CN107243012A - A kind of applications of the 5p of miR 93 that exosomes is loaded in treatment rheumatoid arthritis - Google Patents
A kind of applications of the 5p of miR 93 that exosomes is loaded in treatment rheumatoid arthritis Download PDFInfo
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Abstract
The invention provides a kind of applications of the 5p of miR 93 of exosomes loadings in treatment rheumatoid arthritis, belong to field of biomedicine technology；The method comprises the steps of firstly, preparing the G exosomes for being overexpressed or blocking the 5p of miR 93, and proved by experiment in vitro, be overexpressed the more obvious differentiation for inhibiting Th17 cells of the 5p of miR 93 G exosomes；And by studying the effect for being overexpressed and blocking the 5p of miR 93 G exosomes to be fallen ill for CIA model mices, it is found that protective effects of the overexpression miR 93 5p G exosomes to CIA mouse is stronger；And the 5p of miR 93 G exosomes are blocked then without above-mentioned effect；As a result show that the G exosomes for being overexpressed the 5p of miR 93 can be used for the treatment of rheumatoid arthritis.
Description
Technical field
The invention belongs to field of biomedicine technology, it is related to a kind of miR-93-5p of exosomes loadings in treatment class wind
Application in wet arthritis, and in particular to granulocyte sample marrow source property suppresses the miR-93-5p that cell derived exosomes is loaded
Application in treatment rheumatoid arthritis.
Background technology
Rheumatoid arthritis (rheumatoid arthritis, RA) is a kind of agnogenic chronic progressive and tired out
And the autoimmune disease of whole body multisystem, pathological characters performance mostly hand, the synovial membrane chronic inflammation of sufficient Minor articulus symmetry,
Its body harm caused, has a strong impact on human being's production and life.RA definite pathogenesis is failed to understand, in immunology and epidemic disease
Research think with heredity, hormone, environment, virus infection etc. factor it is relevant.The main purpose that it is treated is at present to mitigate
The inflammation in joint, suppresses the mobility of advancing of disease, as far as possible Saving cortilage and reduction disease.Clinically for RA treatment
Mainly include drug therapy and immunological therapy.Drug therapy mainly includes non-steroidal drug, antirheumatic medicine, sugared cortex
Hormone etc., but the toxic side effect that medicine is brought is more, influences liver kidney metabolic function, easily produces pharmacological dependence, in addition steroids
Medicine can cause the adverse reaction of patient's stomach and intestine, central obesity, hypoimmunity, osteoporosis etc..Immunological therapy is mainly wrapped
Immunologic purging and biological targeting drug therapy are included, wherein immunologic purging can be removed in patient with rheumatoid arthritis blood and itself resisted
Body, immune complex and immunoglobulin etc., but treatment cost is high, complex operation, and wound is larger.Biological targeting medicine is mainly wrapped
TNF-α antagonist and the biological agent for RA inflammatory molecules are included, these medicines have quick suppression inflammatory reaction, reparation has been damaged
Hinder the effect in joint, but price is sufficiently expensive, and there is the patient's tuberculosis resume combustion for causing latent tuberculosis infection, hepatic injury,
Suffer from tumour equivalent risk.Include IL-1 receptor antagonists and IL-6 receptor antagonists otherwise for the biological agent of RA inflammatory molecules
Deng.Conventional medication brings great toxic side effect, and current immunological therapy method is relatively single and expensive, for RA
Treatment still have and many need to study and improvements.
Existing researcher has found the new CD4 of a class+Helper T lymphocyte, thus cell subsets specific secretion IL-17 and by
The referred to as type of helper cell 17 (helper T cell type 17, Th17), it can also secrete the cells such as IL-6, TNF-α because
Son, I L-17 and Th17 cell plays a significant role in the morbidity of RA and its animal model.
Correlative study in recent years shows that MDSCs has huge potential in terms of the treatment of autoimmune disease, existing real
Test using MDSCs treatment mouse collagen Induced Arthritis (collagen-induced arthritis, CIA), and obtain one
Constant current modulation is imitated；But, because MDSCs sources are not enough, storage inconvenience and its application is limited the shortcomings of complicated component.Marrow source property suppresses
Cell (myeloid derived suppressor cells, MDSC) is the heterogeneous population body of derived from bone marrow, in mouse
In, MD SC are the double positive cells of Gr-1 (being made up of Ly6G and Ly6C) and CD11b, according to cellular morphology and Ly6G, Ly 6C
Expression quantity height, and MDSC is divided to for two hypotypes：CD11b+Ly6G+Ly6ClowGranulocyte sample MDSC (G- MDSC) and
CD11b+Ly6G-Ly6ChiMonocyte sample MDSC (M-MDSC), wherein G-MDSCs mainly pass through ARG1 (Arginine
1, Arg-1) and active oxygen (reactive oxygen species, ROS) suppresses activation and the propagation of T cell, M-MDSC is then
Inhibitory action is mainly played by Arg-1 and nitric oxide.
There are some researches show the exosomes (G-exosomes) in G-MDSC sources is for IBD model mice and CIA
Model mice has protective effect, and exosomes is the vesica with bioactivity produced by cell endocytic system,
The various lipids of parcel, protein, nucleic acid in exosomes, and the matter transportation that can be carried plays life to recipient cell
Thing is acted on.Current exosomes application mainly includes pharmaceutical carrier and medical diagnosis on disease, and drug transport is used as using exosomes
Carrier, has that immunogenicity is low, conevying efficiency is high, stability good, target relative to current liposome vectors and polymer support
Tropism is strong and crosses over the advantage of blood-brain barrier, and the medicine using exosomes as carrier mainly has gene class medicine at present
(siRNA, mRNA, miRNA) and anticancer class medicine (adriamycin, taxol etc.) and the immune antigen of enhancing etc..Due to difference
The exosomes of different cell secretions contains different nucleic acid, protein ingredient under disease, so analysis is separated from patient's body fluid
The compositions that are included of exosomes can as medical diagnosis on disease foundation.Increasing research shows exosomes certainly
Played an important role in the inflammation generating process of body immunity disease, but the exosomes complicated components in G-MDSC sources, its
Specific mechanism of action is still unclear.
More research shows that the miRNA that exosomes is carried has the effect for suppressing T cell Proliferation, Differentiation, and such as Treg is thin
The Let-7d that born of the same parents source exosomes is carried can suppress the cell-mediated inflammatory reactions of Th1, and reducing inflammation property in vivo
Enteropathy mouse model disease severity (Okoye IS, Coomes SM, Pelly VS, Czieso S,
Papayannopoulos V,Tolmachova T,et al.MicroRNA-containing T-regulatory-cell-
derived exosomes suppress pat hogenic T helper 1cells[J].Immunity.2014,41(1):
89-103.).Microrna (microRNA, miRNA) is a series of non-coding tiny RNA that length are 18~22 nucleotides, they
Mainly by being specifically bound with the 3 ' of target gene non-translational regions (3 '-UTR), suppress the translation of its target gene or induce it
MRNA degraded, so that the expression of suppression target albumen.
