CN106645748B - A kind of exception lymphatic temperament type impotence disease mammalian serum and testosterone histological difference albumen system and screening technique and application - Google Patents
A kind of exception lymphatic temperament type impotence disease mammalian serum and testosterone histological difference albumen system and screening technique and application Download PDFInfo
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Abstract
The present invention provides the screening techniques that dimension cures the mammalian serum differential protein and testosterone histological difference albumen system and above-mentioned differential protein system of abnormal lymphatic temperament type impotence disease, including 1) Normal group, modeling card marquis group and disease group；2) whole blood, separation serum and testosterone tissue are acquired；3) protein is extracted；4) it is purified after marking；5) finger-print is obtained after Mass Spectrometer Method, proteome analysis is carried out after screening, obtains the syndrome group, disease group and Normal group serum proteins and testosterone tissue protein；6) it counts, compare, screening to obtain tieing up and cure abnormal lymphatic temperament type impotence disease serum differential protein and testosterone histological difference protein, it lays a good foundation further to carry out abnormal lymphatic temperament type impotence (erectile dysfunction) disease Mechanism Study, drug screening, conversion and study on prevention, provides foundation.
Description
Technical field
The invention belongs to biotechnologies, and in particular to a kind of mammal of the abnormal lymphatic temperament type impotence disease of dimension doctor
Serum and testosterone histological difference albumen and its screening technique and application.
Background technology
It is important component in traditional Chinese medicine that Uygur medicine (dimension doctor), which is learned, and dimension medicine body fluid opinion thinks, human body by
4 kinds of bile matter, blood matter, lymphatic temperament and black courage matter body fluid matter compositions, the dynamic equilibrium of above-mentioned 4 kinds of body fluid matter is body health
Physiological foundation, and the variation of the amount or/and matter of a certain body fluid matter, cause body fluid matter dysequilibrium, generate a kind of abnormal humour
There is causality with a variety of diseases in cross-examination (syndrome).Humoral pathology (body fluid opinion) is dimension medical knowledge opinion foundation, is recognized
For in 4 kinds of body fluid, lymphatic temperament body fluid attribute is raw, Chang Yiyin lives, diet and environmental factor, operating pressure, shortage movement etc.
Reason causes internal isohydria to be destroyed, and lymphatic temperament body fluid changes, and generates abnormal lymphatic temperament, causes metabolic water pancake
Low, product accumulation is internal, and then pathology is caused to sexually revise, and causes relevant disease.
Clinical research confirmation, abnormal mucus cross-examination (Abnormal Phlegm) is in impotence (Erectile Dysfunction), morning
It lets out, lack the metabolic diseases such as reproductive diseases, obesity, the hyperlipidemia such as azoospermia, premature ovarian failure, infertile and cardiovascular disease
In sick generating process, it is in leading position, may be also the master that early stage occurs for the complex diseases such as tumour, diabetes, hypertension
Demonstrate,prove type.Erectile dysfunction (Erectile Dysfunction, ED), also known as impotence, refer to man's penis anorthosis and
It cannot maintain to erect and complete satisfied sexual life, belong to dimension doctor's advantage disease.Telotism is by the multiple biological activities factor (packet
Include neurotransmitter, hormone, enzyme, ion channel etc.) nerve-vascular biological activity of comprehensive adjustment.In dimension medicine clinic and base
Plinth research direction, without related Disease Syndrome integrated animal model, leads to abnormal lymphatic temperament type impotence because experimental zoology is started late
Card and its verification prescription Study on mechanism lag always, and seriously constrain research and development of the Wei Yi andrologies in this field,
There are no the research of correlating markings albumen and its protein markers objects system about abnormal lymphatic temperament type impotence disease reports.
In order to further recognize the abnormal lymphatic temperament type impotence of dimension doctor from modern biology of reproduction angle, we are based on dimension doctor's body
Liquid is discussed and modeling theory, has been successfully established abnormal lymphatic temperament syndrome animal model, and test and mated in fact using APO telotisms
The method for testing combination therefrom filters out ED models, to its Biological Characterization, sexual function and mating behavior, gonad axis and NO-CGMP
Change in terms of the biology of reproduction such as signal transduction pathway is studied, but it is single that abnormal lymphatic temperament type impotence disease, which is not,
The disease of reproductive organs, that is, testosterone tissue, but systemic disease, it is therefore desirable to which we use organic conception, from whole body
The visual field, to analyze, study and explore the regularity of occurrence and development of the disease and possible therapy.Dimension doctor's differentiation of symptoms and signs for classification of syndrome rises
Point and core are dimension doctors " body fluid opinion ", and starting point is the external manifestation in body fluid dynamic change in human body, and ties up medicine and pharmacology institute
The diagnosis and treatment based on an overall analysis of the illness and the patient's condition of high praise is also according to the body fluid phenotypic difference occurred in individual integral level, this integrally grinds with systems biology
It is perfectly in harmony to study carefully thinking.
Serum has incorporated the protein multidate information of body multiple organ, tissue and cell, and close with penile erectile function
Relevant secretory protein the only way which must be passed is cut, can reflect the space-time of the relevant global regulation network of the pathophysiological change of body
Property, and drug finally also be reflected among the variation of serum proteins group the effect of any target organ, tissue or cell.Penis is
The effector organ of telotism, corpus cavernosal smooth muscle then be effect tissue, any type pathology triumph change also with the tissue
Protein level dynamic change is inseparable, wherein containing impotence occurs relevant potential protein labeling and the disease medicine
One of the important target spot of object treatment.
Therefore this research is based on abnormal lymphatic temperament syndrome on the basis of Disease Syndrome integrated animal model, has carried out serum and penis
The proteomics research of smooth muscle tissue establishes abnormal lymphatic temperament type impotence disease associated serum and testosterone tissue
Albumen system, Mechanism Study, drug screening, conversion and study on prevention further to carry out abnormal lymphatic temperament type impotence disease are established
Basis is determined.
