CN109207425A - Porphyromonas gingivalis inducing macrophage excretion body rna expression research method - Google Patents
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Abstract
Porphyromonas gingivalis inducing macrophage excretion body rna expression research method of the invention, step includes: that 1) person monocytic cell U937 is successively recovered, cultivates and induced, and obtains macrophage；2) bacterium P.gingivalis W83 is recovered；3) the external inflammatory model of building P.gingivalis W83 infection macrophage；4) the excretion body discharged before and after P.gingivalis W83 infection macrophage is extracted respectively and is identified；5) the excretion body total serum IgE discharged before and after P.gingivalis W83 infection macrophage is extracted respectively, is carried out the high-flux sequence of full transcript profile, is screened mRNA, lncRNA, circRNA of differential expression, carries out confluence analysis.The present invention has studied the variation of rna expression in the excretion body that macrophage is secreted in the simulation inflammatory process infected by porphyromonas gingivalis for the first time, can be caused a disease for excretion body in periodontal according to experimental result and the further research of immunologic process lays a good foundation.
Description
Technical field
The invention belongs to technical field of cell biology, and in particular to a kind of porphyromonas gingivalis (Porphyromonas
Gingivalis, P.gingivalis) inducing macrophage excretion body rna expression research method.
Background technique
It using plaque is to originate the factor, P.gingivalis as principal causative that periodontitis (periodontitis), which is a kind of,
Chronic destructive, diseases associated with inflammation of the generation of bacterium in Periodontal Supporting Tissue[1].Wherein P.gingivalis is a kind of Gram
Negative brevibacterium produces melanin and obligate anaerobic, belongs to a member of red complex.P.gingivalis is to periodontal support group
The destruction knitted and its outer membrane structure are closely related, pili (fimbriae), lipopolysaccharides (lipopolysaccharide, LPS), outer
Memebrane protein (outer membrane protein, OMP), hemagglutinin (hemagglutinin, HA), protease (gum
Element, gingipains) it is the most important virulence factor in the surface P.gingivalis[2], can be identified, stimulate huge by macrophage
Make a variety of pro-inflammatory cytokines of its synthesis and secretion after phagocyte, cell damage causes periodontium to damage and destroy.Have
Person has found that it is special that the LPS stimulating expression of macrophage on Gram-negative bacteria surface can be such that its secretion has in Research on Cardiovascular disease
The excretion body of function, plays effective immunostimulation during immunity of organism[3].So macrophage conduct
Whether one of main inflammatory cell in P.gingivalis pathogenic course, research excretion body participate in Immunization Activities involved in it
And mechanism is of great significance for the clinic diagnosis of periodontosis.
Excretion body (exosomes) is that one kind has surface Special Proteins marker and unique lipid, by cell " endocytosis-
The phospholipid bilayer film property imitated vesicle structure that the active procedures such as fusion-outlet " are formed.Its size is uniform, diameter be 40~
150nm, density are 1.10~1.18g/mL.Through studying, excretion body can remove waste plant protein in cell growth process, thin
Intercellular transmitting biological information, offer antigen to activate T cell, participate in immunity of organism inflammatory reaction process, the new life of blood vessel and
Growth of tumour cell etc..At present in Dendritic Cells, macrophage, lymphocyte, fibroblast, mescenchymal stem cell
Different cell types and most of body fluid such as peripheral blood, urine, saliva, ascites, joint fluid etc. are ok with tumour cell etc.
Detect excretion body from a wealth of sources[4].The constituent of excretion body has: cholesterol abundant, sphingomyelins, tubulin, flesh are dynamic
Albumen, binding protein, four transmembrane proteins, various mRNA, miRNA and siRNA etc.[5].Wherein certain specificity that excretion body carries
Albumen can reflect its biogenesis and cell origin, can be used as mark even one of the index of clinical diagnosis of the identification of excretion body.
Excretion body can mediate information interchange in intercellular trafficking semiochemicals, and domestic and foreign scholars have found it with heart and brain blood under study for action
Generation, development and the injury repair aspect of the systemic diseases such as pipe, liver, kidney and tumour have close connection.
