CN109709326B - Application of the PPM1A in treating asthma and diagnosis - Google Patents
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Abstract
The invention discloses application of the PPM1A in treating asthma and diagnosis.Application of the PPM1A as detection target spot in preparation bronchial asthma diagnostic reagent.Detect application of the reagent of PPM1A in preparation bronchial asthma diagnostic reagent.PPM1A inhibits application of the substance of PPM1A expression in the drug of preparation treatment bronchial asthma as application of the therapy target in the drug for preparing or screening treatment bronchial asthma.The invention discloses the applications of the PPM1A of detection bronchus patients blood plasma patient, find PPM1A for the first time in asthma and the differential expression of normal person, which can be used as the molecular marker of bronchial asthma.The present invention also has found that PPM1A as miR-1165-3p target gene, promotes Th2 differentiation by STAT1, STAT5, Akt access, leads to the generation of asthma for the first time.Therefore, it can be used as the therapy target of asthma.
Description
Technical field
The invention belongs to biomedicine fields, are related to application of the PPM1A in treating asthma and diagnosis.
Background technique
Bronchial asthma is one kind with chronic airway inflammation, Airway Remodeling, the heterogeneous disease that airway hyperreactivity is characterized
Disease.There are about 300,000,000 asthmatic patients, China's asthmatic patient number is more than 3,000,000 in the whole world.In recent years, with industrialized progress, I
The illness rate of state's asthma is still rising year by year, and brings heavy family and burden on society.Due to the heterogeneity of asthma, asthma tool
There are many phenotype and inner mold.Asthma is divided into following several phenotypes by Global Asthma Prevention Initiative (GINA) 2018: allergic asthma,
Non-allergic asthma, late-onset asthma, asthma are fat with fixed flow limitation, asthma concomitant fertilizer.Not isophenic Asthma Population for
The therapeutic response of drug especially inhaled glucocorticoids (ICS) is also not quite similar.The recurrent exerbation of asthma can also cause respectively
Kind complication, such as pulmonary emphysema, pulmonary heart disease, heart failure etc..
Traditional Diagnosing Asthma relies primarily on clinical manifestation, pulmonary function test, peak flow rate detection, but above-mentioned technology is not easy to examine
The unconspicuous asthmatic patient of symptom is measured, with the development of biotechnology, biomarker provides one kind for the diagnosis of asthma
New approach.
PPM1A (the protein phosphatase 1 A αisomer that magnesium ion relies on): PPM1A is the family of fibroin albumen phosphatase PP2C
Family member, karyon are distributed with endochylema, its dephosphorylation can be made in conjunction with multiple protein.It is passing in tumor area pair
The research of PPM1A is relatively more, it is considered to be a kind of cancer suppressorfactor, so far, there are no the passes that people has found PPM1A and asthma
System.
Summary of the invention
The purpose of the present invention is being directed to the above-mentioned deficiency of the prior art, PPM1A answering as asthma biomarker is provided
With.
It is a further object of the present invention to provide a kind of PPM1A as therapy target in preparation or screening treatment bronchial asthma
Drug in application.
The purpose of the present invention can be achieved through the following technical solutions:
Application of the PPM1A as detection target spot in preparation bronchial asthma diagnostic reagent.
Detect application of the reagent of PPM1A in preparation bronchial asthma diagnostic reagent.
Wherein, the reagent of the detection PPM1A is the Elisa kit for detecting PPM1A expression quantity.
Application of the PPM1A as therapy target in the drug for preparing or screening treatment bronchial asthma.
Inhibit application of the substance of PPM1A expression in the drug of preparation treatment bronchial asthma.
Preferred the miR-1165-3p ((AGCAGGCGCAGGGGGUGGUGUGGU of substance for inhibiting PPM1A expression
(SEQ ID NO.1))) or promote miR-1165-3p expression substance.