Invention content
In view of this, the first object of the present invention is to provide the dimension doctor mammal blood of abnormal lymphatic temperament type impotence disease
Clear differential protein system, including following albumen：
Preferably, the mammalian serum differential protein system of the abnormal lymphatic temperament type impotence disease of the dimension of the invention doctor
From model animal；The model animal is rat.
The second object of the present invention is to provide the dimension doctor mammal testosterone of abnormal lymphatic temperament type impotence disease
Histological difference albumen system, including following albumen：
Preferably, the mammal testosterone histological difference of the abnormal lymphatic temperament type impotence disease of dimension doctor of the present invention
Albumen system comes from model animal；The model animal is rat.
The mammalian serum that still a further object of the present invention is the provision of the above-mentioned abnormal lymphatic temperament type impotence disease of dimension doctor is poor
The screening technique of M-band system and testosterone histological difference albumen system, includes the following steps：
1) Normal group (N groups), abnormal lymphatic temperament syndrome group (ZM groups), abnormal lymphatic temperament type disease group (BM are established
Group)；
2) mammalian whole blood that the step 1) obtains is acquired, the serum of mammal is obtained after separation, takes the step
The rapid testosterone tissue for 1) obtaining mammal；
3) protein is extracted in the serum and the testosterone tissue that are obtained from the step 2)；
4) it will be purified after the protein labeling obtained in the step 3), obtain peptide fragment after purification；
5) peptide fragment after purification that the step 4) obtains is obtained into finger-print, institute after screening after Mass Spectrometer Method
It states finger-print to be retrieved using proteome analysis software, be carried out according to the spectrogram after obtained search result and screening
Quantitative analysis examines obtained analysis data in the mammalian proteins database using proteomic image retrieval software
Rope obtains the N groups, ZM groups and BM groups mammalian serum and testosterone histological difference protein；
It 6) will be in the step 5)
The BM groups obtain the serum differential protein of BM/ZM compared with the serum proteins of ZM groups；By BM groups and N
The serum proteins of group compare, and obtain the serum differential protein of BM/N；Count the differential protein of the BM/ZM and BM/N
The type and quantity of common albumen, obtain the serum differential protein of abnormal lymphatic temperament type impotence disease group；
BM groups and compared with the testosterone protein of ZM groups, the testosterone tissue for obtaining BM/ZM is poor
M-band matter；BM groups are compared with the testosterone tissue protein of N groups, obtain BM/N group testosterone histological differences
Protein；The type and quantity for counting the common albumen of the differential protein of the BM/ZM and BM/N obtain abnormal lymphatic temperament type sun
The testosterone histological difference protein of atrophy disease card group；
Preferably, in screening technique of the invention, the mammal is rat.
Preferably, in screening technique of the invention, rats in normal control group is raised in conventional environment in the step 1),
The environment temperature of feeding is 18~22 DEG C, and the relative humidity of the feeding environment is 40~60%, the environment of the feeding of modeling group
Temperature is 8~12 DEG C, the relative humidity 70~80%% of the feeding environment；Normal group is common raises in the step 1)
Material, modeling group feeding feed are raw property feed.
Preferably, in screening technique of the invention, labeling method described in the step 3) is iTRAQ labelling kit marks
Note.
Preferably, in screening technique of the invention, proteome analysis software is Proteome in the step 4)
Discoverer；The proteomic image retrieval software is mascot softwares.
The present invention also provides the mammalian serum differential protein systems that above-mentioned dimension cures abnormal lymphatic temperament type impotence disease
It is tied up in preparation prevention or treatment mammal exception lymphatic temperament type impotence drug with testosterone histological difference proteosome
Using.
From the foregoing, it will be observed that the advantage of the invention is that：
1, there is no the concept of syndrome in modern medicine, commonly use and find differential protein between disease and Normal group, and
In dimension doctor's clinical diagnosis and treatment, then need diagnosis and treatment, and treated using different prescriptions to the impotence patient of different syndrome type, i.e., it is sick
Card is corresponding with prescription.Inventor herein is gained knowledge based on the understanding to syndrome and disease with abundant Uygur medicine, with debating
The mode being combined with the differentiation of disease is demonstrate,proved, by Analogy, between the differential protein and the differential protein of BM and N groups of BM and ZM groups
Common albumen is found, screening has gone out the serum and testosterone tissue candidate protein markers of abnormal lymphatic temperament type impotence disease
Object, and by verification, establish the mammalian serum and testosterone histone mark of abnormal lymphatic temperament type impotence disease
Will objects system, and the system then more accurately reflects annotation of the dimension doctor to disease, embodies the essence of syndrome.And establish macroscopic view
Unified index system is studied with microcosmic cognition cooperation, analysis and synthesis, promotes the diagnosis and treatment level and medicament research and development of impotence
Level is of great significance
2, the present invention provides the mammalian serum differential proteins and 48 of 35 kinds of abnormal lymphatic temperament type impotence diseases of dimension doctor
Kind testosterone histological difference albumen；Show its serum and testosterone group in model animal such as rat by embodiment
It knits the up-regulation of middle differential protein or lowers expression, show that these differential proteins produce centainly the penile erectile function of rat
Influence, be ultimately determined to the biomarker of abnormal lymphatic temperament type impotence disease, can be directed to above-mentioned biomarker carry out
Drug basic research, for further carry out abnormal lymphatic temperament type impotence (erectile dysfunction) disease Mechanism Study, drug screening,
Conversion is laid a good foundation with study on prevention, provides foundation.
Description of the drawings
Fig. 1 be Normal group, abnormal mucus cross-examination marquis group and disease group rat model modeling flow chart；
Fig. 2 is the mammalian serum and testosterone tissue protein group of the abnormal lymphatic temperament type impotence disease of dimension doctor
And its screening process figure of differential protein system；
Fig. 3 is the integrity detection of rat blood serum differential protein and rat testosterone histological difference protein
SDS-PAGE electrophoresis is composed.
Fig. 4 is the ELISA detections that 7 kinds of albumen is arbitrarily chosen in 35 kinds of candidate differential proteins.
