WO2023025161A1 - 来源于药用植物的sRNA及其应用 - Google Patents

来源于药用植物的sRNA及其应用 Download PDF

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WO2023025161A1
WO2023025161A1 PCT/CN2022/114330 CN2022114330W WO2023025161A1 WO 2023025161 A1 WO2023025161 A1 WO 2023025161A1 CN 2022114330 W CN2022114330 W CN 2022114330W WO 2023025161 A1 WO2023025161 A1 WO 2023025161A1
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seq
srna
group
combination
use according
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PCT/CN2022/114330
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French (fr)
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张强
蒋澄宇
张珩
杜芯仪
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北京嘉树佳业科技有限公司
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Priority to CN202410618722.7A priority Critical patent/CN118562793A/zh
Priority to CN202280006412.9A priority patent/CN116472066B/zh
Publication of WO2023025161A1 publication Critical patent/WO2023025161A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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

Definitions

  • the invention belongs to the technical field of medicinal small nucleic acid molecules, in particular to an sRNA isolated from a medicinal plant composition and its application.
  • Osteoporosis is the most common skeletal disease, and it is a systemic bone disease characterized by low bone mass and damage to the microarchitecture of bone tissue, resulting in increased bone fragility and susceptibility to fracture.
  • OP Osteoporosis
  • it is a systemic bone disease characterized by low bone mass and damage to the microarchitecture of bone tissue, resulting in increased bone fragility and susceptibility to fracture.
  • the National Institutes of Health of the United States defined it as a skeletal disease characterized by decreased bone strength and increased fracture risk, suggesting that decreased bone mass is the main risk factor for osteoporotic fractures, but there are other risk factors.
  • OP can occur at any age, but is more common in postmenopausal women and older men.
  • the main therapeutic drugs at this stage include calcium, vitamin D and bone resorption inhibitors (including estrogen, selective estrogen receptor modulators, bisphosphonates, etc.) and other three categories.
  • the long-term use of these drugs will increase the incidence of gynecological cancer, cardiovascular disease, and thrombosis, and there are many problems such as kidney damage, long course of treatment, high cost, and inaccurate curative effect, which makes patients unable to tolerate it during treatment. Response and bear high treatment costs forced to terminate treatment. Therefore, it is still necessary to find new therapeutic measures against osteoporosis.
  • sRNA small RNA, small RNA
  • sRNA is a type of ribonucleic acid fragments with a length less than 200nt. Most of the sRNA is non-coding RNA (non-coding RNA, ncRNA), including: micro sRNA (microRNA, miRNA), nucleolar sRNA ( small nuclear RNA, snoRNA), piwi protein-interacting RNA (piwi-interacting RNA, piRNA), nuclear sRNA (small nuclear RNA, U-RNA), small interfering RNA (small interfering RNA, siRNA), etc.
  • sRNA widely exists in animals, plants and microorganisms, and participates in the regulation of key biological processes such as cell proliferation, differentiation, metabolism and death by inducing gene silencing.
  • Medicinal plants refer to plants that can be used to prevent or treat diseases in medicine. my country is one of the countries with the richest medicinal plant resources, the longest history of use, and the most detailed historical records.
  • active ingredients of medicinal plants are small chemical molecules, small biological molecules (glycosides, alkaloids, etc.) It is difficult for ingredients to replace the role of plants as a whole.
  • miR168a1 from rice, miR29112 from honeysuckle, HJT-sRNA-m73 from Rhodiola, FvmiR168 from strawberry, miR1594 from Arabidopsis and other medicinal plant sRNAs can regulate gene expression and organisms across species. Physiological process. These research results show that sRNA may be a new active ingredient of medicinal plants, which opens up a new field for the study of the mechanism of medicinal plants.
  • a traditional Chinese medicine compound composed of Epimedium, Dipsacus, Salvia, Anemarrhena, Psoralen, and Dihuang, used to treat osteoporosis caused by liver and kidney deficiency and blood stasis blocking collaterals.
  • the traditional Chinese medicine compound mostly uses water extraction or ethanol extraction, and no systematic research has been carried out on its plant-derived sRNA.
  • the object of the present invention is to provide an sRNA derived from a medicinal plant. Specifically, it is the sRNA isolated from the compound Chinese medicine composition composed of Epimedium, Dipsacus, Salvia, Anemarrhena, Psoraleae, and Rehmannia glutinosa.
