CN104147615A - Application of small RNA miR-20b in preparation of anti-inflammatory medicine - Google Patents

Abstract

The invention relates to an application of small non-coded RNA genes miR-20b in preparation of an anti-inflammatory medicine. An inflammatory reaction is mainly caused by reactive Th17 cells which widely participate and play important pathogenic actions. The genes miR-20b have low specific expressions in the reactive Th17 cells; after miR-20b simulated bodies are respectively transfected into na.ve CD<4+>T cells or the negative control of the genes miR-20b is carried out, the miR-20b simulated bodies can be used for obviously reducing expressions of a gene level and a protein level of cytokine interleukin 17A, and the levels of proliferation and apoptosis of the Th17 cells are not changed by the genes miR-20b. Thus, the results show that the genes miR-20b can be used for effectively inhibiting differentiation of the Th17 cells and does not depend on the regulation and control of the numbers of cells.

Description

Little RNA miR-20b is in the application of preparing in anti-inflammatory drug

Technical field

The invention belongs to biological pharmacy technical field, relate to the application of non-coding MicroRNA gene miR-20b on medicine, particularly miR-20b relates to the application of autoimmune disease related drugs and anti-inflammatory drug in preparation.

Background technology

Immune system stable state is the basis that maintains the normal operation of body, it is by producing various immunocytes and secreting various cytokines and antibody resists that exotic is invaded and harassed or regulation and control related immune process, and the abnormal differentiation of immunocyte or the abnormal expression of cytokine can cause the generation of various inflammation.

Th17 cell can secrete cytokines IL-17A and IL-17F, and express in subbreed idiosyncratic transcription factor RORC(mice for ROR γ t).In the production process of Th17 cell, transcription growth factor-β (TGF-β) and inflammatory cytokine IL-6, IL-21, the effect of the performance such as IL-1 β and IL-23 important regulating and controlling.Th17 cell is most important for the removing of the outer pathogen of born of the same parents, comprising reading the pathogen such as Coccus and Klebsiella, but, under given conditions, Th17 cell and responsiveness cytokine IL-17, IL-21, IL-22, GM-CSF and CCL20 etc. also can cause the generation of some autoimmunity and diseases associated with inflammation, such as rheumatoid arthritis, systemic lupus erythematosus (sle), multiple sclerosis, psoriasis, enteritis, allergy and asthma etc.

Microrna (microRNAs, miRNAs) be a huge family, be approximately 21nt(nucleotide) non-coding RNAs of length, thus express and bring into play its regulating and controlling effect by transcriptional level or translation skill regulator gene, be extensively present in the eukaryote including plant and animal.MiRNAs can affect growth promoter, cellular processes (propagation, apoptosis, adhesion, migration etc.) etc., and human cancer generation and immune system are all brought into play to important regulating and controlling effect.

In recent years, the important function of miRNAs in immune system is day by day revealed, the immune growth that confirmed miRNAs wide participation, and various lymphocytic differentiation and antibody produce.In some diseases associated with inflammation, such as rheumatoid arthritis, multiple sclerosis and systemic lupus erythematosus (sle), miRNAs wide participation the initial sum progress of disease.And can effectively alleviate generation and the development of disease by intervening the unconventionality expression of relevant miRNAs.

We think and are tested and appraised and study expression and the effect of miRNAs in some inflammation, thereby can find that the miRNAs of a collection of key can effectively improve diagnosis and the treatment level of this inflammation.

Summary of the invention

The object of the invention is to provide a kind of microRNA gene miR-20b in the application of preparing in anti-inflammatory drug, and this miR-20b can effectively suppress the inflammatory reaction being caused by Th17 cell.

MiR-20b of the present invention is the low expression of specificity in Th17 cell, and gives na ve CD4 respectively ⁺after T cell transfecting miR-20b analogue body or miR-20b negative control, miR-20b analogue body can significantly reduce cytokine interleukin element 17A gene level and protein expression, and miR-20b does not change the level of Th17 cell proliferation and apoptosis, show that miR-20b can effectively suppress the differentiation of Th17 cell and not rely on the regulation and control to cell quantity.