The utility model has the advantages that
The invention discloses detection bronchus patients blood plasma patient PPM1A application, for the first time find PPM1A asthma with
And the differential expression of normal person, the index can be used as the molecular marker of bronchial asthma.Blood plasma is easier to obtain, and belongs to
In woundless testing, the early diagnosis of asthma can be assisted.Therefore, the reagent for detecting PPM1A expression quantity can be applied to preparation branch
San bronchial asthma diagnostic reagent.
The present invention is led to after Th2 cell is overexpressed PPM1A, and STAT1 and Akt phosphorylation level reduce, STAT5 phosphoric acid
Change horizontal raising, but STAT2, STAT3, STAT6 are then without significant change.Flow cytometer detection is the results show that Th2 cell is overexpressed
After PPM1A, GATA3+ ratio is increased, the decline of T-bet+ ratio.Show that PPM1A is promoted by STAT1, STAT5, Akt access
Th2 differentiation, leads to the generation of asthma.By being overexpressed miR-1165-3p in Th2 cell, it is found that its PPM1A is expressed reduces,
PPM1A expresses PPM1A in the CD4+ T cell height of Th2 and mouse asthma, further demonstrates that and is inhibited by miR-1165-3p
PPM1A expression can inhibit the Th2 in asthma to break up, so as to improve asthma symptoms.Therefore, PPM1A can be used as therapy target,
It is applied in the drug for preparing or screening treatment asthma.And the substance for being able to suppress PPM1A expression can also be in preparation treatment branch
It is applied in the drug of san bronchial asthma.
Detailed description of the invention
Fig. 1 asthmatic patient and the blood plasma PPM1A of normal person are horizontal: A is Small Sample Database；B is big-sample data
After Fig. 2 Th2 cell is overexpressed miR-1165-3p, differentiation is suppressed, and Th1 is promoted to break up
(A) miR-1165-3p is overexpressed slow-virus transfection effect
(B) qPCR detects Th1 type cytokines (IFN-γ, IL-12) and Th2 type cytokines (IL-4, IL-5, IL-
13) change
(C) Elisa detects the variation of cell factor
(D) qPCR detects the variation of the main transcription factor of Th1 (T-bet) and the main transcription factor of Th2 (GATA3)
(E) Western blot detects variation and its statistical analysis of transcription factor
(F) nuclear factor detection Th2 differentiation is suppressed, and Th1 is promoted to break up
After Fig. 3 Th1 cell silencing miR-1165-3p, differentiation is suppressed, and Th2 is promoted to break up
(A) miR-1165-3p silencing slow-virus transfection effect
(B) qPCR detects Th1 (IFN-γ, IL-12) and Th2 type cytokines (IL-4, IL-5, IL-13) variation
(C) Elisa detects the variation of cell factor
(D) qPCR detects the variation of Th1 transcription factor (T-bet) and the main transcription factor of Th2 (GATA3)
(E) Western blot detects variation and its statistical analysis of transcription factor
(F) nuclear factor detection Th1 differentiation is suppressed, and Th2 is promoted to break up
Fig. 4 mouse asthma is overexpressed miR-1165-3p, can improve asthma symptoms
(A) asthmatic model excites, and in the 0th day sensitization in the 7th day tail vein injection miR-1165-3p mistake for the 7-11 days
Express slow virus or blank control virus
(B) miR-1165-3p is overexpressed slow virus vivo efficacy
(C) each group lung function situation
(D) each group serum IgE level
(E) the inflammatory cell differential counting of mouse bronchoalveolar lavage fluid
(F) reflect HE dyeing and the semi-quantitative analysis of inflammation
(G) reflect PAS dyeing and the semi-quantitative analysis of goblet cell metaplasia and mucous secretion
Fig. 5 mouse asthma is overexpressed miR-1165-3p, and Th2 differentiation is suppressed
(A) Elisa detects the cell factor in BALF
(B) nuclear factor marks Th1 the and Th2 cell proportion of CD4+ T lymphocyte in spleen and lymph node cells
