CN113337505B

CN113337505B - Application of artificially constructed antisense nucleotide fragment Ri111 in polarization of Th1 cells

Info

Publication number: CN113337505B
Application number: CN202110594248.5A
Authority: CN
Inventors: 张毅; 李雪; 张晗; 刘元林; 刘伟江; 王洋
Original assignee: Academy of Military Medical Sciences AMMS of PLA
Current assignee: Academy of Military Medical Sciences AMMS of PLA
Priority date: 2021-05-07
Filing date: 2021-05-28
Publication date: 2022-04-08
Anticipated expiration: 2041-05-28
Also published as: CN113337505A

Abstract

The invention belongs to the technical field of cell immune regulation and control, and particularly discloses application of an artificially constructed antisense nucleotide fragment Ri111 in polarization of Th1 cells. The invention provides an artificially constructed antisense nucleotide fragment Ri111, wherein the nucleotide sequence of Ri111 is shown in SEQ ID NO. 1 and/or SEQ ID NO. 2; the invention utilizes Ri111 to treat mice

The regulation and control function of the differentiation capacity of the CD4+ T lymphocyte to the Th1 cell, after the Ri111 is introduced, the differentiation capacity of the mouse juvenile CD4+ T lymphocyte to the Th1 cell is obviously reduced; thus, the biological agent for inhibiting the differentiation of the mouse type 1 helper T lymphocyte is provided, and can be applied to the regulation of mice

Description

Application of artificially constructed antisense nucleotide fragment Ri111 in polarization of Th1 cells

Technical Field

The invention belongs to the technical field of cell immune regulation and control, and particularly relates to application of an artificially constructed antisense nucleotide fragment Ri111 in polarization of Th1 cells.

Background

The immune system is an important physiological defense line of organisms, generates immune cells and related cytokines by activation, recognizes and eliminates foreign pathogens and self-generated damaged cells or tumor cells and the like, plays roles in immune monitoring, defense, regulation and the like, maintains the stability of the environment in the organisms, and ensures the health of the organisms.

Uncontrolled or dysfunctional immune responses result from an abnormally activated immune system, and aberrant immune attack is also a major cause of various autoimmune system diseases (e.g., crohn's disease, type 1 diabetes, graft-versus-host disease, etc.). The immune system is maintained to be stable, the abnormally activated immune response is inhibited, and the controllability of the immune response is ensured, so that the immune response has important significance for preventing and treating autoimmune system diseases caused by immune disorder.

The CD4+ T cells are in an important position in acquired immune response and immune regulation, the CD4+ T cells have high plasticity, can be activated into various auxiliary T cell (Th, helper T cell) subgroups such as Th1, Th2, Th17, Treg and Tfh by recognizing different TCR antigen signals and co-stimulation signals, play the diversity of immune regulation and control and maintain the immune steady state of an organism together. The abnormal activation of the type 1 helper T lymphocyte (Th1 cell) is closely related to the occurrence of various immune homeostasis disorders, and is also the root cause of various immune system diseases such as anaphylaxis, systemic lupus erythematosus, acute graft-versus-host disease, multiple sclerosis and the like. IL12 can induce T lymphocyte to differentiate to Th1 cell and activate IFN-gamma expression, is a classic proinflammatory factor, plays an important role in early inflammation generation, and is a key proinflammatory factor for starting inflammation reaction and driving Th1 cell differentiation. At present, the autoimmune system diseases clinically applied to the treatment of the immune disorder are often dependent on hormone drugs or immunosuppressive agents, but are often accompanied by the problems of drug resistance, infection caused by long-term immunosuppression and the like.