Bibliography:
[1] Sudhakar U, Thyagarajan R, Jeyapal B, et al.Indagation of serum and
salivary reactive oxygen metabolite and cortisol levels in chronic
periodontitis and stress-induced chronic periodontitis patients[J].J Indian
Soc Periodontol, 2017,21 (5): 371-5.
[2] Bengtsson T, Zhang B,R, et al.Dual action ofbacteriocin PLNC8
αβ through inhibition of Porphyromonas gingivalis infection andpromotion of
Cell proliferation [J] .Pathog Dis, 2017,75 (5)
[3] Kim OY, Hong BS, Park KS, et al.Immunization with Escherichia coli
outer membrane vesicles protects bacteria-induced lethality via Th1 and Th17
Cell responses [J] .J Immunol, 2013,190 (8): 4092-102.
[4] Kim YS, Ahn JS, Kim S, et al.The potential theragnostic (diagnostic+
therapeutic)application ofexosomes in diverse biomedical fields[J].Korean J
Physiol Pharmacol, 2018,22 (2): 113-25.
[5] Williams C, Royo F, Aizpurua-Olaizola O, et al.Glycosylation of
Extracellular vesicles:current knowledge, tools and clinical perspectives [J] .J
Extracell Vesicles, 2018,7 (1): 1442985.
Summary of the invention
The purpose of the present invention is overcoming above-mentioned the shortcomings of the prior art, it is huge to provide a kind of porphyromonas gingivalis induction
Phagocyte excretion body rna expression research method.
To achieve the above object, the invention adopts the following technical scheme:
Porphyromonas gingivalis inducing macrophage excretion body rna expression research method, comprising the following steps:
(1) person monocytic cell U937 is successively recovered, cultivated and induced, obtain macrophage；
(2) bacterium P.gingivalis W83 is recovered；
(3) the external inflammatory model of building P.gingivalis W83 infection macrophage；
(4) the excretion body discharged before and after P.gingivalis W83 infection macrophage is extracted respectively and is identified；
(5) the excretion body total serum IgE discharged before and after P.gingivalis W83 infection macrophage is extracted respectively, is turned entirely
The high-flux sequence of record group screens mRNA, lncRNA, circRNA of differential expression, and carries out integration point in conjunction with GO and KEGG
Analysis.
Beneficial effects of the present invention:
The present invention has studied what macrophage was secreted in the simulation inflammatory process infected by porphyromonas gingivalis for the first time
The variation of rna expression in excretion body, the further research that can be caused a disease in periodontal with immunologic process for excretion body according to experimental result
It lays a good foundation.
Detailed description of the invention
Fig. 1 is the excretion body of the macrophages secrete in control group under 40000 times of transmission electron microscopes；
Fig. 2 is the excretion body of the macrophages secrete in experimental group under 40000 times of transmission electron microscopes；
Fig. 3 infects the outer of macrophage front and back release through P.gingivalis W83 for what the embodiment of the present invention was finally collected
Secrete the thermal map result of internal mRNA differential expression, wherein SY: experimental group；DZ: control group；
Fig. 4 infects the outer of macrophage front and back release through P.gingivalis W83 for what the embodiment of the present invention was finally collected
Secrete the GO enrichment result of internal mRNA；
Fig. 5 infects the outer of macrophage front and back release through P.gingivalis W83 for what the embodiment of the present invention was finally collected
Secrete the KEGG enrichment result of internal mRNA；
Fig. 6 infects the outer of macrophage front and back release through P.gingivalis W83 for what the embodiment of the present invention was finally collected
Secrete the thermal map result of internal circRNA differential expression, wherein SY: experimental group；DZ: control group；
Fig. 7 infects the outer of macrophage front and back release through P.gingivalis W83 for what the embodiment of the present invention was finally collected
Secrete the GO enrichment result of internal circRNA；
Fig. 8 infects the outer of macrophage front and back release through P.gingivalis W83 for what the embodiment of the present invention was finally collected
Secrete the KEGG enrichment result of internal circRNA；
Fig. 9 infects the outer of macrophage front and back release through P.gingivalis W83 for what the embodiment of the present invention was finally collected
Secrete the thermal map result of internal lncRNA differential expression, wherein SY: experimental group；DZ: control group；
Figure 10 infects what macrophage front and back discharged through P.gingivalis W83 for what the embodiment of the present invention was finally collected
The GO of lncRNA is enriched with result in excretion body；
Figure 11 infects what macrophage front and back discharged through P.gingivalis W83 for what the embodiment of the present invention was finally collected
The KEGG of lncRNA is enriched with result in excretion body.