According to G-exosomes structural and functional characteristic, RA treatment is applied to using the miRNA of its loading, be can reach
The purpose of " safe, targeting is strong ", with good potential applicability in clinical practice, is expected to send out in terms of immunity of organism balance is maintained
Positive role is waved, a kind of brand-new therapy approach is provided for RA treatment.The present invention carries out artificial to miRNA in G-exosomes
Intervene, application effect can be significantly improved after overexpression, to obtain the G-exosomes with more preferably therapeutic effect.
The content of the invention
It is an object of the invention to overcome defect of the prior art, there is provided a kind of overexpression miR-93-5p G-
Exosomes preparation method and its application in the medicine for preparing treatment rheumatoid arthritis.
To reach above-mentioned purpose, the technological means that the present invention takes is as follows：
Present invention firstly provides a kind of applications of miR-93-5p in the medicine for preparing treatment rheumatoid arthritis.
The present invention also provides a kind of miR-93-5p screening technique.
The present invention also provides a kind of overexpression miR-93-5p G-exosomes preparation method.
The present invention also provides a kind of medicine for treating rheumatoid arthritis, and the medicine includes overexpression miR-93-5p
G-exosomes.
Specifically, the technical scheme used is as follows：
Present invention separation and Extraction M-MDSC and G-MDSC first, then extracts and identifies secreted by both cells
Exosomes (G-exosomes and M-exosomes), by experimental verification G-exosomes and M-exosomes for CIA moulds
The influence of type mouse invasion, as a result shows, G-exosomes has the effect for the morbidity for suppressing CIA model mices, passes through G-
Exosomes and M-exosomes is handled Th17 cells, is further demonstrated that, G-exosomes can suppress Th17 cells
Differentiation, and then mitigate mouse invasion, and M-exosomes does not have obvious inhibitory action.
MiRNA extraction sequencing analysis are carried out to G-exosomes and M-exosomes, filter out in G-exosomes and
The first two larger ten miRNA of the absolute difference of content difference in M-exosomes；Further according to miRNA structure and work(
Energy characteristic is screened, and filters out 9 miRNA that may be acted to RA, is respectively：miR-16-5p、 miR-29a-3p、
miR-93-5p、miR-22-3p、miR-26b-5p、Let-7f-5p、Let7g-5p、miR-221-3p、miR-92a-3p。
Primer is designed according to 9 miRNA sequences filtered out, detects 9 miRNA in G-exosomes by qRT-PCR
The change of expression quantity in Th17 cells after processing, as a result filters out expression quantity in the Th17 cells after G-exosomes processing and increases
Plus maximum 3 miRNA are miR-16-5p, miR-29a-3p, miR-93-5p.
By the corresponding mimics of filter out 3 miRNA (miR-16-5p, miR-29a-3p, miR-93-5p) and
Mimics- negative controls transfect T cells, and induce differentiation Th17 cells, are detected by qRT-PCR, as a result show miR-
93-5p can suppress T cells to the differentiation of Th17 cells, also indicate that G-exosomes may be by it and load carrying
MiR-93-5p suppress Th17 cells differentiation.
Further by transfecting mimics and inhibors G-MDSC, the G- for being overexpressed or blocking miR-93-5p is prepared
Exosomes, and proved by experiment in vitro, miR-93-5p G-exosomes is overexpressed relative to original G-exosomes
The more obvious differentiation for inhibiting Th17 cells；And block miR-93-5p G-exosomes then to lose original G-
Inhibitory action of the exosomes to Th17 cell differentiations.As a result show that the miR-93-5p that G-exosomes is carried suppresses in vitro
Th17 differentiation, overexpression miR-93-5p prepared by present invention G-exosomes is relative to original G-exosomes pairs
The rejection ability of Th17 cell differentiations is stronger.
The present invention is overexpressed and blocked what miR-93-5p G-exosomes fell ill for CIA model mices by studying
Effect, find to be overexpressed miR-93-5p G-exosomes relative to original G-exosomes to the protective effects of CIA mouse more
Qiang Liao, and block miR-93-5p G-exosomes then to lose suppression of original G-exosomes to CIA mouse invasions and make
With.As a result the morbidity for the miR-93-5p mitigation CIA model mices that G-exosomes is carried, overexpression prepared by the present invention are shown
MiR-93-5p G-exosomes is stronger to the protective effect of CIA model mices relative to original G-exosomes.
Present invention discover that the miR-93-5p that G-exosomes is loaded can suppress corresponding recipient cell target egg in vivo and in vitro
White STAT3 expression, the inhibitory action to Th17 cell differentiations is played with this.
Advantages of the present invention
(1) present invention firstly discovers that the miR-93-5p that G-exosomes is carried can be by suppressing the differentiation of Th17 cells
Mitigate the morbidity of CIA model mices, new method is provided for RA treatment.
(2) present invention prepare be overexpressed miR-93-5p G-exosomes method take it is short, it is easy to operate, it is contained into
Divide activity stabilized, -80 DEG C of holding times were up to more than 1 year；Structure and active component are also stablized at room temperature, be conducive to clinically should
With.
(3) the overexpression miR-93-5p prepared in the present invention G-exosomes has relative to common G-exosomes
The effect of stronger suppression Th17 cell differentiations and alleviation CIA model mice disease severities.
(4) miR-93-5p that G-exosomes is loaded in the present invention can suppress corresponding recipient cell target in vivo and in vitro
Protein S TAT3 expression, action target spot is clear and definite.
(5) have that immunogenicity is low, conevying efficiency is high, stability good using exosomes as drug delivery carrier, absorb
Hurry up, the advantage that targeting is strong, overexpression miR-93-5p G-exosomes prepared by the present invention is relative to existing treatment RA medicines
Thing, with having no toxic side effect, normal hepatocytes kidney metabolic function is not influenceed, preparation process is easy, the low advantage of required cost, in addition originally
The overexpression miR-93-5p prepared G-exosomes is invented, mechanism of drug action is clear and definite, beneficial to the monitoring of curative effect of medication.
Brief description of the drawings
G-MDSC and M-MDSC Purity result that Fig. 1 is prepared for present invention sorting, wherein A is G-MDSC, and B is
M-MDSC。
G-exosomes and M-exosomes transmission electron microscope picture that Fig. 2 is prepared for present invention sorting, wherein A is G-
Exosomes, B are M-exosomes.
G-exosomes and M-exosomes particle size chart of frequency distribution that Fig. 3 is prepared for present invention sorting, wherein A
For G-exosomes, B is M-exosomes.
Fig. 4 is the significant protein expression result that Westem blot detect G-exosomes and M-exosomes.
Fig. 5 is shadows of the G-exosomes and M-exosomes to average arthritic index in CIA model mice pathogenic processes
Ring result.
Fig. 6 is influence results of the G-exosomes and M-exosomes to CIA model mice toes swelling degree.
A is phosphoric acid in influence results of the Fig. 7 for G-exosomes and M-exosomes in vitro for Th17 cell differentiations, figure
Salt buffer control group Th17 cell proportions；B is N-exosomes treatment group Th17 cell proportions；C is M-exosomes processing
Group Th17 cell proportions；D is G-exosomes treatment group Th17 cell proportions.
Fig. 8 is that the absolute value for the miRNA content differences that G-exosomes and M-exosomes is loaded arranges first 20
miRNA。
Fig. 9 is the result of variations of miRNA expression quantity in the Th17 cells that G-exosomes is handled.