RNA interference (RNAi) technology is a technical method commonly applied to eukaryotic cells at present for gene expression regulation and control, gene expression is inhibited in a base complementary pairing mode with a target sequence, long double-stranded RNA (dsRNA) is cut into small interfering RNA (small interfering RNA) of 21nt per month by Dicer enzyme, the siRNA and Dicer/TRBP/Argonaute2 protein are assembled into a RISC complex, then a guide chain and the target sequence are subjected to base complementary mode to cut the target RNA under the action of nuclease activity Ago2, the target sequence is degraded, and the gene expression is reduced. Similar to RNAi effect of siRNA, microRNA (micro RNA, miRNA) is a non-coding small RNA molecule with gene expression regulation and control effect in organisms, hairpin-shaped precursor of the microRNA is digested into mature miRNA of 21-24nt by Dicer enzyme, mRNA degradation is started and gene expression is reduced in a complementary pairing mode with mRNA base of a target gene, and abnormal expression of miRNA can be related to various diseases. RNAi effect regulation and control play a vital role in the life processes of organism development, apoptosis necrosis, cell proliferation, immunity, nervous system mode formation and the like, and the expression and regulation of miRNA play a role, and the abnormal expression of miRNA can be related to various diseases.

Therefore, the intensive research on the functions and effects of RNAi will help to understand the mechanism of action of biological development and disease development, and there is a great need to develop a drug for preventing and treating autoimmune diseases based on RNAi-related drugs.

Disclosure of Invention

The present invention is intended to solve the problems as described above, and an object of the present invention is to provide an artificially constructed antisense nucleotide fragment Ri111 targeted to interfere with the IL12-IFN- γ pathway for preventing and treating autoimmune diseases by inhibiting the differentiation of T lymphocytes into Th1 cells.

In order to achieve the purpose, the specific technical scheme of the invention is as follows:

in the first aspect of the invention, an artificially constructed antisense nucleotide fragment Ri111 is provided, wherein target genes of the Ri111 are IFN-gamma, STAT4, Tbx21, IL12R beta 1 and/or IL12R beta 2; the nucleotide sequence of Ri111 is shown as SEQ ID NO. 1 and/or SEQ ID NO. 2.

The nucleotide sequences of Ri111 described in the present invention are all capable of specifically targeting the 3' UTR sequences of IFN- γ, STAT4, Tbx21, IL12R β 1 and IL12R β 2, as shown in FIG. 4.

In the second aspect of the invention, the application of the artificially constructed antisense nucleotide fragment Ri111 in preparing products for inhibiting the differentiation of T lymphocytes into Th1 cells is provided.

In some embodiments, the products include (but are not limited to) formulations and pharmaceuticals.

In a third aspect of the invention, a biological agent for inhibiting the differentiation of T lymphocytes into Th1 cells is provided, and the biological agent comprises the artificially constructed antisense nucleotide fragment Ri 111.

In some embodiments, the biological agent further comprises an inhibitor of one or more of the following genes or proteins: IFN-gamma, STAT4, Tbx21, IL12R beta 1 and/or IL12R beta 2.

In the present invention, the inhibitor is capable of inhibiting IFN- γ, STAT4, Tbx21, IL12R β 1 and/or IL12R β 2 activity or expression, which may include small molecule compounds, antibodies and nucleotide sequences such as shRNA (small hairpin RNA), small interfering RNA (sirna), dsRNA, microrna, antisense nucleic acids, or constructs capable of expressing or forming the shRNA, small interfering RNA, dsRNA, microrna, antisense nucleic acids, and the like.

In the fourth aspect of the invention, the application of the biological agent in preparing a pharmaceutical composition for preventing or treating immune system diseases is provided.

In some embodiments, the artificially constructed antisense nucleotide fragment Ri111 inhibits T lymphocyte differentiation to Th1 cell by inhibiting IL12-IFN- γ signaling pathway.

In some embodiments, the immune system disorder is a disorder associated with overactivation of the immune system due to polarization of cells including Th 1.

The diseases related to the excessive activation of the immune system caused by the polarization of the Th1 cells include (but are not limited to) allergic reaction, systemic lupus erythematosus, acute graft-versus-host disease, multiple sclerosis and the like.

In the fifth aspect of the invention, the application of the artificially constructed antisense nucleotide fragment Ri111 in the preparation of an IL 12-IFN-gamma signal pathway inhibitor is provided.

