CN106645746A - Method for screening and confirming kidney-yang deficiency animal model biomarker - Google Patents
Method for screening and confirming kidney-yang deficiency animal model biomarker Download PDFInfo
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- CN106645746A CN106645746A CN201611015606.8A CN201611015606A CN106645746A CN 106645746 A CN106645746 A CN 106645746A CN 201611015606 A CN201611015606 A CN 201611015606A CN 106645746 A CN106645746 A CN 106645746A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
- G01N33/6851—Methods of protein analysis involving laser desorption ionisation mass spectrometry
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
Abstract
The invention provides a method for screening and confirming a kidney-yang deficiency animal model biomarker. The method comprises the following steps: extracting total protein of cancellous bone of a sample; determining the protein concentration; performing two-dimensional gel electrophoresis analysis; detecting gel protein spot and displaying and analyzing differential protein spot; performing MALDI-TOF-MS mass spectrometry for differential protein, retrieving and identifying a protein database; confirming the differential protein; identifying a part of differential protein through Western-Blot and RT-PCR and mutually verifying the detection result for differential protein ELISA in serum; using the differential protein in consistent expression tendency and beyond the normal value scope as a biomarker for identifying a kidney-yang deficiency animal model. The invention is beneficial to the supply of an identifying index to the kidney-yang deficiency in an animal experiment and provides a basis for further discovering the kidney-yang deficiency biomarker.
Description
Technical field
The invention belongs to basic research field, and in particular to a kind of screening of syndrome of deficiency of kidney yang animal model biomarker and
Determine method.
Background technology
" card " is the core of theory system of TCM, the first weight diagnosis and treatment of tcm clinical practice, but dialectical is largely cured
Family itself（Understanding, clinical experience such as to knowledge of TCM）And the impact of patient's subjective factor, it is difficult to objectify and quantitatively
Change, cause dialectical result disunity, affect the evaluation of the establishment, Selection drug and clinical efficacy of therapy, hinder grinding for traditional Chinese medicine
Study carefully and develop.The real processes of disease, the physiology and pathology with " card " can be simulated in TCM basis research using animal model
State is similar, therefore is the only way of research kidney deficiency Syndrome essence by syndrome of deficiency of kidney yang animal model.
The symptom of the syndrome of deficiency of kidney yang rat model that this experiment is simulated using hydrocortisone intramuscular injection, sign and blood
Clear cAMP, cGMP content detection and cAMP/cGMP ratio results meet the identification requirement of syndrome of deficiency of kidney yang model conventional at present, table
It is successful that the kidney-yang deficiency model of bright research makes, but above evaluation method is based on subjective assessment, and evaluation index lacks special
The opposite sex, objectivity, result of study can the science essence for illustrating TCM Syndrome and the animal syndrome model that replicated whether have
It is representative, also relate to the dialectical problem objectified with quantification.Therefore the objective indicator with certain specificity is found
To differentiate whether identification kidney-yang deficiency model is successful, is the key for carrying out follow-up related experiment.
Based on the theory of the traditional Chinese medical science " Shen controls bone ", bone tissue is being given birth to as one of the external manifestation position of " kidney " function in it
The change of thing feature is certainly existed with the functional status of " kidney " and maintained close ties with.Protein is to embody histocyte function and hold
The most direct active material of row vital movement, structure and the functional status of bone tissue are finally embodied in its built-in function protein group
With the expression status of genome.With going deep into protein group correlative study to disease TCM Syndrome, have now realized that：
The performance essence of TCM Syndrome is the expression of the specific functional protein group of diseased individuals and its genome；In disease difference
The essence distinguished between doctor's card type is the difference of functional protein group and its genomic expression level.Therefore, by bone tissue
The detection of differential protein group expression, is expected to from bone tissue functional protein group aspect further disclose traditional Chinese medical science kidney-yang deficiency
The essence of syndrome.
Based on above theoretical foundation and research background, this research is by means of proteomics research thinking and technical excellent
Gesture, with the bone tissue of rat（Cancellous bone）For research object, their cancellous bones under kidney-yang deficiency and normal condition are shown respectively
Proteomic map, compares the differential expression protein existed between them, deeply visits from cancellous bone functional protein group aspect
The essence of traditional Chinese medical science syndrome of deficiency of kidney yang is begged for, further in-depth is to the traditional Chinese medical science " kidney " and the understanding of bone internal association.Meanwhile, these differential expressions
Protein may syndrome of deficiency of kidney yang occur evolution in play a significant role, it may be possible to syndrome of deficiency of kidney yang diagnosis potential source biomolecule
Mark.Tediously long and numerous and diverse process is the discovery that due to biomarker, need to be many from stage construction by kinds of experiments technology
Angle is identified, verifies and confirmed repeatedly.Therefore for the differential protein of preliminary screening, it would be desirable to from protein level, gene level
Further verify to targeting albumen, to evade the limitation of single experimental technique, it is ensured that early stage proteomics assay
Accuracy.Candidate albumen interested how is chosen, to its further research, its close ties with syndrome of deficiency of kidney yang is inquired into,
And while the feasibility for proving this biomarker screening technique is the emphasis of this research.
