WO2020132844A1 - 炎性相关疾病防治的小rna药物及其组合 - Google Patents

炎性相关疾病防治的小rna药物及其组合 Download PDF

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WO2020132844A1
WO2020132844A1 PCT/CN2018/123289 CN2018123289W WO2020132844A1 WO 2020132844 A1 WO2020132844 A1 WO 2020132844A1 CN 2018123289 W CN2018123289 W CN 2018123289W WO 2020132844 A1 WO2020132844 A1 WO 2020132844A1
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small rna
tnf
cell
1beta
alpha
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PCT/CN2018/123289
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English (en)
French (fr)
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蒋澄宇
金宁一
赵丹丹
秦宇豪
张聪
林烨暄
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中国医学科学院基础医学研究所
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Priority to PCT/CN2018/123289 priority Critical patent/WO2020132844A1/zh
Priority to EP18944638.8A priority patent/EP3960858A4/en
Priority to US17/417,321 priority patent/US20230272402A1/en
Priority to CN201880100050.3A priority patent/CN114729354A/zh
Priority to CA3124730A priority patent/CA3124730A1/en
Publication of WO2020132844A1 publication Critical patent/WO2020132844A1/zh

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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1136—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against growth factors, growth regulators, cytokines, lymphokines or hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
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    • C12N2330/00—Production
    • C12N2330/30—Production chemically synthesised
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    • C12N2330/00—Production
    • C12N2330/50—Biochemical production, i.e. in a transformed host cell

Definitions

  • the present invention generally relates to the field of nucleic acid therapeutics, and more specifically relates to small RNA and methods and uses thereof.
  • Inflammation is a very common and important basic pathological process. Trauma infection on the body surface and most common and frequently-occurring diseases of various organs. Inflammation can be infectious inflammation caused by infection (such as pneumonia, myocarditis, acute and chronic gastritis, acute and chronic enteritis, acute and chronic hepatitis, acute and chronic nephritis, dermatitis, encephalitis, lymphitis, conjunctivitis, keratitis, iridocyclitis , Otitis media, etc.), or non-infectious inflammation caused by infection, which is usually closely related to the body's immunity (such as allergic rhinitis, asthma, pulmonary fibrosis, chronic obstructive pulmonary disease, allergic dermatitis, sickle Cystic cell disease, multiple sclerosis, systemic lupus erythematosus, lupus nephritis, etc.), and inflammation is also one of the main triggers of cancer (such as lung cancer,
  • the clinical manifestations of inflammation are redness, swelling, heat, pain and dysfunction.
  • the biochemical indicators of inflammation are usually the high expression of inflammatory factors that participate in the inflammation process and mediate the inflammatory response, such as IL-1beta, IL-6 and TNF-alpha.
  • IL-1beta IL-1beta
  • IL-6 IL-6
  • TNF-alpha inflammatory factor-1beta
  • the prevention and treatment of inflammation is still based on Western medicine, but there are also many Chinese medicine products that have certain anti-inflammatory effects, but each has its own unavoidable shortcomings. Therefore, it is still necessary to find new treatments against inflammation.
  • RNA or its composition in the present application can reduce or down-regulate the expression level of IL-1beta, IL-6 or/and TNF-alpha and rescue cell death due to H5N1 infection.
  • the present invention provides the following:
  • (C) a sequence that hybridizes to the sequence shown in (A), preferably a sequence that hybridizes to the sequence shown in (A) under stringent conditions, which has the ability to inhibit any one or more of the pathways or genes listed in Table 3 ability;
  • (D) A sequence obtained by adding, deleting, replacing, or inserting one or more, such as 2, 3, 4, 5, 6, 7, 8, or 9 bases from the sequence shown in (A), which has inhibition The ability of any one or more pathways or genes listed in Table 3; or
  • the small RNA according to item 1 which has the ability to inhibit the same pathways or genes listed in Table 3, or to prevent and/or treat IL-1beta, IL-6 or/and TNF-alpha related diseases Ability and/or ability to improve cell survival.
  • RNA has the ability to reduce or down-regulate the expression level of IL-1beta, IL-6 or/and TNF-alpha and/or has the ability to rescue due to viruses, such as RNA viruses, For example, the ability of avian influenza viruses, such as cell death caused by H5N1 infection,
  • the small RNA has the ability to reduce or down-regulate the expression level of an inflammatory factor in IL-1beta, IL-6 and TNF-alpha,
  • the IL-1beta, IL-6 or/and TNF-alpha related diseases are selected from any one or more of the IL-1beta, IL-6 or/and TNF-alpha related diseases listed in the specification, preferably Pneumonia, myocarditis, acute and chronic gastritis, acute and chronic enteritis, acute and chronic hepatitis, acute and chronic nephritis, dermatitis, encephalitis, lymphitis, conjunctivitis, keratitis, iridocyclitis, otitis media, allergic rhinitis, asthma, lung fibers Chronic obstructive pulmonary disease, allergic dermatitis, sickle cell disease, multiple sclerosis, systemic lupus erythematosus, lupus nephritis, lung cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, cervical cancer, breast cancer, leukemia, Multiple myeloma, diabetes and g
  • RNA according to any one of items 1-3, wherein the small RNA is in a double-stranded or single-stranded form or a hybrid form of double-stranded and single-stranded.
  • a nucleic acid sequence or a construct comprising the nucleic acid sequence, the nucleic acid sequence comprising a sequence encoding a small RNA according to any one of items 1-6, wherein preferably the construct is a viral construct, preferably Retroviral constructs.
  • a recombinant virus comprising the nucleic acid sequence or construct according to item 7, preferably, the recombinant virus is a retrovirus.
  • An expression vector comprising a sequence encoding the small RNA according to any one of items 1-6.
  • a cell comprising the nucleic acid sequence or construct according to item 7 or transfected with the recombinant virus according to item 8 or comprising the expression vector according to item 9.
  • a method of expressing the small RNA according to any one of items 1-6 which comprises expressing the cell according to any one of the items 10 under suitable conditions and recovering the small RNA according to any one of the items 1-4.
  • a pharmaceutical composition comprising one or more small RNAs according to any one of items 1-6, a nucleic acid sequence or a construct according to item 7, a recombinant virus according to item 8, according to Item 9, the expression vector and/or the cell of Item 10,
  • the pharmaceutical composition is for oral, intravenous administration, such as bolus injection or continuous infusion for a period of time, by subcutaneous, intramuscular, intraarterial, intraperitoneal, intrapulmonary, intracerebrospinal, intraarticular, synovial Intra-membrane, intrathecal, intralesional, or inhalation routes such as intranasal, usually by intravenous or subcutaneous administration of the pharmaceutical composition.
  • intravenous administration such as bolus injection or continuous infusion for a period of time
  • subcutaneous, intramuscular, intraarterial, intraperitoneal, intrapulmonary, intracerebrospinal, intraarticular, synovial Intra-membrane, intrathecal, intralesional, or inhalation routes such as intranasal usually by intravenous or subcutaneous administration of the pharmaceutical composition.
  • composition according to item 12 which comprises any one or more of the mixture 1 to the mixture 43 in Table 2.
  • kits comprising one or more small RNAs according to any one of items 1-6, a nucleic acid sequence or a construct according to item 7, a recombinant virus according to item 8, a according to item
  • the expression vector of 9 and/or the cell of item 10 preferably, the kit further contains one or more drugs listed in the instructions.
  • a method of inhibiting any one or more of the pathways or genes listed in Table 3 in vitro or in vivo which includes administering one or more of the one or more according to any one of items 1-6 to a cell or a subject Small RNA, nucleic acid sequence or construct according to item 7, recombinant virus according to item 8, expression vector according to item 9, cell according to item 10 and/or item 12-15
  • the pharmaceutical composition of any one includes administering one or more of the one or more according to any one of items 1-6 to a cell or a subject Small RNA, nucleic acid sequence or construct according to item 7, recombinant virus according to item 8, expression vector according to item 9, cell according to item 10 and/or item 12-15.
  • a method of reducing or down-regulating the expression level of IL-1beta, IL-6 or/and TNF-alpha in vitro or in vivo and/or improving the cell survival rate which includes administering one or more The small RNA according to any one of items 1-6, the nucleic acid sequence or construct according to item 7, the recombinant virus according to item 8, the expression vector according to item 9, the item 10 Cells and/or the pharmaceutical composition according to any one of items 12-15.
  • the cell survival rate is a virus, such as an RNA virus, such as avian influenza virus, such as the cell survival rate in an H5N1 infection, preferably the improved cell survival rate is by rescuing the virus, for example RNA viruses, such as avian influenza viruses, such as H5N1 infection cause cell death.
  • RNA viruses such as avian influenza viruses, such as H5N1 infection cause cell death.
  • a method of treating or preventing IL-1beta, IL-6 or/and TNF-alpha related diseases and/or viruses, such as RNA viruses, such as avian influenza viruses, such as H5N1 infection in a subject which includes Administration of one or more small RNAs according to any one of items 1-6, nucleic acid sequences or constructs according to item 7, recombinant viruses according to item 8, expression vectors according to item 9 , The cell according to item 10 and/or the pharmaceutical composition according to any one of items 12-15,
  • the IL-1beta, IL-6 or/and TNF-alpha related diseases are selected from the IL-1beta, IL-6 or/and TNF-alpha related diseases listed in the specification, preferably pneumonia, myocarditis, acute and chronic Gastritis, acute and chronic enteritis, acute and chronic hepatitis, acute and chronic nephritis, dermatitis, encephalitis, lymphitis, conjunctivitis, keratitis, iridocyclitis, otitis media, allergic rhinitis, asthma, pulmonary fibrosis, chronic obstructive pulmonary disease Diseases, allergic dermatitis, sickle cell disease, multiple sclerosis, systemic lupus erythematosus, lupus nephritis, lung cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, cervical cancer, breast cancer, leukemia, multiple myeloma, Diabetes and gout.
  • RNA viruses such as avian influenza viruses, such as H5N1 infection
  • the IL-1beta, IL-6 or/and TNF-alpha related diseases are selected from the IL-1beta, IL-6 or/and TNF-alpha related diseases listed in the specification, preferably pneumonia, myocarditis, acute and chronic Gastritis, acute and chronic enteritis, acute and chronic hepatitis, acute and chronic nephritis, dermatitis, encephalitis, lymphitis, conjunctivitis, keratitis, iridocyclitis, otitis media, allergic rhinitis, asthma, pulmonary fibrosis chronic obstructive pulmonary disease , Allergic dermatitis, sickle cell disease, multiple sclerosis, systemic lupus erythematosus, lupus nephritis, lung cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, cervical cancer, breast cancer, leukemia, multiple myeloma, diabetes And gout.
  • RNA viruses such as avian Influenza virus, such as H5N1 infection
  • the IL-1beta, IL-6 or/and TNF-alpha related diseases are selected from the IL-1beta, IL-6 or/and TNF-alpha related diseases listed in the specification, such as pneumonia, myocarditis, acute and chronic Gastritis, acute and chronic enteritis, acute and chronic hepatitis, acute and chronic nephritis, dermatitis, encephalitis, lymphitis, conjunctivitis, keratitis, iridocyclitis, otitis media, allergic rhinitis, asthma, pulmonary fibrosis, chronic obstructive pulmonary disease Diseases, allergic dermatitis, sickle cell disease, multiple sclerosis, systemic lupus erythematosus, lupus nephritis, lung cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, cervical cancer, breast cancer, leukemia, multiple myeloma, Diabetes and gout.
  • the nucleic acid sequence or construct according to item 7 the recombinant virus according to item 8, the expression vector according to item 9,
  • a kit comprising the reagent according to item 23.
  • Figure 1 The expression of the inflammatory factor IL-1beta at the protein level relative to the control group in the cell inflammation model stimulated by LPS stimulated 9 hours after transfection of small RNA (BZL) small RNA as indicated in the THP1 cells 24 hours in advance .
  • BZL small RNA
  • Figure 2 Results of expression of the inflammatory factor IL-1beta at the protein level relative to the control group in a cell inflammation model stimulated with LPS-stimulated 9 hours after TPS1 cells were transfected with Chaihu (CHu) small RNA as indicated in the figure 24 hours in advance.
  • CHu Chaihu
  • Figure 3 The expression of inflammatory factor IL-1beta at the protein level relative to the control group in the cell inflammation model stimulated by LPS stimulated 9 hours after THP1 cells were transfected with Houttuynia cordata (YXC) small RNA as indicated in the figure 24 hours in advance .
  • YXC Houttuynia cordata
  • FIG. 4 THP1 cells were transfected with small RNAs as indicated in the figure 24 hours in advance and expressed the andrographis paniculata (CXL) and dandelion (PGY) small RNA. LPS stimulated 9 hours of cell inflammation model. The inflammatory factor IL-1beta was expressed at the protein level relative to the control group the result of.
  • CXL andrographis paniculata
  • PGY dandelion
  • Figure 5 Results of expression of inflammatory factor IL-6 at the protein level relative to the control group in a model of 9-hour cell inflammation induced by LPS stimulated 9 hours after transfection of small RNA (BZL) small RNA as indicated in THP1 cells.
  • Figure 6 The results of the expression of inflammatory factor IL-6 at the protein level relative to the control group in the THP1 cell transfected with the small RNA of Bupleurum chinense (CHu) as indicated in the figure 24 hours before LPS stimulation to the cell inflammation model for 9 hours.
  • CHu Bupleurum chinense
  • Figure 7 LPS stimulation after transfection of small RNAs such as DDi, Scutellaria baicalensis (HQi), Honeysuckle (JYH), Forsythia (LQi), and Prunella vulgaris (XKC) into THP1 cells 24 hours in advance
  • small RNAs such as DDi, Scutellaria baicalensis (HQi), Honeysuckle (JYH), Forsythia (LQi), and Prunella vulgaris (XKC) into THP1 cells 24 hours in advance
  • RNAs such as DDi, Scutellaria baicalensis (HQi), Honeysuckle (JYH), Forsythia (LQi), and Prunella vulgaris (XKC)
  • FIG. 8-9 THP1 cells were transfected with Houttuynia cordata (YXC) small RNA as indicated in the figure 24 hours in advance. LPS stimulated 9 hours of cellular inflammation model. The inflammatory factor IL-6 was expressed at the protein level relative to the control group. result.
  • YXC Houttuynia cordata
  • THP1 cells were transfected with andrographis paniculata (CXL) and Taraxacum officinale (PGY) small RNA as indicated in the figure 24 hours in advance.
  • LPS stimulated 9 hours of cell inflammation in the model of inflammatory factor IL-6 expressed at the protein level relative to the control group the result of.
  • FIG. 11 THP1 cells were transfected with small RNAs (BZL), Didi (DDi), Scutellaria baicalensis (HQi), Forsythia (LQi), and Prunella vulgaris (XKC) 24 hours before transfection LPS stimulated 9 hours of cell inflammation model, the expression of inflammatory factor TNF-alpha at the protein level relative to the control group.
  • BZL small RNAs
  • DDi Didi
  • HQi Scutellaria baicalensis
  • LQi Forsythia
  • XKC Prunella vulgaris
  • Figure 12 Results of expression of inflammatory factor TNF-alpha at the protein level relative to the control group in the model of 9-hour cell inflammation after LPS stimulated 9 hours of transfection of Bupleurum chinense (CHu) small RNA as indicated in the figure to THP1 cells.
  • CHu Bupleurum chinense
  • FIG. 13-14 THP1 cells were transfected with Houttuynia cordata (YXC) small RNA as indicated in the figure 24 hours before LPS stimulated 9 hours of cell inflammation model.
  • YXC Houttuynia cordata
  • FIG. 15 THP1 cells were transfected with dandelion (PGY) small RNA as indicated in the figure 24 hours in advance after LPS stimulated 9 hours of cell inflammation model.
  • PGY dandelion
  • Figure 16 Results of expression of the inflammatory factor IL-1beta at the mRNA level relative to the control group in a model of 9-hour cell inflammation induced by LPS stimulated 9-hour small RNA (BZL) small RNA as indicated in the figure after transfecting THP1 cells 24 hours in advance.
  • BZL 9-hour small RNA
  • FIG. 17 THP1 cells were transfected 24 hours in advance with Bupleurum (CHu), Forsythia (LQi), and Houttuynia cordata (YXC) small RNA as indicated in the figure.
  • LPS stimulated the inflammatory factor IL- in 9 hours of cell inflammation model The results of 1beta expression at the mRNA level relative to the control group.
  • THP1 cells were transfected with Houttuynia cordata (YXC) small RNA as indicated in the figure 24 hours before LPS stimulated 9 hours of cell inflammation model.
  • Figure 20 The expression of inflammatory factor IL-1beta at the mRNA level relative to the control group in a model of 9-hour cell inflammation induced by LPS stimulation of THP1 cells 24 hours before transfection of dandelion (PGY) small RNA as indicated in the figure.
  • PGY dandelion
  • Figure 21-22 The expression of inflammatory factor IL-6 at the mRNA level relative to the control group in the model of 9-hour cell inflammation stimulated by LPS stimulated with small RNA (BZL) small RNA as indicated in the figure 24 hours in advance to THP1 cells .
  • BZL small RNA
  • Figure 23-24 The expression of inflammatory factor IL-6 at the mRNA level relative to the control group in the model of 9-hour cell inflammation stimulated by LPS stimulated with Chaihu (CHu) small RNA as indicated in the figure 24 hours in advance to THP1 cells.
  • CHu Chaihu
  • FIG. 25 TPS1 cells were transfected with DDI, Scutellaria baicalensis (HQi), Honeysuckle (JYH), Forsythia (LQi), and Prunella vulgaris (XKC) small RNAs 24 hours in advance before transfection 9
  • FIG. 26-27 THP-1 cells were transfected with Houttuynia cordata (YXC) small RNA as indicated in the figure 24 hours before LPS stimulated 9 hours of cell inflammation model. Inflammatory factor IL-6 was expressed at the mRNA level relative to the NC group the result of.
  • YXC Houttuynia cordata
  • FIG. 28 THP1 cells were transfected with andrographis paniculata (CXL) and dandelion (PGY) small RNA as indicated in the figure 24 hours in advance.
  • LPS stimulated 9 hours of cellular inflammation model in the model of inflammatory factor IL-6 at the mRNA level relative to the NC group. result.
  • FIG. 29 THP1 cells were transfected 24 hours in advance with the small RNA of Scutellaria barbata (BZL) and Bupleurum chinense (CHu) as indicated in the figure. LPS stimulated 9 hours of cell inflammation model. The inflammatory factor TNF-alpha at the mRNA level relative to NC The results expressed by the group.
  • BZL Scutellaria barbata
  • CHu Bupleurum chinense
  • Figure 30 LPS stimulated inflammation in a 9-hour cell inflammation model after THP1 cells were transfected with small RNAs as indicated in the figure for 24 hours in advance, as shown in the figure: DDi, Honeysuckle (JYH), Forsythia (LQi) and Prunella vulgaris (XKC). The expression of factor TNF-alpha at the mRNA level relative to the NC group.
  • FIG. 31-32 THP1 cells were transfected with Houttuynia cordata (YXC) small RNA as indicated in the figure 24 hours in advance, and the expression of inflammatory factor TNF-alpha at the mRNA level relative to the NC group in the 9-hour cell inflammation model stimulated by LPS .
  • YXC Houttuynia cordata
  • Figure 33 Expression of the inflammatory factor TNF-alpha at the mRNA level relative to the NC group in THP1 cells transfected with dandelion (PGY) small RNA as indicated in the figure 24 hours before LPS stimulation 9 hours of cell inflammation model.
  • PY dandelion
  • Figure 34A-C BZL small RNA: H5N1 (0.4MOI) infection The results of the rescue of the specified Scutellaria barbata (BZL) small RNA on cell death;
  • Figure 34D-G CHu small RNA: H5N1 (0.4MOI) infection The rescue effect of the small RNA of Bupleurum chinense (CHu) specified in the latter figure on cell death;
  • Figure 34H LQi/XKC small RNA: H5N1 (0.4MOI) infection The specified forsythia (LQi)/Prunella vulgaris (XKC) in the figure after infection The rescue results of small RNA on cell death;
  • Figure 35 Expression of inflammatory factor IL-1beta at the protein level relative to the NC group in THP1 cells transfected with a small RNA mixture as indicated in the figure 24 hours before LPS stimulation 9 hours in the cell inflammation model.
  • FIG. 36-37 THP1 cells were transfected with the small RNA mixture as indicated in the figure 24 hours in advance, and the expression of inflammatory factor IL-6 at the protein level relative to the NC group in the model of cell inflammation stimulated by LPS for 9 hours.
  • Figure 38 Expression of the inflammatory factor IL-1beta at the mRNA level relative to the NC group in THP1 cells transfected with a small RNA mixture as indicated in the figure 24 hours before LPS stimulation for 9 hours.
  • Figure 39-40 The expression of inflammatory factor IL-6 at the mRNA level relative to the NC group in THP1 cells transfected with a small RNA mixture as indicated in the figure 24 hours before LPS stimulation 9 hours in the cell inflammation model.
  • Figure 41 Expression of inflammatory factor TNF-alpha at the mRNA level relative to the NC group in THP1 cells transfected with a small RNA mixture 24 hours in advance and LPS stimulated 9 hours in the cell inflammation model.
  • Figure 42 Mice fed small RNA three days in advance, LPS stimulated animal inflammation model for 9 hours, the expression of inflammatory factor TNF-alpha in alveolar lavage fluid at protein level relative to NC group.
  • Figure 43 Mice fed small RNA three days in advance, LPS stimulated 9 hours of animal inflammation model, the expression of inflammatory factor IL-6 in lung exfoliated cells at the mRNA level relative to the NC group.
  • the invention discloses some small RNAs and their ability to inhibit any one or more of the pathways or genes listed in Table 3 or reduce or down-regulate the expression levels of IL-1beta, IL-6 or/and TNF-alpha in vivo or in vitro Or to treat or prevent the application of IL-1beta, IL-6 or/and TNF-alpha-related diseases and/or H5N1 infection in the subject, those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve.
  • all similar substitutions and modifications will be obvious to those skilled in the art, and they are all considered to be included in the present invention.
  • the nucleic acids and applications of the present invention have been described through preferred embodiments, and it is obvious that relevant persons can modify or appropriately modify and combine the applications described herein without departing from the content, spirit, and scope of the present invention.
  • the invention technology is described through preferred embodiments, and it is obvious that relevant persons can modify or appropriately modify and combine the applications described herein without departing from the content, spirit
  • small RNA RNA
  • sRNA small RNA
  • shRNA small RNA
  • subject refers to, for example, a subject in need of prevention who has inflammation and/or H5N1 infection and needs treatment or has potential signs of inflammation development and/or is susceptible to H5N1 infection.
  • the sequence "identity” is used herein to describe the correlation between two amino acid sequences or between two nucleotide sequences.
  • EMBOSS European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. [Genetics Trends] 16:276-277)
  • Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) implemented in the Needle program to determine the sequence identity between two deoxyribonucleotide sequences.
  • the parameters used were a gap opening penalty of 10, a gap extension penalty of 0.5, and EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix.
  • Needle output labeled "Longest Consistency” obtained using the -non-simplified (–nobrief) option
  • percentage consistency calculate as follows:
  • the present invention covers having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity with the sequence shown in any of SEQ ID NO. 1-222 sexual sequence.
  • stringent conditions may refer to, for example, hybridization in 4 ⁇ SSC at 65° C., and then washing in 0.1 ⁇ SSC at 65° C. several times for about 1 hour in total.
  • stringent hybridization conditions as used herein may also refer to hybridization at 68°C, 0.25M sodium phosphate, 7% SDS, pH 7.2, 1 mM EDTA, and 1% BSA for 16 hours, and then at 68°C with 2 ⁇ SSC Wash twice with 0.1% SDS.
  • One skilled in the art can determine stringent conditions based on the specific sequence.
  • cell survival rate is also called cell survival rate.
  • the cell survival rate can be calculated using the MTS detection kit according to the manufacturer's instructions.
  • IL-1beta, IL-6 or/and TNF-alpha-related diseases refers to diseases characterized by increased expression levels of IL-1beta, IL-6 or/and TNF-alpha.
  • diseases characterized by increased expression levels of IL-1beta, IL-6 or/and TNF-alpha.
  • diseases characterized by increased expression levels of IL-1beta, IL-6 or/and TNF-alpha.
  • the inventors found that the small RNA sequence of the present invention reduces or down-regulates the expression level of IL-1beta, IL-6 or/and TNF-alpha in vivo or in vitro or treats or prevents IL-1beta, IL- 6 or/and TNF-alpha related diseases and/or H5N1 infection (thereby saving cell death due to H5N1 infection).
  • the sequence of the small RNA used in this experiment is shown in Table 1 below.
  • Table 1 The sequences of small RNAs used in this experiment are all commercially synthesized.
  • the use concentration of the above small RNA is 20 uM.
  • synthetic small RNA is used to test its ability to inhibit any one or more of the pathways or genes listed in Table 3, or to reduce or down-regulate IL-1beta, IL-6 or in vitro or in vivo / And TNF-alpha expression levels and/or the ability to improve cell survival and/or treat or prevent IL-1beta, IL-6 or/and TNF-alpha related diseases and/or H5N1 infection in a subject.
  • the small RNA used targets or reduces the same pathway or gene in Table 3.
  • the small RNA used reduces or down-regulates the expression level of one of IL-1beta, IL-6 and TNF-alpha. In one embodiment, the small RNA used increases cell survival. In one embodiment, the cell survival rate is the survival rate of H5N1 infected cells. In one embodiment, small RNA is used to treat or prevent a disease associated with increased expression levels of one of IL-1 beta, IL-6, and TNF-alpha in a subject.
  • the expression vector expressing the small RNA of the present disclosure may be directly introduced into a subject or a test cell, thereby inhibiting any one or more of the pathways or genes listed in Table 3, or used to reduce or down-regulate in vitro or in vivo IL-1beta, IL-6 or/and TNF-alpha expression levels and/or improve cell survival and/or treat or prevent IL-1beta, IL-6 or/and TNF-alpha related diseases and/or in subjects Or H5N1 infection, provided that the expression vector can be expressed in a subject or a test cell.
  • the expression vector can be expressed in a subject or a test cell.
  • those skilled in the art can also prepare constructs that express small RNA in cells, such as retroviral constructs, and transfect the packaging cell line with the constructs to produce recombinant retroviral particles, which can then be used in vitro. Or infect target cells in vivo to inhibit any one or more of the pathways or genes listed in Table 3, reduce or down-regulate the expression levels of IL-1beta, IL-6 or/and TNF-alpha and/or improve cell survival rate , And/or treatment or prevention of IL-1beta, IL-6 or/and TNF-alpha related diseases and/or H5N1 infection in the subject.
  • constructs that express small RNA in cells such as retroviral constructs
  • transfect target cells in vivo to inhibit any one or more of the pathways or genes listed in Table 3, reduce or down-regulate the expression levels of IL-1beta, IL-6 or/and TNF-alpha and/or improve cell survival rate , And/or treatment or prevention of IL-1beta,
  • a person skilled in the art can introduce cells containing an expression vector or construct into a subject or cell in vitro or in vivo to achieve the above object of the present invention.
  • one skilled in the art can isolate the small RNA of the present disclosure from cells by conventional techniques. Therefore, a method of expressing small RNA is encompassed, which includes the steps of expressing cells under suitable conditions and recovering the small RNA.
  • the invention also encompasses reagents for detecting small RNAs, constructs, recombinant viruses, expression vectors, cells and/or pharmaceutical compositions.
  • reagents for detecting small RNAs, constructs, recombinant viruses, expression vectors, cells and/or pharmaceutical compositions Those skilled in the art can also use detection reagents to detect cells from different sources to detect whether the small RNA of the present disclosure is covered therein.
  • the reagent is a primer and/or a probe.
  • the design or use of reagents is well known to those skilled in the art.
  • the small RNA is BZL-sRNA-20.
  • the inventors found that BZL-sRNA-20 has a good effect in inhibiting TNF-alpha, IL-1beta or IL-6 protein or mRNA. Therefore, the inventors chose BZL-sRNA-20 as the base small RNA and combined with other small RNAs to prepare the small RNA mixture described in Table 2.
  • the small RNA mixtures in Table 2 were used for each test.
  • a siRNA mixture is prepared by mixing 20 uM of BZL-sRNA-20 with each 20 uM of other small RNAs in a volume ratio of 2:1.
  • the molar concentration of BZL-sRNA-20 and other small RNAs is 2:1.
  • the molar concentration of total small RNA in the small RNA mixture is 20 uM.
  • the mixture is indicated by the symbol MIX.
  • the small RNA of the present invention can treat IL-6 related diseases.
