WO2018176330A1 - 小rna及其用于预防和/或治疗纤维增生性病症和/或综合征 - Google Patents

小rna及其用于预防和/或治疗纤维增生性病症和/或综合征 Download PDF

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WO2018176330A1
WO2018176330A1 PCT/CN2017/078815 CN2017078815W WO2018176330A1 WO 2018176330 A1 WO2018176330 A1 WO 2018176330A1 CN 2017078815 W CN2017078815 W CN 2017078815W WO 2018176330 A1 WO2018176330 A1 WO 2018176330A1
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polynucleotide
disease
fibrosis
diseases
fibroproliferative
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PCT/CN2017/078815
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English (en)
French (fr)
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蒋澄宇
杜涧超
梁竹
许剑涛
赵妍
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中国医学科学院基础医学研究所
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Priority to CN201780089210.4A priority Critical patent/CN110546261B/zh
Priority to US16/498,435 priority patent/US11471476B2/en
Priority to CN202310119834.3A priority patent/CN116836977A/zh
Priority to JP2019553481A priority patent/JP2020513192A/ja
Priority to EP17903508.4A priority patent/EP3604529A4/en
Publication of WO2018176330A1 publication Critical patent/WO2018176330A1/zh
Priority to JP2021160557A priority patent/JP7401498B2/ja
Priority to US17/936,820 priority patent/US20230293570A1/en
Priority to JP2023206389A priority patent/JP2024028877A/ja

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Definitions

  • the present invention relates to Rhodiola-derived small RNAs, and to their use for the prevention and treatment of fibroproliferative disorders and/or syndromes.
  • Fibrosis is a terminal change of a large class of diseases characterized by fibroblast proliferation and a large number of extracellular matrix aggregation with inflammatory damage and tissue destruction. That is, normal tissue is damaged and abnormally repaired to cause structural abnormalities. Pulmonary fibrosis is caused by toxic substances, spontaneous immune diseases, side effects of drugs, infections, severe trauma and other causes of lung inflammation, persistent alveolar damage, repeated destruction of extracellular matrix, repair, reconstruction and excessive deposition, resulting in A type of disease in which normal lung tissue changes and function is lost. Currently, fibrosis (including pulmonary fibrosis) remains untargeted, safe and effective.
  • IIP interstitial pneumonia
  • Pulmonary fibrosis seriously affects the human respiratory function, manifested as dry cough, progressive dyspnea (consciously insufficient gas), and with the aggravation of the disease and lung damage, the patient's respiratory function is deteriorating.
  • the incidence and mortality of idiopathic pulmonary fibrosis increased year by year, and the average survival after diagnosis was only 2.8 years.
  • Pulmonary fibrosis is one of the four major diseases of respiratory diseases. The etiology is complicated and the pathogenesis is unknown. The existing drugs and methods for treating pulmonary fibrosis are very limited, and the curative effect is unsatisfactory. The prognosis is very poor. The 5-year survival rate is only 50. %.
  • glucocorticoids mainly glucocorticoids and immunosuppressive agents, such as prednisone, cyclophosphamide, and colchicine.
  • immunosuppressive agents such as prednisone, cyclophosphamide, and colchicine.
  • glucocorticoids, antibiotics and immunosuppressants against organ fibrosis can reduce early alveolar inflammation and reduce clinical symptoms.
  • it does not inhibit the development of fibrosis.
  • Long-term use of large doses of hormones and antibiotics not only brings serious complications, but also exacerbates the process of fibrosis.
  • Other treatments, including oxygen only relieve the effect, and can not solve the problem fundamentally; in addition, lung transplantation in extreme cases is also limited by many application conditions, especially in the end-stage lung disease patients with very limited transplant success rate .
  • the inventors have unexpectedly discovered that some sRNAs derived from Rhodiola can effectively inhibit fibrosis-related gene expression in a cell model and/or effectively alleviate lung fibrosis in mice in an animal model.
  • the present invention has been completed based on this.
  • the invention provides a polynucleotide comprising:
  • the invention provides a nucleic acid vector comprising or expressing the polynucleotide of the above first aspect.
  • the present invention provides a pharmaceutical composition comprising the polynucleotide of the first aspect of the invention or the nucleic acid vector of the second aspect.
  • the invention provides a method of preventing and/or treating fibroproliferative diseases and/or synthesis
  • a method of enrolling comprising administering a polynucleotide of the first aspect of the invention, a vector of the second aspect, a pharmaceutical composition of the third aspect, and/or endogenous to a subject in need thereof
  • An elicitor that produces the polynucleotide of the invention is stimulated in vivo.
  • the method produces the polynucleotide of the first aspect of the invention by endogenous challenge in vivo.
  • the present invention also provides polynucleotides, vectors, pharmaceutical compositions, and endogenous elicitors for use as described above, and their use in the preparation of a proliferative disease and/or syndrome for the prevention and/or treatment of fibroproliferative diseases and/or syndromes.
  • the use of the drug is not limited to, but rather to, but rather to, but rather to, but rather to, but rather to, but rather to, a proliferative disease and/or syndrome for the prevention and/or treatment of fibroproliferative diseases and/or syndromes. The use of the drug.
  • the invention also provides an elicitor for endogenously eliciting the production of a polynucleotide of the invention in vivo.
  • the invention provides a method of making the polynucleotide of the first aspect of the invention, comprising: synthesizing and/or expressing a polynucleotide of the invention from a nucleic acid vector, and/or endogenous Cells that have this ability are stimulated to express the polynucleotides of the invention.
  • the present invention at least achieves at least one of the effects of effectively inhibiting expression of one or more fibrosis-related genes at the mRNA and/or protein level; and/or effective prevention and/or treatment of fibrosis sexual disorders and/or syndromes; and/or providing polynucleotides that achieve one or more of the above effects.
  • FIG. 1 and Figure 2 show that Rhodiola-derived sRNA (HJT sRNA) is associated with four fibrosis of ⁇ -SMA, fibronectin, COL1A1 and PAI-1 in a TGF- ⁇ 1-stimulated MRC-5 fibrosis cell model Screening results for mRNA expression levels of genes.
  • HJT sRNA Rhodiola-derived sRNA
  • Figure 1 and Figure 2 show that NC sRNA and HJT sRNA were transfected 48 h earlier, TGF- ⁇ 1 stimulated MRC-5 for 48 h, and the relevant indicators (prevention group) and TGF- ⁇ 1 stimulated MRC-5 for 3 h and then transfected with NC sRNA and HJT sRNA, the results of the two groups of experiments were detected 72 hours after TGF- ⁇ 1 stimulation (treatment group).
  • FIGS. 3 to 6 show that in the TGF- ⁇ 1-stimulated MRC-5 fibrosis cell model, the selected four Rhodiola sRNAs can effectively reduce ⁇ -SMA, fibronectin, COL1A1, PAI-1, TGF. mRNA expression levels of - ⁇ and SMAD4. Specifically, FIGS. 3 to 6 sequentially show the anti-fibrotic effects of HJT-sRNA-m7, HJT-sRNA-a2, HJT-sRNA-h3, and ppe-miR-169c in the MRC-5 fibrosis cell model.
  • Figures 7 to 9 show that in the bleomycin-induced mouse pulmonary fibrosis model, the selected four Rhodiola sRNAs can effectively reduce the mortality of mice, significantly slow down the trend of weight loss in mice, and alleviate The result of symptoms of pulmonary fibrosis in mice.
  • Figure 7 shows that HJT-sRNA-a2 is effective in reducing bleomycin-induced mortality in mice.
  • the weight loss of mice was slowed down (Fig. 7A); HJT-sRNA-h3 effectively reduced the mortality of mice caused by bleomycin, and slowed down the weight of mice (Fig. 7B); ppe-miR-169c was effective
  • the mortality of mice caused by bleomycin was reduced, while the decrease in body weight of mice was slowed down (Fig. 7C).
  • Figure 8 shows the effect of HJT-sRNA-m7 in a bleomycin-induced mouse pulmonary fibrosis model.