Specific implementation mode
In embodiments of the present invention, experimental animal is：30 (bodies of sexual maturing period SD male rat with normal sexuality
Weight 200g or so), female rats 15 (weight 180g or so) are provided by Xinjiang Medicine University's Experimental Animal Center；
In embodiments of the present invention, drug and reagent：Before raw property feed is pressed by Xinjiang Medicine University's Experimental Animal Center
The requirement used in phase research is prepared；Spinach is real to be purchased from Uygur medicine hospital of Xinjiang Uygur Autonomous Regions with medicinal materials such as coriandri,fructus
Herbal medicine room；Yellow Jackets；Physiological saline；Apomorphine (90 μ g/kg)；Estradiol；Progesterone；Predominantly detect reagent：ELISA
Kit, BCA protein quantification kits etc..
In embodiments of the present invention, term is following meanings：
Term APO:Apomorphine (Apomorphine)；
Term iTRAQ：Isotope labelling is opposite and absolute quantitation (isobaric tags for relative and
absolute quantitation)
Term HPLC:：High performance liquid chromatography (HighPerformance Liquid Chromatography)
Term SDS：Lauryl sodium sulfate (sodium dodecyl sulfate, sodium salt)
Biological Characterization is observed：
Qualitative index is observed：The qualitative index is observed skin and hair and observes in the sunlight, and emotional reactions are in a cage
Each rat is observed 30 minutes based on reacting to each other, and behavior state is based on the activity during this, 30 points of observation under quiet environment
Clock, excitement degree with press from both sides tail test when reaction based on, tongue picture tongue fur is observed, is recorded and takes pictures in the sunlight, diet water state
With the same time feeding observation diet water state of each group, when urine was just urinated with each group rat same day based on color, state of defecating
Based in the form of being defecated when the same day positive bowel movement；
Quantitative target is to avoid circadian influence, is fixed on 20 daily:00-22:00 collects every data, wherein body
Weight：It weighs and records in required time daily, with every cage total weight divided by the cage rat number of elements, obtain average weight, and press
Formula is stated to obtain each cage body weight ratio of model group and record, weight ratio=model group average weight-model group original body mass/just
Often group average weight-normally organizes original body mass；Dietary amount：Daily 20:00 addition feed 300g, next day 20:00 claims food surplus,
The corresponding dietary amount of 100g weight is obtained according to following formula and is recorded；Amount of drinking water：Daily 20:00 water supply 500ml, next day
20:00 amount surplus water is calculated the corresponding amount of drinking water of 100g weight and records；Urine volume：Modeling record within first day to
Dry dunnage weight, claim within second day the weight and record of the wet bedding and padding containing urine and stool in required time, then in constant temperature
It weighs again after drying in 2 hours at 100 DEG C of air dry oven, calculates each group urine volume；Stool amount：It is corresponding to calculate 100g weight
Stool amount simultaneously records.
APO erective tests or APO experimental methods are
With reference to the method for Heaton etc., experiment rat is placed in 10~15min in observation case and adapts to environment, is kept quite,
Light is dimmed, (0.5mg/kg vitamin Cs are in middle dissolving apo- in rat neck injection of skin apomorphine (APO) 90 μ g/kg
After coffee, with 5mL/kg physiological saline mixing), it is observed at once after injection, when timer, observes and record in 1800s (0.5h)
The erection incubation period of rat and erection number.The standard of telotism：Rat stretches out in position of crouching of squatting, phallosome end when erection,
It bows to lick and licks glans penis.Erection incubation period is the time erected to first time after injecting.Erection number is big in 1800s after injecting
There is the number of telotism in mouse.If having no erection in 1800s, which is denoted as 0, and incubation period of erecing is denoted as
1800s。
Mating test method：
Male mouse and female mice (having carried out oestrus preparation) are respectively placed in different observation casees and adapt to environment, 10~15 points
Female-male proportion 1 is pressed after clock:2 are put into the same observation case, and the climb back of the body, insertion and the ejaculation for observing male rat in 1800s are hidden
Phase and climb the back of the body, insertion and number.If male rat does not carry out climbing the back of the body in 1800s, the rat climb the back of the body incubation period be denoted as
1800s climbs back of the body number and is denoted as 0.It is inserted into ejaculation behavior similarly.
In embodiments of the present invention, Normal group (N), abnormal lymphatic temperament syndrome group (ZM) and abnormal mucus are built first
Matter type disease group (BM) rat.In embodiments of the present invention, to the no spy of the acquisition methods of the N groups, ZM groups and BM group rats
Different limitation, using the technical solution well known to those skilled in the art for establishing abnormal lymphatic temperament syndrome animal model；
In the embodiment of the present invention, following steps are preferably specifically included：
Select sexual maturing period female mice, raised under normal condition after castration operation, by the female mice oestrus and male mouse into
Row mating test and APO experiment tests obtain the male mouse of normal sexual function.The present invention tests the mating test and APO
Time sequencing be not particularly limited.The mating test and the object of APO experiments are that the Normal group and modeling group are big
Mouse.
In embodiments of the present invention, the specific steps of the mating are preferably that the male mouse and female mice that will carry out oestrus are divided
It is not placed in different observation casees and adapts to environment, female-male proportion 1 is pressed after 10~15min:2 are put into the same observation case, observation
Male rat climbs the back of the body, insertion and ejaculation latency and climbs the back of the body, insertion and number in 1800s, if male rat is not in 1800s
Carry out climbing the back of the body, then the rat climb the back of the body incubation period be denoted as 1800s, climb the back of the body number be denoted as 0；It is inserted into ejaculation behavior similarly.
Male mouse with normal mating ability is included in Normal group and modeling by the present invention according to the result of the mating
Group, to being eliminated without normal mating ability hero mouse.