  • IL-6 related diseases include:
  • obstructive airway diseases including chronic obstructive pulmonary disease (COPD); asthma, such as bronchial, allergic, endogenous, exogenous and dusty asthma, especially chronic or accumulative asthma (such as advanced asthma) And airway hyperresponsiveness); bronchitis; acute, allergic, atrophic rhinitis and chronic rhinitis, including caseous rhinitis, hypertrophic rhinitis, suppurative rhinitis, dry rhinitis and drug-induced rhinitis; membranous rhinitis, including Lubber, fibrinous and pseudomembranous rhinitis, and adenopathy; seasonal rhinitis, including neurorhinitis (hay fever) and vasomotor rhinitis, sinusitis, and idiopathic pulmonary fibrosis (IPF); Sarcoidosis, farmers' lungs and related diseases, adult respiratory distress syndrome, hypersensitivity pneumonia, fibrotic lung,
  • COPD chronic o
  • Rheumatoid arthritis juvenile chronic arthritis, juvenile arthritis systemic onset, seronegative spondyloarthropathy (including ankylosing spondylitis, psoriatic arthritis and Wright's disease), Becht disease, She Glenn syndrome and systemic sclerosis, gout, osteoporosis and osteoarthritis;
  • Cachexia multiple sclerosis, atherosclerosis, acquired immunodeficiency syndrome (AIDS), mesangial proliferative glomerulonephritis, nephrotic syndrome, nephritis, glomerulonephritis, acute Renal failure, hemodialysis, uremia, local or discoid lupus erythematosus, systemic lupus erythematosus, Castleman disease, Hashimoto's thyroiditis, myasthenia gravis, type I diabetes, type B insulin-resistant diabetes, sickle cells Anemia, iridocyclitis/ uveitis/ optic neuritis, nephritis syndrome, eosinophilic fasciitis, high IgE syndrome, systemic vasculitis/ Wegener's granulomatosis, orchitis/vasectomy Reversal procedure (reversal procedure), leprosy, alcohol-induced hepatitis, Se
  • AIDS acquired immunodeficiency
  • Allograft rejection Acute and chronic rejection after kidney, heart, liver, lung, pancreas, bone marrow, bone, small intestine, skin, cartilage, and corneal transplantation; and chronic graft-versus-host disease;
  • Malignant disease Leukemia, acute lymphoblastic leukemia (ALL), acute leukemia, T cells, B cells or FABALL, chronic myelogenous leukemia (CML), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, myelodysplastic syndrome (MDS), lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, malignant lymphoma, Burkitt's lymphoma, multiple myeloma, Kaposi Jeremy's sarcoma, renal cell carcinoma, colorectal cancer, prostate cancer, pancreatic cancer, nasopharyngeal cancer, malignant histiocytosis, tumor-associated syndrome/malignant hypercalcemia, solid tumor, adenocarcinoma, sarcoma, malignant melanoma , Hemangioma, metastatic disease, cancer-related bone resorption, cancer-related bone pain;
  • Cystic fibrosis stroke, reperfusion injury of the heart, brain, peripherallimbs and other organs; burns, trauma/bleeding, ionizing radiation exposure, chronic skin ulcers;
  • Reproductive diseases such as ovulation, menstruation and implantation diseases, premature delivery, preeclampsia, endometriosis); (infection) acute or chronic bacterial infections, acute and chronic parasitic processes or infection processes, including bacteria, viruses and fungi Infection, HIV infection/HIV neuropathy, meningitis, hepatitis (A, B, or C, or other viral hepatitis, etc.), septic arthritis, peritonitis, pneumonia, epiglottis, hemolytic uremic syndrome/thrombotic Thrombocytopenic purpura, malaria, dengue hemorrhagic fever, leishmaniasis, leprosy, toxic shock syndrome, streptococcal myositis, gas gangrene, Mycobacterium tuberculosis, Mycobacterium avium, Pneumocystis carinii pneumonia, pelvic inflammatory disease, orchitis/epididitis, legionella, Lyme disease, influenza A, Ep
  • the small RNA of the present invention can be used in combination with one or more of the following:
  • cytokine function for example, agents acting on cytokine signaling pathways, such as modulators of the SOCS system, such as alpha-, beta-, and/or gamma-interferon; type I Insulin-like growth factor (IGF-1), its receptors and related binding proteins; interleukins (IL), such as one or more of IL-1-33, and/or interleukin antagonists or inhibitors, such as Ana Interleukin; inhibitors of receptors of members of the interleukin family or inhibitors of specific subunits of these receptors; tumor necrosis factor alpha (TNF-alpha) inhibitors, such as anti-TNF monoclonal antibodies (eg, infliximab) Monoclonal antibody; adalimumab and/or CDP-870), and/or TNF receptor antagonists, such as immunoglobulin molecules (such as etanercept) and/or low molecular weight agents, such as pentoxy
  • B-lymphocytes eg CD20 (rituximab) or MRA-aIL16R
  • T-lymphocytes eg CTLA4-Ig, HuMaxIl-15 or Abatacept
  • -Modulators that inhibit osteoclast activity such as antibodies to RANKL;
  • chemokine or chemokine receptor function such as antagonists of the following chemokines: CCR1, CCR2, CCR2A, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10 and CCR11 ( For the CC family); CXCR1, CXCR2, CXCR3, CXCR4 and CXCR5 and CXCR6 (for the CXC family) and CX3CR1 of the C-X3-C family;
  • MMP -Matrix metalloproteinase
  • interstitial lysin interstitial lysin
  • collagenase and gelatinase and aggrecanase
  • MMP-1 collagenase-1
  • MMP-8 collagenase -2
  • MMP-13 collagenase-3
  • MMP-3 interstitial lysin-1
  • MMP-10 interstitial lysin-2
  • MMP-11 interstitial lysin-3
  • MMP-9 and/or MMP-12 such as doxycycline
  • -Leukotriene biosynthesis inhibitors for example: zileuton; ABT-761; fenureton ; Tepoxalin; Abbott-79175; Abbott-85761; N-(5-substituted)-thiophene-2-alkylsulfonamide; 2,6-di-tert-butylphenolhydrazone; methoxytetrahydropyran ,
  • Zenica ZD-2138 compound SB-210661; pyridyl-substituted 2-cyanonaphthalene compounds, such as L-739,010; 2-cyanoquinoline compounds, such as L-746,530; indole and/or quinoline Porphyrin compounds, such as MK-591, MK-886 and/or BAYx1005;
  • -Receptor antagonists of leukotriene (LT) B4, LTC4, LTD4 and LTE4 selected from: phenothiazine-3-1s, such as L-651,392; amidino compounds, such as CGS-25019c; aminobenzoxazole (Benzoxalamines), such as angazolate; benzenecarboximidamides, such as BIIL284/260; and such as zalulast, alulast, montelukast, prenastrel, velustrin ( MK-679), RG-12525, Ro-245913, ilalast (CGP45715A) and BAYx 7195 and other compounds;
  • phenothiazine-3-1s such as L-651,392
  • amidino compounds such as CGS-25019c
  • aminobenzoxazole (Benzoxalamines) such as angazolate
  • benzenecarboximidamides such as BIIL284/260
  • PDE -Phosphodiesterase
  • methylxanthanine such as theophylline and/or aminophylline
  • selective PDE isoenzyme inhibitors such as PDE4 inhibitors and/or the same Type PDE4D inhibitors, and/or PDE5 inhibitors
  • Histamine type 1 antagonists such as cetirizine, loratadine, desloratadine, fexofenadine, atorvastatin, terfenadine, astemizole, azetazole Ting, levocabastine, chlorpheniramine, prorubicin, seclizine, and/or mizolastine (usually oral, topical, or parenteral);
  • adrenergic receptor agonists such as cyclohexylmethanamine, phenylephrine, phenylpropanolamine, ephedrine, pseudoephedrine, naphazoline hydrochloride , Oxymetazoline hydrochloride, tetrahydrozoline hydrochloride, xymetazoline hydrochloride, tramazoline hydrochloride and ethyl norepinephrine hydrochloride;
  • -Anticholinergic drugs such as muscarinic receptor (M1, M2 and M3) antagonists such as atropine, scopolamine, glycopyrrrolate, ipratropium bromide, tiotropium bromide, oxitropium bromide Ammonium, Pirenzepine and Tilzepine;
  • M1, M2 and M3 antagonists such as atropine, scopolamine, glycopyrrrolate, ipratropium bromide, tiotropium bromide, oxitropium bromide Ammonium, Pirenzepine and Tilzepine;
  • -beta-adrenoceptor agonists include beta-receptor subtypes 1-4, such as isoproterenol, salbutamol, formoterol, salmeterol, terbutaline, m-hydroxyisopropyl Epinephrine, ditolidine mesylate and/or pibuterol, such as their chiral enantiomers;
  • -Chromone such as sodium cromoglycate and/or nedocromil sodium
  • -Glucocorticoids such as flunisolide, prednisolone acetone, clomidasone dipropionate, budesonide, fluticasone propionate, ciclesonide and/or mometasone furoate;
  • Ig immunoglobulin
  • Ig preparations or antagonists or antibodies that modulate Ig function such as anti-IgE (eg, omalizumab (Omalizumab));
  • -Other systemic or topical anti-inflammatory drugs such as thalidomide or its derivatives, retinoids, anthratriol and/or calcipotriol;
  • aminosalicylate and sulfapyridine such as sulfasalazine, mesalazine, balsalazide, and olsalazine; immunomodulators, such as thiopurines and corticosteroids, such as budesonide;
  • Antibacterial agents such as penicillin derivatives, tetracyclines, macrolides, beta-lactams, fluoroquinolones, metronidazole and/or inhaled aminoglycosides; and/or antiviral agents such as acyclovir , Famciclovir, valciclovir, ganciclovir, cidofovir; tricyclodecaneamine, amantadine; ribavirin; zanamivir and/or oseltamivir; protease inhibitors, for example Indinavir, nelfinavir, ritonavir, and/or saquinavir; nucleoside reverse transcriptase inhibitors, such as didanosine, lamivudine, stavudine, tazacitabine , Zidovudine; non-nucleoside reverse transcriptase inhibitors, such as nevirapine, efavirenz;
  • -Cardiovascular drugs such as calcium channel blockers, beta-adrenergic receptor blockers, angiotensin converting enzyme (ACE) inhibitors, angiotensin-2 receptor antagonists; lipid-lowering agents, such as statins And/or fibrates; modulators of blood cell morphology, such as pentoxifylline; thrombolytic and/or anticoagulant, such as blood cell aggregation inhibitors;
  • ACE angiotensin converting enzyme
  • lipid-lowering agents such as statins And/or fibrates
  • modulators of blood cell morphology such as pentoxifylline
  • thrombolytic and/or anticoagulant such as blood cell aggregation inhibitors
  • -CNS drugs such as antidepressants (eg sertraline), anti-Parkinson drugs (eg selegiline, levodopa, ropinirole, pramipexole, MAOB inhibitors such as selegine and Rasagiline, comP inhibitors, such as tokapon, A-2 inhibitors, dopamine reuptake inhibitors, NMDA antagonists, nicotinic agonists, dopamine agonists, and/or neuronal nitric oxide synthase inhibitors )
  • anti-Alzheimer's disease drugs such as donepezil, rivastigmine, tacrine, COX-2 inhibitors, pentamylline, or methotrexate;
  • -Agents for the treatment of acute and chronic pain such as centrally or peripherally acting analgesics, such as opioid analogs or derivatives, carbamizine, phenytoin, sodium valproate, amitryptiline or other antidepressants , Acetaminophen or non-steroidal anti-inflammatory drugs;
  • analgesics such as opioid analogs or derivatives, carbamizine, phenytoin, sodium valproate, amitryptiline or other antidepressants , Acetaminophen or non-steroidal anti-inflammatory drugs
  • -Anti-osteoporosis agents such as hormonal drugs such as raloxifene or bisphosphonates such as alendronate;
  • kinase inhibitors such as tyrosine kinase inhibitors (for example, examples of Btk, Itk, Jak3MAP inhibitors may include Gefitinib), methanesulfonate Imatinib acid), serine/threonine kinase (eg, MAP kinases such as inhibitors of p38, JNK, protein kinases A, B, and C, and IKK), or kinases involved in cell cycle regulation (eg, cycle-dependent Protein kinase);
  • glucose-6 phosphate dehydrogenase inhibitors (ix) kinin-B1- and/or B2-receptor antagonists; (x) anti-gout agents, such as colchicine; (xi) xanthine oxidation Enzyme inhibitors, such as allopurinol; (xii) uricosuric agents, such as probenecid, benzenesulfonone, and/or benzbromarone; (xiii) growth hormone secretagogue; (xiv) transforming growth factor ( (TGFbeta); (xv) platelet-derived growth factor (PDGF); (xvi) fibroblast growth factor, such as basic fibroblast growth factor (bFGF); (xvii) granulocyte macrophage colony-stimulating factor (GM- CSF); (xviii) capsaicin cream; (xix) tachykinin NK1 and/or NK3 receptor antagonists, such as NKP-608C, SB-23
  • the small RNA of the present invention can be used in combination with one or more of the following drugs:
  • non-steroidal anti-inflammatory drugs including non-selective cyclooxygenase (COX)-1/COX-2 inhibitors, regardless of local or systemic application (eg, piroxicam, diclofenac, propionic acid, Such as Meproxen, Flurbiprofen, Fenoprofen, Ketoprofen and Ibuprofen, Fennafil, such as Mefenamic acid, Indomethacin, Sulindac, Azaprozine, Pyrazole Ketones, such as bubutazolone, salicylates, such as aspirin; selective COX-2 inhibitors (eg, meloxicam, celecoxib, rofecoxib, valdecoxib, lumarocoxib), (Parecoxib and Etocoxib); cyclooxygenases (CINODs) that inhibit nitric oxide donors; glucocorticoids (whether
  • the small RNA of the present invention can also be used in combination with existing therapeutic agents for treating cancer.
  • Suitable agents that can be used in combination include: (i) antiproliferative/antitumor drugs for medical oncology and combinations thereof, such as Gleevec (imatinib mesylate), alkylating agents (e.g., cis Platinum, carboplatin, cyclophosphamide, nitrogen mustard, melphalan, neocarcinol, busulfan, and nitrosourea); antimetabolites (eg, antifolates, such as fluoropyrimidine, such as 5-fluorouracil, and Fluoride, raltitrexed, methotrexate, cytarabine, hydroxyurea, gemcitabine, and paclitaxel; antitumor antibiotics (eg, anthracycline antibiotics such as doxorubicin, bleomycin, doxorubicin , Daunorubicin,
  • Cytostatic drugs such as anti-estrogens (eg, tamoxifen, toremifene, raloxifene, droloxifene, and iodoxyfene), estrogen receptor down-regulators ( For example, fulvestrant), anti-androgens (for example, bicalutamide, flutamide, nilutamide, and cyproterone acetate), LHRH antagonists or LHRH agonists (for example, Gosere Lin, leuprolide, and buserelin), progestins (eg, megestrol acetate), aromatase inhibitors (eg, anastrozole, letrozole, vorazole, and exemestane) ) And 5 ⁇ -reductase inhibitors, such as finasteride;
  • anti-estrogens eg, tamoxifen, toremifene, raloxifene, droloxifene,
  • Agents that inhibit the invasion of cancer cells for example, inhibitors of metalloproteinases, such as inhibitors of the function of receptors such as marimastat and urokinase plasminogen activator);
  • Inhibitors of growth factor function include growth factor antibodies, growth factor receptor antibodies (eg, anti-erbb2 antibodies, trastuzumab and anti-erbb1 antibodies cetuximab [ C225]), farnesyl transferase inhibitors, tyrosine kinase inhibitors and serine/threonine kinase inhibitors, for example, epidermal growth factor family inhibitors (for example, EGFR family tyrosine kinase inhibitors, such as N -(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinylpropoxy)quinazolin-4-amine (gefitinib, AZD1839), N-( 3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine (erlotinib (erlotinib), OSI-774) and 6-propene Amido-
  • anti-angiogenic agents such as those that inhibit the action of vascular endothelial growth factor, (eg, anti-vascular endothelial growth factor antibody bevacizumab, international patent applications WO97/22596, WO97/30035, WO97/32856 and Compounds disclosed in WO98/13354, each patent is incorporated herein in its entirety) and compounds that function by other mechanisms (eg, linalamide, inhibitors of integrin ⁇ vbeta3 function, and angiostatin);
  • vascular endothelial growth factor eg, anti-vascular endothelial growth factor antibody bevacizumab, international patent applications WO97/22596, WO97/30035, WO97/32856 and Compounds disclosed in WO98/13354, each patent is incorporated herein in its entirety
  • compounds that function by other mechanisms eg, linalamide, inhibitors of integrin ⁇ vbeta3 function, and angiostatin
  • Vascular disrupting agents such as combostatin A4 and the compounds disclosed in international patent applications WO99/02166, WO00/40529, WO00/41669, WO01/92224, WO02/04434, and WO02/08213 (parts by reference The full text of the patent is incorporated into this article);
  • Antisense therapy for example, those against the above targets, such as ISIS2503, anti-ras antisense;
  • Gene therapy methods including, for example, methods for replacing abnormal genes such as abnormal p53 or abnormal BRCA1 or BRCA2, GDEPT (gene-directed enzyme prodrug therapy) methods, such as the use of cytosine deaminase, thymidine kinase or bacteria Methods of nitroreductase and methods of increasing the patient's tolerance to chemotherapy or radiotherapy, such as multi-drug resistance gene therapy; and
  • Immunotherapy methods including, for example, in vitro and in vivo methods to increase the immunogenicity of the patient’s tumor cells, such as transfection with cytokines, such as interleukin 2, interleukin 4, or granulocyte macrophage colony-stimulating factor, to reduce T
  • cytokines such as interleukin 2, interleukin 4, or granulocyte macrophage colony-stimulating factor
  • the method of cell anergy is to use transfected immune cells, such as cytokine-transfected dendritic cells, cytokine-transfected tumor cell lines, and anti-idiotypic antibodies.
  • IL-1beta plays a key role in the pathology associated with various diseases involving immune and inflammatory factors.
  • the small RNA of the present invention can treat IL-1beta-related diseases. These diseases include, but are not limited to: Acquired Immunodeficiency Syndrome; Acquired Immunodeficiency-Related Diseases; Acquired Pernicious Anemia; Acute Coronary Syndrome; Acute and Chronic Pain (different forms of pain); Acute Idiopathic Polyneuropathy Inflammation; acute immune disease associated with organ transplantation; acute or chronic immune disease associated with organ transplantation; acute inflammatory demyelinating polyradiculoneuropathy; acute ischemia; acute liver disease; acute rheumatic fever; acute transversal Myelitis; Addison's disease; adult (acute) respiratory distress syndrome; adult Still's disease; alcoholic cirrhosis; alcohol-induced liver injury; allergic disease; allergy; baldness; alopecia areata; Alz Hammer's disease; allergic reactions; ankylosing
  • the invention encompasses combinations including the small RNA described herein and at least one additional agent listed below.
  • the combination may also include more than one additional agent, for example, 2 or 3 additional agents.
  • Exemplary combinations include the small RNA described herein and non-steroidal anti-inflammatory drugs (NSAIDS), such as ibuprofen.
  • Other exemplary combinations include the small RNA and corticosteroids described herein, including prednisolone.
  • Non-limiting examples of therapeutic agents for rheumatoid arthritis to which the small RNA of the present invention can be combined include the following: cytokine inhibitory anti-inflammatory drugs (CSAIDs); antibodies or antagonists against other human cytokines or growth factors Agents, for example, TNF, LT, IL-1 ⁇ , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-15, IL-16, IL- 18.
  • CSAIDs cytokine inhibitory anti-inflammatory drugs
  • Agents for example, TNF, LT, IL-1 ⁇ , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-15, IL-16,
  • the small RNA of the present invention can be combined with antibodies against cell surface molecules or ligands thereof including CD154 (gp39 or CD40L), such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2), CD90, CTLA.
  • CD154 gp39 or CD40L
  • Exemplary therapeutic agents used in combination with the small RNA of the present invention can interfere at different points in the autoimmune and subsequent inflammatory cascades, such as TNF antagonists, such as chimeric, humanized or human TNF antibodies, D2E7 ( PCT Publication No. WO 97/29131), CA2 (REMICADEa), CDP 571, and soluble p55 or p75 TNF receptors, their derivatives (p75TNFR1gG (ENBRELa) or p55TNFR1gG (Lenercept), and TNF-alpha converting enzyme (TACE) inhibition Agents, and other IL-1 inhibitors (interleukin-1 converting enzyme inhibitors, IL-1RA, etc.).
  • TNF antagonists such as chimeric, humanized or human TNF antibodies, D2E7 ( PCT Publication No. WO 97/29131), CA2 (REMICADEa), CDP 571, and soluble p55 or p75 TNF receptors, their derivatives (p75TNFR1gG (
  • agents used in combination with small RNA include interleukin 11, which functions in parallel with IL-1a function, depending on Agents that are functional in or consistent with IL-1a, such as IL-18 antagonists (eg, IL-18 binding proteins such as antibodies or soluble IL-18 receptors, or antigen-binding fragments thereof.
  • IL-18 antagonists eg, IL-18 binding proteins such as antibodies or soluble IL-18 receptors, or antigen-binding fragments thereof.
  • Additional agents include non-depleting anti-CD4 inhibitors, costimulatory pathway CD80 (B7.1) or CD86 (B7.2) antagonists, including antibodies, soluble receptors, antagonist ligands or antigen-binding fragments thereof.
  • Small RNA can also be combined with agents used to treat rheumatoid arthritis, such as methotrexate, 6-MP, azathioprine sulfasalazine, mesalazine, olsalazine chloroquinine/ Hydroxychloroquine, penicillamine, gold malate sulfide (intramuscular and oral), azathioprine, colchicine, corticosteroids (oral, inhalation and local injection), beta2 adrenergic receptor agonist (salbutamol, special Butaline, salmeterol), xanthine (theophylline, aminophylline), cromoglycate, nadolomib, ketotifen, ipratropium and ethodine, cyclosporine, FK506, Rapamycin, mycophenolate mofetil, leflunomide, NSAIDs such as ibuprofen, corticosteroids such as prednisolone,
  • IL-1beta converting enzyme inhibitors TNF-alpha Invertase (TACE) inhibitors
  • TACE TNF-alpha Invertase
  • T cell signaling inhibitors such as kinase inhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine, 6-mercaptopurine, angiotensin converting enzyme inhibitors, soluble cytokine receptors And its derivatives (for example, soluble p55 or p75 TNF receptors and derivatives p75TNFRIgG (ENBRELTM and p55TNFRIgG (Lenercept)), sIL-1RI, sIL-1RII, sIL-6R), anti-inflammatory cytokines (for example, IL- 4.IL-10, IL-11, IL-13 and TGFbeta), celecoxib, folic acid, hydroxychloroquine sulfate, profenixib
  • TACE TNF-alpha In
  • Non-limiting examples of therapeutic agents for inflammatory bowel disease to which the small RNA of the present invention can be combined include the following: budesonide; epidermal growth factor, corticosteroids, cyclosporin, sulfasalazine, amino water Salicylate, 6-mercaptopurine, azathioprine, metronidazole, lipoxygenase inhibitors, mesalazine, olsalazine, balsalazide, antioxidants, thromboxane inhibitors, IL-1 receptor antagonists Agents, anti-IL-1beta monoclonal antibodies, anti-IL-6 monoclonal antibodies, growth factors, elastase inhibitors, pyridyl-imidazole compounds, antibodies or antagonists against other human cytokines or growth factors, said Cytokines or growth factors such as TNF, LT, IL-1beta, IL-2, IL-6, IL-7, IL-8, IL-15, IL-16,
  • the small RNA of the present invention may be combined with antibodies against cell surface molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, and CD90, and their ligands.
  • Small RNA can also be combined with reagents such as methotrexate, cyclosporin, FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAIDs such as ibuprofen, corticosteroids such as strong Songlong, phosphodiesterase inhibitors, adenosine agonists, anticoagulants, complement inhibitors, adrenergic drugs, agents that interfere with signaling through pro-inflammatory cytokines such as TNF-alpha or IL-1 (e.g.
  • IL-1beta converting enzyme inhibitors such as kinase inhibitors, metalloproteinase inhibitors, sulfasalazine, Azathioprine, 6-mercaptopurine, angiotensin converting enzyme inhibitors, soluble cytokine receptors and their derivatives (such as soluble p55 or p75 TNF receptors, sIL-1RI, sIL-1RII, sIL-6R) and anti- Inflammatory cytokines (eg, IL-4, IL-10, IL-11, IL-13, and TGFbeta).
  • TNF-alpha converting enzyme inhibitors such as kinase inhibitors, metalloproteinase inhibitors, sulfasalazine, Azathioprine, 6-mercaptopurine, angiotensin converting enzyme inhibitors, soluble cytokine receptors and their derivatives (such as soluble p55 or p75 TNF receptors, sIL-1RI
  • TNF antagonists such as anti-TNF antibodies
  • D2E7 PCT Publication No. WO97/29131
  • CA2 CDP 571 TNFR-Ig construct
  • PDE4 inhibitors small RNA can be combined with corticosteroids, such as budesonide and dexamethasone.
  • Small RNAs can also be combined with reagents such as sulfasalazine, 5-aminosalicylic acid and olsalazine, as well as reagents that interfere with the synthesis or action of proinflammatory cytokines such as IL-1, such as IL-1beta converting enzyme Inhibitors and IL-1RA.
  • reagents such as sulfasalazine, 5-aminosalicylic acid and olsalazine
  • reagents that interfere with the synthesis or action of proinflammatory cytokines such as IL-1, such as IL-1beta converting enzyme Inhibitors and IL-1RA.
  • Small RNA can also be used with T cell signaling inhibitors, for example, tyrosine kinase inhibitor 6-mercaptopurine.
  • Small RNA can be combined with IL-11.
  • RNA can be combined with the following reagents: mesalazine, prednisone, azathioprine, mercaptopurine, infliximab, methylprednisolone sodium succinate, diphenoxylate/atropine sulfate, lorazone hydrochloride Piperamine, methotrexate, omeprazole, folate, ciprofloxacin/glucose-water, dihydrocodeinone tartrate/paracetamol, tetracycline hydrochloride, fluocinolone, metronidazole, thimerosal/ Boric acid, cholestyramine/sucrose, ciprofloxacin hydrochloride, scopolamine sulfate, pethidine hydrochloride, midazolam hydrochloride, oxycodone hydrochloride/paracetamol, promethazine hydrochloride, sodium phosphate, sulfamethoxazole/
  • Non-limiting examples of the therapeutic agent with which the small RNA of the present invention can be combined for multiple sclerosis include the following: corticosteroids, prednisolone, methylprednisolone, azathioprine, cyclophosphamide, cyclic Sporin, methotrexate, 4-aminopyridine, tizanidine, interferon-beta1a Biogen), interferon-beta1b Chiron/Berlex), interferon ⁇ -n3) (Interferon Sciences/Fujimoto), interferon- ⁇ (Alfa Wassermann/J&J), interferon beta1A-IF (Serono/Inhale Therapeutics), pegylated interferon (Peginterferon) ⁇ 2b (Enzon/Schering-Plough), copolymer 1 (Cop-1, Teva Pharmaceutical Industries, Inc.), hyperbaric oxygen, intravenous immunoglobulin, clabribine, antibodies or antagonists or inhibitor
  • the small RNA of the present invention can be combined with antibodies against cell surface molecules such as CD2, CD3, CD4, CD8, CD19, CD20, CD25, CD28, CD30, CD40, CD45, CD69, CD80 , CD86, CD90.
  • the small RNA of the present invention can also be combined with reagents such as FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAIDs such as ibuprofen, phosphodiesterase inhibitors, adenosine agonists, Anticoagulants, complement inhibitors, adrenergic drugs, agents that interfere with signaling via pro-inflammatory cytokines such as TNF-alpha or IL-1 (eg IRAK, NIK, IKK, p38 or MAP kinase inhibitors), IL- 1b convertase inhibitors, TACE inhibitors, T cell signaling inhibitors such as kinase inhibitors, metalloproteinase inhibitors, sulfasal
  • the small RNAs of the present invention can also be combined with reagents such as alemtuzumab, dronabinol, Unimed, daclizumab, mitoxantrone, zariroden hydrochloride (xaliproden hydrochloride), 4-aminopyridine, glatiramer acetate, natalizumab, sinnabidol, a-immunokine NNSO3, ABR-215062, AnergiX.MS, Chemokine receptor antagonist, BBR-2778, calagualine, CPI-1189, LEM (liposome-encapsulated mitoxantrone), THC.CBD (cannabinoid agonist), MBP-8298 , Mesupama (PDE4 inhibitor), MNA-715, anti-IL-6 receptor antibody, neuroxin (neurovax), pirfenidone allotrap 1258 (RDP-1258), sTNF-R1, tarampanay ( talampanel),
  • Non-limiting examples of therapeutic agents with which the small RNA of the present invention can be combined for the treatment or prevention of angina include the following: aspirin, nitroglycerin, isosorbide mononitrate, metoprolol succinate, atenolol, Metoprolol tartrate, arotipine sulfonate, diltiazem hydrochloride, isosorbide dinitrate, clopidogrel bisulfate, nifedipine, atorvastatin calcium, potassium chloride, furosemide, simvastatin , Verapamil hydrochloride, digoxin, propranolol hydrochloride, carvedilol, lisinopril, spironolactone, hydrochlorothiazide, enalapril maleate, nadolol, ramipril, Noparin sodium, heparin sodium, valsartan, sotalol hydrochloride, fenof
  • Non-limiting examples of therapeutic agents with which small RNA can be combined to treat or prevent ankylosing spondylitis include the following: ibuprofen, futarin and misoprostol, naproxen, meloxicam, indomethacin Simn, futarin, celecoxib, iprofenoxib, sulfasalazine, methotrexate, azathioprine, minocycline, prednisone, etanercept, and infliximab.
  • Non-limiting examples of therapeutic agents to which the small RNA of the present invention can be combined for the treatment or prevention of asthma include the following: salbutamol, salmeterol/fluticasone, montelukast sodium, fluticasone propionate, budesonide, and Pine, sameterol xinafoate, levosalbuterol hydrochloride, salbutamol sulfate/isotropium, prednisolone sodium phosphate, triamcinolone, beclomethasone dipropionate, isopropyl Propium bromide, azithromycin, pyributerol acetate, prednisolone, anhydrous theophylline, sodium methylprednisolone succinate, clarithromycin, zalastrin, formoterol fumarate, Influenza virus vaccine, methylprednisolone, amoxicillin trihydrate, flunisolide, allergy injection, sodium cromoglycate, fexofenadine
  • Non-limiting examples of therapeutic agents with which the small RNA of the present invention can be combined for the treatment or prevention of COPD include the following: salbutamol sulfate/isotropium, ipratropium bromide, salmeterol/fluticasone, salbutamol, salmetery Tronaphthalate, fluticasone propionate, prednisone, anhydrous theophylline, methylprednisolone sodium succinate, montelukast sodium, budesonide, formoterol fumarate, koji Anzapine, levofloxacin, guaiacol glyceryl ether, azithromycin, beclomethasone dipropionate, levosalbuterol hydrochloride, flunisolide, ceftriaxone sodium, amoxicillin trihydrate, gati Safloxacin, Zarastrin, Amoxicillin/Clavulanate Potassium, Flunisolide/Menthol, Chlorpheni
  • Non-limiting examples of therapeutic agents to which the small RNA of the present invention can be combined for the treatment or prevention of HCV include the following: interferon- ⁇ -2a, interferon- ⁇ -2b, interferon- ⁇ con1, interferon- ⁇ -n1, pegylated interferon- ⁇ -2a, pegylated interferon- ⁇ -2b, ribavirin, pegylated interferon alpha-2b+ribavirin, ursodeoxycholic acid, licorice Acid, thymus fascin, Maxamine, VX-497 and any compound used to treat HCV by interfering with the following targets: HCV polymerase, HCV protease, HCV helicase, HCV IRES (internal ribosome entry site ).
  • Non-limiting examples of therapeutic agents with which small RNA can be combined to treat or prevent idiopathic pulmonary fibrosis include the following: prednisone, azathioprine, salbutamol, colchicine, salbutamol sulfate, digoxin, ⁇ interferon, methylprednisolone sodium succinate (sod susu), lorazepam, furosemide, lisinopril, nitroglycerin, spironolactone, cyclophosphamide, ipratropium bromide, actinomycetes D, alteplase, fluticasone propionate, levofloxacin, metaproterenol sulfate, morphine sulfate, oxycodone hydrochloride, potassium chloride, triamcinolone, tacrolimus anhydrous, calcium , Interferon- ⁇ , methotrexate, mycophenolate mofetil and interferon- ⁇ -1b
  • Non-limiting examples of therapeutic agents to which the small RNA of the present invention can be combined for the treatment or prevention of myocardial infarction include the following: aspirin, nitroglycerin, metoprolol tartrate, enoxaparin sodium, heparin sodium, chloride bisulfate Pigregrave, carvedilol, atenolol, morphine sulfate, metoprolol succinate, warfarin sodium, lisinopril, isosorbide mononitrate, digoxin, furosemide, simvastatin , Ramipril, teniplase, enalapril maleate, torsemide, reteplase, losartan potassium, quinapril hydrochloride/magcarb, bumetanide , Alteplase, enalaprilat, amiodarone hydrochloride, tirofiban hydrochloride monohydrate, diltiazem hydrochloride
  • Non-limiting examples of therapeutic agents to which the small RNA of the present invention can be combined for the treatment or prevention of psoriasis include the following: calcipotriene, clobetasol propionate, triamcinolone, propionate halide Betaxol, tazorotene, methotrexate, fluocinolone, enhanced betamethasone dipropionate, fluocinolone acetate, acitretin, tar shampoo, betamethasone valerate, bran Mometasone acid, ketoconazole, prilocaine/fluocinolone, hydrocortisone valerate, fluoxycortisone, urea, betamethasone, clobetasol propionate/emoll, Fluticasone propionate, azithromycin, hydrocortisone, moist formula, folic acid, hydroxyprednisolone, pimecrolimus, coal tar, diflufenone diacetone, etanercept foli
  • Non-limiting examples of therapeutic agents to which the small RNA of the present invention can be combined for the treatment or prevention of psoriatic arthritis include the following: methotrexate, etanercept, propofol, celecoxib, Folic acid, sulfasalazine, naproxen, leflunomide, methylprednisolone acetate, indomethacin, hydroxychloroquine sulfate, prednisone, sulindac, betamethasone dipropionate, Infliximab, methotrexate, folate, triamcinolone, futarin, dimethyl sulfoxide, piroxicam, diclofenac sodium, ketoprofen, meloxicam, methyl Prednisolone, naproxone, tolmetin sodium, calcipotriol, cyclosporin, diclofenac/misoprostol, fluocinolone, glucosamine sulfate, gold
  • Non-limiting examples of therapeutic agents to which the small RNA of the present invention can be combined for the treatment or prevention of restenosis include the following: sirolimus, paclitaxel, everolimus, tacrolimus, ABT- 578 and paracetamol.
  • Non-limiting examples of therapeutic agents to which the small RNA of the present invention can be combined for the treatment or prevention of sciatica include the following: dihydrocodeinone tartrate/paracetamol, lofenixib, cyclobenzaprine hydrochloride, Methylprednisolone, naproxen, ibuprofen, oxycodone hydrochloride/paracetamol, celecoxib, valdecoxib, methylprednisolone acetate, prednisone, codeine phosphate/paracetamol, trihydrochloride Madol/paracetamol, metaxalone, meloxicam, methocarbamol, lidocaine hydrochloride, diclofenac sodium, gabapentin, dexamethasone, caripold, ketorolac tromethamine Salt, indomethacin, paracetamol, diazepam, naproxen, oxycodone hydrochloride, tizan
  • Non-limiting examples of therapeutic agents in which the small RNA of the present invention can be combined for the treatment or prevention of systemic lupus erythematosus include the following: NSAIDS, for example, futarin, naproxen, ibuprofen, piroxicam, indole Indomethacin, COX2 inhibitors, for example, celecoxib, profenoxib, valdecoxib, antimalarial drugs, for example, hydroxychloroquine, steroids, for example, prednisone, prednisolone, budesonide, dexamethasone Metaxone, cytotoxins, for example, azathioprine, cyclophosphamide, mycophenolate mofetil, methotrexate, PDE4 inhibitors or purine synthesis inhibitors, for example Small RNAs can also be combined with reagents such as sulfasalazine, 5-aminosalicylic acid, olsalazine
  • Small RNAs can also be used with T cell signaling inhibitors, such as tyrosine kinase inhibitors, or molecules that target T cell activation molecules, such as CTLA-4-IgG or anti-B7 family antibodies and anti-PD-1 family antibodies .
  • T cell signaling inhibitors such as tyrosine kinase inhibitors
  • molecules that target T cell activation molecules such as CTLA-4-IgG or anti-B7 family antibodies and anti-PD-1 family antibodies .
  • the small RNA of the present invention can be combined with IL-11 or anti-cytokine antibodies, such as fonotolizumab (anti-IFNg antibody), or anti-receptor receptor antibodies, such as anti-IL-6 receptor antibodies and Antibodies against molecules on the surface of B cells.
  • Small RNA can also be used with the following reagents: LJP 394 (Abetimus), an agent that depletes or inactivates B cells, such as rituximab (anti-CD20 antibody), Lymphast ( lymphostat)-B (anti-BlyS antibody), TNF antagonist, for example, anti-TNF antibody, D2E7 (PCT Publication No. WO 97/29131, CA2 CDP 571, TNFR-Ig construct (p75TNFRIgG And p55TNFRIgG (Lenercept)).
  • LJP 394 Abetimus
  • an agent that depletes or inactivates B cells such as rituximab (anti-CD20 antibody), Lymphast ( lymphostat)-B (anti-BlyS antibody), TNF antagonist, for example, anti-TNF antibody, D2E7 (PCT Publication No. WO 97/29131, CA2 CDP 571, TNFR-Ig construct (p75
  • TNF-alpha has a proven pathophysiological role in various human diseases, especially inflammatory disorders, immune and immunomodulatory disorders, infections that cause sepsis, endotoxin and cardiovascular shock, neurodegenerative diseases and malignancy disease.
  • the small RNA of the present invention can be applied to treat the diseases listed below, which is not considered to be a complete or exclusive list. It also includes other diseases that are not specifically mentioned and are directly or indirectly affected by TNF-alpha.
  • Autoimmune or chronic inflammation general inflammation chronic and/or autoimmune state, general immune-mediated inflammatory disorder, inflammatory CNS disease, affects eyes, joints, skin, mucosa, central nervous system, gastrointestinal tract, urinary tract Inflammatory diseases of the tract or lungs, general uveitis, retinitis, HLA-B27+ uveitis, Behcet's disease, dry eye syndrome, glaucoma, Sjgren syndrome, diabetes (including diabetes Neuropathy), insulin resistance, general arthritis status, rheumatoid arthritis, osteoarthritis, reactive arthritis and Reiter's syndrome (juvenile arthritis), juvenile arthritis, ankylosing spondylitis, multiple Sclerosis, Guillain-Barre syndrome, myasthenia gravis, amyotrophic lateral sclerosis, sarcoidosis, glomerulonephritis, chronic kidney disease, cystitis, psoriasis (including psoriasis joints) Inflammation), suppurative sweat
  • Acute inflammation and/or prevention of postoperative or post-traumatic inflammation and pain prevention of general postoperative inflammation, eye surgery (eg cataract (eye lens replacement) or glaucoma surgery), joint surgery (including arthroscopy surgery), joint-related structures ( (E.g. ligament) surgery, oral and/or dental surgery, minimally interventional cardiovascular procedures (e.g. PTCA, percutaneous transluminal atherectomy, placement of stents), laparoscopy and/or endoscopic abdomen Endo-gynecological procedures, endoscopic urology procedures (eg prostate surgery, ureteroscopy, cystoscopy, interstitial cystitis), general inflammation before and after surgery (prevention).
  • eye surgery eg cataract (eye lens replacement) or glaucoma surgery
  • joint surgery including arthroscopy surgery
  • joint-related structures (E.g. ligament) surgery
  • oral and/or dental surgery minimally interventional cardiovascular procedures (e.g. PTCA, percutaneous transluminal atherectomy, placement of stents), laparo
  • Neuropathy and neurodegenerative diseases Alzheimer's disease (Alzheimer's disease), Parkinson's disease, Huntington's disease, Bell's palsy (Bell'palsy), Creutzfeld-Jakob disease (Creutzfeld-Jakob disease). Cancer: cancer-related osteolysis, cancer-related inflammation, cancer-related pain, cancer-related cachexia, bone metastasis.