Specific embodiment
Person monocytic cell U937 and P.gingivalis W83 bacterium used by the present invention is implemented is big by China Medical
It learns attached stomatological hospital central laboratory to provide, belonging to the public can get material.
The present invention is described in further details with specific embodiment with reference to the accompanying drawing, described is to solution of the invention
It releases rather than limits.
The specific embodiment of the invention provides porphyromonas gingivalis inducing macrophage excretion body rna expression research method,
The following steps are included:
1. common agents are prepared
1.1 U937 cell culture reagents
- 1640 cell culture medium of 10% fetal calf serum: inactivated fetal bovine serum 30min in 55 DEG C of water-baths is cooled to it
After room temperature, -1640 cell culture medium of fetal calf serum, the volume ratio of fetal calf serum and 1640 culture mediums are prepared in superclean bench
Example is 1: 9, after 0.22 μm of filter filtration sterilization, 4 DEG C of refrigerator long-term preservations.
No -1640 cell culture medium of fetal calf serum: preparing 1640 cell culture mediums in superclean bench, is filtered with 0.22 μm
After device filtration sterilization, 4 DEG C of refrigerator long-term preservations.
Cells frozen storing liquid is prepared: by 1640 culture mediums, fetal calf serum, dimethyl sulfoxide (DMSO) three according to 7: 2: 1
Volume ratio is prepared, matching while using, pays attention to being kept in dark place.
1.2P.gingivalis W83 Bacteria Culture reagent
Brain heart infusion (BHI) bacterial liquid culture medium: 3.6gBHI powder and 0.05g yeast are added in 100mL distilled water
Powder is cooled to 55-60 DEG C through high pressure steam sterilization with the NaOH tune PH to 7.0 of lmol/L after being completely dissolved, in ultra-clean work
500 μ L hemins and 100 μ L vitamin Ks are added in platform.
BHI bacterium solid culture medium: 3.6gBHI powder, 0.05g yeast powder and 1.5g agar are added in 100mL distilled water
Powder uses the NaOH tune PH to 7.0 of 1mol/L, through high pressure steam sterilization, when being cooled to 55-60 DEG C, in ultra-clean work after being completely dissolved
Make that 5mL Sheep Blood, 500 μ L hemins and 100 μ L vitamin Ks are added in platform, pour into plate rapidly, after solidification 4 DEG C it is close
Seal long-term preservation.
2. person monocytic cell U937 is successively recovered, cultivate and is induced；
With containing the person monocytic cell frozen before the recovery of -1640 (10%FBS-1640) cell culture medium of 10% fetal calf serum
U937 is subsequently placed in 5%CO2, under the conditions of 37 DEG C culture about 3-4 days in incubator, by the person monocytic cell U937 after culture in
Cell conditioned medium is abandoned after 800rpm centrifugation 5min, collects cell precipitation, then take 18 big culture bottles, is added in each big culture bottle
- 1640 cell culture medium of 10% fetal calf serum of 15ml simultaneously spreads 4.5 × 106U937 cell after a centrifugation, by 100ng/mL's
Person monocytic cell U937 of the concentration into each big culture bottle is added propylene glycol methyl ether acetate (PMA) and is placed in 5%CO2、37℃
Under the conditions of in incubator after culture 48h, observe macrophage morphology, promoting it successfully induce is adherent macrophage, Zhi Houyong
Suction pipe removes the cell culture fluid in each culture bottle, and 15ml is added without 1640 cell culture medium of fetal calf serum, is placed in 5%CO2、
It is cultivated for 24 hours in insulating box under the conditions of 37 DEG C.
3. bacterium P.gingivalis W83 is recovered；
By the P.gingivalis W83 bacteria resuscitation previously frozen on solid BHI culture medium, in anaerobic jar
Then culture 4-5 days takes collarium stroke to take part black colonies, is placed in BHI fluid nutrient medium and increases bacterium, after 12-16h using sterile
It draws bacteria culture media supernatant and is centrifuged 5min in 3500rpm, abandon supernatant, collect P.gingivalis W83 bacterial precipitation, use
Bacterial precipitation, the bacterium P.gingivalis W83 after being recovered, through spectrophotometric is resuspended in 1640 cell culture medium of serum-free
After counting detection bacterium concentration, for use.