In embodiments of the present invention, APO experiment specific steps preferably experimental rat is placed in 10 in observation case~
15min adapts to environment, keeps quite, dims light, in 90 μ g/kg apomorphine APO of rat neck injection of skin, is stood after injection
Observation is carved, when timer, observes and record erection incubation period and the erection number of rat in 1800s；The standard of the erection
Rat stretches out in position of crouching of squatting, phallosome end, bows to lick and lick glans penis when to erect；The erection incubation period is after injection to the
The time once erected；The erection number is that the number of telotism occurs in rat in 1800s after injecting；If in 1800s not
See erection, then the rat erection number is denoted as 0, and incubation period of erecing is denoted as 1800s.
The male mouse with normal penile erection function is included in just according to the APO results tested in the embodiment of the present invention
Normal control group and modeling group, eliminate no normal penile erection function hero mouse.
In embodiments of the present invention, the 90 μ g/kg apomorphine solution preferably includes 90 μ g apomorphines and is dissolved in
In 0.5mg/kg vitamin Cs and physiological saline, adjustment volume is 5ml/kg.
In embodiments of the present invention, the Normal group is raised in conventional environment, and the environment temperature of feeding is 18~
22 DEG C, the relative humidity of the feeding environment is 40~60%；The environment temperature of the feeding of modeling group is 8~12 DEG C, described to feed
Eat the relative humidity 70~80% of environment；Normal group is normal diet, and modeling group feeding feed is raw property feed.
In embodiments of the present invention, the Biological Characterization observation refers to qualitative and quantitative observation and record.Qualitative index
Observation：The qualitative index is observed skin and hair and observes in the sunlight, emotional reactions with each rat in a cage react to each other for
Subjectivity is examined 30 minutes, and behavior state is observed 30 minutes based on the activity during this, under quiet environment, and excitement degree is to press from both sides tail
Based on reaction when experiment, tongue picture tongue fur is observed, is recorded and takes pictures in the sunlight, and diet water state is fed with each group same time
Diet water state is observed, when urine was just urinated with each group rat same day based on color, when stool state positive with same day bowel movement is big
Just based on form；Quantitative target is to avoid circadian influence, is fixed on 20 daily:00-22:00 collects every data,
Middle weight：It weighs and records in required time daily, with every cage total weight divided by the cage rat number of elements, obtain average weight, and
Each cage body weight ratio of model group is obtained by following formula and is recorded, the first initial body of weight ratio=model group average weight-model group
Weight/normally group average weight-normally organize original body mass；Dietary amount：Daily 20:00 addition feed 300g, next day 20:00 claims food
Surplus obtains the corresponding dietary amount of 100g weight according to following formula and records；Amount of drinking water：Daily 20:00 water supply 500ml,
Next day 20:00 amount surplus water is calculated the corresponding amount of drinking water of 100g weight and records；Urine volume：Modeling records for first day
The dry dunnage weight given well claims the weight and record of the wet bedding and padding containing urine and stool for second day, then exists in required time
It weighs again after drying in 2 hours at 100 DEG C of constant temperature blast drying oven, calculates each group urine volume；Stool amount：It is opposite to calculate 100g weight
The stool amount answered simultaneously records.
After obtaining the male mouse of normal sexual function (normal mating ability and penile erectile function), the present invention has what is obtained
The male mouse of normal performance is divided into Normal group and modeling group, and the Normal group is raised under conventional environment, the modeling
Group is raised with raw property forage feed in raw environment, and the Biological Characterization for observing and recording normal group and modeling group is seen.Packet
Include qualitative index and quantitative target.Qualitative index observes the skin and hairs of all groups of rats, emotional reactions, excitement degree, sleep
State, drinking-water state, urine volume property, stool state and tongue picture tongue fur.Quantitative target is the weight for recording all groups of rats, diet
Amount, amount of drinking water, urine volume and stool amount.Find that rat skin hair is slightly dim during modeling, it is matt；Burnout is curled up sleeping few
It is dynamic；Sensitive to stimulation, happiness flocks together, and burnout is drowsiness；Nothing strives water phenomenon；Urine volume is clear, and color is light, and amount is more；It is half congealed when soft when stool；Tongue fur
Secretly, white and greasy fur.And with the extension of modeling time, modeling group body weight increase is slow, dietary amount increases, and amount of drinking water is reduced, greatly
Urine amount significantly increases, and above-mentioned Biological Characterization meets the clinical card Hou Tedian of dimension doctor, big to obtain abnormal mucus cross-examination marquis
Mouse model, and the model has good scientific, reliability and stability.By APO and mating test from syndrome model group
In filter out abnormal lymphatic temperament type impotence disease binding model (Erectile Dysfunction and amixia ability), it is different to obtain
Normal lymphatic temperament type impotence disease animal model.The present invention to the syndrome at mould rate up to 100%, Syndrome model reaches at mould rate
50% or more, the period of Syndrome model is preferably 22~25 weeks.
In embodiments of the present invention, the rat quantity of the N groups is preferably 10.
In embodiments of the present invention, the method raised under the conventional environment is same as above the conventional environment raising side in step
Method.In the present invention, the feeding volume of the N groups is preferably the normal diet of daily 300g.The normal diet be include 500ml water
With 80g bedding and padding.The present invention is not particularly limited the bedding and padding, using bedding and padding well-known to those skilled in the art.
In the present invention, the quantity of the rat of the experimental group is preferably 20.
In the present invention, the raw property feed is the mixture of spinach reality and coriandri,fructus and conventional feed.In the present invention
In embodiment, the raw property feed is preferably：Include 100~200g coriandri,fructus and 100~200g spinach per 1kg conventional feeds
Dish is real, more preferably includes that 150g coriandri,fructus and 150g spinach are real per 1kg conventional feeds.The present invention is not special to conventional feed
Limitation, using the feed well-known to those skilled in the art for raising rat.The present invention is to the dish reality and coriander
Real source is also not particularly limited, using dish well-known to those skilled in the art reality and coriandri,fructus.Of the invention real
It applies in example, the real source with coriandri,fructus of the dish is purchased from Uygur medicine hospital of Xinjiang Uygur Autonomous Regions herbal medicine room.
In the present invention, the feeding volume of the raw property feed is preferably daily 300g.