  • Pain acute and chronic forms of pain, whether these are caused by central or peripheral effects of TNF-alpha and whether they are classified as inflammatory, noxious, or neuropathic forms of pain, sciatica, low back pain, carpal tunnel Syndrome, complex regional pain syndrome (CRPS), gout, postherpetic neuralgia, fibromyalgia, local pain state, chronic pain syndrome due to metastatic tumors, dismenorrhea.
  • Infection bacterial, viral or fungal sepsis, tuberculosis, AIDS.
  • Cardiovascular diseases atherosclerosis, coronary artery disease, hypertension, dyslipidemia, cardiac dysfunction and chronic heart failure.
  • the TNF-alpha-related disease is spondyloarthropathy, lung-related disorders, coronary heart disease, metabolic disorders, anemia, pain, liver disorders, skin disorders, nail disorders, or vasculitis.
  • the TNF-alpha-related disease is age-related cachexia, Alzheimer's disease, cerebral edema, inflammatory brain injury, chronic fatigue syndrome, dermatomyositis, drug response, intraspinal and/or Peripheral edema, family periodic fever, Felty's syndrome, fibrosis, glomerulonephropathy (eg, glomerulonephritis or IgA nephropathy after streptococcal infection), prosthesis relaxation, microscopic polyangiitis, mixed Type connective tissue disorders, multiple myeloma, cancer and cachexia, multiple organ disorders, spinal dysplasia syndrome, orchitism osteolysis, pancreatitis, including acute, chronic, and pancreatic abscesses, periodontal polymy
  • the TNF-alpha-related disease is Crohn's disease-related disease, juvenile arthritis/Steir's disease (JRA), uveitis, sciatica, prostatitis, endometrium Ectopic, choroidal neovascularization, lupus, Sjogren's syndrome, and wet macular degeneration.
  • JRA juvenile arthritis/Steir's disease
  • Non-limiting examples of therapeutic drugs to which the small RNA of the present invention can be combined include the following: non-steroidal anti-inflammatory drugs (NSAIDs); cytokine inhibitory anti-inflammatory drugs (CSAIDs); CDP-571/BAY-10 -3356 (humanized anti-small RNA; Celltech/Bayer); cA2/infuzumab (chimeric anti-small RNA; Centocor); 75kd TNFR-IgG/etanercept (75kD TNF receptor-IgG fusion protein; Immunex; J. Invest. Med. (1996) Vol.
  • NSAIDs non-steroidal anti-inflammatory drugs
  • CSAIDs cytokine inhibitory anti-inflammatory drugs
  • CDP-571/BAY-10 -3356 humanized anti-small RNA; Celltech/Bayer
  • cA2/infuzumab chimeric anti-small RNA; Centocor
  • 75kd TNFR-IgG/etanercept 75kD TNF receptor-I
  • TNF-IgG 55kd TNF receptor-IgG fusion protein; Hoffmann-LaRoche
  • IDEC-CE9.1/SB 210396 non-depleted primate Anti-CD4 antibody; IDEC/SmithKline; DAB 486-IL-2 and/or DAB389-IL-2 (IL-2 fusion protein; Seragen; Anti-Tac (humanized anti-IL-2R ⁇ ; Protein Design Labs/Roche ); IL-4 (anti-inflammatory cytokines; DNAX/Schering); IL-10 (SCH 52000; recombinant IL-10, anti-inflammatory cytokines; DNAX/Schering); IL-4; IL-10 and/or IL- 4 agonist (eg, agonist antibody); IL-1RA (IL-1 receptor antagonist; Synergen/Amgen); TNF-bp/s-TNF (soluble TNF binding protein; R973401 (phosphodiesterase type IV inhibition Agents; MK-966 (COX-2 inhibitor
  • the small RNA of the present invention is used in combination with one of the following drugs to treat rheumatoid arthritis: small molecule inhibitor KDR (ABT-123), small molecule inhibitor of Tie-2; methotrexate; Prednisone; Celecoxib; Folic acid; Hydroxychloroquine sulfate; Rofexixi; etanercept; Infuzumab; Leflunomide; Naproxen; Valdecoxib; Sulfapyridine; Prednisolone methyl; Blow Fen; meloxicam; methylhydroprednisone acetate; gold sodium thiomalate; aspirin; azathiopurine; triamcinolone acetate; propoxyphene naphthylsulfonate/paracetamol; folate; Nabumetone; Diclofenac sodium; Piroxicam; Etodolic acid; Diclofenac sodium; Oxaprozine; Oxycodone hydro
  • the small RNA of the present invention is used in combination with one of the following drugs to treat TNF-alpha related diseases in which TNF-alpha activity is harmful: anti-IL12 antibody (ABT874); anti-IL18 antibody (ABT325); LCK Small molecule inhibitors; COT small molecule inhibitors; anti-Ill antibodies; MK2 small molecule inhibitors; anti-CD 19 antibodies; CXCR3 small molecule inhibitors; CCR5 small molecule inhibitors; CCRll small molecule inhibitors Agent; anti-E/L selectin antibody; small molecule inhibitor of P2X7; small molecule inhibitor of IRAK-4; small molecule agonist of glucocorticoid receptor; anti-C5a receptor antibody; small molecule of C5a receptor Molecular inhibitors; anti-CD32 antibodies; and CD32 as therapeutic proteins.
  • anti-IL12 antibody ABT874
  • anti-IL18 antibody ABT325
  • LCK Small molecule inhibitors COT small molecule inhibitors
  • anti-Ill antibodies MK2 small
  • the small RNA of the present invention is used in combination with antibiotics and anti-infective drugs.
  • Anti-infective drugs include those known in the art to treat viral, fungal, parasitic or bacterial infections.
  • antibiotic refers to a chemical substance that inhibits the growth of microorganisms or kills them.
  • the term includes antibiotics produced by microorganisms known in the art, as well as synthetic antibiotics (eg, analogs).
  • Antibiotics include, but are not limited to, clarithromycin (Biaxin), ciprofloxacin (Cipro), and metronidazole (Flagyl).
  • the small RNA of the present invention is used in combination with additional therapeutic agents for the treatment of sciatica or pain.
  • drugs that can be used to reduce or suppress the symptoms of sciatica or pain include hydrocodone ditartrate/paracetamol, rofexi, cyclobenzaprine hydrochloride, methylprednisone, naproxen, ibuprofen, Hydroxycodone hydrochloride/acetaminophen, celecoxib, valdecoxib, methylprednisolone acetate, prednisone, cocaine phosphate/paracetamol, tramadol hydrochloride/acetaminophen, metaxalone , Meloxicam, methocarbamol, lidocaine hydrochloride, diclofenac sodium, gabapentin, dexamethasone, calipordol, ketorolac, indomethacin, acetaminophen, diazepam, na
  • the small RNA of the present invention is used in conjunction with hemodialysis to prepare TNF-alpha-related diseases.
  • the small RNA of the present invention is used in combination with a drug used to treat Crohn's disease or Crohn's disease-related diseases.
  • Therapeutic drugs that can be used to treat Crohn's disease include mesalazine, prednisone, azathioprine, mercaptopurine, infuzumab, budesonide, azosulfapyridine salicylate, methylprednisolone, phenethyl Piperidine, Loperamide hydrochloride, methotrexate, folate, ciprofloxacin/glucose-water, hydrocodone ditartrate, tetracycline hydrochloride, fluocinol acetate, metronidazole, thimerosal /Boric acid, cholestyramine/sucrose, ciprofloxacin hydrochloride, genipramine sulfate, dilbutine hydrochloride, midazolam hydrochloride,
  • the small RNA of the present invention is used in combination with additional therapeutic drugs used to treat asthma.
  • drugs that can be used to reduce or suppress the symptoms of asthma include the following: salbutamol; salmeterol/fludroisone; sodium; fludroisone propionate; budesonide; prednisone; salmeterol xinafoate; hydrochloric acid Levalbuterol; Sulfate/Ipratropium; Prednisone Sodium Phosphate; Triamcinolone; Beclomethasone Dipropionate; Ipratropium Bromide; Azithromycin; Pibuterol Acetate, Prednisone , Anhydrous theophylline, methylprednisolone, clarithromycin, zalust, formoterol fumarate, influenza virus vaccine, methylprednisolone trihydrate, flunisolide, allergic allergy Injection, sodium cromoglycate, mecamyl ammonium hydrochloride, flunisolide
  • the small RNA of the invention is used in combination with additional therapeutic agents used to treat COPD.
  • drugs that can be used to alleviate or suppress the symptoms of COPD include salbutamol sulfate/ipratropium; ipratropium bromide; salmeterol/flufidone; salbutamol; salmeterol; xinafoate; flutisone propionate; Nisone; anhydrous theophylline; methylhydroprednisone sod succ; montelukast sodium; budesonide; formoterol fumarate; triamcinolone acetonide; levofloxacin; guaifenesin; a Zazimycin; beclomethasone; dipropionic acid; levalbuterol hydrochloride; flunisolide; sodium; trihydrate; gatifloxacin; zalastrin; amoxicillin/clavulanate; flunisolide/ Menthol; chlorphenir
  • the small RNA of the invention is used in combination with additional therapeutic agents used to treat IPF.
  • drugs that can be used to reduce or suppress IPF symptoms include prednisone; azathioprine; salbutamol; colchicine; sulfate; digoxin; interferon gamma; methylprednisolone sodsucc; furosemide Lisinopril; nitroglycerin; spironolactone; cyclophosphamide; ipratropium bromide; actinomycin d; alteplase; fluticasone propionate; levofloxacin; oxenaline sulfate; sulfuric acid Morphine; oxycodone hydrochloride; potassium chloride; triamcinolone acetonide; anhydrous tenamycin; calcium; alpha-interferon; methotrexate; mycophenolate mofetil.
  • the small RNA of the present invention is used in combination with additional therapeutic agents commonly used to treat spinal arthropathy.
  • additional therapeutic agents commonly used to treat spinal arthropathy.
  • NSAIDs non-steroidal anti-inflammatory drugs
  • COX 2 inhibitors including Celebrex, Vioxx; and Bextra, and etoricoxib.
  • Physical therapy is also commonly used to treat spondyloarthropathy, usually in combination with non-steroidal anti-inflammatory drugs.
  • the small RNA of the present invention is used in combination with additional therapeutic drugs used to treat ankylosing spondylitis.
  • additional therapeutic drugs used to treat ankylosing spondylitis.
  • drugs that can be used to reduce or suppress symptoms of ankylosing spondylitis include ibuprofen, diclofenac and misoprostol, naproxen, meloxicam, indomethacin, diclofenac, celecoxib, rofexixi , Azosulfapyridine salicylate, prednisone, methotrexate, azathioprine, metformin, prednisone, etanercept, and infuzumab.
  • the small RNA of the present invention is used in combination with additional therapeutic drugs used to treat arthritis in patients with psoriasis.
  • drugs that can be used to reduce or suppress the symptoms of arthritis in patients with psoriasis include methotrexate; etanercept; rofexixi; celecoxib; folic acid; azosulfapyridine salicylate; naproxen; leflunomide Rice; methylprednisolone acetate; indomethacin; hydroxychloroquine sulfate; sulindac; prednisone; betamethasone diprop increment; infuzumab; methotrexate; folic acid; triamcinolone diclofenac; dimethyl sulfoxide Piroxicam; diclofenac sodium; ketoprofen; meloxicam; prednisone; methylprednisolone; nabumetone; sodium tetrabenzoyl pyrrole
  • the small RNA of the present invention can be used in combination with another therapeutic drug used to treat restenosis.
  • drugs that can be used to reduce or inhibit restenosis include rapamycin, paclitaxel, everolimus, tenamycin, ABT-578, and acetaminophen.
  • the small RNA of the present invention can be used in combination with another therapeutic drug used to treat myocardial infarction.
  • drugs that can be used to reduce or suppress myocardial infarction include aspirin, nitroglycerin, metoprolol tartrate, enoxaparin sodium, heparin sodium, clopidogrel bisulfate, carvedilol, atenolol, sulfate Morphine, metoprolol succinate, warfarin sodium, lisinopril, isosorbide mononitrate, digoxin, furosemide, simvastatin, ramipril, teniplase, maleic acid Enalapril, torsemide, reteplase, losartan potassium, quinapril hydrochloride/magcarb, bumetanide, alteplase, enalaprilat, amiodarone hydrochloride, tirofil hydrochloride Ban m-hydrate, d
  • the small RNA of the present invention can be used in combination with another therapeutic drug used to treat angina pectoris.
  • drugs that can be used to reduce or suppress angina include: aspirin; nitroglycerin; isosorbide mononitrate; metoprolol succinate; atenolol; metoprolol tartrate; arotipine sulfonate, dilitiazem hydropchloride, isosorbide dinitrate, clopidogrel bisulfate; nifedipine; Lipitor; potassium chloride; furosemide; simvastatin; verapamil hydrochloride; digoxin; proprane hydrochloride Carrollol; Carvedilol; Lisinopril; sprionolactone; dihydrochlorothiazide; enalapril maleate; madolol; ramipril; enoxaparin sodium; heparin sodium; valsartan; sota hydroch
  • the small RNA of the invention is administered in combination with drugs commonly used to treat hepatitis C virus.
  • drugs include interferon- ⁇ -2a, interferon- ⁇ -2b, interferon- ⁇ conl, interferon- ⁇ -nl, pegylated interferon- ⁇ -2a, pegylated interferon- ⁇ -2b, Ribavirin, pegylated interferon- ⁇ -2b and ribavirin, androsodeoxycholic acid, glycyrrhizic acid, thymus fascin, Maxamine, and VX-497.
  • the small RNA of the present invention is combined with corticosteroids, vitamin D analogs, and topical or oral retinoic acid, or a combination thereof, for the treatment of psoriasis.
  • the small RNA of the present invention is used in combination with one of the following drugs for treating psoriasis: small molecule inhibitor of KDR (ABT-123), small molecule inhibitor of Tie-2, calcipotriene, propylene Clobetasol acid, triamcinolone acetonide, halobetasol propionate, tazorotene, methotrexate, fluocinolone acetate, fluocinolone, avitamin A, tar shampoo, betamethasone valerate, bran Ritamethasone, ketoconazole, pramoxine/fluroxine, hydrocortisone valerate, fludrocondensate, urea, betamethasone, clobetasol propionate/emoll, flutis
  • the small RNA of the present invention can be used in combination with other drugs for treating skin diseases.
  • the small RNA of the present invention is combined with PUVA therapy.
  • PUVA is a combination of psoralen (P) and long-wave ultraviolet rays, used to treat many different skin diseases.
  • the small RNA of the present invention can also be combined with pimecrolimus.
  • the antibody of the present invention is used to treat psoriasis, wherein the antibody is used in combination with vanamycin.
  • the tenamycin and the small RNA of the invention are administered in combination with methotrexate and/or cyclosporine.
  • the inventive small RNA of the invention is administered in combination with excimer laser treatment for psoriasis.
  • Non-limiting examples of other therapeutic drugs with which the small RNA of the present invention can be combined to treat skin diseases or nail diseases include UVA and UVB phototherapy.
  • Other non-limiting examples that can be used in combination with the small RNA of the present invention include anti-IL-12 and anti-IL-18 therapeutic drugs, including antibodies.
  • the small RNA of the present invention is used in combination with additional therapeutic agents for treating Bechet's disease.
  • Additional therapeutic drugs that can be used to treat Bechet's disease include, but are not limited to, prednisone, cyclophosphamide (Cytoxan), azathioprine (also known as azathioprine imuran), methotrexate, timethoprim/ Sinomin (also known as compound sinomin tablets or synergistic sulfamethoxazole) and folic acid.
  • any of the above-mentioned therapeutic drugs can affect patients suffering from TNF-alpha-related diseases in which TNF-alpha activity is harmful.
  • ELISA enzyme-linked immunosorbent assay
  • RT-qPCR real-time fluorescence quantitative PCR
  • RPMI 1640 medium (MACGENE, cat. CM10041), fetal bovine serum (FETAL BOVINE SERUM) (GE, cat. SV30160.03) are added to the medium at a ratio of 10%
  • RNA total RNA rapid extraction kit (Shanghai Feijie Biotechnology Co., Ltd., cat.no.220011), TRIZOL REAGENT (SIGMA, T9424-200ml), reverse transcription kit (High Capacity cDNA Reverse Transcription Kit, Thermo, 4368813 ), LightCycler 480 SYBR Green I Master (Roche, 04877352001)
  • ELISA test kit (DuoSet Human IL-1beta/IL-6/TNF-alpha, R&D, DY201/DY206/DY210), protease inhibitor (TargetMol, cat.no.C0001)
  • THP-1 cells mononuclear macrophages, purchased from the Cell Center of the Institute of Basic Medical Sciences of the Chinese Academy of Medical Sciences) in RPMI 1640 medium containing fetal bovine serum to the logarithmic growth phase and divide them into 12-well plates. 1ml culture medium/well, incubate at 37 degrees Celsius overnight and then conduct subsequent experiments
  • Blank group that is, blank group, refers to untreated cells, this group serves as a blank control
  • LPS group the group is treated as follows: 2ul RNAimax is diluted with 200ul Opti-MEM, added to the cells and stimulated with LPS, this group serves as a negative control;
  • NC group Random nonsense sequence 5’UUCUCCGAACGUGACACGUTT-3 (double-stranded, Genepharma) was added to the cells at the same concentration and same transfection as the experimental group sequence and stimulated with LPS, this group served as a negative control.
  • RNAimax RNAimax 2ul/100ul Opti-MEM
  • small RNA (20uM) 5ul/100ul Opti-MEM mix the above liquids and incubate at room temperature for 10 minutes, then add to the cells.
  • THP-1 cells mononuclear macrophages, purchased from the Cell Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) in RPMI 1640 medium containing fetal bovine serum to the logarithmic growth phase and divide into 12-well plates. 1ml culture medium/well, incubate at 37 degrees Celsius overnight and then conduct subsequent experiments.
  • Blank group refers to untreated cells, this group serves as a blank control
  • LPS group this group was diluted with 200ul Opti-MEM, 2ul RNAimax was added to the cells and stimulated with LPS, this group served as a negative control;
  • NC group Random nonsense sequence 5'UUC, UCC, GAA, CGU, GUC, ACG, UTT-3 (Genepharma) was added to the cells at the same concentration and same transfection as the experimental group sequence and stimulated with LPS. This group served as a negative control.
  • RNAimax 2ul/100ul Opti-MEM
  • small RNA (20uM) 5ul/100ul Opti-MEM mix the above liquids and incubate at room temperature for 10 minutes, then add to the cells.
  • RNA into cDNA Through reverse transcription kit (High-Capacity cDNA Reverse Transcription Kits, Applied Biosystems, cat.no.4368813) according to the manufacturer's instructions, reverse transcription of small RNA into cDNA, the reverse transcription system is as follows: Template RNA (150ng/ ⁇ l) 10 ⁇ l, 10X RT Buffer 2.0 ⁇ l, 25X dNTP Mix (100mM) 0.8 ⁇ l, 10X Random Primer (included in the kit) 2.0ul, MultiScribeTM reverse transcriptase 1.0 ⁇ l, RNase inhibitor 1.0 ⁇ l, Nuclease -free 1.2 Hl of H2O, after instantaneous centrifugation, put it into the PCR instrument to react, the reaction conditions are as follows: (1) 25 °C, 10min; (2) 37 °C, 120min; (3) 85 °C, 5min; (4) 4 °C, Terminate the reaction. After the reaction, add 20 ⁇ l RNase Free dH2
  • the total volume of the qPCR reaction system is 10 ⁇ l, including: 5 ⁇ L 2 ⁇ SYBR Green Master Mix, 0.5 ⁇ l forward primer (10 ⁇ M), 0.5 ⁇ l reverse primer (10 ⁇ M), 1 ⁇ l reverse transcription cDNA, 3 ⁇ l RNase Free dH2O.
  • the PCR reaction conditions are: 95°C, pre-denaturation for 5 minutes, and start to enter the PCR amplification cycle: (1) 95°C, 10s; (2) 55°C, 10s; (3) 72°C, 20s; 40 cycles in total; last 40s for 10s to cool down.
  • Both the forward and reverse primers of the amplification reaction were designed and synthesized by Beijing Qingke Xinye Biotechnology Co., Ltd.
  • the primer sequences used are as follows:
  • the primers for UBC are:
  • the primers for IL-1beta are:
  • the primers for IL-6 are:
  • the primers for TNF-alpha are:
  • F12 medium Hyclone
  • FBS Gibico
  • Model establishment and transfection synthetic small RNA, RNAimax, opti-MEM, H5N1 virus (A/Jilin/9/2004);
  • MTS cell activity detection kit MTS cell activity detection kit
  • A549 cells human lung adenocarcinoma epithelial cells, purchased from the American Model Culture Collection (ATCC, Rockville, MD, USA) were cultured in Ham's F12 nutrient medium (HyClone, Logan, UT, USA), divided into 96-well plates, 100 ⁇ L of cell-containing medium per well);
  • THP-1 cells monoonuclear macrophages, purchased from the Cell Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) to the logarithmic growth phase, divide into 12-well plates, 1ml medium/well, and incubate at 37 degrees Celsius overnight Conduct follow-up experiments.
  • Blank group refers to untreated cells, this group serves as a blank control
  • LPS group this group was diluted with 200ul Opti-MEM, 2ul RNAimax was added to the cells and stimulated with LPS, this group served as a negative control;
  • NC (native) group Random nonsense sequence 5'UUC, UCC, GAA, CGU, GUC, ACG, UTT-3 (double-stranded, Genepharma) was added to the cells at the same concentration and same transfection as the experimental group sequence and stimulated by LPS. Negative control.
  • RNAimax to transfect artificially synthesized plant small RNA mixture (Table 2)
  • the volume ratio of BZL-sRNA-20 to other small RNAs is 2:1 (the initial concentration of various small RNAs is 20uM), RNAimax 2ul/100ul Opti-MEM, small RNA mixed solution (20uM) 10ul/100ul Opti-MEM, mixed the above liquids and incubated at room temperature for 10 minutes, then added to the cells.
  • THP-1 cells mononuclear macrophages, purchased from the Cell Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) to the logarithmic growth phase, divide into 12-well plates, 1ml medium/well, and incubate at 37 degrees Celsius overnight Conduct follow-up experiments.
  • Blank group refers to untreated cells, this group serves as a blank control
  • LPS group this group was diluted with 200ul Opti-MEM, 2ul RNAimax was added to the cells and stimulated with LPS, this group served as a negative control;
  • NC group Random nonsense sequence 5’UUC, UCC, GAA, CGU, ACG, UTT-3 (double-stranded, Genepharma) was added to the cells at the same concentration and same transfection as the experimental group sequence and stimulated with LPS. This group served as a negative control.
  • RNAimax to transfect artificially synthesized plant small RNA mixture (Table 2), the volume ratio of BZL-sRNA-20 to other small RNA is 2:1, RNAimax 2ul/100ul Opti-MEM, small RNA mixture (20uM) 10ul /100ul Opti-MEM, mix the above liquids, incubate at room temperature for 10 minutes, and add to the cells.
  • LPS stimulation was added 24 hours after transfection, and the final concentration of LPS was 1ug/ml.
  • RNA sample 5.6 0.5ml TRI Reagent (sigma, T9424-200ML) lysed cells, 12,000rpm, 4°C, centrifuge for 5min, discard the pellet, add chloroform at a ratio of 200 ⁇ l/mlTRIzol, shake well and mix, leave at room temperature for 15min, 12,000rpm, 4 Centrifuge at 15°C for 15 minutes, draw the upper aqueous phase into another centrifuge tube, draw the upper aqueous phase into another new EP tube, add isopropyl alcohol at 0.5ml/ml TRIzol, mix well, let stand at room temperature for 5-10min, 12,000rpm , 4°C, centrifuge for 10min, discard the supernatant, add 1ml of 75% ethanol, gently shake the centrifuge tube, suspend the pellet, 8000g, 4°C, centrifuge for 5min, discard the supernatant as much as possible, dry at room temperature for 5-10min, and treat with 20 ⁇ l DEPC H2O dissolves the
  • RNA into cDNA Reverse transcription of small RNA into cDNA through reverse transcription kit (High-Capacity cDNA Reverse Transcription Kits, Applied Biosystems, cat.no.4368813), the reverse transcription system is as follows: template RNA (150ng/ ⁇ l) 10 ⁇ l, 10X RT Buffer 2.0 ⁇ l, 25X dNTP Mix (100mM) 0.8 ⁇ l, 10X Random Primer (included in the kit) 2.0ul, MultiScribeTM reverse transcriptase 1.0 ⁇ l, RNase inhibitor 1.0 ⁇ l, Nuclease-free H2O 1.2 ⁇ l After instantaneous centrifugation, the reaction conditions were as follows: (1) 25°C, 10min; (2) 37°C, 120min; (3) 85°C, 5min; (4) 4°C, to terminate the reaction. After the reaction, add 20 ⁇ l RNase Free dH2O to make up the final volume to 40 ⁇ l.
  • reverse transcription kit High-Capacity cDNA Reverse Tra
  • the total volume of the qPCR reaction system is 10 ⁇ l, including: 5 ⁇ L 2 ⁇ SYBR Green Master, 0.5 ⁇ l forward primer (10 ⁇ M), 0.5 ⁇ l reverse primer (10 ⁇ M), 1 ⁇ l cDNA obtained by reverse transcription, 3 ⁇ l RNase FreeH2O.
  • the PCR reaction conditions are: 95°C, pre-denaturation for 5 minutes, and start to enter the PCR amplification cycle: (1) 95°C, 10s; (2) 55°C, 10s; (3) 72°C, 20s; 40 cycles in total; last 40s for 10s to cool down.
  • Both the forward and reverse primers of the amplification reaction were designed and synthesized by Beijing Qingke Xinye Biotechnology Co., Ltd.
  • the UBC gene was used as an internal reference gene.
  • the primer sequences used are as follows:
  • mice weighing 20-23g at 7 weeks of age are divided into 4 groups, one of which remains untreated throughout the experiment, that is, the blank group.
  • BZL-sRNA-20 or NC small RNA doses of 1 nmol/mouse were given to mice by gavage 3 days, 2 days, and 1 day respectively, which were BZL-sRNA-20 or NC group (native group).
  • mice were given tracheal injection with a dose of LPS (1mg/ml) 50ul, 50ug/mouse, of which only the LPS treatment group was expressed as the LPS group.
  • the resulting lavage fluid was 800g, centrifuged for 5 minutes to obtain lung exfoliated cells, lysed the cells with 0.5ml Trizol (Thermo), centrifuged at 12,000rpm, 4°C for 5min, discarded the pellet, added chloroform at the ratio of 200 ⁇ l/mlTRIzol, and mixed thoroughly Keep at room temperature for 15min, 12,000rpm, 4°C, centrifuge for 15min, draw the upper aqueous phase into another centrifuge tube, draw the upper aqueous phase into another new EP tube, add 0.5ml/mlTRIZol and add isopropanol to mix Keep at room temperature for 5-10min, 12,000rpm, 4°C, centrifuge for 10min, discard the supernatant, add 1ml of 75% ethanol, gently shake the centrifuge tube, suspend the pellet, 8000g, 4°C, centrifuge for 5min, discard the supernatant as far as possible, and dry at room temperature Dry for 5-10 minutes and dissolve the
  • RNA into cDNA Reverse transcription of small RNA into cDNA by reverse transcription kit (High-Capacity cDNA Reverse Transcription Kits, Applied Biosystems, cat.no.4368813), the reverse transcription system is as follows: template RNA (150ng/ ⁇ l) 10 ⁇ l, 10X RT Buffer 2.0 ⁇ l, 25X dNTP Mix (100mM) 0.8 ⁇ l, 10X Random Primer (included in the kit) 2.0ul, MultiScribeTM reverse transcriptase 1.0 ⁇ l, RNase inhibitor 1.0 ⁇ l, Nuclease-free H2O 1.2 ⁇ l After instantaneous centrifugation, the reaction conditions were as follows: (1) 25°C, 10min; (2) 37°C, 120min; (3) 85°C, 5min; (4) 4°C, to terminate the reaction. After the reaction, add 20 ⁇ l RNase Free dH2O to make up the final volume to 40 ⁇ l.
  • reverse transcription kit High-Capacity cDNA Reverse Tra
  • the total volume of the qPCR reaction system is 10 ⁇ l, including: 5 ⁇ L 2 ⁇ SYBR Green Master, 0.5 ⁇ l forward primer (10 ⁇ M), 0.5 ⁇ l reverse primer (10 ⁇ M), 1 ⁇ l cDNA obtained by reverse transcription, 3 ⁇ l RNase FreeH2O.
  • the PCR reaction conditions are: 95°C, pre-denaturation for 5 minutes, and start to enter the PCR amplification cycle: (1) 95°C, 10s; (2) 55°C, 10s; (3) 72°C, 20s; 40 cycles in total; last 40s for 10s to cool down.
  • Both the forward and reverse primers of the amplification reaction were designed and synthesized by Beijing Qingke Xinye Biotechnology Co., Ltd.
  • the GAPDH gene was used as an internal reference gene.
  • the primer sequences used are as follows:
  • Blank group refers to untreated cells, this group serves as a blank control
  • LPS group this group was diluted with 200ul Opti-MEM, 2ul RNAimax was added to the cells and stimulated with LPS, this group served as a negative control;
  • NC group Random nonsense sequence 5'UUC, UCC, GAA, CGU, GUC, ACG, UTT-3 (Genepharma) was added to the cells at the same concentration and same transfection as the experimental group sequence and stimulated with LPS. This group served as a negative control.
  • Xftp version Xftp 5.0
  • XShell version XShell 5.0
  • step 4 select genes in each sample whose gene expression level FC is reduced by more than 1.5 times compared with the control group NC, and upload these genes to the Metacore database for analysis.
  • Select parameters ignore first line; species: homo sapiens; type: down register; p-value, FDR: no limit
  • Figure 1 The expression of the inflammatory factor IL-1beta at the protein level relative to the control group in the cell inflammation model stimulated by LPS stimulated 9 hours after transfection of small RNA (BZL) small RNA as indicated in the THP1 cells 24 hours in advance .
  • Figure 2 Results of expression of the inflammatory factor IL-1beta at the protein level relative to the control group in a cell inflammation model stimulated with LPS-stimulated 9 hours after TPS1 cells were transfected with Chaihu (CHu) small RNA as indicated in the figure 24 hours in advance.
  • BZL small RNA
  • FIG 3 The expression of inflammatory factor IL-1beta at the protein level relative to the control group in the cell inflammation model stimulated by LPS stimulated 9 hours after THP1 cells were transfected with Houttuynia cordata (YXC) small RNA as indicated in the figure 24 hours in advance .
  • Figure 4 THP1 cells were transfected with small RNAs as indicated in the figure 24 hours in advance and expressed the andrographis paniculata (CXL) and dandelion (PGY) small RNA. LPS stimulated 9 hours of cell inflammation model. The inflammatory factor IL-1beta was expressed at the protein level relative to the control group the result of.
  • CXL andrographis paniculata
  • PGY dandelion
  • Figure 5 Results of expression of inflammatory factor IL-6 at the protein level relative to the control group in a model of 9-hour cell inflammation induced by LPS stimulated 9 hours after transfection of small RNA (BZL) small RNA as indicated in THP1 cells.
  • Figure 6 The results of the expression of inflammatory factor IL-6 at the protein level relative to the control group in the THP1 cell transfected with the small RNA of Bupleurum chinense (CHu) as indicated in the figure 24 hours before LPS stimulation to the cell inflammation model for 9 hours.
  • CHu Bupleurum chinense
  • FIG 7 LPS stimulation after transfection of small RNAs such as DDi, Scutellaria baicalensis (HQi), Honeysuckle (JYH), Forsythia (LQi), and Prunella vulgaris (XKC) into THP1 cells 24 hours in advance
  • small RNAs such as DDi, Scutellaria baicalensis (HQi), Honeysuckle (JYH), Forsythia (LQi), and Prunella vulgaris (XKC) into THP1 cells 24 hours in advance
  • inflammatory factor IL-6 at the protein level relative to the control group in the 9-hour cellular inflammation model.
  • Figure 8-9 THP1 cells were transfected with Houttuynia cordata (YXC) small RNA as indicated in the figure 24 hours in advance. LPS stimulated 9 hours of cellular inflammation model. The inflammatory factor IL-6 was expressed at the protein level relative to the control group. result.
  • FIG. 10 THP1 cells were transfected with andrographis paniculata (CXL) and Taraxacum officinale (PGY) small RNA as indicated in the figure 24 hours in advance.
  • LPS stimulated 9 hours of cell inflammation in the model of inflammatory factor IL-6 expressed at the protein level relative to the control group the result of.
  • "*" indicates that statistical analysis is considered statistically significant by Unpaired test P ⁇ 0.05, indicating that it has the effect of inhibiting the expression of inflammatory factors in in vitro experiments.
  • the experimental results show that the small RNA as shown in Figures 5-10 is significantly higher than the NC group in reducing IL-6 protein expression.
  • the values in Figures 5-10 are all normalized relative to the NC group.
  • BZL-sRNA-20 inhibits IL-6 protein levels well.
  • FIG. 11 THP1 cells were transfected with small RNAs (BZL), Didi (DDi), Scutellaria baicalensis (HQi), Forsythia (LQi), and Prunella vulgaris (XKC) 24 hours before transfection LPS stimulated 9 hours of cell inflammation model, the expression of inflammatory factor TNF-alpha at the protein level relative to the control group.
  • Figure 12 Results of expression of inflammatory factor TNF-alpha at the protein level relative to the control group in the model of 9-hour cell inflammation after LPS stimulated 9 hours of transfection of Bupleurum chinense (CHu) small RNA as indicated in the figure to THP1 cells.
  • CHu Bupleurum chinense
  • FIG. 13-14 THP1 cells were transfected with Houttuynia cordata (YXC) small RNA as indicated in the figure 24 hours before LPS stimulated 9 hours of cell inflammation model. The expression of inflammatory factor TNF-alpha at the protein level relative to the control group .
  • Figure 15 THP1 cells were transfected with dandelion (PGY) small RNA as indicated in the figure 24 hours before transfection. LPS stimulated 9 hours of cell inflammation model. The expression of inflammatory factor TNF-alpha at the protein level relative to the control group. "*" indicates that statistical analysis is considered statistically significant by Unpaired test P ⁇ 0.05, indicating that it has the effect of inhibiting the expression of inflammatory factors in in vitro experiments.
  • FIG 16 THP1 cells were transfected with small RNA of Scutellaria barbata (BZL) as indicated in the figure 24 hours in advance. LPS stimulated 9 hours of cellular inflammation model. Inflammatory factor IL-1beta at the mRNA level (IL-1beta relative to UBC) The expression level) is relative to the expression of the control group.