  • Figure 9 shows that HJT-sRNA-m7 effectively attenuates lung fibrosis symptoms and reduces collagen, fibronectin and ⁇ -SMA expression in a mouse model of pulmonary fibrosis induced by bleomycin.
  • Fig. 9A shows hydroxyproline content in the right lung of the mouse ( ⁇ g/right lung);
  • Fig. 9B shows hematoxylin-eosin staining (H&E staining) of mouse lung tissue;
  • Fig. 9C shows horseshoe staining, ⁇ - Immunohistochemical results of SMA, fibronectin and COL3A1;
  • Figure 9D shows the pathological statistics corresponding to Figure 9C.
  • Figures 10 to 12 show the results of the luciferase reporter gene experiment. Specifically, Figure 10 shows that the luciferase reporter gene assay validates the target gene of HJT-sRNA-m7; Figure 11 shows that the luciferase reporter gene assay validates the target gene of HJT-sRNA-a2; Figure 12 shows luciferase The reporter gene assay verified the target gene of HJT-sRNA-h3.
  • Figures 13 to 14 show the screening of Rhodiola-derived sRNA (HJT sRNA) for protein expression levels of two fibrosis-associated genes, ⁇ -SMA and fibronectin, in a TGF- ⁇ 1-stimulated MRC-5 fibrosis cell model. result.
  • HJT sRNA Rhodiola-derived sRNA
  • small RNAs small RNAs
  • sRNAs small RNAs
  • small RNA (sRNA) refers to various non-coding small RNAs including siRNA and miRNA.
  • RNA may be non-native, such as synthetic or expressed by an artificial vector.
  • non-natural means that the target substance is not naturally occurring in nature, and this does not exclude that the non-natural substance has the same structure and/or composition as the naturally occurring substance.
  • fibrosis refers to the process and state of increased fibrous connective tissue, parenchymal cells in tissues/organs, which can occur in a variety of tissues/organs, and sustained progression can result in organ structural damage and dysfunction, and even exhaustion, a serious threat. Human health and life.
  • the term "ability for preventing/treating fibrosis” means that the target substance itself can prevent/treat fibrosis, or a substance capable of preventing/treating fibrosis can be produced based thereon. That is, this The ability does not need to be directly achieved by the target substance itself, but may be a further application of the consequences of the target substance.
  • inhibiting means that the target activity is at least partially reduced or completely eliminated via a particular treatment.
  • the invention provides a polynucleotide comprising:
  • B) and A) are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 a sequence of %, 94%, 95%, 96%, 97%, 98% or 99% identity having the ability to prevent/treat fibrosis;
  • sequence of A is selected from the group consisting of SEQ ID NOs: 3, 10, 13, and 16.
  • the polynucleotide is DNA or RNA, such as RNA, preferably a small RNA.
  • the polynucleotide is 10-50 nucleotides in length, 12-40 nucleotides, such as 16-35 or 18-30 nucleotides; more specifically, the above polynucleotide is 10 in length , 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 , 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • the polynucleotide is single stranded or double stranded, preferably single stranded. In another specific embodiment, the polynucleotide is non-native, such as synthetic or expressed by an artificial vector.
  • the invention provides a nucleic acid vector comprising or expressing the polynucleotide of the above first aspect.
  • the nucleic acid vector may be DNA, but the expressed polynucleotide may be RNA, such as sRNA.
  • the present invention provides a pharmaceutical composition comprising the polynucleotide of the first aspect or the nucleic acid vector of the second aspect.
  • the pharmaceutical composition further comprises an additional anti-fibrotic agent.
  • the additional anti-fibrotic agent may be selected from any one or more of the following: glucocorticoids such as cortisone acetate, hydrocortisone, prednisolone, dexamethasone, betamethasone , Qu Anxilong, triamcinolone acetonide, beclomethasone; immunosuppressive agents such as cyclophosphamide, azathioprine, methotrexate; antioxidants such as Fulu Shi, carboxymethyl statin; anticoagulants such as low molecular weight heparin; And colchicine, interferon, ACEI and statins.
  • the present invention provides a method of preventing and/or treating a fibroproliferative disease and/or syndrome comprising administering a polynucleotide of the first aspect of the invention to a subject in need thereof,
  • the vector of claim 2 the pharmaceutical composition of the third aspect, and/or the endogenous agonist to produce an elicitor of the polynucleotide of the invention in vivo.
  • the method produces the polynucleotide of the first aspect of the invention by endogenous challenge in vivo.
  • the present invention also provides polynucleotides, vectors, pharmaceutical compositions, and endogenous elicitors for use in the fourth aspect, and their use in the preparation for the prevention and/or treatment of fibroproliferative diseases and/or synthesis The use of the drug.
  • the polynucleotides, vectors, pharmaceutical/cosmetic compositions of the invention may be formulated for non-invasive administration (eg, topical administration) and/or injection administration, for example, for transgestal, trans-respiratory And/or administration by injection, for example oral, inhalation and/or injection.
  • non-invasive administration eg, topical administration
  • injection administration for example, for transgestal, trans-respiratory And/or administration by injection, for example oral, inhalation and/or injection.
  • an invasive route of administration eg, administration by injection, including intramuscular, subcutaneous, intravenous, intraarterial, intraperitoneal, intra-target injection
  • non-invasive use is preferred. Route of administration.
  • the fibroproliferative disease and/or syndrome is selected from the group consisting of: fibroproliferative diseases of the lung, cardiovascular system, liver, pancreas, kidney, spleen, eyes, nervous system, bone marrow, and skin and/or Or syndrome.
  • the fibroproliferative disease and/or syndrome is selected from the group consisting of:
  • Inorganic dust occupational diseases including silicosis, asbestosis and coal lungs; organic dust and allergic pneumonia, including farmer lungs, air-conditioning humidifier lungs, pigeon pigeon lungs and bagasse pneumoconiosis; drug/treatment-related diseases, drug selection From: antibiotics, non-steroidal anti-inflammatory preparations, cardiovascular drugs, anti-tumor drugs, oral drops Sugar and morphine; infectious diseases, including tuberculosis, viral pneumonia and pulmonary cysticercosis; secondary lung diseases, including heart failure, congenital heart disease, adult respiratory distress syndrome, chronic heart failure, transplantation Repulsive-related lung diseases; primary pulmonary diseases, including idiopathic interstitial pneumonia, obliterative bronchiolitis with organizing pneumonia and pulmonary lymphangioleiomyoma; pulmonary diseases associated with collagen vascular disease, Includes lung diseases associated with systemic lupus erythematosus, rheumatoid arthritis, progressive systemic sclerosis, polymyosit
  • Ischemic heart disease including alternative and interstitial fibrosis after myocardial infarction; hypertensive heart disease; inflammatory cardiomyopathy, including viral myocarditis; metabolic cardiomyopathy, including hemochromatosis, amyloid Cardiomyopathy, glycogen accumulating cardiomyopathy and diabetic cardiomyopathy; Keshan disease; dilated cardiomyopathy; hypertrophic cardiomyopathy, restrictive cardiomyopathy; arrhythmogenic right ventricular cardiomyopathy;
  • Viral cirrhosis including hepatitis B, C and D viral hepatitis; schistosomiasis cirrhosis; alcoholic cirrhosis; biliary cirrhosis, including primary biliary cirrhosis, secondary gallstones, inflammation around the portal; Metabolic cirrhosis, including hepatolenticular degeneration, hemochromatosis; toxic cirrhosis, including organophosphate poisoning, carbon tetrachloride poisoning, hepatotoxic drugs such as isoniazid, tetracycline, chlorpromazine poisoning; malnutrition liver Hardening; cardiogenic cirrhosis, including chronic congestive heart failure;
  • pancreatic ischemia Acute pancreatitis; pancreatic duct obstruction; chronic alcoholism; Oddi sphincter dysfunction; pancreatic ischemia;
  • Vascular renal fibroproliferative diseases and/or syndromes including hypertension; immune renal fibroproliferative diseases and/or syndromes, including glomerulonephritis, systemic lupus erythematosus, scleroderma, renal transplant rejection; infection Renal fibroproliferative diseases and/or syndromes, including pyelonephritis, kidney stones; metabolic renal fibroproliferative diseases and/or syndromes, including hyperlipidemia, diabetes, hyperuricemia, hypercalciuria;
  • Fibroproliferative diseases and/or syndrome after spinal trauma stroke scar formation, Alzheimer's disease;
  • Idiopathic and drug-induced myelofibrosis polycythemia vera, chronic myeloid leukemia, and Hodgkin's disease;
  • Dermal fibroproliferative disorders and/or syndromes including oral mucosal fibrosis, scarring, cramps, and pachydermia.