Current science and technology is just stepping into the epoch of cloud media, big data, and various information swarm forward, and Jing cloud computings, big data are dug
Pick, frequently online protein interaction database (PPI Database) meets the tendency of for some powerful, abundant in content, renewals
And give birth to, it has also become one of research prediction protein function important means.This part research and utilization free online PPI Database
- String10 identifies known albumen to early stage and carries out the comprehensive analysis of system, and screening candidate albumen is for further study.The number
It is fast according to storehouse broad covered area, degree of integration height, renewal frequency, and the relation directly or indirectly interacted between protein be able to can be done
Go out certain prediction, so as to can be tentatively in bioinformatics aspect to the differential protein identified in disease effect carry out it is pre-
Survey, to provide thinking to follow-up confirmatory study.Pointed out by means of the biological information that String database retrievals are provided, together
When we consult pertinent literature with reference to early-stage Study result, it is presumed that the change of syndrome of deficiency of kidney yang sings and symptoms may with it is a few
The regulation and control of individual key difference albumen place protein regulation network are closely related.Therefore we pick NTx α -1 chains and GDP
Dissociation inhibiting factor 1 is used as identifying object.
Additionally, in infrastest, the skeleton specimen of animal model is difficult to obtain, and comparatively blood specimen is obtained more just
Prompt and easily preservation.Blood testing has been clinically one of prevention and the most common supplementary means of diagnosis and treatment disease.Blood is almost contained
The albumen from each organ-tissue of body is covered, since in kidney-yang deficiency and normal rat bone tissue（Cancellous bone）Between have differences egg
White matter, then whether there is also this species diversity in blood.In consideration of it, we are intended to extract two groups of experimental rat blood measurings
The content of corresponding protein, and analyze its concentration and bone tissue（Cancellous bone）The uniformity of corresponding protein expression level, reference is faced
The definition of " medical reference scope " on bed：By the ELISA measurement results of rat models of kidney-yang-deficiency syndrome and the number of normal rats
Value is compared, and whether beyond the fluctuation range of normal rat corresponding index, we are provided Observe and measure value from normal distribution
The formula of the material reference range of bilateral 95%（±1.96S）Calculate the fluctuation of corresponding protein content in normal rats serum
Scope, as the important references for differentiating measurement result, seeks the potential blood markers thing of syndrome of deficiency of kidney yang diagnosis.
Based on the above, this research is with the bone tissue of rat（Cancellous bone）For research object, in early stage proteomics
On experiment basis, the differential protein that syndrome of deficiency of kidney yang occurs to be played an important role in evolution is chosen at, is used
Western-Blot and RT-PCR technology carry out double verification from albumen and gene level, further the bone group of clear and definite syndrome of deficiency of kidney yang
Knit（Cancellous bone）Differential protein.Meanwhile, corresponding albumen is determined using ELISA and is contained in blood of the syndrome of deficiency of kidney yang with normal rat
Amount, based on test in the evaluation of syndrome of deficiency of kidney yang model establish Research foundation.
The content of the invention
The purpose of the present invention is to overcome the shortcomings of that the dialectical technology of current syndrome of deficiency of kidney yang does not objectify, using two-way gel electricity
Swimming（2DE）And MALDI-TOF/MS technologies, obtain syndrome of deficiency of kidney yang and normal rats bone tissue protein graphical spectrum, Jing mass spectral analyses and
Identification, filters out the albumen that expression is had differences in cancellous bone, and chooses and may send out in syndrome of deficiency of kidney yang occurs evolution
Waving the Partial Protein of important function carries out Western-Blot and RT-PCR technology from albumen and gene level double verification, and ties
The ELISA detections of GAP-associated protein GAP in serum are closed, finally targeting can become the potential source biomolecule mark of syndrome of deficiency of kidney yang animal model.
For achieving the above object, the present invention is adopted the following technical scheme that：
A kind of screening of syndrome of deficiency of kidney yang animal model biomarker and method, it is comprised the following steps that：
（1）The foundation of syndrome of deficiency of kidney yang rat model；Rat for building animal model is WISTAR rats.
（2）The screening of differential protein.Specifically include following steps：
A) extraction of rat cancellous bone total protein；
B) protein concentration is determined；
C) Two Dimensional Gel Electrophoresis；
D) detection of gel protein point shows analysis with differential protein spot；
E) the MALDI-TOF-MS mass spectral analyses of differential protein；
F) protein database search and identification, determine differential protein.
（3）The checking of part variation albumen.The checking of differential protein is specifically carried out using following two methods：
A) using Western-Blot methods mark is verified from protein level；
B) using RT-PCR method mark is verified from gene level.
（4）The determination of syndrome of deficiency of kidney yang animal model biomarker.It determines that method is：The ELISA of haemocyanin is examined
Survey result and detect comparative analysis with Western-Blot, RT-PCR, selection expression trend is unanimously and outside range of normal value
Differential protein is used as biomarker.
For than prior art, it is an advantage of the current invention that：
（1）It is theoretical according to the traditional Chinese medical science " Shen controls bone ", with syndrome of deficiency of kidney yang state sending down the fishbone setup action research object, using protein science etc.
Advanced research method, has deepened to " Shen controls bone " theoretical scientific knowledge.