  • Figure 17 THP1 cells were transfected 24 hours in advance with Bupleurum (CHu), Forsythia (LQi), and Houttuynia cordata (YXC) small RNA as indicated in the figure. LPS stimulated the inflammatory factor IL- in 9 hours of cell inflammation model The results of 1beta expression at the mRNA level relative to the control group.
  • CHu Bupleurum
  • Forsythia LiQi
  • YXC Houttuynia cordata
  • FIG. 18-19 THP1 cells were transfected with Houttuynia cordata (YXC) small RNA as indicated in the figure 24 hours before LPS stimulated 9 hours of cell inflammation model.
  • Figure 20 The expression of inflammatory factor IL-1beta at the mRNA level relative to the control group in a model of 9-hour cell inflammation induced by LPS stimulation of THP1 cells 24 hours before transfection of dandelion (PGY) small RNA as indicated in the figure.
  • "*" indicates that statistical analysis is considered statistically significant by Unpaired test P ⁇ 0.05, indicating that it has the effect of inhibiting the expression of inflammatory factors in in vitro experiments.
  • Figure 21-22 The expression of inflammatory factor IL-6 at the mRNA level relative to the control group in the model of 9-hour cell inflammation stimulated by LPS stimulated with small RNA (BZL) small RNA as indicated in the figure 24 hours in advance to THP1 cells .
  • Figure 23-24 The expression of inflammatory factor IL-6 at the mRNA level relative to the control group in the model of 9-hour cell inflammation stimulated by LPS stimulated with Chaihu (CHu) small RNA as indicated in the figure 24 hours in advance to THP1 cells.
  • CHu Chaihu
  • FIG. 25 TPS1 cells were transfected with DDI, Scutellaria baicalensis (HQi), Honeysuckle (JYH), Forsythia (LQi), and Prunella vulgaris (XKC) small RNAs 24 hours in advance before transfection 9
  • Figure 26-27 THP-1 cells were transfected with Houttuynia cordata (YXC) small RNA as indicated in the figure 24 hours before LPS stimulated 9 hours of cell inflammation model. Inflammatory factor IL-6 was expressed at the mRNA level relative to the NC group the result of.
  • FIG 28 THP1 cells were transfected with andrographis paniculata (CXL) and dandelion (PGY) small RNA as indicated in the figure 24 hours in advance.
  • LPS stimulated 9 hours of cellular inflammation model in the model of inflammatory factor IL-6 at the mRNA level relative to the NC group. result.
  • "*" indicates that statistical analysis is considered statistically significant by Unpaired test P ⁇ 0.05, indicating that it has the effect of inhibiting the expression of inflammatory factors in in vitro experiments.
  • the results showed that the small RNA as shown in Figures 21-28 was significantly higher in reducing IL-6 mRNA expression than the NC group.
  • the values in Figures 21-22 are all values obtained by normalization with respect to the NC group.
  • BZL-sRNA-20 has a lower value in the inflammatory factor IL-6qPCR, indicating that it has a better effect on inhibiting IL-6mRNA levels in the small RNA tested.
  • FIG 29 THP1 cells were transfected with small RNA (BZL) and Bupleurum chinense (CHu) small RNA as indicated in the figure 24 hours in advance.
  • LPS stimulated 9 hours of inflammatory factor TNF-alpha at the mRNA level (TNF- Alpha (relative to UBC) (relative expression) relative to the expression of the NC group.
  • Figure 30 LPS stimulated inflammation in a 9-hour cell inflammation model after THP1 cells were transfected with small RNAs as indicated in the figure for 24 hours in advance, as shown in the figure: DDi, Honeysuckle (JYH), Forsythia (LQi) and Prunella vulgaris (XKC).
  • FIG. 31-32 THP1 cells were transfected with Houttuynia cordata (YXC) small RNA as indicated in the figure 24 hours in advance, and the expression of inflammatory factor TNF-alpha at the mRNA level relative to the NC group in the 9-hour cell inflammation model stimulated by LPS .
  • Figure 33 Expression of the inflammatory factor TNF-alpha at the mRNA level relative to the NC group in THP1 cells transfected with dandelion (PGY) small RNA as indicated in the figure 24 hours before LPS stimulation 9 hours of cell inflammation model.
  • PGY dandelion
  • Figure 34A-C BZL: H5N1 (0.4M.O.I) infection results in the rescue of cell death by the small RNA of Scutellaria barbata (BZL) specified in the figure after infection.
  • Figure 34D-G CHu:H5N1 (0.4M.O.I) infection results of the rescue of cell death by the small RNA of Bupleurum (CHu) specified in the figure after infection.
  • Figure 34H LQi/XKC: H5N1 (0.4M.O.I) infection The results of small forsythia (LQi)/Prunella vulgaris (XKC) small RNA specified in the figure after rescue for cell death.
  • Figure 34I XKC/YXC: H5N1 (0.4M.O.I) infection
  • XKC Prunella vulgaris
  • YXC Houttuynia cordata
  • YXC: H5N1 (0.4M.O.I) infection results in the rescue of cell death by Houttuynia cordata (YXC) small RNA specified in the figure.
  • the statistical analysis in Figure 34 is considered statistically significant by Unpaired test P ⁇ 0.05, indicating that it has a salvage effect on cell death caused by H5N1 infection. "*” means P ⁇ 0.05 in Unpaired test, "**” means P ⁇ 0.01 in Unpaired test.
  • FIGS. 34A-N compared with the NC group, the small RNA specified in the figure significantly improves the cell survival rate, showing a more obvious effect of rescuing cell death.
  • the values in Figs. 34A-N are all values normalized with respect to the NC group. Among them, BZL-sRNA-20 is very effective in saving cell death.
  • Figure 35 Expression of inflammatory factor IL-1beta at the protein level relative to the NC group in THP1 cells transfected with a mixture of small RNAs 24 hours in advance and LPS stimulation for 9 hours.
  • the results showed that the mixture of small RNAs as shown in Fig. 28 was significantly higher than the NC group in reducing IL-1beta protein levels, of which MIX20, 24, 33, 36, 42 were higher than BZL-sRNA in reducing IL-1beta protein levels.
  • the values in Fig. 35 are all values obtained by normalization with respect to the NC group.
  • the molar concentration of BZL-sRNA-20 small RNA in the test cell fluid in the mixture is much lower than that in the BZL-sRNA-20 group The molar concentration of this indicates that the various small RNAs in the mixture also have a good synergistic effect in reducing the level of IL-1beta protein.
  • Figure 36-37 Expression of inflammatory factor IL-6 at the protein level relative to the NC group in the THP1 cell transfected with a small RNA mixture 24 hours in advance and LPS stimulated 9 hours in the cell inflammation model.
  • BZL-sRNA-20 and other small The RNA mixing ratio is 2:1 (v/v), "*" indicates that statistical analysis is considered statistically significant with Unpaired test P ⁇ 0.05, indicating that it has the effect of inhibiting the expression of inflammatory factors in in vitro experiments.
  • Figure 38 The expression of inflammatory factor IL-1beta at the mRNA level relative to the NC group in THP1 cells transfected with a small RNA mixture 24 hours in advance and LPS stimulated 9 hours in the cell inflammation model.
  • the results showed that the mixture of small RNAs as shown in FIG.
  • Fig. 38 was significantly higher than the NC group in reducing IL-1beta mRNA levels, and MIX23, 42, 43 were higher in reducing IL-1beta mRNA levels than the BZL-sRNA-20 group.
  • the values in Fig. 38 are all values obtained by normalization with respect to the NC group.
  • the molar concentration of the BZL-sRNA-20 small RNA in the test cell fluid in the mixture is much lower than that of the BZL-sRNA-20 group Concentration, which indicates that the various small RNAs in the mixture also have a very good synergistic effect in reducing IL-1beta mRNA levels.
  • Figure 39-40 The expression of inflammatory factor IL-6 at the mRNA level relative to the NC group in the THP1 cell transfected with a small RNA mixture 24 hours in advance and LPS stimulated 9 hours in the cell inflammation model, of which BZL-sRNA-20 and other small The RNA mixing ratio is 2:1 (v/v), "*" indicates that statistical analysis is considered statistically significant by Unpaired test P ⁇ 0.05, indicating that it has the effect of inhibiting the expression of inflammatory factors in in vitro experiments. The results showed that the mixture of small RNAs as shown in Figure 31 was significantly higher than the NC group in reducing IL-6mRNA levels.
  • MIX10 and 14 were removed in Figure 39, and MIX25-27, 30, 31, and 38 in Figure 40 were decreasing IL- 6mRNA level is higher than BZL-sRNA-20 group.
  • the values in Figures 39-40 are all normalized values relative to the NC group. Compared with the BZL-sRNA-20 group in reducing IL-6 mRNA levels, the molar concentration of BZL-sRNA-20 small RNA in the test cell fluid in the mixture is much lower than that in the BZL-sRNA-20 group This indicates that various small RNAs in the mixture have a good synergistic effect in reducing IL-6mRNA levels.
  • Figure 41 Expression of inflammatory factor TNF-alpha at the mRNA level relative to the NC group in THP1 cells transfected with a small RNA mixture 24 hours before LPS stimulation for 9 hours in a cell inflammation model, in which BZL-sRNA-20 was mixed with other small RNAs The ratio is 2:1 (v/v), "*" indicates that statistical analysis is considered statistically significant by Unpaired test P ⁇ 0.05, indicating that it has the effect of inhibiting the expression of inflammatory factors in in vitro experiments.
  • Figure 42 Mice fed small RNA three days in advance, LPS stimulated 9 hours of animal inflammation model, the expression of inflammatory factor TNF-alpha in alveolar lavage fluid at the protein level relative to the NC group, "*" indicates statistical analysis Unpaired test P ⁇ 0.05 is considered statistically significant, indicating that BZL-sRNA-20 can inhibit the expression of inflammatory factor TNF-alpha in in vivo experiments.
  • Figure 43 Mice fed small RNA 3 days in advance, LPS stimulated 9 hours of animal inflammation model, the expression of inflammatory factor IL-6 in the lung exfoliated cells at the mRNA level relative to the NC group, "**" statistical analysis Unpaired test P ⁇ 0.01 is considered statistically significant, indicating that it has the effect of inhibiting the expression of inflammatory factor IL-6 in in vivo experiments.
  • Example 4 Verification of the transcriptome sequencing results of artificially synthesized small RNA according to the LPS-stimulated THP-1 cell model. Calculation of the main target base of the down-regulation of small RNA and the classification of its pathway or biological process
  • 192 small RNA target genes and their associated pathways or biological processes are classified and tabulated.
  • Table 3 Select 192 small RNAs from Table 1 for transcriptome sequencing.

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Abstract

提供了小RNA、包含小RNA的组合物以及其使用方法和用途。所述小RNA或组合物可以抑制表3中列出的任一种或多种通路或基因的能力或在体内或体外降低或下调IL-1beta、IL-6或/和TNF-alpha的表达水平或治疗或预防受试者中IL-1beta、IL-6或/和TNF-alpha相关疾病和/或提高细胞生存率。

Description

炎性相关疾病防治的小RNA药物及其组合 技术领域
本发明一般涉及核酸治疗剂领域，更具体涉及小RNA及其使用方法和用途。
背景技术
炎症是十分常见而又重要的基本病理过程，体表的外伤感染和各器官的大部分常见病和多发病。炎症可以是感染引起的感染性炎症(如肺炎、心肌炎、急慢性胃炎、急慢性肠炎、急慢性肝炎、急慢性肾炎、皮炎、脑炎、淋巴炎、结膜炎、角膜炎、虹膜睫状体炎、中耳炎等)，也可以是不是由于感染引起的非感染性炎症，这种炎症通常与机体免疫密切相关(如过敏性鼻炎、哮喘、肺纤维化、慢性阻塞性肺疾病、过敏性皮炎、镰状细胞病、多发性硬化、系统性红斑狼疮、狼疮性肾炎等)，同时炎症也是癌症发病的主要诱发因素之一(如肺癌、胃癌、结直肠癌、肝癌、胰腺癌、宫颈癌、乳腺癌、白血病、多发性骨髓瘤等)，还有研究表明部分炎症因子还与代谢疾病相关(如糖尿病、痛风等)。通常情况下，炎症是有益的，是人体的自动的防御反应，但是有的时候，炎症也是有害的，例如对人体自身组织的攻击、发生在透明组织的炎症等等。
炎症的临床表现为红、肿、热、痛和功能障碍等。而炎症的生化指标通常是在炎症过程中参与、介导炎症反应的炎症因子的高表达，例如IL-1beta,IL-6和TNF-alpha等。目前对于炎症的预防和治疗仍以西药为主，但也有较多的中药产品有一定的抗炎作用，但都有其各自无法避免的缺点。因此，寻找新型的对抗炎症的治疗措施仍然是十分必要的。
发明内容
本申请部分基于发明人对一系列小RNA的发现。出乎意料地，发明人发现了本申请中的小RNA或其组合物可以降低或下调IL-1beta、IL-6或/和TNF-alpha的表达水平和挽救由于H5N1感染导致的细胞死亡。
本发明提供以下内容：
1.小RNA，其包含：
(A)以SEQ ID NO.1-222中任一项所示的序列，优选SEQ ID NO.20的序列，或其互补序列；
(B)与(A)所示序列具有至少80％-98％同一性的序列，其具有抑制表3中列出的任一种或多种通路或基因的能力；
(C)与(A)所示序列杂交的序列，优选地在严格条件下与(A)所示序列杂交的序列，其具有抑制表3中列出的任一种或多种通路或基因的能力；
(D)由(A)所示序列经添加、缺失、替换或插入一个或更多个，诸如2、3、4、5、6、7、8或9个碱基得到的序列，其具有抑制表3中列出的任一种或多种通路或基因的能力；或
(E)由(A)、(B)、(C)或(D)所示序列的前体或经修饰的变体，其具有抑制表3中列出的任一种或多种通路或基因的能力。
2.根据项1所述的小RNA，其具有抑制表3中列出的相同通路或基因的能力，或者具有预防和/或治疗IL-1beta、IL-6或/和TNF-alpha相关疾病的能力和/或提高细胞生存率的能力。
3.根据项2所述的小RNA，其中所述小RNA具有降低或下调IL-1beta、IL-6或/和TNF-alpha的表达水平的能力和/或具有挽救由于病毒，例如RNA病毒，例如禽流感病毒，例如H5N1感染导致的细胞死亡的能力，
优选地，所述小RNA具有降低或下调IL-1beta、IL-6和TNF-alpha中一种炎性因子的表达水平的能力，
优选地，所述IL-1beta、IL-6或/和TNF-alpha相关疾病选自说明书中列出的任一种或多种IL-1beta、IL-6或/和TNF-alpha相关疾病，优选肺炎、心肌炎、急慢性胃炎、急慢性肠炎、急慢性肝炎、急慢性肾炎、皮炎、脑炎、淋巴炎、结膜炎、角膜炎、虹膜睫状体炎、中耳炎、过敏性鼻炎、哮喘、肺纤维化慢性阻塞性肺疾病、过敏性皮炎、镰状细胞病、多发性硬化、系统性红斑狼疮、狼疮性肾炎、肺癌、胃癌、结直肠癌、肝癌、胰腺癌、宫颈癌、乳腺癌、白血病、多发性骨髓瘤、糖尿病和痛风。
4.根据项1-3中任一项所述的小RNA，其中所述小RNA为双链或单链形式或双链和单链的杂合形式。
5.根据项1-4中任一项所述的小RNA，其中所述小RNA为非天然的小RNA。
6.根据项1-5中任一项所述的小RNA，其中所述非天然的小RNA为经过人工合成或由人工载体表达获得的小RNA。
7.核酸序列或包含所述核酸序列的构建体，所述核酸序列包含编码根据项1-6中任一项所述的小RNA的序列，其中优选地所述构建体是病毒构建体，优选逆转录病毒构建体。
8.重组病毒，其包含根据项7所述的核酸序列或构建体，优选地，所述重组病毒是逆转录病毒。
9.表达载体，其包含编码根据项1-6中任一项所述的小RNA的序列。
10.细胞，其包含根据项7所述的核酸序列或构建体或用根据项8所述的重组病毒转染或包含根据项9所述的表达载体。
11.表达项1-6中任一项所述的小RNA的方法，其包括在合适的条件下表达项10所述的细胞并且回收根据项1-4中任一项所述的小RNA。
12.药物组合物，其包含一种或多种根据项1-6中任一项所述的小RNA、根据项7所述的核酸序列或构建体、根据项8所述的重组病毒、根据项9所述的表达载体和/或所述项10所述的细胞，
优选地，所述药物组合物是用于口服、静脉内施用，如推注或通过连续灌注一段时间，通过皮下、肌肉内、动脉内、腹膜内、肺内、脑脊髓内、关节内、滑膜内、鞘内、损伤内、或吸入路径如鼻内，通常通过静脉内或皮下施用的药物组合物。
13.根据项12所述的药物组合物，其包含表2中的混合物1至混合物43中的任一种或多种。
14.根据项13所述的药物组合物，其中在所述药物组合物中，包含以SEQ ID NO.20所示的序列的小RNA与组合物中的其它小RNA的摩尔浓度比率为约2:1。
15.根据项12-14中任一项的药物组合物，其还包含说明书中列出的一种或多种药物。
16.试剂盒，其包含一种或多种根据项1-6中任一项所述的小RNA、根据项7所述的核酸序列或构建体、根据项8所述的重组病毒、根据项9所述的表达载体和/或所述项10所述的细胞，优选地，所述试剂盒还包含 说明书中列出的一种或多种药物。
17.在体外或体内抑制表3中列出的任一种或多种通路或基因的方法，其包括对细胞或受试者施用一种或多种根据项1-6中任一项所述的小RNA、根据项7所述的核酸序列或构建体、根据项8所述的重组病毒、根据项9所述的表达载体、所述项10所述的细胞和/或根据项12-15中任一项的药物组合物。
18.在体外或体内降低或下调IL-1beta、IL-6或/和TNF-alpha的表达水平和/或提高细胞生存率的方法，其包括对细胞或受试者施用一种或多种根据项1-6中任一项所述的小RNA、根据项7所述的核酸序列或构建体、根据项8所述的重组病毒、根据项9所述的表达载体、所述项10所述的细胞和/或根据项12-15中任一项的药物组合物。
19.根据项18所述的方法，其中细胞生存率是病毒，例如RNA病毒，例如禽流感病毒，例如H5N1感染中的细胞生存率，优选地所述提高细胞生存率是通过挽救由于病毒，例如RNA病毒，例如禽流感病毒，例如H5N1感染导致的细胞死亡实现。
20.治疗或预防受试者中IL-1beta、IL-6或/和TNF-alpha相关疾病和/或病毒，例如RNA病毒，例如禽流感病毒，例如H5N1感染的方法，其包括对受试者施用一种或多种根据项1-6中任一项所述的小RNA、根据项7所述的核酸序列或构建体、根据项8所述的重组病毒、根据项9所述的表达载体、所述项10所述的细胞和/或根据项12-15中任一项的药物组合物，
优选地，所述IL-1beta、IL-6或/和TNF-alpha相关疾病选自说明书中列出的IL-1beta、IL-6或/和TNF-alpha相关疾病，优选肺炎、心肌炎、急慢性胃炎、急慢性肠炎、急慢性肝炎、急慢性肾炎、皮炎、脑炎、淋巴炎、结膜炎、角膜炎、虹膜睫状体炎、中耳炎、过敏性鼻炎、哮喘、肺纤维化、慢性阻塞性肺疾病、过敏性皮炎、镰状细胞病、多发性硬化、系统性红斑狼疮、狼疮性肾炎、肺癌、胃癌、结直肠癌、肝癌、胰腺癌、宫颈癌、乳腺癌、白血病、多发性骨髓瘤、糖尿病和痛风。
21.根据项1-6中任一项所述的小RNA、根据项7所述的核酸序列或构建体、根据项8所述的重组病毒、根据项9所述的表达载体、所述项10所述的细胞和/或根据项12-15中任一项的药物组合物，用于在体外或体内 降低或下调IL-1beta、IL-6或/和TNF-alpha的表达水平和/或提高细胞生存率和/或治疗或预防受试者中IL-1beta、IL-6或/和TNF-alpha相关疾病和/或病毒，例如RNA病毒，例如禽流感病毒，例如H5N1感染，