Of the present invention being applied as little RNA miR-20b is in the application of preparing in anti-inflammatory drug, the medicine of this treatment antiinflammatory be treatment by the wide participation of reactive Th17 cell and bring into play the medicine of the inflammatory reaction of important pathogenic effects, relate to autoimmune disease and the relevant medicine of inflammation as special participation.

MiR-20b provided by the invention is made up of following nucleotide: 5 '-CAAAGUGCUCAUAGUGCAGGUAG-3 '.

The present invention breaks up first in vitro by the infantilism of selected by flow cytometry apoptosis (na ve) CD4 ⁺t cell, thereby obtain neutrality (neutral), Th1, Th2, Th17, induction type regulatory T (Treg) cell of vitro differentiation, and utilize the expression of real-time quantitative PCR (real-time PCR) technical Analysis miR-20b, found that respectively compared with neutral noble cells, miR-20b in Th17 cell than Th1, Th2 and the low expression of Treg cell-specific.

Secondly, airflow classification na ve CD4 in the present invention ⁺t cell, differentiation culture under Th17 condition, give respectively after cell transfecting miR-20b negative control (miR-20b NC) and analogue body (miR-20b mimics), miR-20b mimics can significantly reduce cytokine interleukin element 17A gene level and protein expression, shows that miR-20b can effectively suppress the differentiation of Th17 cell.

Meanwhile, airflow classification na ve CD4 in the present invention ⁺t cell, differentiation culture under Th17 condition, give respectively after cell transfecting miR-20b negative control (miR-20b NC) and analogue body (miR-20b mimics), detect find miR-20 mimics in vitro to the proliferation and apoptosis of Th17 cell be do not have influential.Illustrate that miR-20b has produced inhibition to the differentiation of Th17 cell in itself, and can not change the quantity of cell.

Therefore, miR-20b has great potential in preparation by special relating to aspect autoimmune disease and the relevant medicine of inflammation of participating in of Th17 cell.

For above and other objects of the present invention, feature and advantage can be become apparent, preferred embodiment cited below particularly, and coordinate accompanying drawing, be described in detail below.

Brief description of the drawings

Fig. 1: the detection of expression of miR-20b in external different differentiation direction CD4+ T cells;

Fig. 2: Flow cytometry miR-20b suppresses Th17 cellular expression IL-17A in vitro;

Fig. 3: real-time round pcr detects miR-20b and suppresses in vitro Th17 cellular expression IL-17A;

Fig. 4: elisa technique detects miR-20b and suppresses in vitro Th17 emiocytosis IL-17A;

Fig. 5: miR-20b is the statistical analysis on the impact of Th17 cell proliferation in vitro;

Fig. 6: Flow cytometry miR-20b does not affect the propagation of Th17 cell in vitro;

Fig. 7: Flow cytometry miR-20b does not affect the apoptosis of Th17 cell in vitro.

Detailed description of the invention

Embodiment 1:

Choose 6-8 week wild-type mice, prepare spleen and lymph node single cell suspension, use APC-anti CD4, Pe-Cy5-anti CD44, PE-anti CD62L antibody, 4 DEG C of lucifuge padding 15min; RPMI 1640 culture medium, wash cell twice for 4 DEG C; Cell concentration is adjusted into 5 × 10 by RPMI 1640 culture medium ⁷cell/ml, airflow classification CD4 ⁺cD44 ^lowcD62L ^hingbe na ve CD4 ⁺t cell; Obtain na ve CD4 ⁺before T cell, with 10 μ g/ml CD3 antibody wrapper sheets, 37 DEG C, 2h is for subsequent use; Press cytokine concentrations irritation cell below, make it to neutral (cultivating 48 hours), Th1 (cultivating 48 hours), Th2 (cultivating 48 hours), Th17 (cultivating 72 hours) and the differentiation of Treg (cultivating 72 hours) direction.