  • Another object of the present invention is to provide the use of sRNA derived from the above-mentioned medicinal plants.
  • the use includes the use of preventing, treating or improving bone metabolism diseases of subjects, and the use of preparing pharmaceutical compositions.
  • Another object of the present invention is to provide a genetic engineering expression vector for preparing the above-mentioned sRNA derived from a medicinal plant, and a host cell comprising the genetic engineering expression vector.
  • the present invention provides a kind of sRNA, described sRNA comprises the sRNA shown in any one of the following sequences or its combination:
  • the sRNA has the RNA shown in any one of SEQ ID NO.1 to SEQ ID NO.84 or a combination thereof.
  • the sRNA comprises 1-5 or 1-3 nucleosides truncated at the 5' end and/or 3' end of the sRNA shown in any one of SEQ ID NO.1 to SEQ ID NO.84 acidic sRNA or a combination thereof.
  • the sRNA has 1-5 or 1-3 cores truncated at the 5' end and/or 3' end of the sRNA shown in any one of SEQ ID NO.1 to SEQ ID NO.84 nucleotide RNA or a combination thereof.
  • the truncated sRNA has the same or related biological effect as the sequence corresponding to SEQ ID NO.1 to SEQ ID NO.84.
  • the sRNA comprises an sRNA or a combination thereof that is greater than or equal to 80%, 85%, 90%, 95% homology to the sRNA shown in any sequence of SEQ ID NO.1 to SEQ ID NO.84 .
  • the sRNA has an sRNA whose homology with the sRNA shown in any sequence of SEQ ID NO.1 to SEQ ID NO.84 is greater than or equal to 80%, 85%, 90%, 95%, or combination.
  • the homologous sRNA has the same or related biological effect as the sequence corresponding to SEQ ID NO.1 to SEQ ID NO.84.
  • the sRNA comprises a fusion RNA of any sRNA sequence in SEQ ID NO.1 to SEQ ID NO.84 or a combination thereof.
  • the fusion sRNA has the same or related biological effect as the sequence corresponding to SEQ ID NO.1 to SEQ ID NO.84.
  • the sRNA comprises any sRNA complementary or hybridized to any of the RNAs of SEQ ID NO. 1 to SEQ ID NO. 84, or a combination thereof.
  • the complementary or hybrid sRNA has the same or related biological effect as the sequence corresponding to SEQ ID NO.1 to SEQ ID NO.84.
  • the sRNA comprises any sRNA in SEQ ID NO.1 to SEQ ID NO.84 and a nucleic acid sequence capable of hybridizing with the sRNA, a fully complementary double-stranded sRNA or a partially complementary double-stranded RNA or a combination thereof.
  • the sRNA has any sRNA in SEQ ID NO.1 to SEQ ID NO.84 and a fully complementary double-stranded sRNA or partially complementary double-stranded sRNA formed by hybridization with a nucleic acid sequence capable of hybridizing with the sRNA. Stranded RNA or combinations thereof.
  • the fully complementary or partially complementary double-stranded sRNA has the same or related biological effects as the corresponding sequences in SEQ ID NO.1 to SEQ ID NO.84.
  • the sRNA is any sRNA in SEQ ID NO.1 to SEQ ID NO.84 and contains a hairpin structure or a combination thereof. In another embodiment, the sRNA has any sRNA in SEQ ID NO.1 to SEQ ID NO.84 and contains a hairpin structure sRNA or a combination thereof.
  • the sRNA is any sRNA of SEQ ID NO.1 to SEQ ID NO.84 and contains 2'-fluoro-modified nucleotides, 2'-methoxyethyl-modified nucleotides, 2'-deoxy-modified nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides , 2'-hydroxy-modified nucleotides, 2'-O-alkyl-modified nucleotides or combinations thereof; preferably 2'-O-methyl-modified nucleotides, more preferably the 3' end of RNA 2'-O-methyl-modified nucleotides.
  • the sRNA using the above-mentioned modified nucleotides has the same or associated biological effects as the corresponding sequences in SEQ ID NO.1 to SEQ ID NO.84.