Figure 10 for transfected corresponding mimics to after T cells, induce miR-93-5p in the Th17 cells of differentiation,
MiR-16-5p, miR-29a-3p expression quantity result of variations.
Figure 11 is right to have transfected the corresponding mimics of miR-93-5p, miR-16-5p, miR-29a-3p to after T cells
A is mimics- negative control group Th17 cell proportions in the influence result of induction differentiation Th17 cells, figure；B is miR-16-
5p-mimics treatment group Th17 cell proportions；C is miR-29a-3p-mimics treatment group Th17 cell proportions；D is miR-
93-5p-mimics treatment group Th17 cell proportions.
Figure 12 expresses miR-93- for miR-93-5p mimics or inhibitors are transfected into after G-MDSC to it
5p influence.
MiR-93-5p mimics or inhibitors is are transfected into after G-MDSC by Figure 13, G-exosomes expression
MiR-93-5p expression quantity result figure.
Figure 14 is overexpression or blocks A in influence results of the miR-93-5p G-exosomes to Th17 cell differentiations, figure
For negative control group Th17 cell proportions, B is phosphate buffer control group Th17 cell proportions, and C is G-exosomes processing
Group Th17 cell proportions, D is mimics-NC treatment group Th17 cell proportions, and E is mimic treatment group Th17 cell proportions, and F is
Inhibitors-NC treatment group Th17 cell proportions, G is inhibitors treatment group Th17 cell proportions.
Figure 15 is overexpression or blocks miR-93-5p G-exosomes to miR-93- in cell in Th17 induction systems
5p expression quantity.
Figure 16 is overexpression or blocks influences of the miR-93-5p G-exosomes to CIA model mice arthritis index
Degree.
Figure 17 is overexpression or blocks miR-93-5p G-exosomes to the influence journey of CIA model mice toes swelling
Spend picture directly perceived.
The HE dyeing of toes after G-exosomes processing CIA model mices of the Figure 18 to be overexpressed or blocking miR-93-5p
Section result, wherein A is PBS group, and B is G-exosomes groups, and C is mimics-NC-exosomes groups, and D is
Mimics-exosomes groups, E is inhibors-NC-exosomes groups, and F is inhibors-exosomes groups.
Figure 19 is thin to be overexpressed or blocking miR-93-5p G-exosomes to handle CIA model mice Hou popliteal nest lymph nodes
The streaming result of Th17 cell proportions in born of the same parents.
Figure 20 is overexpression or blocks miR-93-5p G-exosomes to CIA model mice draining lymph nodes CD4+T is thin
The result of miR-93-5p expression quantity in born of the same parents.
Figure 21 is miR-93-5p and STAT3 possibility binding site.
Figure 22 is overexpression or blocks miR-93-5p G-exosomes to the total STAT3 of cell in Th17 induction systems
Expression quantity, wherein, A is Western blot testing results, and B is statistical analysis figure.
Figure 23 is overexpression or blocks miR-93-5p G-exosomes to CIA model mice draining lymph nodes CD4+T is thin
The total STAT3 of born of the same parents expression quantity, wherein, A is Western blot testing results, and B is statistical analysis figure.
Embodiment
Technical scheme is further described with reference to specific embodiment, but the present invention is not limited to these
Embodiment.
Embodiment 1：G-MDSC and M-MDSC extraction and identification
(1) bearing mouse model is built：Using containing 10% calf serum (being purchased from Gibco companies of the U.S.), pH 7.3
The culture of DMEM nutrient solutions (is purchased from Gibco companies of the U.S.), 37 DEG C, 5%CO2Under the conditions of culture Mice Bearing Lewis lung adenocarcinoma cell system
(being purchased from Shanghai school of life and health sciences).The form of close observation cell, adherent situation, density, nutrient solution color in incubation
Change, when cell growth to density reaches 85% or so of culture dish bottom, (doctor's moral is purchased from biological with 0.25% pancreatin
Engineering Co., Ltd) digest and pass on.Take the logarithm growth period Lewis cell, by the right abdominal part hypodermic 3.0 of every mouse ×
106The method of individual cell, transplantable tumor is carried out to 6-8 week old male C57BL/6 mouse (being purchased from Jiangsu University's Experimental Animal Center)
Structure, close observation mouse animation and tumor size in model construction process.
(2) tumor-bearing mice splenocyte is separated：In Lewis cell infusions to the 30th day after mouse, eyeball takes blood and neck
Mouse is put to death in dislocation, after with 75% alcohol-pickled mouse 5min, and mouse is dissected in super-clean bench and obtains spleen；Use 0.22 μm of hole
Footpath screen filtration grinding after spleen, with PBS (phosphate buffer) rinse, collect suspension under the conditions of 4 DEG C, 500g from
Heart 5min, abandons supernatant；In the cell of precipitation add 5mL ACK erythrocyte cracked liquids (ACK Lysis Buffer, ACK) (on
Hai Ruigu bio tech ltd), after mixing, splitting erythrocyte 5min；Plus the RPMI-1640 trainings containing 10% calf serum
Nutrient solution (Gibco companies of the U.S.) centrifuges 5min under the conditions of 4 DEG C, 500g after mixing, abandons supernatant to 10mL；Cell be resuspended to
Leave and take and counted under a small amount of microcytoscope during 10mL.
(3) magnetic bead sorting G-MDSC：Every 108Individual splenocyte is resuspended with 350 μ L PBE, adds 50 μ L FcR Blocking
Reagent (German Mei Tian Ni companies), is placed in after mixing and is incubated 10min on ice；Every 108Individual splenocyte adds 40 μ L anti-
Ly-6G Biotin (German Mei Tian Ni companies), are placed in after mixing and are incubated 30min on ice, are mixed once per 10min；Add
10mL PBE, centrifuge 5min in 4 DEG C, 500g by suspension after mixing, abandon supernatant；Every 108Individual splenocyte adds 50 μ L anti-Ly-
6G-Biotin Beads (German Mei Tian Ni companies), are placed in after mixing and are incubated 30min on ice, are mixed once per 10min；Add
10mL PBE, centrifuge 5min under the conditions of 4 DEG C, 500g by suspension after mixing, abandon supernatant；500 μ L PBE are added, are mixed；Will
MACS sorting posts add splenocyte on the VarioMACS sorters (German Mei Tian Ni companies), plus after 3mL PBE rinses 1 time
Suspension, washs sorting post after last drop cell suspension flows out, then with 3mL PBE, is repeated 3 times；Withdrawn from from sorter point
Post is selected, sorting post is placed on 10mL centrifuge tubes, the PBE of 5mL precoolings is added, piston is promoted, the cell in splitter, weight is extruded
It is multiple 1 time, 10mL cell suspension is obtained, as G-MDSC leaves and takes and counted under a small amount of microscope.
