WO2017063196A1 - c-Rel特异性siRNA及其用于防治自身免疫性银屑病的应用 - Google Patents

c-Rel特异性siRNA及其用于防治自身免疫性银屑病的应用 Download PDF

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WO2017063196A1
WO2017063196A1 PCT/CN2015/092124 CN2015092124W WO2017063196A1 WO 2017063196 A1 WO2017063196 A1 WO 2017063196A1 CN 2015092124 W CN2015092124 W CN 2015092124W WO 2017063196 A1 WO2017063196 A1 WO 2017063196A1
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rel
psoriasis
sirel
seq
small interfering
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阮庆国
陈有海
范婷婷
马轶凡
蔡林涛
王绍文
万晓春
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深圳先进技术研究院
宾夕法尼亚大学
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Priority to CN201580001128.2A priority patent/CN107250360B/zh
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Priority to US15/680,449 priority patent/US10081810B2/en

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Definitions

  • the present invention relates to the treatment of autoimmune psoriasis with the NF- ⁇ B family member c-Rel as a target, in particular to c-Rel specific siRNA and its use for preventing (preventing and/or treating) autoimmune psoriasis
  • the application of the present invention can effectively prevent autoimmune psoriasis while hardly affecting the function of the normal function of the immune system.
  • Psoriasis is one of the three most widely affected autoimmune diseases, with a global incidence of about 3%, with one-third of patients reaching moderate to severe levels. Psoriasis is characterized by thickening of the skin and extensive damage that can cause itching, scaling and pain, and severely affect the patient's quality of life, mental health and social relationships. In addition, people with severe illnesses are prone to complications such as arthritis, heart disease and diabetes and are at risk of death.
  • autoimmune psoriasis is mainly mediated by the IL-23/IL-17A inflammation axis (van der Fits, L. et al. Imiquimod-induced psoriasis-like skin inflammation in mice is Mediated via the IL-23/IL-17 axis.
  • pathogenic factors eg genetic, environmental, infection, physical damage, etc.
  • congenital immune cells such as keratinocytes, natural killer cells, etc.
  • pro-inflammatory factors such as TNF- ⁇ , IL-1 ⁇ and IL-6
  • dendritic cells Wait for innate immune cells.
  • Activated dendritic cell migration In immune organs such as lymph nodes, it presents antigen and secretes pro-inflammatory factors such as IL-23, which promotes differentiation of naive CD4+ T cells into Th17.
  • the differentiated autoreactive Th17 migrates from the capillaries of the skin and infiltrates into the site of inflammation in the skin. After re-stimulation of self-antigen, it proliferates and secretes various inflammatory factors such as IL-17A.
  • Factors such as IL-17A activate keratinocytes, stimulate their proliferation, and form symptoms of psoriasis.
  • activated keratinocytes secrete antibacterial peptides (such as LL-37 antibacterial peptide and ⁇ -defensin), pro-inflammatory factors (TNF- ⁇ , IL-1 ⁇ and IL-6), and chemokines (CXCL8).
  • IL-17A plays an important bridging role in the inflammatory circuit of innate immunity and adaptive immunity of psoriasis.
  • IL-23/IL-17A axis of inflammation is only an important part of the pathological process of psoriasis.
  • ⁇ T cells and macrophages can also secrete IL-17A to promote the development of psoriasis (Cai, Y., Fleming, C. & Yan, J.
  • Th22 cells can secrete IL-22 and aggravate the development of psoriasis, and the remaining pathological links remain to be discovered (Benham, H. Et al. Th17 and Th22 cells in psoriatic arthritis and psoriasis. Arthritis research & therapy 15, R136, doi: 10.1186/ar4317 (2013)).
  • psoriasis is mainly classified according to the severity of the disease. For mild to moderate psoriasis, it is often treated with topical drugs, such as glucocorticoids, vitamin D3 analogues, retinoic acid, eucalyptus, and tar. Ineffective or topical psoriasis, especially erythrodermic psoriasis, generalized pustular psoriasis and arthritic psoriasis, often using systemic therapy such as muscle or intravenous drip Small molecule immunosuppressive agents such as methotrexate and cyclosporine.
  • topical drugs such as glucocorticoids, vitamin D3 analogues, retinoic acid, eucalyptus, and tar.
  • topical drugs such as glucocorticoids, vitamin D3 analogues, retinoic acid, eucalyptus, and tar.
  • topical drugs such as glucocortic
  • the mammalian NF- ⁇ B family consists of five members: c-Rel, RelA (p65), RelB, NF- ⁇ B1 (p50/p105), and NF- ⁇ B2 (p52/p100).
  • the amino terminus of these proteins has a highly conserved region consisting of approximately 300 amino acid residues, termed the Rel homology region (RHD), which functions as dimerization, interaction with I ⁇ B, nuclear localization, and Binding to DNA, etc.; in contrast, the carboxy terminus of these proteins is not conserved, wherein the carboxy terminus of c-Rel, RelA and RelB has a transactivation domain.
  • RHD Rel homology region
  • the newly synthesized NF- ⁇ B is usually stored in the cytoplasm in a state in which it is bound to I ⁇ B in the form of a homologous or heterodimer.
  • NF- ⁇ B NF- ⁇ B kinase
  • NGF nerve growth factor
  • T cell and B cell antigen receptors T cell and B cell antigen receptors
  • Toll-like receptors activate NF- ⁇ B upon binding to related ligands.
  • IKK I ⁇ B kinase
  • I ⁇ B protein degradation or processing eg, p100.
  • NF- ⁇ B Once NF- ⁇ B is activated, It enters the nucleus as a free dimer and binds to 9-10 base pairs on the target gene promoter to regulate gene expression.
  • Activated NF- ⁇ B can be down-regulated by various mechanisms (such as feedback pathway), combined with newly synthesized I ⁇ B, and stored in the cytoplasm at rest.
  • NF- ⁇ B plays a key role in the development of autoimmune diseases, in addition to its role in the maintenance of physiological functions and pathological conditions.
  • type 1 diabetes In mice and humans, the development of type 1 diabetes is often accompanied by a high degree of activation of NF- ⁇ B in dendritic cells and monocytes, as well as infiltration of these cells in tissues. Inhibition of NF- ⁇ B activation can effectively inhibit the development of type 1 diabetes in NOD mice, CD1 mice and C57BL/6 mice (Campbell, IK, Gerondakis, S., O'Donnell, K. & Wicks, IPDistinct). Roles for the NF-kappaB1 (p50) and c-Rel transcription factors in inflammatory arthritis.
  • Liou et al. used c-Rel and p50 knockout mice as experimental subjects to study type I diabetes.
  • the incidence of arthritis and encephalomyelitis BAHilliard, N. Mason, L. Xu, J. Sun, SELamhamedi-Cherradi, HC Liou, C. Hunter, YHChen, Critical roles of c-Rel in autoimmune inflammation and Helper T cell differentiation.
  • c-Rel knockout mice develop normally and have a structurally normal immune system that does not produce spontaneous infectious diseases. When attacked by high doses of pathogens, they can normally clear pathogens or have a slightly reduced ability to clear. At the same time, c-Rel knockout mice are resistant to autoimmune diseases such as encephalomyelitis, type I diabetes, and arthritis. These phenomena suggest that c-Rel is closely related to the development of autoimmune diseases.
  • c-Rel APC, T cells, and B cells in autoimmune mice are often associated with excessive activation of c-Rel, whereas c-Rel knockout mice are resistant to autoimmune diseases, suggesting that c-Rel is an autoimmune disease. It plays a key regulatory role in the pathological process. The study found that c-Rel is involved in the regulation of the expression of various inflammatory factors and the differentiation and development of Th17 in antigen-presenting cells. Mainly manifested in:
  • BMDC bone marrow-derived dendritic cells
  • IL-23p19 in c-Rel knockout BMDC was significantly reduced compared to wild-type BMDC (Carmody, RJ, Ruan, Q., Liou, HC). &Chen, YHEssential roles of c-Rel in TLR-induced IL-23 p19 gene expression in dendritic cells. Journal of immunology (Baltimore, Md.: 1950) 178, 186-191 (2007)).
  • c-Rel can interact with IL-23p19 during Toll-like receptor (TLR) signaling.
  • TLR Toll-like receptor
  • the two promoter sites on the gene promoter specifically bind to each other and form an enhancer with other transcription factors to directly regulate the expression of IL-23p19.
  • the p19 gene is not activated when only other transcription factors (the p19 promoter also contains a site where AP-1, C/EBP, and IRF may bind) bind to the p19 gene promoter, indicating The expression of the IL-23p19 gene is completely dependent on c-Rel.
  • Nicola Mason et al. found that c-Rel directly regulates IL-12p40 in the form of c-Rel/p50 heterodimers in macrophages and dendritic cells during inflammatory stimuli such as LPS. Expression (N. Mason, J. Aliberti, JCCaamano, HC Liou, CAHunter, Cutting edge: identification of c-Rel-dependent and-independent pathways of IL-12 production during infectious and inflammatory stimuli. Journal of immunology (Baltimore) Md.: 1950) 168, 2590-2594 (2002)).
  • c-Rel is also involved in the regulation of the expression of pro-inflammatory factors such as IL-6 in APC (JRTumang, CYHsia, W.Tian, JFBromberg, HCLiou, IL-6 rescues the hyporesponsiveness of c-Rel deficient B cells independent of Bcl-xL, Mcl-1, and Bcl-2. Cellular immunology 217, 47-57 (2002)).
  • Th1 cells are the main pathogenic cells in the pathogenesis of autoimmune diseases.
  • Th17 cells play a more critical pathogenic role in a variety of autoimmune diseases, including multiple sclerosis, psoriasis, and rheumatoid arthritis.
  • Th17 cells mainly produce cytokines such as IL-17A, IL-17F and IL-22, and play a role in inflammatory diseases and against extracellular bacterial infections.
  • Th17 lineage-specific factors include ROR ⁇ t, ROR ⁇ , and STAT3.
  • c-Rel affects Th17 differentiation and development through two aspects.