优选地，所述IL-1beta、IL-6或/和TNF-alpha相关疾病选自说明书中列出的IL-1beta、IL-6或/和TNF-alpha相关疾病，优选肺炎、心肌炎、急慢性胃炎、急慢性肠炎、急慢性肝炎、急慢性肾炎、皮炎、脑炎、淋巴炎、结膜炎、角膜炎、虹膜睫状体炎、中耳炎、过敏性鼻炎、哮喘、肺纤维化慢性阻塞性肺疾病、过敏性皮炎、镰状细胞病、多发性硬化、系统性红斑狼疮、狼疮性肾炎、肺癌、胃癌、结直肠癌、肝癌、胰腺癌、宫颈癌、乳腺癌、白血病、多发性骨髓瘤、糖尿病和痛风。
22.根据项1-6中任一项所述的小RNA、根据项7所述的核酸序列或构建体、根据项8所述的重组病毒、根据项9所述的表达载体、所述项10所述的细胞和/或根据项12-15中任一项的药物组合物在制备药物中的用途，所述药物用于在体外或体内降低或下调IL-1beta、IL-6或/和TNF-alpha的表达水平和/或提高细胞生存率和/或治疗或预防受试者中的IL-1beta、IL-6或/和TNF-alpha相关疾病和/或病毒，例如RNA病毒，例如禽流感病毒，例如H5N1感染，
优选地，所述IL-1beta、IL-6或/和TNF-alpha相关疾病选自说明书中列出的IL-1beta、IL-6或/和TNF-alpha相关疾病，例如肺炎、心肌炎、急慢性胃炎、急慢性肠炎、急慢性肝炎、急慢性肾炎、皮炎、脑炎、淋巴炎、结膜炎、角膜炎、虹膜睫状体炎、中耳炎、过敏性鼻炎、哮喘、肺纤维化、慢性阻塞性肺疾病、过敏性皮炎、镰状细胞病、多发性硬化、系统性红斑狼疮、狼疮性肾炎、肺癌、胃癌、结直肠癌、肝癌、胰腺癌、宫颈癌、乳腺癌、白血病、多发性骨髓瘤、糖尿病和痛风。
23.用于检测根据项1-6中任一项所述的小RNA、根据项7所述的核酸序列或构建体、根据项8所述的重组病毒、根据项9所述的表达载体、所述项10所述的细胞和/或根据项12-15中任一项的药物组合物的试剂，其中优选地，所述试剂是引物和/或探针。
24.试剂盒，其包含根据项23的试剂。
25.使用根据项23所述的试剂或根据项24所述的试剂盒检测不同来源的细胞是否含有根据项1-6中任一项所述的小RNA的方法，其中所述细 胞优选是植物细胞。
附图说明
图1：向THP1细胞中提前24小时转染如图中指定的半枝莲(BZL)小RNA后LPS刺激9小时的细胞炎症模型中炎症因子IL-1beta在蛋白水平相对于对照组表达的结果。
图2：向THP1细胞中提前24小时转染如图中指定的柴胡(CHu)小RNA后LPS刺激9小时的细胞炎症模型中炎症因子IL-1beta在蛋白水平相对于对照组表达的结果。
图3：向THP1细胞中提前24小时转染如图中指定的鱼腥草(YXC)小RNA后LPS刺激9小时的细胞炎症模型中炎症因子IL-1beta在蛋白水平相对于对照组表达的结果。
图4：向THP1细胞中提前24小时转染如图中指定的穿心莲(CXL)和蒲公英(PGY)小RNA后LPS刺激9小时细胞炎症模型中炎症因子IL-1beta在蛋白水平相对于对照组表达的结果。
图5：向THP1细胞中提前24小时转染如图中指定的半枝莲(BZL)小RNA后LPS刺激9小时细胞炎症模型中炎症因子IL-6在蛋白水平相对于对照组表达的结果。
图6：向THP1细胞中提前24小时转染如图中指定的柴胡(CHu)小RNA后LPS刺激9小时细胞炎症模型中炎症因子IL-6在蛋白水平相对于对照组表达的结果。
图7：向THP1细胞中提前24小时转染如图中指定的地丁(DDi)、黄芩(HQi)、金银花(JYH)、连翘(LQi)、和夏枯草(XKC)小RNA后LPS刺激9小时细胞炎症模型中炎症因子IL-6在蛋白水平相对于对照组表达的结果。
图8-9：向THP1细胞中提前24小时转染如图中指定的鱼腥草(YXC)小RNA后LPS刺激9小时细胞炎症模型中炎症因子IL-6在蛋白水平相对于对照组表达的结果。
图10：向THP1细胞中提前24小时转染如图中指定的穿心莲(CXL)和蒲公英(PGY)小RNA后LPS刺激9小时细胞炎症模型中炎症因子IL-6在蛋白水平相对于对照组表达的结果。
图11：向THP1细胞中提前24小时转染如图中指定的半枝莲(BZL)、地丁(DDi)、黄芩(HQi)、连翘(LQi)、和夏枯草(XKC)小RNA后LPS刺激9小时细胞炎症模型中炎症因子TNF-alpha在蛋白水平相对于对照组表达的结果。
图12：向THP1细胞提前24小时转染如图中指定的柴胡(CHu)小RNA后LPS刺激9小时细胞炎症模型中炎症因子TNF-alpha在蛋白水平相对于对照组表达的结果。
图13-14：向THP1细胞提前24小时转染如图中指定的鱼腥草(YXC)小RNA后LPS刺激9小时细胞炎症模型中炎症因子TNF-alpha在蛋白水平相对于对照组表达的结果。
图15：向THP1细胞提前24小时转染如图中指定的蒲公英(PGY)小RNA后LPS刺激9小时细胞炎症模型中炎症因子TNF-alpha在蛋白水平相对于对照组表达的结果。
图16：向THP1细胞提前24小时转染如图中指定的半枝莲(BZL)小RNA后LPS刺激9小时细胞炎症模型中炎症因子IL-1beta在mRNA水平相对于对照组表达的结果。
图17：向THP1细胞提前24小时转染如图中指定的柴胡(CHu)、连翘(LQi)、和鱼腥草(YXC)小RNA后LPS刺激9小时细胞炎症模型中炎症因子IL-1beta在mRNA水平相对于对照组表达的结果。
图18-19：向THP1细胞提前24小时转染如图中指定的鱼腥草(YXC)小RNA后LPS刺激9小时细胞炎症模型中炎症因子IL-1beta在mRNA水平相对于对照组表达的结果。
图20：向THP1细胞提前24小时转染如图中指定的蒲公英(PGY)小RNA后LPS刺激9小时细胞炎症模型中炎症因子IL-1beta在mRNA水平相对于对照组表达的结果。
图21-22：向THP1细胞提前24小时转染如图中指定的半枝莲(BZL)小RNA后LPS刺激9小时细胞炎症模型中炎症因子IL-6在mRNA水平相对于对照组表达的结果。
图23-24：向THP1细胞提前24小时转染如图中指定的柴胡(CHu)小RNA后LPS刺激9小时细胞炎症模型中炎症因子IL-6在mRNA水平相对于对照组表达的结果。
图25：向THP1细胞提前24小时转染如图中指定的地丁(DDi)、黄芩(HQi)、金银花(JYH)、连翘(LQi)、和夏枯草(XKC)小RNA后LPS刺激9小时细胞炎症模型中炎症因子IL-6在mRNA水平相对于对照组表达的结果。
图26-27：向THP-1细胞提前24小时转染如图中指定的鱼腥草(YXC)小RNA后LPS刺激9小时细胞炎症模型中炎症因子IL-6在mRNA水平相对于NC组表达的结果。
图28：向THP1细胞提前24小时转染如图中指定的穿心莲(CXL)和蒲公英(PGY)小RNA后LPS刺激9小时细胞炎症模型中炎症因子IL-6在mRNA水平相对于NC组表达的结果。
图29：向THP1细胞提前24小时转染如图中指定的半枝莲(BZL)和柴胡(CHu)小RNA后LPS刺激9小时细胞炎症模型中炎症因子TNF-alpha在mRNA水平相对于NC组表达的结果。
图30：向THP1细胞提前24小时转染如图中指定的地丁(DDi)、金银花(JYH)、连翘(LQi)和夏枯草(XKC)小RNA后LPS刺激9小时细胞炎症模型中炎症因子TNF-alpha在mRNA水平相对于NC组表达的结果。
图31-32：向THP1细胞提前24小时转染如图中指定的鱼腥草(YXC)小RNA后LPS刺激9小时细胞炎症模型中炎症因子TNF-alpha在mRNA水平相对于NC组表达的结果。
图33：向THP1细胞提前24小时转染如图中指定的蒲公英(PGY)小RNA后LPS刺激9小时细胞炎症模型中炎症因子TNF-alpha在mRNA水平相对于NC组表达的结果。
图34A-C：BZL小RNA:H5N1(0.4M.O.I)感染后图中指定的半枝莲(BZL)小RNA对细胞死亡的挽救结果；图34D-G：CHu小RNA:H5N1(0.4M.O.I)感染后图中指定的柴胡(CHu)小RNA对细胞死亡的挽救结果；图34H:LQi/XKC小RNA:H5N1(0.4M.O.I)感染后图中指定的连翘(LQi)/夏枯草(XKC)小RNA对细胞死亡的挽救结果；图34I:XKC/YXC小RNA:H5N1(0.4M.O.I)感染后图中指定的夏枯草(XKC)/鱼腥草(YXC)小RNA对细胞死亡的挽救结果；图34J-N:YXC小RNA:H5N1(0.4M.O.I)感染后图中指定的鱼腥草(YXC)小RNA对细胞死亡的挽救结果。
图35：THP1细胞中提前24小时转染如图中指定的小RNA混合物后LPS 刺激9小时细胞炎症模型中炎症因子IL-1beta在蛋白水平相对于NC组表达的结果。
图36-37：THP1细胞中提前24小时转染如图中指定的小RNA混合物后LPS刺激9小时细胞炎症模型中炎症因子IL-6在蛋白水平相对于NC组表达的结果。
图38：THP1细胞中提前24小时转染如图中指定的小RNA混合物后LPS刺激9小时细胞炎症模型中炎症因子IL-1beta在mRNA水平相对于NC组表达的结果。
图39-40：THP1细胞中提前24小时转染如图中指定的小RNA混合物后LPS刺激9小时细胞炎症模型中炎症因子IL-6在mRNA水平相对于NC组表达的结果。
图41：THP1细胞中提前24小时转染小RNA混合物后LPS刺激9小时细胞炎症模型中炎症因子TNF-alpha在mRNA水平相对于NC组表达的结果。
图42：小鼠提前3天小RNA灌胃，LPS刺激9小时动物炎症模型，肺泡灌洗液中炎症因子TNF-alpha在蛋白水平相对于NC组表达的结果。
图43：小鼠提前3天小RNA灌胃，LPS刺激9小时动物炎症模型，肺脱落细胞中炎症因子IL-6在mRNA水平相对于NC组表达的结果。
具体实施方式
发明公开了一些小RNA及其在抑制表3中列出的任一种或多种通路或基因的能力或体内或体外降低或下调IL-1beta、IL-6或/和TNF-alpha的表达水平或治疗或预防受试者中IL-1beta、IL-6或/和TNF-alpha相关疾病和/或H5N1感染方面的应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明所述核酸和应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的应用进行改动或适当变更与组合，来实现和应用本发明技术。
通常，人们把siRNA，miRNA及其它非编码小RNA不加区分地称之为小RNA(sRNA)。除非特别指明，在本文中，术语“小RNA(sRNA)”是指 包括siRNA和miRNA在内的各种非编码小RNA。该术语中的“小”并非限定RNA具有特定的大小。
术语“包括”、“包含”、“含有”是指，除了列出的特征要素以外，还可以有其他附加的特征要素。特别地，也可以仅由所列出的特征要素组成。
术语“受试者”是指例如患有炎症和/或H5N1感染并需要进行治疗或有潜在炎症发展迹象和/或易于H5N1感染的需要预防的受试者。
在本文中用序列“同一性”来描述两个氨基酸序列之间或两个核苷酸序列之间的相关性。出于本发明的目的，使用如在EMBOSS包(EMBOSS：欧洲分子生物学开放软件套件，Rice等人，2000，Trends Genet.[遗传学趋势]16:276-277))(优选5.0.0版或更新版本)的Needle程序中所实施的Needleman-Wunsch算法(Needleman和Wunsch,1970，同上)来确定两个脱氧核糖核苷酸序列之间的序列一致性。所使用的参数是空位开放罚分10，空位延伸罚分0.5和EDNAFULL(NCBI NUC4.4的EMBOSS版本)取代矩阵。将标记为“最长一致性”的Needle输出(使用-非简化(–nobrief)选项获得)用作百分比一致性并且计算如下：
(相同的脱氧核糖核苷酸X 100)/(比对长度-比对中的空位总数)。
例如，本发明涵盖与SEQ ID NO.1-222中任一项所示的序列具有至少80％、85％、90％、95％、96％、97％、98％、99％或99.9％同一性的序列。
在本文中，术语“严格条件”可以指例如65℃下4×SSC中杂交，然后在65℃下0.1×SSC中洗涤若干次，大约共1小时。此处所用的术语”严格杂交条件也可以指68℃下，0.25M磷酸钠、pH为7.2的7％SDS、1mM EDTA和1％BSA中杂交16小时，然后在68℃下，以2×SSC和0.1％SDS中洗涤两次。本领域技术人员可以根据具体的序列确定严格条件。
术语“细胞生存率”又称为细胞存活率。在一个实施方案中，细胞生存率可以使用MTS检测试剂盒，根据制造商的说明计算。
术语“IL-1beta、IL-6或/和TNF-alpha相关疾病”指以IL-1beta、IL-6或/和TNF-alpha的表达水平升高为特征的疾病。如肺炎、心肌炎、急慢性胃炎、急慢性肠炎、急慢性肝炎、急慢性肾炎、皮炎、脑炎、淋巴炎、结膜炎、角膜炎、虹膜睫状体炎、中耳炎、过敏性鼻炎、哮喘、肺纤维化、过敏性皮炎、多发性硬化、系统性红斑狼疮、肺癌、胃癌、结直肠癌、肝癌、宫颈癌、乳腺癌、白血病、糖尿病、痛风等。
发明人经过长期研究，发现本发明的小RNA序列在体内或体外降低或下调IL-1beta、IL-6或/和TNF-alpha的表达水平或治疗或预防受试者中IL-1beta、IL-6或/和TNF-alpha相关疾病和/或H5N1感染(从而挽救由于H5N1感染导致的细胞死亡)。本实验中使用的小RNA的序列如下表1中所示。
表1：本实验中使用的小RNA的序列，均为商业合成。
Figure PCTCN2018123289-appb-000001
Figure PCTCN2018123289-appb-000002
Figure PCTCN2018123289-appb-000003
Figure PCTCN2018123289-appb-000004
Figure PCTCN2018123289-appb-000005
Figure PCTCN2018123289-appb-000006
在本发明中，上述小RNA的使用浓度为20uM。在一个实施方案中，使用合成的小RNA测试其抑制表3中列出的任一种或多种通路或基因的能力，或用于在体外或体内降低或下调IL-1beta、IL-6或/和TNF-alpha的表达水平和/或提高细胞生存率和/或治疗或预防受试者中IL-1beta、IL-6或/和TNF-alpha相关疾病和/或H5N1感染的能力。在一个实施方案中，使用的小RNA靶向或降低表3中的相同通路或基因。在一个实施方案中，使用的小RNA降低或下调IL-1beta、IL-6和TNF-alpha之一的表达水平。在一个实施方案中，使用的小RNA提高细胞生存率。在一个实施方案中，细胞生存率是H5N1感染的细胞的生存率。在一个实施方案中，使用的小RNA治疗或预防受试者中与IL-1beta、IL-6和TNF-alpha之一的表达水平升高相关的疾病。
应当理解，本领域技术人员可以根据本公开的小RNA制备其编码核酸，并且可以将编码核酸导致合适的表达载体中。可以将表达本公开的小RNA的表达载体直接导入受试者或者受试细胞中，从而抑制表3中列出的任一种或多种通路或基因，或用于在体外或体内降低或下调IL-1beta、IL-6或/和TNF-alpha的表达水平和/或提高细胞生存率和/或治疗或预防受试者中IL-1beta、IL-6或/和TNF-alpha相关疾病和/或H5N1感染，条件是所述表达载体可以在受试者或受试细胞中表达。例如，参见美国专利US2017/0342410，其通过引用并入本文。
此外，本领域技术人员也可以将制备成在细胞中表达小RNA的构建体，例如逆转录病毒构建体，并且通过用该构建体转染包装细胞系，产生重组逆转录病毒颗粒，进而在体外或在体内感染靶细胞来抑制表3中列出的任一种或多种通路或基因，降低或下调IL-1beta、IL-6或/和TNF-alpha的表达水平和/或提高细胞生存率，和/或治疗或预防受试者中IL-1beta、IL-6或/和TNF-alpha相关疾病和/或H5N1感染。例如，参见美国专利US2017/0342410，其通过引用并入本文。
本领域技术人员可以将含有表达载体或构建体的细胞在体外或在体内导入受试者或细胞来实现本发明的上述目的。或者，本领域技术人员可以通过常规技术从细胞中分离出本公开的小RNA。因此，涵盖表达小RNA的方法，其包括在合适的条件下表达细胞并且回收小RNA的步骤。
本发明还涵盖用于检测小RNA、构建体、重组病毒、表达载体、细胞和/或药物组合物的试剂。本领域技术人员也可以使用检测试剂来检测不同来源的细胞以检测其中是否涵盖本公开的小RNA。优选地，试剂是引物和/或探针。试剂的设计或使用对于本领域技术人员是公知的。
在一个实施方案中，小RNA是BZL-sRNA-20。发明人发现BZL-sRNA-20在抑制TNF-alpha、IL-1beta或IL-6的蛋白质或mRNA中具有很好的效果。因此，发明人选择BZL-sRNA-20作为基础小RNA，并且与其它小RNA组合制备表2中所述的小RNA混合物。
表2：本发明中使用的小RNA混合物(实验结果在图35-图41中体现)
Figure PCTCN2018123289-appb-000007
Figure PCTCN2018123289-appb-000008
Figure PCTCN2018123289-appb-000009
在一个实施方案中，使用表2中的小RNA混合物进行各项测试。在一个实施方案中，通过将20uM的BZL-sRNA-20与各20uM的其它小RNA以2:1的体积比制备siRNA混合物。在小RNA混合物的一个实施方案中，BZL-sRNA-20与其它小RNA的摩尔浓度为2:1。在小RNA混合物的一个实施方案中，小RNA混合物中的总小RNA的摩尔浓度为20uM。在附图中以符号MIX表示混合物。
IL-6相关疾病
本发明的小RNA可以治疗IL-6相关疾病。IL-6相关疾病包括：
(呼吸道)阻塞性气道疾病，包括慢性阻塞性肺病(COPD)；哮喘，如支气管、变应性、内源性、外源性和尘埃性哮喘，特别是慢性或积习性哮喘(例如晚期哮喘和气道高反应性)；支气管炎；急性、变应性、萎缩性鼻炎和慢性鼻炎，包括干酪性鼻炎、肥厚性鼻炎、化脓性鼻炎、干燥性鼻炎和药物性鼻炎；膜性鼻炎，包括格鲁布性、纤维蛋白性和假膜性鼻炎，以及腺病性鼻炎；季节性鼻炎，包括神经性鼻炎(枯草热)和血管运动性鼻炎、鼻窦 炎、特发性肺纤维化(IPF)；结节病、农民肺和相关疾病、成人呼吸窘迫综合征、超敏性肺炎、纤维化肺和特发性间质性肺炎；
(骨和关节)类风湿性关节炎、青少年慢性关节炎、青少年关节炎全身发病、血清阴性脊柱关节病(包括强直性脊柱炎、银屑病关节炎和莱特病)、贝切特病、舍格伦综合征和全身性硬化症、痛风、骨质疏松和骨关节炎；
(皮肤)银屑病、特应性皮炎、接触性皮炎和其它湿疹性皮肤病、变应性接触性皮炎、脂溢性皮炎、扁平苔藓、硬皮病、天疱疮、大疱性类天疱疮、大疱性表皮松解症、荨麻疹、干皮病(angiodermas)、血管炎、红斑、皮肤嗜酸性细胞增多、葡萄膜炎、斑秃、变应性结膜炎和春季结膜炎(vernalvemalconjunctivitis)；
(胃肠道)胃溃疡、腹部疾病、直肠炎、嗜酸性肠胃炎、肥大细胞增生病、炎性肠病、克罗恩氏病、溃疡性结肠炎、抗磷脂综合征))、产生远离内脏的影响，如偏头痛、鼻炎和湿疹的食品相关性变态反应；
(其它组织和全身性疾病)恶病质、多发性硬化、动脉粥样硬化、获得性免疫缺陷综合征(AIDS)、系膜增生性肾小球肾炎、肾病综合征、肾炎、肾小球肾炎、急性肾衰竭、血液透析、尿毒症、局部或盘状红斑狼疮、系统性红斑狼疮、卡斯尔曼病、桥本甲状腺炎、重症肌无力、I型糖尿病、B型胰岛素抗性糖尿病、镰状细胞贫血、虹膜睫状体炎/葡萄膜炎/视神经炎、肾炎综合征、嗜酸细胞增多性筋膜炎、高IgE综合征、全身性血管炎/韦格纳肉芽肿病、睾丸炎/输精管切除术逆转术(reversalprocedure)、瘤型麻风、酒精诱导的肝炎、塞扎里综合征和特发性血小板减少紫癜；术后粘连、肾病、全身性炎症反应综合征、败血症综合征、革兰阳性败血症、革兰阴性败血症、培养阴性败血症、真菌性败血症、中性粒细胞减少性发热、急性胰腺炎、尿脓毒症、格拉夫斯病、雷诺病、抗体介导的细胞毒型、III型超敏反应、POEMS综合征(多神经病、器官巨大症、内分泌病、单克隆丙种球蛋白病和皮肤改变综合征)、混合型结缔组织病、特发性阿狄森病、糖尿病、慢性活动性肝炎、原发性胆汁性肝硬化、白癜风、MI后(心切开术)综合征、IV型超敏症、胞内生物体引起的肉芽肿、威尔逊病、血色病、α-I-抗胰蛋白酶缺陷、糖尿病视网膜病、桥本甲状腺炎、下丘脑-垂体-肾上腺轴评估、甲状腺炎、脑脊髓炎、新生儿慢性肺病、家族性噬血淋巴组织细胞增多病(familialhematophagocytic lymphohistiocytosis)、脱发、放疗(包括 例如但不限于：虚弱、贫血、恶病质等)、慢性水杨酸中毒、睡眠性呼吸暂停、肥胖、心力衰竭和脑膜炎球菌血症；
(同种异体移植物排斥)肾脏、心脏、肝脏、肺、胰腺、骨髓、骨、小肠、皮肤、软骨和角膜移植后的急性和慢性排斥；和慢性移植物抗宿主病；
(恶性疾病)白血病、急性成淋巴细胞性白血病(ALL)，急性白血病，T细胞、B细胞或FABALL，慢性髓细胞性白血病(CML)、急性髓细胞性白血病(AML)、慢性淋巴细胞性白血病(CLL)、毛细胞性白血病、骨髓异常增生综合征(MDS)、淋巴瘤、霍奇金病、非霍奇金淋巴瘤、恶性淋巴瘤、伯基特淋巴瘤、多发性骨髓瘤、卡波济氏肉瘤、肾细胞癌、结直肠癌、前列腺癌、胰腺癌、鼻咽癌、恶性组织细胞增多病、肿瘤伴随综合征/恶性高钙血症、实体瘤、腺癌、肉瘤、恶性黑色素瘤、血管瘤、转移病、癌症相关性骨再吸收、癌症相关性骨痛；癌症转移的抑制；癌症恶病质的改进；
囊性纤维化病，中风，心脏、脑、四肢(peripherallimbs)和其它器官的再灌注损伤；烧伤、外伤/出血、电离辐射照射、慢性皮肤溃疡；
生殖疾病(如排卵、月经和着床的疾病，早产，先兆子痫，子宫内膜异位)；(感染)急性或慢性细菌感染，急性和慢性寄生过程或感染过程，包括细菌、病毒和真菌感染、HIV感染/HIV神经病、脑膜炎、肝炎(甲型、乙型或丙型、或者其它病毒性肝炎等)、脓毒性关节炎、腹膜炎、肺炎、会厌炎、溶血尿毒症综合征/血栓性血小板减少性紫癜、疟疾、登革出血热、利什曼病、麻风病、中毒性休克综合征、链球菌性肌炎、气性坏疽、结核杆菌(Mycobacteriumtuberculosis)、鸟-胞内分枝杆菌、卡氏肺囊虫(pneumocystiscarinii)性肺炎、盆腔炎疾病、睾丸炎/附睾炎、军团菌、莱姆病、甲型流感、爱波斯坦-巴尔病毒、致命相关性噬血综合征(vital-associatedhemaphagocytic syndrome)、病毒性脑炎/无菌性脑膜炎等。
本发明的小RNA可以与以下一种或多种组合使用：
-细胞因子或者细胞因子功能的激动剂或拮抗剂(例如，作用于细胞因子信号传导途径的药剂，例如SOCS系统的调节剂)，例如α-、beta-和/或γ-干扰素；I型胰岛素样生长因子(IGF-1)、其受体和相关结合蛋白；白介素(IL)，例如IL-1-33中的一种或多种，和/或白介素拮抗剂或抑制剂，例如阿那白滞素；白介素家族成员的受体的抑制剂或这些受体的特定亚单位的抑制剂；肿瘤坏死因子alpha(TNF-alpha)抑制剂，例如抗-TNF单克隆抗体 (例如，英夫利昔单抗；阿达木单抗和/或CDP-870)、和/或TNF受体拮抗剂，例如免疫球蛋白分子(例如依那西普)和/或低分子量药剂，例如已酮可可碱(pentoxyfylline)；
-B细胞调节剂，例如靶向B-淋巴细胞(例如CD20(利妥昔单抗)或MRA-aILl6R)或T-淋巴细胞(例如，CTLA4-Ig、HuMaxIl-15或阿巴西普(Abatacept))的单克隆抗体；
-抑制破骨细胞活性的调节剂，例如RANKL的抗体；
-趋化因子或趋化因子受体功能的调节剂，例如以下趋化因子的拮抗剂：CCR1、CCR2、CCR2A、CCR2B、CCR3、CCR4、CCR5、CCR6、CCR7、CCR8、CCR9、CCR10和CCR11(就C-C家族而言)；CXCR1、CXCR2、CXCR3、CXCR4和CXCR5及CXCR6(就C-X-C家族而言)和C-X3-C家族的CX3CR1；
-基质金属蛋白酶(MMP)，即以下一种或多种的抑制剂：间质溶解素、胶原酶和明胶酶以及聚集蛋白聚糖酶；特别是胶原酶-1(MMP-1)、胶原酶-2(MMP-8)、胶原酶-3(MMP-13)、间质溶解素-1(MMP-3)、间质溶解素-2(MMP-10)和/或间质溶解素-3(MMP-11)和/或MMP-9和/或MMP-12，例如强力霉素等药剂；
-白三烯生物合成抑制剂、5-脂肪氧合酶(5-LO)抑制剂或5-脂肪氧合酶活化蛋白(FLAP)拮抗剂，例如：齐留通；ABT-761；芬留顿；替泊沙林；Abbott-79175；Abbott-85761；N-(5-取代的)-噻吩-2-烷基磺酰胺；2,6-二叔丁基苯酚腙；甲氧基四氢吡喃，例如泽尼卡ZD-2138；化合物SB-210661；吡啶基-取代的2-氰基萘化合物，例如L-739,010；2-氰基喹啉化合物，例如L-746,530；吲哚和/或喹啉化合物，例如MK-591、MK-886和/或BAYx1005；
-白三烯(LT)B4、LTC4、LTD4和LTE4的受体拮抗剂，选自：吩噻嗪-3-1s，例如L-651,392；脒基化合物，例如CGS-25019c；氨基苯并恶唑类(benzoxalamines)，例如昂唑司特；苄脒类(benzenecarboximidamides)，例如BIIL284/260；和诸如扎鲁司特、阿鲁司特、孟鲁司特、普仑司特、维鲁司特(MK-679)、RG-12525、Ro-245913、伊拉司特(CGP45715A)和BAYx 7195等化合物；
-磷酸二酯酶(PDE)抑制剂，例如甲基黄嘌呤(methylxanthanine)，如茶 碱和/或氨茶碱；和/或选择性PDE同工酶抑制剂，例如PDE4抑制剂和/或同种型PDE4D的抑制剂，和/或PDE5抑制剂；
-1型组胺受体拮抗剂，例如西替立嗪、氯雷他定、地氯雷他定、非索非那定、阿伐斯汀、特非那定、阿司咪唑、氮卓斯汀、左卡巴司汀、氯苯那敏、普鲁本近、赛克利嗪和/或咪唑斯汀(通常口服、局部或胃肠外应用)；
-质子泵抑制剂(例如奥美拉唑)或胃保护性组胺2型受体拮抗剂；
-组胺4型受体拮抗剂；
-α-1/α-2肾上腺素受体激动剂、血管收缩剂、拟交感神经药，例如环己丙甲胺、苯福林、苯丙醇胺、麻黄素、伪麻黄碱、盐酸萘甲唑啉、盐酸氧甲唑啉、盐酸四氢唑啉、盐酸木甲唑啉、盐酸曲马唑啉和盐酸乙基去甲肾上腺素；
-抗胆碱能药，例如毒蕈碱受体(M1、M2和M3)拮抗剂，例如阿托品、东莨菪碱、甘罗溴铵(glycopyrrrolate)、异丙托溴铵、噻托溴铵、氧托溴铵、哌仑西平和替仑西平；
-beta-肾上腺素受体激动剂(包括beta受体亚型1-4)，例如异丙肾上腺素、柳丁氨醇、福莫特罗、沙美特罗、叔丁喘宁、间羟异丙肾上腺素、双甲苯喘定甲磺酸盐和/或吡布特罗，例如它们的手性对映体；
-色酮，例如色甘酸钠和/或奈多罗米钠；
-糖皮质激素，例如氟尼缩松、羟氢化泼尼松缩丙酮、二丙酸氯地米松、布地奈德、丙酸氟替卡松、环索奈德和/或糠酸莫米松；
-调节核激素受体的药剂，例如PPAR；
-免疫球蛋白(Ig)或Ig制品或调节Ig功能的拮抗剂或抗体，例如抗-IgE(例如，奥玛立珠单抗(奥马珠单抗))；
-其它全身性或局部应用的抗炎药，例如沙利度胺或其衍生物、类维生素A、蒽三酚和/或卡泊三醇；
-氨基水杨酸盐和磺胺吡啶的组合，例如柳氮磺吡啶、美沙拉秦、巴柳氮和奥沙拉秦；免疫调节剂，例如巯基嘌呤(thiopurines)和皮质类固醇，例如布地奈德；
-抗菌剂，例如青霉素衍生物、四环素、大环内酯类、beta-内酰胺、氟喹诺酮、甲硝哒唑和/或吸入性氨基糖苷类；和/或抗病毒剂，例如阿昔洛维、泛昔洛韦、缬昔洛韦、更昔洛韦、西多福韦；三环癸烷胺、金刚乙胺； 利巴韦林；扎那米韦和/或奥塞米韦；蛋白酶抑制剂，例如印地那韦、那非那韦、利托那韦和/或沙奎那韦；核苷逆转录酶抑制剂，例如地达诺新、拉米夫定、司他夫定、扎西他滨、齐多夫定；非核苷逆转录酶抑制剂，例如奈韦拉平、依法韦仑；
-心血管药物，例如钙通道阻断剂、beta-肾上腺素受体阻断剂、血管紧张素转化酶(ACE)抑制剂、血管紧张素-2受体拮抗剂；降脂剂，例如他汀类和/或贝特类；血细胞形态的调节剂，例如已酮可可碱；溶解血栓和/或抗凝剂，例如血细胞聚集抑制剂；
-CNS药，例如抗抑郁药(例如舍曲林)、抗-帕金森药(例如塞利吉林、左旋多巴、罗平尼咯、普拉克索、MAOB抑制剂，如塞乐金(selegine)和雷沙吉兰、comP抑制剂，如托卡朋、A-2抑制剂、多巴胺再摄取抑制剂、NMDA拮抗剂、烟碱激动剂、多巴胺激动剂和/或神经元一氧化氮合酶抑制剂)和抗阿尔茨海默病药物，例如多奈哌齐、利凡斯的明、他克林、COX-2抑制剂、丙戊茶碱或美曲磷酯；
-治疗急性和慢性疼痛的药剂，例如中枢或外周作用的镇痛剂，例如阿片类似物或衍生物、卡巴米嗪、苯妥英、丙戊酸钠、阿米替林(amitryptiline)或其它抗抑郁药、对乙酰氨基酚或非类固醇抗炎药；
-胃肠外或局部-应用的(包括吸入的)局部麻醉剂，例如利诺卡因或其类似物；
-抗-骨质疏松剂，例如激素药物，如雷洛昔芬或双膦酸盐，如阿伦膦酸盐；
-(i)胰蛋白酶抑制剂；(ii)血小板活化因子(PAF)拮抗剂；(iii)白介素转换酶(ICE)抑制剂；(iv)IMPDH抑制剂；(v)粘附分子抑制剂，包括VLA-4拮抗剂；(vi)组织蛋白酶；(vii)激酶抑制剂，例如酪氨酸激酶抑制剂(例如，Btk、Itk、Jak3MAP抑制剂的例子可包括吉非替尼(Gefitinib)、甲磺酸伊马替尼)、丝氨酸/苏氨酸激酶(例如，MAP激酶，如p38、JNK、蛋白激酶A、B和C及IKK的抑制剂)、或参与细胞周期调节的激酶(例如，依赖周期蛋白的激酶)；
(viii)葡萄糖-6磷酸脱氢酶抑制剂；(ix)激肽-B1-和/或B2–受体拮抗剂；(x)抗-痛风药剂，例如秋水仙素；(xi)黄嘌呤氧化酶抑制剂，例如别嘌呤醇；(xii)排尿酸剂，例如，丙磺舒、苯磺唑酮和/或苯溴马隆；(xiii)生长激素促 分泌素；(xiv)转化生长因子(TGFbeta)；(xv)血小板衍生的生长因子(PDGF)；(xvi)成纤维细胞生长因子，例如碱性成纤维细胞生长因子(bFGF)；(xvii)粒细胞巨噬细胞集落刺激因子(GM-CSF)；(xviii)辣椒碱乳膏；(xix)速激肽NK1和/或NK3受体拮抗剂，例如NKP-608C、SB-233412(他奈坦)和/或D-4418；(xx)弹性蛋白酶抑制剂，例如UT-77和/或ZD-0892；(xxi)TNF-alpha转化酶抑制剂(TACE)；(xxii)诱导型一氧化氮合酶(iNOS)抑制剂或(xxiii)表达在TH2细胞上的化学引诱物受体-同源分子，(例如，CRTH2拮抗剂)；(xxiv)P38的抑制剂；(xxv)调节Toll-样受体(TLR)的功能的药剂和(xxvi)调节嘌呤能受体活性的药剂，例如P2X7；(xxvii)转录因子活化的抑制剂，例如NFkB、API和/或STATS。
为了治疗炎性疾病，本发明的小RNA可以与一种或多种以下药物联用：
例如，非类固醇抗炎药(以下称为NSAID)，包括无选择性环加氧酶(COX)-1/COX-2抑制剂，无论局部或全身性应用(例如吡罗昔康、双氯芬酸、丙酸，如甲氧萘丙酸、氟比洛芬、非诺洛芬、酮洛芬和布洛芬、芬那酯，如甲芬那酸、吲哚美辛、舒林酸、阿扎丙宗、吡唑酮，例如苯丁唑酮、水杨酸盐，例如阿司匹林)；选择性COX-2抑制剂(例如，美洛昔康、塞来考昔、罗非考昔、伐地考昔、陆玛考昔(lumarocoxib)、帕瑞考昔和艾托考昔)；抑制一氧化氮供体的环加氧酶(CINODs)；糖皮质激素(无论通过局部、口服、肌肉内、静脉内或关节内途径)；甲氨蝶呤、来氟米特；羟氯喹、d-青霉胺、金诺芬或其它胃肠外或口服金制品；镇痛剂；双醋瑞因；关节内治疗，例如透明质酸衍生物；和营养添加剂，例如葡糖胺。本发明的小RNA还可与治疗癌症的现有治疗剂组合使用。可组合使用的合适药剂，包括：(i)用于医学肿瘤学的抗增殖/抗肿瘤药物和它们的组合，例如格列卫(甲磺酸伊马替尼)、烷化剂(例如，顺铂、碳铂、环磷酰胺、氮芥、美法仑、瘤可宁、白消安和亚硝基脲)；抗代谢物(例如，抗叶酸剂，如氟嘧啶，如5-氟尿嘧啶和替加氟、雷替曲塞、甲氨蝶呤、阿糖胞苷、羟基脲、吉西他滨和紫杉醇；抗肿瘤抗生素(例如，蒽环类抗生素，如阿霉素、博来霉素、多柔比星、道诺霉素、表柔比星、伊达比星、丝裂霉素-C、更生霉素和光辉霉素)；抗(有丝分)裂剂(例如，长春花碱，如长春新碱、长春碱、长春地辛和长春瑞滨及紫杉烷，如紫杉酚和泰索帝)；和拓扑异构酶抑制剂(例如， 鬼臼乙叉甙，如依托泊苷和替尼泊苷、安吖啶、托泊替康及喜树碱)；
(ii)细胞抑制药，例如抗雌激素(例如，他莫昔芬、托瑞米芬、雷洛昔芬、屈洛昔芬和吲哚昔芬(iodoxyfene))、雌激素受体下调剂(例如，氟维司群)、抗雄激素(例如，比卡鲁胺、氟他胺、尼鲁米特和醋酸环丙氯地孕酮)、LHRH拮抗剂或LHRH激动剂(例如，戈舍瑞林、亮丙瑞林和布舍瑞林)、孕激素(例如，醋酸甲地孕酮)、芳香酶抑制剂(例如，阿那曲唑、来曲唑、伏氯唑(vorazole)和依西美坦)和5α-还原酶的抑制剂，例如非那雄胺；
(iii)抑制癌细胞侵袭的药剂(例如，金属蛋白酶抑制剂，如马立马司他和尿激酶纤维蛋白溶酶原激活剂受体功能的抑制剂)；
(iv)生长因子功能的抑制剂，例如，这种抑制剂包括生长因子抗体、生长因子受体抗体(例如，抗-erbb2抗体，曲妥珠单抗和抗-erbb1抗体西妥昔单抗[C225])、法尼基转移酶抑制剂、酪氨酸激酶抑制剂和丝氨酸/苏氨酸激酶抑制剂，例如，表皮生长因子家族抑制剂(例如，EGFR家族酪氨酸激酶抑制剂，如N-(3-氯-4-氟苯基)-7-甲氧基-6-(3-吗啉基丙氧基)喹唑啉-4-胺(吉非替尼、AZD1839)、N-(3-乙炔基苯基)-6,7-二(2-甲氧基乙氧基)喹唑啉-4-胺(埃罗替尼(埃罗替尼)、OSI-774)和6-丙烯基酰氨基-N-(3-氯-4-氟苯基)-7-(3-吗啉基丙氧基)喹唑啉-4-胺(CI1033))，例如，血小板衍生的生长因子家族的抑制剂和例如，肝细胞生长因子家族的抑制剂；
