CN106645748A - Mammalian differential serum protein and differentially expressed protein system of penile smooth muscle tissue of abnormal and phlegmatic type of impotence syndrome and screening method and application - Google Patents
Mammalian differential serum protein and differentially expressed protein system of penile smooth muscle tissue of abnormal and phlegmatic type of impotence syndrome and screening method and application Download PDFInfo
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Abstract
The invention provides a mammalian differential serum protein and a differentially expressed protein system of the penile smooth muscle tissue of an abnormal and phlegmatic type of impotence syndrome in uighur medicine, and a screening method of the differentially expressed protein system. The method comprises the steps that 1, a normal control group is formed, a model of a syndrome group and a disease syndrome group is built; 2, whole blood is collected, the serum and the penile smooth muscle tissue are separated; 3, protein is extracted; 4, after the labeling, purification is conducted; 5, after the detection of mass spectrum, a fingerprint spectrum is obtained, proteomics analysis is conducted after the screening, and the protein of serum and the protein of penile smooth muscle tissue of the syndrome group, the disease syndrome group and the normal control group are obtained; 6, differentially expressed protein of serum and differentially expressed protein of penile smooth muscle tissue of abnormal and phlegmatic type of impotence syndrome in uighur medicine are obtained by statistic measurements, comparisons and screening, and a foundation and a reference are provided for further mechanism research, medicine screening, conversion, and prevention and cure research on abnormal and phlegmatic type of impotence syndrome.
Description
Technical field
The invention belongs to biological technical field, and in particular to a kind of mammal of dimension doctor exception lymphatic temperament type impotence disease
Serum and testosterone histological difference albumen and its screening technique and application.
Background technology
It is important component part in traditional Chinese medicine that Uygur medicine (dimension doctor) is learned, and dimension medical science body fluid opinion thinks, human body by
Bile matter, blood matter, 4 kinds of body fluid matter compositions of lymphatic temperament and black courage matter, the dynamic equilibrium of above-mentioned 4 kinds of body fluid matter is body health
Physiological foundation, and the amount of a certain body fluid matter or/and the change of matter, cause body fluid matter dysequilibrium, produce a kind of abnormal humour
, there is causality with various diseases in cross-examination (syndrome).Humoral pathology (body fluid opinion) is dimension medical knowledge opinion foundation, and it is recognized
In for 4 kinds of body fluid, lymphatic temperament body fluid attribute is raw, Chang Yiyin lives, diet and environmental factor, operating pressure, shortage motion etc.
Reason causes internal isohydria to be destroyed, and lymphatic temperament body fluid changes, and produces abnormal lymphatic temperament, causes metabolic water pancake
Low, product accumulation is internal, and then causes pathology to sexually revise, and causes relevant disease.
Clinical research confirmation, abnormal mucus cross-examination (Abnormal Phlegm) is in impotence (Erectile Dysfunction), morning
Let out, lack the metabolic diseases such as reproductive disease, obesity, the hyperlipidemia such as azoospermia, premature ovarian failure, infertile and cardiovascular disease
May be also the master of the Complex Diseases such as tumour, diabetes, hypertension generation early stage in leading position in sick generating process
Want card type.Erectile dysfunction (Erectile Dysfunction, ED), also known as impotence, refer to man's penis anorthosis and
Can not maintain to erect and complete satisfied sexual life, category dimension doctor's advantage disease.Telotism is by the multiple biological activities factor (bag
Include neurotransmitter, hormone, enzyme, ion channel etc.) nerve-vascular biological activity of comprehensive adjustment.In dimension medicine clinic and base
Plinth research direction, because experimental zoology is started late, without related Disease Syndrome integrated animal model, causes abnormal lymphatic temperament type impotence
Card and its verification side's medicine Study on mechanism are delayed always, and seriously constrain research and development of the Wei Yi andrologies in this field,
The research report of correlating markings albumen and its protein markers objects system more independent of abnormal lymphatic temperament type impotence disease.
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 is tested 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 abnormal lymphatic temperament type impotence disease is not single
Reproductive organs is the disease of testosterone tissue, but systemic disease, it is therefore desirable to we use organic conception, from whole body
The visual field come the regularity of occurrence and development and possible treatment method analyzing, study and explore the disease.Dimension cures rising for differentiation of symptoms and signs for classification of syndrome
Point and core are dimensions doctor " body fluid opinion ", and its starting point is the interior external manifestation in body fluid dynamic change of 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 that this integrally grinds with systems biology according to the body fluid phenotypic difference occurred in individual integral level
Study carefully thinking perfectly in harmony.
Serum has incorporated the protein multidate information of body multiple organ, tissue and cell, is also close with penile erectile function
Secretory protein the only way which must be passed that cut is closed, can reflect the space-time of the related global regulation network of the pathophysiological change of body
Property, and effect of the medicine to any target organ, tissue or cell is finally also be reflected among the change of serum proteins group.Penis is
The effector organ of telotism, corpus cavernosal smooth muscle is then effect tissue, its any pathology triumph change also with the tissue
Protein level dynamic change is inseparable, wherein containing impotence there is related potential protein labeling, it is also the disease medicine
One of important target spot of thing treatment.