In the present invention, the temperature of the raw environment is preferably 8~12 DEG C, and the humidity of the raw environment is preferably 70~
80%.The present invention is not particularly limited the facility for providing the raw environment, is carried using well-known to those skilled in the art
For the facility of raw environment.In the present invention in example, the facility for providing raw environment is growth cabinet.
In the present invention, the time of the raw property forage feed is preferably in Beijing time 09:00 to 21:00 is put into,
Its time is positioned in the identical feeding environment of control rats and raises.
In the present invention, the APO tests the method with mating test and verification result is tested with above-mentioned APO and mating test
Method and verification result.
The present invention is to preferably anaesthetizing rat before the blood sampling.The present invention does not have special limit to the method for the anesthesia
System, using anesthesia well-known to those skilled in the art.In the embodiment of the present invention, the anesthesia is abdominal cavity
Injection.The present invention is preferably yellow Jackets to the anesthetic used in the anaesthesia process.The yellow Jackets come
Source is purchased from and medicine company Co., Ltd of field Uygur.
In embodiments of the present invention, the specific method of the acquisition peripheral blood is preferably by postanesthetic rat quickly from abdomen
Aorta takes blood.
In embodiments of the present invention, fresh rat whole blood is preferably set 4 DEG C of environment by the method for the separation serum immediately,
5~10min is centrifuged with the speed of 3000~3500rpm after 30~40min, by obtained supernatant under the conditions of 4 DEG C, with 3000
The speed of~3500rpm centrifuges again, obtains serum, and finally centrifuging obtained serum, to be positioned over -80 DEG C of ultra low temperature freezers standby
With.4 DEG C of environment are preferably 4 DEG C of refrigerators.
The method for obtaining testosterone tissue is specifically：Testosterone tissue is taken from root, washes away bloodstain, and remove
Remaining skin, fascia take epimere testosterone tissue, are positioned in cryopreservation tube that be positioned over -80 DEG C of ultra low temperature freezers spare.
After obtaining serum and testosterone tissue, the embodiment of the present invention in serum and tissue to extracting protein, detection,
Obtain known concentration serum and testosterone tissue protein.
In the embodiment of the present invention, sample mixing is preferably carried out respectively according to group before extracting albumen, obtains three groups of blood serum samples
With three groups of testosterone tissue samples.
The embodiment of the present invention is not particularly limited the method for extracting proteins, ripe using those skilled in the art institute
The method for extracting proteins known.In the embodiment of the present invention, the method for extracting proteins is TCA- acetone precipitations.
In the embodiment of the present invention, the detection preferably includes the detection to protein content and the inspection to protein integrity
It surveys.In the embodiment of the present invention, the detection method to protein content is preferably Bradford methods, described complete to protein
The detection method of property is preferably SDS-PAGE methods.
After obtaining the protein of known concentration, the embodiment of the present invention marks protein iTRAQ labelling kits,
The protein marked, it is purified, obtain peptide fragment after purification.
In embodiments of the present invention, preferably the protein of obtained known concentration is digested before the label.This
Inventive embodiments are not particularly limited the method for the digestion, using protein digestibility side well-known to those skilled in the art
Method.The digestion described in the embodiment of the present invention uses trypsinization.
The embodiment of the present invention does not have special limitation to the source of the iTRAQ labelling kits, using art technology
The commercial goods of iTRAQ labelling kits known to personnel.The kit that the embodiment of the present invention uses is
Reagent-8Plex Multiplex Kit (Applied Biosystem) 8 mark iTRAQ reagents, including 8 kinds of equivalent
(reporter group quality 113-121 does not include that 120), peptide fragment after protein digestion can be marked, to tie to isotope reagent
The methods of tandem mass spectrum is closed, accurate identification can be carried out to peptide fragment and is quantified.
In embodiments of the present invention, the purifying preferably uses the purifying of HPLC progress peptide fragments.The instrument purified
Device uses model (inventor's offer) Agilent conventional liquid phase.SCX strong cat ion exchange columns point are used in the embodiment of the present invention
From, in pH=3 phosphate buffers, using 20%-30% gradient eluent (10mM KH2PO4,2M KCl, 25%ACN,
PH=3), elution requirement and gradient reference instrument and consumptive material concrete operations explanation, elute postdigestive peptide fragment.After elution
It jumps section and uses C18 reverse-phase chromatography desalting processings.
After obtaining purifying peptide fragment, the embodiment of the present invention obtains mass spectrum total figure to the peptide fragment of the purifying after Mass Spectrometer Method,
The finger-print is screened in PD softwares, in embodiments of the present invention, the instrument of the Mass Spectrometer Method uses model
The mass spectrograph of Thermo fisher Q-Exactive.
In embodiments of the present invention, the version of the PD softwares is Proteome Discoverer 1.3, is purchased from the U.S.
Thermo companies.
In the embodiment of the present invention, the parameter of mass spectrum screening is as follows：
| Parent ion mass range | 350Da-6000Da |
| Minimum peak number in second order ms figure | 10 |
| Signal-to-noise ratio S/N thresholdings | 1.5 |
Spectrogram after PD extractions, is searched for mascot, obtains search result, using PD softwares according to mascot search results
Spectrogram after being screened with the first step carries out quantitative analysis.
Retrieval by header parameter is as follows：
Note：Fixed modification are fixed modifications；Carbamidomethyl (C) be reductive alkylation processing after,
The urea that cysteine generates methylates.Variable modification are variable modifications；Oxidation (M) is methionine
Oxidation；Gln → Pyro-Glu (N-term Q) refers to peptide fragment N-terminal there are when glutamine, may generate recirculation, become
Glutamic acid；ITRAQ 8plex (K), iTRAQ 8plex (Y), iTRAQ 8plex (N-term) are iTRAQ reagents in different location
Combination.Peptide tol are the precision of first mass spectrometric.MS/MS tol are the precision of second order ms.Maxmissed
Cleavages is maximum allowable when being enzymolysis to leak the number cut.Enzyme is enzyme class used in experiment.Database is retrieval
The database used.Time files compressed are the Database time.Number of sequences are data
Sequence number in library.