In some embodiments, the inhibitor reduces the expression of a gene of interest in the IL12-IFN- γ signaling pathway; preferably, the genes include IFN- γ, STAT4, Tbx21, IL12R β 1 and IL12R β 2.

In a sixth aspect of the invention, there is provided a method for modulating CD4+ naive T lymphocytes for non-therapeutic purposes

CD4+ T lymphocytes) to Th1 cells, comprising the steps of:

separating and extracting splenic lymphocyte of mouse, separating by magnetic bead

CD4+ T lymphocyte, and introducing the artificially constructed antisense nucleotide fragment Ri111 into the cells

CD4+ T lymphocyte is induced to differentiate to Th1 cell under the action of IL12/IL2/anti CD28/anti IL4/anti CD3 epsilon mixed factor.

In some embodiments, the concentrations of each factor IL12/IL2/anti CD28/anti IL4/anti CD3 epsilon are: IL 12: 10 ng/ml; IL 2: 5 ng/ml; anti CD 28: 0.5 mug/ml; anti IL 4: 1 mu g/ml; anti CD3 ε: 1. mu.g/ml.

In some embodiments, the Ri111 is introduced

The method for CD4+ T lymphocytes is as follows: coating Ri111 with liposome, inoculating to magnetic bead

CD4+ T lymphocytes;

the artificially constructed antisense nucleotide fragment Ri111,

wherein the sense strand is 5'-UCAGUGAAGAAACGGUUCAAUU-3' and the antisense strand is 5'-UUGAACCGUUUCUUCACUGAUU-3'.

In some embodiments, the transfection method of liposome-encapsulated Ri 111: control Ri111 Final concentration to 100nM, mice

The culture density of CD4+ T lymphocytes is 1 × 10⁶mL, culture time 72-96 hours, transfection medium is 1640 medium containing 10% FBS, no antibiotics.

In some embodiments, the mouse is a human mouse

The introduction of the CD4+ T lymphocyte into Ri111 can block a pathway of mixed factor induced Th1 cell differentiation, and the Ri111 can target and inhibit IL 12-IFN-gamma mediated Th1 cell polarization.

In some embodiments, the method for isolating and extracting splenic lymphocytes of mice comprises: aseptically placing spleen of six-week-old C57BL/6 mouse in 1640 culture medium, cutting spleen with ophthalmic scissors, transferring to 200 mesh sterile sieve, grinding with glass rod, dripping into 1640 culture medium while grinding until all grinded and dispersed spleen cells flow out through the sieve, collecting spleen cells, centrifuging for 10min at 450g, removing supernatant, resuspending, adding equal volume lymphocyte separation solution, centrifuging for 30min at 450g, sucking lymphocyte layer, adding PBS for resuspension, centrifuging for 10min at 400g, washing with PBS, adding erythrocyte lysate, standing for 5min, centrifuging for 10min at 1500rpm, collecting cell count, and using Miltenyi

CD4+ T cell isolation kit for isolation of splenic lymphocytes

CD4+ T lymphocytes, counted at 1X 10⁶The cells are planted in a 6-well plate, mixed factors are added to induce the cells to differentiate into Th1 cells, and the culture time is 72-96 h.

Based on the technical scheme, the invention has the following beneficial effects:

1. according to the invention, an antisense nucleotide fragment Ri111 of a targeted interference IL 12-IFN-gamma pathway is artificially constructed for the first time, the Ri111 nucleotide sequence has high specific targeted combination with 3' UTR sequences of IFN-gamma, STAT4, Tbx21, IL12R beta 1 and IL12R beta 2, mRNA transcription is inhibited, a sense chain or an antisense chain has certain matching degree, no potential interference on other gene expression is found through mouse whole genome range blast comparison, and expression of Th1 polarization related genes IFN-gamma, STAT4, Tbx21, IL12R beta 1 and IL12R beta 2 can be specifically targeted.