  • the inventors of the present study found that c-Rel indirectly regulates Th17 cell development by directly regulating IL-23 production in antigen-presenting cells (Carmody, RJ, Ruan, Q., Liou, HC & Chen, YHEssential roles of c-Rel in TLR-induced IL-23 p19 gene expression in dendritic cells. Journal of immunology (Baltimore, Md.: 1950) 178, 186-191 (2007)); on the other hand, the inventor of the present invention found that c-Rel can also Th17 differentiation can be directly regulated in T cells (Ruan, The Th. i.
  • the inventors of the present invention stimulated IL-17A messenger RNA (mRNA) and IL-17A protein when CD4+ T cells isolated from c-Rel knockout mice were stimulated with anti-CD3 mAb and anti-CD28 mAb.
  • the expression was significantly reduced compared with the wild type; when CD4+ T cells isolated from c-Rel knockout mice were cultured and stimulated in vitro, the number of Th17 was found to be as high as 70% (Ruan, Q. et al.
  • the Th17 immune response is Controlled by the Rel-RORgamma-RORgamma T transcriptional axis.
  • Th17 differentiation condition TCR, costimulatory molecules on CD4+ T cells, and cytokine receptors such as IL-1, IL-23, IL-6, etc.
  • Th17 differentiation condition an activation signal is transmitted intracellularly, and a plurality of transcription factors are released into the nucleus.
  • the free c-Rel/p65 dimer binds to different promoters of the Ror ⁇ gene and interacts with other transcription factors released into the nucleus (such as NFAT and Stat) to form a Ror ⁇ -specific enhancer, which initiates transcription of the Ror ⁇ gene. , thereby driving Th17 cell differentiation.
  • the c-Rel/p65 transcription factor is the only transcription factor found to bind to the Ror ⁇ gene promoter and activate transcription. Other transcription factors that bind to the Ror ⁇ gene promoter remain to be discovered.
  • drugs targeting the entire NF- ⁇ B family can be used to treat autoimmune psoriasis, such as protease inhibitors (such as FDA-approved PS-341), NF- ⁇ B decoy oligonucleotides, NBD polypeptides and glucocorticoids, etc.
  • protease inhibitors such as FDA-approved PS-341
  • NF- ⁇ B decoy oligonucleotides such as FDA-approved PS-341
  • NBD polypeptides and glucocorticoids etc.
  • NF-kappaB activation by a peptide that blocks the interaction of NEMO with the IkappaB kinase complex. Science 289, 1550-1554 (2000); De Stefano , D. Oligonucleotides decoy to NF-kappaB: becoming a reality? Discovery medicine 12, 97-105 (2011)).
  • NF- ⁇ B family proteins are ubiquitously expressed in many cells of the body and are associated with the maintenance of normal physiological functions of the body, such as congenital and adaptive immune regulation, inflammatory response, anti-apoptosis, cell proliferation, etc. during infection. Lead to the side effects of these drugs Poor, can only be used in the short term to control acute allergic reactions. Therefore, drugs that target the entire NF- ⁇ B family cannot be used to treat chronic inflammatory diseases such as autoimmune psoriasis.
  • One object of the present invention is to inhibit c-Rel biosynthesis by using c-Rel-specific small interfering ribonucleic acid (siRNA) to prevent autoimmunity by inhibiting the IL-23/IL-17A inflammation axis in psoriasis mice. Sexual psoriasis.
  • siRNA small interfering ribonucleic acid
  • Another object of the present invention is to provide a c-Rel-specific small interfering ribonucleic acid which inhibits c-Rel biosynthesis in an individual.
  • the individual is a mammal (e.g., a mouse) or a human.
  • Another object of the present invention is to provide a method for controlling autoimmune psoriasis by delivering a c-Rel-specific small interfering ribonucleic acid with a nanomaterial.
  • siRel The c-Rel-specific small interfering ribonucleic acid is referred to as siRel in the present invention.
  • the present invention proposes to use the c-Rel-specific small interfering ribonucleic acid only as a target of the NF- ⁇ B family member c-Rel. (siRel) to inhibit the biosynthesis of c-Rel to prevent (prevent and/or treat) autoimmune psoriasis by intervening in the IL-23/IL-17A inflammation axis in individuals with psoriasis.
  • the present invention employs a c-Rel-specific small interfering ribonucleic acid (siRel) to inhibit the biosynthesis of the NF- ⁇ B family member c-Rel, thereby preventing autoimmunity by interfering with the IL-23/IL-17A axis of inflammation.
  • siRel small interfering ribonucleic acid
  • the invention provides a c-Rel specific small interfering ribonucleic acid (siRel).
  • the c-Rel-specific small interfering ribonucleic acid (siRel) can be used to inhibit the biosynthesis of c-Rel.
  • the c-Rel-specific small interfering ribonucleic acid (siRel) sequence is:
  • Antisense strand 5'AGACGGGUAUGUCCGGUUGdTdT 3' (SEQ ID No. 2)
  • siRel small interfering ribonucleic acid sequence described in the human body is:
  • Antisense strand 5'AGAAGGGUAUGUUCGGUUGdTdT 3' (SEQ ID No. 4)
  • the present invention also provides a method of preventing autoimmune psoriasis by delivering c-Rel-specific small interfering ribonucleic acid (siRel) with a nanomaterial.
  • the nano-micelle eg, PEG-PLL-PLLeu triblock copolymer nanomicelle
  • the nano-micelle can be used to transport siRel to dendritic cells in vivo to achieve Preventive and / or therapeutic purposes.
  • the present invention provides a small interfering ribonucleic acid specific for c-Rel in the preparation of a pharmaceutical composition for inhibiting the axis of IL-23/IL-17A inflammation in a psoriasis individual by inhibiting biosynthesis of c-Rel Applications.
  • the c-Rel-specific small interfering ribonucleic acid has a sequence as shown in SEQ ID Nos. 1 to 2 or SEQ ID Nos. 3 to 4.
  • the pharmaceutical composition may further comprise a nanomaterial (e.g., PEG-PLL-PLLeu triblock copolymer micelle) for carrying a small interfering ribonucleic acid specific for c-Rel and transporting it to the cytoplasm.
  • a nanomaterial e.g., PEG-PLL-PLLeu triblock copolymer micelle
  • the present invention provides a pharmaceutical composition for preventing (preventing and/or treating) autoimmune psoriasis, the composition comprising an effective amount of said c-Rel-specific small interfering ribonucleic acid .
  • the pharmaceutical composition further comprises a PEG-PLL-PLLeu triblock copolymer nanomicelle.
  • the pharmaceutical compositions can include one or more pharmaceutically acceptable excipients.
  • the pharmaceutical composition of the present invention may be an external preparation for preventing autoimmune psoriasis of an individual (may be in the form of a nanomicelle suspension for applying the skin), wherein c- The Rel-specific small interfering ribonucleic acid concentration is 10 to 100 nM, and the nanomicelle concentration is 10 to 20 ⁇ g/ml.
  • the individual can be a mammal (e.g., a mouse) or a human.
  • the present invention also provides a method for preventing (preventing and/or treating) autoimmune psoriasis, which comprises administering to an individual an effective amount of c-Rel-specific small interfering ribonucleic acid to inhibit c-Rel organisms.
  • the process of synthesis to inhibit the axis of inflammation of IL-23/IL-17A in individuals with psoriasis.
  • the invention provides a method of controlling autoimmune psoriasis using nanomicelles to deliver c-Rel specific siRNA.
  • the present invention employs PEG-PLL-PLLeu triblock copolymer micelles to carry siRNA and transport it into the cytoplasm.
  • the inventors of the present invention have shown that the triblock copolymer micelles are biodegradable and have high transfection efficiency (Deng, J. et al. Self-assembled cationic micelles based on PEG-PLL-PLLeu hybrid polypeptides as highly effective gene vectors.
  • mice In vivo in mice, for light silver shavings Treatment of the disease Each mouse was intraperitoneally given 500 pmol of siRel, corresponding to 100 ⁇ g of nanomicelles, once every 2 days; for the treatment of moderate psoriasis, each mouse was given 500 pmol of siRel, Should 100 ⁇ g micelles, administration frequency is once a day.
  • the dose of siRel administered intravenously was 0.3 mg/kg
  • the dose of nanomicelle was 4.5 mg/kg
  • the frequency of administration was once every three weeks
  • the dose of siRel administered intradermally was 8 mg
  • the dose of nanomicelle was 8 mg.
  • the administration volume is 2 ml, the frequency of administration is once a week; the dose of siRel is 16 mg subcutaneously, the dose of nano-micelle is 16 mg, the dosage volume is 4 ml, and the frequency of administration is once a week; the concentration of siRel on the skin is 10 nM.
  • the corresponding nanomicelle dose is 10 ⁇ g/ml, the frequency of administration is 1 to 3 times per day, and 0.5 ml of siRel nanomicelle suspension is rubbed every 500 square centimeters each time.
  • the effectiveness of the c-Rel-specific siRNA of the present invention i.e., the siRel
  • the siRel is first confirmed in the NIH3T3, BMDC and RAW264.7 cell lines. .
  • the present inventors have found that siRel can significantly reduce the mRNA and protein expression levels of c-Rel and IL-23p19.
  • siRel in a specific in vivo experiment of the present invention, it was found that siRel can significantly control the development of psoriasis in mice with mild psoriasis, and significantly reduce the production of IL-17A in mouse spleen cells, and It inhibited the expression of IL-1 ⁇ , IL-6, TNF- ⁇ , IL-23p19 and IL-17A in mouse skin lesions and achieved therapeutic effects.
  • the H&E staining results of the skin slices of the two groups of mice showed that the skin epidermis of the mice in the treatment group was thinner than that of the control group, and the infiltrating inflammatory cells in the skin of the treatment group were significantly reduced.
  • the siRel drug was evaluated on moderate psoriasis mice, and the siRel drug was also found to have a certain therapeutic effect.
  • nano-micelle can effectively transport c-Rel-specific siRNA to dendritic cells, which can significantly reduce the expression of IL-23/IL-17 inflammation-related inflammatory factors, and achieve prevention and treatment.
  • Figure 1 Schematic representation of the preparation of siRNA/PEG-PLL-PLLeu micellar suspension.
  • Figure 3 In vitro silencing effect of siRel/PEG-PLL-PLLeu nanomicelles.