  • the method further comprises applying additional anti-fibrotic agents separately and/or together to the subject in need thereof, both temporally and/or spatially.
  • the additional anti-fibrotic agent may be selected from any one or more of the following: glucocorticoids such as cortisone acetate, hydrocortisone, prednisolone, dexamethasone, betamethasone , Qu Anxilong, triamcinolone acetonide, beclomethasone; immunosuppressive agents such as cyclophosphamide, azathioprine, methotrexate; antioxidants such as Fulu Shi, carboxymethyl statin; anticoagulants such as low molecular weight heparin; And colchicine, interferon, ACEI and statins.
  • glucocorticoids such as cortisone acetate, hydrocortisone, prednisolone, dexamethasone, betamethasone , Qu Anxilong, triamcinolone acetonide
  • the method comprises endogenously stimulating the production of the polynucleotide of the first aspect of the invention in vivo.
  • the invention also provides an elicitor that endogenously stimulates the production of the polynucleotide in vivo.
  • the present invention provides a skin rejuvenation cosmetic method comprising administering to a subject in need thereof a polynucleotide, a carrier, a pharmaceutical/cosmetic composition, and/or an endogenous excitatory in vivo production.
  • An activator of the polynucleotide of the invention is administered by non-invasive means, such as topical application.
  • the invention provides a method of producing the polynucleotide of the first aspect, comprising: synthesizing and/or expressing a polynucleotide of the first aspect of the invention from a nucleic acid vector.
  • Rhodiola rosea Small RNA extraction of Rhodiola rosea was performed according to the manufacturer's instructions for the miRNeasy Mini Kit (QIAGEN #217004).
  • Chloroform was added in a ratio of 200 ⁇ l/ml TRIzol, and the mixture was shaken well and allowed to stand at room temperature for 15 minutes.
  • step 4 add an equal amount of chloroform to the upper aqueous phase, thoroughly homogenize and let stand for 10 min at room temperature, 12,000 rpm, 4 ° C, and centrifuge for 15 min.
  • Reverse transcription of sRNA into cDNA Reverse transcription of sRNA into cDNA by the stem-loop method (Stem-loop method) by High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems, cat. no. 4368813).
  • the reverse transcription system was as follows: template RNA (150 ng/ ⁇ l) 10 ⁇ l, 10X RT Buffer 2.0 ⁇ l, 25 ⁇ dNTP Mix (100 mM) 0.8 ⁇ l, U6RT primer (10 ⁇ M) 2.0 ⁇ l, HJT-sRNA-m7RT primer (10 ⁇ M) 2.0 ⁇ l, MultiScribe TM reverse transcriptase 1.0 ⁇ l, RNase inhibitor 1.0 ⁇ l, Nuclease-free H 2 O 1.2 ⁇ l, after transient centrifugation, put into a PCR reactor reaction, the reaction conditions are as follows: (1) 25 ° C, 10 min; (2) 37 ° C, 120 min; (3) 85 ° C, 5 min; (4) 4 ° C, the reaction was terminated. After the reaction, 20 ⁇ l of RNase Free dH 2 O was added to make up the final volume to 40 ⁇ l.
  • the primer sequences used are as follows:
  • HJT-sRNA-m7RT GTCGTATCCAGTGCACGCTCCGAGGTATTCGCACTGGATACGACGCTTACAA.
  • Quantitative PCR amplification reaction qPCR reaction system total volume 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 of cDNA obtained by reverse transcription, 3 ⁇ l RNase Free dH 2 O.
  • the PCR reaction conditions were: 95 ° C, pre-denaturation for 5 min, and began to enter the PCR amplification cycle: (1) 95 ° C, 10 s; (2) 55 ° C, 10 s; (3) 72 ° C, 20s; a total of 40 cycles; the last 40 ° C for 10s to cool down.
  • Both the forward and reverse primers of the amplification reaction were designed and synthesized by Beijing Qingke Biotechnology Co., Ltd.
  • the primer sequences used are as follows:
  • HJT-sRNA-m7F TCCCGCTGAGGTAGTAGGTT;
  • HJT-sRNA-m7R GTGCACGCTCCGAGGT.
  • Gluing The lanes were made of 10% separation gel (lower layer glue) and 5% concentration of concentrated glue (upper layer glue) and 15-hole comb. The protein loading of each lane sample was equal.
  • Sample treatment The sample was added to an equal volume of 2 ⁇ loading beffer, and placed in an ice bath at 97 ° C for 10 minutes, and then placed on ice for use.
  • mice Male C57BL/6 mice, 6-8 weeks old and weighing 20-25 g, were selected for anesthesia
  • the tube was perfused with bleomycin solution (3.5 U/kg) prepared in 100 ⁇ L of physiological saline, and 100 ⁇ L of physiological saline was administered to the control group.
  • the body weight and mortality of the mice were recorded every day, and the mice were treated on the 21st day.
  • the right lung was taken for the determination of hydroxyproline.
  • the left lung was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned for hematoxylin-eosin staining (H&E staining), horseshoe trichrome staining and immunization group. Detection.
  • the results of histopathological section and fibrosis index were combined to evaluate whether the bleomycin-induced pulmonary fibrosis model was successful.
  • DMEM medium Dulbecco's Modified Eagle's Medium
  • FBS fetal bovine serum
  • penicillin 100 U/ml
  • streptomycin 100 mg/ml
  • transfection reagent RNAiMax (Invitrogen) and Lipofectamine 2000 (Invitrogen)
  • psiCHECK2 Promega C2081
  • the agarose gel electrophoresis showed that the RNA fragment was ⁇ 20 nt.
  • Small RNA fragment small RNA, sRNA.
  • SE36, Illumina HiSeq2500 was performed on A549 cells 24 h after the addition of Rhodiola-derived RNA.
  • RNA fragments entering mouse lung, human blood or A549 cells are identified according to the following conditions: (1) Rhodiola-derived small RNA present in human blood, mouse lung tissue or A549 cells; 2) Cannot be compared to the human or mouse genome for Rhodiola-derived small RNA.
  • Rhodiola-derived small RNAs see Table 1 that entered human blood, and were sequentially named HJT-sRNA-h1 ⁇ 3 according to their relative abundance in human blood.
  • Rhodiola-derived small RNAs entering the lung tissue of mice (see Table 2), and ranked according to their relative abundance in mouse lung tissue, named HJT-sRNA-m1 ⁇ 8; Rhodiola from A549 cells are derived from small RNAs and are sequentially named HJT-sRNA-a1 ⁇ 2 according to their relative abundance in A549 cells (see Table 3).
  • the inventors also screened out four small RNAs in Rhodiola that can be aligned with the miRbase database for subsequent experiments (see Table 4).
  • Rhodiola sources can be compared to the sRNA sequence and nomenclature of the miRbase database
  • RNA samples were taken and the fibrosis-related genes ⁇ -SMA and fibronectin were detected by RT-PCR. Relative mRNA expression of COL1A1, PAI-1, SMAD4 and TGF- ⁇ .
  • ⁇ -SMA, Fibronectin, COL1A1 and PAI-1 are four fibrosis-related genes.
  • the TGF- ⁇ 1-stimulated MRC-5 fibrosis cell model was treated with Rhodiola-derived sRNA to detect the expression of mRNA levels of the above four fibrosis-related genes, thereby entering small human blood, mouse lung and A549 cells.