Quantitative retrieval parameter is as follows：
Note:Protein ratio Type：For quantitative peptide fragment obtaining value method, median refers to taking the side of middle position
Method.Minimum peptides：For quantitative minimum unique peptide number Normalisation method：Correction
Method, median refer to choosing in one group of sample all can the median of Quantitative Western correct.P-value albumen is credible
Degree assessment represents albumen confidence level less than 0.05 and is more than 95%.
According to the spectrogram quantitative analysis after described search result and screening, obtained analysis data are in rat database
Retrieval in Uniprot-2014-rat (serum) and Uniprot-2015-rat (tissue), total serum protein are 318 kinds, and tissue is total
Albumen is 844 kinds, FDR<1%, specifically it see the table below.
Note：All spectra are the spectrogram number of whole；Matched spectra are the spectrogram number matched；Peptide
For peptide fragment species number；Protein Group are the protein total matched；FDR is false positive rate, and the present embodiment uses vacation
Positive rate carrys out filter result less than 1%.
According to software operating instruction, using identical parameters and method, evaluation and screening obtain 318 kinds of rat blood serum protein and
44 kinds of testosterone cathepsin 18.
After obtained three groups of (N, ZM and BM) serum and tissue protein, the embodiment of the present invention to the three histones matter into
The sieve of the mammalian serum differential protein and testosterone histological difference albumen of the abnormal lymphatic temperament type impotence disease of dimension of practising medicine
Choosing.
The embodiment of the present invention is to the abnormal lymphatic temperament type disease group serum and testosterone histological difference protein
The screening technique of differential protein be specifically：
The BM groups obtain the serum differential protein of BM/ZM compared with the serum proteins of ZM groups；By BM groups with
The serum proteins of N groups compare, and obtain the serum differential protein of BM/N；Count the differential protein of the BM/ZM and BM/N
Common albumen type and quantity, obtain the serum differential protein of abnormal lymphatic temperament type impotence disease group；
BM groups and compared with the testosterone protein of ZM groups, obtain BM/ZM group testosterone difference eggs
White matter；The protein of BM groups is compared with the testosterone protein of N groups, obtains the testosterone histological difference of BM/N
Protein；The type and quantity for counting the common albumen of the differential protein of the BM/ZM groups and BM/N groups, obtain abnormal lymphatic temperament
The testosterone histological difference protein of type impotence disease group；
The technical solution further illustrated the present invention below by way of specific embodiment.
Embodiment 1
1, modeling and grouping
Sexual maturing period female mice 15 is selected, is raised under normal condition after castration operation, mating test is spare.By male rat
Through carrying out mating test with oestrus female mice and APO erection experiments being combined to confirm it with normal sexual function (no normal sexual function
Person eliminates), it takes 30 to only enter experiment, therefrom randomly selects 10 rats again and be set as Normal group, raise under conventional environment,
18~22 DEG C of temperature, temperature are fed, envionmental humidity 40~60%, remaining 20 pressed with raw property forage feed for modeling group
Uygur's medicine carries out modeling to the view of environment using growth cabinet, and temperature is arranged：8~12 DEG C, humidity is 70~80%,
Raising that day alternates with night in 12/12 hour, other time are positioned in the identical feeding environment of normal rats and raise.When modeling group mouse
It is slightly dim to there is skin and hair, it is matt；Burnout is curled up sleeping few dynamic；Sensitive to stimulation, happiness flocks together, and burnout is drowsiness；It is existing without water is striven
As；Urine volume is clear, and color is light, and amount is more；It is half congealed when soft when stool；Tongue fur is dark, white and greasy fur.And with the extension of modeling time, and compare
Group is compared, and modeling group body weight increase is slow, and dietary amount increases, and amount of drinking water is reduced, and stool and urine amount significantly increases, above-mentioned biology table
Sign meets the clinical card Hou Tedian of dimension doctor, and to obtain abnormal mucus cross-examination marquis's rat model, and the model has good section
The property learned, reliability and stability.No penile erectile function and mating are filtered out from syndrome model group by APO and mating test
Ability male rat obtains abnormal lymphatic temperament type impotence disease animal model.The present invention reaches 50% to the mould rate, the above disease
The period of model of a syndrome is preferably 22~25 weeks.The Biological Characterization for recording rat weekly since modeling first week, according to biology
It learns characterization and determines abnormal lymphatic temperament syndrome model into mould situation, the period that the present invention reaches the syndrome mould rate 100% is preferred
It is 8-10 weeks.And on this basis, an APO experiment and mating test are monthly carried out, APO experiments are by counting the number to erect
With incubation period of erecing, judge that penile erectile function, mating test are climbed by incubation period/number by statistics, are inserted into incubation period/number
The mating ability that rat is judged with ejaculation latency/index of number this several, when occur Erectile Dysfunction and mating energy
Power obstacle after that is, impotence disease reaches 50% at mould rate, is tested and is mated by APO from abnormal lymphatic temperament syndrome model group
Experiment screening impotence disease rat, and BM groups are included in, remaining is ZM groups.
2, peripheral blood acquisition, tissue sample materials and preservation：After yellow Jackets intraperitoneal anesthesia, quickly taken from abdominal aorta
Fresh rat whole blood is set 4 DEG C of refrigerators by blood immediately, after 30 minutes with the speed of 3000rpm centrifuge 5min, Aspirate supernatant, then
High speed low temperature centrifugal machine is set, under the conditions of 4 DEG C, after the speed centrifugation of 3000rpm again Aspirate supernatant, -80 DEG C of separating device is low
Temperature refrigerator saves backup.Testosterone tissue is taken from root, washes away bloodstain, and removes remaining skin, fascia, takes epimere penis
Smooth muscle tissue, is positioned in cryopreservation tube that be positioned over -80 DEG C of ultra low temperature freezers spare.
3, the preparation of protein group sample
Protein extracting method uses TCA- ice acetone extractions.