(4) magnetic bead sorting M-MDSC：The cell suspension of sorting post outflow when collecting sorting G-MDSC, 4 DEG C, under the conditions of 500g
5min is centrifuged, supernatant is abandoned；Every 108Individual splenocyte adds 40 μ L anti-Gr-1Biotin (German Mei Tian Ni companies), after mixing
It is placed in and is incubated 30min on ice, is mixed once per 10min；Add 10mL PBE, 4 DEG C after mixing, 500g centrifugation 5min, abandon
Clearly；Every 108Individual splenocyte adds 50 μ L anti-Gr-1-Biotin Beads (German Mei Tian Ni companies), and ice is placed in after mixing
Upper incubation 30min, is mixed once per 10min；10mL PBE is added, 5min is centrifuged 4 DEG C after mixing, under the conditions of 500g, abandons
Clearly；500 μ L PBE is added, is mixed；By MACS sorting posts on sorter (German Mei Tian Ni companies), plus 3mL PBE moistens
Wash 1 time, then add above-mentioned cell suspension to be sorted, treat that last drip is use up, add 3mL PBE washing sorting posts, repeat
3 times；Withdraw from sorting post to be placed on 10mL centrifuge tubes, add 5mL PBE, promote piston, extrude cell in post, be repeated 1 times, obtain
To 10mL cell suspension, as M-MDSC leaves and takes and counted under a small amount of microscope.
(5) G-MDSC and M-MDSC Purity：The G-MDSC 1 × 10 for taking step (4) and step (5) to obtain respectively6
Individual and M-MDSC 1 × 106Individual 1mL PBS is resuspended, and 5min is centrifuged under the conditions of 4 DEG C, 500g in EP pipes, is abandoned after supernatant and is remained
Remaining about 100 μ L PBS；Add 0.5 μ L anti-Ly-6G antibody (U.S. eBioscience public affairs after mixing in G-MDSC EP pipes
Department) and 0.5 μ L anti-CD11b antibody (eBioscience companies of the U.S.), M-MDSC EP pipes plus 0.5 μ L anti-Ly-6C are anti-
The anti-CD11b antibody (eBioscience companies of the U.S.) of body (eBioscience companies of the U.S.) and 0.5 μ L；Incubated respectively at 4 DEG C
Educate 30min；5min is centrifuged 4 DEG C after 1mL PBS resuspensions, under the conditions of 500g, supernatant is abandoned, plus 200 μ L PBS is resuspended, fluidic cell
Instrument (U.S. company BD) is detected.As a result as shown in figure 1, the G-MDSC purity obtained in the present invention is 97.6%, M-MDSC purity
For 95.3%.
Embodiment 2：G-exosomes and M-exosomes extraction and determination of protein concentration
(1) G-MDSC and M-MDSC of sorting are resuspended in containing 10% calf serum (Gibco companies of the U.S., serum respectively
After 4 DEG C, 100000g centrifugation 16h processing, leave and take serum upper strata 2/3, remove excretion body in serum) RPMI-1640 trainings
In nutrient solution (Gibco companies of the U.S.), with every hole 1.5 × 106Individual cell, and per hole 1mL volume kind in 24 orifice plates；37
DEG C, 5%CO2Under conditions of culture 24 hours after, collect culture supernatant.
(2) G-MDSC of collection and M-MDSC culture supernatant is centrifuged under the conditions of 4 DEG C, 300g in 20min, collection
Clearly；4 DEG C of supernatant, 1000g are centrifuged into 30min again, supernatant is collected；4 DEG C of supernatant, 10000g are centrifuged into 30min again, supernatant is collected；
Supernatant is added in 100kDa super filter tubes (German Milipore companies), 4 DEG C, 1000g centrifugation 30min are collected in pipe and concentrated
Liquid.
(3) ExoQuick-TC is passed throughTMExosome kits (SBI companies of the U.S.) extract exosomes：By concentrate with
ExoQuick-TCTMExosome reagents by volume 5:1 mixing, it is slight to mix, more than 16h is stood in 4 DEG C of refrigerators；4 DEG C,
30min is centrifuged under the conditions of 1000g, supernatant discarding collects sediment, as G-exosomes or M-exosomes.Preparation is obtained
The exosomes obtained is dissolved with PBS, is dispensed into EP pipes, -80 DEG C of preservations.
(4) exosomes lysate and isometric lysate (RIPA:PMSF=250:1) after mixing, 1 is placed on ice
Hour, during which shake 1min on every 10min earthquake devices；4 DEG C, 12000g centrifugation 15min, collect supernatant.By BCA traces of albumin
Protein concentration in the method detection exosomes cracking supernatants of quantification kit (Beijing health is century Co., Ltd) specification,
To determine the content for extracting exosomes.As a result show, each G-MDSC or M-MDSC obtained in the present invention is in culture 24h
After can extract about 3 × 10-6μ g exosomes.
Embodiment 3：G-exosomes and M-exosomes identification
(1) transmission electron microscope observing G-exosomes form：20 μ L G-exosomes or M-exosomes suspensions are taken to be added dropwise
In on diameter 3mm load sample copper mesh, 2min is stored at room temperature；Liquid is gently blotted with filter paper, 2% phosphotungstic acid that pH 6.8 is added dropwise is molten
Liquid (Shanghai Jun Rui Science and Technology Ltd.s) redyes lmin on copper mesh；Filter paper is blotted to be dried under dye liquor, incandescent lamp, transmission electron microscope
Observed under (Dutch PHILIPS Co.), as a result as shown in Fig. 2 observing G-exosomes and M- under transmission electron microscope
Exosomes is circular or ellipse microcapsule structure, there is complete coating, and intracavitary is low electron density composition；Fig. 3 is respectively G-
Exosomes and M-exosomes particle size frequency disribution, as a result shows G-exosomes and M-exosomes particle diameter
It is mainly distributed between 20-120nm.
(2) Western blot detect protein molecular CD9, CD63 and line grain that G-exosomes and M-exosomes is included
The calnexin molecule Calnexin that body is included：Prepare 5% concentration glue and 12% separation gel, with RIPA protein lysates (on
Hai Bi skies Bioisystech Co., Ltd) cracking G-exosomes and M-exosomes, 250 are pressed by the G-exosomes after cracking
μ g Tot Prot loadings, after 100V constant pressure electrophoresis；After turning 90min through 350mA Constant Electric Currents again, 5% skim milk closing PVDF
Film lh；With the monoclonal antibody of anti-CD 63 (eBioscience companies of the U.S.) and the anti-Calnexin monoclonal antibodies (U.S.
EBioscience companies) 4 DEG C be incubated overnight；After taking-up film, 10min are washed with TBS/T (Shanghai is through chemical Science and Technology Ltd. of section)
× 3 times, with 37 DEG C of incubation 30min of secondary antibody (eBioscience companies of the U.S.) of horseradish peroxidase-labeled；Used after taking-up
TBS/T (Shanghai is through chemical Science and Technology Ltd. of section) washes film, 10min × 3 time；The gel imaging systems of ImageQuant LAS 4000
Unite (GE companies of the U.S.) exposure colour developing, as a result as shown in figure 4, G-exosomes, M-exosomes specificity overexpression CD9,
CD63 molecules, but G-exosomes and M-exosomes does not include the calnexin being positioned on endocytoplasmic reticulum film.In view of carrying
The G-exosomes and M-exosomes form that take, size, protein molecular expression of results, it was demonstrated that the present embodiment is successfully extracted
G-exosomes and M-exosomes.
