CN107688057A - A kind of evaluation method of Passive-Heymann Nephritis model rat model - Google Patents
A kind of evaluation method of Passive-Heymann Nephritis model rat model Download PDFInfo
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Abstract
The invention discloses a kind of evaluation method of Passive-Heymann Nephritis model rat model.The evaluation method of the present invention is to gather rat urine in Passive-Heymann Nephritis model rat model using LC MS instruments, pass through the content of 10 biomarkers in Marker view software analysis urines, the content of 10 biomarkers meets the requirements, then Passive-Heymann Nephritis model rat model successfully constructs.The change that the present invention passes through endogenous biological label content in body end product urine, evaluate Passive-Heymann Nephritis model rat model, so as to establish unified evaluation body change comprehensively, it can quantify, compared with tradition takes blood examination to survey biochemical indicator or taking internal organ observation pathological section, there is the advantages of efficient, quick, hurtless measure, high specificity.
Description
Technical field
The present invention relates to a kind of evaluation method of Passive-Heymann Nephritis model rat model.
Background technology
Passive-Heymann Nephritis model (Passive Heymann Nephritis, PHN), it is to be used to study mankind's film
Animal model the most classical in nephrosis, its Development process and symptom are in close proximity to the membranous nephropathy of the mankind, and this
The advantages of model is to fall ill relatively rapidly, and whole pathological process is also more stable, while the model is easy to repeat, and is for detecting
The ideal animals model of medication effect.In order to preferably carry out experimental study, screening treatment passive-type Heymann kidneys are promoted
The progress of scorching medicine, the duplication evaluation of Passive-Heymann Nephritis model model turn into key problem in technology.Passive-type Heymann kidneys at present
The judgement of scorching model copy success or not is mainly according to histopathologic examination, and the observation result of nephridial tissue form is as auxiliary.
The mechanism coherent detection index such as Urine proteins, urea nitrogen, creatinine is also monitored by a large number of researchers.Tradition take blood examination survey biochemical indicator or
Taking internal organ observation pathological section evaluation method needs the long period, while brings wound to animal pattern.Therefore, it is a kind of that there is system
First, the method for being used to evaluate the rat model of Passive-Heymann Nephritis model the features such as quantization, high specificity, quick, hurtless measure
Urgently build.
Metabolism group is a kind of brand-new omics technology to grow up after the protein science, using high separating efficiency,
The advanced analysis instrument of high sensitivity and low test limit, with the dynamic rule of the small molecule endogenous metabolism thing in organism
Organism physiology pathological change trend is illustrated from overall, in qualitative and quantitative analysis study of disease progress rule and medicine efficacy relation etc.
Aspect has a wide range of applications.
The content of the invention
The present invention is to solve existing Passive-Heymann Nephritis model rat model to lack unification, quantization, high specificity, fast
A kind of the problem of speed and the evaluation method of hurtless measure, there is provided evaluation method of Passive-Heymann Nephritis model rat model.
A kind of evaluation method of Passive-Heymann Nephritis model rat model of the present invention, through the following steps that carry out：Adopt
Rat urine in Passive-Heymann Nephritis model rat model is gathered with LC-MS instruments, it is big by Marker view software analysis
The content of 10 biomarkers in mouse urine；
Wherein 10 biomarkers are：Tryptophan、L-Glutamate、Succinic acid、Creatinite、
Phytosphingosine, Taurine, Kynuric acid, Phenylpyruvate, Citric acid and Uridine；
If Tryptophan integral area mean drops to 5.268- from the 7.416 of normal rat in rat model urine
7.812；
L-Glutamate integral area mean drops to 1.200~1.430 from the 2.930 of normal rat；
Succinic acid integral area mean rises to 3.742~3.924 from the 1.383 of normal rat；
Creatinite integral area mean drops to 1.832~2.102 from the 2.993 of normal rat；
Phytosphingosine integral area mean rises to 6.023~6.546 from the 3.900 of normal rat；
Taurine integral area mean drops to 2.101~2.635 from the 4.931 of normal rat；
Kynuric acid integral area mean rises to 1.204~1.763 from the 1.113 of normal rat；
Phenylpyruvate integral area mean drops to 0.928~1.310 from the 1.810 of normal rat；
Citric acid integral area mean drops to 3.00~3.445 from the 5.238 of normal rat；
Uridine integral area mean drops to 0.704~1.026 from the 1.360 of normal rat；
Content of the wherein 7 kinds of labels in rat model urine all drops in described number range, 3 kinds of labels
Content in rat model urine is all risen in described number range, then proves Passive-Heymann Nephritis model rat mould
Type successfully constructs.