（2）The present invention is set up on complete proteomics methodology basis, through verifying repeatedly and screening, is picked out
Testing protein can react the pathomechanism of syndrome of deficiency of kidney yang.
（3）Blood is covered almost from the protein of whole body body tissue, therefore blood testing is clinically most universal
One of assisting in diagnosis and treatment means；And elisa technique can be directly used for detecting macromolecular antigen and specific antibody etc. in body fluid, tool
Have the advantages that quick, sensitive, easy, carrier is easy to standardization.The present invention organically combines the two, the syndrome of deficiency of kidney yang for filtering out
Marker protein, can detect its content in blood using ELISA method, and with the reference value model of normal distribution data bilateral 95%
The formula for enclosing（±1.96S）The fluctuation range of corresponding protein content in normal rats serum is calculated, is determined as differentiating
As a result important references, thus can very simple and effective ground judge that the state of animal used as test kidney-yang deficiency screening meets to be needed
The animal model asked.
（4）The present invention can further promote tcm diagnosis it is convenient, objectify, promote animal model in infrastest
Scientific Assessment.
Description of the drawings
The dry cancellous bone differential protein point identification of Fig. 1 normal groups-kidney-yang deficiency group rat femur.
Fig. 2 GDP dissociation inhibiting factors 1（Arhgdia）MAIDI-TOF-TOF/MS mass spectrograms.
Fig. 3 NTx α -1 chains（Col1a1）MAIDI-TOF-TOF/MS mass spectrograms.
The comparison of Col1a1 and Arhgdia protein expressions in Fig. 4 normal groups-kidney-yang deficiency group rat cancellous bone.
The comparison of Col1a1 mRNA expression in Fig. 5 normal groups-kidney-yang deficiency group rat cancellous bone.
The comparison of Arhgdia mRNA expression in Fig. 6 normal groups-kidney-yang deficiency group rat cancellous bone.
The comparison that Col1a1 and Arhgdia content tables reach in Fig. 7 normal groups-kidney-yang deficiency group rat blood serum.
Specific embodiment
Present invention is described in detail with reference to embodiment：
Embodiment one：
The foundation and identification of syndrome of deficiency of kidney yang rat model：
1 experiment consumptive material
1.1 animal used as test
Male 3 monthly ages health SPF（Specific Pathogen Free）Level WISTAR rats totally 40, the g of body weight 200 ~ 240,
Bought on behalf in Shanghai Slac Experimental Animal Co., Ltd., animal used as test license by Fujian University of Traditional Chinese Medicine's Experimental Animal Center
Card number：SCXK（Shanghai）2012-0002, Quality of Experimental Animals quality certification numbering：2007000548015.Fujian University of Traditional Chinese Medicine's reality
Test animal center animal used as test and use credit number：SYXK（Fujian）2014-0001.
1.2 main agents and medicine
The main agents of table 1 and medicine
1.3 key instrument
The key instrument of table 2
2 experimental techniques
2.1 animal packet
40 WISTAR rats adaptability are fed one week, and using random digits table normal group, kidney-yang deficiency group are divided into, per group 20
Only.After the continuous d of modeling 15 of rats with deficiency of kidney-Yang, model stability observes 21 d, and two groups of rats are normally raised.
2.2 syndrome of deficiency of kidney yang rat model modeling methods
2.2. 1 syndrome of deficiency of kidney yang rat model
After hydrocortisone parenteral solution is fully mixed, according to 2.5 mg/100g ratios in rat hindlimb intramuscular injection, one time a day,
Normal diet, drinking-water, continuous 15d.
2.3 rat models are identified and model stability observational technique
2.3.1 general state observation
Observing and nursing rat general state, daily routines, the state of mind, be quick on the draw, hunchbacked situation, hair onyx color and luster, excrement
Just the aspect such as quality and the change of rat body weight and body temperature is recorded.
2.3.2 the change detection of rat blood serum Cyclic Nucleotide system
At the end of modeling, kidney-yang deficiency and normal rats are taken, after etherization, vena orbitalis posterior clump carries out the 1-1.5 ml that take a blood sample.4℃
Refrigerator is placed after 4 h, and 2000 rpm are centrifuged 10 min and take supernatant, and using cAMP and cGMP ELISA kits rat blood serum is detected
Middle cAMP, cGMP content.Concrete operation step is operated by kit specification.
Modeling terminates rear 21 d, abdominal aorta 3 ~ 5 ml of blood sampling.At the end of concrete detection method is with modeling.
The acquisition of 2.4 spongiosa bone specimens
Modeling terminates rear 21 d, takes the stable rat of kidney-yang deficiency model and normal rat, and intraperitoneal anesthesia, sacrificed by exsanguination is fast on ice
Speed cuts each group rats with bilateral condyle of femur, half-and-half cuts condyle of femur open, and curet scraping cancellous bone is put into 5 ml EP pipes, marks rearmounted
In liquid nitrogen container, quick unloading is standby in -80 DEG C of refrigerators.