4, iTRAQ labels and HPLC
(1) iTRAQ is marked：Each group rat blood serum and testosterone tissue are taken, after group sample mixing, extracts each histone
Matter is detected the integrality of albumen by SDS and is quantified to albumen using Bradford methods.
Protein after Trypsin digests with ITRAQ labelling kits (Reagent-8Plex
Multiplex Kit (Applied Biosystem)) it is marked, carry out peptide fragment by HPLC (Agilent conventional liquid phase)
Purifying；The concentration of the serum proteins of three groups of rats is as shown in table 1, and the concentration of tissue protein is as shown in table 2.From attached drawing 3
From the point of view of PAGE gel electrophoresis picture, three histone matter integralities of extraction are preferable, can be used for downstream experiment.
1 Bradford methods of table carry out quantitative result to rat blood serum albumen
| Sample ID | N | BM | ZM |
| Concentration (μ g/ μ L) | 0.26 | 0.39 | 0.42 |
2 Bradford methods of table carry out quantitative result to rat testosterone histone
| Sample ID | N | BM | ZM |
| Concentration (μ g/ μ L) | 1.75 | 1.29 | 1.46 |
After quantitative, after each group protein digestibility, with ITRAQ labelling kits.The peptide fragment of liquid phase after purification is through mass spectrum
Finger-print is obtained after (Thermo fisher Q-Exactive mass spectrographs) detection, mass spectrogram is input to PD (Proteome
Discoverer 1.3, thermo) after software, software first carries out screening and identification, type, title, after PD extractions to mass spectrogram
Spectrogram scanned for mascot, after search, PD softwares screened according to mascot search results and the first step after spectrum
Figure carries out quantitative analysis, finally retrieves rat database, and acquisition finally identifies rat blood serum and testosterone histone
Matter is respectively 318 kinds and 844 kinds, and design parameter is as follows.
In the embodiment of the present invention, the parameter of mass spectrum screening is as follows：
| Parent ion mass range | 350Da-6000Da |
| Minimum peak number in second order ms figure | 10 |
| Signal-to-noise ratio S/N thresholdings | 1.5 |
Spectrogram after PD extractions, is searched for mascot, obtains search result, using PD softwares according to mascot search results
Spectrogram after being screened with the first step carries out quantitative analysis.
Retrieval by header parameter is as follows：
Note：Fixed modification are fixed modifications；Carbamidomethyl (C) be reductive alkylation processing after,
The urea that cysteine generates methylates.Variable modification are variable modifications；Oxidation (M) is methionine
Oxidation；Gln → Pyro-Glu (N-term Q) refers to peptide fragment N-terminal there are when glutamine, may generate recirculation, become
Glutamic acid；ITRAQ 8plex (K), iTRAQ 8plex (Y), iTRAQ 8plex (N-term) are iTRAQ reagents in different location
Combination.Peptide tol are the precision of first mass spectrometric.MS/MStol is the precision of second order ms.Max missed
Cleavages is maximum allowable when being enzymolysis to leak the number cut.Enzyme is enzyme class used in experiment.Database is retrieval
The database used.Time files compressed are the Database time.Number of sequences are data
Sequence number in library.
Quantitative retrieval parameter is as follows：
| Parameter name | Test option |
| Protein ratio Type | median |
| Minimum peptides | 1 |
| Normalisation method | median |
| P-value | <0.05 |
Note:Protein ratio Type：For quantitative peptide fragment obtaining value method, median refers to taking the side of middle position
Method.Minimum peptides：For quantitative minimum unique peptide number Normalisation method：Correction
Method, median refer to choosing in one group of sample all can the median of Quantitative Western correct.P-value albumen is credible
Degree assessment represents albumen confidence level less than 0.05 and is more than 95%.
According to the spectrogram quantitative analysis after described search result and screening, obtained analysis data are in rat database
Retrieval in (Uniprot-2014-rat (serum) and Uniprot-2015-rat (tissue)), it is 318 kinds to obtain total serum protein,
Organize 844 kinds of total protein, FDR<1%, specifically it see the table below.
Note：All spectra are the spectrogram number of whole；Matched spectra are the spectrogram number matched；Peptide
For peptide fragment species number；Protein Group are the protein total matched；FDR is false positive rate, and the present embodiment uses vacation
Positive rate carrys out filter result less than 1%.
According to software operating instruction, using identical parameters and method, evaluation and screening obtains 318 kinds of rat blood serum protein,
44 kinds of cathepsin 18.