2. Inhibition of mice by Ri111 transfection

CD4+ T lymphocyte, and finds that Ri111 targets and inhibits IL 12-IFN-gamma pathway so as to inhibit

The differentiation of CD4+ T lymphocytes to Th1 lymphocytes obviously inhibits the polarization effect of Th1 cells, so that a biological preparation for inhibiting the differentiation of mouse type 1 helper T lymphocytes is provided, the biological preparation can be used for inhibiting the over-activation of an immune system caused by the polarization of Th1 cells, and an RNAi-related medicine is developed for preventing and treating autoimmune diseases.

3. The method for inhibiting polarization of Th1 cells of mice provided by the invention is to transfect the mice by using liposome carried small molecule compound Ri111

CD4+ T lymphocytes, the method is simple and convenient to operate, and Ri111 expression can be stably up-regulated; the polarization culture condition of Th1 cells and Ri111 transfection system are preferred, and the transfection and cell culture can be carried out quickly, conveniently and efficiently.

Drawings

FIG. 1 mouse

Differentiation results of CD4+ T lymphocytes to Th1 lymphocytes are that Th1 cells are induced to be cultured for 72h to 96h under the action of mixed factors of IL12/IL2/anti CD28/anti IL4/anti CD3 epsilon, IFN-gamma and CD4 double positive cell proportion is detected by flow cytometry, wherein Th1 polarization can be normally induced to reach 74.5% (left), and the polarization proportion of Th1 is reduced to 68.0% (right) after Ri111 transfection.

FIG. 2 is a q-PCR method used for detecting the expression levels of intracellular IFN-gamma, STAT1, STAT4, Tbx21, IL12R beta 1 and IL12R beta 2 genes of a Th1 differentiation group and an Ri111 treatment group after induction of differentiation culture of Th1 cells.

FIG. 3 Western Blot was used to detect the intracellular pi-STAT4 and STAT4 protein levels in the Th1 differentiation group and the Ri111 treatment group after induction of differentiation culture of Th1 cells.

FIG. 4 is a diagram of bioinformatics predicting the target sequences of Ri111 to IFN-gamma, STAT4, Tbx21, IL12R beta 1 and IL12R beta 2 of IL 12-IFN-gamma pathway components, wherein Ri111 targets the pathway components specifically.

FIG. 5 Dual luciferase reporter genes to detect expression of IFN-. gamma.STAT 4, Tbx21, IL 12R. beta.1 and IL 12R. beta.2.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

All materials, reagents and the like in the following examples are commercially available unless otherwise specified.

Materials mainly used in the invention

Fetal Bovine Serum (FBS) is a product of PAN company; flow cytometry-related anti-murine monoclonal antibodies: anti-mouse CD4-PE, Anti-mouse IFN-gamma-APC, Isotype Rat-IgG2a-APC, Isotype Rat-IgG2a-PE, all of which are products of eBioscience; PBS (8.0g NaCl, 2.9g Na)₂HPO₄·12H₂O、0.2g KCl、0.24g KH₂PO₄Dissolving the mixture in 1000mL of deionized water at constant volume, and adjusting the pH value to 7.4, wherein PBS is used as the formula in the specification); 1640 Medium (2.4g HEPES, 2.2g NaHCO)₃10g 1640 medium dissolved in 1000mL deionized water to adjust the pH to 7.4, not specifically indicated, the 1640 medium used herein is the same formulation); anti-IL-14, anti-CD28, anti-CD3 epsilon, IL-12 and IL-2 are products of TONBO corporation of America; NEAA products from Gibco, Inc. in the United states; the mouse spleen tissue lymphocyte separation solution is purchased from China tertiary ocean science and technology limited; miltenyi

The CD4+ T cell isolation kit was purchased from America and whirlwind, Germany; TRIzol is available from Sigma, USA; the RT-PCR kit is a product of Takara company; the Dual-Luciferase kit is purchased from Promega, USA; ri111, primers purchased from Shanghai Biotechnology engineering (Shanghai) Inc., and the specific sequences are shown in Table 1.