  • RNA was screened for anti-fibrotic function tests. The results are shown in Figures 1 and 2. In both groups of prophylactic and therapeutic tests, a variety of Rhodiola-derived sRNAs were able to inhibit the expression of fibrosis-related genes in mRNA levels in MRC-5 cells.
  • NC sRNA and HJT sRNA were transfected 24 h in advance, and TGF- ⁇ 1 stimulated MRC-5 for 72 h. Detect relevant indicators.
  • TGF- ⁇ 1-stimulated MRC-5 fibrosis cell model the above four sRNAs were effective in reducing ⁇ -SMA, fibronectin, COL1A1, PAI-1, TGF- ⁇ and mRNA expression level of SMAD4.
  • FIG. 13 and Figure 14 show the results of experiments with the relevant indicators (prevention group) after transfection of NC sRNA and HJT sRNA 24 h in advance, and TGF- ⁇ 1 stimulation of MRC-5 for 72 h. As shown in Figure 13 and Figure 14 It was shown that a variety of Rhodiola-derived sRNAs were able to inhibit the expression of fibrosis-related genes at the protein level in MRC-5 cells.
  • the HJT sRNA with SEQ ID NO: 4, 5, 6, 8, and 10 can significantly reduce the protein expression level of fibronectin, and the HJT corresponding to SEQ ID NOs: 3, 5, 6, 8, 10, 16, and 17. sRNA can reduce the protein expression level of ⁇ -SMA.
  • the luciferase reporter gene system was used to detect intracellular target genes in the sRNA derived from Rhodiola.
  • HJT-sRNA-m7 can exert anti-fibrotic function by directly targeting ⁇ -SMA, fibronectin and COL3A1;
  • HJT-sRNA-a2 can directly target COL1A1, COL3A1, TGF - ⁇ and SMAD4 exert anti-fibrotic function;
  • HJT-sRNA-h3 can exert anti-fibrotic function by directly targeting COL1A1 and COL3A1.

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Abstract

提供了红景天来源的小RNA以及其在用于预防与治疗纤维增生性病症和/或综合征的用途。

Description

小RNA及其用于预防和/或治疗纤维增生性病症和/或综合征
交叉引用
本申请要求于2017年3月29日提交至中国专利局的发明名称为“小RNA及其用于预防和/或治疗纤维增生性病症和/或综合征”的中国专利申请(申请号尚未收到)的优先权，其全部内容在此通过引用并入本申请中。
技术领域
本发明涉及红景天来源的小RNA，还涉及其用于预防与治疗纤维增生性病症和/或综合征的用途。
背景技术
纤维化是以成纤维细胞增殖及大量细胞外基质聚集并伴炎症损伤、组织结构破坏为特征的一大类疾病的终末期改变，也就是正常的组织被损坏后经过异常修复导致结构异常。肺纤维化疾病是由于有毒物质、自发免疫疾病、药物副作用、感染、严重外伤等多种不同原因引起的肺部炎症，肺泡持续性损伤、细胞外基质反复破坏、修复、重建并过度沉积，导致正常肺组织结构改变、功能丧失的一类疾病。目前，纤维化(包括肺纤维化)仍然没有针对性的、安全有效的治疗方案。
绝大部分肺纤维化病人病因不明(特发性肺纤维化)，这组疾病称为特发性间质性肺炎(IIP)，是间质性肺病中一大类。肺纤维化严重影响人体呼吸功能，表现为干咳、进行性呼吸困难(自觉气不够用)，且随着病情和肺部损伤的加重，患者呼吸功能不断恶化。特发性肺纤维化发病率和死亡率逐年增加，诊断后的平均生存期仅2.8年。
肺纤维化患者肺部肺泡逐渐被纤维性物质取代，导致肺组织变硬变厚，肺脏气体交换能力逐步丧失，导致患者不同程度缺氧而出现呼吸困难，最后因呼吸衰竭而死亡。肺纤维化是呼吸病四大病种之一，病因复杂，发病机制不明，目前已有的治疗肺纤维化的药物和方法十分有限，且疗效差强人意，预后极差，5年生存率仅为50％。
目前肺纤维化的治疗以糖皮质激素、免疫抑制剂为主，如泼尼松、环磷酰胺、秋水仙碱等。近年来的临床实践已经证实，使用糖皮质激素、抗生素及免疫抑制剂对抗器官纤维化虽可减轻肺泡早期炎症，减轻患者临床症状， 但是并不能抑制纤维化的发展，长期大剂量使用激素、抗生素不仅带来了严重的并发症，还会加剧纤维化进程。其它治疗方法包括氧气给予，仅仅起到缓解作用，并不能从根本上解决问题；另外在极端情况下的肺移植也受到诸多应用条件的限制，尤其是在末期肺病患者中十分有限的移植成功率。
由于病因和发病机理不清，纤维化的治疗一直是医学领域的难题之一，虽然不断地研发新药，但仍然没有令人满意的治疗或预防药物和有效治疗方案。
因此，当前仍然十分需要有效的治疗或预防纤维化的药物。
发明概述
发明人意外发现，红景天来源的一些sRNA能够在细胞模型上有效地显著抑制纤维化相关基因表达，和/或在动物模型上有效缓解小鼠肺纤维化。基于此完成了本发明。
在一个方面，本发明提供一种多核苷酸，其包含：
A)如SEQ ID NO：1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16和17中任一项所示的序列，或其互补序列；
B)与A)所示序列具有至少80％、85％、90％、95％、96％、97％或98％同一性的序列，其具有用于预防/治疗纤维化的能力；
C)在严格条件下与A)所示序列杂交的序列，其具有用于预防/治疗纤维化的能力；
D)由A)所示序列经添加、缺失、替换一个或更多个核苷酸得到的序列，其具有用于预防/治疗纤维化的能力；或
E)由A)、B)、C)或D)所示序列的前体或经修饰变体，其具有用于预防/治疗纤维化的能力。
在另一方面，本发明提供一种核酸载体，其包含或表达以上第一个方面所述的多核苷酸。
在第三个方面，本发明提供一种药物组合物，其包含本发明第一个方面所述的多核苷酸或者第二个方面所述的核酸载体。