  • Each image, AE, NIH3T3, RAW264.7, and BMDC were treated with siS or siRel for 24 hours after LPS stimulation or no stimulation for 4 hours, real-time quantitative PCR was used to detect the relative expression of c-Rel or IL23p19;
  • F BMDC was treated in siNC or siRel After 48 hours, LPS stimulated or did not stimulate for 4 hours, and the protein level of IL23 in the supernatant was detected by ELISA;
  • GH the siRel can reduce the expression of c-Rel at the protein level, wherein the picture H is a quantitative map of the western blot.
  • FIG. 4 Construction of an IMQ-induced mouse psoriasis model.
  • A IMQ induced the psoriasis phenotype on day 6 of psoriasis in BALB/c mice (Day 0-3: 65 mg/day; Day 4-5: 80 mg/day; Day 6: 100 mg/day)
  • FIG. 5 siRel treatment can effectively control the development of mild psoriasis.
  • FIG. 6 siRel treatment can effectively improve the symptoms of moderate psoriasis lesions.
  • A psoriasis treatment course
  • B control group (siNC) and treatment group (siRel) mouse skin lesions H&E staining.
  • Figure 9 Q-PCR detection of inflammatory factor expression levels in mouse skin at day 6 after treatment in moderate psoriasis mice.
  • NIH3T3 and RAW264.7 cell lines were purchased from ATCC. Further, bone marrow-derived dendritic cells (BMDC) were used as the research object to confirm the silencing effect of the siRel.
  • BMDC bone marrow-derived dendritic cells
  • c-Rel-specific small interfering ribonucleic acid (siRel) is used to inhibit the synthesis of c-Rel.
  • siRel small interfering ribonucleic acid
  • Antisense strand 5'AGACGGGUAUGUCCGGUUGdTdT 3' (SEQ ID No. 2)
  • siRel sequence was synthesized by Shanghai Jima Co., Ltd., and siNC was a universal negative control.
  • the FAM-labeled siRel was also synthesized by Shanghai Jima Company.
  • PEG-PLL-PLLeu is a polyethylene glycol-polylysine-polyleucine triblock copolymer synthesized by collaborators Ma Yifan and Cai Lintao using NCA ring-opening polymerization (Deng, J. et al .Self-assembled cationic micelles based on PEG-PLL-PLLeu hybrid polypeptides as highly effective gene vectors. Biomacromolecules 13, 3795-3804, doi: 10.1021/bm3012538 (2012)).
  • the amphiphilic triblock copolymer can self-assemble to form micelles having a particle size of about 150 nm.
  • the polyleucine segment aggregates to form a hydrophobic core of the micelle
  • the polyethylene glycol forms a shell of the micelle
  • the poly polylysine segment in the middle is protonated by containing a large amount of primary amino groups, thereby making the micelle
  • the zeta potential is approximately 43 mV.
  • an appropriate amount of PEG-PLL-PLLeu lyophilized powder was dissolved in deionized water to prepare a 1 mg/ml copolymer micelle suspension, which was sterilized by filtration at 0.22 ⁇ m.
  • the OPTI-MEM diluted siRel was mixed with an equal volume of the copolymer suspension according to different N/P ratios (molar ratio of the copolymer micelle primary amino group to the siRel phosphate group), and allowed to stand at room temperature for 30 minutes to obtain a copolymer of SiRel.
  • Compound micelle (siRel/PEG-PLL-PLLeu) suspension The micelles were dropped into a cell culture plate, and the mixture was gently patted to transfect the cells.
  • the different N/P ratio prepared siRel/PEG-PLL-PLLeu prepared as described above was applied to a 2% agarose gel, electrophoresed at 120 mV for 20 minutes using TAE buffer, and then observed with an ultraviolet gel imager. Gel retardation results.
  • the particle size and zeta potential of the nanomicelles were measured at room temperature using a dynamic light scattering instrument (Nano-ZSZEN3600), and the results were analyzed using Malvem Dispersion Technology Software 4.2 software.
  • A shows the ability of a gel retardation assay to detect micellar binding to siRNA.
  • the copolymer micelles can fully load the siRNA when the N/P ratio is greater than or equal to 2.
  • the results show that when the copolymer micelles and siRNA are mixed at a ratio of N/P ratio of 15, the particle size of the micelle after the loading of siRel is detected. From about 148 nm to about 180 nm, the zeta potential is reduced from about 42 mV to about 38 mV.
  • C and D in Figure 2 show the results of assays for the efficiency of transfection of BMDC or BM20 with different N/P ratio mixed siRNA-loaded micelle mixtures.
  • Flow cytometry results showed that there was no significant difference in efficiency between transfected BMDC and BM20 when the N/P ratio was 10, 15, or 20.
  • the N/P ratio is 15, the nanomicelle-transfected BMDC has an efficiency of about 45%, and the transfection BM20 has an efficiency of about 60%.
  • NIH3T3 and RAW264.7 cell lines were purchased from ATCC and cultured according to standard procedures.
  • BMDC is induced by primary separation.
  • the BALB/c mice were sacrificed by cervical dislocation, and the femur and tibia were taken under aseptic conditions.
  • Bone marrow cells were washed out with a 1 ml syringe and pipetted with 2% FBS in PBS. The cell suspension was collected, resuspended in erythrocyte lysate after centrifugation, and lysed for 1 min and then washed three times with medium.
  • the cells were suspended in X-Vivo medium containing 20 ng/ml recombinant mouse GM-CSF and 10 ng/ml recombinant mouse IL-4, and prepared into 2 ⁇ 10 6 cells/ml cell suspension, and seeded in 24-well culture plates, each well. 1ml.
  • BMDC bone marrow-derived dendritic cells
  • BMDCs were harvested by LPS for 6 hours, and the nuclear proteins were extracted. After protein concentration was detected by BCA method, Western blotting was performed to detect the amount of c-Rel protein in the nucleus.
  • Figure 3 shows the in vitro silencing effect when siRel was delivered using PEG-PLL-PLLeu nanomicelle.
  • the picture A-C showed that siRel could effectively reduce the level of c-Rel mRNA in NIH 3T3 (P ⁇ 0.01); and the siRel could effectively reduce the mRNA levels of c-Rel and IL23p19 in RAW 264.7 cell line (P ⁇ 0.05).
  • the picture D-E showed that siRel significantly decreased the mRNA expression of c-Rel and IL23p19 in BMDC at mRNA level (P ⁇ 0.05).
  • Panel F shows that the siRel also reduced IL23 expression at the protein level (P ⁇ 0.01).
  • Figure G-H shows that this siRel can reduce the expression of c-Rel at the protein level, where picture H is a quantified map of the Western blot.
  • the treatment of psoriasis is divided into early, middle and late treatment, and the severity of psoriasis is mild, moderate and severe.
  • the effects of siRel treatment were evaluated in mice with mild to moderate psoriasis.
  • the IMQ-induced psoriasis model has more similarities with the pathological changes of human psoriasis and is an ideal model for studying psoriasis.
  • IMQ is an agonist of Toll like receptor (TLR) 7/8, which is applied to the skin of mice to activate dendritic cells and macrophages via the TLR pathway and TLR-independent pathway to activate keratin
  • TLR Toll like receptor
  • the cells promote the secretion of pro-inflammatory factors such as IFN- ⁇ and TNF- ⁇ , and recruit inflammatory cells to the skin to produce psoriasis-like lesions and histological changes.
  • pro-inflammatory factors such as IFN- ⁇ and TNF- ⁇
  • the dose of IMQ administered is increased, which is caused by the difference in the manufacturer of the IMQ drug and the different feeding environment of the mouse.
  • the dose required for psoriasis is different.
  • mice were anesthetized by intraperitoneal injection of 10% chloral hydrate (300 mg/kg), and their back hairs were removed to form a bare area of about 2 cm X 3 cm.
  • IMQ was applied daily (day 0-3: 65 mg/day; day 4-day 5: 80 mg/day; day 6-day 7: 100 mg/day;), and the skin of the mice was observed daily and the scale formation was scored with a scale of 0. , no; 1, mild; 2, moderate; 3, severe; 4, extremely severe.
  • the scores of the mice in each group were averaged and the trend line was drawn. The changes of skin lesions in each group were observed.
  • siRNA drug treatment was given when the mouse silver stain score was 0.5.
  • Psoriasis mice were randomly divided into control group (siNC) and treatment group (siRel), and were administered intraperitoneally to the control group and experimental group siNC/PEG-PLL-PLLeu and siRel/PEG on days 1, 2, 4, and 6 respectively.
  • mice On the 8th day, the skin of the mice was taken, and the tissues of the same parts of the mice in each group were cut according to the nine-grid method, and fixed in 4% paraformaldehyde. After OCT was embedded, frozen sections were performed at a thickness of 7 ⁇ m. Then H&E staining was performed to observe changes in epidermal thickness of each group of mice.
  • siRNA drug treatment was given when the mouse silver stain score reached 2.
  • Psoriasis mice were randomly divided into control group (siNC) and treatment group (siRel), and the control group and experimental group siNC/PEG-PLL-PLLeu and siRel/ were administered intraperitoneally on days 3, 4, 5, 6, and 7, respectively.
  • mice On the 8th day, the skin of the mice was taken, and the tissue at the same site of the mice in each group was cut according to the nine-grid method, and fixed in 4% paraformaldehyde. After OCT was embedded, frozen sections were performed at a thickness of 7 ⁇ m. Then H&E staining was performed to observe changes in epidermal thickness of each group of mice.
  • Figure 4 shows the construction of a psoriasis mouse model using IMQ.
  • picture A shows the sputum phenotype of the control group and the IMQ-induced group on the 7th day, and it was observed that the mice in the induction group had obvious scales on the back.
  • Panel B shows the scoring of scaly symptoms during the induction of psoriasis using IMQ. It can be observed that the scaly score of the induction group has been increasing, while the control group has almost no scaly symptoms and has the 4th, 5th, and 6th days. Significant difference (P ⁇ 0.05).
  • Panel C shows the H&E staining results of the skin sections of the control group and the IMQ-induced group on the 6th day. It can be observed that the epidermal thickness of the IMQ-induced group was significantly thicker than that of the control group.