The metabonomic technology means that the present invention is utilized are considered as " the biochemical phenotype " of organism allomeric function state, energy
The response of enough instant, sensitive, exact representations organism allomeric function state under the stimulation of various extraneous factors and regulation situation.This
Patent utilization metabonomic technology method finds rat Passive-Heymann Nephritis model model organism label, and the present invention passes through machine
The change of endogenous biological label content in body end product urine, Passive-Heymann Nephritis model rat model is evaluated, so as to
Unified evaluation body change comprehensively is established, can be quantified, while for the specific model evaluation side of Passive-Heymann Nephritis model
Method.The present invention utilizes metabolism group method, to the LC-MS spectrograms of rat urine are metabolized before Heymann ephritis modelings and after end
Profile carries out non-supervisory model PCA and analyzed, and checks the variation track of rat urine metabolic profile before and after modeling；Then pass through
There are PLS (PLS-DA) discriminant analysis of supervised recognition, the variable weight value obtained according to PLS-DA models
(VIP), using VIP values more than 1 variable as biomarker candidate variables, in order to verify the candidate found in multidimensional statistics
Whether variable in unit statistically has marked difference, is examined in experiment using T, wherein P<0.05 has significant difference；With reference to
Load diagram screens candidate variables, by the Information in Mass Spectra of the compound representated by these variables, METLIN, HMDB, KEGG,
Scan for, match and speculate in the databases such as PubChem, finally determine possible biomarker；All data use
SPSS20.0 statistical analysis softwares are handled, and each group of data is represented with (Mean ± SD), are sentenced using One-wayANOVA methods
Otherness between other group.This patent finally identifies 10 potential source biomolecule labels of rat Passive-Heymann Nephritis model model,
Be respectively Tryptophan, L-Glutamate, Succinic acid, Creatinite, Phytosphingosine,
Taurine, Kynuric acid, Phenylpyruvate, Citric acid and Uridine；10 biology marks are illustrated simultaneously
Remember that thing changes in the relative amount of different groups, Tryptophan integral area mean is from normal big in rat model urine
The integral area mean that the 7.416 of mouse drop to 5.324, L-Glutamate drops to 1.310 from the 2.930 of normal rat,
Succinic acid integral area mean is from the 1.383 of the normal rat integral areas for rising to 3.844, Creatinite
Mean drops to 1.958, Phytosphingosine integral area mean from normal rat from the 2.993 of normal rat
The 3.900 integral area means for rising to 6.373, Taurine drop to 2.446, Kynuric from the 4.931 of normal rat
Acid integral area mean from the 1.113 of normal rat rise to 1.553, Phenylpyruvate integral area mean from
The integral area mean that the 1.810 of normal rat drop to 1.191, Citric acid drops to from the 5.238 of normal rat
3.336, Uridine integral area mean drops to 0.885 from the 1.360 of normal rat.
10 biomarkers can be turned into the pass of Passive-Heymann Nephritis model rat model urine metabolism group significant change
The validity of key metabolite is verified that the change of 10 biomarker expressions is certain in urine before and after discovery modeling
Passive-Heymann Nephritis model rat model urine metabolism trail change trend is reflected in degree.The present invention takes blood examination with tradition
Survey biochemical indicator or taking internal organ observation pathological section is compared, there is the advantages such as efficient, quick, hurtless measure, high specificity.
Brief description of the drawings
The PCA dynamic trend figures of rat model urine during Fig. 1 is model construction
A represents Normal group, B representative model groups；
Fig. 2 is rat model urine PLS-DA analysis plane shot charts
A represents Normal group, B representative model groups；
Fig. 3 is the relative amount change that biomarker difference group is identified in rat urine metabolism；
Fig. 4 is Normal group and model group rats renal pathology figure；
Embodiment
By combination accompanying drawing described further below it will be further appreciated that the features and advantages of the invention.The implementation provided
Example is only explanation to the inventive method, remaining content without limiting the invention in any way announcement.
Rat in present embodiment is SD rats, and male, 7 week old, cleaning grade, is in Wuhan University experimental animal
Center is commercially available.