Therefore, on the basis of this research is based on abnormal lymphatic temperament syndrome and Disease Syndrome integrated animal model, carry 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, is that further the Mechanism Study of development exception lymphatic temperament type impotence disease, drug screening, conversion are established with study on prevention
Basis is determined.
The content of the invention
In view of this, the first object of the present invention is to provide dimension doctor the mammal blood of abnormal lymphatic temperament type impotence disease
Clear differential protein system, including following albumen：
Preferably, the mammalian blood 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 dimension doctor the 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 is from model animal；The model animal is rat.
The mammalian blood serum that still a further object of the present invention there are provided the abnormal lymphatic temperament type impotence disease of above-mentioned dimension doctor is poor
The screening technique of M-band system and testosterone histological difference albumen system, comprises the following steps：
1) Normal group (N groups), abnormal lymphatic temperament syndrome group (ZM groups), abnormal lymphatic temperament type disease group (BM are set up
Group)；
2) step 1 is gathered) mammalian whole blood that obtains, the serum of mammal is obtained after separation, take the step
The rapid testosterone tissue for 1) obtaining mammal；
3) from the step 2) serum that obtains and extract protein in testosterone tissue；
4) by the step 3) in purify after the protein labeling that obtains, obtain peptide fragment after purification；
5) by the step 4) peptide fragment after purification that obtains obtains finger-print Jing after Mass Spectrometer Method, institute after screening
State finger-print and enter line retrieval using proteome analysis software, carried out according to the spectrogram after the Search Results and screening for obtaining
Quantitative analysis, by the analyze data for obtaining proteomic image retrieval software inspection used in the mammalian proteins database
Rope, obtains the N groups, ZM groups and BM groups mammalian blood serum and testosterone histological difference protein；
6) by the step 5) in
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, BM/N group testosterone histological differences are obtained
Protein；The type and quantity of the common albumen of the differential protein of the BM/ZM and BM/N are counted, abnormal lymphatic temperament type sun is obtained
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, the step 1) in rats in normal control group raise in conventional environment,
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；The step 1) in Normal group be common feeding
Material, modeling group feeding feed is raw property feed.
Preferably, in screening technique of the invention, the step 3) described in labeling method be iTRAQ labelling kit marks
Note.
Preferably, in screening technique of the invention, the step 4) in proteome analysis software be Proteome
Discoverer；The proteomic image retrieval software is mascot softwares.
Present invention also offers the mammalian blood serum differential protein system of above-mentioned dimension doctor exception lymphatic temperament type impotence disease
Preparation prevention is tied up to testosterone histological difference proteosome or treat in mammal exception lymphatic temperament type impotence medicine
Using.
From the foregoing, it will be observed that it is an advantage of the current invention that：
1st, the concept without syndrome in modern medicine, commonly uses and differential protein is found between disease and Normal group, and
In dimension doctor's clinical diagnosis and treatment, then diagnosis and treatment, and the impotence patient to different card types is needed to treat using different square medicines, i.e. disease
Card is corresponding with square medicine.Inventor herein, is gained knowledge based on the understanding to syndrome and disease with abundant Uygur medicine, is used and is debated
Mode of the card in combination with the differentiation of disease, by Analogy, between the differential protein of BM and ZM groups and the differential protein of BM and N groups
Common albumen is found, examination has gone out the serum of abnormal lymphatic temperament type impotence disease and testosterone tissue candidate protein markers
Thing, and by checking, establish the mammalian blood 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 set up macroscopic view
With the index system that microcosmic cognition coordinates, analysis and synthesis research is unified, the diagnosis and treatment level and medicament research and development of impotence are lifted
Level is significant
2nd, the invention provides the mammalian blood serum differential protein and 48 of 35 kinds of dimension doctor's exception lymphatic temperament type impotence diseases
Plant testosterone histological difference albumen；Its serum and testosterone group in model animal such as rat is shown by embodiment
Knit the rise of middle differential protein or lower expression, show that these differential proteins are generated necessarily to the penile erectile function of rat
Impact, be ultimately determined to the biomarker of abnormal lymphatic temperament type impotence disease, can carry out for above-mentioned biomarker
Medicine basic research, be 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, there is provided foundation.
Description of the drawings
Fig. 1 is the modeling flow chart of Normal group, abnormal mucus cross-examination marquis group and disease group rat model；
Fig. 2 is the mammalian blood 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 is composed.
Fig. 4 is the ELISA detections that 7 kinds of albumen is arbitrarily chosen in 35 kinds of candidate's differential proteins.