4. constructing the external inflammatory model of P.gingivalis W83 infection macrophage
It will be trained in step 2 with the macrophage inner cell that -1640 cell culture medium of serum-free has cultivated 18 big bottle for 24 hours
Feeding base is removed, and every bottle is changed to new -1640 cell culture medium of 15ml serum-free, wherein 9 bottles are used as experimental group, is in addition made for 9 bottles
For blank control group, 4.5 × 10 are added in every bottle of experimental group8A P.gingivalis W83 bacterium (MOI=100: 1), blank
Isometric -1640 cell culture medium of serum-free is only added in every bottle of control group, cell-bacterium is obtained after mixing gently and is suspended
Liquid is put in 5%CO2, cultivate in 37 DEG C of insulating boxs.
5. the extraction and identification of the excretion body of macrophage release
Macrophages secrete is outer when 5.1 premenstruum (premenstrua) preliminary experiments proof P.gingivalis W83 infection macrophage 48h
It secretes that the scale of construction is most, therefore collects bacterium-cell suspension of corresponding experimental group and blank control group after cultivating 48h, it is every 3 bottles thin
Bacterium-cell suspension is collected as 1 sample, altogether collect 3 samples of 3 samples of experimental group and blank control group, with 3000rcf from
Heart 15min collects supernatant and is filtered with 0.22 μm of disposable filter, uses ExoQuick kit afterwards, will according to its specification
In bacterium-cell suspension and kit precipitating reagent by volume 5: 1 ratio after mixing, under the conditions of 4 DEG C overnight；Second
It by mixture with 1500rcf be centrifuged 30min, abandon supernatant, then 1500rcf be centrifuged 5min, be sucked out all liq, precipitating with
100pLPBS is resuspended, and saves backup in -80 DEG C.
The spare excretion body of 5.2 taking-ups freezes sample and is placed on ice chest, after slowly melting, drips on the copper mesh in the aperture 2nm
20 μ L excretion body suspensions simultaneously stand 3min at room temperature, lightly blot liquid in strainer side with filter paper, pH6.8 is added dropwise
2% or 3% Salkowski's solution, 30 μ L negative staining 5min at room temperature；With filter paper blot the negative staining liquid on copper mesh surface again and in room temperature
Under the conditions of it is dry after, be placed in the sample room of TEM, observation excretion volume morphing simultaneously shoots electromicroscopic photograph, in 40000 times of transmitted electrons
The excretion body of macrophages secrete is as shown in Figure 1 in control group under microscope；The experimental group under 40000 times of transmission electron microscopes
The excretion body of middle macrophages secrete is as shown in Figure 2.
5.3 take experimental group and 20 μ L excretion body suspension of control group and 5 μ L people CD9, CD81 colloid pearls (to belong to above respectively
The public can get material) it is incubated for 30min at room temperature after mixing, add 500 μ L PBS, slowly shakes 1h.Under the conditions of 4 DEG C
After 200rcf is centrifuged 30min, the sweet ammonia of 100 μ L 100mM is added and closes 15min, after cleaning 2 times (600rcf*10min) with PBS,
It is separately added into 20 μ L people's CD9, CD81 antibody again and is protected from light incubation 1h, after 200 μ L PBS resuspension is added, it is thin to carry out streaming for upper machine immediately
Born of the same parents' detection, obtains the content ratio of extracted sample interior excretion body Specific Marker Proteins CD9, CD81, repeats this experiment three times.
5.4 dilute excretion body suspension with appropriate PBS, with sample injections after the appropriate dilution of 1mL syringe absorption to detection slot
In, bubble is avoided, how much sample dilutions are adjusted according to the granule number of Scanning NTA-ZetaView instrument record, with view
30~40, Yezhong particle is advisable, and to machine automatic reading, generates NTA examining report.Repeat this experiment three times；It collects in report
Data simultaneously arrange.