Embodiment two：
The screening of differential protein, it is comprised the following steps：
1. the extraction of cancellous bone total protein
1. fresh femoral shaft will be freezed to be cut off with bone shears, it is with ultra-pure water that bone marrow irrigation is clean, and with curet by femoral shaft spongiosa
Bone is gently scraped into mortar（Liquid nitrogen precooler in advance）In, liquid nitrogen is added toward mortar and is ground rapidly, repeat grinding 3～5 times,
Take powder and weigh quality, then according to 1 g bone tissues add the ratio of 1.8 mL lysates to add protein sample lysate, be placed in
The h of albumen 4 is extracted in 4 DEG C of refrigerator cracking（1 time is fully mixed every 1h）；2. the EP pipes for filling bone tissue suspension are placed in into low temperature
In supercentrifuge, 4 DEG C of 100000 rpm is centrifuged 1 h, Aspirate supernatant；Plus nuclease 3.：Every 1 ml lysates add 2 ul
Dnase and 2 ul Rnase, places on ice 15 min；4. it is ultrasonic：Till causing liquid not sticky with ultrasonic cleaning instrument ultrasound.Then
In setting low warm supercentrifuge, 4 DEG C of 100000 rpm is centrifuged 30 min, Aspirate supernatant；5. 2-D clean-up kits are carried
Pure protein concentrate.
2 Bradford methods determine protein concentration and calculate loading albumen volume
With reference to the operation of Bio-Rad company Bradford protein quantification kits operational manual, it is according to albumen loading gross mass
2500 ug calculate desirable proteins sample volume：2500ug/ protein concentrations.
The protein quantification calibration curve loading system of table 3
3. two dimensional gel electrophore- sis
Dielectrophoresis is operated referring especially to the 2-DE operating guidances of Bio-Rad companies of the U.S..
3.1 applied sample amounts and deposition condition are drawn（2500ug/ protein concentrations）Albumen sample and appropriate aquation sample-loading buffer
It is sufficiently mixed, cumulative volume is 380 ul.Adhesive tape under 50 V low voltage conditions after the h of swelling 12, carries out isoelectric focusing at 20 DEG C.Bubble
Swollen and isoelectric focusing parameter is shown in Table 4.
The IEF parameter settings of table 4
3.2 record 12% vertical panel PAGE gel prepares gel solution by formula rate in table 5.
The 12%SDS-PAGE gel formulas of table 5
3.3 adhesive tape are balanced respectively in adhesive tape level pad、Middle balance rinse 15 minutes, is filtered dry and is transferred to two to gel
On.
3.4 second to PAGE gel electrophoresis
Arranging 10 DEG C of circulating water temperature carries out SDS-PAGE, and deposition condition is：Switch on power, originally with the constant voltage 40 of 100 V
Min, after use the h of 300 V constant voltages 4.5 instead and carry out electrophoresis, stop electrophoresis when bromophenol blue is run at the cm of gel bottom about 1.
4. it is gel-colored to show with scanning
Electrophoresis takes out gel after terminating, and performs mark.2- is obtained after coomassie brilliant blue staining with Image Scanner scanners
DE gel images.
5. the detection of gel protein point shows analysis with differential protein spot
The analysis softwares of PD Quest 8.0 provided using Bio-Rad companies carry out bone tissue（Cancellous bone）Dielectrophoresis gel figure
Spectrin point is detected and differential protein spot shows analysis.Faint spot, small spot and tri- ginsengs of large spot are set
Counting and remove background and transverse and longitudinal striped etc. carries out the automatic detection of protein site.It is to be detected finish after, the matching analysis of set-point ginseng
Number, using the volume of point as the measured value of protein site content, the relative normalized Volume Changes of setting protein site are up to more than 1.5 times works
Basic parameter for the matching analysis of differential protein spot and to arrange t inspections up to 99% be statistically significant.Preliminary matches are completed
Afterwards, manual manual synchronizing is carried out, observation is amplified to the protein site that each is matched, to exclude some fict protein sites.
6. mass spectrum sample preparation and the MALDI-TOF-MS mass spectral analyses of differential protein
Differential protein spot is cut manually glue, is digested, Matrix-assisted laser desorption ionization is carried out after desalination point sample
（MALDI-TOF-MS）The protein of cancellous bone differential expression between technology qualitative each group, and retrieved to difference by bioinformatics
The function of expressing protein carries out initial analysis.
7. protein database search and identification
Using MASCOT（V3.2, Matrix Science, London, U.K）Search one of library software to each differential protein sample
Level and second order mses data carry out protein database search and identify protein.
The bioinformatics retrieval of 8 protein correlation functions
The relevant information of the known albumen that Mass Spectrometric Identification is obtained, including egg are searched in Uniprot and NCBI Protein Data Banks
White Gene Name, protein family, amino acid sequence, major function etc..