【Embodiment 1】The foundation of rat syndrome of deficiency of heart qi model
(1) preparation of resist proximal tubule brush border antigen (Fx1A)
By healthy male SD rat with after 3% amobarbital (50mg/kg) intraperitoneal anesthesia, aseptically, along abdomen just
Middle opening abdominal cavity, takes out double kidneys of rat both sides, and isolates cortex renis, by 1:30 (v/w) ratios add 50mmol/L sweet dews
Alcohol/2mmol/L Tris-HCl (pH 7.0), is made homogenate in ice bath.Add 1mol/L CaCl2 to final concentration of 10mmol/
L, at 4 DEG C 3000g centrifuge 15min, take supernatant, 43000g centrifuges 20min again, takes precipitation, with 50mmol/L mannitol/
2mmol/L Tris-HCl (pH 7.0) repeated washing 1 time.It is by the 0.01mol/L PBS dissolvings precipitated with pH 7.4
Fx1A, it is standby to be placed in -20 DEG C of refrigerators.
(2) the sero-fast preparations of rabbit-anti rat Fx1A
Fx1A solution is mixed with appropriate Freund's adjuvant carry out it is fully emulsified after, be expelled to neck, back, the abdomen of White Rabbit
Portion and the foot pad of four limbs.It is immune repeatedly after, take blood 1mL from White Rabbit auricular vein, centrifuge after serum, with exempting from indirectly
Epidemic disease fluorescence method or double diffusion determine sero-fast potency, up to 1:2000 or 1:More than 32 be qualified.If antibody titer
It is unqualified can injections of antigens again, until potency is qualified.Through abdominal aortic blood after potency is qualified, 57 DEG C of water-bath 30min are placed in
Inactivate complement.Subsequent 5000g centrifugations 10min, it is rabbit-anti mouse Fx1A antiserums to draw supernatant, divides tubule to preserve, is placed in -20 DEG C
It is standby in refrigerator.(3) foundation of PHN models
In SPF level Animal Houses, start to test after 12 male SD rat adaptability are raised one week, during which ad lib
Drinking-water and natural lighting.Normal group, model group (each 6) are randomly divided into, wherein, the rat of model group is by the anti-blood of Fx1A
It is clear to press 8ml/kg dose delivery tail vein injection, while normal saline is expelled to Normal group.Generation is used after ten days
Thank to cage and collect all rat 24h urines respectively to detect urine protein content.
【Embodiment 2】
Using metabolism group method, to the LC- of rat urine before the Heymann ephritis modelings in embodiment 1 and after terminating
MS spectrograms metabolic profile carries out non-supervisory model PCA and analyzed, and checks the change rail of rat urine metabolic profile before and after modeling
Mark.In the PCA and PLS-DA of SIMCIA-P12.0 softwares plane shot chart, the coordinate representation sample each put is in master
Correlation in composition 1 and principal component 2.Abscissa represents principal component t [1], and ordinate represents principal component t [2].SIMCIA-P
Software stereoscopic three-dimensional shot chart is used to indicate that the correlation between principal component.If there is obvious difference between two samples
It is different, then this position of two coordinate points on shot chart can relatively far away from, and vice versa.Take Normal group and model group rats
24h urine specimens carry out PCA analyses, as a result such as Fig. 1, rats in normal control group and Heymann moulds are shown on PCA shot charts
The sample point of type rat is scattered in different regions, and in obvious difference trend, this explanation is using the injection sero-fast sides of Fx1A
It is feasible that method, which establishes model,.The sample point of Normal group compares concentration, is mainly distributed on second and third quadrant, has certain
Cluster, illustrate that its group difference is smaller, the metabolism state of endogenous material is relatively stable, or has consistent change to become
Gesture；The sample point of model group is concentrated mainly on first, fourth quadrant, the probability distribution of samples points relative distribution, and the sample point of the group is all
Clearly away from Normal group, this explanation is compared with Normal group, and rat is after medicine modeling, in its urine
Endogenous material metabolism state there occurs certain change.It is right because PCA is a kind of pattern recognition analysis method of no inspection
The individual difference XOR of sample in itself is disturbed caused difference None- identified by extraneous factor.In order to reduce or delete group difference
Interference, this experimental data additionally use the PLS discriminant analysis (PLS- of supervised recognition to caused by classification
DA).This method introduces a new variable (Y variables) compared with PCA, mainly during analysis, and this variable has
Classification information.As a result Fig. 2 is seen, after the analysis of PLS-DA methods, compared with the result that principal component analysis obtains, the plan of the model
Right more preferable, the sample point in each group also tends to concentrate, and separates between Normal group and model group farther.