Fig. 6 IL-13 and PPM1A are the target genes of miR-1165-3p
(A) thermal map of biotin-miRNA pull down experimental differences mRNA
(B) the volcano figure of biotin-miRNA pull down experimental differences mRNA
(C) intersection of difference mRNA and raw letter analysis target gene
(D) the Dual-Luciferase experiment of IL-13
(E) differential expression of the PPM1A in normal and mouse asthma CD4+ T cell
(F) PPM1A induces the differential expression of the Th1 and Th2 cell of differentiation in vitro
(G) the Dual-Luciferase experiment of PPM1A
(H) after qPCR detects Th2 cell transfecting miR-1165-3p overexpression slow virus, PPM1A expression variation
(I) after Western blot detects Th2 cell transfecting miR-1165-3p overexpression slow virus, PPM1A expression variation
Fig. 7 PPM1A regulates and controls Th2 differentiation by STAT and Akt access
(A) Th2 cell electricity turns PPM1A overexpression plasmid, the downstream variation of access
(B) experiment is remedied, nuclear factor label detection PPM1A can remedy the inhibition that miR-1165-3p breaks up Th2
Specific embodiment
The protein level detection of PPM1A in 1 peripheral blood from asthma patients of embodiment
Main agents
People's PPM1A Elisa detection kit (Wuhan Bio-swamp)
Key instrument
Microplate reader, centrifuge
Main method
It the acquisition of peripheral blood sample and freezes
The periphery blood specimen of 20 asthmatic patients is from Jiangsu Prov. People's Hospital, and the sample of 20 Healthy Peoples is from south
Capital medical university student enrollment.In the peripheral blood and anticoagulant tube for acquiring normal person and asthmatic patient, 1000rpm/min centrifugation
5 minutes, upper plasma is collected, is frozen in -80 DEG C
With the expression of PPM1A in enzyme-linked immunosorbent assay detection human plasma
This kit is using people's Mg2+/Mn2+ dependence protein phosphatase 1 A in double antibody sandwich method measurement sample
(PPM1A) horizontal.It is coated with microwell plate with people's Mg2+/Mn2+ dependence protein phosphatase 1 A (PPM1A) antibody of purifying, is made solid
Phase antibody sequentially adds plasma sample (PPM1A) into the micropore of coating monoclonal antibody, then the Mg2+/Mn2+ dependence with HRP label
Protein phosphatase 1 A (PPM1A) antibody combines, and forms antibody-antigene-hrp-antibody complex, and substrate is added after thoroughly washing
TMB colour developing.TMB converts au bleu under the catalysis of HRP enzyme, and is converted to final yellow under the action of an acid.The depth of color
Mg2+/Mn2+ dependence protein phosphatase 1 A (PPM1A) in shallow and sample is positively correlated.With microplate reader under 450nm wavelength
It measures absorbance (OD value), passes through standard
Curve calculates people Mg2+/Mn2+ dependence protein phosphatase 1 A (PPM1A) concentration in sample.
(1) dilution of standard items: preparing small test tube 6, successively finishes number, and it is dilute that standard items are first added in each small test tube
Liquid 100ul is released, then takes original content standard items 100ul to be added one in the test tube for the number of finishing, mixes well；Again in the test tube
In take 100ul be added second test tube in, mix well；100ul is taken to be added in third test tube in the test tube again, it is sufficiently mixed
It is even；It takes 100ul to be added in the 4th test tube in the test tube again, mixes well；100ul is taken to be added the 5th in the test tube again
In test tube, mix well；Then 100ul is taken in the test tube, discarded.6th test tube is as No. 0 standard items.It is respectively managed after dilution
Concentration is respectively: 20ng/ml, 10ng/ml, 5ng/ml, 2.5ng/ml, 1.25ng/ml, 0ng/ml.It is set on enzyme mark coating plate
Standard sample wells sequentially adds the standard items 50ul (it is recommended that each concentration does 2 parallel holes) of various concentration.