Embodiment 4：Experiment proves G-exosomes and M-exosomes immune suppression function
(1) CIA model mices are built：
It is immune for the first time：With the Freund's complete adjuvant of 2mg/mL ox Type Ⅱ collagen (sigma companies of the U.S.) and 2mg/mL
(Sai Mofei companies of the U.S.) is mixed in equal volume, and Water-In-Oil state is ground on ice；To every DBA/1 mouse, (Shanghai Si Laike is real
Test animal Co., Ltd) root of the tail portion be subcutaneously injected 0.1mL above-mentioned mixed liquor；
Second immune：Not exclusively helped with the Freund of 2mg/mL ox Type Ⅱ collagen (sigma companies of the U.S.) and 2mg/mL
Agent (Sai Mofei companies of the U.S.) is mixed in equal volume, and Water-In-Oil state is ground on ice, and the 21st day after first time is immune is to every
0.1mL above-mentioned mixed liquor is subcutaneously injected in DBA/1 mouse (Shanghai Slac Experimental Animal Co., Ltd.) root of the tail portion.
(2) exosomes (N-exosomes) in the ripe neutrophil leucocyte source of mouse is prepared：
To 6-8 week old male C57BL/6 mouse (Jiangsu University's Experimental Animal Center) extract eyeball take blood, be added drop-wise to containing
EDTA-Na2Anticoagulant blood-collecting pipe (Suzhou Lingyan Crag Medical Devices Co., Ltd.) in, 1000rpm centrifugation eyeball blood 10min, suction out
Blood plasma, haemocyte is resuspended with 500 μ L PBS.By 1mL single karyolymph cell separation liquid (Tianjin Hao oceans biological products science and technology
Co., Ltd) it is added in the teat glass of 10mL capacity, blood cell suspension is slowly added drop-wise in teat glass along tube wall, used
Horizontal centrifuge, 4 DEG C, 2000rpm centrifugations 15min.Other cells in addition to red blood cell layer are slowly drawn from top to bottom with suction pipe
Layer is simultaneously discarded, and adds 5mL ACK (Shanghai Rui Gu bio tech ltd), and piping and druming is mixed, and is stored at room temperature 5min, adds 5mL
RPMI 1640 culture mediums (Gibico companies of the U.S.) containing 10% calf serum terminate cracking, 4 DEG C after mixing, 500g from
Heart 5min.Supernatant is abandoned, is washed with 10mLPBE 2 times, obtains neutrophil leucocyte.Then N- is extracted according to method in embodiment 2
exosomes。
(3) experiment packet：
PBS control group：The 18th day and 24 days after first time is immune is to every mouse tail vein injection PBS respectively
100μL；
N-exosomes groups：The 18th day and 24 days after first time is immune is to every μ of mouse tail vein injection 100 respectively
The N-exosomes of L PBS dissolvings, wherein 100 μ LPBS dissolve 100 μ gN-exosomes；
G-exosomes groups：The 18th day and 24 days after first time is immune is to every μ of mouse tail vein injection 100 respectively
The G-exosomes of L PBS dissolvings, wherein 100 μ LPBS dissolve 100 μ gG-exosomes；
M-exosomes groups：The 18th day and 24 days after first time is immune is to every μ L of mouse tail vein note 100 respectively
The M-exosomes of PBS dissolvings, wherein 100 μ LPBS dissolve 100 μ gM-exosomes.
(4) it is monitored in second of immune rear every 3 days toes situation to each group mouse, records the joint of every mouse
Scorching fraction (arthritic score, AS), AS represents the summation of all lesion joint fractions of every mouse, and each arthropathy
Fraction obtains 0 point, joint is red and pneumonedema obtains 1 point, and joint mild redness obtains 2 points, and joint moderate is red using 5 grades of point systems without red and swollen
Swell to obtain 3 points, joint severe is red and swollen simultaneously to obtain 4 points with dysfunction.Calculate the average arthritic index (mean of each group mouse
Arthritic index, MAI), MAI is by each group mouse AS summation divided by the number of each group mouse.As shown in figure 5, G-
The average arthritic index of exosomes treatment group mouse is reduced with respect to N-exosomes treatment groups, and PBS, N-
No significant difference between exosomes, M-exosomes treatment group；Fig. 6 represents the toes of G-exosomes treatment group mouse only
There is slight redness, joint motion is normal；But PBS, the toes of N-exosomes, M-exosomes treatment group mouse
Mostly red and swollen substantially joint motion is limited, and these results show that G-exosomes has the work for suppressing the morbidity of CIA model mices
With, and M-exosomes is without the effect for substantially suppressing the morbidity of CIA model mices.
(5) Th17 cell induction differentiated systems are set up：With containing the 2 μ g/mL anti-mouse CD3 monoclonal antibodies (U.S.
EBioscience companies) the μ L of coating buffer 200 be coated with 24 hole round bottom culture plates, 4 DEG C overnight；The coating buffer in each hole is suctioned out,
PBS is washed 1 time, and sorting kit by the T cells of German Mei Tian Ni companies sub-elects T cells, by every hole 1.5 ×
106T cells are inoculated in 24 orifice plates by the density of individual cell, are 1mL per hole cumulative volume；Each hole adds final concentration of 2 μ g/
ML anti-mouse CD28 monoclonal antibodies (eBioscience companies of the U.S.), final concentration of 5ng/mL mouse restructuring TGF-β
It is (eBioscience companies of the U.S.), final concentration of 30ng/mL IL-6 (eBioscience companies of the U.S.), final concentration of
30ng/mL IL-23 (eBioscience companies of the U.S.), final concentration of 5 μ g/mL anti-IL-4 (U.S. eBioscience
Company), final concentration of 5 μ g/mL anti-IFN- γ (eBioscience companies of the U.S.)；With containing the 15% hyclone (U.S.
Gibico companies, 16h processing is centrifuged by 100000g), pH 7.2-7.4 RPMI-1640 cell culture fluids (U.S. gibico
Company), 37 DEG C, 5%CO2Under the conditions of cultivate 72h.
(6) experiment packet：Control group：100 μ L PBS is added as control；Remaining is organized each hole and adds final concentration of 60 μ g/
ML N-exosomes, G-exosomes and M-exosomes, as shown in fig. 7, the ratio of G-exosomes treatment group Th17 cells
The relative N-exosomes treatment groups of example are reduced, and without bright between PBS, N-exosomes, M-exosomes treatment group
Significant difference is different.These results show that G-exosomes is played compared to M-exosomes in CIA model mices and mitigate mouse invasion
Effect, the external differentiation for suppressing Th17 cells, G-exosomes is with respect to showing more powerful immune suppression for M-exosomes
Function processed.
Embodiment 5：MiRNA sequencing result and analysis in G-exosomes and M-exosomes
(1) commission Guangzhou Rui Bo companies carry out extraction sequencing to the miRNA carried in G-exosomes and M-exosomes
Analysis, G-exosomes and M-exosomes as well known to those skilled in the art include many specific miRNA, and in expression quantity
It is upper that there is some difference.Analyzed according to sequencing result, with reference to the information of correlation miRNA on TargetScan websites, determine miRNA
Classification；As shown in figure 8, the absolute value of G-exosomes and the M-exosomes related miRNA contents differences included the first two ten
Position is miR-148a-3p (MIMAT0000516), miR-340-5p (MIMAT0004651), miR-16-5p
(MIMAT0000527)、miR-22-3p(MIMAT0000531)、miR-27a-3p(MIMAT0000537)、miR-92a-3p
(MIMAT0000539)、miR-140-3p(MIMAT0000152)、miR-27b-3p(MIMAT0000126)、miR-24-3p
(MIMAT0000219)、miR-26b-5p(MIMAT0000534)、miR-423-3p(MIMAT0000516)、Let-7f-5p
(MIMAT0000525)、miR-29a-3p(MIMAT0000535)、miR-7a-5p(MIMAT0000677)、miR-140-5p
(MIMAT0000151)、Let7g-5p(MIMAT0000121)、miR-26a-5p(MIMAT0000533)、miR-221-3p
(MIMAT0000669) numbering is in, miR-93-5p (MIMAT0000540), miR-148b-3p (MIMAT0000580), bracket
MiRNA to be screened is used as according to the corresponding number information of MIRBASE web analytics, and using them.