The variable weight value (VIP) obtained according to PLS-DA models, using variable of the VIP values more than 1 as biomarker
Candidate variables, in order to verify whether the candidate variables found in multidimensional statistics statistically have marked difference in unit, experiment
It is middle to be examined using T, wherein P<0.05 has significant difference；Candidate variables are screened with reference to load diagram, pass through these variable institute's generations
The Information in Mass Spectra of the compound of table, scan for, match and speculate in the databases such as METLIN, HMDB, KEGG, PubChem,
Finally determine possible biomarker；All data are handled using SPSS20.0 statistical analysis softwares, each group of data with
(Mean ± SD) is represented, differentiates the otherness between group using One-wayANOVA methods.It is passive that the present invention finally identifies rat
10 potential source biomolecule labels of type Heymann Nephritis Models, are Tryptophan, L-Glutamate, Succinic respectively
acid、Creatinite、Phytosphingosine、Taurine、Kynuric acid、Phenylpyruvate、Citric
Acid and Uridine.
The relative amount that 10 biomarkers are illustrated simultaneously in different groups changes, in rat model urine
Tryptophan integral area mean drops to 5.324 from the 7.416 of normal rat,
L-Glutamate integral area mean drops to 1.310 from the 2.930 of normal rat,
Succinic acid integral area mean rises to 3.844 from the 1.383 of normal rat,
Creatinite integral area mean drops to 1.958 from the 2.993 of normal rat,
Phytosphingosine integral area mean rises to 6.373 from the 3.900 of normal rat,
Taurine integral area mean drops to 2.446 from the 4.931 of normal rat,
Kynuric acid integral area mean rises to 1.553 from the 1.113 of normal rat,
Phenylpyruvate integral area mean drops to 1.191 from the 1.810 of normal rat,
Citric acid integral area mean drops to 3.336 from the 5.238 of normal rat,
Uridine integral area mean drops to 0.885 from the 1.360 of normal rat.
【Embodiment 3】
Sample in the present embodiment is both from the rat (each 6 of Normal group, model group) in embodiment 1.In profit
The evaluation method obtained with embodiment 2 continue on the basis of the Passive-Heymann Nephritis model model that checking successfully constructs
Tests below and analysis.
The urine metabolism of control group and model group identifies that Fig. 3 is shown in the relative amount change of biomarker difference group；
Fig. 3 abscissa is biomarker information in control group and model group, and ordinate is biomarker relative amount, by scheming
3 understand, compare control group, in Passive-Heymann Nephritis model rat model urine Succinic, Phytosphingosin and
Kynuric acid contents raise, and difference has conspicuousness；And remaining 7 biomarker content reduces, difference has notable
Property.
Collect each group experimental rat weekly 24 it is small caused by urines and record volume.The urine being collected into is placed in addition
In the EP pipes of 200 μ L1% sodium azide, it is placed in -70 DEG C of refrigerator and stores, for detecting the content of twenty-four-hour urine albumen.
All rats of each group are put to death in the last day anesthesia of experiment, and 3mL or so is taken by the way of Culling heart blood, rapidly at 4 DEG C,
Centrifuged under 4000rmin-1 several minutes, isolated serum, by kit method determine blood plasma in seralbumin (ALB),
Urea nitrogen (BUN), creatinine (Cr), T-CHOL (TCh) and total triglycerides (TG) content.When putting to death all rats of each group, cut open
Abdominal cavity is opened, the kidney profile variation situation for the rat that detects by an unaided eye simultaneously records, while aseptically wins rat rapidly
Double kidneys, fixed respectively with formaldehyde, glutaraldehyde after removing coating, carry out renal pathology and Electronic Speculum detection.