6. macrophage and blank control group that experimental group is infected through P.gingivalis W83 are without P.gingivalis
The high-flux sequence of full transcript profile in the excretion body of the macrophage release of W83 infection:
48h in the Exosomal RNA Extraction Kit kit operating procedure extraction step 2 of reference SBI company
When blank control group and experimental group macrophage release the intracorporal total serum IgE of excretion, use NanoDrop ND-1000 instrument
(Thermo Fisher Scientific, Waltham, MA, USA) measures the RNA concentration of each sample.With OD260/OD280
Value is used as RNA purity index, if OD260/OD280 value range, in 1.8-2.1, RNA purity is qualified.Through after the assay was approved, making
Library Quality is detected with Agilent2100 Bioanalyzer instrument.It prepares and is sequenced followed by the library RNA, use
Ribo-Zero rRNA Removal Kits (Illumina, the U.S.) removes the rRNAs in sample total serum IgE, uses TruSeq afterwards
Stranded Total RNA Library Prep Kit (Illumina, the U.S.) pre-processes RNA and constructs sequencing library.It uses
BioAnalyzer2100 instrument (Agilent Technologies, the U.S.) carries out library Quality Control and quantifies.According to Illumina
Sequencing explanation, the library 10pM be denaturalized as single strand dna, is captured on Illumina flowcell, in situ to expand as cluster
(cluster), 150cycle sequencing and on Illumina HiSeq sequenator is carried out using both-end mode (PE mode).
By 4000 sequencer of Illumina HiSeq, both-end reads is harvested.Quality Control is carried out using Q30, is used
Cutadapt (vl.9.3) software removes connector, removes low quality reads, obtains high quality reads.CircRNA uses STAR software
High quality reads is compared onto reference genome/transcript profile, carries out circular rna detection and identification using DCC software.And make
The circular rna identified is annotated with circBase database and Circ2Traits.Use total comparison reads number pair
The comparison montage reads (junction reads) of each sample is standardized, and log2 is converted.Use Volcano plot
The difference circular rna s identification for carrying out two groups of samples, is identified using the difference circular rna s that multiple variation carries out two sample rooms,
And carry out GO and the KEGG analysis of difference circular rna s derived genes.LncRNA: using hisat2 software (v2.0.4), will be high-quality
Reads is measured to compare to the mankind with reference on genome (UCSC HG19).Then, it under the guidance of gtf gene annotation file, uses
FPKM (the Fragments of cuffdiff software (a part of cufflinks software suite) acquisition transcript degree LncRNA
Per kilobase ofexon per fragments mapped) value, as the express spectra of LncRNA, and calculate two groups of samples
Between multiple variation and p-value to screen the LncRNA of differential expression.According to the relationship of closing on, the target gene of LncRNA is predicted,
Carry out GO and the KEGG analysis of target gene.
7. interpretation of result
Fig. 3 provide the macrophage infected through P.gingivalis W83 that the present embodiment is finally collected and without
The thermal map of mRNA differential expression is as a result, infect front and back in the excretion body of the macrophage release of P.gingivalis W83 infection
The original express spectra of mRNA obtains the FPKM expression value of transcript degree using cuffdiff software (cufflinks module), and counts
Multiple variation and p-value are calculated to screen differential expression mRNA.According to 2 times of multiple expression variation and p value 0.05 in the present embodiment
As threshold value, screening obtains differential expression mRNA.Dendrogram is done using heatmap2 R language pack for differential gene, is used
Each mRNA is carried out data normalization conversion to FPKM on the direction be expert in different samples by scale parameter, be expert at
The direction of column is clustered using Euclidean distance (Euclidean distance), adjusts the suitable of row and column according to relationship of closing on
The difference mRNA expression value of upper downward is drawn thermal map by sequence.It is two-way to be carried out with differential expression mRNA with sample as the result is shown
Dendrogram, red represent expression quantity height, and green represents that expression quantity is low, and abscissa is sample number.SY-1:48hTreat, SY-2:
48hTreat, SY-3:48hTreat: 3 samples of experimental group；DZ-1:48hControl, DZ-2:48hControl, DZ-3:
48hControl: 3 samples of control group, ordinate are mRNA title.P.gingivalis W83 infection macrophage is discharged
The mRNA raised in excretion body has 5658, is respectively as follows: mRNAGPR143, mRNAPPY, mRNAIL23A, mRNAGRP,
MRNAC18orf21, mRNAIGFBP1, mRNASERP2, mRNADDX4, mRNABTG3, mRNAGOLGA6A etc..The mRNA of downward
There are 61, be respectively as follows: mRNASNURF, mRNAIDH3G, mRNAMT-ND6, mRNAACSL4, mRNAC12orf39,
MRNACOMMD3, mRNAMTRNR2L2, mRNASPARC, mRNAPF4, mRNASKAP2 etc..