在第四个方面，本发明提供一种预防和/或治疗纤维增生性疾病和/或综合 征的方法，其包括向有此需要的对象施用本发明第一方面所述的多核苷酸、第二方面所述的载体、第三个方面所述的药物组合物、和/或内源性激发体内产生本发明所述多核苷酸的激发剂。在一个实施方案中，所述方法通过内源性激发体内产生本发明第一方面所述的多核苷酸。相应地，本发明还提供用于上述用途的多核苷酸、载体、药物组合物以及内源性激发剂，以及它们用于制备用于预防和/或治疗纤维增生性疾病和/或综合征的药物的用途。
此外，相应地在第五个方面，本发明还提供内源性激发体内产生本发明所述多核苷酸的激发剂。
在第六个方面，本发明提供制备本发明第一方面所述多核苷酸的方法，其包括：合成和/或从核酸载体上表达本发明所述的多核苷酸，和/或内源性激发有此能力的细胞表达本发明的多核苷酸。
通过以上任一个方面，本发明至少能够实现以下至少一个方面的效果：有效抑制一个或更多个纤维化相关基因在mRNA和/或蛋白质水平的表达；和/或有效预防和/或治疗纤维增生性病症和/或综合征；和/或提供能实现以上一种或更多种效果的多核苷酸。
附图说明
图1和图2显示出红景天来源的sRNA(HJT sRNA)在TGF-β1刺激的MRC-5纤维化细胞模型中对α-SMA、纤连蛋白、COL1A1和PAI-1四个纤维化相关基因的mRNA表达水平的筛选结果。图1和图2分别示出了提前48h转染NC sRNA和HJT sRNA，TGF-β1刺激MRC-5 48h后检测相关指标(预防组)和TGF-β1刺激MRC-5 3h后转染NC sRNA和HJT sRNA，TGF-β1刺激后72h检测相关指标(治疗组)两组实验的结果。
图3至图6显示出在TGF-β1刺激的MRC-5纤维化细胞模型中，选定的四种红景天sRNA均可有效降低α-SMA、纤连蛋白、COL1A1、PAI-1、TGF-β及SMAD4的mRNA表达水平。具体地，图3至图6依次显示出HJT-sRNA-m7、HJT-sRNA-a2、HJT-sRNA-h3和ppe-miR-169c在MRC-5纤维化细胞模型中的抗纤维化作用。
图7至图9显示出在博来霉素诱导的小鼠肺纤维化模型中，选定的四种红景天sRNA均可有效降低小鼠死亡率，显著减缓小鼠体重下降的趋势，缓解小鼠肺纤维化症状的结果。
具体地，图7表明HJT-sRNA-a2有效降低博来霉素导致的小鼠死亡率， 同时减缓小鼠体重的下降情况(图7A)；HJT-sRNA-h3有效降低博来霉素导致的小鼠死亡率，同时减缓小鼠体重的下降情况(图7B)；ppe-miR-169c有效降低博来霉素导致的小鼠死亡率，同时减缓小鼠体重的下降情况(图7C)。
图8表明HJT-sRNA-m7在博来霉素诱导的小鼠肺纤维化模型中的作用。
图9表明在博莱霉素导致的肺纤维化小鼠模型中，HJT-sRNA-m7有效缓解肺组织纤维化症状，降低胶原、纤连蛋白及α-SMA表达。具体地，图9A表示鼠右肺中羟脯氨酸含量(μg/右肺)；图9B表示小鼠肺组织苏木精-伊红染色(H&E染色)；图9C表示马松染色、α-SMA、纤连蛋白(fibronectin)和COL3A1的免疫组织化学结果；图9D表示与图9C相对应的病理统计结果。
图10至图12表示荧光素酶报告基因实验的结果。具体地，图10显示出荧光素酶报告基因实验验证HJT-sRNA-m7的靶基因；图11显示出荧光素酶报告基因实验验证HJT-sRNA-a2的靶基因；图12显示出荧光素酶报告基因实验验证HJT-sRNA-h3的靶基因。
图13至14显示出红景天来源的sRNA(HJT sRNA)在TGF-β1刺激的MRC-5纤维化细胞模型中对α-SMA、纤连蛋白两个纤维化相关基因的蛋白表达水平的筛选结果。
具体实施方式
下面对本发明作进一步的说明，但不以任何方式对本发明加以限制，基于本发明教导所作的任何变换，均落入本发明的保护范围。
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。
在本文中，小RNA可以是非天然的，例如是合成的或者由人工载体表达的。术语“非天然的”是指目标物质不是自然界天然存在的，这并不排除所述非天然物质与天然存在的物质具有相同的结构和/或组成。
术语“纤维化”是指组织/器官内纤维结缔组织增多、实质细胞减少的过程和状态，其可发生于多种组织/器官，持续进展可致器官结构破坏和功能减退，乃至衰竭，严重威胁人类健康和生命。
术语“用于预防/治疗纤维化的能力”表示目标物质自身能够预防/治疗纤维化，或者可以以其为基础产生能够预防/治疗纤维化的物质。也就是说，这 种能力不需要由所述目标物质自身直接实现，而可以是该目标物质所产生后果的进一步应用。
术语“抑制”是指经由特定处理使目标活性至少一部分地降低或者完全消除。
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。
在一个方面中，本发明提供一种多核苷酸，其包含：
A)如SEQ ID NO：1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16和17中任一项所示的序列，或其互补序列；
B)与A)所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，其具有用于预防/治疗纤维化的能力；
C)在严格条件下与A)所示序列杂交的序列，其具有用于预防/治疗纤维化的能力；
D)由A)所示序列经添加、缺失、替换一个或更多个核苷酸得到的序列，其具有用于预防/治疗纤维化的能力；或
E)由A)、B)、C)或D)所示序列的前体或经修饰变体，其具有用于预防/治疗纤维化的能力。
在一个实施方案中，对于本发明的多核苷酸，其中A)所示序列选自SEQ ID NO：3、10、13和16。
在另一个实施方案中，所述多核苷酸是DNA或RNA，例如是RNA，优选地是小RNA。具体地，所述多核苷酸长度为10-50个核苷酸，12-40个核苷酸，例如16-35或18-30个核苷酸；更具体地，上述多核苷酸长度为10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49或50个核苷酸。
在一个具体实施方案中，所述多核苷酸是单链或双链，优选地是单链。在另一个具体实施方案中，所述多核苷酸是非天然的，例如是合成的或由人工载体表达的。
在第二方面，本发明提供一种核酸载体，其包含或表达以上第一个方面所述的多核苷酸。例如，具体地，所述核酸载体可以是DNA，但表达的多核苷酸可以是RNA，例如sRNA。
在第三个方面，本发明提供一种药物组合物，其包含第一方面中所述的多核苷酸或者第二方面中所述的核酸载体。
在一个实施方案中，所述药物组合物还包含另外的抗纤维化剂。具体地，所述另外的抗纤维化剂可以选自以下的任何一种或更多种：糖皮质激素如醋酸可的松、氢化可的松、氢化泼尼松、地塞米松、倍他米松、曲安西龙、曲安奈德、倍氯米松；免疫抑制剂如环磷酰胺、硫唑嘌呤、甲氨蝶呤；抗氧化剂如富露施、羧甲司坦；抗凝剂如低分子肝素；以及秋水仙碱、干扰素、ACEI和他汀类药物。
在第四方面中，本发明提供一种预防和/或治疗纤维增生性疾病和/或综合征的方法，其包括向有此需要的对象施用本发明第一方面所述的多核苷酸、第二方面所述的载体、第三个方面所述的药物组合物、和/或内源性激发体内产生本发明所述多核苷酸的激发剂。在一个实施方案中，所述方法通过内源性激发体内产生本发明第一方面所述的多核苷酸。相应地，本发明还提供用于第四方面用途的多核苷酸、载体、药物组合物以及内源性激发剂，以及它们用于制备用于预防和/或治疗纤维增生性疾病和/或综合征的药物的用途。
在一个实施方案中，可以将本发明的多核苷酸、载体、药物/化妆品组合物配制成供非侵入式施用(如表面施用)和/或注射施用，例如配制成供经消化道、经呼吸道和/或注射施用，例如口服、吸入和/或注射施用。在一些情况下，优选使用侵入式施用途径(如注射施用，包括肌肉注射、皮下注射、静脉注射、动脉注射、腹腔注射、目标组织内注射)；而在另一些情况下，则优选使用非侵入式施用途径。