Neutral: anti-CD28 1μg/ml; rmIL-2 2ng/ml; anti-IL-4 10μg/ml; anti-IFN-γ 10μg/ml。

Th1: anti-CD28 1μg/ml; rmIL-2 2ng/ml; rmIL-12 5 ng/mL; anti-IL-4 10μg/ml。

Th2: anti-CD28 1μg/ml; rmIL-2 2ng/ml; IL-4 20 ng/mL; anti-IFN-γ 10μg/ml。

Th17: anti-CD28 1μg/ml; TGF-β 2ng/mL; IL-6 10 ng/mL; anti-IL-4 10μg/ml; anti-IFN-γ 10μg/ml。

Treg: anti-CD28 1μg/ml; rmIL-2 2ng/ml; TGF-β 2 ng/mL。

Cultured cell extracts cell total rna by Trizol method above, getting respectively the each 10ng of total RNA is template, then utilizing TaqMan MicroRNA Reverse Transcription Kit(Life Technologies) reverse transcription is cDNA, reaction system is as follows: dNTPs (100 mM) 0.15 μ l; MultiScribe ^tMreverse transcriptase 1 μ l; 10 × RT buffer, 1.5 μ l; (20 U/ μ are 0.19 μ l l) for RNase inhibitor; RT primer 2.0 μ l; Total RNA 5 μ l; DEPC H ₂o mends to cumulative volume 15 μ l.Reaction condition: 16 DEG C of 30 min, 42 DEG C of 30 min, 85 DEG C of 5 min makes enzyme deactivation, 4 DEG C of preservations.

Again taking cDNA as template, utilize Premix Ex Taq (Probe qPCR) (Takara) test kit carry out Real-time PCR.Reaction system is as follows: 2 × PCR mix, 10 μ l; CDNA 2 μ l; Probe 0.4 μ l; DEPC H ₂o 7.6 μ l.Reaction condition: 95 DEG C, 2 min; 95 DEG C of 15 sec, 60 DEG C of 30 sec, 40 circulations, the contrast taking U6 as internal reference.Adopt 2 ^{-△ △ CT}relative quantification method is carried out deal with data.

Real-time PCR result shows, with respect to neutral noble cells, miR-20b is high expressed in Th1, Th2, Treg noble cells, is adjusted to 1.54,3.07 and 1.53 times on respectively, the low expression of specificity in Th17 noble cells, lowers and reaches 0.53 times (Fig. 1).

Embodiment 2:

Obtain na ve CD4 according to method sorting in embodiment 1 ⁺t cell.MiR-20b mimics and the nc of FAM labelling are synthesized by Shanghai JiMa pharmacy Technology Co., Ltd, and sequence is as follows respectively:

miR-20b mimics：

5’-CAAAGUGCUCAUAGUGCAGGUAG-3’

5’-ACCUGCACUAUGAGCACUUUGUU-3

miR-20b nc：

5’-UUCUCCGAACGUGUCACGUTT-3’

5’-ACGUGACACGUUCGGAGAATT-3’

Gained na ve CD4 ⁺t cell makes it to Th17 cell direction differentiation according to method in embodiment 1, and by the miR-20b mimics of FAM labelling and nc, transfection is to cell above respectively simultaneously, and transfection reagent is the Lipofectamine of invitrogen company of the U.S. ^tM2000 reagent, continue to cultivate after 72h, and 4-6h before harvesting adds PMA, Inomycin and Golgi Plug to answer cytokine to reach detection abundance with irritation cell Secretion in fresh culture medium.Draw cell in 5ml BD streaming pipe, add 1 ice-cold × PBS, 4 DEG C, the centrifugal 7min of 1400rpm, abandons supernatant, 1ml 2% formaldehyde re-suspended cell, and room temperature lucifuge is fixed 30min.Fixing end, with after twice, ice-cold PBS cleaning cell, resuspended with 2ml 0.5% Saponin, room temperature lucifuge punching 30min.Thereafter 4 DEG C, the centrifugal 7min of 1400rpm, abandons supernatant, adds FITC-anti-mouse IFN-γ, PE-anti-mouse IL-17A, APC-anti-mouse CD4, and 4 DEG C, lucifuge dyeing 30min.Dyeing finishes, and fills 0.5% Saponin, and 4 DEG C, the centrifugal 7min of 1400rpm, abandons supernatant.Resuspended with 4ml 1 × PBS, 4 DEG C, after the centrifugal 7min of 1400rpm, detect CD4 with U.S. company BD FACS Calibur Flow Cytometer instrument ⁺iL-17A ⁺cell proportion.