Fig. 4 provide the macrophage infected through P.gingivalis W83 that the present embodiment is finally collected and without
The GO of mRNA is enriched with as a result, to difference mRNA target gene in the excretion body of the macrophage release of P.gingivalis W83 infection
It is predicted, GO enrichment analysis is carried out to target gene, as the result is shown the bar chart of GO enrichment, before indicating enrichment number from big to small
10 (Top10): primary metabolic process (main metabolic process), cellular component
Organization or biogenesis (cell component tissue or biology occur), metabolic process (were metabolized
Journey), organic substance metabolic process (organic substance metabolic process), cellular component
Organization (cell component tissue), cellular metabolic process (cellular process), cellular
Protein modification process (cell protein modification), protein modification process
(protein modification process), macromolecule modification (macromolecular modification), regulation of
Metabolic process (adjusts metabolic process).
Fig. 5 provide the macrophage infected through P.gingivalis W83 that the present embodiment is finally collected and without
The KEGG of mRNA is enriched with as a result, to difference mRNA target base in the excretion body of the macrophage release of P.gingivalis W83 infection
Because being predicted, KEGG enrichment analysis is carried out to target gene, as the result is shown the bar chart of KEGG enrichment, indicate enrichment number from big
To small preceding 10 (Top10): (glycosyl sphingolipid biology closes Glycosphingolipid biosynthesis-ganglio series
At-neuromere series), Lysosome (lysosome), (longevity is adjusted Longevity regulating pathway-mammal
Access-mammal), (protein in endoplasmic reticulum adds Protein processing in endoplasmic reticulum
Work), ABC transporters (ABC transport), Steroid biosynthesis (steroids biosynthesis),
Phosphatidylinositol signaling system (phosphatidylinositols signal system), Basal transcription
Factors (basal transcription factor), Morphine addiction (morphine addiction) Glycosaminoglycan
Biosynthesis-heparan sulfate/heparin (glycosaminoglycan biosynthesis-Heparan sulfate/heparin).
Fig. 6 provide the macrophage infected through P.gingivalis W83 that the present embodiment is finally collected and without
The thermal map result of circRNA differential expression in the excretion body of the macrophage release of P.gingivalis W83 infection.It uses
STAR software compares the circRNA of high quality reads onto reference genome/transcript profile, is carried out using DCC software cyclic annular
RNA detection and identification, and the circular rna identified is annotated using circBase database and Circ2Traits.It uses
Total comparison reads number is standardized the comparison montage reads (junction reads) of each sample, and log2 turns
It changes, is identified using the difference circular rna s that Volcano plot carries out two groups of samples.According to multiple expression variation 2 in the present embodiment
It is used as threshold value again, screening obtains differential expression circRNA.Dendrogram is done using heatmap2 R language pack for differential gene,
Data normalization is carried out to logCPM on the direction that each circRNA is expert in different samples using scale parameter to turn
It changes, is clustered in the direction of row and column using Euclidean distance (Euclidean distance), gone according to relationship adjustment is closed on
With the sequence of column, the difference circRNA expression value of upper downward is drawn into thermal map.Red represents expression quantity height, and green represents expression
Measure low, abscissa is sample number.SY-1:48hTreat, SY-2:48hTreat, SY-3:48hTreat: 3 samples of experimental group
This；DZ-1:48hControl, DZ-2:48hControl, DZ-3:48hControl: 3 samples of control group, ordinate are
CircRNA title.P.gingivalis W83 infects circRNA in the discharged excretion body of the adjustable macrophage of macrophage
Expression quantity, the circRNA of downward has 299, is respectively as follows: hsa_circ_0000437, hsa_circ_0004771, hsa_
Circ_0027463, hsa_circ_0001824, hsa_circ_0126525, hsa_circ_0001073, hsa_circ_
0121178, hsa_circ_0000117, hsa_circ_0000284, hsa_circ_0027464, etc..The circRNA of up-regulation has
6, respectively hsa_circ_0007854, hsa_circ_0006956, etc..