Embodiment three：
, from albumen and gene level double verification, it is comprised the following steps for Western Blot and RT-PCR technology：
1. the detection of the Western Blot of the dry and soft matter bone GAP-associated protein GAP of each group rat femur
The extraction of 1.1 cancellous bone total proteins
The dry cancellous bone of 8 rat femurs is randomly selected per group from 80 DEG C of refrigerator-freezers of ﹣ to take out, is placed on ice, rapidly will with rongeur
Bone tissue is shredded, and in being put into the mill of prior precooling, powder is ground to rapidly, adds liquid nitrogen cold in process of lapping in good time
But, in being then transferred to Eppendorf pipes, weight is weighed；In proportion 1 g bone tissues add 1 ml lysates, the shake of micro-whirlpool device
5 s are swung, 4 DEG C of refrigerators are then placed in, concussion is vortexed once every 30 min, taken out after 4 h；In proceeding to low-temperature and high-speed centrifuge,
4 DEG C of setting program, 14000 rpm are centrifuged 20 min, Aspirate supernatant.
1.2 BCA methods determine protein concentration
（1）Prepare the molten product of protein standard（25 mg/ml）：0.8 ml protein standards are prepared into liquid and adds 20 mg bovine serum albumins
（BSA）After being completely dissolved, dispense standby；
（2）Diluted protein standard items（0.5 mg/ml）：Take the protein standard substance for having configured in right amount（25 mg/ml）, it is slow with phosphoric acid
Rush salting liquid（PBS）It is diluted to 0.5 mg/ml concentration；
（3）Prepare BCA working solutions：According to the quantity of required sample, BCA reagent A liquid, B liquid are pressed into 50:1 ratio, is fully vortexed mixed
It is even；
（4）By 0,1,2,4,8,12,16,20 l is by standard items（0.5 mg/ml）It is sequentially added 96 orifice plate protein standard sample wells
In, per hole PBS is supplied to 20 l；
（5）Plus appropriate protein sample, 5 times, 10 times, 20 times are diluted with PBS, complement to 20 l per hole；
（6）Each hole adds 200 l BCA working solutions；
（7）37 DEG C of 30 min of incubation；
（8）The nm of wavelength 570 is determined, absorbance is read（OD）Value；
（9）Establishing criteria curve calculates the protein concentration of sample.
1.3 cancellous bone total protein denaturation
Appropriate protein sample is taken by 5:1 ratio and SDS-PAGE albumen sample-loading buffers（6×）It is vortexed and mixes, puts constant-temperature metal bath
100 DEG C of 5 min, is immediately placed in cooled on ice after denaturation, be stored in -80 DEG C of refrigerators standby.
The preparation of 1.4 solution
（1）Lysate takes appropriate RIPA lysates and PMSF（l00 mM）By 99:1 ratio is well mixed, and makes that PMSF's is final dense
Spend for 1 mM, preserve on ice stand-by.
（2）Electrophoresis liquid by 5 × electrophoresis liquid dilute 5 times with ultra-pure water, be made into 1 × electrophoresis liquid.
（3）The g ammonium persulfates of 10% ammonium persulfate 0.1 add ultra-pure water to be settled to 1 ml, are completely dissolved mixing, now with the current；
（4）TBST cleaning solutions are by 50 ml TBS（20×）It is placed in together in beaker with 1 ml tweens and mixes, uses ultra-pure water constant volume
To 1 L, fully mix, 4 DEG C of Refrigerator stores are standby.
（5）BeyoECL Plus working solutions press 1:1 ratio takes respectively appropriate BeyoECL Plus A liquid and B liquid is mixed, existing
With existing use, lucifuge during operation.
1.5 PAGE gels are prepared
The gel reagents of respective volume are sequentially added according to table 6, after about 20 min are solidified under room temperature, is placed in electrophoresis liquid and is stored in 4
DEG C refrigerator is standby, typically carries the previous day and prepares PAGE gel.
The PAGE gel of table 6 prepares required each component volume（ml）
The sample concentration that 1.6 loadings are determined according to BCA, the loading volume as needed for the sample-adding gauge of the ug of every hole 30 calculates sample,
Testing protein is slowly at the uniform velocity loaded successively, and adds the ul of standard items Marker 4.The edge hole of non-loading adds SDS-PAGE
Albumen sample-loading buffer（1×）10 ul.
1.7 protein electrophoresises cover electrode cap, switch on power, and initially running 10 min with the V of constant pressure 20 guarantees to be floated in duct
Albumen sink, then with the V of constant pressure 60 run 20 min, finally with 80 V run 100 min, treat Bromophenol Blue dye reach glue bottom,
Stop electrophoresis.
1.8 half-dried turns under the V of constant voltage 25 according to membrane area setting electric current, monoblock glue limits the A of electric current 1.3,1
The A of electric current 0.7 during glue.Transferring film duration is adjusted according to destination protein molecular weight（It is shown in Table 7）；After transferring film terminates, glue is placed in into coomassie
Incubator overnight in light blue dye liquor dye, is decolourized to transparent for second day, checks whether albumen shifts completely.
The destination protein transferring film duration of table 7
After the closing transferring film of 1.9 films terminates, at tweezers folder marker, take out pvdf membrane and marked front, rinsed with ultra-pure water
5 min × 3 time, pvdf membrane is put in the valve bag equipped with 5 ml confining liquids, is placed in room temperature, and horizontal shaker is incubated 2 h.
1.10 1 anti-incubations are shown in Table a 8 anti-extension rate according to specification, anti-with the anti-diluteds one of WB mono-, dilute
Release rear cumulative volume and be about 5 ml, the pvdf membrane after closing is placed in the valve bag containing an anti-working solution, 4 on horizontal shaker
DEG C overnight incubation.