Result shows that miR-20b mimics can significantly suppress CD4 ⁺iL-17A ⁺the ratio (Fig. 2) of cell.

Embodiment 3:

Obtain na ve CD4 according to method in embodiment 2 completely ⁺t cell, make simultaneously its to Th17 cell direction differentiation and by miR-20b inhibitor (inhibitor), mimics and nc respectively transfection to above in cell, cultivate after 72h, harvesting, obtain total RNA of cell according to method in embodiment 1, and analyze IL-17A and IL-17F expression according to Real-time PCR method in embodiment 1.

MiR-20b inhibitor sequence is as follows:

5’-CUACCUGCACUAUGAGCACUUUG-3’

Result shows that miR-20b mimics can significantly suppress the expression (Fig. 3) of IL-17A and IL-17F.

Embodiment 4:

Obtain na ve CD4 according to method in embodiment 3 ⁺t cell, make simultaneously its to Th17 cell direction differentiation and by miR-20binhibitor, mimics and nc respectively transfection to above in cell, cultivate after 72h, results supernatant, uses the Mouse IL-17 ELISA kits test kit of BioLegend company of the U.S. according to the secreting, expressing of ELISA methods analyst IL-17A.

Result shows that miR-20b mimics can significantly suppress the secreting, expressing (Fig. 4) of IL-17A.

Embodiment 5:

Detect the impact of miR-20b on Th17 cell proliferation with the APC BrdU Flow Kit of U.S. company BD.Obtain na ve CD4 according to method in embodiment 2 ⁺t cell, make simultaneously its to Th17 cell direction differentiation and by FAM fluorescent labeling miR-20b mimics and nc respectively transfection to above in cell.Be that at cultured cell, initial or harvesting added BrdU to hatch to 10 μ M in cells and supernatant before 1 hour according to final concentration.After transfection 72 hours, the PE-anti-mouse CD4 antibody 15min that dyes on ice for cell.Then cell is fixed and punches with Cytofix/Cytoperm Buffer and Cytoperm Permeabilization Buffer Plus, and with the BrdU antibody staining of APC labelling, finally detect cell proliferation with U.S. company BD FACS Calibur Flow Cytometer instrument.

Result shows that miR-20b does not affect (Fig. 5 and Fig. 6) to Th17 cell proliferation.

Embodiment 6:

Obtain na ve CD4 according to method in embodiment 2 ⁺t cell, make simultaneously its to Th17 cell direction differentiation and by FAM fluorescent labeling miR-20b mimics and nc respectively transfection to above in cell.After cell transfecting 72 hours, use the Annexin V-APC Cell Apoptosis Analysis Kit of Tianjin Sungene Biotechnology Co., Ltd. to detect miR-20b to the apoptotic impact of Th17.

Result shows that miR-20b does not affect (Fig. 7) to Th17 apoptosis.

Although the present invention discloses as above with preferred embodiment; but it is not in order to limit the present invention; any person of ordinary skill in the field; not departing from spirit and scope of the invention; when doing a little change and improvement, therefore the present invention's protection domain is when being as the criterion depending on the claim person of defining.

SEQUENCE LISTING

<110> Nankai University

The little RNA miR-20b of <120> is in the application of preparing in anti-inflammatory drug

<130>

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 23

<212> RNA

<213> synthetic

<400> 1

caaagugcuc auagugcagg uag 23

Claims (3)

1. little RNA miR-20b is in the application of preparing in anti-inflammatory drug.

2. application according to claim 1, is characterized in that described anti-inflammatory drug is that treatment is by the wide participation of reactive Th17 cell and bring into play the medicine of the inflammatory reaction of important pathogenic effects.

3. application according to claim 1 and 2, is characterized in that described miR-20b is made up of following nucleotide: 5 '-CAAAGUGCUCAUAGUGCAGGUAG-3 '.