(2) be loaded: setting blank well respectively, (the anti-PPM1A of sample, enzyme marking reagent and biotin labeling is not added in blank control wells
Antibody, remaining each step operation are identical), sample to be tested hole.It is first loaded 40 μ l of product in sample to be tested hole on enzyme mark coating plate, then
Again plus the 10 μ l of anti-PPM1A antibody of biotin labeling.Sample is added on ELISA Plate hole bottom by sample-adding, does not touch hole wall as far as possible, gently
Light shake mixes.
(3) enzyme: every hole is added 50 μ l of enzyme marking reagent, except blank well.
(4) it incubates: being incubated 30 minutes with 37 DEG C of sealing plate film sealing plate postposition.
(5) match liquid: will be spare after 30 times of concentrated cleaning solutions, 30 times of distilled water dilutions.
(6) it washs: carefully taking sealing plate film off, discard liquid, dry, cleaning solution is filled it up in every hole, is discarded after standing 30 seconds, such as
This is repeated 5 times, and pats dry.
(7) develop the color: color developing agent A50 μ l is first added in every hole, adds color developing agent B50 μ l, and gently concussion mixes, and 37 DEG C are protected from light
Colour developing 10 minutes.
(8) terminate: every hole adds 50 μ l of terminate liquid, terminates reaction (blue is vertical at this time turns yellow).
(9) it measures: being returned to zero with blank well, 450nm wavelength sequentially measures the absorbance (OD value) in each hole.Measurement should add end
Only carried out within 15 minutes after liquid.
As a result
The blood plasma PPM1A level of asthmatic patient is higher than normal person, statistically significant.(Figure 1A)
Embodiment 2 increases sample size and detects PPM1A expression again
Main agents
People's PPM1A Elisa detection kit (Wuhan Bio-swamp)
Key instrument
Microplate reader, centrifuge
Main method
It the acquisition of peripheral blood sample and freezes
The periphery blood specimen of 50 asthmatic patients is from Jiangsu Prov. People's Hospital, and the sample of 50 Healthy Peoples is from south
Capital medical university student enrollment.In the peripheral blood and anticoagulant tube for acquiring normal person and asthmatic patient, 1000rpm/min centrifugation
5 minutes, upper plasma is collected, is frozen in -80 DEG C
With the expression of PPM1A in enzyme-linked immunosorbent assay detection human plasma
With embodiment 1
As a result
The blood plasma PPM1A level of asthmatic patient is higher than normal person, statistically significant (Figure 1B).
Regulation (cellular level) of the embodiment 3miR-1165-3p to Th2 cell differentiation
Main agents:
Cell factor (peprotech), function antibody (ebioscience), serum (Gibco), primer (Jin Sirui),
Sybr green (takara), miRNA reverse transcription reagents (takara), 1640 culture mediums (Gibco), Elisa kit
(Biolegend) Western antibody (Abcam, CST), streaming antibody and reagent (ebioscience), Trizol and tiny RNA
It extracts reagent (Thermo), magnetic bead sorting kit
Key instrument
Cell incubator, magnet stand, flow cytometer detection machine, electrophoresis transferring film instrument, exposure instrument, PCR instrument and qPCR instrument, microplate reader
Main method
The induction differentiation of Th1/Th2 cells in vitro
1. coating: the anti-CD3e of 100ul 5ug/ml is added in every hole, and 4 DEG C overnight.