The antibody extension rate of table 8
1.11 2 it is anti-incubation one it is anti-incubation terminate after, film is put into into washing in bellows equipped with TBST, at room temperature on horizontal shaker
Rinse 5 min × 3 time；Wash after film terminates, corresponding two anti-working solution is prepared according to an anti-source, film is placed in into two anti-working solutions
In, slowly shake on horizontal shaker, be incubated at room temperature 1 h.
The anti-incubation of 1.12ECL chemiluminescence detections two rinses min × 3 time of filter membrane 5 after terminating with TBST, washes away uncombined
Two resist.Lucifuge prepares BeyoECL Plus working solutions（A liquid：B liquid=1：1）, fully mix, will on chemiluminescence imaging instrument
Film is overlying on development pad, is blotted surplus liquid with filter paper, and with roller bearing membrane removal bubble is removed, and developer solution is uniformly dripped in film
On, develop after film reacts 1 min at room temperature.
1.13 quantitative analyses adopt the software analysis protein bands of Bio-Rad image lab 3.0, calculate each group purpose egg
Develop the color in vain the Reinhoit Zahl for integrating with corresponding β-actin albumen, i.e., each destination protein relative expression quantity.
2. the genetic test of the dry and soft matter bone GAP-associated protein GAP of each group rat femur
2.1 bone tissues process bone tissue and are ground to the same protein extraction of powder process, rapidly with 1.5 ml without RNase
Eppendorf is managed（Following Eppendorf pipes are without RNase）Bone meal is plucked out along alms bowl bottom, weighs about 100 mg.
2.2 extract total serum IgE with Trizol methods
The detection of 2.3 total rna concentrations clicks Nucleic Acid in ultramicron nucleic acid-protein analyzer key frame, after zeroing, plus 1
μ l testing samples determine the absorbance ratio of 260/280 nm on detecting platform, draw the concentration of extracted RNA.
2.4 reverse transcription reaction（Reverse Transcription, RT）Total RNA is dense measured by more than
Degree, calculates the loading volume V needed for 2 g total RNAsRNA, by the system of table 9 reverse transcription reaction system is prepared；In 42 DEG C
2 min of lower reaction.Reverse transcription step is set：25 DEG C of 10min, react 30min, 85 DEG C of fire extinguishing 5min at 50 DEG C, be statically placed in 4 DEG C
Under.
The reverse transcription reaction system of table 9
The design of 2.5 primers and synthesis
Genes of interest primer, the synthesis of GAPDH RT-PCR primers are by the limited public affairs of Shanghai life work bioengineering share used by this experiment
Department's design synthesis（Table 10）.
Table 10 tests relevant primer list
2.6 PCR（Polymerase Chain Reaction PCR）In strict accordance with PCR kit specification
Operation, will above react the cDNA for obtaining for preparing PCR amplification system（Table 11）.
The PCR amplification system of table 11
Note：Taq is included in 2 × AceTaqMaster MixTMArchaeal dna polymerase, MgCl2, dNTPs, reaction buffer
The reaction system for preparing is mixed and is put in gene-amplificative instrament after centrifugation, response parameter is set by following reaction condition：
94 DEG C of denaturations 3min；94 DEG C of denaturation 30s, anneal 30s（Each system annealing temperature is the annealing temperature of added primer）, 72 DEG C are prolonged
Stretch 60s, 35 circulations；72 DEG C extend eventually 10min, preserve at 4 DEG C.
2.7 agarose gel electrophoresis take each 3 l of PCR primer and enter row agarose gel electrophoresis, and deposition condition is 90 V, and 15
min；Electrophoresis poststaining simultaneously adopts the software analysis images of 2000 gel imaging system Quantity One of GEL DOC 465, obtains
The OD value and corresponding internal reference gene of corresponding protein expression（GAPDH）The ratio of OD value.
Example IV：
ELISA determines GAP-associated protein GAP content in blood normally with rats with deficiency of kidney-Yang, comprises the following steps：
1. the preparation of each group rat blood serum
Prepared by each group rat blood serum, 21 d after terminating with modeling.See the 2.3.2 of embodiment one.
2. in each group rat blood serum GAP-associated protein GAP content detection
2.1.ELISA the preparation of method main agents：Operate according to ELISA kit specification.
2.2.ELISA detection method
（1）Sample-adding：Blank well is set respectively（Blank control wells are not added with sample and enzyme marking reagent, and remaining each step operation is identical）, it is to be measured
Sample well.First add the μ l of sample diluting liquid 40 in testing sample hole on enzyme mark coating plate, the μ l of testing sample 10 are then added again（Sample
The final dilution factor of product is 5 times）Sample is added on ELISA Plate bottom hole portion by sample-adding, and hole wall is not touched as far as possible, gently rocks mixing；
（2）Incubate：37 DEG C of 40 min is stood with the rearmounted incubate box of shrouding film shrouding；
（3）Board-washing：Carefully take shrouding film off, discard liquid, dry, cleaning solution is filled it up with per hole, stand and discarded after 30 s, so weight
It is multiple 5 times, print dry on filter paper；
（4）It is enzyme-added：The μ l of enzyme marking reagent 50 are added per hole, in addition to blank well；
（5）Incubate：Method is same（2）；
（6）Board-washing：Method is same（3）；
（7）Colour developing：Colour developing A, each 50 μ l of colour developing B, gently concussion is successively added to mix, be statically placed in 37 DEG C of incubator and keep away per hole
Light is incubated, and develop the color 15 min；
（8）Terminate：The μ l of terminate liquid 50 are added per hole, now color transition is by Lan Zhuanhuang, terminating reaction；
（9）Determine：With blank zeroing, the nm of wavelength 450 is determined, read absorbance.It is complete within 15 min after Ying Jia terminate liquids
Into.