(v)抗血管生成药剂，例如抑制血管内皮生长因子的作用的那些药剂，(例如，抗血管内皮生长因子抗体贝伐珠单抗、国际专利申请WO97/22596、WO97/30035、WO97/32856和WO98/13354公开的化合物，各份专利全文纳入本文)和以其它机制起作用的化合物(例如，利诺胺、整联蛋白αvbeta3功能的抑制剂和血管他丁)；
(vi)血管破坏剂，例如考布他汀A4和国际专利申请WO99/02166、WO00/40529、WO 00/41669、WO01/92224、WO02/04434和WO02/08213中公开的化合物(通过引用将各份专利全文纳入本文)；
(vii)反义治疗，例如，针对上述靶标的那些，如ISIS2503、抗-ras反义；
(viii)基因治疗方法，包括例如，替代异常基因，如异常p53或异常BRCA1或BRCA2的方法、GDEPT(基因指导的酶前药治疗)方法，例如利用胞嘧啶脱氨酶、胸苷激酶或细菌硝基还原酶的那些方法和增加患者对化疗或放疗耐受性的方法，例如多药抗性基因治疗；和
(ix)免疫治疗方法，包括例如，离体和体内方法以增加患者肿瘤细胞的免疫原性，如用细胞因子，如白介素2、白介素4或粒细胞巨噬细胞集落刺激因子转染，降低T细胞无反应性的方法，利用转染的免疫细胞，例如细胞因子转染的树突细胞的方法，利用细胞因子转染的肿瘤细胞系的方法和利用抗独特型抗体的方法。
IL-1beta相关疾病
IL-1beta在与多种涉及免疫和炎症要素的疾病有关的病理学中起到关键作用。本发明的小RNA可以治疗IL-1beta相关疾病。这些疾病包括但不限于：获得性免疫缺陷病综合征；获得性免疫缺陷相关病；获得性恶性贫血；急性冠状动脉综合征；急性和慢性痛(不同形式的疼痛)；急性特发性多神经炎；与器官移植相关的急性免疫性疾病；与器官移植相关的急性或慢性免疫性疾病；急性炎性脱髓鞘性多发性神经根性神经病；急性缺血；急性肝病；急性风湿热；急性横贯性脊髓炎；阿狄森氏病；成人(急性)呼吸窘迫综合征；成人Still氏病；酒精性肝硬变；酒精诱导的肝损伤；变应性疾病；变态反应；秃头；斑秃；阿尔茨海默氏病；过敏反应；强直性脊柱炎；强直性脊柱炎相关性肺病；抗磷脂抗体综合征；再生障碍性贫血；动脉硬化；关节病；哮喘；动脉粥样化疾病/动脉硬化；动脉粥样硬化；特应性变态反应；特应性湿疹；特应性皮炎；萎缩性自身免疫性甲状腺功能减退；自身免疫性大疱性疾病；自身免疫性皮炎；自身免疫性糖尿病；与链球菌感染相关的自身免疫性病症；自身免疫性肠病；自身免疫性溶血性贫血；自身免疫性肝炎；自身免疫性听力丧失；自身免疫性淋巴细胞增生综合征(ALPS)；自身免疫介导的低血糖；自身免疫性心肌炎；自身免疫性嗜中性白细胞减少症；自身免疫性过早卵巢衰竭；自身免疫性血小板减少症(AITP)；自身免疫性甲状腺病；自身免疫性葡萄膜炎；闭塞性细支气管炎；Behcet氏病；睑炎；支气管扩张；大疱性类天疱疮；恶病质；心血管病；灾变性抗磷脂综合征；乳糜泻；颈椎关节强硬；衣原体；胆汁郁积(choleosatatis)；慢性活动性肝炎；慢性嗜酸性肺炎；慢性疲乏综合征；与器官移植相关的慢性免疫性疾病；慢性缺血；慢性肝病；慢性粘膜皮肤念珠菌病；疤痕性类天疱疮；具有多发性硬化风险的临床孤立综合征(CIS)；常见的各种免疫缺陷(常见的可变低丙种球蛋白血症)；结缔组织病相关性间质性肺病；结膜炎；Coombs阳性溶血性贫血；儿童期发病性精神病； 慢性阻塞性肺部疾病(COPD)；克罗恩病；隐原性自身免疫性肝炎；隐原性纤维化肺泡炎；泪囊炎；抑郁；皮炎硬皮病；皮肌炎；皮肌炎/多肌炎相关性肺病；糖尿病性视网膜病；糖尿病；扩张型心肌病；盘状红斑狼疮；椎间盘突出；盘脱垂；弥散性血管内凝血；药物诱导的肝炎；药物诱导的间质性肺病；药物诱导的免疫性溶血性贫血；心内膜炎；子宫内膜异位；眼内炎；肠病性滑膜炎；巩膜外层炎；多形性红斑；重型多形性红斑；女性不育；纤维化；纤维化肺疾病；妊娠期类天疱疮；巨细胞性动脉炎(GCA)；肾小球肾炎；甲状腺肿性自身免疫性甲状腺功能减退(桥本氏病)；Goodpasture氏综合征；痛风性关节炎；移植物抗宿主病(GVHD)；Grave氏病；B群链球菌(BGS)感染；格林巴利综合征(BGS)；含铁血黄素沉着病相关性肺病；枯草热；心力衰竭；溶血性贫血；过敏性紫癜；乙型肝炎；丙型肝炎；Hughes综合征；亨廷顿氏舞蹈病；甲状腺机能亢进；甲状旁腺机能减退；特发性白细胞减少；特发性血小板减少症；特发性帕金森氏病；特发性间质性肺炎；特应性肝病；IgE介导的变态反应；免疫性溶血性贫血；内含体肌炎；感染病；感染性眼炎性疾病；炎性肠病；炎性脱髓鞘疾病；炎性心脏病；炎性肾病；胰岛素依赖性糖尿病；间质性肺炎；IPF/UIP；虹膜炎；青少年慢性关节炎；青少年性恶性贫血；青少年类风湿性关节炎(JRA)；川崎氏病；角膜炎；干燥性角膜结膜炎；Kussmaul病或Kussmaul-Meier病；Landry氏麻痹；朗格汉斯细胞组织细胞增多症；线性IgA疾病；网状青斑；莱姆关节炎；淋巴细胞性浸润性肺病；黄斑变性；特发性男性不育症或NOS；恶性肿瘤；肾显微血管炎；显微镜下多脉管炎；混合型结缔组织病相关性肺病；白赫铁列夫症(Morbus Bechterev)；运动神经元病症；粘膜类天疱疮；多发性硬化(所有亚型：原发进行性，继发进行性，复发缓和性(relapsing remitting)等)；多器官衰竭；肌痛脑炎/Royal Free疾病；重症肌无力；脊髓异常增生综合征；心肌梗塞；心肌炎；肾病综合征；神经根障碍；神经病；非酒精性脂肪性肝炎；非甲非乙型肝炎；视神经炎；器官移植排斥；骨关节炎；骨质溶解；卵巢癌；卵巢衰竭；胰腺炎；寄生虫病；帕金森氏病；少关节的JRA；类天疱疮；落叶状天疱疮；寻常天疱疮；周围动脉闭塞性疾病(PAOD)；周围血管疾病(PVD)；外周动脉疾病(PAD)；晶状体性葡萄膜炎；静脉炎；结节性多动脉炎(或结节性动脉外膜炎)；多软骨炎；风湿性多肌痛；白发症；多关节的JRA；多发性内分泌 缺陷综合征；多肌炎；多腺性I型缺乏和多腺性II型缺乏；风湿性多肌痛(PMR)；感染后间质性肺病；炎症后间质性肺病；泵后综合征；过早卵巢衰竭；原发性胆汁性肝硬变；原发性粘液性水肿；原发性帕金森综合征；原发性硬化性胆管炎；原发性硬化性肝炎；原发性血管炎；前列腺和直肠癌以及造血恶性肿瘤(白血病和淋巴瘤)；前列腺炎；牛皮癣；1型牛皮癣；2型牛皮癣；牛皮癣性关节炎；牛皮癣性关节病；结缔组织病继发的肺动脉高压；结节性多动脉炎的肺表现；单纯红细胞再生障碍；原发性肾上腺机能不足；放射性纤维化；反应性关节炎；Reiter氏病；复发型视神经脊髓炎；肾脏病NOS；再狭窄；类风湿性关节炎；类风湿性关节炎相关性间质性肺病；风湿性心脏病；SAPHO(滑膜炎、痤疮、脓疱病、骨肥厚和骨炎)；肉状瘤病；精神分裂症；Schmidt氏综合征；硬皮病；继发性淀粉样变性；休克肺；巩膜炎；坐骨神经痛；继发性肾上腺机能不足；脓毒病综合征；脓毒性关节炎；脓毒性休克；血清反应阴性关节病；聚硅氧烷相关的结缔组织病；斯耶格伦氏病相关性肺病；斯耶格伦氏综合征；Sneddon-Wilkinson皮肤病；精子自身免疫性；脊椎关节病；关节强硬性脊椎炎；Stevens-Johnson综合征(SJS)；Still氏病；中风；交感性眼炎；全身性炎症应答综合征；系统性红斑狼疮；系统性红斑狼疮相关性肺病；全身性硬皮病；全身性硬皮病相关性间质性肺病；高安氏病/动脉炎；颞动脉炎；Th2型和Th1型介导的疾病；甲状腺炎；中毒性休克综合征；弓形体视网膜炎；中毒性表皮坏死松解；横贯性脊髓炎；TRAPS(肿瘤坏死因子受体I型(TNFR)相关性周期综合征)；具有黑棘皮症的B型胰岛素抵抗；1型变态反应；1型自身免疫性肝炎(传统自身免疫性或狼疮样肝炎)；2型自身免疫性肝炎(抗LKM抗体肝炎)；II型糖尿病；溃疡性结肠炎性关节病；溃疡性结肠炎；荨麻疹；普通型间质性肺炎(UIP)；葡萄膜炎；脉管炎性弥散性肺病；脉管炎；春季结膜炎；病毒性视网膜炎；白癜风；Vogt-Koyanagi-Harada综合征(VKH综合征)；韦格纳氏肉芽肿病；湿黄斑变性；伤口愈合；耶尔森氏菌和沙门氏菌相关性关节病。
本发明涵盖包括本文描述的小RNA和下文列出的至少一种另外的试剂的组合。组合还可以包括超过一种另外的试剂，例如，2种或3种另外的试剂。
示例性组合包括本文描述的小RNA和非类固醇消炎药(NSAIDS)，例 如布洛芬。其他示例性组合包含本文描述的小RNA和皮质类固醇，包括强的松龙。本发明的小RNA可以与之组合用于类风湿性关节炎的治疗剂的非限制性例子包括下述：细胞因子抑制性消炎药(CSAIDs)；针对其他人细胞因子或生长因子的抗体或拮抗剂，例如，TNF、LT、IL-1α、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-15、IL-16、IL-18、IL-21、干扰素、EMAP-II、GM-CSF、FGF和PDGF。本发明的小RNA可以与针对细胞表面分子或其配体包括CD154(gp39或CD40L)的抗体组合，所述细胞表面分子例如CD2、CD3、CD4、CD8、CD25、CD28、CD30、CD40、CD45、CD69、CD80(B7.1)、CD86(B7.2)、CD90、CTLA。
用于与本发明的小RNA组合的示例性治疗剂可以在自身免疫和后续炎症级联中的不同点上进行干扰，例如TNF拮抗剂，如嵌合、人源化或人TNF抗体，D2E7(PCT公开号WO 97/29131)、CA2(REMICADEa)、CDP 571、和可溶性p55或p75 TNF受体，其衍生物(p75TNFR1gG(ENBRELa)或p55TNFR1gG(Lenercept)，以及TNF-alpha转换酶(TACE)抑制剂，和其他IL-1抑制剂(白细胞介素-1转换酶抑制剂，IL-1RA等)。用于与小RNA组合的其他试剂包括白细胞介素11，与IL-1a功能平行作用、依赖于IL-1a功能或与IL-1a功能一致的试剂，例如IL-18拮抗剂(例如IL-18结合蛋白例如抗体或可溶性IL-18受体，或其抗原结合片段。用于与小RNA组合的另外试剂包括非耗尽性抗CD4抑制剂、共刺激途径CD80(B7.1)或CD86(B7.2)拮抗剂，包括抗体、可溶性受体、拮抗性配体或其抗原结合片段。
小RNA还可以与用于治疗类风湿性关节炎的试剂组合，所述试剂例如氨甲蝶呤、6-MP、硫唑嘌呤柳氮磺吡啶、美沙拉秦、奥沙拉嗪氯喹(chloroquinine)/羟氯喹、青霉胺、硫化苹果酸金(肌内和经口)、硫唑嘌呤、秋水仙碱、皮质类固醇(经口、吸入和局部注射)、beta2肾上腺素受体激动剂(沙丁胺醇、特布他林、沙美特罗)、黄嘌呤(茶碱、氨茶碱)、色甘酸盐、萘多罗米、酮替芬、异丙托铵和乙东碱、环孢菌素、FK506、雷帕霉素、霉酚酸酯、来氟洛米、NSAIDs例如布洛芬、皮质类固醇例如强的松龙、磷酸二酯酶抑制剂、腺苷激动剂、抗凝剂、补体抑制剂、肾上腺素能药、干扰经由促炎细胞因子例如TNF-alpha或IL-1的发信号的试剂(例如IRAK、NIK、IKK、p38和MAP激酶抑制剂)、IL-1beta转换酶抑制剂、 TNF-alpha转换酶(TACE)抑制剂、T细胞发信号抑制剂例如激酶抑制剂、金属蛋白酶抑制剂、柳氮磺吡啶、硫唑嘌呤、6-巯基嘌呤、血管紧张素转换酶抑制剂、可溶性细胞因子受体及其衍生物(例如，可溶性p55或p75 TNF受体和衍生物p75TNFRIgG(ENBRELTM和p55TNFRIgG(Lenercept))、sIL-1RI、sIL-1RII、sIL-6R)、抗炎细胞因子(例如，IL-4、IL-10、IL-11、IL-13和TGFbeta)、塞来昔布、叶酸、硫酸羟氯喹、洛芬昔布、依那西普、英夫利昔单抗、萘普生、伐地考昔、柳氮磺吡啶、甲基强的松龙、美洛昔康、乙酸甲基强的松龙、硫代苹果酸金钠、阿司匹林、曲安缩松、萘磺酸右丙氧芬/扑热息痛、叶酸盐、萘普酮、扶他林、吡罗昔康、依托度酸、双氯酚酸钠、奥沙普嗪、盐酸羟可酮、重酒石酸二氢可待因酮/扑热息痛、双氯酚酸钠/米索前列醇、芬太尼、阿那白滞素(anakinra)、人重组体、盐酸曲马多、双水杨酸酯、舒林酸、氰钴胺素/fa/吡哆醇、扑热息痛、阿仑膦酸钠、强的松龙、硫酸吗啡、盐酸利多卡因、吲哚美辛、硫酸葡糖胺(glucosamine sulf)/软骨素、盐酸阿米替林、磺胺嘧啶、盐酸羟可酮/扑热息痛、盐酸奥洛帕定、米索前列醇、甲氧萘丙酸钠、奥美拉唑、环磷酰胺、利妥希玛、IL-1TRAP、MRA、CTLA4-IG、IL-18BP、抗IL-18、抗IL15、BIRB-796、SCIO-469、VX-702、AMG-548、VX-740、罗氟司特(Roflumilast)、IC-485、CDC-801和美苏帕玛(Mesopram)。
本发明的小RNA可以与之组合用于炎性肠病的治疗剂的非限制性例子包括下述：布地奈德；表皮生长因子、皮质类固醇、环孢菌素、柳氮磺吡啶、氨基水杨酸盐、6-巯基嘌呤、硫唑嘌呤、甲硝唑、脂肪加氧酶抑制剂、美沙拉秦、奥沙拉嗪、巴柳氮、抗氧化剂、血栓烷抑制剂、IL-1受体拮抗剂、抗-IL-1beta单克隆抗体、抗-IL-6单克隆抗体、生长因子、弹性蛋白酶抑制剂、吡啶基-咪唑化合物、针对其他人细胞因子或生长因子的抗体或拮抗剂，所述细胞因子或生长因子例如TNF、LT、IL-1beta、IL-2、IL-6、IL-7、IL-8、IL-15、IL-16、IL-17、IL-18、EMAP-II、GM-CSF、FGF和PDGF。本发明的小RNA可以与针对细胞表面分子及其配体的抗体组合，所述细胞表面分子例如CD2、CD3、CD4、CD8、CD25、CD28、CD30、CD40、CD45、CD69、CD90。小RNA还可以与试剂组合，所述试剂例如氨甲蝶呤、环孢菌素、FK506、雷帕霉素、霉酚酸酯、来氟洛米、NSAIDs例如布洛芬、皮质类固醇例如强的松龙、磷酸二酯酶抑制剂、腺苷激动剂、 抗凝剂、补体抑制剂、肾上腺素能药、干扰经由促炎细胞因子例如TNF-alpha或IL-1的发信号的试剂(例如IRAK、NIK、IKK、p38或MAP激酶抑制剂)、IL-1beta转换酶抑制剂、TNF-alpha转换酶抑制剂、T细胞发信号抑制剂例如激酶抑制剂、金属蛋白酶抑制剂、柳氮磺吡啶、硫唑嘌呤、6-巯基嘌呤、血管紧张素转换酶抑制剂、可溶性细胞因子受体及其衍生物(例如可溶性p55或p75 TNF受体，sIL-1RI、sIL-1RII、sIL-6R)和抗炎细胞因子(例如，IL-4、IL-10、IL-11、IL-13和TGFbeta)。
其中如本文描述的小RNA可以组合用于克罗恩病的治疗剂的示例性例子包括下述：TNF拮抗剂，例如抗TNF抗体，D2E7(PCT公开号WO97/29131；
Figure PCTCN2018123289-appb-000010
CA2
Figure PCTCN2018123289-appb-000011
CDP 571，TNFR-Ig构建体，(p75TNFRIgG
Figure PCTCN2018123289-appb-000012
和p55TNFRIgG(Lenercept))抑制剂和PDE4抑制剂。小RNA可以与皮质类固醇组合，例如布地奈德和地塞米松。小RNA还可以与试剂组合，所述试剂例如柳氮磺吡啶、5-氨基水杨酸和奥沙拉嗪，以及干扰促炎细胞因子例如IL-1合成或作用的试剂，例如IL-1beta转换酶抑制剂和IL-1RA。小RNA还可以与T细胞发信号抑制剂一起使用，例如，酪氨酸激酶抑制剂6-巯基嘌呤。小RNA可以与IL-11组合。小RNA可以与下述试剂组合：美沙拉秦、强的松、硫唑嘌呤、巯基嘌呤、英夫利昔单抗、甲基强的松龙琥珀酸钠、地芬诺酯/硫酸阿托品、盐酸洛哌丁胺、氨甲蝶呤、奥美拉唑、叶酸盐、环丙沙星/葡萄糖-水、重酒石酸二氢可待因酮/扑热息痛、盐酸四环素、氟轻松、甲硝唑、硫柳汞/硼酸、消胆胺/蔗糖、盐酸环丙沙星、硫酸莨菪碱、盐酸哌替啶、盐酸咪达唑、盐酸羟可酮/扑热息痛、盐酸异丙嗪、磷酸钠、磺胺甲异唑/甲氧苄啶、塞来昔布、聚卡波非、萘磺酸右丙氧芬、氢化可的松、多种维生素、巴柳氮二钠、磷酸可待因/扑热息痛、盐酸考来维仑(colesevelam hcl)、氰钴胺素、叶酸、左氟沙星、甲基强的松龙、那他珠单抗和干扰素γ。
本发明的小RNA可以与之组合用于多发性硬化的治疗剂的非限制性例子包括下述：皮质类固醇，强的松龙，甲基强的松龙，硫唑嘌呤，环磷酰胺，环孢菌素，氨甲蝶呤，4-氨基吡啶，替扎尼定，干扰素-beta1a
Figure PCTCN2018123289-appb-000013
Biogen)，干扰素-beta1b
Figure PCTCN2018123289-appb-000014
Chiron/Berlex)，干扰素α-n3)(Interferon Sciences/Fujimoto)，干扰素-α(Alfa Wassermann/J&J)，干扰素beta1A-IF(Serono/Inhale Therapeutics)，聚乙二醇 化干扰素(Peginterferon)α2b(Enzon/Schering-Plough)，共聚物1(Cop-1，
Figure PCTCN2018123289-appb-000015
Teva Pharmaceutical Industries，Inc.)，高压氧，静脉内免疫球蛋白，克拉屈滨(clabribine)，针对其他人细胞因子或生长因子及其受体的抗体或拮抗剂或抑制剂，例如，TNF、LT、IL-1beta、IL-2、IL-6、IL-7、IL-8、IL-1A、IL-15、IL-16、IL-18、EMAP-II、GM-CSF、FGF和PDGF。本发明的小RNA可以与针对细胞表面分子或其配体的抗体组合，所述细胞表面分子例如CD2、CD3、CD4、CD8、CD19、CD20、CD25、CD28、CD30、CD40、CD45、CD69、CD80、CD86、CD90。本发明的小RNA还可以与试剂组合，所述试剂例如FK506、雷帕霉素、霉酚酸酯、来氟洛米、NSAIDs例如布洛芬、磷酸二酯酶抑制剂、腺苷激动剂、抗凝剂、补体抑制剂、肾上腺素能药、干扰经由促炎细胞因子例如TNF-alpha或IL-1的发信号的试剂(例如IRAK、NIK、IKK、p38或MAP激酶抑制剂)、IL-1b转换酶抑制剂、TACE抑制剂、T细胞发信号抑制剂例如激酶抑制剂、金属蛋白酶抑制剂、柳氮磺吡啶、硫唑嘌呤、6-巯基嘌呤、血管紧张素转换酶抑制剂、可溶性细胞因子受体及其衍生物(例如可溶性p55或p75 TNF受体，sIL-1RI、sIL-1RII、sIL-6R)、抗炎细胞因子(例如IL-4、IL-10、IL-13和TGFbeta)、
Figure PCTCN2018123289-appb-000016
和半胱天冬酶抑制剂例如半胱天冬酶-1抑制剂。
本发明的小RNA还可以与试剂组合，所述试剂例如阿来组单抗、屈大麻酚、尤迈(Unimed)、达克珠单抗(daclizumab)、米托蒽醌、盐酸扎利罗登(xaliproden hydrochloride)、4-氨基吡定、乙酸格拉太咪尔、那他珠单抗、辛纳必醇(sinnabidol)、a-免疫因子(a-immunokine)NNSO3、ABR-215062、AnergiX.MS、趋化因子受体拮抗剂、BBR-2778、卡拉古林(calagualine)、CPI-1189、LEM(脂质体被囊化的米托蒽醌)、THC.CBD(大麻素激动剂)、MBP-8298、美苏帕玛(PDE4抑制剂)、MNA-715、抗IL-6受体抗体、神经素(neurovax)、哌非尼酮allotrap 1258(RDP-1258)、sTNF-R1、他仑帕奈(talampanel)、特立氟胺(teriflunomide)、TGF-beta2、替利莫肽(tiplimotide)、VLA-4拮抗剂(例如，TR-14035、VLA4 Ultrahaler、Antegran-ELAN/Biogen)、干扰素γ拮抗剂、IL-4激动剂。
本发明的小RNA可以与之组合用于治疗或预防心绞痛的治疗剂的非限制性例子包括下述：阿司匹林、硝酸甘油、单硝酸异山梨酯、琥珀酸美托洛尔、阿替洛尔、酒石酸美托洛尔、阿罗地平磺酸盐、盐酸地尔硫、硝 酸异山梨酯、重硫酸氯吡格雷、硝苯地平、阿托伐他汀钙、氯化钾、呋塞米、斯伐他汀、盐酸维拉帕米、地高辛、盐酸普萘洛尔、卡维地洛、赖诺普利、螺内酯、氢氯噻嗪、马来酸依那普利、纳多洛尔、雷米普利、依诺肝素钠、肝素钠、缬沙坦、盐酸索他洛尔、非诺贝特、依泽替米贝(ezetimibe)、布美他尼、氯沙坦钾、赖诺普利/氢氯噻嗪、非洛地平、卡托普利和富马酸比索洛尔。
小RNA可以与之组合用于治疗或预防强直性脊柱炎的治疗剂的非限制性例子包括下述：布洛芬、扶他林和米索前列醇、萘普生、美洛昔康、吲哚美辛、扶他林、塞来昔布、洛芬昔布、柳氮磺吡啶、氨甲蝶呤、硫唑嘌呤、米诺环素、强的松、依那西普和英夫利昔单抗。
本发明的小RNA可以与之组合用于治疗或预防哮喘的治疗剂的非限制性例子包括下述：沙丁胺醇、沙美特罗/氟替卡松、孟鲁司特钠、丙酸氟替卡松、布地奈德、强的松、沙美特罗羟萘甲酸盐(sameterol xinafoate)、盐酸左旋沙丁胺醇、硫酸沙丁胺醇/异丙托铵、强的松龙磷酸钠、曲安缩松、倍氯美松双丙酸酯、异丙托溴铵、阿奇霉素、醋酸吡布特罗、强的松龙、无水茶碱、甲基强的松龙琥珀酸钠、克拉霉素、扎鲁司特、富马酸福莫特罗、流感病毒疫苗、甲基强的松龙、阿莫西林三水合物、氟尼缩松、变态反应注射、色甘酸钠、盐酸非索那丁、氟尼缩松/薄荷醇、阿莫西林/克拉维酸钾、左氟沙星、吸入器辅助装置、愈创木酚甘油醚、地塞米松磷酸钠、盐酸莫西沙星、盐酸多西环素、愈创木酚甘油醚/d-甲吗喃、p-麻黄素/cod/氯苯那敏(chlorphenir)、加替沙星、盐酸西替立嗪、糠酸莫米他松、沙美特罗羟萘甲酸盐、苯佐那酯、头孢氨苄、二氢可待因酮/氯苯那敏、盐酸西替立嗪/伪麻黄碱(pseudoephed)、苯福林/异丙嗪、可待因/异丙嗪、头孢罗齐、地塞米松、愈创木酚甘油醚/伪麻黄碱、氯苯那敏/二氢可待因酮、奈多罗米钠、硫酸特布他林、肾上腺素、甲基强的松龙和硫酸间羟异丙肾上腺素。
本发明的小RNA可以与之组合用于治疗或预防COPD的治疗剂的非限制性例子包括下述：硫酸沙丁胺醇/异丙托铵、异丙托溴铵、沙美特罗/氟替卡松、沙丁胺醇、沙美特罗羟萘甲酸盐、丙酸氟替卡松、强的松、无水茶碱、甲基强的松龙琥珀酸钠、孟鲁司特钠、布地奈德、富马酸福莫特罗、曲安缩松、左氟沙星、愈创木酚甘油醚、阿奇霉素、倍氯美松双丙酸酯、盐酸左旋沙丁胺醇、氟尼缩松、头孢曲松钠、阿莫西林三水合物、加 替沙星、扎鲁司特、阿莫西林/克拉维酸钾、氟尼缩松/薄荷醇、氯苯那敏/二氢可待因酮、硫酸间羟异丙肾上腺素、甲基强的松龙、糠酸莫米他松、p-麻黄素/cod/氯苯那敏、醋酸吡布特罗、p-麻黄素/氯雷他定、硫酸特布他林、噻托溴铵(tiotropium bromide)、(R,R)-福莫特罗、TgAAT、西洛司特和罗氟司特。
本发明的小RNA可以与之组合用于治疗或预防HCV的治疗剂的非限制性例子包括下述：干扰素-α-2a、干扰素-α-2b、干扰素-αcon1、干扰素-α-n1、聚乙二醇化干扰素-α-2a、聚乙二醇化干扰素-α-2b、利巴韦林、聚乙二醇干扰素α-2b+利巴韦林、熊脱氧胆酸、甘草酸、胸腺法新、马克胺(Maxamine)、VX-497以及通过干扰下述靶用于治疗HCV的任何化合物：HCV聚合酶、HCV蛋白酶、HCV解旋酶、HCV IRES(内部核糖体进入位点)。
小RNA可以与之组合用于治疗或预防特发性肺纤维化的治疗剂的非限制性例子包括下述：强的松、硫唑嘌呤、沙丁胺醇、秋水仙碱、硫酸沙丁胺醇、地高辛、γ干扰素、甲基强的松龙琥珀酸钠(sod succ)、劳拉西泮、呋塞米、赖诺普利、硝酸甘油、螺内酯、环磷酰胺、异丙托溴铵、放线菌素d、阿替普酶、丙酸氟替卡松、左氟沙星、硫酸间羟异丙肾上腺素、硫酸吗啡、盐酸羟可酮、氯化钾、曲安缩松、无水他克莫司、钙、干扰素-α、氨甲蝶呤、霉酚酸酯和干扰素-γ-1b。
本发明的小RNA可以与之组合用于治疗或预防心肌梗塞的治疗剂的非限制性例子包括下述：阿司匹林、硝酸甘油、酒石酸美托洛尔、依诺肝素钠、肝素钠、重硫酸氯吡格雷、卡维地洛、阿替洛尔、硫酸吗啡、琥珀酸美托洛尔、华法林钠、赖诺普利、单硝酸异山梨酯、地高辛、呋塞米、斯伐他汀、雷米普利、替尼普酶、马来酸依那普利、托拉塞米(torsemide)、瑞替普酶、氯沙坦钾、盐酸喹那普利/mag carb、布美他尼、阿替普酶、依那普利拉、盐酸胺碘酮、盐酸替罗非班一水合物、盐酸地尔硫、卡托普利、依贝沙坦、缬沙坦、盐酸普萘洛尔、福辛普利钠、盐酸利多卡因、表非替得、头孢唑啉钠、硫酸阿托品、氨基已酸、螺内酯、干扰素、盐酸索他洛尔、氯化钾、多库酯钠、盐酸多巴酚丁胺、阿普唑仑、普伐他丁钠、阿托伐他汀钙、盐酸咪达唑、盐酸哌替啶、硝酸异山梨酯、肾上腺素、盐酸多巴胺、比伐卢定、罗苏伐他汀(rosuvastatin)、依泽替米贝/斯伐他汀、阿伐 麦布(avasimibe)和卡立泊来德(cariporide)。
本发明的小RNA可以与之组合用于治疗或预防牛皮癣的治疗剂的非限制性例子包括下述：卡泊三醇(calcipotriene)、丙酸氯倍他索、曲安缩松、丙酸卤倍他索、他佐罗汀、氨甲蝶呤、氟轻松、增强型二丙酸倍他米松、醋酸氟轻松、阿昔曲丁、焦油(tar)洗发剂、戊酸倍他米松、糠酸莫米他松、酮康唑、丙吗卡因/氟轻松、戊酸氢化可的松、氟羟可舒松、尿素、倍他米松、丙酸氯倍他索/润滑药(emoll)、丙酸氟替卡松、阿奇霉素、氢化可的松、湿润配方、叶酸、丙缩羟强龙、吡美莫司(pimecrolimus)、煤焦油、双醋二氟松、叶酸依那西普、乳酸、8-甲氧基补骨质素、hc/次五倍子酸铋(bismuth subgal)/znox/resor、乙酸甲基强的松龙、强的松、遮光剂、氯氟舒松、水杨酸、蒽地酚、氯可托龙、煤提取物、煤焦油/水杨酸、煤焦油/水杨酸/硫、去羟米松、地西泮、润滑药、氟轻松/润滑药、矿物油/蓖麻油/na lact、矿物油/花生油、石油/肉豆蔻酸异丙酯、补骨脂素、水杨酸、皂/三溴沙仑、硫柳汞/硼酸、塞来昔布、英夫利昔单抗、环孢菌素、阿来塞普、依法利珠单抗、他克莫司、吡美莫司、PUVA、UVB和柳氮磺吡啶。
本发明的小RNA可以与之组合用于治疗或预防牛皮癣性关节炎的治疗剂的非限制性例子包括下述：氨甲蝶呤、依那西普、洛芬昔布、塞来昔布、叶酸、柳氮磺吡啶、萘普生、来氟洛米、乙酸甲基强的松龙、吲哚美辛、硫酸羟氯喹、强的松、舒林酸、增强型二丙酸倍他米松、英夫利昔单抗、氨甲蝶呤、叶酸盐、曲安缩松、扶他林、二甲基亚砜、吡罗昔康、双氯酚酸钠、酮基布洛芬、美洛昔康、甲基强的松龙、萘普酮、托美汀钠、卡泊三醇、环孢菌素、双氯酚酸钠/米索前列醇、氟轻松、硫酸葡糖胺、硫代苹果酸金钠、重酒石酸二氢可待因酮/扑热息痛、布洛芬、利塞膦酸钠、磺胺嘧啶、硫鸟嘌呤、伐地考昔、阿来塞普和依法利珠单抗。
本发明的小RNA可以与之组合用于治疗或预防再狭窄的治疗剂的非限制性例子包括下述：西罗莫司、紫杉醇、依维莫司(everolimus)、他克莫司、ABT-578和扑热息痛。
本发明的小RNA可以与之组合用于治疗或预防坐骨神经痛的治疗剂的非限制性例子包括下述：重酒石酸二氢可待因酮/扑热息痛、洛芬昔布、盐酸环苯扎林、甲基强的松龙、萘普生、布洛芬、盐酸羟可酮/扑热息痛、塞来昔布、伐地考昔、乙酸甲基强的松龙、强的松、磷酸可待因/扑热息痛、 盐酸曲马多/扑热息痛、美他沙酮、美洛昔康、美索巴莫、盐酸利多卡因、双氯酚酸钠、加巴喷丁、地塞米松、卡立普多、酮咯酸氨基丁三酸醇盐、吲哚美辛、扑热息痛、地西泮、萘普酮、盐酸羟可酮、盐酸替扎尼定、双氯酚酸钠/米索前列醇、萘磺酸右丙氧芬/扑热息痛、asa/oxycod/羟可酮、布洛芬/二氢可待因酮bit、盐酸曲马多、依托度酸、盐酸丙氧芬、盐酸阿米替林、卡立普多/磷酸可待因/asa、硫酸吗啡、多种维生素、甲氧萘丙酸钠、柠檬酸奥芬那君和替马西泮。
其中本发明的小RNA可以组合用于治疗或预防系统性红斑狼疮(SLE)的治疗剂的非限制性例子包括下述：NSAIDS，例如，扶他林、萘普生、布洛芬、吡罗昔康、吲哚美辛，COX2抑制剂，例如，塞来昔布、洛芬昔布、伐地考昔，抗疟药，例如，羟氯喹，类固醇，例如，强的松、强的松龙、布地奈德、地塞米松，细胞毒素，例如，硫唑嘌呤、环磷酰胺、霉酚酸酯、氨甲蝶呤，PDE4抑制剂或嘌呤合成抑制剂，例如
Figure PCTCN2018123289-appb-000017
小RNA还可以与试剂组合，所述试剂例如柳氮磺吡啶、5-氨基水杨酸、奥沙拉嗪、依木兰，以及干扰促炎细胞因子例如IL-1合成、生产或作用的试剂，例如胱天蛋白酶抑制剂，如IL-1beta转换酶抑制剂和IL-1ra。小RNA还可以与T细胞发信号抑制剂一起使用，例如酪氨酸激酶抑制剂，或靶向T细胞活化分子的分子，例如CTLA-4-IgG或抗B7家族抗体和抗PD-1家族抗体。本发明的小RNA可以与IL-11或抗细胞因子抗体组合，例如芬突利珠单抗(fonotolizumab)(抗IFNg抗体)，或抗受体受体抗体，例如抗IL-6受体抗体和针对B细胞表面分子的抗体。小RNA还可以与下述试剂一起使用：LJP 394(阿贝莫司(abetimus))，耗尽或灭活B细胞的试剂，例如利妥昔单抗(抗CD20抗体)，淋弗斯特(lymphostat)-B(抗BlyS抗体)，TNF拮抗剂，例如，抗TNF抗体，D2E7(PCT公开号WO 97/29131，
Figure PCTCN2018123289-appb-000018
CA2
Figure PCTCN2018123289-appb-000019
CDP 571，TNFR-Ig构建体(p75TNFRIgG
Figure PCTCN2018123289-appb-000020
和p55TNFRIgG(Lenercept))。
TNF-alpha相关疾病
TNF-alpha在各种人类疾病中具有被证实的病理生理作用，尤其是炎症性紊乱、免疫和免疫调节的紊乱、引起脓毒性、内毒素性和心血管性休克的感染、神经变性疾病和恶性病。本发明的小RNA可以应用于治疗下列中所列疾病，其不被认为是完整或排他的列表。也包括没有具体提及的 直接或间接受TNF-alpha影响的其他疾病。
自身免疫或慢性炎症：一般的炎症慢性和/或自身免疫状态、一般的免疫介导的炎性紊乱、炎性CNS病、影响眼睛、关节、皮肤、粘膜、中枢神经系统、胃肠道、泌尿道或肺的炎性疾病、一般的眼色素层炎状态、视网膜炎、HLA-B27+眼色素层炎、Behcet氏病、干眼综合征、青光眼、干燥综合征(Sjgren syndrome)、糖尿病(包括糖尿病神经病变)、胰岛素抵抗、一般的关节炎状态、类风湿性关节炎、骨关节炎、反应性关节炎和瑞特氏综合征(Reiter′s syndrome)、少年关节炎、强直性脊柱炎、多发性硬化、格林-巴利综合征(Guillain-Barre syndrome)、重症肌无力、肌萎缩性侧索硬化、结节病、肾小球性肾炎、慢性肾病、膀胱炎、银屑病(包括牛皮癣关节炎)、化脓性汗腺炎、脂膜炎、坏疽性脓皮病、SAPHO综合征(滑膜炎、痤疮、脓疱病、骨肥大和骨炎)、痤疮、Sweet综合征、天疱疮、克罗恩病(包括肠外表现)、溃疡性结肠炎、支气管哮喘、过敏性肺炎、一般的变态反应、过敏性鼻炎、过敏性鼻窦炎、慢性阻塞性肺病(COPD)、肺部纤维化、Wegener肉芽肿病、川崎综合征、巨细胞性动脉炎、Churg-Strauss血管炎、结节性多动脉炎、烧伤、移植物抗宿主疾病、宿主抗移植物反应、器官或骨髓移植后排斥发作、一般的全身或局部血管炎状态、系统性和盘状红斑狼疮、多发性肌炎和皮肌炎、硬皮病、先兆子痫、急性和慢性胰腺炎、病毒性肝炎、酒精性肝炎。急性炎症和/或预防术后或外伤后炎症和疼痛：预防一般的术后炎症、眼手术(例如白内障(眼晶体置换)或青光眼手术)、关节手术(包括关节镜手术)、关节相关结构(例如韧带)的手术、口腔和/或牙科手术、最低限度的介入心血管程序(例如PTCA、经皮腔内斑块旋切术(atherectomy)、放置支架)、腹腔镜和/或内窥镜腹内和妇科程序、内窥镜泌尿科程序(例如前列腺手术、输尿管镜检查、膀胱镜检查、间质性膀胱炎)、一般的手术前后炎症(预防)。神经病和神经变性疾病：阿尔茨海默病(Alzheimer disease)、帕金森氏病、亨廷顿舞蹈病、贝尔麻痹(Bell′palsy)、克-雅病(Creutzfeld-Jakob disease)。癌症：癌症相关骨质溶解、癌症相关炎症、癌症相关疼痛、癌症相关恶病质、骨转移。疼痛：急性和慢性形式的疼痛，无论这些是由TNF-alpha的中枢还是外周作用引起的和无论它们被分为炎性、伤害性还是神经性形式的疼痛、坐骨神经痛、腰背痛、腕管综合征、复杂性区域疼痛综合征(CRPS)、痛风、带状疱疹后神经痛、纤维肌痛、局部疼痛状态、 由于转移性肿瘤引起的慢性疼痛综合征、dismenorrhea。感染：细菌、病毒或真菌性脓毒病、结核、AIDS。心血管疾病：动脉粥样硬化、冠状动脉病、高血压、血脂障碍(dyslipidemia)、心功能不全和慢性心力衰竭。在一个实施方案中，TNF-alpha-相关疾病是脊柱关节病，与肺有关的病症，冠心病，代谢病症，贫血，疼痛，肝脏病症，皮肤病症，指甲病症，或脉管炎。在另一个实施方案中，TNF-alpha-相关疾病是与年龄有关的恶病质，早老性痴呆，脑水肿，炎性脑损伤，慢性疲劳综合征，皮肌炎，药物反应，脊髓之内和/或周围的水肿，家族周期性发热，Felty′s综合征，纤维化，肾小球性肾病(例如链球菌感染后肾小球肾炎或IgA肾病)，假体松弛，显微多脉管炎，混合型结缔组织病症，多发性骨髓瘤，癌症和恶病质，多器官失调，脊髓发育不良综合征，orchitism骨质溶解，胰腺炎，包括急性，慢性，和胰腺脓肿，牙周病多肌炎，进行性肾衰竭，假痛风，坏疽性脓皮病，复发性多软骨炎，风湿性心脏病，肉样瘤病，硬化胆管炎，中风，胸腹主动脉动脉瘤修补(TAAA)，TNF受体相关周期性综合征(TRAPS)，与黄热病疫苗接种相关的综合征，与耳，慢性耳炎，或儿科耳炎相关的炎性疾病。在本发明的另一个实施方案中，TNF-alpha-相关疾病是克隆氏病-相关疾病，少年关节炎/斯提耳氏病(JRA)，葡萄膜炎，坐骨神经痛，前列腺炎，子宫内膜异位，脉络膜新血管生成，狼疮，斯耶格伦氏综合征，和湿黄斑变性。