Specific embodiment
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 (body weight 180g or so) are provided by Xinjiang Medicine University's Experimental Animal Center；
In embodiments of the present invention, medicine and reagent：Raw property feed is by Xinjiang Medicine University's Experimental Animal Center by front
Requirement used in phase research is prepared；The medicinal material such as spinach reality and coriandri,fructus is purchased from Uygur medicine hospital of Xinjiang Uygur Autonomous Regions
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 marks are relative 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 with a cage
Each rat is observed 30 minutes based on reacting to each other, and behavior state is based on activity interior during this, 30 points are observed under quiet environment
Clock, excitement degree with press from both sides tail test when reaction based on, tongue picture tongue fur is observed in the sunlight, recorded and takes pictures, 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 on form of defecating during bowel movement positive with the same day；
Quantitative target is fixed on daily 20 to avoid circadian impact:00-22:00 collects every data, wherein body
Weight：Weigh in required time and record daily, with every cage TBW divided by the cage rat number of elements, draw average weight, and press
State formula to draw each cage body anharmonic ratio value of model group and record, body weight ratio=model group average weight-model group original body mass/just
Often organize average weight-normal group original body mass；Dietary amount：Daily 20:00 addition feed 300g, next day 20:00 claims food surplus,
The corresponding dietary amount of 100g body weight is drawn according to following formula and record；Amount of drinking water：Daily 20:00 feedwater 500ml, next day
20:00 amount surplus water, calculates the corresponding amount of drinking water of 100g body weight and records；Urine volume：Modeling record within first day to
Dry dunnage weight, the weight of the wet bedding and padding containing urine and stool is claimed within second day in required time and is recorded, then in constant temperature
Weigh again after drying in 2 hours at 100 DEG C of air dry oven, calculate each group urine volume；Stool amount：Calculate 100g body weight corresponding
Stool is measured and records.
APO erective tests or APO experimental techniques are
With reference to the method for Heaton etc., test rat is placed in into 10～15min in inspection box and adapts to environment, 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, mix with 5mL/kg physiological saline), observe at once after injection, during timer, observe and record in 1800s (0.5h)
The erection incubation period of rat and erection number of times.The standard of telotism：Rat crouches position in crouching during erection, and phallosome end is stretched out,
Bow to lick and lick glans penis.Erection incubation period is the time erected to first time after injection.Erection number of times is big in 1800s after injection
There is the number of times of telotism in mouse.If having no erection in 1800s, the rat erection number of times is designated as 0, and incubation period of erecing is designated as
1800s。
Mating test method：
Male mouse and raettin (having carried out oestrus preparation) are respectively placed in different inspection boxes and adapt to environment, 10～15 points
Female-male proportion 1 is pressed after clock:2 are put into same inspection box, and the climb back of the body, insertion and the ejaculation for observing male rat in 1800s is hidden
Phase and climb the back of the body, insertion and number of times.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 designated as
1800s, climbs back of the body number of times and is designated as 0.Insertion is with ejaculation behavior in the same manner.
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 acquisition methods of the N groups, ZM groups and BM group rats
Different restriction, using the technical scheme for setting up abnormal lymphatic temperament syndrome animal model well known to those skilled in the art；
In the embodiment of the present invention, following steps are preferably specifically included：
Sexual maturing period raettin is selected, is raised under castration Post operation normal condition, the raettin is entered in oestrus with male mouse
Row mating test and APO experiment tests, obtain the male mouse of normal sexual function.The present invention is to the mating test and APO experiments
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 concrete steps of the mating are preferably will carry out male mouse and the raettin point in oestrus
It is not placed in different inspection boxes and adapts to environment, female-male proportion 1 is pressed after 10～15min:2 are put into same inspection box, observation
In 1800s male rat climb the back of the body, insertion and ejaculation latency and climb the back of the body, insert and number of times, 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 designated as 1800s, climb the back of the body number of times be designated as 0；Insertion is with ejaculation behavior in the same manner.
Male mouse with normal mating ability is included Normal group and modeling by the present invention according to the result of the mating
Group, to eliminating without normal mating ability hero mouse.
In embodiments of the present invention, APO experiment concrete steps be preferably experimental rat is placed in 10 in inspection box～
15min adapts to environment, keeps quite, and dims light, in the μ g/kg apomorphine APO of rat neck injection of skin 90, stands after injection
Observation is carved, during timer, erection incubation period and the erection number of times of rat in 1800s is observed and record；The standard of the erection
Rat crouches position in crouching during to erect, and phallosome end is stretched out, bows to lick and lick glans penis；The erection incubation period for after injection to the
The time once erected；The erection number of times is that the number of times of telotism occurs in rat in 1800s after injection；If in 1800s not
See erection, then the rat erection number of times is designated as 0, incubation period of erecing is designated as 1800s.