  • Figure 5 shows in vivo treatment of mild psoriasis with PEG-PLL-PLLeu nanomicelle delivery of siRel.
  • picture A shows the treatment plan of psoriasis in BALB/c mice.
  • Panel B shows the symptoms of psoriasis in mice at day 8 and it can be observed that the silver-like lesions in the treated group are clearly controlled.
  • Panel C shows the score of mice with silver-like symptoms. It can be observed that the silver stain score of the mice in the treatment group has been maintained at a low level, while the silver-stain score of the control mice is on the rise, at the 7th and 8th. There was a significant difference in days (P ⁇ 0.05).
  • Panel D shows the H&E staining results of the skin sections of the treated group and the control group. It can be observed that the skin epidermis of the treated group is thinner than that of the control group, and the infiltrating inflammatory cells in the treated group are significantly reduced. These data indicate that siRel can effectively control the development of mild autoimmune psoriasis in mice.
  • Figure 6 shows the in vivo treatment of moderate psoriasis effects when siRel is delivered using PEG-PLL-PLLeu nanomicelles.
  • picture A shows the treatment plan of psoriasis in BALB/c mice.
  • Picture B shows the score of mice with silver-like symptoms. It can be observed that the psoriasis score of the mice in the treatment group has been decreasing, while the psoriasis score of the control mice has remained at about 2 points, and there is a significant difference on the 8th day. (P ⁇ 0.001).
  • Panel C shows the H&E staining results of skin sections of the treated and control mice. It can be observed that the skin epidermis of the treated group is thinner than the control group.
  • mice were sacrificed on the 2nd, 6th, and 8th day, and the spleens of the psoriasis mice in the treatment group and the control group were taken and ground into cell suspensions. 10 6 were inoculated in 48-well plates, 500 ul culture system. Supernatants were harvested 48 h after stimulation with or without 0.5 ug/ml anti-CD3 antibody, anti-CD28 antibody, and the level of IL-17A was detected by enzyme-linked immunosorbent assay.
  • the enzyme-linked immunosorbent assay kit was purchased from Ebioscience, and the specific procedure was followed according to the instructions.
  • Figure 7 shows the level of IL-17A production by mouse spleen cells on days 2, 6, and 8 during the treatment of mild psoriasis mice by ELISA.
  • the results showed that on the 2nd, 6th and 8th day, the secretion of IL17A from the spleen cells of the treatment group was significantly lower than that of the control group (P ⁇ 0.05). This indicates that siRel can effectively reduce the level of IL-17A produced by spleen cells in psoriasis mice.
  • RNA in the skin was cut according to the nine-grid method, immediately placed in liquid nitrogen, then ground into a powder, and added with Trizol reagent for extraction. Total RNA in the skin. Reverse transcription was performed using oligo dT as a primer and M-MLV Reverse Transcriptase. The specific experimental procedures were carried out according to the promega product specifications.
  • Figure 8 shows the expression levels of inflammatory factors in mouse skin at day 2 and 6 during the treatment of mild psoriasis mice by Q-PCR.
  • the results showed that the expression level of TNF- ⁇ in the skin of the treated group and the control group was slightly decreased on the second day (P ⁇ 0.05), but no difference was detected on the 8th day.
  • the levels of IL23p19 and IL6 in the skin of the treated mice were significantly decreased on the 2nd day and the 6th day (P ⁇ 0.05).
  • the present invention detects that the expression levels of IL17A and IL-1 ⁇ in the skin of the treated group and the control group are not different on the second day, but the treatment group is significantly reduced on the sixth day (P ⁇ 0.01).
  • Figure 9 shows Q-PCR detection of expression levels of inflammatory factors in mouse skin at 6 days after treatment in moderate psoriasis mice.
  • the results showed that the expression levels of IL-1 ⁇ , IL-6, IL-23p19, IL-17A and IFN- ⁇ in the skin of mice treated with moderate psoriasis were significantly higher than those in the control group. Decrease (P ⁇ 0.05). However, there was no significant difference in the expression levels of IL-12p35 and TNF- ⁇ .
  • the above results indicate that the inflammatory response in the skin of mice treated with moderate psoriasis is significantly improved.

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Abstract

提供了c-Rel特异性siRNA及其用于防治自身免疫性银屑病的应用。具体而言，所述c-Rel特异性siRNA具有如SEQ ID Nos.1～2或SEQ ID Nos.3～4所示序列，本发明采用c-Rel特异的小干扰核糖核酸来抑制c-Rel的生物合成，并通过抑制IL-23/IL-17A炎症轴相关炎症因子来防治自身免疫性银屑病。

Description

c-Rel特异性siRNA及其用于防治自身免疫性银屑病的应用 技术领域
本发明涉及以NF-κB家族成员c-Rel为靶点来治疗自身免疫性银屑病，具体涉及c-Rel特异性siRNA及其用于防治(预防和/或治疗)自身免疫性银屑病的应用，本发明能有效防治自身免疫性银屑病，同时几乎不影响免疫系统正常功能的发挥。