1. biochemical indicator detects
Each group rat 24h Urine proteins, ALB, BUN, Cr, TCh and TG changes of contents (Mean ± SD, n=before and after the modeling of table 1
6)
Note：The * P compared with Normal group<0.05**P<0.01
Compared with blank group, BUN, Cr, TCh, TG content are equal in Passive-Heymann Nephritis model rat 24h Urine proteins, blood plasma
Rise and difference has significant (P<0.01), ALB contents are then slightly decreased, but equally have significant difference (P<
0.01)。
2. renal pathology changes
Visually observe, the rat kidney size and form of Normal group (Fig. 4 A) are normal, and color is normally in dark red
Color.The obvious enlargement of rat kidney of model group, different from normal size, color is in dark gray.Om observation after HE dyeing, normally
The renal tissues of rats mesonephric glomerulus structure and size of control group are normal.Renal tubule size, arrangement situation and form are normal,
Do not find that crystal deposits in urinary cast intracavitary.Compared with Normal group, kidney in the renal tissues of rats of model group (Fig. 4 B)
Bead volume is slightly bigger than normal, and the phenomenon that diffusivity thickens occurs in glomerular basement membrane.There is obvious vacuole in renal cells
Denaturation, discovery have cloudy swelling, it was observed that there is the epithelial cell for the necrosis that comes off in tube chamber, renal tubule obvious atrophy occurs and showed
As.Fibrous connective tissue hyperplasia can be observed in renal interstitial, and swelling occurs in sertoli cell.
From control group result, the rat Passive-Heymann Nephritis model model construction success of this experiment.This experiment is tested
Group has the advantages of efficient, quick, hurtless measure, high specificity, test group utilizes metabonomic technology method compared with control group
Rat Passive-Heymann Nephritis model model organism label is found, is marked by endogenous biological in body end product urine
The change of thing content, Passive-Heymann Nephritis model model is evaluated, so as to establish unified evaluation body change comprehensively, can quantified, together
When for Passive-Heymann Nephritis model specific model evaluation method.
Claims (2)
1. a kind of evaluation method of Passive-Heymann Nephritis model rat model, it is characterised in that through the following steps that carrying out
's：Rat urine in Passive-Heymann Nephritis model rat model is gathered using LC-MS instruments, passes through Marker view softwares point
Analyse the content of 10 biomarkers in rat urine；
Wherein 10 biomarkers are：Tryptophan、L-Glutamate、Succinic acid、Creatinite、
Phytosphingosine, Taurine, Kynuric acid, Phenylpyruvate, Citric acid and Uridine；
If Tryptophan integral area mean drops to 5.324-7.812 from the 7.416 of normal rat in rat model urine；
L-Glutamate integral area mean drops to 1.200 ~ 1.430 from the 2.930 of normal rat；
Succinic acid integral area mean rises to 3.742 ~ 3.924 from the 1.383 of normal rat；
Creatinite integral area mean drops to 1.832 ~ 2.102 from the 2.993 of normal rat；
Phytosphingosine integral area mean rises to 6.023 ~ 6.546 from the 3.900 of normal rat；
Taurine integral area mean drops to 2.101 ~ 2.635 from the 4.931 of normal rat；
Kynuric acid integral area mean rises to 1.204 ~ 1.763 from the 1.113 of normal rat；
Phenylpyruvate integral area mean drops to 0.928 ~ 1.310 from the 1.810 of normal rat；
Citric acid integral area mean drops to 3.00 ~ 3.445 from the 5.238 of normal rat；
Uridine integral area mean drops to 0.704 ~ 1.026 from the 1.360 of normal rat；
Then Passive-Heymann Nephritis model rat model successfully constructs.
A kind of 2. evaluation method of Passive-Heymann Nephritis model rat model according to claim 1, it is characterised in that
Through the following steps that carry out：Rat urine in Passive-Heymann Nephritis model rat model is gathered using LC-MS instruments, led to
Cross the content of 10 biomarkers in Marker view software analysis rat urines；
Wherein 10 biomarkers are：Tryptophan、L-Glutamate、Succinic acid、Creatinite、
Phytosphingosine, Taurine, Kynuric acid, Phenylpyruvate, Citric acid and Uridine；
If Tryptophan integral area mean drops to 5.324 from the 7.416 of normal rat in rat model urine,
L-Glutamate integral area mean drops to 1.310 from the 2.930 of normal rat,
Succinic acid integral area mean rises to 3.844 from the 1.383 of normal rat,
Creatinite integral area mean drops to 1.958 from the 2.993 of normal rat,
Phytosphingosine integral area mean rises to 6.373 from the 3.900 of normal rat,
Taurine integral area mean drops to 2.446 from the 4.931 of normal rat,
Kynuric acid integral area mean rises to 1.553 from the 1.113 of normal rat,
Phenylpyruvate integral area mean drops to 1.191 from the 1.810 of normal rat,
Citric acid integral area mean drops to 3.336 from the 5.238 of normal rat,
Uridine integral area mean drops to 0.885 from the 1.360 of normal rat；
Then Passive-Heymann Nephritis model rat model successfully constructs.
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Application publication date: 20180213 |