Embodiment five：
Verified repeatedly by above-mentioned experiment, the ELISA testing results of haemocyanin and Western-Blot, RT-PCR are examined
Comparative result analysis is surveyed, expression trend is chosen unanimously and the differential protein differential protein outside range of normal value is used as kidney-yang deficiency
Card biomarker.
Above-mentioned test can obtain following experimental result：
1. the foundation of animal model and qualification result
Modeling terminates in rear 21d, and 3 rats of kidney-yang deficiency group are excessively weak due to physique, the factor such as feeding and amount of drinking water deficiency, consumption
Exhaust and die.So last normal group quantity is constant, kidney-yang deficiency group mores than 17.Modeling terminates rear 21d, compares with normal rats,
Kidney-yang deficiency group rat remains unchanged auricle onyx without color, and the happiness back of a bow is curled, flocked together, and activity is few, loose stool, loses weight, body temperature drop
Low, serum cAMP contents and cAMP/cGMP ratios decline.Above symptom, sign and serum cAMP and cAMP/cGMP ratio knot
Fruit meets the identification requirement of syndrome of deficiency of kidney yang model, points out the modeling of this animal used as test to be successful.
2. differential protein the selection result
The identification of 2.1 gel protein point differences and analysis
2.1.1 by Bio-Rad companies PDQuest software analysis, each gel protein collection of illustrative plates can be recognized for the detection of gel protein point
Protein site be 560.
2.1.2 the identification of gel protein point difference and analysis are by normal group and kidney-yang deficiency group bone tissue（Cancellous bone）
Two-dimensional Gel Electrophoresis gel pattern comparative analysis, detects the protein spots of 29 notable difference expression, is with normal group
With reference to, the protein spots of differential expression are marked, point 3,7 is downward expressing protein（See Fig. 1）.
The MALDI-TOF-MS Mass Spectrometric Identifications of 2.2 differential proteins
29 differential expression protein points are cut by hand from gel, Jing after trypsin digestion digestion MALDI-TOF- is carried out
MS mass spectral analyses.The peptide mass fingerprinting spectrum signal of acquisition is strong and baseline is more steady, and noise is low, is adapted for database retrieval.
It is exemplified below MS the and MS/MS mass spectrogram information of part peptide fragment（See Fig. 2,3）, searching library software using MASCOT carries out protein mirror
It is fixed, table 12 for Mass Spectrometric Identification Partial Protein point relevant information, including encoded register, species, isoelectric point, the molecule of protein
Amount, score.
The part variation protein site MALDI-TOF-MS qualification results of table 12
The bioinformatics retrieval of 2.3 differential protein correlation functions
It is related albumen to be carried out in the differential protein title input UniProt and NCBI Protein Data Banks that Mass Spectrometric Identification is established
The bioinformatics retrieval of function, the results detailed in Table 13.Point out by the biological information that String database retrievals are provided, together
When we consult pertinent literature binding result, it is presumed that the change of syndrome of deficiency of kidney yang symptom and sign may with GDP dissociation press down
The factor processed 1, NTx α -1 chains are relevant.
The bioinformatics retrieval result of the partially protein correlation function of table 13
3. in normal group-kidney-yang deficiency group rat femur cancellous bone Colla1 and Arhgdia protein expressions detection
3.1 Western bolt detections show：Compare with normal group, the expression of kidney-yang deficiency group Colla1 is reduced, and has significant difference
（P＜ 0.05）；Compare with normal group, the expression of kidney-yang deficiency group Arhgdia is reduced, and has significant difference（P＜ 0.05）.It is shown in Table 14,
Fig. 4.
The comparison of Col1a1 and Arhgdia protein expressions in 14 two groups of rat cancellous bones of table（±S）
Note：Compare with normal group, a P＜ 0.05.
The detection of 3.2 normal groups-kidney-yang deficiency group rat femur dry cancellous bone Col1a1 and Arhgdia mRNA expression
The expression of the mRNA of two groups of visible Colla1.Compare with normal group, kidney-yang deficiency group rat Colla1 expression declines, and has aobvious
Work sex differernce (P＜ 0.05)；Compare with normal group, rats with deficiency of kidney-Yang Arhgdia expression decline, have significant difference (P＜
0.05).Be shown in Table 15, Fig. 5,6.
The comparison of Col1a1 and Arhgdia mRNA expression in 15 two groups of rat cancellous bones of table（±S）
Note：Compare with normal group, a P＜ 0.05.