背景技术
银屑病是影响最为广泛的三大自身免疫病之一，其全球发病率约为3％，其中三分之一的患者达到中度至重度的程度。银屑病病征表现为皮肤增厚和广泛性损害，可引起瘙痒、鳞屑和疼痛，并严重影响患者的生活质量、心理健康以及社会关系。此外，病症较重者易患合并症，如关节炎、心脏病和糖尿病，具有死亡风险。
1自身免疫性银屑病
1.1自身免疫性银屑病致病机理研究进展
在1980年之前，研究人员仅仅看到银屑病银屑症状的表象并认为银屑病是由于皮肤角质细胞过度增殖引起。随后的20年里，随着免疫学的发展，研究人员检测到银屑病患者皮肤内p40蛋白的显著增多，而p40在当时被认为仅仅是IL-12p35p40的亚基，并且IL-12p35p40是驱使naive CD4+T细胞向Th1细胞分化的重要因子。因此，当时的学者认为银屑病是Th1细胞异常活化所介导的炎症性疾病。然而，在2000年，Oppmann B等人发现了p19蛋白，其可与p40聚合形成IL-23p19p40。于是，研究人员意识到之前基于p40表达差异所提出的Th1介导银屑病的理论并不可靠。果然，在进一步验证时，Lee E等人发现银屑病患者皮肤内IL-23p19、p40蛋白均明显增多，但IL-12p35却无显著变化。在2005年左右，Langrish CL、Harrington LE以及Park H等人发现IL-23能促使naive CD4+T细胞向Th17发育，其主要通过分泌IL-17A来调控炎症反应。目前，Leslie van der Fits等人已经证实自身免疫性银屑病主要由IL-23/IL-17A炎症轴介导(van der Fits,L.et al.Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis.Journal of immunology(Baltimore,Md.:1950)182,5836-5845,doi:10.4049/jimmunol.0802999(2009))，其病理过程主要为：致病因素(如遗传、环境、感染以及物理损伤等)导致先天性免疫细胞(如角质细胞、自然杀伤细胞等)分泌TNF-α、IL-1β和IL-6等促炎性因子，激活树突状细胞等先天免疫细胞。活化的树突状细胞迁移 到皮肤淋巴结等免疫器官中，提呈抗原并分泌IL-23等促炎性因子，促使naive CD4+T细胞向Th17分化。分化的自身反应性Th17从皮肤的毛细血管中迁移出，浸润到皮肤中的炎症部位。在获得自身抗原的再刺激后，持续增殖并分泌IL-17A等多种炎性因子。IL-17A等因子可激活角质细胞，刺激其增殖，形成银屑症状。另一方面，活化的角质细胞又可分泌抗菌肽(如LL-37抗菌肽和β-防御素)、促炎性因子(TNF-α、IL-1β和IL-6)、趋化因子(CXCL8-11、CCL20)以及S100蛋白等，这些因子又可激活先天性免疫细胞，导致炎症恶性循环，从而维持并加重银屑病发展。可见，IL-17A在银屑病先天免疫和适应性免疫的炎症回路中扮演着重要的桥联作用。当然，IL-23/IL-17A炎症轴仅仅是银屑病病理过程中的某一重要的环节。近期研究发现γδT细胞以及巨噬细胞也可分泌IL-17A而促进银屑病的发生发展(Cai,Y.,Fleming,C.&Yan,J.Dermal gammadelta T cells--a new player in the pathogenesis of psoriasis.International immunopharmacology 16,388-391,doi:10.1016/j.intimp.2013.02.018(2013))，Th22细胞可分泌IL-22而加重银屑病的发展，其余病理环节还有待发现(Benham,H.et al.Th17 and Th22 cells in psoriatic arthritis and psoriasis.Arthritis research&therapy 15,R136,doi:10.1186/ar4317(2013))。
1.2自身免疫性银屑病的治疗现状
目前，对银屑病的治疗主要按照疾病的严重程度予以分类治疗。对于轻中度银屑病常局部予以外用药物来治疗，如糖皮质激素、维生素D3类似物、维A酸、蒽林、焦油类等。外用药物无效或者重度银屑病，尤其是红皮病型银屑病、泛发性脓疱型银屑病及关节病型银屑病等，常采用系统给药疗法，如肌肉或静脉滴注甲氨蝶呤、环孢素等小分子免疫抑制剂。近几年，一些生物制剂也被FDA批准用于治疗重度银屑病，如以T细胞CD2或CD11a、TNF-α、IL-12和IL-23为靶点的单抗(表1)。
表1.近几年银屑病药物研发进展
Figure PCTCN2015092124-appb-000001
Figure PCTCN2015092124-appb-000002
这些生物制剂疗效显著，但安全性不明确，有待临床的长期考证。此外，各大医药公司还正积极开发以IL-23、IL-17或IL-17受体等为靶点的单抗药物，其中诺华公司研发的Secukinumab在2015年年初获欧盟批准，系全球首个IL-17单抗。遗憾的是，出于安全性考虑，中轻度患者在短期内不能使用这些单抗药物。除大分子单抗药物外，一些小分子抑制剂也被FDA批准用于治疗银屑病，如以PDE4为靶点的口服小分子抑制剂，以TLR7&9、JAK为靶点的小分子抑制剂也在进行临床试验。
2 NF-κB家族及其成员c-Rel
2.1 NF-κB家族及其成员
现有基础研究中已有关于NF-κB家族及其成员c-Rel与自身免疫病关系的相关报道。哺乳动物NF-κB家族由五名成员组成：c-Rel，RelA(p65)，RelB，NF-κB1(p50/p105)，和NF-κB2(p52/p100)。这些蛋白的氨基末端具有由约300个氨基酸残基组成的一个高度保守的区域，称为Rel同源区(RHD)，此同源区的功能包括二聚化、与IκB相互作用、核定位以及与DNA结合等；相比之下，这些蛋白的羧基末端则是不保守的，其中，c-Rel、RelA以及RelB的羧基末端具有反式激活功能域。
新合成的NF-κB通常以同源或异源二聚体的形式与IκB相结合形成无活性的状态储存于细胞质中。到目前为止，哺乳动物IκB家族已发现有九种成员：IκBα、IκBβ、IκBε、IκBζ、IκBη、BCL-3、IkB-NS、p52的前体蛋白p100以及p50的前体蛋白p105。它们主要通过覆盖NF-κB家族成员的核定位序列来阻止NF-κB向核内迁移。一些受体，包括肿瘤坏死因子、IL-1和神经生长因子(NGF)的受体、T细胞和B细胞抗原受体以及Toll样受体，在与相关配体结合后均能活化NF-κB。NF-κB的活化需由IκB激酶(IKK)介导IκB磷酸化以及随后的IκB蛋白降解或加工处理(如p100)。一旦NF-κB被活化， 其会以游离的二聚体形式进入细胞核，并结合到靶基因启动子上的9-10个碱基对来调控基因的表达。活化的NF-κB又可通过多种机制(如反馈通路)下调，与新合成的IκB相结合，并以静息状态储存于细胞质中。
2.2 c-Rel与自身免疫病的发展密切相关
研究表明，NF-κB除在生理功能以及病理状态的维持方面发挥作用外，其在自身免疫病的发展中也发挥着关键作用。在小鼠以及人体中，1型糖尿病的发生常伴随着树突状细胞和单核细胞中NF-κB的高度活化，以及组织中这些细胞的浸润。而抑制NF-κB的活化能有效抑制1型糖尿病在NOD小鼠、CD1小鼠和C57BL/6小鼠中的发生(Campbell,I.K.,Gerondakis,S.,O'Donnell,K.&Wicks,I.P.Distinct roles for the NF-kappaB1(p50)and c-Rel transcription factors in inflammatory arthritis.The Journal of clinical investigation 105,1799-1806,doi:10.1172/jci8298(2000)；)。为了直接研究NF-κB在自身免疫病的发展中所起的作用，Liou等人(包括本案发明人所在实验室)以c-Rel和p50基因敲除鼠为实验对象，研究了I型糖尿病、关节炎和脑脊髓炎的发病情况(B.A.Hilliard,N.Mason,L.Xu,J.Sun,S.E.Lamhamedi-Cherradi,H.C.Liou,C.Hunter,Y.H.Chen,Critical roles of c-Rel in autoimmune inflammation and helper T cell differentiation.The Journal of clinical investigation 110,843-850(2002)；published online EpubSep(10.1172/jci15254)；S.E.Lamhamedi-Cherradi,S.Zheng,B.A.Hilliard,L.Xu,J.Sun,S.Alsheadat,H.C.Liou,Y.H.Chen,Transcriptional regulation of type I diabetes by NF-kappa B.Journal of immunology(Baltimore,Md.:1950)171,4886-4892(2003)；Q.Ruan,V.Kameswaran,Y.Zhang,S.Zheng,J.Sun,J.Wang,J.DeVirgiliis,H.C.Liou,A.A.Beg,Y.H.Chen,The Th17 immune response is controlled by the Rel-RORgamma-RORgamma T transcriptional axis.The Journal of experimental medicine 208,2321-2333(2011)；published online EpubOct 24(10.1084/jem.20110462))。研究发现，c-Rel敲除的小鼠发育正常，具有结构正常免疫系统，它们不会产生自发的传染病，当受到高剂量的病原体攻击时，它们能正常清除病原体或者清除能力略有降低。同时，c-Rel敲除的小鼠对自身免疫病具有耐受力，如脑脊髓炎、I型糖尿病、关节炎。这些现象表明c-Rel与自身免疫病的发展密切相关。
3 c-Rel与自身免疫病中的炎症反应
自身免疫病小鼠体内APC、T细胞、B细胞常伴有c-Rel的过度活化，而c-Rel敲除的小鼠能抵抗自身免疫病的发生，这说明c-Rel在自身免疫病的病理过程中发挥着关键的调控作用。研究发现，c-Rel参与调控抗原提呈细胞中多种炎性因子的表达和Th17的分化发育。主要表现在：
3.1 c-Rel直接调控抗原递呈细胞中多种促炎性因子的表达