In the embodiment of the present invention the male mouse with normal penile erection function is included just according to the result of APO tests
Normal control group and modeling group, to eliminating without 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
The relative humidity 70～80% of food 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 as
Subjectivity is examined 30 minutes, and behavior state is based on activity interior during this, observe 30 minutes under quiet environment, and excitement degree is pressing from both sides tail
Based on reaction during experiment, tongue picture tongue fur is observed in the sunlight, recorded and takes pictures, and diet water state is with the same time feeding of each group
Observation diet water state is big during stool positive with the same day bowel movement of state when urine was just urinated with each group rat same day based on color
Just based on form；Quantitative target is fixed on daily 20 to avoid circadian impact:00-22:00 collects every data, its
Middle body weight：Weigh in required time and record daily, with every cage TBW divided by the cage rat number of elements, draw average weight, and
The each cage body anharmonic ratio value of model group is drawn by following formula and record, the first initial body of body weight ratio=model group average weight-model group
Weight/normal group average weight-normal group original body mass；Dietary amount：Daily 20:00 addition feed 300g, next day 20:00 claims food
Surplus, draws the corresponding dietary amount of 100g body weight and records according to following formula；Amount of drinking water：Daily 20:00 feedwater 500ml,
Next day 20:00 amount surplus water, calculates the corresponding amount of drinking water of 100g body weight and records；Urine volume：Modeling is recorded for first day
The dry dunnage weight given well, claims in required time the weight of the wet bedding and padding containing urine and stool for second day and records, Ran Hou
Weigh again after drying in 2 hours at 100 DEG C of constant temperature blast drying oven, calculate each group urine volume；Stool amount：Calculate 100g body weight relative
The stool for answering is measured and records.
It is of the invention by having for obtaining after obtaining the male mouse of normal sexual function (normal mating ability and penile erectile function)
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.Bag
Include qualitative index and quantitative target.Qualitative index observes skin and hair, emotional reactions, excitement degree, the sleep of all groups of rats
State, drinking-water state, urine volume property, stool state and tongue picture tongue fur.Quantitative target is body weight, the diet for recording all groups of rats
Amount, amount of drinking water, urine volume and stool amount.Find that rat skin hair is slightly dim during modeling, it is matt；Burnout, curls up sleeping few
It is dynamic；Sensitive to stimulating, happiness flocks together, and burnout is drowsiness；Nothing strives water phenomenon；Urine volume is clear, and color is light, measures many；It is half congealed when soft during stool；Tongue fur
Secretly, white and greasy fur.And as the prolongation of modeling time, modeling group body weight increase are 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 so as to obtain abnormal mucus cross-examination marquis
Mouse model, and the model has good science, 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 so as to obtain
Normal lymphatic temperament type impotence disease animal model.To the syndrome into mould rate up to 100%, Syndrome model reaches the present invention into mould rate
More than 50%, the cycle 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 conventional environment conventional environment raising side ibid 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 is to include 500ml water
With 80g bedding and padding.The present invention is not particularly limited to 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 described experimental group is preferably 20.
In the present invention, described raw property feed is real and coriandri,fructus and conventional feed the mixture of spinach.In the present invention
In embodiment, the raw property feed is preferably：Include 100～200g coriandri,fructuses and 100～200g spinach per 1kg conventional feeds
Dish reality, more preferably includes 150g coriandri,fructuses and 150g spinach realities per 1kg conventional feeds.The present invention is not special to conventional feed
Limit, using the feed for raising rat well-known to those skilled in the art.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
In applying 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 described 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 to 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, there is provided the facility of raw environment is growth cabinet.
In the present invention, the time of the raw property forage feed was preferably in Beijing time 09:00 to 21:00 is put into, its
Its time is positioned in control rats identical feeding environment and raises.
In the present invention, the method and the result of the APO experiments and mating test are tested and mating test with above-mentioned APO
Method and the result.
The present invention is anaesthetized to preferred pair 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, described anesthesia is abdominal cavity
Injection.The present invention is preferably yellow Jackets to the anesthetic used in the anaesthesia process.Described yellow Jackets
Source is purchased from and medicine company Co., Ltd of field Uygur.
In embodiments of the present invention, the concrete grammar of the collection peripheral blood is preferably postanesthetic rat quickly from abdomen
Sustainer takes blood.
In embodiments of the present invention, the method for separating serum is preferably and for fresh rat whole blood to put 4 DEG C of environment immediately,
With the 5～10min of centrifugation of 3000～3500rpm after 30～40min, by the supernatant for obtaining under the conditions of 4 DEG C, with 3000
The speed of～3500rpm is centrifuged again, obtains serum, and the serum that obtains will be finally centrifuged, and to be positioned over -80 DEG C of ultra low temperature freezers standby
With.4 DEG C of described environment are preferably 4 DEG C of refrigerators.
Obtain the method for testosterone tissue specifically：Testosterone tissue is taken from root, bloodstain is washed away, and is removed
Remaining skin, manadesma, take epimere testosterone tissue, are positioned in cryopreservation tube that to be positioned over -80 DEG C of ultra low temperature freezers standby.
After obtaining serum and testosterone tissue, the embodiment of the present invention is detected to extracting protein in serum and tissue,
Obtain concentration known serum and testosterone tissue protein.
In the embodiment of the present invention, preferably sample mixing is carried out respectively according to group, obtain three groups of blood serum samples before albumen is extracted
With three groups of testosterone tissue samples.