2. mouse Naive CD4+ T cell separates.(the 0th day)
1. 6~8 weeks female C57/B6 mouse cervical dislocations is taken to put to death, 75% ethyl alcohol impregnates 3 minutes, takes out mouse and is placed in nothing
On bacterium ware, mouse spleen, two sides inguinal lymph nodes, lymphonodi mesenterici and two sides para-aortic lymph node are taken, 40 μ are put into
It is placed on m in the culture dish for filling 1640 serum-free mediums and gently grinds pressure with pestle, the flushing of 1640 serum-free mediums obtains thin
Born of the same parents' suspension, about 10ml, with lymphocyte separation medium sub-department lymphocyte
2. waiting time, prepare the culture mediums such as corresponding Th1, Th2 and change liquid culture medium (change liquid culture medium do not need plus
Anti-CD3e and anti-CD28)
3. magnetic bead sorting naive CD4+Cell, cell density 1-2*10^ is resuspended with the culture medium prepared in T cell5/ hole
3.Th1 cell is then in first day addition 1ul lentil-III-mir-Off-Control virus (scramble)
(for the control virus of silencing virus) (1.47*10^9IU/ml)/LentimiRa-off-miRNA-1165-3p Virus
(inhibitor) (for miR-1165-3p silencing virus) (6.25*10^9IU/ml), Th2 cell was then added at first day
1ulLentimiRa-GFp-miR-1165-3p virus (enhancer) (being overexpressed virus for miR-1165-3p) (4.4*10^
9IU/ml)/Lenti-III-mir-GFP control virus (blank) (compares disease for miR-1165-3p over-express vector
Poison) (5.9*10^9IU/ml)
4. the third day in culture is partly changed liquid to cell
5. the 5th day harvest cell collects supernatant, qPCR, western blotting, Elisa and the record of streaming consideration convey are done
Factor marker.
RNA is extracted and real-time quantitative pCR
CDNA is obtained after taking equivalent RNA reverse transcription with trizol small RNA extraction total serum IgE and miRNA., is used
After the RNase free water dilution of respective volume, SYBR Green method carries out fluorescence quantitative PCR detection, and 3, each sample multiple
Hole.The expression quantity of different transcription factors and cell factor is counted using β-actin as the Δ Δ CT value of internal reference standard,
The expression quantity of miR-1165-3p is counted using U6 as the Δ Δ CT value of internal reference standard.
Enzyme-linked immunosorbent assay
Cell conditioned medium is collected, according to kit specification, is coated with, closed, be loaded, detected, according to absorbance
Calculate IL-4, IL-5, IL-13 and IFN-γ concentration in supernatant.
Protein immunoblot experiment
With RIPA lysate extract albumen, it is denatured by boiling, carry out electrophoresis, transferring film, closing, apply primary antibody, wash film, apply secondary antibody,
Film, exposure are washed, using β-actin as internal reference albumen, compares the expression variation of transcription factor
Nuclear factor label
Cell is harvested, life or death dye marker anyway, marks main transcription factor after marking cd4 cell, rupture of membranes to break core, detects
The variation of Th1/Th2 differentiation
As a result
After Th2 cell miR-1165-3p is overexpressed, Th1 type cytokines IFN-γ, IL-12 expression increase, and Th2 class is thin
The expression of intracellular cytokine IL-4, IL-5, IL-13 reduce, and the main Transcription Factor T-bet expression of Th1 increases, the main transcription factor of Th2
GATA3 expression reduces (Fig. 2), and after Th1 cell mir-1165-3p silencing, variation tendency then opposite (Fig. 3), shows miR-
The differentiation of 1165-3p inhibition Th2 cell.
Embodiment 4miR-1165-3p improves asthma (integral level)
Main agents
Dermatophagoides pteronyssinus (HDM) (Greer), miR-1165-3p are overexpressed slow virus (love must dream), methacholine, ELISA examination
Agent box (Biolegend)), streaming antibody (Thermo), PAS dyestuff, HE dyestuff, count microballoon (Thermo) etc.
Key instrument
Lung function instrument, microplate reader, PCR instrument and qPCR instrument, microplate reader, flow cytometer detection instrument
Main method
Mouse HDM acute asthma model
32 SPF grades of C57/B6 female mices are randomly divided into 4 groups, every group 8.Control group (control), HDM group
(HDM), HDM+blank group (HDM+Lenti-III-mir-GFP control virus), HDM+enhancer group (HDM+
LentimiRa-GFp-miR-1165-3p virus).HDM processing group: the HDM that the 0th day beginning tracheae gives 100ug/40ul is molten
Liquid Intratracheal administration is with sensitization, and the 7th, 8,9,10, the 11 continuous HDM solution Intratracheal administrations for giving 10ug/40ul for 5 days are to excite.Its
The slow virus dilution that middle HDM+enahncer group and HDM+blank group were prepared in the 7th day tail vein injection 100ul.Each group is small
Mouse is in the 14th heaven-made subsequent detection.