Example 1 mice

Isolation of CD4+ T lymphocytes

The method for separating and extracting the splenic lymphocytes of the mice comprises the following steps: aseptically placing spleen of six-week-old C57BL/6 mouse in 1640 culture medium, cutting spleen with ophthalmic scissors, transferring to 200 mesh sterile sieve, grinding with glass rod, dripping into 1640 culture medium while grinding until all grinded and dispersed spleen cells flow out through the sieve, collecting spleen cells, centrifuging for 10min at 450g, removing supernatant, resuspending, adding equal volume of lymphocyte separation solution, centrifuging for 30min at 450g, sucking lymphocyte layer, adding PBS, resuspending, centrifuging for 10min at 400g, washing with PBS, adding erythrocyte lysate, standingCentrifuging at 1500rpm for 10min for 5min, collecting cell count, and collecting cell count by using Miltenyi

CD4⁺T cell isolation kit for isolation of splenic lymphocytes

CD4⁺T lymphocytes.

Example 2 induced differentiation of mouse Th1 cells

CD4⁺After T cell sorting, the cells were counted at 1X 10⁶The cells were seeded in 6-well plates, placed in 1640 complete medium containing 10% FBS, and mixed factors were added to induce differentiation of Th1 cells, at the respective factor concentrations: IL 12: 10ng/mL, IL 2: 5ng/mL, anti CD 28: 0.5. mu.g/mL, anti IL 4: 1. mu.g/mL, anti CD3 ε: NEAA and 55 mu M beta-mercaptoethanol are added into the mixture at the concentration of 1 mu g/ml, and the culture time is 72-96 h. At 36h of cell culture, half-exchange was performed, 1mL of original medium was removed, and 2 fold concentration factor, NEAA, and 55 μ M β -mercaptoethanol were mixed with 1mL of 10% FBS in 1640 complete medium.

Example 3 design preparation and transfection of artificially constructed antisense nucleotide fragment Ri111

1. Preparation of artificially constructed antisense nucleotide fragment Ri111

In order to reduce RNAi cascade amplification reaction caused by mRNA degradation, the inventor sets RNAi interference targets in a 3 ' UTR region of a target gene, and searches repeated matching fragments aiming at the 3 ' UTR regions of IFN-gamma, STAT4, Tbx21, IL12R beta 1 and IL12R beta 2 to ensure that the fragments can be combined with the target 3 ' UTR in a highly specific targeting manner and simply inhibit mRNA transcription; meanwhile, a sense strand or an antisense strand of the fragment is designed to be matched with a target 3' UTR to a certain extent, potential interference on other gene expression is not found through blast comparison in a mouse whole genome range, expression of Th1 polarization related genes IFN-gamma, STAT4, Tbx21, IL12R beta 1 and IL12R beta 2 can be specifically targeted, reliability of the design is verified through various means such as in-vitro induction of Th1 cell polarization, identification of target gene expression level through qPCR detection and the like, and an antisense nucleotide fragment is finally screened and named as Ri 111.

Ri111 at 1.00OD260 was diluted with 125. mu.L DEPC water to a final concentration of 20. mu.M. Wherein the sequence of Ri111 is shown in Table 1, which is available from Shanghai Biotechnology engineering (Shanghai) Inc.

TABLE 1 RNA and primer sequences

2. Transfection

CD4+ T cells at 1X 10⁶Perml in 6-well plate, after adding 1640 complete medium and Th1 inducer. mu.L of Ri111 was diluted in 100. mu.L of Jet Prime Buffer, 6. mu.L of Jet Prime was added, mixed gently, and allowed to stand at room temperature for 15min to form a complex. The liposome complex was added to a six-well plate with 100. mu.L transfection system and 100nM final Ri111 concentration. And changing the liquid in the left and right half parts for 36 hours.