5, the screening technique of the abnormal lymphatic temperament type impotence disease rat model serum of dimension doctor and the candidate differential protein of tissue
(1) screening technique of the abnormal lymphatic temperament type impotence disease rat model serum differential protein of dimension doctor
BM groups are compared with the serum proteins of ZM groups, (take difference to be more than or equal to 1.2 times, p value is small using above-mentioned software
In equal to the serum differential protein for 0.05), comparing to obtain BM/ZM；BM groups are compared with the serum proteins of N groups, it uses
Above-mentioned software (takes difference to be more than or equal to 1.2 times, p value is less than or equal to 0.05), obtain the serum differential protein of BM/N；Statistics institute
The type and quantity for stating the common albumen of the differential protein of BM/ZM and BM/N, the serum for obtaining abnormal lymphatic temperament type disease group are poor
M-band matter is 35 kinds total, such as the following table 3, wherein up-regulated expression protein 31 kind, lowers 4 kinds of albumen of expression：
3 abnormal phlegm type impotence disease rat mould disease group serum differential protein of table
(2) screening technique of the abnormal lymphatic temperament type impotence disease rat model testosterone histological difference albumen of dimension doctor
(difference is taken to be more than or equal to 1.2 times, p value is less than or equal to 0.05), by the penis of BM groups and ZM groups using above-mentioned software
Smooth muscle tissue's protein compares, and obtains the testosterone histological difference protein of BM/ZM；By the penis of BM groups and N groups
Smooth muscle tissue's differential protein compares, and obtains the testosterone histological difference protein of BM/N；Count the BM/ZM and
The type and quantity of the common albumen of the differential protein of BM/N, the testosterone tissue for obtaining abnormal lymphatic temperament type disease group are poor
M-band matter is 48 kinds total, wherein 35 kinds of up-regulated expression albumen, lowers 13 kinds of albumen of expression.As shown in table 4：
4 abnormal phlegm type impotence disease rat model tissue disease group candidate's differential protein of table
6, the ELISA detections of abnormal phlegm type impotence disease rat model serum candidate's differential protein
Randomly choose 7 kinds of serum candidate's differential proteins, to abnormal lymphatic temperament type impotence disease rat model and control group into
Row ELISA detections, to verify serum candidate differential protein of the present invention whether can be as the mark of abnormal lymphatic temperament type impotence disease
Will object：
That is random selection detection o.11 albumen P48199 (c reactive proteins)；No. 13 albumen P26644 (β -2- glycoprotein
1)；No. 15 albumen P20059 (Hemopexin)；No. 24 albumen P04916 (retinol-binding proteins)；No. 26
Albumen P02767 (transthyretin)；No. 27 albumen P02764 (α -1- acidoglycoproteins) and No. 33 albumen
O35460 (Ang-1) uses double antibodies sandwich kit (rat c reactive protein (CRP) double antibodies sandwich kit respectively：
Yi Lai Retts bio tech ltd；Rat beta2 Glycoprotein 1 (APOH) double antibodies sandwich kit：Your excellent raw commerce and trade of Wuhan are limited
Company；Rat Hemopexin (HPx) double antibodies sandwich kit：Abcam Ai Bo resist (Shanghai) trade Co., Ltd；Rat
Retinol-binding proteins (RBP4) double antibodies sandwich kit：Yi Lai Retts bio tech ltd；Rat thyroid element delivers
Albumen (TTR) double antibodies sandwich kit：Abnova Yanuofa Biotechnology Co., Ltd.；1 acidoglycoproteins of rat α (α 1AG) are double
Resist sandwich kit：Abcam Ai Bo resist (Shanghai) trade Co., Ltd；Rat aorta generates plain 1 (Ang-1) double antibodies sandwich reagent
Box：Raybiotech Rui Boao (Guangzhou) bio tech ltd；According to kit specification to Normal group and disease
Group rat blood serum carries out ELSA detections.
Measurement result is as shown in Fig. 4 A-G, and as shown in Figure 4, each exception lymphatic temperament type impotence disease rat model serum is waited
In sortilin in addition to the content of Ang-1 is substantially less than control group, other oroteins content is all remarkably higher than control group,
This is consistent with iTRAQ results, illustrates that the method for the present invention acquisition protein marker can be as abnormal lymphatic temperament type impotence disease
The biomarker of serum.
It is consistent with iTRAQ results, illustrating can be as the biomarker of abnormal lymphatic temperament type impotence disease serum, specifically
As a result it see the table below：
Protein content in each group rat blood serum
The * P compared with N groups<0.05
7. abnormal phlegm type impotence disease rat model testosterone Immunohistochemical detection
Randomly choose 4 kinds of testosterone tissue candidate's differential proteins, to abnormal lymphatic temperament type impotence disease rat with it is right
Immunohistochemical detection is carried out according to group rat testosterone tissue, is with the candidate differential protein in verification present invention tissue
It is no can be as the marker of abnormal lymphatic temperament impotence disease：
That is No. 12 protein Q 9Z2L0 (voltage dependence anion selectivity channel protein 1) of random selection detection；30th
Number albumen P02625 (parvalbumin α)；No. 36 albumen P04904 (glutathione S-transferase α -3) and No. 44 albumen
Using the antibody of above-mentioned albumen, (all antibody are purchased to be had P47853 (biglycan) in Abcam (Shanghai) trade respectively
Limit company), Normal group and disease group rat testosterone tissue are detected according to antibody specification.With optics
Micro- sem observation and statistical result.Photo amplification factor is object lens multiple × eyepiece multiple.Coloration result method of counting is as follows：
In the case that amplification factor is 400, each sample randomly chooses the 6-10 visual field, is not overlapped completely between each visual field, counts sun
Property cell, every group is generally 10 different samples, then carries out the comparison between group.Methods of marking is divided into two aspects, i.e., positive
Range and tinctorial strength.Percentage wherein in the visual field shared by positive cell is positive range 5%-100%；Positive cell dyeing
The depth be tinctorial strength, be divided into 4 grades, i.e., (there are coloring in 0 (almost not colored or slightly have a Point Coloring), 1, but compare
It is shallower), 2 (having coloring, deep), 3 (having coloring, very deeply)；Final result is (positive range × tinctorial strength)/visual field
Number.
Measurement result is as shown in table 5, and as shown in Table 5, the penis of each exception lymphatic temperament type impotence disease rat model is flat
The content of voltage dependence anion selectivity channel protein 1 and parvalbumin α are significantly higher than just in sliding muscular tissue candidate albumen
The content of normal control group, glutathione S-transferase α -3 and biglycan is substantially less than Normal group, this and iTRAQ
As a result it is consistent, illustrates that the method for the present invention acquisition protein marker can be as the testosterone of abnormal lymphatic temperament type impotence disease
The biomarker of tissue.
Protein expression result in 5 each group rat penis of table
* compared with N<0.05
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (5)
1. a kind of abnormal lymphatic temperament type impotence disease mammalian serum differential protein combination of dimension doctor, which is characterized in that the egg
White combination includes following 35 kinds of albumen：
2. mammalian serum differential protein combination according to claim 1, which is characterized in that the protein combination comes from
Model animal；The model animal is rat.
3. a kind of mammal testosterone histological difference protein combination of the abnormal lymphatic temperament type impotence disease of dimension doctor, feature
It is, the protein combination, including following 48 kinds of albumen：
4. mammal testosterone histological difference protein combination according to claim 3, which is characterized in that the egg
White combination comes from model animal；The model animal is rat.
5. the protein combination according to claim 1-3 any one is in preparing abnormal lymphatic temperament type impotence disease drug
Using.
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