本发明的小RNA可以与之联合使用的治疗药物的非限制性例子包括如下：非甾族化合物抗炎药(NSAIDs)；细胞因子抑制性抗炎药(CSAIDs)；CDP-571/BAY-10-3356(人源化抗-小RNA；Celltech/Bayer)；cA2/英夫单抗(嵌合抗-小RNA；Centocor)；75kd TNFR-IgG/etanercept(75kD TNF受体-IgG融合蛋白；Immunex；J Invest.Med.(1996)Vol.44，235A)；55kd TNF-IgG(55kD TNF受体-IgG融合蛋白；Hoffmann-LaRoche)；IDEC-CE 9.1/SB 210396(非耗尽型灵长类化抗-CD4抗体；IDEC/SmithKline；DAB 486-IL-2和/或DAB389-IL-2(IL-2融合蛋白；Seragen；Anti-Tac(人源化抗-IL-2Rα；Protein Design Labs/Roche)；IL-4(抗炎细胞因子；DNAX/Schering)；IL-10(SCH 52000；重组IL-10，抗炎细胞因子；DNAX/Schering)；IL-4；IL-10和/或IL-4激动剂(例如，激动剂抗体)；IL-1RA(IL-1受体拮抗剂；Synergen/Amgen)；TNF-bp/s-TNF(可溶性TNF结合蛋白质；R973401(磷酸二酯酶IV型抑制剂；MK-966(COX-2抑制剂)；伊洛前列；甲氨喋呤；萨利多胺和萨利多胺-相 关药物(例如，Celgen)；来氟洛米(抗炎剂和细胞因子抑制剂)；凝血酸(纤溶酶原激活的抑制剂；T-614(细胞因子抑制剂)；前列腺素El；替尼达普(非甾族抗炎药)；萘普生(非甾族抗炎药)；莫比可(非甾族抗炎药)；布洛芬(非甾族抗炎药)；吡罗昔康(非甾族抗炎药)；双氯酚酸钠(非甾族抗炎药)；消炎痛(非甾族抗炎药)；水杨酸偶氮磺胺吡啶；硫唑嘌呤；ICE抑制剂(酶白介素-1beta转化酶的抑制剂)；zap-70和/或Ick抑制剂(酪蛋白激酶的抑制剂zap-70或lck)；VEGF抑制剂和/或VEGF-R抑制剂(血管内皮细胞生长因子或血管内皮细胞生长因子受体；血管生成的抑制剂)；皮质类固醇抗炎药(例如，SB203580)；TNF-转化酶抑制剂；抗-IL-12抗体；抗-IL-18抗体；白介素-11；白介素-13；白介素-17抑制剂；金；青霉素；氯喹；羟氯喹；苯丁酸氮芥；环磷酰胺；环孢菌素；总的淋巴细胞照射法；抗胸腺细胞球蛋白；抗-CD4抗体；CD5-毒素；口服给药的肽和胶原；羟苯基氯氨茴酸二钠；细胞因子调节剂(CRAs)HP 228和HP 466(Houghten Pharmaceuticals，Inc.)；ICAM-1抗敏硫代磷酸酯寡脱氧核苷酸(ISIS 2302；Isis Pharmaceuticals，Inc.)；可溶性补体受体1(TP10；T Cell Sciences，Inc.)；泼尼松；奥古蛋白；糖胺聚糖多硫酸盐；二甲胺四环素；抗-IL2R抗体；海产品和植物脂质(fish and plant seed fatty acids)；金诺芬；保泰松；抗炎酸；氟灭酸；静脉内免疫球蛋白；zileuton；霉酚酸(RS-61443)；他克莫司(FK-506)；西罗莫司(瑞帕霉素)；阿普劳斯amiprilose(阿普劳斯therafectin)；克拉屈滨(2-氯脱氧腺苷)；氮尿苷；甲氨喋呤；抗病毒剂；和免疫调节剂。上面提到的药物的任一种可以与本发明的小RNA联合用药治疗TNF-alpha相关疾病。
在一个实施方案中，本发明的小RNA与下面一种药物联合用药治疗类风湿性关节炎：小分子抑制剂KDR(ABT-123)，Tie-2的小分子抑制剂；甲氨喋呤；泼尼松；塞来昔布；叶酸；羟氯喹硫酸盐；罗非克西；etanercept；英夫单抗；来氟洛米；萘普生；valdecoxib；磺胺吡啶；甲基氢化泼尼松；布洛芬；美洛昔康；甲基氢化泼尼松乙酸盐；硫羟苹果酸金钠；阿司匹林；氮杂硫代嘌呤；醋酸去炎松；萘磺酸丙氧芬/扑热息痛；叶酸盐；萘丁美酮；双氯酚酸钠；吡罗昔康；依托度酸；双氯酚酸钠；奥沙普嗪；羟可待酮盐酸盐；氢可酮二酒石酸盐/扑热息痛；双氯酚酸钠/米索普特；芬太尼；anakinra，人重组体；盐酸曲马多；双水杨酯；舒林酸；维生素B12/fa/维生素B6；对乙酰氨基酚；阿仓膦酸钠；氢化泼尼松；硫酸吗啡；盐酸利多 卡因；消炎痛；氨基葡萄糖硫酸盐/软骨素；环孢素；盐酸阿米替林；磺胺嘧啶；羟可待酮盐酸盐/对乙酰氨基酚；盐酸奥洛帕定；米索普特；甲氧萘丙酸钠；奥美拉唑；霉酚酸酯；环磷酰胺；利妥希玛；IL-1TRAP；MRA；CTLA4-IG；IL-8BP；ABT-874；ABT-324(抗-IL18)；抗-IL15；BIRB-796；SCIO-469；VX-702；AMG-548；VX-740；Roflumilast；IC-485；CDC-801；和mesopram。在另一个实施方案中，本发明的小RNA与上面提到的用于治疗类风湿性关节炎的药物的一种联合用药，用于治疗TNF-alpha相关疾病。
在一个实施方案中，本发明的小RNA与下面一种药物联合用药治疗其中TNF-alpha活性有害的TNF-alpha相关疾病：抗-IL12抗体(ABT874)；抗-IL18抗体(ABT 325)；LCK的小分子抑制剂；COT的小分子抑制剂；抗-Ill抗体；MK2的小分子抑制剂；抗-CD 19抗体；CXCR3的小分子抑制剂；CCR5的小分子抑制剂；CCRll的小分子抑制剂；抗-E/L选择蛋白抗体；P2X7的小分子抑制剂；IRAK-4的小分子抑制剂；糖皮质激素受体的小分子激动剂；抗-C5a受体抗体；C5a受体的小分子抑制剂；抗-CD32抗体；和CD32作为治疗性蛋白质。
在另一个实施方案中，本发明的小RNA与抗生素和抗感染药联合用药。抗感染药包括本领域公知的用来治疗病毒，真菌，寄生虫或细菌感染。这里使用的术语“抗生素”指抑制微生物生长或者处死微生物的化学物质。该术语包括本领域公知的微生物产生的抗生素，以及合成的抗生素(例如，类似物)。抗生素包括但不限于，克拉霉素(Biaxin)，环丙沙星(Cipro)，和灭滴灵(Flagyl)。
在另一个实施方案中，本发明的小RNA与治疗坐骨神经痛或疼痛的另外的治疗药物联合用药。能用来减轻或抑制坐骨神经痛或疼痛症状的药物的例子包括氢可酮二酒石酸盐/扑热息痛，罗非克西，盐酸环苯扎林，甲基泼尼松，萘普生，布洛芬，羟可待酮盐酸盐/对乙酰氨基酚，塞来昔布，valdecoxib，乙酸甲基泼尼松，泼尼松，磷酸可卡因/扑热息痛，盐酸曲马多/对乙酰氨基酚，美他沙酮，美洛昔康，美索巴莫，盐酸利多卡因，双氯芬酸钠，加巴喷丁，地塞米松，卡利普多，酮咯酸，消炎痛，对乙酰氨基酚，安定，萘丁美酮，盐酸氧可酮，盐酸替扎尼定，双氯酚酸钠/米索前列醇，丙氧芬萘磺酸盐/扑热息痛，布洛芬/氢可酮少量；盐酸曲马多，依托 度酸；盐酸丙氧芬，盐酸阿米替林，卡利普多/磷酸可待因，硫酸吗啡，多种维生素，甲氧萘丙酸钠，柠檬酸邻甲苯海拉明，和替马西泮。
在另一个实施方案中，与血液透析联合使用本发明的小RNA制备TNF-alpha-相关疾病。
在另一个实施方案中，本发明的小RNA与用来治疗克隆氏病或克隆氏病相关疾病的药物联合使用。能用来治疗克隆氏病的治疗性药物包括美沙拉秦，泼尼松，硫唑嘌呤，巯基嘌呤，英夫单抗，布地奈德，水杨酸偶氮磺胺吡啶，甲基泼尼龙，苯乙哌啶，盐酸洛派丁胺，甲氨喋呤，，叶酸盐，环丙沙星/葡萄糖-水，氢可酮二酒石酸盐，盐酸四环素，氟轻松醋酸酯，灭滴灵，硫汞撒/硼酸，考来烯胺/蔗糖，盐酸环丙沙星，硫酸天仙子胺，盐酸度冷丁，盐酸咪达唑仑，羟可待酮盐酸盐/对乙酰氨基酚，盐酸普鲁米近，磷酸钠，新诺明/甲氧苄定，塞来昔布，聚丙烯酸树脂，丙氧芬萘磺酸盐，氢化可的松，多种维生素，巴柳氮二钠，磷酸可卡因/扑热息痛，colesevelan盐酸盐，维生素B12，叶酸，左氟沙星，甲基氢化泼尼松，natalizumab，和γ-干扰素。
在另一个实施方案中，本发明的小RNA与用来治疗哮喘的另外的治疗药物联合用药。能用来减轻或抑制哮喘症状的药物的例子包括如下：沙丁醇胺；沙美特罗/氟地松；钠；丙酸氟地松；布地缩松；泼尼松；沙美特罗xinafoate；盐酸levalbuterol；硫酸盐/异丙托品；磷酸泼尼松钠；曲安奈德；倍氯美松双丙酸酯；异丙托品溴化物；阿齐霉素；醋酸吡布特罗，泼尼松，无水茶碱，甲基泼尼龙，克拉霉素，扎鲁斯特，富马酸福莫特罗，流感病毒疫苗，甲基泼尼龙三水合物，氟尼缩松，变应性变态反应注射，色甘酸钠，盐酸甲美芳铵，氟尼缩松/薄荷醇，阿莫西林/克拉维酸钾，左氟沙星，吸入辅助装置，愈创甘油醚，地塞米松磷酸钠；盐酸莫西沙星；hyclate；愈创木酚甘油醚/d-甲吗喃；扑尔敏；加替沙星；盐酸西替利嗪；糠酸毛他松；沙美特罗xinafoate；退咳露；头孢氨苄；氢可酮/扑尔敏；盐酸西替利嗪/伪麻黄碱；苯基麻黄碱/异丙嗪；可待因/异丙嗪；头孢罗齐；地塞米松；愈创木酚甘油醚/伪麻黄碱；扑尔敏/氢可酮，奈多米罗纳，硫酸特布他林，肾上腺素和甲基泼尼松，硫酸奥西那林。
在另一个实施方案中，本发明的小RNA与用来治疗COPD的另外的治疗药物联合用药。能用来减轻或抑制COPD症状的药物的例子包括硫酸 沙丁胺醇/异丙托品；异丙托溴胺；沙美特罗/氟地松；沙丁胺醇；沙美特罗；xinafoate；丙酸氟地松；泼尼松；无水茶叶碱；甲基氢化泼尼松sod succ；孟鲁司特钠；布地奈德；富马酸福莫特罗；曲安奈德；左氟沙星；愈创甘油醚；阿齐霉素；倍氯米松；二丙酸；盐酸levalbuterol；氟尼缩松；钠；三水合物；加替沙星；扎鲁司特；阿莫西林/克拉维酸钾；氟尼缩松/薄荷醇；氯苯吡胺/氢可酮；硫酸奥西那林；甲泼尼龙；糠酸盐；-麻黄碱/cod/扑尔敏；盐酸吡布特罗；-麻黄碱/氯雷他定；硫酸特布他林；tiotropium bromide；(R，R)-福莫特罗；TgAAT；Cilomilast和Roflumilast。
在另一个实施方案中，本发明的小RNA与用来治疗IPF的另外的治疗药物联合用药。能用来减轻或抑制IPF症状的药物的例子包括泼尼松；硫唑嘌呤；沙丁醇胺；秋水仙碱；硫酸盐；地高辛；γ干扰素；甲泼尼龙sod succ；呋塞米；赖诺普利；硝酸甘油；螺内酯；环磷酰胺；溴化异丙托品；放线菌素d；阿替普酶；丙酸氟地松；左氟沙星；硫酸奥西那林；硫酸吗啡；盐酸羟可酮；氯化钾；曲安奈德；无水藤霉素；钙；α-干扰素；甲氨喋呤；霉酚酸mofetil。
在一个实施方案中，本发明的小RNA与一般用来治疗脊柱关节病的另外的治疗药物联合用药。这样的药物的例子包括非甾族抗炎药(NSAIDs)，COX 2抑制剂，包括Celebrex，Vioxx；和Bextra，和etoricoxib。还通常用物理疗法来治疗脊柱关节病，通常与非甾族抗炎药联合使用。
在另一个实施方案中，本发明的小RNA与用来治疗强直性脊柱炎的另外的治疗药物联合用药。能用来减轻或抑制强直性脊柱炎症状的药物的例子包括布洛芬，双氯芬酸和米索前列醇，萘普生，美洛昔康，消炎痛，双氯芬酸，塞来昔布，罗非克西，水杨酸偶氮磺胺吡啶，泼尼松，甲氨喋呤，硫唑嘌呤，二甲胺四环素，泼尼松，etanercept，和英夫单抗。
在另一个实施方案中，本发明的小RNA与用来治疗牛皮癣患者关节炎的另外的治疗药物联合用药。能用来减轻或抑制牛皮癣患者关节炎症状的药物的例子包括甲氨喋呤；etanercept；罗非克西；塞来昔布；叶酸；水杨酸偶氮磺胺吡啶；萘普生；来氟洛米；醋酸甲泼尼龙；消炎痛；硫酸羟氯喹；舒林酸；泼尼松；倍他米松diprop增量；英夫单抗；甲氨喋呤；叶酸；曲安奈德；双氯芬酸；二甲亚砜；吡罗昔康；双氯芬酸钠；酮洛芬；美洛昔康；泼尼松；甲泼尼龙；萘丁美酮；四苯酰吡咯乙酸钠；钙泊三烯； 环孢菌素；双氯芬酸；钠/米索前列醇；氟轻松醋酸酯；硫酸氨基葡萄糖；硫羟苹果酸金钠；氢可酮；二酒石酸盐/扑热息痛；布洛芬；利塞膦酸钠；磺胺嘧啶；硫代鸟嘌呤；valdecoxib；alefacept；和efalizumab。
本发明的小RNA能与用来治疗再狭窄的另外的治疗药物联合用药。能用来减轻或抑制再狭窄的药物的例子包括瑞帕霉素，紫杉醇，everolimus，藤霉素，ABT-578，和对乙酰氨基酚。
本发明的小RNA能与用来治疗心肌梗塞的另外的治疗药物联合用药。能用来减轻或抑制心肌梗塞的药物的例子包括阿司匹林，硝酸甘油，酒石酸美托洛尔，依诺肝素钠，肝素钠，氯吡格雷硫酸氢盐，卡维地洛，阿替洛尔，硫酸吗啡，琥珀酸美托洛尔，华法令钠，赖诺普利，异山梨醇一硝酸盐，地高辛，呋塞米，辛伐他汀，雷米普利，替尼普酶，马来酸依那普利，torsemide，瑞替普酶，氯沙坦钾，盐酸喹那普利/mag carb，布美他尼，阿替普酶，依那普里拉，盐酸胺碘酮，盐酸替罗非班m-水合物，盐酸地尔硫翁，卡托普利，吉加，缬沙坦，盐酸普萘洛尔，福辛普利钠，盐酸利多卡因，表非替得，头孢唑啉钠，硫酸阿托品，亮氨酸，螺内酯，干扰素，盐酸索他洛尔，氯化钾，琥珀新酯钠，盐酸多巴酚丁胺，阿普唑仑，普伐他丁钠，立普妥，盐酸咪达唑仑，盐酸度冷丁，异山梨醇二硝酸酯，肾上腺素，盐酸多巴胺，比伐卢定，rosuvastatin，ezetimibe/辛伐他汀，avasimibe，阿昔单抗，和cariporide。
本发明的小RNA能与用来治疗心绞痛的另外的治疗药物联合用药。能用来减轻或抑制心绞痛的药物的例子包括：阿司匹林；硝酸甘油；异山梨醇一硝酸酯；琥珀酸美托洛尔；阿替洛尔；酒石酸美托洛尔；阿罗地平磺酸盐，dilitiazem hydropchloride，异山梨醇二硝酸酯，氯吡格雷硫酸氢盐；硝苯地平；立普妥；氯化钾；呋塞米；辛伐他汀；盐酸维拉帕米；地高辛；盐酸普萘洛尔；卡维地洛；赖诺普利；sprionolactone；二氢氯噻；马来酸依那普利；madolol；雷米普利；依诺肝素钠；肝素钠；缬沙坦；盐酸索他洛尔；非诺贝特；ezetimibe；布美他尼；氯沙坦钾赖诺普利/氢氯噻嗪；非洛地平；卡托普利；和富马酸比索洛尔。
在本发明的一个实施方案中，本发明的小RNA与通常用来治疗丙型肝炎病毒的药物联合施药。这样的药物的例子包括干扰素-α-2a，干扰素-α-2b，干扰素-αconl，干扰素-α-nl，PEG化干扰素-α-2a，PEG化干扰素-α-2b， 利巴韦林，PEG化干扰素-α-2b和利巴韦林，雄去氧胆酸，甘草酸，胸腺法新，Maxamine，和VX-497。
本发明的小RNA与皮质类固醇，维生素D类似物，和局部或口服视黄酸，或者它们的组合，联合用药，用于治疗牛皮癣。另外，本发明的小RNA与用于治疗牛皮癣的下面的药物中的一种联合用药：KDR的小分子抑制剂(ABT-123)，Tie-2的小分子抑制剂，钙泊三烯，丙酸氯倍他索，曲安奈德，卤倍他索丙酸酯，他佐罗汀，甲氨喋呤，氟轻松醋酸酯，氟轻松，阿维A，焦油香波，戊酸倍他米松，糠酸毛他松，酮康唑，普拉莫星/氟轻松，戊酸氢化可的松，氟氢缩松，脲，倍他米松，丙酸氯倍他索/emoll，丙酸氟地松，阿齐霉素，氢化可的松，增加水份的处方，叶酸，地奈德，煤焦油，醋酸二氟拉松，etanercept，叶酸盐，乳酸，甲氧沙林，hc/次没食子酸铋/znox/resor，醋酸甲泼尼龙，泼尼松，防晒物质，水杨酸，哈西奈德，地蒽酚，新戊酸氯可托龙，煤提取物，煤焦油/水杨酸，煤焦油/水杨酸/硫，去羟米松，安定，润滑药，pimecrolimus润滑药，氟轻松醋酸酯/润滑药，矿物油/蓖麻油/na lact，矿物油/花生油，石油十四酸异丙基酯，补骨脂素，水杨酸，肥皂/三溴沙仑，硫汞撒/硼酸，塞来昔布，英夫单抗，alefacept，efalizumab，藤霉素，pimecrolimus，PUVA，UVB，和水杨酸偶氮磺胺吡啶。
本发明的小RNA能与治疗皮肤病的其他药物联合使用。例如，本发明的小RNA与PUVA疗法联合。PUVA是补骨脂素(P)和长波紫外射线的联合，用来治疗很多不同的皮肤病。本发明的小RNA还能和pimecrolimus联合。在另一个实施方案中，本发明的抗体被用来治疗牛皮癣，其中抗体与藤霉素联合用药。在另一个实施方案中，藤霉素和本发明的小RNA与甲氨喋呤和/或环孢菌素联合给药。在另一个实施方案中，本发明的本发明的小RNA与治疗牛皮癣的受激准分子激光治疗联合施用。
本发明的小RNA可以与之联合来治疗皮肤病或指甲病的其他治疗性药物的非限制性例子包括UVA和UVB光线疗法。能与本发明的小RNA联合使用的其他非限制性例子包括抗-IL-12和抗-IL-18治疗药物，包括抗体。
在一个实施方案中，本发明的小RNA与治疗贝切特氏病的另外的治疗药物联合用药。能用来治疗贝切特氏病的另外的治疗药物包括但不限于， 泼尼松，环磷酰胺(Cytoxan)，硫唑嘌呤(也称作硫唑嘌呤imuran)，甲氨喋呤，timethoprim/新诺明(也称作复方新诺明片或增效磺胺甲基异唑)和叶酸。
上面提到的治疗药物中的任何一种，单独地或者联合地，与本发明的小RNA联合，能对患有其中TNF-alpha活性有害的TNF-alpha-相关疾病的患者。
下面结合实施例，进一步阐述本发明。本领域技术人员应当理解这些实施例仅仅是示例性的而非限制性的。本发明的范围由所附权利要求书限定。
实施例
实验材料与方法
ELISA(酶联免疫吸附测定)和RT-qPCR(实时荧光定量PCR)
主要实验仪器及器材：10cm细胞培养皿、12孔细胞培养板、移液器、移液管、光学显微镜
主要实验试剂：
细胞培养：RPMI 1640培养基(MACGENE，cat.CM10041)、胎牛血清(FETAL BOVINE SERUM)(GE，cat.SV30160.03)10％比例加入培养基
模型建立及转染：表1中所示的人工合成小RNA(双链，Genepharma)、转染试剂(RNAimax，invitrogen，17338-150)、Opti-MEM(gibco，31985-070500ml)、LPS(sigma，cat.L4391-1MG)
RNA：总RNA极速抽提试剂盒(上海飞捷生物技术有限公司，cat.no.220011)、TRIZOL REAGENT(SIGMA，T9424-200ml)、逆转录试剂盒(High Capacity cDNA Reverse Transcription Kit，Thermo，4368813)、LightCycler 480 SYBR Green I Master(Roche，04887352001)
ELISA检测试剂盒：(DuoSet Human IL-1beta/IL-6/TNF-alpha，R&D，DY201/DY206/DY210)、蛋白酶抑制剂(TargetMol，cat.no.C0001)
1.应用LPS刺激的THP-1细胞模型验证人工合成的小RNA在蛋白水平上的功 能实验
1.1 在含有胎牛血清的RPMI 1640培养基中培养THP-1细胞(单核巨噬细 胞，购于购自中国医学科学院基础医学研究所细胞中心)至对数生长期，分至12孔板，1ml培养基/孔，37摄氏度孵育过夜后进行后续实验。
1.2 实验分组如下：
Blank组，即空白组，指未经处理过的细胞，该组作为空白对照；
LPS组：该组如下处理：用200ul Opti-MEM稀释2ul RNAimax，加入细胞中并予以LPS刺激，该组作为阴性对照；
NC组：将随机无义序列5’UUC UCC GAA CGU GUC ACG UTT-3(双链，Genepharma)以与实验组序列相同浓度相同转染方式加入细胞中并予以LPS刺激，该组作为阴性对照。
1.3 应用RNAimax转染人工合成植物小RNA，RNAimax 2ul/100ul Opti-MEM，小RNA(20uM)5ul/100ul Opti-MEM，将上述液体混合后室温孵育10分钟，加入细胞中。
1.4 转染后24小时后加入LPS刺激，LPS的终浓度为1ug/ml。
1.5 LPS刺激后9小时收取细胞上清，加入蛋白酶抑制剂浓度为10ul/ml。
1.6 ELISA试剂盒检测IL-1beta/IL-6/TNF-alpha三种因子的表达情况。
2.应用LPS刺激的THP-1细胞模型验证人工合成的小RNA在mRNA水平上的 功能实验
2.1 在含有胎牛血清的RPMI 1640培养基中培养THP-1细胞(单核巨噬细胞，购于购自中国医学科学院基础医学研究所细胞中心)至对数生长期，分至12孔板，1ml培养基/孔，37摄氏度孵育过夜后进行后续实验。
2.2 实验分组如下：
Blank组：空白组，指未经处理过的细胞，该组作为空白对照；
LPS组：该组用200ul Opti-MEM稀释2ul RNAimax加入细胞中并予以LPS刺激，该组作为阴性对照；
NC组：将随机无义序列5’UUC UCC GAA CGU GUC ACG UTT-3(Genepharma)以与实验组序列相同浓度相同转染方式加入细胞中并予以LPS刺激，该组作为阴性对照。
2.3 应用RNAimax转染人工合成植物小RNA，RNAimax 2ul/100ul  Opti-MEM，小RNA(20uM)5ul/100ul Opti-MEM，将上述液体混合后室温孵育10分钟，加入细胞中。
2.4 转染后24小时后加入LPS刺激，LPS的终浓度为1ug/ml。
2.5 LPS刺激后9小时，转速800g离心5分钟收取细胞。
2.6 总RNA极速抽提试剂盒根据制造商的说明提取细胞总RNA。
2.7 将RNA逆转录为cDNA：通过逆转录试剂盒(High-Capacity cDNA Reverse Transcription Kits，Applied Biosystems，cat.no.4368813)根据制造商的说明，将小RNA逆转录为cDNA，逆转录体系如下：模板RNA(150ng/μl)10μl，10X RT Buffer 2.0μl，25X dNTP Mix(100mM)0.8μl，10X Random Primer(试剂盒内包含)2.0ul，MultiScribeTM逆转录酶1.0μl，RNase抑制剂1.0μl，Nuclease-free H2O1.2μl，瞬时离心后，放入PCR仪反应，反应条件如下：(1)25℃，10min；(2)37℃，120min；(3)85℃，5min；(4)4℃，终止反应。反应结束后加入20μl RNase Free dH2O，补足终体积至40μl。
2.8 定量PCR扩增反应：qPCR反应体系总体积10μl，包括：5μL2×SYBR Green Master Mix，0.5μl正向引物(10μM)，0.5μl反向引物(10μM),1μl逆转录得到的cDNA,3μl RNase Free dH2O。使用LightCycler 480荧光定量PCR仪，PCR反应条件是：95℃，持续5min预变性，开始进入PCR扩增循环：(1)95℃，10s；(2)55℃，10s；(3)72℃，20s；总共进行40个循环；最后40℃持续10s降温。扩增反应正向引物和反向引物均由北京擎科新业生物技术有限公司设计和合成。所用引物序列如下：
针对UBC的引物为：
Has-UBC-For CTGGAAGATGGTCGTACCCTG
Has-UBC-Rev GGTCTTGCCAGTGAGTGTCT
针对IL-1beta的引物为：
Has-IL-1beta-For CTCGCCAGTGAAATGATGGCT
Has-IL-1beta-Rev GTCGGAGATTCGTAGCTGGAT
针对IL-6的引物为：
Has-IL-6-For GGTACATCCTCGACGGCATCT
Has-IL-6Rev GTGCCTCTTTGCTGCTTTCAC
针对TNF-alpha的引物为：
Has-TNF-alpha For CTGCCCCAATCCCTTTATT
Has-TNF-alpha Rev CCCAATTCTCTTTTTGAGCC
2.9 利用2-ΔΔCt法计算相对表达量
3.MTS细胞活性检测
3.1.主要实验仪器及器材：10cm细胞培养皿、96well细胞培养板、移液器、移液管、光学显微镜、1.5ml离心管、酶标仪，MTS检测试剂盒(Promega,Celltiter 96 AQueous One Solution Cell Proliferation Assay,REF:G3581,LOT:0000064219)；
3.2.主要实验试剂：
细胞培养：F12培养基(Hyclone)、FBS(Gibico)；
模型建立及转染：人工合成小RNA、RNAimax、opti-MEM、H5N1病毒(A/Jilin/9/2004)；
细胞生存率：MTS细胞活性检测试剂盒；
3.3 应用H5N1来源于2004吉林株A/Jilin/9/2004感染的A549细胞模型验证人工合成的中药饮片来源的小RNA在抵抗H5N1感染、缓解细胞死亡上的功能。
3.3.1 用10厘米细胞培养皿培养A549细胞(人肺腺癌上皮细胞，购自美国模式培养物集存库(ATCC,Rockville,MD,USA)培养于Ham’s F12营养培养基(HyClone,Logan,UT,USA)，分至96孔板，每孔100μL含细胞的培养基)；
3.3.2 光学显微镜下观察到细胞生长至9成满时(约12h)，应用转染试剂转染人工合成植物小RNA，转染试剂0.2微升/毫升，小RNA 100纳摩尔/毫升；
3.3.3 转染24小时后感染H5N1病毒，攻毒量为0.4M.O.I；
3.3.4 攻毒48h后用MTS试剂盒检测细胞死亡状况，将MTS检测相关试剂按照：无血清培养基：A液：B液＝100：20：1充分混合，吸去96孔板中的细胞上清，将MTS检测试剂混合液加入96孔板中，100μL/孔，加入完毕后于37℃烘箱中孵育30min(避光)；
3.3.5 用酶标仪检测细胞的存活情况：检测492nm吸光度值，每板检测三次，以第三次的结果为准。
4.应用LPS刺激的THP-1细胞模型验证人工合成的小RNA混合物在 蛋白水平上的功能实验
4.1 培养THP-1细胞(单核巨噬细胞，购于购自中国医学科学院基础医学研究所细胞中心)至对数生长期，分至12孔板，1ml培养基/孔，37摄氏度孵育过夜后进行后续实验。
4.2 实验分组如下：
Blank组：空白组，指未经处理过的细胞，该组作为空白对照；
LPS组：该组用200ul Opti-MEM稀释2ul RNAimax加入细胞中并予以LPS刺激，该组作为阴性对照；
NC(native)组：将随机无义序列5’UUC UCC GAA CGU GUC ACG UTT-3(双链，Genepharma)以与实验组序列相同浓度相同转染方式加入细胞中并予以LPS刺激，该组作为阴性对照。
4.3 应用RNAimax转染人工合成植物小RNA混合液(表2)，BZL-sRNA-20与其他小RNA的体积比为2:1(各种小RNA的初始浓度均为20uM)，RNAimax 2ul/100ul Opti-MEM，小RNA混合液(20uM)10ul/100ul Opti-MEM，将上述液体混合后室温孵育10分钟，加入细胞中。
4.4 转染后24小时后加入LPS刺激，LPS的终浓度为1ug/ml。
4.5 LPS刺激后9小时收取细胞上清，加入蛋白酶抑制剂浓度为10ul/ml。
4.6 ELISA试剂盒检测IL-1beta/IL-6/TNF-alpha三种因子的表达情况。
5.应用LPS刺激的THP-1细胞模型验证人工合成的小RNA混合物在 mRNA水平上的功能实验
5.1 培养THP-1细胞(单核巨噬细胞，购于购自中国医学科学院基础医学研究所细胞中心)至对数生长期，分至12孔板，1ml培养基/孔，37摄氏度孵育过夜后进行后续实验。
5.2 实验分组如下：
Blank组：空白组，指未经处理过的细胞，该组作为空白对照；
LPS组：该组用200ul Opti-MEM稀释2ul RNAimax加入细胞中并予以LPS刺激，该组作为阴性对照；
NC组：将随机无义序列5’UUC UCC GAA CGU GUC ACG UTT-3(双 链，Genepharma)以与实验组序列相同浓度相同转染方式加入细胞中并予以LPS刺激，该组作为阴性对照。
5.3 应用RNAimax转染人工合成植物小RNA混合液(表2)，BZL-sRNA-20与其他小RNA的体积比为2:1,RNAimax 2ul/100ul Opti-MEM，小RNA混合液(20uM)10ul/100ul Opti-MEM，将上述液体混合后室温孵育10分钟，加入细胞中。
5.4转染后24小时后加入LPS刺激，LPS的终浓度为1ug/ml。
5.5 LPS刺激后9小时，转速800g离心5分钟收取细胞。
5.6 0.5ml TRI Reagent(sigma,T9424-200ML)裂解细胞，12,000rpm，4℃，离心5min，弃沉淀，按200μl/ml TRIzol的比例加入氯仿，充分振荡混匀，室温放置15min，12,000rpm，4℃，离心15min，吸取上层水相，至另一离心管中，吸取上层水相至另一新EP管中，按0.5ml/ml TRIzol加入异丙醇混匀，室温放置5-10min，12,000rpm，4℃，离心10min，弃上清，加入1ml 75％乙醇，温和振荡离心管，悬浮沉淀，8000g，4℃，离心5min，尽量弃上清，室温晾干5-10min，用20μl DEPC处理过的H2O溶解RNA样品。
5.7 将RNA逆转录为cDNA：通过逆转录试剂盒(High-Capacity cDNA Reverse Transcription Kits，Applied Biosystems，cat.no.4368813)，将小RNA逆转录为cDNA，逆转录体系如下：模板RNA(150ng/μl)10μl，10X RT Buffer 2.0μl，25X dNTP Mix(100mM)0.8μl，10X Random Primer(试剂盒内包含)2.0ul，MultiScribeTM逆转录酶1.0μl，RNase抑制剂1.0μl，Nuclease-free H2O 1.2μl，瞬时离心后，放入PCR仪反应，反应条件如下：(1)25℃，10min；(2)37℃，120min；(3)85℃，5min；(4)4℃，终止反应。反应结束后加入20μl RNase Free dH2O，补足终体积至40μl。
5.8 定量PCR扩增反应：qPCR反应体系总体积10μl，包括：5μL 2×SYBR Green Master Mix，0.5μl正向引物(10μM)，0.5μl反向引物(10μM),1μl逆转录得到的cDNA,3μl RNase Free dH2O。使用LightCycler 480荧光定量PCR仪，PCR反应条件是：95℃，持续5min预变性，开始进入PCR扩增循环：(1)95℃，10s；(2)55℃，10s；(3)72℃，20s；总共进行40个循环；最后40℃持续10s降温。扩增反应正向引物和反向引物均由北京擎科新业生物技术有限公司设计和合成。使用UBC基因作为内参基因。所用引物序列如 下：
Has-UBC-For CTGGAAGATGGTCGTACCCTG
Has-UBC-Rev GGTCTTGCCAGTGAGTGTCT
Has-IL-1beta-For CTCGCCAGTGAAATGATGGCT
Has-IL-1beta-Rev GTCGGAGATTCGTAGCTGGAT
Has-IL-6-For GGTACATCCTCGACGGCATCT
Has-IL-6Rev GTGCCTCTTTGCTGCTTTCAC
Has-TNF-alpha For CTGCCCCAATCCCTTTATT
Has-TNF-alpha Rev CCCAATTCTCTTTTTGAGCC
5.9 利用2-ΔΔCt法计算相对表达量
6.验证BZL-sRNA-20体内抗炎作用的实验
6.1 C57小鼠雄性7周龄体重20-23g，分成4组，其中一组在整个实验期间保持未处理，即空白组。
6.2 分别于提前3天、2天、1天灌胃给予小鼠BZL-sRNA-20或NC小RNA剂量1nmol/只，分别为BZL-sRNA-20或NC组(native组)。
6.3 0h 1％戊巴比妥钠麻醉后，对小鼠行气管注射术，剂量LPS(1mg/ml)50ul,50ug/只，其中仅LPS处理组表示为LPS组。
6.4 9h 1％戊巴比妥钠麻醉后，800ul行肺泡灌洗术，共行术2次，每次PBS 800ul反复吹吸3次。
6.5所得灌洗液800g，离心5分钟，获取肺脱落细胞以0.5ml Trizol(Thermo)裂解细胞，12,000rpm，4℃，离心5min，弃沉淀，按200μl/ml TRIzol的比例加入氯仿，充分振荡混匀，室温放置15min，12,000rpm，4℃，离心15min，吸取上层水相，至另一离心管中，吸取上层水相至另一新EP管中，按0.5ml/ml TRIzol加入异丙醇混匀，室温放置5-10min，12,000rpm，4℃，离心10min，弃上清，加入1ml 75％乙醇，温和振荡离心管，悬浮沉淀，8000g，4℃，离心5min，尽量弃上清，室温晾干5-10min，用20μl DEPC处理过的H2O溶解RNA样品。
6.6 将RNA逆转录为cDNA：通过逆转录试剂盒(High-Capacity cDNA Reverse Transcription Kits，Applied Biosystems，cat.no.4368813)，将小RNA逆转录为cDNA，逆转录体系如下：模板RNA(150ng/μl)10μl， 10X RT Buffer 2.0μl，25X dNTP Mix(100mM)0.8μl，10X Random Primer(试剂盒内包含)2.0ul，MultiScribeTM逆转录酶1.0μl，RNase抑制剂1.0μl，Nuclease-free H2O 1.2μl，瞬时离心后，放入PCR仪反应，反应条件如下：(1)25℃，10min；(2)37℃，120min；(3)85℃，5min；(4)4℃，终止反应。反应结束后加入20μl RNase Free dH2O，补足终体积至40μl。
6.7 定量PCR扩增反应：qPCR反应体系总体积10μl，包括：5μL 2×SYBR Green Master Mix，0.5μl正向引物(10μM)，0.5μl反向引物(10μM),1μl逆转录得到的cDNA,3μl RNase Free dH2O。使用LightCycler 480荧光定量PCR仪，PCR反应条件是：95℃，持续5min预变性，开始进入PCR扩增循环：(1)95℃，10s；(2)55℃，10s；(3)72℃，20s；总共进行40个循环；最后40℃持续10s降温。扩增反应正向引物和反向引物均由北京擎科新业生物技术有限公司设计和合成。使用GAPDH基因作为内参基因。所用引物序列如下：