3. Flow cytometry analysis

Detection of

The flow antibodies used for differentiating the CD4+ T cells into the Th1 cells are Anti-mouse CD4-PE, Anti-mouse IFN-gamma-APC, Isotype Rat-IgG2a-APC and Isotype Rat-IgG2 a-PE. Collecting cells of a Th1 differentiation group and a Ri111 treatment group after induction of Th1 cell differentiation culture, adding a flow antibody Anti-mouse CD4-PE, incubating for 30min at4 ℃ in the dark, washing with PBS, centrifuging, fixing with 4% neutral formaldehyde for 10min, breaking membranes for 10min with 0.1% Triton, washing with 0.1% Tween-20, centrifuging, incubating for 30min at4 ℃ with the flow antibody Anti-mouse IFN-gamma-APC, washing, centrifuging, adding PBS, and detecting on a machine.

The results are shown in FIG. 1: the double positive cell proportion of IFN-gamma and CD4 is detected by flow cytometry, wherein the polarization of Th1 can be normally induced to 74.5% (left), and the polarization proportion of Th1 is reduced to 68.0% (right) after Ri111 transfection.

4. RNA extraction and detection

Collecting cells of a Th1 differentiation group and an Ri111 treatment group after inducing the differentiation culture of the Th1 cells, extracting total RNA by a TRIzol method, preparing 20 mu L of reverse transcription mixed reaction liquid by utilizing 1 mu g of total RNA, RNA free water, Oligo dT 1 mu L, Random 1 mu L, 5 xM-MLV Buffer 4 mu L, DTT 2 mu L, dNTP 1 mu L, M-MLV 1 mu L and RNase inhibitor 1 mu L, carrying out reverse transcription reaction on a PCR amplification instrument (70 ℃, 10 min; 42 ℃, 1 h; 70 ℃, 10min), and after the reaction is finished, placing the reverse transcription product cDNA on ice or storing at-20 ℃ for storage. And (3) detecting the gene expression quantity of IL12R beta 1, IL12R beta 2, Tbx21, STAT1, STAT4 and IFN-gamma by using q-PCR. Wherein the RT-PCR primer sequences are shown in Table 1. Reaction system: 1 uL of cDNA, 1 uL of PCR upstream and downstream primers (SEQ ID NO:5-16, 10 uM), 10 uL of PT-PCR Master Mix and 8 uL of RNA free water; reaction conditions are as follows: at 95 ℃ for 10min, at 95 ℃ for 15s and at 60 ℃ for 1min for 40 cycles; 95 ℃ for 15s,60 ℃ for 1min and 95 ℃ for 15 s. GAPDH was used as an internal reference and data were taken

The method is used for analysis.

The changes in intracellular gene expression of the Th1 differentiation group and the Ri111 treatment group after induction of differentiation culture of Th1 cells are shown in FIG. 2. After Ri111 treatment, the gene expression level of each component IL12R beta 1, IL12R beta 2, Tbx21, STAT4, STAT1 and IFN-gamma of the IL 12-IFN-gamma related signal pathway is obviously lower than that of the Th1 differentiation group.

5. Western protein detection

Western blot detects changes in levels of STAT4, phosphorylated STAT4(pi-STAT 4). Collecting cells of a Th1 differentiation group and a Ri111 treatment group after the differentiation culture of the Th1 cells, washing for 2 times by PBS, cracking the cells for 15min at4 ℃ by cell lysate, centrifuging for 10min at 13000rpm and 4 ℃, and detecting the protein content by a BCA protein concentration determination reagent. After 30 mu L of protein sample is added with loading buffer solution to be boiled and denatured, the protein sample is separated by 10% SDS-PAGE and transferred to a PVDF membrane, and the PVDF membrane is sealed for 1h at room temperature and contains 5% skimmed milk powder. The primary antibody is incubated at4 ℃ overnight, the membrane is washed 3 times by TBST, the secondary antibody is added for incubation for 1h at room temperature, the membrane is washed 3 times by TBST, and ECL chemiluminescence is developed.

As shown in fig. 3, compared to the group inducing Th1 differentiation, the levels of the critical transcription factor STAT4 in Th1 differentiation were significantly down-regulated after Ri111 transfection, and the pi-STAT4 protein level was significantly lower than that in the Th1 differentiation group.