树突状细胞和巨噬细胞均是机体先天性免疫系统中的专职抗原递呈细胞，它们能启动适应性免疫系统。本案发明人在对骨髓来源的树突状细胞(BMDC)的研究中发现c-Rel敲除BMDC中IL-23p19的表达较野生型BMDC明显减少(Carmody,R.J.,Ruan,Q.,Liou,H.C.&Chen,Y.H.Essential roles of c-Rel in TLR-induced IL-23 p19 gene expression in dendritic cells.Journal of immunology(Baltimore,Md.:1950)178,186-191(2007))。通过体外荧光素酶实验(luciferase)、凝胶迁移实验(EMSA)以及体内染色质免疫共沉淀(ChIP)技术发现，在Toll样受体(TLR)信号传导时，c-Rel可与IL-23p19基因启动子上两个结合位点特异性结合，并与其它转录因子形成增强体来直接调控IL-23p19的表达。最为重要的是，在只有其它转录因子(p19启动子上还包含有AP-1、C/EBP和IRF可能结合的位点)结合到p19基因启动子上时，p19基因并不活化，这表明IL-23p19基因的表达完全依赖于c-Rel。另外，Nicola Mason等人研究发现，在炎性刺激(如LPS)时，c-Rel在巨噬细胞以及树突状细胞中以c-Rel/p50异源二聚体的形式直接调控IL-12p40的表达(N.Mason,J.Aliberti,J.C.Caamano,H.C.Liou,C.A.Hunter,Cutting edge:identification of c-Rel-dependent and-independent pathways of IL-12 production during infectious and inflammatory stimuli.Journal of immunology(Baltimore,Md.:1950)168,2590-2594(2002))。此外，研究发现c-Rel还参与调控APC中IL-6等促炎性因子的表达(J.R.Tumang,C.Y.Hsia,W.Tian,J.F.Bromberg,H.C.Liou,IL-6 rescues the hyporesponsiveness of c-Rel deficient B cells independent of Bcl-xL,Mcl-1,and Bcl-2.Cellular immunology 217,47-57(2002))。
3.2 c-Rel直接或间接调控Th17细胞分化发育
早期，研究人员普遍认为Th1细胞是自身免疫病病理过程中的主要致病细胞。然而，近几年更深入的研究发现Th17细胞在多种自身免疫病中均发挥着更为关键的致病作用，包括多发性硬化症、银屑病、类风湿性关节炎等。不同于Th1细胞，Th17细胞主要产生IL-17A、IL-17F和IL-22等细胞因子，并在炎性疾病以及抵抗细胞外细菌感染中发挥作用。Th17谱系特异性因子包括RORγt、RORα和STAT3。
c-Rel通过两方面作用来影响Th17分化发育。一方面，本案发明人研究发现c-Rel通过在抗原提呈细胞中直接调控IL-23的产生来间接调控Th17细胞发育(Carmody,R.J.,Ruan,Q.,Liou,H.C.&Chen,Y.H.Essential roles of c-Rel in TLR-induced IL-23 p19 gene expression in dendritic cells.Journal of immunology(Baltimore,Md.:1950)178,186-191(2007))；另一方面，本案发明人发现c-Rel还可在T细胞中可以直接调控Th17分化(Ruan, Q.et al.The Th17 immune response is controlled by the Rel-RORgamma-RORgamma T transcriptional axis.The Journal of experimental medicine 208,2321-2333,doi:10.1084/jem.20110462(2011))。本案发明人Ruan,Q等人在使用抗CD3单抗和抗CD28单抗刺激从c-Rel敲除小鼠体内分离的CD4+T细胞时，IL-17A信使RNA(mRNA)以及IL-17A蛋白的表达较野生型明显减少；从c-Rel敲除小鼠体内分离的CD4+T细胞在体外培养并刺激时，发现Th17数量高达70％减少(Ruan,Q.et al.The Th17 immune response is controlled by the Rel-RORgamma-RORgamma T transcriptional axis.The Journal of experimental medicine 208,2321-2333,doi:10.1084/jem.20110462(2011)；Ruan,Q.et al.Development of Foxp3(+)regulatory t cells is driven by the c-Rel enhanceosome.Immunity 31,932-940,doi:10.1016/j.immuni.2009.10.006(2009))。进一步研究发现RORγ和RORγt的表达在c-Rel敲除的T细胞中显著降低，而RORγ和RORγT的重建能恢复c-Rel敲除T细胞中Th17的分化缺陷。此外，通过染色质免疫共沉淀技术，Ruan,Q等人发现c-Rel/p65通过结合并分别激活两种不同的Rorγ启动子来调控RORγT和RORγmRNA的表达。基于以上研究结果，本案发明人Ruan,Q等人提出了Th17分化理论：CD4+T细胞上TCR、共刺激分子以及IL-1、IL-23、IL-6等细胞因子受体在与相应配体结合时(Th17分化条件)，向胞内传导活化信号，释放多种转录因子入核。其中，游离的c-Rel/p65二聚体与Rorγ基因的不同启动子结合，并与其它释放入核的转录因子(如NFAT和Stat)相互作用形成Rorγ特异性增强体，启动Rorγ基因的转录，从而驱动Th17细胞分化。值得注意的是，c-Rel/p65转录因子是目前所发现的唯一与Rorγ基因启动子结合并激活转录的转录因子，其它与Rorγ基因启动子结合的转录因子还有待发现。
4以整个NF-κB家族为靶点来治疗自身免疫性银屑病
理论上，以整个NF-κB家族为靶点的药物可发挥治疗自身免疫性银屑病的功效，如蛋白酶抑制剂(如FDA批准的PS-341)、NF-κB的诱饵寡核苷酸、NBD多肽以及糖皮质激素等(Vanderlugt,C.L.,Rahbe,S.M.,Elliott,P.J.,Dal Canto,M.C.&Miller,S.D.Treatment of established relapsing experimental autoimmune encephalomyelitis with the proteasome inhibitor PS-519.Journal of autoimmunity 14,205-211,doi:10.1006/jaut.2000.0370(2000)；May,M.J.et al.Selective inhibition of NF-kappaB activation by a peptide that blocks the interaction of NEMO with the IkappaB kinase complex.Science 289,1550-1554(2000)；De Stefano,D.Oligonucleotides decoy to NF-kappaB:becoming a reality？Discovery medicine 12,97-105(2011))。但是，大多数NF-κB家族蛋白在机体许多细胞中普遍表达，并与机体正常生理功能的维持相关，如感染过程中先天性以及适应性免疫的调控、炎症反应、抗凋亡、细胞增殖等，导致这些药物副作用大、特异性 差，只能在短期内使用来控制急性过敏反应。因此，不能使用以整个NF-κB家族为靶点的药物来治疗慢性炎症性疾病，如自身免疫性银屑病。
发明内容
本发明一个目的在于采用c-Rel特异性的小干扰核糖核酸(siRNA)来抑制c-Rel的生物合成，从而通过抑制银屑病小鼠体内IL-23/IL-17A炎症轴来防治自身免疫性银屑病。
本发明另一目的在于提供一种抑制个体体内c-Rel生物合成的c-Rel特异的小干扰核糖核酸。所述的个体为哺乳动物(例如小鼠)或人类。
本发明另一目的在于提供一种以纳米材料递送c-Rel特异性小干扰核糖核酸防治自身免疫性银屑病的方法。
本发明中将所述c-Rel特异性小干扰核糖核酸称为siRel。
鉴于c-Rel在炎症反应中的重要作用以及自身免疫性银屑病的病理过程，本发明提出仅以NF-κB家族成员c-Rel为靶点，采用c-Rel特异性的小干扰核糖核酸(siRel)来抑制c-Rel的生物合成，从而通过干预银屑病个体体内IL-23/IL-17A炎症轴来防治(预防和/或治疗)自身免疫性银屑病。
一方面，本发明采用c-Rel特异性的小干扰核糖核酸(siRel)来抑制NF-κB家族成员c-Rel的生物合成，从而通过干预IL-23/IL-17A炎症轴来防治自身免疫性银屑病的方法。
根据本发明的具体实施方案，本发明提供了一种c-Rel特异的小干扰核糖核酸(siRel)。可以采用c-Rel特异的小干扰核糖核酸(siRel)来抑制c-Rel的生物合成。具体地，在小鼠体内，所述的c-Rel特异的小干扰核糖核酸(siRel)序列为：
正义链5’CAACCGGACAUACCCGUCUdTdT 3’(SEQ ID No.1)
反义链5’AGACGGGUAUGUCCGGUUGdTdT 3’(SEQ ID No.2)
对应地，在人体内所述的c-Rel特异的小干扰核糖核酸(siRel)序列为：
正义链5’CAACCGAACAUACCCUUCUdTdT 3’(SEQ ID No.3)
反义链5’AGAAGGGUAUGUUCGGUUGdTdT 3’(SEQ ID No.4)
另一方面，本发明还提供了一种以纳米材料递送c-Rel特异性小干扰核糖核酸(siRel)防治自身免疫性银屑病的方法。具体而言，可以是以纳米胶束(如，PEG-PLL-PLLeu三嵌段共聚物纳米胶束)为载体，体内输送siRel至树突状细胞，达到 预防和/或治疗目的。
另一方面，本发明还提供了c-Rel特异性的小干扰核糖核酸在制备通过抑制c-Rel的生物合成从而抑制银屑病个体体内IL-23/IL-17A炎症轴的药物组合物中的应用。具体而言，其中，所述c-Rel特异性的小干扰核糖核酸具有如SEQ ID Nos.1～2或SEQ ID Nos.3～4所示序列。所述的药物组合物还可包括用来携载c-Rel特异性的小干扰核糖核酸并将其转运至细胞质中的纳米材料(如，PEG-PLL-PLLeu三嵌段共聚物胶束)。
另一方面，本发明还提供了一种防治(预防和/或治疗)自身免疫性银屑病的药物组合物，该组合物包括有效量的所述的c-Rel特异性的小干扰核糖核酸。优选地，所述药物组合物还包括PEG-PLL-PLLeu三嵌段共聚物纳米胶束。此外，所述的药物组合物可包括一种或多种药学上可接受的辅料。在本发明的一具体实施方案，本发明的所述药物组合物可以是防治个体自身免疫性银屑病的外用制剂(可以是用于涂抹皮肤的纳米胶束悬液的形式)，其中c-Rel特异性的小干扰核糖核酸浓度为10～100nM，纳米胶束浓度为10～20μg/ml。所述的个体可以为哺乳动物(例如小鼠)或人类。