(2) present invention miRNA to be screened is further screened, due to miR-16-5p, miR-29a-3p, miR-93-5p,
The development of Let7g-5p and osteoarthritis and the propagation of Gegenbaur's cell, differentiation, migration are related；MiR-22-3p and miR-26b-5p
Participate in RA morbidity；MiR-29a-3p, miR-93-5p, miR-221-3p and T cell propagation, differentiation, immune response etc. are relevant；
MiR-16-5p, miR-29a-3p, Let-7f-5p, miR-92a-3p potential target Smad7, Smad6, FOS, PIK3R1,
STAT3, EMOS, PIK3R1 etc. and the Proliferation, Differentiation of T cell are closely related.Thus the present invention further filter out miR-16-5p,
miR-29a-3p、miR-93-5p、miR-22-3p、miR-26b-5p、Let-7f-5p、Let7g-5p、miR-221-3p、 miR-
92a-3p is used as candidate miRNA.
(3) the multiple change of detection candidate miRNA expression quantity in Th17 inducing cells after G-exosomes processing：Collect
Th17 inducing cell of the control group with adding G-exosomes processing in the step of embodiment 4 (6), with the 1mL Trizol (U.S.
Invitrogen companies) by 1 × 106Individual Th17 inducing cell cytolysis, mixing is fully blown and beaten with pipettor, adds precooling chloroform
(Solution on Chemical Reagents in Shanghai company) 200 μ L, shake 15~30s immediately, and normal temperature stands 10min；Under the conditions of 4 DEG C, 12000g centrifugations
15min；Liquid is layered in EP pipes, is drawn upper strata aqueous phase layer (containing RNA) into new clean EP pipes, is added 500 μ L's
Isopropanol, is gently mixed using pipettor, and normal temperature stands 5~10min；Under the conditions of 4 DEG C, 12000g centrifugation 15min, now
RNA is sunken to ttom of pipe, and be translucent shape；Supernatant discarding, plus 1mL 75% ethanol (Solution on Chemical Reagents in Shanghai company) are washed；In 4 DEG C of bars
Under part, 7800g centrifugations 5min；Room temperature is placed in super-clean bench to dry up to RNA and is translucent shape；Plus 5 μ L DEPC (the green clouds in Shanghai
Its Co., Ltd) water dissolving；The concentration that silent winged detector detects RNA is matched with the U.S..
(4) reverse transcription is carried out to the RNA extracted in step (3) according to the requirement of Japanese Takara Reverse Transcriptase kits, according to
MiRNA sequence design primer, then entrust Guangzhou Rui Bo companies synthesis miR-16-5p, miR-29a-3p, miR-93-5p,
MiR-22-3p, miR-26b-5p, Let-7f-5p, Let7g-5p, miR-221-3p, miR-92a-3p's is front and rear to primer, leads to
Cross qRT-PCR kits detection candidate miRNA Th17 inductions after G-exosomes processing of Shanghai BIO-RAD companies purchase
Expression quantity change in cell, as a result as shown in figure 9, Th17 is induced in noble cells after G-exosomes processing, candidate miRNA
Expression quantity increase it is maximum be miR-16-5p, miR-29a-3p and miR-93-5p.
(5) by EntranceTM-R transfection reagents (Beijing Engreen companies) by miR-16-5p, miR-29a-3p and
MiR-93-5p mimics and mimics- negative controls (Guangzhou Rui Bo bio tech ltd) are transfected into embodiment 4 respectively
The T cells that step (5) is sub-elected, are then induced to differentiate into Th17 cells according to method in embodiment 4, pass through qRT-PCR
Detection, as a result such as Figure 10, transfects miR-16-5p, miR-29a-3p and miR-93-5p expression quantity phase in mimics Th17 cells
It should improve；Such as Figure 11, the ratio for having transfected miR-93-5p mimics T cells to Th17 cell differentiations is significantly lower than
The T cells of mimics- negative controls have been transfected, and it is corresponding with miR-29a-3p to have transfected miR-16-5p correspondences mimics
Mimics T cells are then without significant change.These results indicate that can to suppress initial T thin by exogenous miR-93-5p
Born of the same parents are to the differentiation of Th17 cells, and this prompting G-exosomes may dividing by the miR-93-5p suppression Th17 cells of its carrying
Change.
Embodiment 6：Prepare the G-exosomes for being overexpressed or blocking miR-93-5p
(1) by miR-93-5p mimics, mimics- negative control (mimics-negitive control,
Mimics-NC) and inhibitors, inhibitors- negative control (inhibitors-negitive control,
Inhibitors-NC after freeze-dried powder (Guangzhou Rui Bo bio tech ltd) centrifugation), dissolved with sterile DEPC water, piping and druming
Final concentration of 20 μM of storing liquid is mixed into, packing is stored in -80 DEG C of refrigerators；
(2) inoculating cell, the inoculation 1 × 10 per hole in 24 orifice plates6Individual G-MDSC, with the RPMI of 10% calf serum
1640 nutrient solutions (Gibico companies of the U.S.) supply system for 450 μ L；
(3) mimics, mimics-NC and inhibitors, the preparation of inhibitors-NC dilutions：Take clean
EP is managed, and is separately added into mimics and mimics-NC 50nM, then be separately added into 1.25 μ L mimics or mimics-NC storage
Liquid, then complements to 25 μ L with the RPMI 1640 culture mediums (Gibico companies of the U.S.) of serum-free；Clean EP is taken to manage again, point
Not Jia Ru inhibitors and inhibitors-NC100nM, then be separately added into 2.5 μ L inhibitors, inhibitors-
NC storing liquid, then complements to 25 μ L with the RPMI 1640 culture mediums (Gibico companies of the U.S.) of serum-free；
(4) preparation of EntranceTM-R transfection reagents dilution：A clean EP pipe is taken, respective amount is added
EntranceTM-R transfection reagents (Beijing Engreen companies), according to mimics, mimics-NC, inhibitors,
Inhibitors-NC amount controls the consumption of transfection reagent, 1 μ L transfection reagents is added per 50nM, then with the 1640 of serum-free
Culture medium (Gibico companies of the U.S.) complements to 25 μ L, is stored at room temperature 5min；
(5) preparation of transfection composite：EntranceTM-R transfection reagents (Beijing Engreen companies) dilution is added
Into corresponding mimics, mimics-NC or inhibitors, inhibitors-NC dilutions, fully mix immediately after,
It is stored at room temperature 30min；
(6) 50 μ L transfection composite is added drop-wise in the inoculating cell in step (2), gently mixed；
(7) cell state is observed after transfecting 6 hours, if cell state is good, G-MDSC is collected by centrifugation；
(8) prepared and be overexpressed using the G-MDSC for having transfected mimics or inhibitors according to method in embodiment 2
Or block miR-93-5p G-exosomes.