The detection of Col1a1 and Arhgdia contents in 3.3 normal groups-kidney-yang deficiency group rat blood serum
ELISA method detects that Colla1 assay situations show in normal group and kidney-yang deficiency group rat blood serum：Two groups can examine
Determine the expression of Colla1.Compare with normal group, rats with deficiency of kidney-Yang Colla1 expression decline, have significant difference (P＜
0.05) ；Compare with normal group, rats with deficiency of kidney-Yang Arhgdia expression decline, have significant difference (P＜ 0.05).16 are shown in Table, figure
7。
The comparison of Col1a1 and Arhgdia protein expressions in 16 two groups of rat bloods of table（±S）
Note：Compare with normal group, aP＜ 0.05.
The reference range of Col1a1, Arhgdia content in 3.4 normal rats serum
According to the ELISA testing results of three albumen in normal group, we calculate reference range.It is shown in Table 17.
The reference range of the content of Col1a1, Arhgdia in the rat blood of table 17（pmol•mL-1）
4. preliminary screening confirms the albumen that can be used as syndrome of deficiency of kidney yang mark.
Show through Western blot and RT-PCR the results：Organize in kidney-yang deficiency and normal rats Grafting Cancellous Bone Bolt
In, Colla1, Arhgdia are consistent with early stage proteomics result in albumen and gene level expression trend, show as：Kidney
Colla1, Arhgdia are in conspicuousness low expression in deficiency of yang group.Show the accuracy reliability of early-stage Study result, can be used as kidney
Biomarker of the yang deficiency syndrome in bone tissue.
ELISA testing results show, Colla1 and Arhgdia and protein expression one in femoral shaft cancellous bone in peripheral blood
Cause, comparing with normal group has significant difference, content is below normal reference value, point out Colla1 and Arhgdia to decline
May be closely related with syndrome of deficiency of kidney yang, can be used as the potential mark of blood of syndrome of deficiency of kidney yang diagnosis.
The foregoing is only presently preferred embodiments of the present invention, all protein screenings done according to scope of the present invention patent and
It is determined that, should all belong to the covering scope of the present invention.
SEQUENCE LISTING
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<120>A kind of screening of syndrome of deficiency of kidney yang animal model biomarker and determination method
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Claims (6)
1. a kind of syndrome of deficiency of kidney yang animal model biomarker screening and determine method, it is characterised in that required concrete steps
It is as follows：
（1）The foundation of syndrome of deficiency of kidney yang rat model；
（2）The screening of differential protein；
（3）Checking to part and syndrome of deficiency of kidney yang relevant difference albumen；
（4）The determination of syndrome of deficiency of kidney yang animal model biomarker.
2. a kind of screening of syndrome of deficiency of kidney yang animal model biomarker according to claim 1 and method is determined, it is special
Levy and be, the used rat for building animal model is WISTAR rats.
3. a kind of screening of syndrome of deficiency of kidney yang animal model biomarker according to claim 1 and method is determined, it is special
Levy and be, what the differential protein was screened comprises the following steps that：
(1) extraction of rat cancellous bone total protein；
(2) protein concentration is determined；
(3) Two Dimensional Gel Electrophoresis；
(4) detection of gel protein point shows analysis with differential protein spot；
(5) the MALDI-TOF-MS mass spectral analyses of differential protein；
(6) protein database search and identification, determine differential protein.
4. a kind of screening of syndrome of deficiency of kidney yang animal model biomarker according to claim 1 and method is determined, it is special
Levy and be, the verification method of the part variation albumen is as follows：
(1) using Western-Blot methods mark is verified from protein level；
(2) using RT-PCR method mark is verified from gene level.
5. a kind of screening of syndrome of deficiency of kidney yang animal model biomarker according to claim 1 and method is determined, it is special
Levy and be, the determination method of the syndrome of deficiency of kidney yang animal model biomarker is：By the ELISA testing results of haemocyanin with
Western-Blot, RT-PCR testing result comparative analysis, chooses expression trend unanimously and the difference outside range of normal value
Albumen is used as biomarker.
6. utilize a kind of screening of the syndrome of deficiency of kidney yang animal model biomarker described in claim 5 and determine what method was obtained
It is NTx α -1 chains as syndrome of deficiency of kidney yang mark（Colla1）And GDP dissociation inhibiting factors 1（Arhgdia）.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001077385A1 (en) * | 2000-04-11 | 2001-10-18 | Takara Bio Inc. | Method of detecting cancer |
| CN101960309A (en) * | 2008-01-17 | 2011-01-26 | 东丽株式会社 | Composition and method for diagnosis or detection of gastric cancer |
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| WO2001077385A1 (en) * | 2000-04-11 | 2001-10-18 | Takara Bio Inc. | Method of detecting cancer |
| CN101960309A (en) * | 2008-01-17 | 2011-01-26 | 东丽株式会社 | Composition and method for diagnosis or detection of gastric cancer |
Non-Patent Citations (4)
| Title |
|---|
| 刘希成，等: "肾阳虚证候的人血清比较蛋白质组学分析", 《中国生物化学与分子生物学报》 * |
| 张子怡，等: "肾阳虚、肾阴虚证大鼠模型的建立与稳定性观察", 《福建中医药》 * |
| 王艺茹，等: "肾阳虚大鼠股骨皮质骨差异蛋白质表达分析", 《福建中医药》 * |
| 邹叶强: "单侧肾上腺切除对哮喘大鼠嗜铬细胞转变的影响及补肾方的干预作用", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
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