  • the sRNA preferably comprises sRNA or a combination thereof shown in any of the following sequences: SEQ NO.2, SEQ NO.5, SEQ NO.9, SEQ NO.11, SEQ NO.13, SEQ NO.14 , SEQ NO.18, SEQ NO.20, SEQ NO.30, SEQ NO.34, SEQ NO.38, SEQ NO.39, SEQ NO.45, SEQ NO.46, SEQ NO.51, SEQ NO.58 , SEQ NO.64, SEQ NO.67, SEQ NO.69, SEQ NO.70, SEQ NO.71, SEQ NO.73, SEQ NO.75, SEQ NO.76, SEQ NO.77, SEQ NO.78 , SEQ NO.80, SEQ NO.81, SEQ NO.82, SEQ NO.83, SEQ NO.84.
  • the sRNA preferably comprises sRNA or a combination thereof shown in any of the following sequences: SEQ NO.1, SEQ NO.2, SEQ NO.3, SEQ NO.4, SEQ NO.5, SEQ NO.7 , SEQ NO.8, SEQ NO.10, SEQ NO.12, SEQ NO.13, SEQ NO.14, SEQ NO.15, SEQ NO.16, SEQ NO.17, SEQ NO.18, SEQ NO.19 , SEQ NO.20, SEQ NO.21, SEQ NO.22, SEQ NO.23, SEQ NO.24, SEQ NO.25, SEQ NO.26, SEQ NO.27, SEQ NO.28, SEQ NO.29 , SEQ NO.30, SEQ NO.31, SEQ NO.32, SEQ NO.34, SEQ NO.35, SEQ NO.36, SEQ NO.37, SEQ NO.38, SEQ NO.39, SEQ NO.42 , SEQ NO.44, SEQ NO.46,
  • the sRNA preferably comprises sRNA or a combination thereof shown in any of the following sequences: SEQ NO.2, SEQ NO.5, SEQ NO.13, SEQ NO.14, SEQ NO.18, SEQ NO.20 , SEQ NO.30, SEQ NO.34, SEQ NO.38, SEQ NO.39, SEQ NO.46, SEQ NO.58, SEQ NO.64, SEQ NO.67, SEQ NO.69, SEQ NO.71 , SEQ NO.73, SEQ NO.76, SEQ NO.77, SEQ NO.78, SEQ NO.83.
  • the present invention provides a kind of sRNA described in formula (I),
  • Any one of X1 is independently selected from G-, UGGG-, CUGGG-, GCUGGG-, GGCUGGG-, GGGCUGGG-, AGGGCUGGG- or a combination thereof;
  • Any one of X2 is independently selected from -C, -CG, -CGC, -CGCG or a combination thereof;
  • n 0, 1, 2, or 3;
  • the sRNA has an sRNA whose homology with the structural sRNA of formula (I) is greater than or equal to 80%, 85%, 90% or 95%;
  • the sRNA is the complementary sequence of the sRNA with the structure of formula (I).
  • 1-5 nucleotides are further fused to the 5' end and/or 3' end of the sRNA of formula (I).
  • the sRNA of formula (I) further comprises fully complementary double-stranded sRNA or partially complementary double-stranded sRNA or a combination thereof formed by hybridization with a nucleic acid sequence capable of hybridizing with the sRNA.
  • the sRNA of formula (I) contains a hairpin structure.
  • the formula (I) sRNA contains 2'-fluoro-modified nucleotides, 2'-methoxyethyl-modified nucleotides, 2'-deoxy-modified nucleotides, 2'- Amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxy-modified nucleotides, 2' -O-alkyl-modified nucleotides, preferably 2'-O-methyl-modified nucleotides, preferably 2'-O-methyl-modified nucleotides at the 3' end of the sRNA.
  • the sRNA of formula (I) includes the sRNA shown in SEQ NO.2, 4-18, 30, 33-36, 42-46 or a combination thereof. In other embodiments, the sRNA of formula (I) has the sRNA shown in SEQ NO.2, 4-18, 30, 33-36, 42-46 or a combination thereof.
  • the present invention provides an sRNA having 1-9 nucleotides truncated at the 5' end of the sRNA shown in SEQ ID NO.42, and/or 1-4 nucleotides truncated at the 3' end.
  • the specific number of truncations is 1, 2, 3, 4, 5, 6, 7, 8 or 9.