Embodiment 7：Checking is overexpressed or blocked miR-93-5p G-exosomes
(1) each treatment group 1 × 10 in the step of embodiment 6 (6) is collected6Individual G-MDSC, according in the step of embodiment 5 (3)
Method extracts each treatment group G-MDSC total serum IgE, and detects its concentration；
(2) fluorescent quantitation inspection is carried out to each treatment group G-MDSC miR-93-5p content according to the method in embodiment 5
Survey, as a result miR-93-5p as shown in figure 12 mimics or inhibitors Successful transfections have arrived G-MDSC.
(3) each treatment group every 3 × 10 in the step of embodiment 6 (8) is collected7The G-exosomes of individual G-MDSC secretions, is used
1mL Trizol (Invitrogen companies of the U.S.) dissolvings.
(4) miR-93-5p included according to operating method in embodiment 5 to each group G-exosomes carries out quantitative analysis,
Relative to mimics-NC groups in the G-exosomes for the G-MDSC secretions for having transfected miR-93-5p mimics as shown in figure 13
MiR-93-5p expression quantity significantly improves (P<0.01) miR-93-5p inhibitors G-MDSC secretions, have been transfected
Expression quantity in G-exosomes relative to inhibitors-NC groups miR-93-5p reduces (P<0.05).These result explanations
The present embodiment is successfully extracted the overexpression prepared in the G-exosomes for being overexpressed or blocking miR-93-5p, the present embodiment
MiR-93-5p G-exosomes is compared with original G-exosomes, and miR-93-5p content has increased about 15
Times.
Embodiment 8：The miR-93-5p that G-exosomes is loaded can suppress the differentiation of Th17 cells in vitro
(1) G- that 60 μ g/mL are overexpressed or block miR-93-5p is added in Th17 cell induction differentiated systems
Exosomes, as shown in figure 14, be overexpressed miR-93-5p G-exosomes treatment groups (transfection mimics groups) relative to
The ratio of mimics-NC group Th17 cells significantly reduces (P<0.01) miR-93-5p G-exosomes processing, is blocked
Group (transfection inhibitors groups) increases (P relative to the ratio of inhibitors-NC group Th17 cells<0.05).These knots
Fruit shows that the miR-93-5p that G-exosomes is carried can suppress the differentiation of Th17 cells in vitro, and is overexpressed miR-93-
5p G-exosomes is stronger for the rejection ability of Th17 cell differentiations.MiR-93-5p G- is overexpressed in the present embodiment
Exosomes is stronger for the rejection ability of Th17 cell differentiations compared with original G-exosomes, Th17 cells
Ratio reduces one times.
(2) after the G-exosomes processing for being overexpressed or blocking miR-93-5p according to operating method detection in embodiment 5
The miR-93-5p of cell expression quantity in Th17 induction systems, as shown in figure 15, is overexpressed miR-93-5p G-exosomes
Treatment group can make inducing cell miR-93-5p expressions significantly improve (P compared with mimics-NC groups<0.01), hinder
Disconnected miR-93-5p G-exosomes treatment group miR-93-5p expressions then significantly drop relative to inhibitors-NC groups
Low (P<0.001).The miR-93-5p that these results illustrate G-exosomes and can carried is delivered to recipient cell, from
And play and suppress function, and the G-exosomes transmission miR-93-5p of the overexpression miR-93-5p of the invention prepared efficiency
It is higher.
Embodiment 9：G-exosomes load miR-93-5p can with the morbidity of significantly more efficient suppression CIA model mices,
Alleviate the order of severity of disease
(1) the CIA model mices built in embodiment 4 are utilized；
(2) tail vein injection overexpression or resistance were carried out to CIA model mices in 18th day and the 24th day after first time is immune
Disconnected miR-93-5p G-exosomes.
(3) experiment packet：
PBS group：Respectively at immune the 18th day and 24 days to every μ L of mouse tail vein injection PBS 100；
G-exosomes groups：It is molten in the 18th day and 24 days PBS to every μ L of mouse tail vein injection 100 being immunized respectively
The G-exosomes of solution；
Mimics-NC-exosomes groups：Respectively at immune the 18th day and 24 days to every μ of mouse tail vein injection 100
The G-exosomes that G-MDSC secretes after the transfection mimic-NC of L PBS dissolvings；
Mimics-exosomes groups：Respectively at immune the 18th day and 24 days to every μ L's of mouse tail vein injection 100
The G-exosomes that G-MDSC secretes after the transfection mimic of PBS dissolvings；
Inhibitors-NC-exosomes groups：Respectively at immune the 18th day and 24 days to every mouse tail vein injection
The G-exosomes that G-MDSC secretes after the transfection inhibitors-NC of 100 μ L PBS dissolvings；
Inhibitors-exosomes groups：Respectively at immune the 18th day and 24 days to every mouse tail vein injection 100
The G-exosomes that G-MDSC secretes after the transfection inhibitors of μ L PBS dissolvings.
In above-mentioned each group, the G-exosomes of 100 μ g different disposal groups is dissolved in 100 μ L PBS.
(4) as shown in figure 16, the average arthritic index of mimics-exosomes treatment groups mouse is with respect to mimics-
NC-exosomes treatment groups substantially reduce (P<0.05), the average arthritic of inhibitors-exosomes treatment groups mouse refers to
The relative inhibitors-NC-exosomes treatment groups of number significantly raise (P<0.01).As shown in figure 17, G-exosomes,
The toes of mimics-NC-exosomes, mimics-exosomes, inhibitors-NC-exosomes group mouse only have slightly
Redness, joint motion is normal；But PBS group, the toes of inhibitors-exosomes group mouse are mostly red and swollen bright
Aobvious, joint motion is limited.As shown in figure 18, G-exosomes, mimics-NC-exosomes, mimics-exosomes,
The toes joint bone structure of inhibitors-NC-exosomes group mouse is relatively complete, and joint space is relatively normal, rare
Inflammatory cells infiltrate；But PBS group, the toes joint bone structure of inhibitors-exosomes group mouse meet with
To heavy damage, joint space reduces, and has a large amount of inflammatory cells to infiltrate.These results show the miR- that G-exosomes is carried
93-5p can suppress the morbidity of CIA model mices, alleviate the order of severity of disease, and be overexpressed miR-93-5p G-
Exosomes has stronger protective effect to CIA model mices.
In the present embodiment be overexpressed miR-93-5p G-exosomes compared with original G-exosomes for
The protective effect of CIA mouse is stronger, relative to original G-exosomes, is overexpressed miR-93-5p G-exosomes processing
The average arthritic index of mouse, which have dropped, to be connect by about one time, and toes swelling degree substantially mitigates, and articulation structure is more complete.