本发明还提供了一种防治(预防和/或治疗)自身免疫性银屑病的方法，该方法包括施予个体有效量的c-Rel特异性的小干扰核糖核酸来抑制c-Rel的生物合成从而抑制银屑病个体体内IL-23/IL-17A炎症轴的过程。根据本发明的具体实施方案，本发明提供了使用纳米胶束输送c-Rel特异性的siRNA来防治自身免疫性银屑病的方法。由于siRNA药物存在转染效率低，在生物体内易被酶解、半衰期短等缺点，本发明采用PEG-PLL-PLLeu三嵌段共聚物胶束来携载siRNA并将其转运至细胞质中。本案发明人研究表明这种三嵌段共聚物胶束生物可降解、转染效率高(Deng,J.et al.Self-assembled cationic micelles based on PEG-PLL-PLLeu hybrid polypeptides as highly effective gene vectors.Biomacromolecules 13,3795-3804,doi:10.1021/bm3012538(2012))，最重要的是，本案发明人研究发现这种PEG-PLL-PLLeu三嵌段共聚物胶束主要富集于脾脏中并被其中树突状细胞摄取(Luo,Z.et al.Cationic polypeptide micelle-based antigen delivery system:A simple and robust adjuvant to improve vaccine efficacy.Journal of Controlled Release 170,259-267,doi:http://dx.doi.org/10.1016/j.jconrel.2013.05.027(2013)。体外实验中，使用siRel浓度为10～100nM，纳米胶束浓度为10～20μg/ml。在小鼠体内实验中，对于轻度银屑病的治疗每只小鼠腹腔给予500pmol siRel，对应100μg纳米胶束，给药频率为每2天一次；对于中度银屑病的治疗每只小鼠腹腔给予500pmol siRel，对应100μg纳米胶束，给药频率为每天一次。在人体内，采用静脉给药或皮损区皮内、皮下或皮肤 上涂抹给药。具体而言，静脉给予siRel剂量为0.3mg/kg，对于纳米胶束剂量为4.5mg/kg，给药频率为每三周一次；皮内给予siRel剂量为8mg，对应纳米胶束剂量为8mg，给药体积为2ml，给药频率为每周一次；皮下给予siRel剂量为16mg，对应纳米胶束剂量为16mg，给药体积为4ml，给药频率为每周一次；皮肤上涂抹siRel浓度为10nM，对应纳米胶束剂量为10μg/ml，给药频率为每日1～3次，每次按每500平方厘米擦涂0.5ml siRel纳米胶束悬液。
根据本发明的具体实施方案，在本发明的一具体的体外实验中，先在NIH3T3、BMDC和RAW264.7细胞系中确证了本发明的c-Rel特异性siRNA即所述的siRel的有效性。本发明发现siRel可显著降低c-Rel和IL-23p19的mRNA及蛋白表达水平。
根据本发明的具体实施方案，在本发明的一具体的体内实验中，发现siRel可明显控制轻度银屑病小鼠银屑病的发展，显著降低小鼠脾细胞IL-17A的产生，并抑制小鼠皮损组织中IL-1β、IL-6、TNF-α、IL-23p19以及IL-17A的表达，达到了治疗效果。此外，两组小鼠皮肤切片H&E染色结果显示，治疗组小鼠皮肤表皮较对照组薄，且治疗组小鼠皮肤内浸润的炎性细胞明显减少。另外，在中度银屑病小鼠上测评该siRel药物，发现该siRel药物也具有一定的治疗疗效。
综上所述，本发明中，采用纳米胶束可有效输送c-Rel特异性的siRNA至树突状细胞，可显著减少IL-23/IL-17炎症轴相关炎性因子的表达，达到防治自身免疫性银屑病的作用。
附图说明
图1：siRNA/PEG-PLL-PLLeu胶束悬液制备示意图。
图2：siRNA/PEG-PLL-PLLeu纳米胶束表征。各图片：A，凝胶阻滞实验检测胶束结合siRNA的能力；B，载siRNA胶束混合物(N/P＝15)的粒径大小和表面电势；C和D，不同N/P比混合的载siRNA胶束混合物转染BMDC或BM20效率的测定。数值显示为均值±标准差(n＝3)。
图3：siRel/PEG-PLL-PLLeu纳米胶束体外沉默效果。各图片，A-E，NIH3T3、RAW264.7以及BMDC在siNC或siRel处理24小时后LPS刺激或不刺激4小时，采用实时定量PCR检测c-Rel或IL23p19的相对表达；F，BMDC在siNC或siRel处理48小时后LPS刺激或不刺激4小时，通过ELISA检测上清中IL23的蛋白水平；G-H，该siRel能在蛋白水平降低c-Rel的表达，其中图片H为蛋白印迹的量化图。数值显示为均 值±标准差(n＝3),*：P<0.05；**：P<0.01；***：P<0.001。
图4：IMQ诱导小鼠银屑病模型的构建。各图片：A，IMQ诱导BALB/c小鼠银屑病第6天银屑表型(第0-3天：65mg/天；第4-5天：80mg/天；第6天：100mg/天)；B，IMQ诱导过程中，每日对小鼠进行皮损评分(0-4)；C，第6天时，对照组与IMQ诱导组小鼠皮损区皮肤H&E染色。数值显示为均值±标准差(n＝3)，非配t检验，*：P<0.05。
图5：siRel处理可有效控制轻度银屑病的发展。各图片：A，BALB/c小鼠银屑病治疗疗程图，8-11周龄小鼠连续8天每日于背部涂抹咪喹莫特乳膏(第0-3天：65mg/天；第4-5天：80mg/天；第6-7天：100mg/天)，第1、2、4、6天时腹腔给予siNC或siRel，第8天处死小鼠并取材；B，第八天时对照组和治疗组小鼠的表型；C，治疗过程中，每日对各组小鼠进行皮损打分(0-4)；D，对照组与治疗组小鼠皮损区皮肤H&E染色。数值显示为均值±标准差(n＝13)，非配t检验，*：P<0.05；***：P<0.001。
图6：siRel处理可有效改善中度银屑病皮损症状。各图片：A，银屑病治疗疗程图；B，对照组(siNC)与治疗组(siRel)小鼠皮损区皮肤H&E染色。
图7：siRel处理组小鼠脾细胞，ELISA检测IL17A的蛋白水平。图中数值显示为均值±标准差(n＝5)，非配对t检验，*：P<0.05；**：P<0.01；***：P<0.001。
图8：siRel处理后采用实时定量PCR检测IL-23p19、IL-17A、IL-1β、IL-6和TNF-α的表达。数值显示为均值±标准差(n＝5)，非配对t检验，*：P<0.05；**：P<0.01；***：P<0.001。
图9：Q-PCR检测中度银屑病小鼠治疗后第6天时小鼠皮肤中炎症因子表达水平。
具体实施方式
为了更清楚地理解本发明的实质，下面通过具体实施例并配合附图进一步详细说明本发明，但本发明并不因此而受到任何限制。下列实施例中未注明具体条件的实验方法，通常按照所属领域的常规操作或按照制造厂商所建议的条件进行。
实施例1
本实施例，通过体外实验，先在NIH3T3和RAW264.7细胞系(NIH3T3和RAW264.7细胞系购自ATCC)中确证c-Rel特异性siRNA即siRel的有效性。进一步，以骨髓来源的树突状细胞(BMDC)为研究对象来确证该siRel的沉默效果。具体操作如下：
1、c-Rel特异的小干扰核糖核酸siRNA的设计与合成
本实施例中，采用c-Rel特异的小干扰核糖核酸(siRel)来抑制c-Rel的合成，在 小鼠体内，该核酸序列为：
正义链5’CAACCGGACAUACCCGUCUdTdT 3’(SEQ ID No.1)
反义链5’AGACGGGUAUGUCCGGUUGdTdT 3’(SEQ ID No.2)
该siRel序列由上海吉玛公司合成，siNC为通用阴性对照。FAM标记的siRel(siRel-FAM)亦由上海吉玛公司合成。
2、siRNA/PEG-PLL-PLLeu胶束悬液的制备
PEG-PLL-PLLeu为聚乙二醇-多聚赖氨酸-多聚亮氨酸三嵌段共聚物，由合作者马轶凡和蔡林涛老师采用NCA开环聚合法合成(Deng,J.et al.Self-assembled cationic micelles based on PEG-PLL-PLLeu hybrid polypeptides as highly effective gene vectors.Biomacromolecules 13,3795-3804,doi:10.1021/bm3012538(2012))。在水溶液中，大于临界胶束浓度时，该两亲性三嵌段共聚物可自组装形成粒径约150nm的胶束。其中，多聚亮氨酸段聚集组成胶束的疏水内核，聚乙二醇形成胶束的外壳，中间的多聚赖氨酸段因含有大量的伯胺基而发生质子化，使该胶束zeta电位约为43mV。通过电性作用，该纳米胶束可以结合带负电的siRNA，形成带正电的荷载siRNA的胶束混悬液。另外，由于细胞膜带负电，荷载siRNA纳米胶束通过电性作用吸附于细胞膜上，促进细胞对该胶束的內吞。
请参见图1，称取适量PEG-PLL-PLLeu冻干粉溶于去离子水中，制备成1mg/ml的共聚物胶束悬液，0.22μm过滤除菌。按不同N/P比(共聚物胶束伯氨基与siRel磷酸基的摩尔比)，将OPTI-MEM稀释的siRel与等体积的共聚物悬液混合，室温下静置30min，获得载siRel的共聚物胶束(siRel/PEG-PLL-PLLeu)混悬液。将该胶束悬液滴入细胞培养板中，轻拍混合均匀，以转染细胞。
3、siRNA/PEG-PLL-PLLeu纳米胶束表征
将如上所述制备的不同N/P比混合的siRel/PEG-PLL-PLLeu上样于2％琼脂糖凝胶，使用TAE缓冲液在120mV电压下电泳20分钟，后用紫外凝胶成像仪观察凝胶阻滞结果。纳米胶束的粒径及zeta电位使用动态光散射仪(Nano-ZSZEN3600)于室温下测定，结果使用Malvem Dispersion Technology Software 4.2软件进行分析。
图2中A显示凝胶阻滞实验检测胶束结合siRNA的能力。从图中可以看出，当N/P比大于等于2时该共聚物胶束能将siRNA全部荷载。
图2中B显示载siRNA胶束混合物(N/P＝15)的粒径大小和表面电势。结果表明，当以N/P比为15的比例混合共聚物胶束和siRNA时，检测到荷载siRel后胶束粒径由 约148nm增大至180nm左右，zeta电位则由约42mV降低至38mV左右。
图2中C和D显示不同N/P比混合的载siRNA胶束混合物转染BMDC或BM20效率的测定结果。流式结果表明，当N/P比为10、15或20时，转染BMDC以及BM20效率没有显著区别。当N/P比为15时，该纳米胶束转染BMDC效率约为45％，转染BM20效率约为60％。
4、siRel/PEG-PLL-PLLeu纳米胶束体外沉默实验
方法：NIH3T3以及RAW264.7细胞系购自ATCC，按标准程序培养。BMDC为原代分离诱导而来。将BALB/c小鼠脱颈处死，无菌条件下取股骨和胫骨，使用1ml注射器吸取含2％FBS的PBS液冲洗出骨髓细胞。收集细胞悬液，离心后采用红细胞裂解液重悬，裂解1min之后使用培养基洗涤三次。采用含20ng/ml重组鼠GM-CSF和10ng/ml重组鼠IL-4的X-Vivo培养基悬浮细胞，配制成2X 106个/ml的细胞悬液，接种于24孔培养板，每孔1ml。第三天和第五天时半量换液，第六天即可获得骨髓来源的树突状细胞(BMDC)。在对siRel体外沉默效果评估时，将NIH3T3、RAW264.7或BMDC种植于24孔板中，滴入配制好的荷载siNC或siRel的纳米胶束，并且培养液中siRNA浓度为100nM。转染24h后，LPS刺激或不刺激6h后收集细胞，采用Trizol试剂从细胞或组织中萃取总RNA，以oligo dT为引物，采用M-MLV Reverse Transcriptase进行逆转录，具体实验操作按promega公司产品说明书进行。随后采用Thunderbird SYBR qPCR Mix进行实时定量PCR，以GAPDH为内参，检测c-Rel、IL-23p19mRNA的表达量。BMDC在转染48小时后，使用LPS刺激6h，收集上清，采用ELISA检测IL23p19的浓度，具体流程按Ebioscience公司说明书进行。BMDC在转染48小时后，使用LPS刺激6h后收集细胞，提取细胞核蛋白，采用BCA法检测蛋白浓度后，进行蛋白印迹实验，检测细胞核中c-Rel蛋白量。