The embodiment of the present invention is not particularly limited to 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
Survey.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 concentration known, the embodiment of the present invention is marked to the protein with iTRAQ labelling kits,
The protein for marking is obtained, it is purified, obtain peptide fragment after purification.
In embodiments of the present invention, preferably the protein of the concentration known for obtaining is digested before the mark.This
Inventive embodiments are not particularly limited to the method for the digestion, using protein digestibility side well-known to those skilled in the art
Method.Trypsinization is adopted in digestion described in the embodiment of the present invention.
The embodiment of the present invention originating without special restriction to 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 is used is
Reagent-8Plex Multiplex Kit (Applied Biosystem) 8 mark iTRAQ reagents, including 8 kinds of equivalent
(reporter group quality 113-121, not including 120), can be marked, so as to tie isotope reagent to peptide fragment after protein digestion
The method such as tandem mass spectrum is closed, accurate identification and quantitative can be carried out to peptide fragment.
In embodiments of the present invention, the purifying preferably carries out the purifying of peptide fragment using HPLC.The instrument for being purified
Device adopts model (inventor's offer) Agilent conventional liquid phase.SCX strong cat ion exchange columns point 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 wash-out
Jump section and use C18 reverse-phase chromatography desalting processings.
Obtain purifying after peptide fragment, the embodiment of the present invention obtains mass spectrum total figure Jing after Mass Spectrometer Method to the peptide fragment of the purifying,
The finger-print is screened in PD softwares, in embodiments of the present invention, the instrument of the Mass Spectrometer Method adopts 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, 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 mses figure | 10 |
| Signal to noise ratio S/N thresholding | 1.5 |
Spectrogram after PD extractions, is searched for mascot, Search Results is obtained, using PD softwares according to mascot Search Results
Quantitative analysis is carried out with the spectrogram after first step screening.
Retrieval by header parameter is as follows：
Note：Fixed modification are to fix modification；Carbamidomethyl (C) be reductive alkylation process after,
The urea that cysteine is produced methylates.Variable modification are variable modifications；Oxidation (M) is methionine
Oxidation；When Gln → Pyro-Glu (N-term Q) refers to that peptide fragment N-terminal has glutamine, recirculation may be produced, be become
Glutamic acid；ITRAQ 8plex (K), iTRAQ 8plex (Y), iTRAQ 8plex (N-term) are iTRAQ reagents in diverse location
Combination.Peptide tol are the precision of first mass spectrometric.MS/MS tol are the precision of second order mses.Maxmissed
Cleavages is maximum allowable when being enzymolysis to leak the number cut.Enzyme is the used enzyme class of experiment.Database is retrieval
The database for using.Time files compressed are the Database time.Number of sequences are data
Sequence number in storehouse.
Quantitative search argument is as follows：
Note:Protein ratio Type：For quantitative peptide fragment obtaining value method, median refers to take the side of middle position
Method.Minimum peptides：For quantitative minimum unique peptide number Normalisation method：Correction
Method, median refer to choose in one group of sample all can Quantitative Western median correcting.P-value albumen is credible
Degree assessment, albumen confidence level is represented less than 0.05 more than 95%.
According to the spectrogram quantitative analysis after the Search Results and screening, the analyze data for obtaining is in rat database
Retrieval in Uniprot-2014-rat (serum) and Uniprot-2015-rat (tissue), total serum protein is 318 kinds, and tissue is total
Albumen is 844 kinds, FDR<1%, specifically see the table below.
Note：All spectra are whole spectrogram numbers；Matched spectra are the spectrogram number for matching；Peptide
For peptide fragment species number；Protein Group are the protein total for matching；FDR is false positive rate, and the present embodiment uses vacation
Positive rate carrys out filter result less than 1%.
According to software operational procedure, using identical parameters and method, evaluation and screening obtain 318 kinds of rat blood serum protein and
44 kinds of testosterone cathepsin 18.
After three groups of (N, the ZM and BM) serum for obtaining and tissue protein, the embodiment of the present invention is entered to the three histones matter
The mammalian blood serum differential protein of the abnormal lymphatic temperament type impotence disease of dimension of practising medicine and the sieve of testosterone histological difference albumen
Choosing.
The embodiment of the present invention is to the abnormal lymphatic temperament type disease group serum and testosterone histological difference protein
Differential protein screening technique 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, the testosterone histological difference of BM/N is obtained
Protein；The type and quantity of the common albumen of the differential protein of the BM/ZM groups and BM/N groups are counted, abnormal lymphatic temperament is obtained
The testosterone histological difference protein of type impotence disease group；
Technical scheme is further illustrated below by way of specific embodiment.