Fig. 7 provide the macrophage infected through P.gingivalis W83 that the present embodiment is finally collected and without
The GO of circRNA is enriched with as a result, to difference circRNA in the excretion body of the macrophage release of P.gingivalis W83 infection
Derived genes are predicted, carry out GO enrichment analysis to derived genes, the bar chart of GO enrichment, indicates enrichment number as the result is shown
From big to small preceding 10 (Top10): peptidyl-threonine phosphorylation (peptidyl-threonine phosphorylation),
Peptidyl-threonine modification (peptidyl-threonine modification), organelle organization (cell
Device tissue), single-organism organelle organization (single creature organelle tissue), regulation
Of Ras GTPase activity (adjusts Ras GTP enzymatic activity), chromatin modification (chromatin modification),
Cellular component organization (cell component tissue), cellular component organization
Or biogenesis (cell component tissue or biology occur), cytoskeleton organization (cytoskeleton group
Knit), positive regulation ofRas GTPase activity (positive regulator of Ras GTP enzymatic activity).
Fig. 8 provide the macrophage infected through P.gingivalis W83 that the present embodiment is finally collected and without
The KEGG of circRNA is enriched with as a result, to difference in the excretion body of the macrophage release of P.gingivalis W83 infection
CircRNA derived genes are predicted, carry out KEGG enrichment analysis to derived genes, as the result is shown the bar chart of KEGG enrichment,
Indicate enrichment number from big to small preceding 10 (Top10): Bacterial invasion of epithelial cells (bacterium
Invade epithelial cell), Chemokine signaling pathway (chemotactic factor (CF) signal path), Colorectal cancer
(colorectal cancer), Amoebiasis (amcbiasis), Focal adhesion (adhesion strength), Regulation of actin
Cytoskeleton (modulate actin cytoskeleton), Leukocyte transendothelial migration are (white thin
Born of the same parents are across endothelial migration), Thyroid hormone signaling pathway (thyroid hormone signal path), N-Glycan
Biosynthesis (biosynthesis of N- glycan), MAPK signaling pathway (MAPK signal path).
Fig. 9 provide the macrophage infected through P.gingivalis W83 that the present embodiment is finally collected and without
The thermal map of lncRNA differential expression is as a result, lncRNA is former in the excretion body of the macrophage release of P.gingivalis W83 infection
Beginning express spectra obtains the FPKM expression value of transcript degree using cuffdiff software (cufflinks module), and calculates multiple
Variation and p-value are to screen differential expression lncRNA.According to 2 times of multiple expression variation and 0.05 conduct of p value in the present embodiment
Threshold value, screening obtain differential expression lncRNA.Dendrogram is done using heatmap2 R language pack for differential gene, is used
Data normalization conversion is carried out to FPKM on the direction that each lncRNA is expert at by scale parameter in different samples, is expert at
It is clustered with the direction of column using Euclidean distance (Euclidean distance), the suitable of row and column is adjusted according to relationship of closing on
The difference lncRNA expression value of upper downward is drawn thermal map by sequence.It is as the result is shown to be carried out with differential expression lncRNA and sample
Bidirectional clustering figure, red represent expression quantity height, and green represents that expression quantity is low, and abscissa is sample number.SY-1:48hTreat,
SY-2:48hTreat, SY-3:48hTreat: 3 samples of experimental group；DZ-1:48hControl, DZ-2:48hControl, DZ-
3:48hControl: 3 samples of control group, ordinate are LncRNA title.P.gingivalis W83 infects macrophage can
To adjust in the discharged excretion body of macrophage, the lncRNA of up-regulation has 756, is respectively as follows:
LncRNAENST00000363008, lncRNAENST00000363328, lncRNAENST00000363838,
LncRNAENST00000365481, lncRNAENST00000397864, lncRNAENST00000411018,
LncRNAENST00000411050, lncRNAENST00000411291, lncRNAENST00000412149,
LncRNAENST00000412218 etc..The lncRNA of downward has 32, is respectively as follows: TCONS_00022987, uc004coz.1,
ENST00000363120, uc022bqs.1, ENST00000565493, ENST00000365241, ENST00000411389,
ENST00000434988, ENST00000497032, ENST00000551629 etc..