在另一个实施方案中，所述纤维增生性疾病和/或综合征选自：肺、心血管系统、肝、胰腺、肾、脾、眼、神经系统、骨髓和皮肤的纤维增生性疾病和/或综合征。
在一个具体实施方案中，所述纤维增生性疾病和/或综合征选自：
无机粉尘职业病，包括矽肺、石棉肺和煤肺；有机粉尘和过敏性肺炎，包括农民肺、空调湿化器肺、饲鸽者肺和蔗渣尘肺；与药物/治疗相关的疾病，所述药物选自：抗生素类、非甾体抗炎制剂、心血管药、抗肿瘤药、口服降 糖药和吗啡类；感染性疾病，包括肺结核、病毒性肺炎和肺囊虫感染；继发性肺疾病，包括与心力衰竭、先天性心脏病、成人呼吸窘迫综合症、慢性心功能不全、移植排斥反应相关的肺部疾病；肺原发性疾病，包括特发性间质性肺炎、闭塞性细支气管炎伴机化性肺炎和肺淋巴管平滑肌瘤；胶原血管病相关的肺部疾病，包括与系统性红斑狼疮、类风湿性关节炎、进行性系统硬化症、多肌炎、皮肌炎、混合型结缔组织病相关的肺部疾病；肺泡充填性疾病，包括弥漫性肺泡出血综合征、肺泡蛋白沉积症、嗜酸粒细胞性肺炎、肺血管炎、淋巴细胞间质性肺炎、坏死性结节性肉芽肿、家族性肺纤维化；
缺血性心脏疾病，包括心肌梗死后的替代性和间质性纤维化；高血压性心脏病；炎症性心肌病，包括病毒性心肌炎；代谢性心肌病，包括血色病性心肌病、淀粉样变心肌病、糖原累积性心肌病和糖尿病性心肌病；克山病；扩张性心肌病；肥厚性心肌病、限制性心肌病；致心律失常性右室心肌病；
病毒性肝硬化，包括乙、丙和丁型病毒性肝炎；血吸虫性肝硬化；酒精性肝硬化；胆汁性肝硬化，包括原发性胆汁性肝硬化、继发胆结石、门管周围炎；代谢性肝硬化，包括肝豆状核变性、血色病；中毒性肝硬化，包括有机磷中毒、四氯化碳中毒、肝毒性药物如异烟肼、四环素、氯丙嗪中毒；营养不良性肝硬化；心源性肝硬化，包括慢性充血性心力衰竭；
急性胰腺炎；胰管梗阻；慢性酒精中毒；Oddi括约肌功能失调；胰腺缺血；
血管性肾脏纤维增生性疾病和/或综合征，包括高血压；免疫性肾脏纤维增生性疾病和/或综合征，包括肾小球肾炎、系统性红斑狼疮、硬皮病、肾移植排斥；感染性肾脏纤维增生性疾病和/或综合征，包括肾盂肾炎、肾结石；代谢性肾脏纤维增生性疾病和/或综合征，包括高血脂、糖尿病、高尿酸尿症、高钙尿症；
脾纤维增生疾病；
眼睛外伤和手术后眼睛纤维增生性疾病和/或综合征，糖尿病视网眼膜纤维增生；
脊髓外伤后纤维增生性疾病和/或综合征、脑卒中瘢痕形成、老年痴呆症；
特发性和药物引起的骨髓纤维化、真性红细胞增多症、慢性髓细胞性白血病和何杰金氏病；和
皮肤纤维增生性病症和/或综合征，包括口腔粘膜纤维化、疤痕、疙瘩和厚皮病。
在另一个实施方案中，所述方法还包括向有此需要的对象在时间和/或空间上分开地和/或一起地施用另外的抗纤维化剂。具体地，所述另外的抗纤维化剂可以选自以下的任何一种或更多种：糖皮质激素如醋酸可的松、氢化可的松、氢化泼尼松、地塞米松、倍他米松、曲安西龙、曲安奈德、倍氯米松；免疫抑制剂如环磷酰胺、硫唑嘌呤、甲氨蝶呤；抗氧化剂如富露施、羧甲司坦；抗凝剂如低分子肝素；以及秋水仙碱、干扰素、ACEI和他汀类药物。
在另一个实施方案中，所述方法包括内源性激发体内产生本发明第一方面所述的多核苷酸。为此，本发明还提供内源性激发体内产生所述多核苷酸的激发剂。
在另一个方面，本发明提供一种嫩肤美容方法，其包括向有此需要的对象施用本发明所述的多核苷酸、载体、药物/化妆品组合物、和/或内源性激发体内产生本发明所述多核苷酸的激发剂。在一个实施方案中，通过非侵入式方式施用上述物质，例如表面施用。
在另一个方面，本发明提供制备第一方面中所述多核苷酸的方法，其包括：合成和/或从核酸载体上表达本发明第一方面所述的多核苷酸。
实施例
以下实施例结合附图仅为举例说明本文公开的发明，在任何情况下都不应解释为对所附权利要求保护范围的限制。
1.实验相关方法和流程
1.1红景天RNA的提取与纯化
新鲜红景天的小RNA提取根据miRNeasy Mini Kit(QIAGEN#217004)制造商说明书进行。
汤汁RNA的提取：
(1)红景天汤汁200μl，加入1ml CTAB裂解液，再加入20μlβ-巯基乙醇，剧烈震荡。
(2)65℃，30min，期间要不停振荡涡旋。
(3)12,000rpm，4℃，离心7min后，取800μl上清，加入380μl乙醇，混匀。
(4)4℃，放置20min。
(5)12,000rpm，4℃，离心15min，取800μl上清，加入0.8倍体积的氯仿，剧烈混匀。
(6)放置10min，12,000rpm，4℃，离心15min。
(7)取600μl上清，加入600μl预冷的异丙醇，混匀后-20℃放置20min。
(8)12,000rpm，4℃，离心10min，弃上清后75％乙醇洗涤沉淀2次。
(9)用DEPC处理过的H2O溶解RNA。
1.2人血、小鼠肺及细胞总RNA的提取及高通量测序
(1)向细胞中加入TRIzol裂解液，室温放置5分钟，使其充分裂解(对于小鼠肺组织，100mg组织中加入1.0ml TRIzol裂解液，用匀浆器研磨，12,000rpm，4℃，离心10min，去除未能匀浆充分的组织沉淀)。
(2)12,000rpm，4℃，离心5min，弃沉淀。
(3)按200μl/ml TRIzol的比例加入氯仿，充分振荡混匀，室温放置15min。
(4)12,000rpm，4℃，离心15min，吸取上层水相，至另一离心管中。
(5)重复步骤4，按上层水相加入等量的氯仿，充分匀后室温放置10min，12,000rpm,4℃，离心15min。
(6)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。
(7)12,000rpm，4℃，离心10min，弃上清。
(8)加入1ml 75％乙醇，温和振荡离心管，悬浮沉淀。
(9)8000g，4℃，离心5min，尽量弃上清。
(10)室温晾干5-10min，用50μl DEPC处理过的H2O溶解RNA样品。
1.3RT-qPCR检测
1)将sRNA逆转录为cDNA：通过逆转录试剂盒(High-Capacity cDNA Reverse Transcription Kits，Applied Biosystems，cat.no.4368813)，用茎环法(Stem-loop法)将sRNA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，U6RT引 物(10μM)2.0μl，HJT-sRNA-m7RT引物(10μM)2.0μl，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。所用引物序列如下：
人U6RT：GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAAAATATG；
HJT-sRNA-m7RT：GTCGTATCCAGTGCACGCTCCGAGGTATTCGCACTGGATACGACGCTTACAA。
2)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。所用引物序列如下：
人U6F：GCGCGTCGTGAAGCGTTC；
人U6R：GTGCAGGGTCCGAGGT；
HJT-sRNA-m7F：TCGCGCTGAGGTAGTAGGTT；
HJT-sRNA-m7R：GTGCACGCTCCGAGGT。
3)利用2-ΔCt法计算相对表达量。
1.4蛋白样品的收集与BCA法浓度测定
(1)弃去培养基，PBS缓冲液清洗两遍，加入适量预冷的RIPA裂解液，将细胞用枪头刮下并转移到离心管中，置冰上裂解20min。
(2)将BCA试剂A与B(50:1，v/v)充分混匀，配制BCA工作液。