Embodiment 1
1st, modeling and packet
From sexual maturing period raettin 15, raise under castration Post operation normal condition, mating test is standby.By male rat
Jing carries out mating test and erects to test with reference to APO to confirm that it has normal sexual function (without normal sexual function with oestrus raettin
Person eliminates), take 30 and only enter experiment, 10 rats are therefrom randomly selected again is set to Normal group, raise under conventional environment,
18～22 DEG C of temperature, temperature is fed, envionmental humidity 40～60%, remainings 20 for modeling group, with raw property forage feed, is pressed
Uygur's medical science carries out modeling to the view of environment using growth cabinet, arranges temperature：8～12 DEG C, humidity is 70～80%,
Day alternates with night within 12/12 hour raises, and other time is positioned in normal rats identical feeding environment and raises.When modeling group mouse
There is skin and hair slightly dim, it is matt；Burnout, curls up sleeping few dynamic；Sensitive to stimulating, happiness flocks together, and burnout is drowsiness；Show without water is striven
As；Urine volume is clear, and color is light, measures many；It is half congealed when soft during stool；Tongue fur is dark, white and greasy fur.And with the prolongation 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
Levy and meet the clinical card Hou Tedian of dimension doctor, so as to obtain abnormal mucus cross-examination marquis's rat model, and the model has good section
The property learned, reliability and stability.Filtered out from syndrome model group without penile erectile function and mating 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, above disease
The cycle of model of a syndrome is preferably 22～25 weeks.Start within first week the Biological Characterization for recording rat weekly from modeling, according to biology
Learn to characterize and determine abnormal lymphatic temperament syndrome model into mould situation, the cycle that the present invention reaches 100% to the syndrome mould rate is preferred
It is all for 8-10.And on this basis, monthly carry out an APO experiment and mating test, the number of times that APO experiments are erected by statistics
With incubation period of erecing, judge penile erectile function, mating test is climbed by incubation period/number of times, inserts incubation period/number of times by statistics
With the mating ability that ejaculation latency/index of number of times this several judges rat, when occur Erectile Dysfunction and mating energy
Power obstacle, i.e. impotence disease are reached after 50% into mould rate, are tested and are mated by APO from abnormal lymphatic temperament syndrome model group
Experiment screening impotence disease rat, and BM groups are included, it is remaining for ZM groups.
2nd, peripheral blood collection, tissue sample are drawn materials and preservation：After yellow Jackets intraperitoneal anesthesia, quickly take from abdominal aorta
Blood, by fresh rat whole blood 4 DEG C of refrigerators are put immediately, with the centrifugation 5min of 3000rpm after 30 minutes, Aspirate supernatant, then
Put high speed low temperature centrifugal machine, under the conditions of 4 DEG C, with the centrifugation of 3000rpm again Aspirate supernatant after, -80 DEG C of separating device is low
Temperature refrigerator is saved backup.Testosterone tissue is taken from root, bloodstain is washed away, and removes remaining skin, manadesma, take epimere penis
Smooth muscle tissue, is positioned in cryopreservation tube that to be positioned over -80 DEG C of ultra low temperature freezers standby.
3rd, the preparation of protein group sample
Protein extracting method adopts TCA- ice acetone extractions.
4th, iTRAQ marks and HPLC
(1) iTRAQ marks：Each group rat blood serum and testosterone tissue are taken, after group sample mixing, each histone is extracted
Matter, is carried out quantitatively by the integrality of SDS detection albumen and using Bradford methods to albumen.
Protein Jing Trypsin digestion after with ITRAQ labelling kits (Reagent-8Plex
Multiplex Kit (Applied Biosystem)) it is marked, carry out peptide fragment by HPLC (Agilent conventional liquid phase)
Purifying；As shown in table 1, the concentration of tissue protein is as shown in table 2 for the concentration of the serum proteins of three groups of rats.From accompanying drawing 3
From the point of view of PAGE gel electrophoresis picture, three histone matter integralities of extraction preferably, can be used for downstream experiment.
The Bradford methods of table 1 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 |
The Bradford methods of table 2 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, by each group protein digestibility after, use ITRAQ labelling kits.Liquid phase peptide fragment Jing mass spectrums after purification
Finger-print is obtained after (Thermo fisher Q-Exactive mass spectrographs) detection, mass spectrogram is input into PD (Proteome
Discoverer 1.3, thermo) after software, software first carries out Screening and Identification, species, title, after PD is extracted to mass spectrogram
Spectrogram scanned for mascot, search terminate after, PD softwares according to mascot Search Results and the first step screening after spectrum
Figure carries out quantitative analysis, finally retrieves rat database, is finally identified 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 mses figure | 10 |
| Signal to noise ratio S/N thresholding | 1.5 |
Spectrogram after PD extractions, is searched for mascot, Search Results is obtained, using PD softwares according to mascot Search Results
Quantitative analysis is carried out with the spectrogram after first step screening.
Retrieval by header parameter is as follows：
Note：Fixed modification are to fix modification；Carbamidomethyl (C) be reductive alkylation process after,
The urea that cysteine is produced methylates.Variable modification are variable modifications；Oxidation (M) is methionine
Oxidation；When Gln → Pyro-Glu (N-term Q) refers to that peptide fragment N-terminal has glutamine, recirculation may be produced, be become
Glutamic acid；ITRAQ 8plex (K), iTRAQ 8plex (Y), iTRAQ 8plex (N-term) are iTRAQ reagents in diverse location
Combination.Peptide tol are the precision of first mass spectrometric.MS/MStol is the precision of second order mses.Max missed
Cleavages is maximum allowable when being enzymolysis to leak the number cut.Enzyme is the used enzyme class of experiment.Database is retrieval
The database for using.Time files compressed are the Database time.Number of sequences are data
Sequence number in storehouse.