Figure 10 provide the macrophage infected through P.gingivalis W83 that the present embodiment is finally collected and without
The GO enrichment of lncRNA is as a result, carry out difference lncRNA in the excretion body of the macrophage release of P.gingivalisW83 infection
Source gene predicted, carries out GO enrichment analysis to derived genes, as the result is shown the bar chart of GO enrichment, indicate enrichment number from
It arrives greatly small preceding 10 (Top10): primary metabolic process (main metabolic process), organic substance
Metabolic process (organic substance metabolic process), the vesicle-mediated transport (fortune that vesica mediates
It is defeated), tRNA methylation (tRNA methylation), metabolic process (metabolic process), Golgi vesicle
Transport (Golgi saccule transport), Growth (growth), cellular metabolic process (cell metabolism mistake
Journey), negative regulation of protein metabolic process (negative regulation of protein metabolism process),
Regulation ofmetabolic process (adjusts metabolic process).
Figure 11 provide the macrophage infected through P.gingivalis W83 that the present embodiment is finally collected and without
The KEGG of lncRNA is enriched with as a result, to difference lncRNA in the excretion body of the macrophage release of P.gingivalis W83 infection
Derived genes are predicted, carry out KEGG enrichment analysis to derived genes, the bar chart of KEGG enrichment, indicates enrichment as the result is shown
Number preceding 10 (Top10): Adherens junction (adhesive connection), ErbB signaling pathway from big to small
(ErbB signal path), Leukocyte transendothelial migration (leucocyte is across endothelial migration),
Neurotrophin signaling pathway (neurotrophic factor signal path), Axon guidance (aixs cylinder guidance),
Osteoclast differentiation (osteoclast differentiation), Signaling pathways regulating
Pluripotency of stem cells (versatility of signal path adjusting stem cell), Phospholipase D
(Jak-STAT signal is logical by signaling pathway (phospholipase D signal path), Jak-STAT signaling pathway
Road), Tuberculosis (tuberculosis).
To sum up, 4.5 × 10 are being used8A P.gingivalis W83 (MOI=100: 1) stimulating expression of macrophage is simultaneously put in
5%CO2, cultivate in 37 DEG C of insulating boxs respectively 6h, 12h, for 24 hours, after 48h, 5658 in the excretion body that causes the cell to discharge
MRNA up-regulation, 61 mRNA lower (Fig. 3), 874 mRNA correlation Go terms enrichment up-regulations (Fig. 4), 42 mRNA correlation letters
Number access difference up-regulation (Fig. 5), 299 circRNA are lowered, 6 circRNA raise (Fig. 6), 469 circRNA correlation Go
Terms enrichment is lowered, (Fig. 7), 43 circRNA associated signal paths differences lower (Fig. 8), 756 lncRNA are raised, 32
LncRNA lowers (Fig. 9), 199 lncRNA correlation Go terms enrichment up-regulations (Figure 10), 19 lncRNA associated signal paths
Difference raises (Figure 11).
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is not considered as limiting the invention, those skilled in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, modifies, replacement and variant.
Claims (1)
1. porphyromonas gingivalis inducing macrophage excretion body rna expression research method, which is characterized in that including following step
It is rapid:
(1) person monocytic cell U937 is successively recovered, cultivated and induced, obtain macrophage；
(2) bacterium P.gingivalis W83 is recovered；
(3) the external inflammatory model of building P.gingivalis W83 infection macrophage；
(4) the excretion body discharged before and after P.gingivalis W83 infection macrophage is extracted respectively and is identified；
(5) the excretion body total serum IgE discharged before and after P.gingivalis W83 infection macrophage is extracted respectively, carries out full transcript profile
High-flux sequence, screen mRNA, lncRNA, circRNA of differential expression, and GO and KEGG is combined to carry out confluence analysis.
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