(3)分别取25μl新鲜配制的BSA标准液和待测样品，加入到96孔板中，每孔中加入200μl BCA工作液，并充分混匀。
(4)37℃孵育30min后冷却至室温或室温放置2h。
(5)用紫外分光光度计(Synergy 4多功能酶标仪)于562nm处检测其吸光度，根据标准曲线计算出样品中的蛋白浓度。
(6)用RIPA裂解液调节样品浓度，使各样品浓度一致。
1.5蛋白免疫印迹法检测(Western blot)
(1)制胶：采用10％浓度分离胶(下层胶)和5％浓度的浓缩胶(上层胶)，15孔梳子所做泳道，每个泳道样品蛋白上样量相等。
(2)样品处理：将样品加入到等体积的2×loading beffer中，97℃金属浴10分钟后置于冰上待用。
(3)蛋白电泳：加入电泳缓冲液，电泳起始电压80V；当溴酚兰染料到分离胶后，提高电压至120V继续电泳，直至溴酚兰染料达到分离胶底部或全部泳出凝胶。
(4)湿法转膜：按照(负极)海绵—滤纸—凝胶—PVDF膜—滤纸—海绵(正极)的顺序进行组装；按照说明书进行安装，并将整个转膜装置置于4℃冷室；恒定电流300mA，转膜120min。
(5)封闭：转膜结束后置于3％BSA封闭液中，室温封闭1h。
(6)一抗孵育：将封闭后的PVDF膜转移至塑料袋中，加入含有一抗的3％BSA封闭液(一抗浓度根据抗体说明而定)，赶出袋中气泡，密封后4℃过夜孵育。
(7)洗膜：将PVDF膜取出，用TBST洗膜3次，每次10min。
(8)二抗孵育：弃去TBST，加入含有二抗的3％BSA封闭液，室温孵育2小时。
(9)洗膜：将PVDF膜取出，用TBST洗膜3次，每次10min。
(10)显影：配制Western显色液，并将配制好的显色液均匀滴加于膜结合蛋白的一侧；用保鲜膜小心的将膜包好，置于X射线摄影暗匣中压片10—20min，最后加显影液、定影液作用后观察。
(11)扫描与分析：将负片用Quantity One进行分析和处理，Image J进行灰度值分析。
1.6博来霉素导致小鼠肺纤维化模型建立
选取6-8周龄，体重为20-25g的雄性C57BL/6小鼠，麻醉状态下经支气 管灌注100μL生理盐水配制的博来霉素溶液(3.5U/kg)，对照组给100μL生理盐水，每天记录小鼠体重及死亡率，第21d时处理小鼠。取右肺用于羟脯氨酸的测定，左肺经4％多聚甲醛内固定后石蜡包埋、切片进行苏木精-伊红染色(H&E染色)、马松三色法染色和免疫组化检测。组织病理切片和纤维化指标的结果相结合评价博来霉素致小鼠肺纤维化模型构建是否成功。
1.7荧光素酶报告基因检测靶基因
实验材料
人胚胎肾细胞293T；DMEM培养基(Dulbecco's Modified Eagle's Medium)，加入10％(v/v)胎牛血清(FBS)；青霉素(100U/ml)与链霉素(100mg/ml)；转染试剂：RNAiMax(Invitrogen)和脂质体Lipofectamine 2000(Invitrogen)；双荧光素酶报告基因系统：psiCHECK2(Promega C2081)
实验方法
(1)含有10％FBS的DMEM培养基培养的293T细胞，分至48孔板中，每孔约为3x104个细胞，贴壁后用RNAiMax(Invitrogen)转染100nM的NC sRNA/sRNA。
(2)24h后，用Lipofectamine 2000(Invitrogen)转染psiCHECK2-3’-UTR或psiCHECK2-3’-mUTR的质粒。
(3)转染质粒后8h、14h或24h后按照Dual-luciferase Assay System(Promega E1910)的手册中使用方法检测其荧光强度。
2.试验实施例
2.1鉴定红景天来源的sRNA
参照以上1.1项，利用试剂盒和改进的CTAB裂解法，分别从新鲜的红景天和红景天中药饮片煎煮的汤剂中提取出RNA，琼脂糖凝胶电泳显示，RNA片段为～20nt的小RNA片段(small RNA，sRNA)。接下来的试验中，分别对连续饮用红景天汤剂七天后0h和24h的人的全血，连续三天灌胃给红景天来源的RNA后12h、24h和48h后小鼠的肺组织，以及加入红景天来源的RNA后24h的A549细胞进行高通量测序(SE36，Illumina HiSeq2500)。通过生物信息学方法，根据以下条件找出进入小鼠肺、人血或者A549细胞的小RNA片段：(1)存在于人血、小鼠肺组织或者A549细胞的红景天来源小RNA；(2) 不能与人或者小鼠基因组比对上的红景天来源小RNA。通过上述方法，发明人意外地发现3条进入人血的红景天来源小RNA(见表1)，并根据其在人血中的相对丰度排序，依次命名为HJT-sRNA-h1～3；8条进入小鼠肺组织的红景天来源小RNA(见表2)，并根据其在小鼠肺组织中的相对丰度排序，依次命名为HJT-sRNA-m1～8；2条进入A549细胞的红景天来源小RNA，并根据其在A549细胞中的相对丰度排序，依次命名为HJT-sRNA-a1～2(见表3)。此外，发明人还筛选出红景天中可与miRbase数据库比对上的4条小RNA进行后续实验(见表4)。
表1人血中红景天来源sRNA序列及命名
Figure PCTCN2017078815-appb-000001
表2小鼠肺中红景天来源sRNA序列及命名
Figure PCTCN2017078815-appb-000002
表3A549细胞中红景天来源sRNA序列及命名
Figure PCTCN2017078815-appb-000003
Figure PCTCN2017078815-appb-000004
表4红景天来源可比对上miRbase数据库的sRNA序列及命名
Figure PCTCN2017078815-appb-000005
2.2sRNA的抗纤维化活性
2.2.1在TGF-β1刺激的MRC-5纤维化细胞模型中筛选并验证红景天来源的sRNA
TGF-β1 3ng/ml刺激MRC-5细胞48h(图1)或72h(图2至6)后，收取RNA样品，用RT-PCR方法检测纤维化相关的基因α-SMA、纤连蛋白(Fibronectin)、COL1A1、PAI-1、SMAD4和TGF-β的mRNA相对表达。
α-SMA、纤连蛋白(Fibronectin)、COL1A1和PAI-1是四个纤维化相关基因。用红景天来源的sRNA处理经TGF-β1刺激的MRC-5纤维化细胞模型，检测上述四个纤维化相关基因的mRNA水平的表达，从而对进入人血、小鼠肺和A549细胞的小RNA进行抗纤维化功能试验的筛选。结果如图1和2所示，在预防和治疗的两组试验中，多种红景天来源的sRNA均能够抑制MRC-5细胞中纤维化相关基因在mRNA水平的表达。
选择HJT-sRNA-m7、HJT-sRNA-h3、HJT-sRNA-a2以及ppe-miR169c四种sRNA序列进行后续验证实验，提前24h转染NC sRNA和HJT sRNA，TGF-β1刺激MRC-5 72h后检测相关指标。分别如图3-6所示，在TGF-β1刺激的MRC-5纤维化细胞模型中，上述四种sRNA均能有效降低α-SMA、纤连蛋白、COL1A1、PAI-1、TGF-β及SMAD4的mRNA表达水平。
发明人筛选了红景天来源的sRNA(HJT sRNA)在TGF-β1刺激的MRC-5纤维化细胞模型中对α-SMA、纤连蛋白两个纤维化相关基因的蛋白质表达水平。图13和图14分别示出了提前24h转染NC sRNA和HJT sRNA，TGF-β1刺激MRC-5 72h后检测相关指标(预防组)实验的结果。如图13和图14所 示，多种红景天来源的sRNA均能够抑制MRC-5细胞中纤维化相关基因在蛋白质水平的表达。其中与SEQ ID NO：4、5、6、8、10的HJT sRNA可以显著降低纤连蛋白的蛋白表达水平，与SEQ ID NO：3、5、6、8、10、16、17对应的HJT sRNA可以降低α-SMA的蛋白表达水平。