Quantitative search argument is as follows：
| Parameter name | Experiment 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 take the side of middle position
Method.Minimum peptides：For quantitative minimum unique peptide number Normalisation method：Correction
Method, median refer to choose in one group of sample all can Quantitative Western median correcting.P-value albumen is credible
Degree assessment, albumen confidence level is represented less than 0.05 more than 95%.
According to the spectrogram quantitative analysis after the Search Results and screening, the analyze data for obtaining is in rat database
Retrieval in (Uniprot-2014-rat (serum) and Uniprot-2015-rat (tissue)), it is 318 kinds to obtain total serum protein,
844 kinds of total protein of tissue, FDR<1%, specifically see the table below.
Note：All spectra are whole spectrogram numbers；Matched spectra are the spectrogram number for matching；Peptide
For peptide fragment species number；Protein Group are the protein total for matching；FDR is false positive rate, and the present embodiment uses vacation
Positive rate carrys out filter result less than 1%.
According to software operational procedure, using identical parameters and method, evaluation and screening obtains 318 kinds of rat blood serum protein,
44 kinds of cathepsin 18.
5th, the screening technique of the abnormal lymphatic temperament type impotence disease rat model serum of dimension doctor and tissue candidate's differential protein
(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, (difference is taken more than or equal to 1.2 times, p value is little using above-mentioned software
In equal to 0.05), compare the serum differential protein for obtaining BM/ZM；BM groups are compared with the serum proteins of N groups, use
Above-mentioned software (takes difference more than or equal to 1.2 times, p value obtains the serum differential protein of BM/N less than or equal to 0.05)；Statistics institute
The type and quantity of the common albumen of the differential protein of BM/ZM and BM/N are stated, the serum for obtaining abnormal lymphatic temperament type disease group is poor
M-band matter amounts to 35 kinds, such as table 3 below, wherein up-regulated expression protein 31 kind, lowers 4 kinds of expressing protein：
The abnormal phlegm type impotence disease rat mould disease group serum differential protein of table 3
(2) screening technique of the abnormal lymphatic temperament type impotence disease rat model testosterone histological difference albumen of dimension doctor
Using above-mentioned software (difference is taken more than or equal to 1.2 times, 0.05) p value is less than or equal to, by BM groups and the penis of ZM groups
Smooth muscle tissue's protein compares, and obtains the testosterone histological difference protein of BM/ZM；By BM groups and the penis of 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 is poor
M-band matter amounts to 48 kinds, wherein 35 kinds of up-regulated expression albumen, lowers 13 kinds of expressing protein.As shown in table 4：
The abnormal phlegm type impotence disease rat model of table 4 organizes disease group candidate's differential protein
6th, the ELISA detections of abnormal phlegm type impotence disease rat model serum candidate's differential protein
7 kinds of serum candidate's differential proteins of random selection, enter to abnormal lymphatic temperament type impotence disease rat model with control group
Row ELISA is detected, to verify whether serum candidate differential protein of the present invention can be used as the mark of abnormal lymphatic temperament type impotence disease
Will thing：
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 respectively double antibodies sandwich kit (rat c reactive protein (CRP) double antibodies sandwich kit：
Yi Lairuite bio tech ltd；Rat beta2 Glycoprotein 1 (APOH) double antibodies sandwich kit：Wuhan your raw commerce and trade excellent 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 Lairuite bio tech ltd；Rat thyroid element delivery
Albumen (TTR) double antibodies sandwich kit：Abnova Yanuofa Biotechnology Co., Ltd.；The acidoglycoproteins of rat α 1 (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 as shown in Fig. 4 A-G, as shown in Figure 4, wait by each abnormal lymphatic temperament type impotence disease rat model serum
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 inventive method acquisition protein marker can be used 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 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
4 kinds of testosterones of random selection organize candidate's differential proteins, to abnormal lymphatic temperament type impotence disease rat with it is right
Carry out Immunohistochemical detection according to group rat testosterone tissue, to verify present invention tissue in candidate's differential protein be
It is no can be used as the mark of abnormal lymphatic temperament impotence disease：
That is No. 12 PQ 9Z2L0 of random selection detection (voltage dependence anion selectivity channel protein 1)；30th
Number albumen P02625 (parvalbumin α)；No. 36 albumen P04904 (glutathione S-transferase α -3) and No. 44 albumen
Respectively using the antibody of above-mentioned albumen, (all antibody are purchased to be had P47853 (biglycan) in Abcam (Shanghai) trade
Limit company), Normal group and disease group rat testosterone tissue are detected according to antibody specification.With optics
Micro- sem observation and statistics.Photo multiplication factor is object lens multiple × eyepiece multiple.Coloration result method of counting is as follows：
In the case that multiplication factor is 400, each sample randomly chooses the 6-10 visual field, does not overlap completely between each visual field, counts sun
Property cell, per group of generally 10 different samples subsequently carry out the comparison between group.Methods of marking is divided into two aspects, i.e., positive
Scope and tinctorial strength.Percentage in the visual field shared by its positive cells is positive scope 5%-100%；Positive cell dyeing
The depth be tinctorial strength, be divided into 4 grades, i.e., (there is coloring 0 (almost not colored or somewhat have a Point Coloring), 1, but compares
It is shallower), 2 (having coloring, deep), 3 (having coloring, very deeply)；Final result is (positive scope × tinctorial strength)/visual field
Number.
