molding method
Essence and blood loss card.
Claims (9)
1. a kind of method for building up of liver-kidney deficiency card animal model, it is characterized in that：Appropriate rat is chosen, the daily power that carries out exhausts trip
Swimming, rest 1.5h after swimming suspend 2h in midair, weekly modeling 6 days.In modeling the 22nd day, the 25th day rat skin lower injection acetylphenylhydrazine
20mg/kg, intraperitoneal injection of cyclophosphamide 10mg/kg from the 25th day, continuous 4 days.By modeling in 28 days, liver kidney can be successfully, reproduced
Loss card animal model.
2. the method for building up of liver-kidney deficiency card animal model according to claim 1, it is characterized in that：Rat adaptability is raised
1 week, during adaptability is raised, carry out an adaptability swimming daily since the 5th day.
3. the method for building up of liver-kidney deficiency card animal model according to claim 1, it is characterized in that：During modeling, rat
The daily power that carries out exhausts swimming 1 time, every time no less than 2h, weekly modeling 6 days.
4. the method for building up of liver-kidney deficiency card animal model according to claim 3, it is characterized in that：The power exhausts swimming appliance
Body mode is：Rat is put in the drum went swimming of high 630mm, diameter 500mm, 32 ± 2 DEG C of water temperature, and depth of water 500mm makes rat not
Bottom of cylinder can be touched, entering water swimming with rat reaches after power exhausts standard, pulls rat out immediately, and its fur is dried up.
5. the method for building up of liver-kidney deficiency card animal model according to claim 1, it is characterized in that：After power exhausts swimming
1.5h, by rat hindlimb immobilization with adhesive tape (tail can be freely movable), by rat, tail suspends 2h in midair upward upside down again later, during which
Fasting water.
6. the method for building up of liver-kidney deficiency card animal model according to claim 1, it is characterized in that：In modeling the 22nd day,
25th day be subcutaneously injected acetylphenylhydrazine 20mg/kg, intraperitoneal injection of cyclophosphamide 10mg/kg from the 25th day, continuous 4 days.
7. the method for building up of liver-kidney deficiency card animal model according to claim 1, it is characterized in that：The modeling time is
28 days.
8. the method for building up of liver-kidney deficiency card animal model according to claim 1, it is characterized in that：The animal is weight
The rat (SD, Wistar) of 150~250g.
9. demonstrate,proving the method for building up of animal model according to any one of claim 1~8 liver-kidney deficiency, it is characterised in that：It is described
During modeling, macro-indicators scoring is carried out to rat, influence of the four methods of diagnosis quantizating index dynamic analysis modeling method to rat uses
Peripheral red blood cells counting, content of hemoglobin measurement, organ coefficient and bone marrow smear pathologic finding, it was demonstrated that modeling rat belongs to
Liver-kidney deficiency is demonstrate,proved.There is macro-indicators 13~15 points of score of scoring in modeling rat, and peripheral red blood cells quantity is less than 5.5 × 1012/
L, content of hemoglobin are less than 5.5g/L, and anxiety degree is more than+50, and mobility is less than -30, can determine whether Animal Model success.
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| CN101849526A (en) * | 2010-05-21 | 2010-10-06 | 广东药学院 | Qi-deficiency fever animal model and preparation method and application thereof |
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| CN101849526A (en) * | 2010-05-21 | 2010-10-06 | 广东药学院 | Qi-deficiency fever animal model and preparation method and application thereof |
| CN103027906A (en) * | 2011-10-08 | 2013-04-10 | 鲁南制药集团股份有限公司 | Application of arctigenin in treating anemia |
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