(5) each group little Shu popliteal nest lymph nodes are separated, single cell suspension, flow cytometry each group as shown in figure 19 is prepared
In Th17 cell proportions in little Shu popliteal nest lymph node cells, mimics-exosomes treatment group mouse popliteal nest lymph node cells
Th17 cell proportions significantly reduce (P relative to mimics-NC-exosomes treatment groups<0.01), inhibitors-
Exosomes treatment groups significantly raise (P relative to inhibitors-NC-exosomes treatment group Th17 cell proportions<
0.001), PBS treatment groups and mimics-NC-exosomes treatment groups or inhibitors-NC-exosomes processing in addition
Group compares no significant difference (P>0.05).QRT-PCR detection each group mouse draining lymph nodes CD4 as shown in figure 20+T cell miR-
93-5p expression quantity, mimics-exosomes treatment group mouse draining lymph nodes CD4+T cell miR-93-5p expression quantity
(P is significantly raised relative to mimics-NC-exosomes treatment groups<0.01), inhibitors-exosomes treatment groups are relative
(P is substantially reduced in inhibitors-NC-exosomes treatment groups miR-93-5p expression quantity<0.01), PBS is buffered in addition
Liquid group no significant difference compared with mimics-NC-exosomes treatment groups or inhibitors-NC-exosomes treatment groups
(P>0.05).These results illustrate that G-exosomes can suppress CIA models small by transporting miR-93-5p to recipient cell
The ratio of mouse draining lymph node pathogenic T h17 cells, alleviates the order of severity of disease, and overexpression prepared by the present invention
MiR-93-5p G-exosomes has higher transport miR-93-5p efficiency, for the protective effect of CIA model mices
Also it is stronger.
In the present embodiment be overexpressed miR-93-5p G-exosomes compared with original G-exosomes for
The protective effect of CIA mouse is stronger, relative to original G-exosomes, is overexpressed miR-93-5p G-exosomes processing
The ratio of Th17 cells have dropped one times in little Shu popliteal nest lymph node cells, and the reduction of pathogenic T h17 cell proportions is for suppressing
The morbidity of CIA mouse is most important.
Embodiment 10：The miR-93-5p that G-exosomes is loaded can reduce target proteinses STAT3 expression
(1) according to Targetscan web analytics, as shown in figure 21,3 ' that miR-93-5p can be with target gene STAT3
Two regiospecificities of 248-254,495-501 of UTR region are combined.
(2) cell in Th17 induction systems after the G-exosomes processing for being overexpressed or blocking miR-93-5p is collected,
Wes tern blot detect each treatment group cell STAT3 expression, as shown in figure 22, are overexpressed miR-93-5p G-
Exoso mes treatment groups reduce (P relative to mimics-NC groups STAT3 expression<0.05) miR-93-5p, is blocked
G-exosomes treatment groups increase (P relative to inhibitors-NC groups STAT3 expression<0.05).These knots
Fruit shows that the miR-93-5p that G-exosomes is carried can suppress recipient cell STAT3 expression, so as to suppress Th17 cells
Differentiation, there is overexpressions miR-93-5p G-exosomes prepared by the present invention stronger suppression target Protein S TAT3 to express
Effect, so as to play stronger immune suppression function.Overexpression miR-93-5p prepared by present invention G-exosomes has
The effect of stronger suppression target Protein S TAT3 expression, compared with original G-exosomes, STAT3 expressions decline
One times, so as to play the function of stronger suppression Th17 cell differentiations.
(3) CIA model mice draining lymph nodes after the G-exosomes processing for being overexpressed or blocking miR-93-5p are collected
CD4+T cell, Western blot detect each treatment group cell STAT3 expression, as shown in figure 23, are overexpressed miR-
93-5p G-exosomes treatment groups significantly reduce (P relative to mimics-NC groups STAT3 expression<0.01), and
MiR-93-5p G-exosomes treatment groups are blocked to increase (P relative to inhibitorss-NC groups STAT3 expression
<0.05).These results illustrate that the miR-93-5p that G-exosomes is carried can reduce recipient cell in CIA model mices inside and outside
Born of the same parents' target proteinses STAT3 expression, and the G-exosomes of the overexpression miR-93-5p of the invention prepared has stronger suppression
The ability of target proteinses STAT3 expression processed, so as to play CIA model mices stronger protective effect.Mistake prepared by the present invention
Expression miR-93-5p G-exosomes has the ability of stronger suppression target Protein S TAT3 expression, relative to original G-
Exosomes is compared, and STAT3 expressions have dropped nearly 2 times, so as to play CIA model mices stronger protective effect.
Claims (10)
1. a kind of applications of miR-93-5p in treatment medicine for treating rheumatoid arthritis is prepared.
2. a kind of applications of overexpression miR-93-5p G-exosomes in treatment medicine for treating rheumatoid arthritis is prepared.
3. a kind of miR-93-5p is preparing the application in suppressing medicine for treating rheumatoid arthritis.
4. a kind of overexpression miR-93-5p G-exosomes is preparing the application in suppressing medicine for treating rheumatoid arthritis.
5. the application according to claim any one of 1-4, it is characterised in that the application is suppression Th17 cell differentiations.
6. the application according to claim any one of 1-4, it is characterised in that the application is the corresponding recipient cell targets of suppression
Mark Protein S TAT3 expression.
7. a kind of medicine for treating rheumatoid arthritis, it is characterised in that the medicine includes miR-93-5p.
8. medicine according to claim 7, it is characterised in that the medicine includes the G- for being overexpressed miR-93-5p
exosomes。
9. a kind of screening technique for the miR-93-5p for treating rheumatoid arthritis, it is characterised in that enter in accordance with the following steps
OK：
(1) separation and Extraction M-MDSC and G-MDSC first, then extracts the exosomes (G- identified secreted by both cells
Exosomes and M-exosomes)；
(2) miRNA extraction sequencing analysis are carried out to G-exosomes and M-exosomes, are filtered out in G-exosomes and M-
The first two larger ten miRNA of the absolute difference of content difference in exosomes；
(3) further screened according to miRNA structural and functional characteristic, filtering out 9 may act to RA
miRNA；
(4) according to the 9 miRNA sequences design primer filtered out, detect 9 miRNA at G-exosomes by qRT-PCR
The change of expression quantity in Th17 cells after reason, as a result filters out expression quantity increase in the Th17 cells after G-exosomes processing
3 miRNA of maximum；
(5) mimics and mimics- negative controls corresponding filter out 3 miRNA are transfected into T cells, and induced
Break up Th17 cells, detected by qRT-PCR, filter out miR-93-5p most obvious to the differentiation effect for suppressing Th17 cells.
10. screening technique according to claim 9, it is characterised in that 20 miRNA described in step (2) are
miR-148a-3p、miR-340-5p、miR-16-5p、miR-22-3p、miR-27a-3p、miR-92a-3p、miR-140-3p、
miR-27b-3p、miR-24-3p、miR-26b-5p、miR-423-3p、Let-7f-5p、miR-29a-3p、miR-7a-5p、
miR-140-5p、Let7g-5p、miR-26a-5p、miR-221-3p、miR-93-5p、miR-148b-3p；Described in step (3)
9 miRNA be miR-16-5p, miR-29a-3p, miR-93-5p, miR-22-3p, miR-26b-5p, Let-7f-5p,
Let7g-5p、miR-221-3p、miR-92a-3p；3 miRNA described in step (4) be miR-16-5p, miR-29a-3p,
miR-93-5p。
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