Measurement result as shown in table 5, as shown in Table 5, put down by the penis of each abnormal lymphatic temperament type impotence disease rat model
The content of voltage dependence anion selectivity channel protein 1 and parvalbumin α is 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 inventive method acquisition protein marker can be used as the testosterone of abnormal lymphatic temperament type impotence disease
The biomarker of tissue.
Protein expression result in each group rat penis of table 5
* compare with N<0.05
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of dimension cures abnormal lymphatic temperament type impotence disease mammalian blood serum differential protein system, it is characterised in that the egg
Lean type system includes following albumen：
2. mammalian blood serum differential protein system according to claim 1, it is characterised in that the albumen system from
Model animal；The model animal is rat.
3. a kind of dimension cures the mammal testosterone histological difference albumen system of abnormal lymphatic temperament type impotence disease, its feature
It is, the albumen system, including following albumen：
4. testosterone histological difference albumen system according to claim 3, it is characterised in that the albumen system is come
Self mode animal；The model animal is rat.
5. a kind of mammalian blood serum differential protein system and testosterone of the abnormal lymphatic temperament type impotence disease of dimension doctor is organized
The screening technique of differential protein system, it is characterised in that comprise the following steps：
1) Normal group (N groups), abnormal lymphatic temperament syndrome group (ZM groups), abnormal lymphatic temperament type disease group (BM groups) lactation are set up
Animal；
2) step 1 is gathered) mammalian whole blood that obtains, the serum of mammal is obtained after separation, take the step 1)
Obtain the testosterone tissue of mammal；
3) from the step 2) serum that obtains and extract protein in testosterone tissue；
4) by the step 3) in purify after the protein labeling that obtains, obtain peptide fragment after purification；
5) by the step 4) peptide fragment after purification that obtains obtains finger-print Jing after Mass Spectrometer Method, the finger after screening
Line collection of illustrative plates enters line retrieval using proteome analysis software, is carried out quantitatively according to the spectrogram after the Search Results and screening for obtaining
Analysis, by the analyze data for obtaining proteomic image retrieval software retrieval used in the mammalian proteins database,
Obtain the N groups, ZM groups, the serum proteins of BM group mammals and testosterone tissue protein；
6) by the step 5) in
The BM groups obtain the serum differential protein of BM/ZM compared with the serum proteins of ZM groups；By BM groups and N groups
Serum proteins compare, and obtain the serum differential protein of BM/N；Count the BM/ZM and BM/N differential protein it is common
The type and quantity of 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, obtain the testosterone histological difference egg of BM/ZM
White matter；BM groups are compared with the testosterone tissue protein of N groups, BM/N group testosterone histological difference albumen is obtained
Matter；The type and quantity of the common albumen of the differential protein of the BM/ZM and BM/N are counted, abnormal lymphatic temperament type impotence is obtained
The testosterone histological difference protein of card group.
6. screening technique according to claim 5, it is characterised in that the mammal is rat.
7. screening technique according to claim 5, it is characterised in that the step 1) in Normal group in conventional environment
Middle raising, the environment temperature of feeding is 18～22 DEG C, and the relative humidity of the feeding environment is 40～60%；The feeding of modeling group
Environment temperature be 8～12 DEG C, the relative humidity 70～80% of the feeding environment；The step 1) in Normal group for general
Logical feed, modeling group feeding feed is raw property feed.
8. screening technique according to claim 5, it is characterised in that the step 3) described in labeling method be iTRAQ
Labelling kit is marked.
9. screening technique according to claim 5, it is characterised in that the step 4) in proteome analysis software be
Proteome Discoverer；The proteomic image retrieval software is mascot softwares.
10. the albumen system according to claim 1-4 any one further carries out abnormal lymphatic temperament type impotence (erection work(
Can obstacle) disease Mechanism Study, drug screening, conversion lay a good foundation with study on prevention, there is provided foundation.
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| CN109613268A (en) * | 2019-01-09 | 2019-04-12 | 新疆医科大学 | Differential protein system and its screening technique and application in a kind of Erectile Dysfunction patients serum |
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| WO2021123323A1 (en) * | 2019-12-20 | 2021-06-24 | Linnane Pharma Ab | Lon protease, alpha-hemolysin, ck1-alpha-1; c-myb inhibitor or a cebp-delta inhibitor as therapeutics |
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