Mouse pulmonary function detection
FinePointe RC mouse lung function instrument is opened, instrument is connected, checks air-tightness and school zero, setting instrument is extremely examined
Survey lung resistance and dynamic lung compliance module.With new prepared 1% amobarbital intraperitoneal anesthesia mouse (every mouse 150ul),
Postanesthetic mouse is fixed in surgical console, cuts off mouse skin of neck with scissors, then with after surgical forceps blunt separation
Exposure tracheae.Tracheae is first stabbed into an osculum with a fine needle, is placed in mouse trachea cannula tube core along osculum, suture is fixed to be intubated,
The mouse of trachea cannula is connected to lung function instrument, again detection device airtightness.Start software, PBS cleaning atomization.To baseline
After numerical stability, record respectively mouse atomization sucking 0,3.125,6.25,12.5, after methacholine, mouse lung impedance value,
Record 3min, after be averaged lung resistance value for the concentration.
Eyeball takes blood
4 groups of mouse Culling heart bloods after pulmonary function test stand 30min or more, 1000rpm, 4 DEG C, are centrifuged 15min, receive
Collection serum is placed in -80 DEG C of preservations after packing.
Collect bronchoalveolar lavage fluid (BALF)
After the blood sampling of 4 groups of mouse hearts, fix tracheae with vessel forceps on annular cartilage, under make row notch, merging connects
The silicone tube for connecing syringe is tightened tracheae and silicone tube with filament, is slowly injected into PBS in three times, and each injection rate is
O.4mL, recycling is placed in centrifuge tube immediately after each lavation, is measured, and is stored in 4 DEG C, makees cell separation in 2h.BALF in
1000g is centrifuged 5min, and supernatant dispenses -80 DEG C and freezes the ELISA measurement for giving over to cytokine levels.Cell precipitation makees streaming point
Class counts.
Classified counting of leucocyte
After cell precipitation contaminates life or death with FVD, streaming dyestuff is added and counts microballoon, CD45+Ly6G+ neutrophil leucocyte,
CD45+F4/80+siglecF+ mononuclear macrophage, CD45+CD3+ lymphocyte, CD45+Anti-SiglecF+F4/80- are thermophilic
Eosinophil.Carry out cell absolute counting.
Enzyme-linked immunosorbent assay
BALF is collected, according to kit specification, is coated with, closed, be loaded, detected, calculated according to absorbance
IL-4, IL-5, IL-13 and IFN-γ concentration in supernatant, while detecting the concentration of serum immune globulin E (IgE).
RNA is extracted and real-time quantitative pCR
It is extracted after total mouse lung tissue miRNA. takes equivalent RNA reverse transcription with small RNA and obtains cDNA, use respective volume
RNasefree water dilution after, SYBR Green method carry out fluorescence quantitative PCR detection, 3 multiple holes of each sample.miR-
The expression quantity of 1165-3p is counted using U6 as the Δ Δ CT value of internal reference standard.
Mouse lung tissue pathological staining
After row bronchoalveolar lavage, thoracic cavity is opened, atrium dextrum rushes PBS, until lung bleaches.Inferior lobe of right lung is taken to organize, in 4%
Property formalin it is fixed overnight.The lobe of the lung is taken out, dehydrated alcohol is immersed and is dehydrated 5 minutes, transparence in dimethylbenzene, paraffin embedding.Point
It carry out not HE and PAS dyeing.
CD4+ T lymphocyte transcription factor label
Mouse spleen, two sides inguinal lymph nodes, lymphonodi mesenterici and two sides para-aortic lymph node are taken, is put into 40
It is placed on μm in the culture dish for filling 1640 serum-free mediums and gently grinds pressure with pestle, the flushing of 1640 serum-free mediums obtains thin
Born of the same parents' suspension, about 10ml.