2.2.2红景天来源的sRNA在博来霉素诱导的小鼠肺纤维化模型中的作用
根据以上1.6项，在博来霉素诱导的小鼠肺纤维化模型中，测试上述四种红景天来源的sRNA对纤维化的作用。C57BL/6J小鼠气管注射博来霉素(BLM,Nippon Kayaku,Tokyo,Japan)剂量是3.5U/kg同时以气管给入NC sRNA、HJT-sRNA-m7、HJT-sRNA-a2、HJT-sRNA-h3和ppe-miR-169c的agomir(苏州吉玛基因股份有限公司定制)，剂量是8mg/kg，用生理盐水稀释到100μl总体积。给博来霉素后第7天、第13天和第16天分别腹腔注射NC sRNA、HJT-sRNA-m7、HJT-sRNA-a2、HJT-sRNA-h3和ppe-miR-169c的agomir，剂量是4mg/kg。如图7-9所示，发明人发现，选定的四种红景天来源的sRNA均能令人惊奇地显著降低小鼠的死亡率，显著减缓小鼠体重下降的趋势，缓解小鼠的肺纤维化症状。
2.2.3荧光素酶报告基因实验检测红景天来源的sRNA的抗纤维化靶标
根据以上1.7项，利用荧光素酶报告基因系统检测红景天来源的sRNA中细胞内的靶标基因。如图10-12所示，HJT-sRNA-m7可通过直接靶向α-SMA、纤连蛋白和COL3A1来发挥抗纤维化的功能；HJT-sRNA-a2可通过直接靶向COL1A1、COL3A1、TGF-β和SMAD4来发挥抗纤维化的功能；HJT-sRNA-h3可通过直接靶向COL1A1和COL3A1来发挥抗纤维化的功能。

Claims (19)

  1. 一种多核苷酸，其包含：
    A)如SEQ ID NO：1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16和17中任一项所示的序列，或其互补序列；
    B)与A)所示序列具有至少80％、85％、90％、95％、96％、97％或98％同一性的序列，其具有用于预防/治疗纤维化的能力；
    C)在严格条件下与A)所示序列杂交的序列，其具有用于预防/治疗纤维化的能力；
    D)由A)所示序列经添加、缺失、替换一个或更多个核苷酸得到的序列，其具有用于预防/治疗纤维化的能力；或
    E)由A)、B)、C)或D)所示序列的前体或经修饰变体，其具有用于预防/治疗纤维化的能力。
  2. 权利要求1所述的多核苷酸，其中A)所示序列选自SEQ ID NO：3、10、13和16。
  3. 权利要求1或2所述的多核苷酸，其是DNA或RNA，例如是RNA，优选地是小RNA。
  4. 权利要求1至3中任一项所述的多核苷酸，其中所述多核苷酸长度为12-40个核苷酸，例如16-35或18-30个核苷酸。
  5. 权利要求1至4中任一项所述的多核苷酸，其中所述多核苷酸是单链或双链，优选地是单链。
  6. 权利要求1至5中任一项所述的多核苷酸，其中所述多核苷酸是非天然的，例如是合成的或由人工载体表达的。
  7. 一种核酸载体，其包含或表达权利要求1至6中任一项所述的多核苷酸。
  8. 一种药物/化妆品组合物，其包含权利要求1至6中任一项所述的多核苷酸或者权利要求7所述的核酸载体。
  9. 权利要求8所述的药物/化妆品组合物，其还包含另外的抗纤维化剂。
  10. 一种预防和/或治疗纤维增生性疾病和/或综合征的方法，其包括向有此需要的对象施用权利要求1至6中任一项所述的多核苷酸、权利要求7所述的载体、权利要求8或9所述的药物组合物、和/或内源性激发体内产生权利要求1至6中任一项所述多核苷酸的激发剂。
  11. 权利要求10所述的方法，其中所述纤维增生性疾病和/或综合征选自：肺、心血管系统、肝、胰腺、肾、脾、眼、神经系统、骨髓和皮肤的纤维增生性疾病和/或综合征。
  12. 权利要求10或11所述的方法，其中所述纤维增生性疾病和/或综合征选自：
    无机粉尘职业病，包括矽肺、石棉肺和煤肺；有机粉尘和过敏性肺炎，包括农民肺、空调湿化器肺、饲鸽者肺和蔗渣尘肺；与药物/治疗相关的疾病，所述药物选自：抗生素类、非甾体抗炎制剂、心血管药、抗肿瘤药、口服降糖药和吗啡类；感染性疾病，包括肺结核、病毒性肺炎和肺囊虫感染；继发性肺疾病，包括与心力衰竭、先天性心脏病、成人呼吸窘迫综合症、慢性心功能不全、移植排斥反应相关的肺部疾病；肺原发性疾病，包括特发性间质性肺炎、闭塞性细支气管炎伴机化性肺炎和肺淋巴管平滑肌瘤；胶原血管病相关的肺部疾病，包括与系统性红斑狼疮、类风湿性关节炎、进行性系统硬化症、多肌炎、皮肌炎、混合型结缔组织病相关的肺部疾病；肺泡充填性疾病，包括弥漫性肺泡出血综合征、肺泡蛋白沉积症、嗜酸粒细胞性肺炎、肺血管炎、淋巴细胞间质性肺炎、坏死性结节性肉芽肿、家族性肺纤维化；
    缺血性心脏疾病，包括心肌梗死后的替代性和间质性纤维化；高血压性心脏病；炎症性心肌病，包括病毒性心肌炎；代谢性心肌病，包括血色病性心肌病、淀粉样变心肌病、糖原累积性心肌病和糖尿病性心肌病；克山病；扩张性心肌病；肥厚性心肌病、限制性心肌病；致心律失常性右室心肌病；
    病毒性肝硬化，包括乙、丙和丁型病毒性肝炎；血吸虫性肝硬化；酒精性肝硬化；胆汁性肝硬化，包括原发性胆汁性肝硬化、继发胆结石、门管周围炎；代谢性肝硬化，包括肝豆状核变性、血色病；中毒性肝硬化，包括有机磷中毒、四氯化碳中毒、肝毒性药物如异烟肼、四环素、氯丙嗪中毒；营养不良性肝硬化；心源性肝硬化，包括慢性充血性心力衰竭；
    急性胰腺炎；胰管梗阻；慢性酒精中毒；Oddi括约肌功能失调；胰腺缺血；
    血管性肾脏纤维增生性疾病和/或综合征，包括高血压；免疫性肾脏纤维增生性疾病和/或综合征，包括肾小球肾炎、系统性红斑狼疮、硬皮病、肾移植排斥；感染性肾脏纤维增生性疾病和/或综合征，包括肾盂肾炎、肾结石；代谢性肾脏纤维增生性疾病和/或综合征，包括高血脂、糖尿病、高尿酸尿症、高钙尿症；
    脾纤维增生疾病；
    眼睛外伤和手术后眼睛纤维增生性疾病和/或综合征，糖尿病视网眼膜纤维增生；
    脊髓外伤后纤维增生性疾病和/或综合征、脑卒中瘢痕形成、老年痴呆症；
    特发性和药物引起的骨髓纤维化、真性红细胞增多症、慢性髓细胞性白血病和何杰金氏病；和
    皮肤纤维增生性病症和/或综合征，包括粘膜纤维化、疤痕、疙瘩和厚皮病。
  13. 权利要求10至12中任一项所述的方法，还包括向有此需要的对象时间和/或空间上分开地和/或一起地施用另外的抗纤维化剂。
  14. 权利要求13所述的方法，其中所述另外的抗纤维化剂选自以下的任何一种或更多种：糖皮质激素如醋酸可的松、氢化可的松、氢化泼尼松、地塞米松、倍他米松、曲安西龙、曲安奈德、倍氯米松；免疫抑制剂如环磷酰胺、硫唑嘌呤、甲氨蝶呤；抗氧化剂如富露施、羧甲司坦；抗凝剂如低分子肝素；以及秋水仙碱、干扰素、ACEI和他汀类药物。
  15. 一种嫩肤美容方法，其包括向有此需要的对象施用权利要求1至6中任一项所述的多核苷酸、权利要求7所述的载体、权利要求8或9所述的药物/化妆品组合物、和/或内源性激发体内产生权利要求1至6中任一项所述多核苷酸的激发剂。
  16. 权利要求15所述的美容方法，其中通过非侵入式方式施用，例如表面施用。
  17. 内源性激发体内/细胞内产生权利要求1-6中任一项所述多核苷酸的激发剂。
  18. 一种抑制一个或更多个纤维化相关基因表达的方法，其包括使细胞接触。
  19. 制备权利要求1至6中任一项所述多核苷酸的方法，其包括：合成和/或从核酸载体上表达权利要求1至6中任一项所述的多核苷酸，和/或内源性激发有此能力的细胞表达权利要求1至6中任一项所述的多核苷酸。
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