图3显示采用PEG-PLL-PLLeu纳米胶束输送siRel时的体外沉默效果。其中，图片A-C显示siRel能有效降低NIH 3T3中c-Rel mRNA的水平(P<0.01)；且该siRel能有效降低RAW 264.7细胞系中c-Rel以及IL23p19的mRNA水平(P<0.05)。在采用PEG-PLL-PLLeu胶束运输siRNA至BMDC，评估siRel的沉默效果时，图片D-E显示，siRel能在mRNA水平显著降低BMDC中c-Rel和IL23p19的mRNA表达(P<0.05)。图片F显示，该siRel亦能在蛋白水平降低IL23的表达(P<0.01)。图片G-H显示该siRel能在蛋白水平降低c-Rel的表达，其中图片H为蛋白印迹的量化图。
实施例2
银屑病的治疗分为早期、中期、晚期治疗，对应银屑病的严重程度为轻度、中度、重度。在本实验中，分别在轻、中度银屑病小鼠体内进行了siRel治疗效果的测评。IMQ诱导的银屑病模型与人类银屑病的病理变化有较多相似之处，是研究银屑病的理想模型。IMQ是Toll样受体(Toll like receptor，TLR)7/8的激动剂，将其涂抹于小鼠的皮肤上，可通过TLR途径激活树突状细胞和巨噬细胞以及TLR非依赖途径激活角质细胞，促进IFN-α、TNF-α等促炎性因子的分泌，募集炎性细胞到皮肤，产生银屑病样皮损及组织学改变。并且，Leslie van der Fits等人已经证实IL-23/IL-17A炎症轴在IMQ诱导的银屑病模型中发挥着关键的作用。与现有技术的报道有所不同的是，本发明在本实施例的实验中，所给予的IMQ的剂量是递增的，这是由于IMQ药品的厂家不同、小鼠的饲养环境不同会导致诱导银屑病时所需的剂量不同。
1、IMQ诱导银屑病模型的构建
BALB/c小鼠经10％水合氯醛(300mg/kg)腹腔注射进行麻醉后，脱去其背部毛，形成约2cm X 3cm的裸露区。每日涂抹IMQ(day0-3：65mg/day；day4-day5：80mg/day；day6-day7：100mg/day；)，并且每天观察小鼠皮肤并对鳞屑的形成进行打分，评分标准为：0，无；1，轻度；2，中度；3，重度；4，极重度。各组小鼠积分取平均值后绘制趋势线，观察各组小鼠皮损的变化情况。
2、siRel治疗小鼠轻度银屑病
银屑病小鼠模型构建过程中，在小鼠银屑评分为0.5时给予siRNA药物治疗。将银屑病小鼠随机分为对照组(siNC)和治疗组(siRel)，于第1、2、4、6天分别腹腔给予对照组和实验组siNC/PEG-PLL-PLLeu和siRel/PEG-PLL-PLLeu胶束混悬液，其中siRNA剂量为500pmol，胶束剂量为100ug。每天观察小鼠皮肤并对鳞屑的形成进行打分，于第8天取小鼠皮肤，按照九格法剪取各组小鼠相同部位皮损处组织，固定于4％多聚甲醛。OCT包埋后，以7μm的厚度进行冰冻切片。而后进行H&E染色，观察各组小鼠表皮厚度变化。
3、siRel治疗小鼠中度银屑病
银屑病小鼠模型构建过程中，在小鼠银屑评分达到2时给予siRNA药物治疗。将银屑病小鼠随机分为对照组(siNC)和治疗组(siRel)，第3、4、5、6、7天分别腹腔给予对照组和实验组siNC/PEG-PLL-PLLeu和siRel/PEG-PLL-PLLeu胶束混悬液，其中siRNA剂量为500pmol，胶束剂量为100ug。每天观察小鼠皮肤并对鳞屑的形成进行打分，于 第8天取小鼠皮肤，按照九格法剪取各组小鼠相同部位皮损处组织，固定于4％多聚甲醛。OCT包埋后，以7μm的厚度进行冰冻切片。而后进行H&E染色，观察各组小鼠表皮厚度变化。
图4显示使用IMQ构建银屑病小鼠模型。其中，图片A显示第7天对照组与IMQ诱导组小鼠银屑表型，可以观察到诱导组小鼠背部有明显的鳞屑。图片B显示使用IMQ诱导银屑病的过程中，对鳞屑样症状的评分，可以观察到诱导组小鼠的鳞屑评分一直上升，而对照组几乎无鳞屑症状，在第4、5、6天时具有明显差异(P<0.05)。图片C显示第6天时对照组与IMQ诱导组小鼠皮肤切片H&E染色结果，可以观察到IMQ诱导组小鼠表皮厚度较对照组明显增厚。这些数据表明，本实验中使用的IMQ剂量可成功诱导出小鼠银屑病模型。
图5显示采用PEG-PLL-PLLeu纳米胶束输送siRel时的体内治疗轻度银屑病效果。其中，图片A显示BALB/c小鼠银屑病治疗疗程图。图片B显示day8时小鼠的银屑症状，可以观察到治疗组小鼠银屑样皮损得到了明显的控制。图片C显示小鼠银屑样症状的评分，可观察到治疗组小鼠的银屑评分一直维持在较低的水平，而对照组小鼠的银屑评分则处于上升状态，在第7、8天时具有明显差异(P<0.05)。图片D显示治疗组与对照组小鼠皮肤切片H&E染色结果，可观察到治疗组小鼠皮肤表皮较对照组薄，且治疗组小鼠皮肤内浸润的炎性细胞明显减少。这些数据表明，siRel能有效控制小鼠轻度自身免疫性银屑病的发展。
图6显示采用PEG-PLL-PLLeu纳米胶束输送siRel时的体内治疗中度银屑病效果。其中，图片A显示BALB/c小鼠银屑病治疗疗程图。图片B显示小鼠银屑样症状的评分，可观察到治疗组小鼠的银屑评分一直下降，而对照组小鼠的银屑评分则一直维持在2分左右，在第8天时具有明显差异(P<0.001)。图片C显示治疗组与对照组小鼠皮肤切片H&E染色结果，可观察到治疗组小鼠皮肤表皮较对照组薄，这些数据表明，siRel能有效改善小鼠中度自身免疫性银屑病的症状。
4、ELISA检测小鼠脾细胞产生IL-17A的水平
在轻度银屑病小鼠的治疗过程中，于第2、6、8天将小鼠脱颈处死，取治疗组和对照组银屑病小鼠的脾，研磨成细胞悬液，按2X 106个接种于48孔板中，500ul培养体系。使用或不使用0.5ug/ml anti-CD3抗体、anti-CD28抗体刺激48h后收集上清，通过酶联免疫吸附法检测IL-17A的水平。酶联免疫吸附法试剂盒购自Ebioscience公司，具体流程按说明书操作。
图7显示ELISA检测轻度银屑病小鼠治疗过程中第2、6、8天时小鼠脾细胞产生IL-17A的水平。结果表明，在第2、6、8天时，治疗组小鼠脾细胞分泌IL17A较对照组明显降低(P<0.05)。这说明siRel可有效降低银屑病小鼠体内脾细胞产生IL-17A的水平。
5、Q-PCR检测小鼠皮肤中IL-23/IL-17A炎症轴相关炎性因子的表达
轻度银屑病小鼠治疗过程中，第2、6天按照九格法剪取各组小鼠相同部位皮损处组织，立即放入液氮中，然后研磨成粉末，加入Trizol试剂以萃取皮肤中总RNA。以oligo dT为引物，采用M-MLV Reverse Transcriptase进行逆转录，具体实验操作按promega公司产品说明书进行。随后采用Thunderbird SYBR qPCR Mix进行实时定量PCR，以GAPDH为内参，检测IL-23p19、IL-17A、IL-1β、IL-6、TNF-α等炎性因子的相对表达。
图8显示Q-PCR检测轻度银屑病小鼠治疗过程中第2、6天时小鼠皮肤中炎症因子的表达水平。结果表明，治疗组小鼠与对照组小鼠皮肤内TNF-α的表达水平在第二天时稍减少(P<0.05)，而第8天时未检测到差异。另外，治疗组小鼠皮肤内IL23p19和IL6的水平在第2天以及第6天时均明显降低(P<0.05)。此外，本发明检测到治疗组小鼠与对照组小鼠皮肤内IL17A、IL-1β的表达水平在第2天时无差异，但在第6天时治疗组明显减少(P<0.01)。以上结果表明轻度银屑病小鼠治疗组小鼠皮肤内的炎症反应得到了一定的控制。图9显示Q-PCR检测中度银屑病小鼠治疗后第6天时小鼠皮肤中炎症因子的表达水平。结果表明，中度银屑病小鼠在治疗第六天后，治疗组小鼠皮肤内IL-1β、IL-6、IL-23p19、IL-17A、IFN-γ的表达水平较对照组小鼠明显减少(P<0.05)。但IL-12p35、TNF-α表达水平则无明显差别。以上结果说明中度银屑病小鼠治疗组小鼠皮肤内的炎症反应得到了明显的改善。
以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本领域的技术人员在本发明所揭露的技术范围内，可不经过创造性劳动想到的变化或替换，都应涵盖在本发明的保护范围之内。

Claims (14)

  1. c-Rel特异性的小干扰核糖核酸，其具有如下SEQ ID Nos.1～2或SEQ ID Nos.3～4所示序列：
    正义链5’CAACCGGACAUACCCGUCUdTdT 3’(SEQ ID No.1)
    反义链5’AGACGGGUAUGUCCGGUUGdTdT 3’(SEQ ID No.2)；或
    正义链5’CAACCGAACAUACCCUUCUdTdT 3’(SEQ ID No.3)
    反义链5’AGAAGGGUAUGUUCGGUUGdTdT 3’(SEQ ID No.4)。
  2. c-Rel特异性的小干扰核糖核酸在制备通过抑制c-Rel的生物合成从而抑制银屑病个体体内IL-23/IL-17A炎症轴的药物组合物中的应用。
  3. 根据权利要求2所述的应用，其中，所述c-Rel特异性的小干扰核糖核酸具有如SEQ ID Nos.1～2或SEQ ID Nos.3～4所示序列。
  4. 根据权利要求2或3所述的应用，其中，所述的药物组合物包括用来携载c-Rel特异性的小干扰核糖核酸并将其转运至细胞质中的纳米材料。
  5. 根据权利要求4所述的应用，其中，所述纳米材料为PEG-PLL-PLLeu三嵌段共聚物胶束。
  6. 一种防治自身免疫性银屑病的药物组合物，该组合物包括权利要求1所述的c-Rel特异性的小干扰核糖核酸。
  7. 根据权利要求6所述的药物组合物，该组合物还包括PEG-PLL-PLLeu三嵌段共聚物纳米胶束。
  8. 根据权利要求7所述的药物组合物，该组合物中，根据权利要求4所述的应用，其中，所述药物组合物为防治自身免疫性银屑病的外用制剂，其中c-Rel特异性的小干扰核糖核酸浓度为10～100nM，纳米胶束浓度为10～20μg/ml。
  9. 一种防治自身免疫性银屑病的方法，该方法包括施予个体有效量的c-Rel特异性的小干扰核糖核酸来抑制c-Rel的生物合成从而抑制银屑病个体体内IL-23/IL-17A炎症轴的过程。
  10. 根据权利要求9所述的方法，其中，是以纳米材料携载c-Rel特异性的小干扰核糖核酸并将其转运至细胞质中。
  11. 根据权利要求10所述的方法，其中，所述纳米材料为PEG-PLL-PLLeu三嵌段共聚物胶束。
  12. 根据权利要求9所述的方法，其中，所述个体为哺乳动物或人类。
  13. 根据权利要求9所述的方法，该方法是用于防治人类自身免疫性银屑病，其中采用静脉给药或皮损区皮内、皮下或皮肤上涂抹给药。
  14. 根据权利要求13所述的方法，其中，静脉给予siRel剂量为0.3mg/kg，对于纳米胶束剂量为4.5mg/kg，给药频率为每三周一次；皮内给予siRel剂量为8mg，对应纳米胶束剂量为8mg，给药体积为2ml，给药频率为每周一次；皮下给予siRel剂量为16mg，对应纳米胶束剂量为16mg，给药体积为4ml，给药频率为每周一次；皮肤上涂抹siRel浓度为10nM，对应纳米胶束剂量为10μg/ml，给药频率为每日1～3次，每次按每500平方厘米擦涂0.5ml siRel纳米胶束悬液。
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