4 DEG C of centrifugation 5min of 500g abandon liquid, and 5ml 1X erythrocyte cracked liquid is added, and incubation at room temperature 5min. adds 20-
30mlPBS, 500g are centrifuged 5min.Mark anyway, marks CD4 later, breaks core mark transcription factor.
As a result (Fig. 4-5)
After mouse asthma tail vein injection miR-1165-3p is overexpressed slow virus, lung function, SERUM IgE, BALF inflammation
Cell count, inflammatory cell infiltration, goblet cell mucous secretion are suppressed, and Th2 type cytokines and Th2 cell ratio
Example decline, Th1 type cytokines and Th1 cell proportion increase.Show that miR-1165-3p can inhibit the Th2 in asthma points
Change, so as to improve asthma symptoms.
Embodiment 5 PPM1A and IL-13 is the target gene of miR-1165-3p
Main agents
Liposome (assist is holy), protein lysate (millipore), magnetic bead (Thermo), RNA extracts kit (QIAGEN)
Deng
Key instrument
Cell incubator, magnet stand, 360 ° of gyroscopes, supercentrifuge
Main method
The miRNA-pull down of biotin labeling is tested
Mouse embryonic fibroblasts (NIH-3T3) are used as vehicles cells, are inoculated in 10cm Tissue Culture Dish, to
When its density reaches 90%, the miR-1165-3p of liposome transfection biotin labeling and corresponding control (NC), transfection 48 are small
Shi Hou harvests cell, UV crosslinking, and cracking supernatant and magnetic bead are incubated for by lytic cell, is extracted therewith with RNA extracts kit
In conjunction with RNA, give Guangzhou Rui Bo company and be sequenced.
The raw letter analysis of sequencing result
The difference mRNA (change>2 P<0.05, Fold) that Biotin-miR-1165-3p and NC control group is screened
It is made into thermal map and volcano figure (5A-5B), and tri- softwares of difference results and miRanda, PITA and RNAhybrid are carried out
The result of the microRNA target prediction of miRNA takes intersection (5C), and conjecture PPM1A and IL-13 may be its target.
Dual-Luciferase experiment
With Bibiserv2 (https: //bibiserv.cebitec.uni-bielefeld.de/
SessionTimeout.jsf the binding site of target gene Ppm1a and IL-13 and miR-1165-3p, commission Zhenjiang love) are predicted
Must dream company synthesize PPM1A, 3 ' UTR MUT plasmids of 3 ' UTR WT of IL-13 and corresponding binding site deletion mutation, commission
Shanghai Ji Ma company synthesizes miR-1165-3p mimics and NC mimics, will be on plasmid and mimic corotation human embryo kidney
Skin (HEK297T) cell after corotation 48h, detects change in fluorescence with Promega Dual-Luciferase laboratory report kit, carries out
Statistical analysis.
RNA is extracted and real-time quantitative pCR
RNA the and TH2 cell of normal or mouse asthma RNA, Th1/Th2 is always taken to be overexpressed miR- with trizol
The RNA of 1165-3p obtains cDNA after taking equivalent RNA reverse transcription, after the RNasefree water dilution of respective volume, SYBR
Green method carries out fluorescence quantitative PCR detection, 3 multiple holes of each sample.PPM1A expression quantity is used as internal reference mark using β-actin
Quasi- Δ Δ CT value counts
As a result (Fig. 6)
By biology prediction and the miRNA-pull down experiment screening target gene of biotin labeling, and by double
The experiment of fluorescein element enzyme, confirmation IL-13 and PPM1A are the target gene of miR-1165-3p, and find that Th2 cell is overexpressed
After miR-1165-3p, PPM1A expression is reduced, and PPM1A expresses PPM1A in the CD4+ T cell height of Th2 and mouse asthma,
Further miR-1165-3p regulates and controls Th2 differentiation by PPM1A.
Embodiment 6PPM1A regulates and controls Th2 differentiation by Akt and STAT access
Main agents
Electricity turns liquid (Thermo), PPM1A is overexpressed plasmid (Origene), and streaming antibody (ebioscience) is fixed and broken
Film liquid (ebioscience) sorts magnetic bead kit etc.