Mus-IL-1beta-For GTTCCCATTAGACAACTGC
Mus-IL-1beta-Rev GATTCTTTCCTTTGAGGC
Mus-IL-6-For TAGTCCTTCCTACCCCAATTTCC
Mus-IL-6-Rev TTGGTCCTTAGCCACTCCTTC
Mus-TNF-For CCTGTAGCCCACGTCGTAG
Mus-TNF-Rev GGGAGTAGACAAGGTACAACCC
Mus-GAPDH-For CACTCACGGCAAATTCAACGGCAC
Mus-GAPDH-Rev GACTCCACGACATACTCAGCAC
6.8 利用2-ΔΔCt法计算相对表达量
6.9 上清12000rpm，离心10分钟，去细胞碎片后用ELISA试剂盒(DuoSet Mouse IL-1beta/IL-6/TNF-alpha，R&D，DY401/DY406/DY410)检测验证因子表达。
7. 应用LPS刺激的THP-1细胞模型验证人工合成的小RNA的转录组 测序
7.1 测序用RNA制备与提取
7.1.1 实验分组
Blank组：空白组，指未经处理过的细胞，该组作为空白对照；
LPS组：该组用200ul Opti-MEM稀释2ul RNAimax加入细胞中并予 以LPS刺激，该组作为阴性对照；
NC组：将随机无义序列5’UUC UCC GAA CGU GUC ACG UTT-3(Genepharma)以与实验组序列相同浓度相同转染方式加入细胞中并予以LPS刺激，该组作为阴性对照。
7.1.2应用RNAimax转染人工合成植物小RNA，RNAimax 2ul/100ul Opti-MEM，小RNA(20uM)5ul/100ul Opti-MEM，将上述液体混合后室温孵育10分钟，加入细胞中。
7.1.3 转染后24小时后加入LPS刺激，LPS的终浓度为1ug/ml。
7.1.4 LPS刺激后9小时，转速800g离心5分钟收取细胞。
7.1.5 用0.5ml Trizol Reagent(sigma)充分裂解细胞，加入100ul氯仿混匀，4℃，13200rpm离心15min，取上清280ul并加入等量异丙醇，混匀后-40℃静置30min4℃，13200rpm离心25min，弃上清，用75％DEPC水配置的乙醇清洗两次，晾干沉淀后用20ulDEPC水溶解沉淀。
7.1.6 获得的RNA溶液送公司测序
7.2 数据分析
7.2.1 上传测序数据
在WINDOW10平台使用Xftp(版本Xftp 5.0)作为传输工具，XShell(版本XShell 5.0)为安全终端模拟软件，以SSH协议上传至222.28.163.113端口222生物信息学服务器，共计194个样本数据(包含2个NC)。
7.2.2 数据库数据准备和测序数据计算
将数据上传后进行下一步处理。下载UCSC的人类基因组hg19版本，使用bowtie2(版本bowtie2 2.1.0)进行建库，注释文件使用UCSC数据库中与hg19相匹配的Annotation(注释)文件。使用shell脚本运行Tophat(版本2.0.11)与cufflink(版本2.2.1)，使测序的150bp的片段匹配到各个被注释区段的基因名的人类基因组文件，并且完成每个基因的表达量计数统计。
Tophat运行参数：
成对的每一个测序片段之间的平均间隔距离-r：150
间隔距离的标准偏差--mate-std-dev：149
library-type(链特异性)：fr-secondstrand
线程数-p：16
7.2.3 测序数据结果汇总
使用python(版本3.6.1)脚本把通过Tophat整理的测序数据写入一个新的文本中。
7.2.4 194个测序结果样本统计差异基因
使用R(版本3.3.2)的DEGseq，编写运行脚本。把每一个样本，每个基因的的表达量整理出来，并且与NC组的各个基因表达量进行比较，计算FC(Fold Change，倍数变化)。
7.2.5 差异下调基因统计与聚类
处理第4步的结果，筛选每个样本中与对照组NC相比，基因表达水平FC下调大于1.5倍的基因,把这些基因上传至Metacore数据库进行分析。选择参数(ignore first line；species：homo sapiens；type：down regulate；p-value、FDR：no limit)
7.2.6 差异下调基因数量统计
在python(3.6.1)使用脚本统计192个小RNA样本，计算基因下调的表达量(下调水平：Fold Change>1.5)，得到192个样本的小RNA能下调的基因。
7.2.7 192条小RNA靶基因及所属通路或生物过程分类制表
查阅PUBMED，KEGG数据库，并结合7.2.5的192个样本的Metacore数据库中每个样本下调的基因(条件：FC>1.5)的聚类结果，依据每个样本下调基因所属的通路或者生物过程分为6类并制表。
实施例1：表1中的小RNA的效果验证
1.如上文“应用LPS刺激的THP-1细胞模型验证人工合成的小RNA在蛋 白水平上的功能实验”所述，用如图1-15中指定的小RNA进行实验。
图1：向THP1细胞中提前24小时转染如图中指定的半枝莲(BZL)小RNA后LPS刺激9小时的细胞炎症模型中炎症因子IL-1beta在蛋白水平相对于对照组表达的结果。图2：向THP1细胞中提前24小时转染如图中指定的柴胡(CHu)小RNA后LPS刺激9小时的细胞炎症模型中炎症因子IL-1beta在蛋白水平相对于对照组表达的结果。图3：向THP1细胞中提前24小时转染如图中指定的鱼腥草(YXC)小RNA后LPS刺激9小时的细胞炎症模型中炎症因子IL-1beta在蛋白水平相对于对照组表达的结果。图4：向THP1细胞中提前24小时转染如图中指定的穿心莲(CXL)和蒲公英(PGY)小RNA后LPS刺激9小时细胞炎症模型中炎症因子IL-1beta在蛋白水平相对于对照组表达的结果。在图1-4中，“*”表 示统计学分析以Unpaired t test P<0.05认为有统计学意义，表明其在体外实验中具有抑制炎症因子表达的作用。实验结果表明如图1-4中所示的小RNA在降低IL-1beta的蛋白质表达上显著高于NC组。图1-4中的数值均为相对于NC组进行标准化获得的数值。BZL-sRNA-20在炎症因子IL-1beta ELISA中数值最小，表明其在测试的小RNA中抑制IL-1beta蛋白水平的效果最佳。
图5：向THP1细胞中提前24小时转染如图中指定的半枝莲(BZL)小RNA后LPS刺激9小时细胞炎症模型中炎症因子IL-6在蛋白水平相对于对照组表达的结果。图6：向THP1细胞中提前24小时转染如图中指定的柴胡(CHu)小RNA后LPS刺激9小时细胞炎症模型中炎症因子IL-6在蛋白水平相对于对照组表达的结果。图7：向THP1细胞中提前24小时转染如图中指定的地丁(DDi)、黄芩(HQi)、金银花(JYH)、连翘(LQi)、和夏枯草(XKC)小RNA后LPS刺激9小时细胞炎症模型中炎症因子IL-6在蛋白水平相对于对照组表达的结果。图8-9：向THP1细胞中提前24小时转染如图中指定的鱼腥草(YXC)小RNA后LPS刺激9小时细胞炎症模型中炎症因子IL-6在蛋白水平相对于对照组表达的结果。图10：向THP1细胞中提前24小时转染如图中指定的穿心莲(CXL)和蒲公英(PGY)小RNA后LPS刺激9小时细胞炎症模型中炎症因子IL-6在蛋白水平相对于对照组表达的结果。“*”表示统计学分析以Unpaired t test P<0.05认为有统计学意义，表明其在体外实验中具有抑制炎症因子表达的作用。实验结果表明如图5-10中所示的小RNA在降低IL-6的蛋白质表达上显著高于NC组。图5-10中的数值均为相对于NC组进行标准化获得的数值。BZL-sRNA-20很好地抑制IL-6蛋白水平。
图11：向THP1细胞中提前24小时转染如图中指定的半枝莲(BZL)、地丁(DDi)、黄芩(HQi)、连翘(LQi)、和夏枯草(XKC)小RNA后LPS刺激9小时细胞炎症模型中炎症因子TNF-alpha在蛋白水平相对于对照组表达的结果。图12：向THP1细胞提前24小时转染如图中指定的柴胡(CHu)小RNA后LPS刺激9小时细胞炎症模型中炎症因子TNF-alpha在蛋白水平相对于对照组表达的结果。图13-14：向THP1细胞提前24小时转染如图中指定的鱼腥草(YXC)小RNA后LPS刺激9小时细胞炎症模型中炎症因子TNF-alpha在蛋白水平相对于对照组表达的结果。图15：向THP1细胞提前24小时转染如图中指定的蒲公英(PGY)小RNA后LPS刺激9小时细胞炎症模型中炎症因子TNF-alpha在蛋白水平相对 于对照组表达的结果。“*”表示统计学分析以Unpaired t test P<0.05认为有统计学意义，表明其在体外实验中具有抑制炎症因子表达的作用。实验结果表明如图11-15中所示的小RNA在降低TNF-alpha的蛋白质表达上显著高于NC组。图11-15中的数值均为相对于NC组进行标准化获得的数值。BZL-sRNA-20在炎症因子TNF-alpha ELISA中数值最小，表明其在测试的小RNA中抑制TNF-alpha蛋白水平的效果最佳。
2.如上文“应用LPS刺激的THP-1细胞模型验证人工合成的小RNA在 mRNA水平上的功能实验”所述，用如图14-33中指定的小RNA进行实验。
图16：向THP1细胞提前24小时转染如图中指定的半枝莲(BZL)小RNA后LPS刺激9小时细胞炎症模型中炎症因子IL-1beta在mRNA水平(IL-1beta相对于UBC的相对于表达量)相对于对照组表达的结果。图17：向THP1细胞提前24小时转染如图中指定的柴胡(CHu)、连翘(LQi)、和鱼腥草(YXC)小RNA后LPS刺激9小时细胞炎症模型中炎症因子IL-1beta在mRNA水平相对于对照组表达的结果。图18-19：向THP1细胞提前24小时转染如图中指定的鱼腥草(YXC)小RNA后LPS刺激9小时细胞炎症模型中炎症因子IL-1beta在mRNA水平相对于对照组表达的结果。图20：向THP1细胞提前24小时转染如图中指定的蒲公英(PGY)小RNA后LPS刺激9小时细胞炎症模型中炎症因子IL-1beta在mRNA水平相对于对照组表达的结果。“*”表示统计学分析以Unpaired t test P<0.05认为有统计学意义，表明其在体外实验中具有抑制炎症因子表达的作用。实验结果表明如图16-20中所示的小RNA在降低IL-1beta的mRNA表达上显著高于NC组。图16-20中的数值均为相对于NC组进行标准化获得的数值。BZL-sRNA-20在炎症因子IL-1beta qPCR中数值最小，表明其在测试的小RNA中抑制IL-1beta mRNA水平的效果最佳。
图21-22：向THP1细胞提前24小时转染如图中指定的半枝莲(BZL)小RNA后LPS刺激9小时细胞炎症模型中炎症因子IL-6在mRNA水平相对于对照组表达的结果。图23-24：向THP1细胞提前24小时转染如图中指定的柴胡(CHu)小RNA后LPS刺激9小时细胞炎症模型中炎症因子IL-6在mRNA水平相对于对照组表达的结果。图25：向THP1细胞提前24小时转染如图中指定的地丁(DDi)、黄芩(HQi)、金银花(JYH)、连翘(LQi)、和夏枯草(XKC)小RNA后LPS刺激9小时细胞炎症模型中炎症因子IL-6在mRNA水平相对于对照组表达的 结果。图26-27：向THP-1细胞提前24小时转染如图中指定的鱼腥草(YXC)小RNA后LPS刺激9小时细胞炎症模型中炎症因子IL-6在mRNA水平相对于NC组表达的结果。图28：向THP1细胞提前24小时转染如图中指定的穿心莲(CXL)和蒲公英(PGY)小RNA后LPS刺激9小时细胞炎症模型中炎症因子IL-6在mRNA水平相对于NC组表达的结果。“*”表示统计学分析以Unpaired t test P<0.05认为有统计学意义，表明其在体外实验中具有抑制炎症因子表达的作用。结果表明如图21-28中所示的小RNA在降低IL-6的mRNA表达上显著高于NC组。图21-22中的数值均为相对于NC组进行标准化获得的数值。BZL-sRNA-20在炎症因子IL-6qPCR中数值较低，表明其在测试的小RNA中抑制IL-6mRNA水平的效果较好。
图29：向THP1细胞提前24小时转染如图中指定的半枝莲(BZL)和柴胡(CHu)小RNA后LPS刺激9小时细胞炎症模型中炎症因子TNF-alpha在mRNA水平(TNF-alpha相对于UBC的相对于表达量)相对于NC组表达的结果。图30：向THP1细胞提前24小时转染如图中指定的地丁(DDi)、金银花(JYH)、连翘(LQi)和夏枯草(XKC)小RNA后LPS刺激9小时细胞炎症模型中炎症因子TNF-alpha在mRNA水平相对于NC组表达的结果。图31-32：向THP1细胞提前24小时转染如图中指定的鱼腥草(YXC)小RNA后LPS刺激9小时细胞炎症模型中炎症因子TNF-alpha在mRNA水平相对于NC组表达的结果。图33：向THP1细胞提前24小时转染如图中指定的蒲公英(PGY)小RNA后LPS刺激9小时细胞炎症模型中炎症因子TNF-alpha在mRNA水平相对于NC组表达的结果。“*”表示统计学分析以Unpaired t test P<0.05认为有统计学意义，表明其在体外实验中具有抑制炎症因子表达的作用。结果表明如图29-33中所示的小RNA在降低TNF-alpha的mRNA表达上显著高于NC组。图29-33中的数值均为相对于NC组进行标准化获得的数值。BZL-sRNA-20在炎症因子TNF-alpha qPCR中数值最低，表明其在测试的小RNA中抑制TNF-alpha mRNA水平的效果最佳。
3.用如图34A-N所示的mRNA进行上述MTS细胞活性(cell viability)检测
图34A-C：BZL:H5N1(0.4M.O.I)感染后图中指定的半枝莲(BZL)小RNA对细胞死亡的挽救结果。图34D-G：CHu:H5N1(0.4M.O.I)感染后图中指定的柴胡(CHu)小RNA对细胞死亡的挽救结果。图34H:LQi/XKC: H5N1(0.4M.O.I)感染后图中指定的连翘(LQi)/夏枯草(XKC)小RNA对细胞死亡的挽救结果。图34I:XKC/YXC:H5N1(0.4M.O.I)感染后图中指定的夏枯草(XKC)/鱼腥草(YXC)小RNA对细胞死亡的挽救结果。图34J-N:
YXC:H5N1(0.4M.O.I)感染后图中指定的鱼腥草(YXC)小RNA对细胞死亡的挽救结果。图34中统计学分析以Unpaired t test P<0.05认为有统计学意义，表明其对于H5N1感染导致的细胞死亡具有的挽救作用。“*”表示在Unpaired t test中P<0.05，“**”表示在Unpaired t test中P<0.01。如图34A-N所示，相对于NC组，图中指定的小RNA显著提高细胞存活率，呈现较为明显的挽救细胞死亡的效果。图34A-N中的数值均为相对于NC组进行标准化获得的数值。其中，BZL-sRNA-20在挽救细胞死亡的效果很好。
实施例2：表2中的混合物的效果验证
如上文“应用LPS刺激的THP-1细胞模型验证人工合成的小RNA混合物 在蛋白水平上的功能实验”所述对表2中的混合物进行效果验证。
图35：THP1细胞中提前24小时转染小RNA混合物后LPS刺激9小时细胞炎症模型中炎症因子IL-1beta在蛋白水平相对于NC组表达的结果，BZL-sRNA-20与其他小RNA混合比例为2:1(v/v),“*”表示统计学分析以Unpaired t test P<0.05认为有统计学意义，表明其在体外实验中具有抑制炎症因子表达的作用。结果表明如图28中所示的小RNA混合物在降低IL-1beta蛋白质水平上显著高于NC组，其中MIX20、24、33、36、42在降低IL-1beta蛋白质水平上高于BZL-sRNA-20组。图35中的数值均为相对于NC组进行标准化获得的数值。对于图中与BZL-sRNA-20组在降低IL-1beta蛋白质水平上相当的混合物，由于混合物中BZL-sRNA-20小RNA在测试细胞液体中的摩尔浓度远低于BZL-sRNA-20组中的摩尔浓度，这表明混合物中的各种小RNA在降低IL-1beta蛋白质水平上也具备很好的协同效果。
图36-37：THP1细胞中提前24小时转染小RNA混合物后LPS刺激9小时细胞炎症模型中炎症因子IL-6在蛋白水平相对于NC组表达的结果，其中BZL-sRNA-20与其他小RNA混合比例为2:1(v/v),“*”表示统计学分析以Unpaired t test P<0.05认为有统计学意义，表明其在体外实验中具有抑制炎症因子表达的作用。结果表明如图36-37中所示的小RNA混合物在降低IL-6蛋白质水平上显著高于NC组，其中图36中除MIX10，图37MIX22、25-28、30-33、 37-39在降低IL-6蛋白质水平上高于BZL-sRNA-20组。图36-37中的数值均为相对于NC组进行标准化获得的数值。混合物10与BZL-sRNA-20组在降低IL-1beta蛋白质水平上相当。由于混合物10中BZL-sRNA-20小RNA在测试细胞液体中的摩尔浓度远低于BZL-sRNA-20组中的摩尔浓度，这表明混合物10中的各种小RNA在降低IL-6蛋白质水平上也具备很好的协同效果。
图38：THP1细胞中提前24小时转染小RNA混合物后LPS刺激9小时细胞炎症模型中炎症因子IL-1beta在mRNA水平相对于NC组表达的结果，BZL-sRNA-20与其他小RNA混合比例为2:1(v/v),“*”表示统计学分析以Unpaired t test P<0.05认为有统计学意义，表明其在体外实验中具有抑制炎症因子表达的作用。结果表明如图38中所示的小RNA混合物在降低IL-1beta mRNA水平上显著高于NC组，其中MIX23、42、43在降低IL-1beta mRNA水平上高于BZL-sRNA-20组。图38中的数值均为相对于NC组进行标准化获得的数值。对于与BZL-sRNA-20组在降低IL-1beta mRNA水平上相当的混合物，由于混合物中BZL-sRNA-20小RNA在测试细胞液体中的摩尔浓度远低于BZL-sRNA-20组中的摩尔浓度，这表明混合物中的各种小RNA在降低IL-1beta mRNA水平上也具备很好的协同效果。
图39-40：THP1细胞中提前24小时转染小RNA混合物后LPS刺激9小时细胞炎症模型中炎症因子IL-6在mRNA水平相对于NC组表达的结果，其中BZL-sRNA-20与其他小RNA混合比例为2:1(v/v),“*”表示统计学分析以Unpaired t test P<0.05认为有统计学意义，表明其在体外实验中具有抑制炎症因子表达的作用。结果表明如图31中所示的小RNA混合物在降低IL-6mRNA水平上显著高于NC组，其中图39中除MIX10、14，图40中MIX25-27、30、31、38在降低IL-6mRNA水平上高于BZL-sRNA-20组。图39-40中的数值均为相对于NC组进行标准化获得的数值。与BZL-sRNA-20组在降低IL-6 mRNA水平上相当的混合物，由于混合物中BZL-sRNA-20小RNA在测试细胞液体中的摩尔浓度远低于BZL-sRNA-20组中的摩尔浓度，这表明混合物中的各种小RNA在降低IL-6mRNA水平上具备很好的协同效果。
图41：THP1细胞中提前24小时转染小RNA混合物后LPS刺激9小时细胞炎症模型中炎症因子TNF-alpha在mRNA水平相对于NC组表达的结果，其中BZL-sRNA-20与其他小RNA混合比例为2:1(v/v),“*”表示统计学分析以 Unpaired t test P<0.05认为有统计学意义，表明其在体外实验中具有抑制炎症因子表达的作用。结果表明如图41中所示的小RNA混合物在降低TNF-alpha mRNA水平上显著高于NC组，其中MIX32、33、40、42在降低TNF-alpha mRNA水平上高于BZL-sRNA-20组。图41中的数值均为相对于NC组进行标准化获得的数值。与BZL-sRNA-20组在降低TNF-alpha mRNA水平上相当的混合物。由于混合物中BZL-sRNA-20小RNA在测试细胞液体中的摩尔浓度远低于BZL-sRNA-20组中的摩尔浓度，这表明混合物中的各种小RNA在降低TNF-alpha mRNA水平上也具备很好的协同效果。
实施例3：BZL-sRNA-20的体内效果
如上文“验证BZL-sRNA-20体内抗炎作用的实验”所述进行实验。
图42：小鼠提前3天小RNA灌胃，LPS刺激9小时动物炎症模型，肺泡灌洗液中炎症因子TNF-alpha在蛋白水平相对于NC组表达的结果,“*”表示统计学分析以Unpaired t test P<0.05认为有统计学意义，表明BZL-sRNA-20在体内实验中具有抑制炎症因子TNF-alpha表达的作用。
图43：小鼠提前3天小RNA灌胃，LPS刺激9小时动物炎症模型，肺脱落细胞中炎症因子IL-6在mRNA水平相对于NC组表达的结果,“**”统计学分析以Unpaired t test P<0.01认为有统计学意义，表明其在体内实验中具有抑制炎症因子IL-6表达的作用。
实施例4：根据LPS刺激的THP-1细胞模型验证人工合成的小RNA的转录组测序结果计算小RNA下调的主要靶基及所属通路或生物过程分类
如上文所述对192条小RNA靶基因及所属通路或生物过程分类制表。
表3：从表1中选取192条小RNA进行转录组测序，计算得到的小RNA靶基因及所属通路或生物过程分类表。该表阐释了小RNA抑制炎性因子和/或H5N1感染通过：趋化因子相关信号通路、JAK/STAT的信号转导、氨基酸代谢、mRNA激活并发挥作功能、辅酶代谢、小核仁RNA等的作用机制、作用靶点及作用途径，并且提示出不同小RNA在相关信号通路以及靶基因中存在协同作用的可能性。
Figure PCTCN2018123289-appb-000021
Figure PCTCN2018123289-appb-000022
以下表格的基因对应于上表中相应行的途径。
Figure PCTCN2018123289-appb-000023
Figure PCTCN2018123289-appb-000024
Figure PCTCN2018123289-appb-000025
Figure PCTCN2018123289-appb-000026
Figure PCTCN2018123289-appb-000027
Figure PCTCN2018123289-appb-000028
Figure PCTCN2018123289-appb-000029
Figure PCTCN2018123289-appb-000030
Figure PCTCN2018123289-appb-000031
Figure PCTCN2018123289-appb-000032
Figure PCTCN2018123289-appb-000033
Figure PCTCN2018123289-appb-000034
Figure PCTCN2018123289-appb-000035
Figure PCTCN2018123289-appb-000036
Figure PCTCN2018123289-appb-000037
Figure PCTCN2018123289-appb-000038
Figure PCTCN2018123289-appb-000039
Figure PCTCN2018123289-appb-000040
Figure PCTCN2018123289-appb-000041
Figure PCTCN2018123289-appb-000042
Figure PCTCN2018123289-appb-000043
Figure PCTCN2018123289-appb-000044
Figure PCTCN2018123289-appb-000045
Figure PCTCN2018123289-appb-000046
Figure PCTCN2018123289-appb-000047
Figure PCTCN2018123289-appb-000048
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (25)

  1. 小RNA，其包含：
    (A)以SEQ ID NO.1-222中任一项所示的序列，优选SEQ ID NO.20的序列，或其互补序列；
    (B)与(A)所示序列具有至少80％-98％同一性的序列，其具有抑制表3中列出的任一种或多种通路或基因的能力；
    (C)与(A)所示序列杂交的序列，优选地在严格条件下与(A)所示序列杂交的序列，其具有抑制表3中列出的任一种或多种通路或基因的能力；
    (D)由(A)所示序列经添加、缺失、替换或插入一个或更多个，诸如2、3、4、5、6、7、8或9个碱基得到的序列，其具有抑制表3中列出的任一种或多种通路或基因的能力；或
    (E)由(A)、(B)、(C)或(D)所示序列的前体或经修饰的变体，其具有抑制表3中列出的任一种或多种通路或基因的能力。
  2. 根据权利要求1所述的小RNA，其具有抑制表3中列出的相同通路或基因的能力，或者具有预防和/或治疗IL-1beta、IL-6或/和TNF-alpha相关疾病的能力和/或提高细胞生存率的能力。
  3. 根据权利要求2所述的小RNA，其中所述小RNA具有降低或下调IL-1beta、IL-6或/和TNF-alpha的表达水平的能力和/或具有挽救由于病毒，例如RNA病毒，例如禽流感病毒，例如H5N1感染导致的细胞死亡的能力，
    优选地，所述小RNA具有降低或下调IL-1beta、IL-6和TNF-alpha中一种炎性因子的表达水平的能力，
    优选地，所述IL-1beta、IL-6或/和TNF-alpha相关疾病选自说明书中列出的任一种或多种IL-1beta、IL-6或/和TNF-alpha相关疾病，优选肺炎、心肌炎、急慢性胃炎、急慢性肠炎、急慢性肝炎、急慢性肾炎、皮炎、脑炎、淋巴炎、结膜炎、角膜炎、虹膜睫状体炎、中耳炎、过敏性鼻炎、哮喘、肺纤维化慢性阻塞性肺疾病、过敏性皮炎、镰状细胞病、多发性硬化、系统性红斑狼疮、狼疮性肾炎、肺癌、胃癌、结直肠癌、肝癌、胰腺癌、宫颈癌、乳腺癌、白血病、多发性骨髓瘤、糖尿病和痛风。
  4. 根据权利要求1-3中任一项所述的小RNA，其中所述小RNA为双 链或单链形式或双链和单链的杂合形式。
  5. 根据权利要求1-4中任一项所述的小RNA，其中所述小RNA为非天然的小RNA。
  6. 根据权利要求1-5中任一项所述的小RNA，其中所述非天然的小RNA为经过人工合成或由人工载体表达获得的小RNA。
  7. 核酸序列或包含所述核酸序列的构建体，所述核酸序列包含编码根据权利要求1-6中任一项所述的小RNA的序列，其中优选地所述构建体是病毒构建体，优选逆转录病毒构建体。
  8. 重组病毒，其包含根据权利要求7所述的核酸序列或构建体，优选地，所述重组病毒是逆转录病毒。
  9. 表达载体，其包含编码根据权利要求1-6中任一项所述的小RNA的序列。
  10. 细胞，其包含根据权利要求7所述的核酸序列或构建体或用根据权利要求8所述的重组病毒转染或包含根据权利要求9所述的表达载体。
  11. 表达权利要求1-6中任一项所述的小RNA的方法，其包括在合适的条件下表达权利要求10所述的细胞并且回收根据权利要求1-4中任一项所述的小RNA。
  12. 药物组合物，其包含一种或多种根据权利要求1-6中任一项所述的小RNA、根据权利要求7所述的核酸序列或构建体、根据权利要求8所述的重组病毒、根据权利要求9所述的表达载体和/或所述权利要求10所述的细胞，
    优选地，所述药物组合物是用于口服、静脉内施用，如推注或通过连续灌注一段时间，通过皮下、肌肉内、动脉内、腹膜内、肺内、脑脊髓内、关节内、滑膜内、鞘内、损伤内、或吸入路径如鼻内，通常通过静脉内或皮下施用的药物组合物。
  13. 根据权利要求12所述的药物组合物，其包含表2中的混合物1至混合物43中的任一种或多种。
  14. 根据权利要求13所述的药物组合物，其中在所述药物组合物中，包含以SEQ ID NO.20所示的序列的小RNA与组合物中的其它小RNA的摩尔浓度比率为约2:1。
  15. 根据权利要求12-14中任一项的药物组合物，其还包含说明书中 列出的一种或多种药物。
  16. 试剂盒，其包含一种或多种根据权利要求1-6中任一项所述的小RNA、根据权利要求7所述的核酸序列或构建体、根据权利要求8所述的重组病毒、根据权利要求9所述的表达载体和/或所述权利要求10所述的细胞，优选地，所述试剂盒还包含说明书中列出的一种或多种药物。
  17. 在体外或体内抑制表3中列出的任一种或多种通路或基因的方法，其包括对细胞或受试者施用一种或多种根据权利要求1-6中任一项所述的小RNA、根据权利要求7所述的核酸序列或构建体、根据权利要求8所述的重组病毒、根据权利要求9所述的表达载体、所述权利要求10所述的细胞和/或根据权利要求12-15中任一项的药物组合物。
  18. 在体外或体内降低或下调IL-1beta、IL-6或/和TNF-alpha的表达水平和/或提高细胞生存率的方法，其包括对细胞或受试者施用一种或多种根据权利要求1-6中任一项所述的小RNA、根据权利要求7所述的核酸序列或构建体、根据权利要求8所述的重组病毒、根据权利要求9所述的表达载体、所述权利要求10所述的细胞和/或根据权利要求12-15中任一项的药物组合物。
  19. 根据权利要求18所述的方法，其中细胞生存率是病毒，例如RNA病毒，例如禽流感病毒，例如H5N1感染中的细胞生存率，优选地所述提高细胞生存率是通过挽救由于病毒，例如RNA病毒，例如禽流感病毒，例如H5N1感染导致的细胞死亡实现。
  20. 治疗或预防受试者中IL-1beta、IL-6或/和TNF-alpha相关疾病和/或病毒，例如RNA病毒，例如禽流感病毒，例如H5N1感染的方法，其包括对受试者施用一种或多种根据权利要求1-6中任一项所述的小RNA、根据权利要求7所述的核酸序列或构建体、根据权利要求8所述的重组病毒、根据权利要求9所述的表达载体、所述权利要求10所述的细胞和/或根据权利要求12-15中任一项的药物组合物，
    优选地，所述IL-1beta、IL-6或/和TNF-alpha相关疾病选自说明书中列出的IL-1beta、IL-6或/和TNF-alpha相关疾病，优选肺炎、心肌炎、急慢性胃炎、急慢性肠炎、急慢性肝炎、急慢性肾炎、皮炎、脑炎、淋巴炎、结膜炎、角膜炎、虹膜睫状体炎、中耳炎、过敏性鼻炎、哮喘、肺纤维化、慢性阻塞性肺疾病、过敏性皮炎、镰状细胞病、多发性硬化、系统性红斑 狼疮、狼疮性肾炎、肺癌、胃癌、结直肠癌、肝癌、胰腺癌、宫颈癌、乳腺癌、白血病、多发性骨髓瘤、糖尿病和痛风。
  21. 根据权利要求1-6中任一项所述的小RNA、根据权利要求7所述的核酸序列或构建体、根据权利要求8所述的重组病毒、根据权利要求9所述的表达载体、所述权利要求10所述的细胞和/或根据权利要求12-15中任一项的药物组合物，用于在体外或体内降低或下调IL-1beta、IL-6或/和TNF-alpha的表达水平和/或提高细胞生存率和/或治疗或预防受试者中IL-1beta、IL-6或/和TNF-alpha相关疾病和/或病毒，例如RNA病毒，例如禽流感病毒，例如H5N1感染，
    优选地，所述IL-1beta、IL-6或/和TNF-alpha相关疾病选自说明书中列出的IL-1beta、IL-6或/和TNF-alpha相关疾病，优选肺炎、心肌炎、急慢性胃炎、急慢性肠炎、急慢性肝炎、急慢性肾炎、皮炎、脑炎、淋巴炎、结膜炎、角膜炎、虹膜睫状体炎、中耳炎、过敏性鼻炎、哮喘、肺纤维化慢性阻塞性肺疾病、过敏性皮炎、镰状细胞病、多发性硬化、系统性红斑狼疮、狼疮性肾炎、肺癌、胃癌、结直肠癌、肝癌、胰腺癌、宫颈癌、乳腺癌、白血病、多发性骨髓瘤、糖尿病和痛风。
  22. 根据权利要求1-6中任一项所述的小RNA、根据权利要求7所述的核酸序列或构建体、根据权利要求8所述的重组病毒、根据权利要求9所述的表达载体、所述权利要求10所述的细胞和/或根据权利要求12-15中任一项的药物组合物在制备药物中的用途，所述药物用于在体外或体内降低或下调IL-1beta、IL-6或/和TNF-alpha的表达水平和/或提高细胞生存率和/或治疗或预防受试者中的IL-1beta、IL-6或/和TNF-alpha相关疾病和/或病毒，例如RNA病毒，例如禽流感病毒，例如H5N1感染，
    优选地，所述IL-1beta、IL-6或/和TNF-alpha相关疾病选自说明书中列出的IL-1beta、IL-6或/和TNF-alpha相关疾病，例如肺炎、心肌炎、急慢性胃炎、急慢性肠炎、急慢性肝炎、急慢性肾炎、皮炎、脑炎、淋巴炎、结膜炎、角膜炎、虹膜睫状体炎、中耳炎、过敏性鼻炎、哮喘、肺纤维化、慢性阻塞性肺疾病、过敏性皮炎、镰状细胞病、多发性硬化、系统性红斑狼疮、狼疮性肾炎、肺癌、胃癌、结直肠癌、肝癌、胰腺癌、宫颈癌、乳腺癌、白血病、多发性骨髓瘤、糖尿病和痛风。
  23. 用于检测根据权利要求1-6中任一项所述的小RNA、根据权利要 求7所述的核酸序列或构建体、根据权利要求8所述的重组病毒、根据权利要求9所述的表达载体、所述权利要求10所述的细胞和/或根据权利要求12-15中任一项的药物组合物的试剂，其中优选地，所述试剂是引物和/或探针。
  24. 试剂盒，其包含根据权利要求23的试剂。
  25. 使用根据权利要求23所述的试剂或根据权利要求24所述的试剂盒检测不同来源的细胞是否含有根据权利要求1-6中任一项所述的小RNA的方法，其中所述细胞优选是植物细胞。
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