6. Dual luciferase reporter System construction

Bioinformatics analysis shows that the 3 'UTR sequences of all components of IL 12-IFN-gamma pathway, IFN-gamma, STAT4, Tbx21, IL12R beta 1 and IL12R beta 2, which are necessary for Th1 polarization by Ri111, have specific binding targets (figure 4), so that clones of the 3' UTR sequences (psi-wt) of IFN-gamma, STAT4, Tbx21, IL12R beta 1 and IL12R beta 2 are respectively constructed by using psiCHECK2 dual-luciferase reporter vectors. The psi-wt vectors and Ri111 or NC (SEQ ID NO:3 and/or SEQ ID NO:4) are respectively transfected into Hela cells to carry out a dual-luciferase report system, and whether the Ri111 has certain targeting on an IL 12-IFN-gamma pathway is detected.

Hela cells were seeded in 96-well plates at a density of 5X 10 as high as 1d before transfection³Per well, 100. mu.L of DMEM complete medium containing 10% FBS was added, transfection was performed at a cell density of about 50%, 0.025. mu.g of vector and 1. mu.L of Ri111 were diluted in 20. mu.L of Jet Prime Buffer, 2. mu.L of Jet Prime was added and mixed gently, and the mixture was allowed to stand at room temperature for 15min to form a transfection complex. The liposome complex was added to 80 μ L DMEM complete medium with 100 μ L transfection system in 96 well plates to a final Ri111 (or NC) concentration of 100 nM. The supernatant was discarded after about 12 hours, and 100. mu.L of DMEM complete medium containing 10% FBS was added. 5% CO at 37 ℃₂Culturing in an incubator for 36 h.

FIG. 5 shows that the dual-Luciferase reporter system has certain targeting property for Ri111 to IFN-gamma, STAT4, Tbx21, IL12R beta 1 and IL12R beta 2 of each component of the pathway, and the Ri111 can be treated to target and down-regulate the expression of reporter gene Renilla-Luciferase.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

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Claims

1. An artificially constructed double-stranded RNA fragment Ri111, wherein the target genes of the Ri111 are IFN-gamma, STAT4, Tbx21, IL12R beta 1 and IL12R beta 2; the nucleotide sequence of the Ri111 sense strand is shown as SEQ ID NO. 1, and the nucleotide sequence of the Ri111 antisense strand is shown as SEQ ID NO. 2.

2. The use of the artificially constructed double-stranded RNA segment Ri111 of claim 1 in the preparation of a product for inhibiting the differentiation of T lymphocytes into Th1 cells.

3. A biological agent for inhibiting T lymphocyte differentiation to Th1 cell, comprising the artificially constructed double-stranded RNA fragment Ri111 of claim 1.

4. The biological agent of claim 3, further comprising an inhibitor of one or more of the following genes or proteins: IFN-gamma, STAT4, Tbx21, IL12R beta 1 and IL12R beta 2.

5. Use of the biological agent of claim 3 or 4 for the preparation of a pharmaceutical composition for the prevention or treatment of a disease associated with over-activation of immune cells due to polarization of Th1 cells.

6. The use of claim 5, wherein the artificially constructed double-stranded RNA segment Ri111 inhibits differentiation of T lymphocytes into Th1 cells by inhibiting the IL12-IFN- γ signaling pathway.

7. The use of the artificially constructed double-stranded RNA fragment Ri111 of claim 1 in the preparation of an inhibitor of IL12-IFN- γ signaling pathway; the inhibitor can reduce the expression of related genes in an IL 12-IFN-gamma signal pathway; the genes include IFN-gamma, STAT4, Tbx21, IL12R beta 1 and IL12R beta 2.

8. Inhibition of non-therapeutic purposes

A method of differentiating CD4+ T lymphocytes into Th1 cells comprising the steps of:

CD4+ T lymphocyte, the artificial double-stranded RNA segment Ri111 of claim 1 is introduced