Key instrument
Electroporation (Thermo), magnet stand, flow cytometer detection instrument
Main method
T cell electricity turns
Th2 culture medium and D-PBS are preheated, the culture medium without antibiont that will be prepared is added 100ul, in 96 holes, is placed on
Incubator preheating.The naive CD4+ T cell sorted out to be cleaned with D-PBS, 100-400g room temperature is centrifuged 5min,
Cell, cell density 2*10^7/ml is resuspended in Resuspension Buffer T, this step solves in 15minTube
2.5mL Electrolytic Buffer E is added, did not had electrode, sets electricity and turns condition, carries out electricity and turns, in repetition in the 3rd day
Step is stated, the 5th day harvest cell carries out follow-up test.
Protein immunoblot experiment
With RIPA lysate extract albumen, it is denatured by boiling, carry out electrophoresis, transferring film, closing, apply primary antibody, wash film, apply secondary antibody,
Film, exposure are washed, using β-actin as internal reference albumen, compares the expression variation of pathway protein
Nuclear factor label
Cell is harvested, life or death dye marker anyway, marks main transcription factor after marking cd4 cell, rupture of membranes to break core, detects
The variation of Th1/Th2 differentiation
As a result (Fig. 7)
After Th2 cell is overexpressed PPM1A, STAT1 and Akt phosphorylation level are reduced, STAT5 phosphorylation level liter
Height, but STAT2, STAT3, STAT6 are then without significant change.Flow cytometer detection the results show that Th2 cell be overexpressed PPM1A after,
GATA3+ ratio increases, the decline of T-bet+ ratio.Show that PPM1A promotes Th2 differentiation by STAT1, STAT5, Akt access.
Sequence information involved in above embodiments
| β-actin—F | GAGAAGCTGTGCTATGTTGCT |
| β-actin—R | CTCCAGGGAGGAAGAGGATG |
| GATA3-F | GAACCGCCCCTTATCAAG |
| GATA3-R | CAGGATGTCCCTGCTCTCCTT |
| T-bet-F | AATCGACAACAACCCCTTTG |
| T-bet-R | AACTGTGTTCCCGAGGTGTC |
| IL-4F | AACTCCATGCTTGAAGAAGAACTC |
| IL-4R | CCAGGAAGTCTTTCAGTGATGTG |
| IL-5F | GCAATGAGACGATGAGGCTTC |
| IL-5F | GCAATGAGACGATGAGGCTTC |
| IL-13F | TGAGCAACATCACACAAGACC |
| IL-13R | GGCCTTGCGGTTACAGAGG |
| IFN-γ-F | ACAATGAACGCTACACACTGC |
| IFN-γ-R | CTTCCACATCTATGCCACTTGAG |
| IL-12F | GATGACATGGTGAAGACGGC |
| IL-12R | AGGCACAGGGTCATCATCAA |
| PPM1A-F | CGCACGTTAGCCAGTGAGAA |
| PPM1A-R | CGCAGAATCAGTGTCGTCATT |
| U6-F | CTCGCTTCGGCAGCACA |
| U6-R | AACGCTTCACCAATTGCGT |
| miR-1165-3p-5' | AGCAGCGCAGGGGGTGGTGTGGT |
Sequence table
<110>Jiangsu Prov. People's Hospital
<120>application of the PPM1A in treating asthma and diagnosis
<160> 1
<170> SIPOSequenceListing 1.0
<210> 2
<211> 24
<212> RNA
<213>mankind (Homo sapiens)
<400> 2
agcaggcgca ggggguggug uggu 24
Claims (3)
- Application of the antibody of 1.PPM1A in preparation bronchial asthma Blood diagnosis reagent.
- 2. detecting application of the Elisa kit of PPM1A expression quantity in preparation bronchial asthma Blood diagnosis reagent.
- Application of the 3.PPM1A as target spot in the drug for preparing or screening treatment bronchial asthma.
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