  • the present invention provides a genetic engineering expression vector capable of transcribing the sRNA described in the present invention.
  • sRNA is any of the aforementioned unmodified sRNA, modified sRNA, 5' and/or 3' truncated sRNA, sRNA with homology greater than or equal to 80%, 85%, 90%, 95%, fusion sRNA, complementary or hybrid sRNA, fully complementary double-stranded RNA or partially complementary double-stranded RNA, sRNA containing a hairpin structure, sRNA represented by formula (I).
  • the genetic engineering expression vector expresses the corresponding RNA in the subject, exerts the corresponding pharmacological effect and realizes gene therapy.
  • the present invention provides a host cell comprising a genetically engineered expression vector.
  • the genetically engineered expression vector expresses the corresponding sRNA in the subject (for example, a patient with a bone metabolic disease or osteoporosis), and then exerts a corresponding pharmacological effect to realize gene therapy.
  • the corresponding sRNA is transcribed from the genetic engineering expression vector in vitro, which can be transcribed by host cells or cell-free systems, and administered to the subject after extraction (purification).
  • the pharmaceutical composition is suitable for gavage, oral, intravenous, subcutaneous, transdermal, intramuscular, intraarterial, intraperitoneal, intrapulmonary, intracerebrospinal, intraarticular, intrasynovial, Pharmaceutical dosage forms administered by intrathecal, intraventricular, or inhalation routes.
  • a drug delivery complex which contains a drug delivery vector and the sRNA of the present invention, a recombinant expression vector, and a host cell.
  • the drug delivery vehicle is a liposome.
  • the present invention provides a use of the sRNA, its genetically engineered expression vector, its host cell or its pharmaceutical composition in the preparation of drugs for preventing, treating or improving bone metabolism diseases in subjects.
  • the present invention provides a use of the sRNA, its genetically engineered expression vector, its host cell or its pharmaceutical composition in preventing, treating or improving a bone metabolic disease in a subject.
  • the bone metabolic disease is prevented, treated or improved by increasing the expression or activity of alkaline phosphatase gene, and/or reducing the expression or activity of tartrate-resistant acid phosphatase.
  • the bone metabolic disease is prevented, treated or improved by promoting osteoblast differentiation, and/or inhibiting osteoclast differentiation.
  • the sRNA that improves alkaline phosphatase gene expression or activity, or promotes osteoblast differentiation is selected from: SEQ NO.2, SEQ NO.5, SEQ NO.9, SEQ NO.11, SEQ NO .13, SEQ NO.14, SEQ NO.18, SEQ NO.20, SEQ NO.30, SEQ NO.34, SEQ NO.38, SEQ NO.39, SEQ NO.45, SEQ NO.46, SEQ NO .51, SEQ NO.58, SEQ NO.64, SEQ NO.67, SEQ NO.69, SEQ NO.70, SEQ NO.71, SEQ NO.73, SEQ NO.75, SEQ NO.76, SEQ NO. .77, SEQ NO.78, SEQ NO.80, SEQ NO.81, SEQ NO.82, SEQ NO.83, SEQ NO.84.
  • the sRNA that reduces the expression or activity of tartrate-resistant acid phosphatase, or inhibits osteoclast differentiation is selected from the group consisting of: SEQ NO.1, SEQ NO.2, SEQ NO.3, SEQ NO.4, SEQ NO .5, SEQ NO.7, SEQ NO.8, SEQ NO.10, SEQ NO.12, SEQ NO.13, SEQ NO.14, SEQ NO.15, SEQ NO.16, SEQ NO.17, SEQ NO. .18, SEQ NO.19, SEQ NO.20, SEQ NO.21, SEQ NO.22, SEQ NO.23, SEQ NO.24, SEQ NO.25, SEQ NO.26, SEQ NO.27, SEQ NO.
  • the sRNA is preferably SEQ NO.2, SEQ NO.5, SEQ NO.13, SEQ NO.14, SEQ NO.18, SEQ NO.20, SEQ NO.30, SEQ NO.34, SEQ NO.38, SEQ NO.39, SEQ NO.46, SEQ NO.58, SEQ NO.64, SEQ NO.67, SEQ NO.69, SEQ NO.71, SEQ NO.73, SEQ NO.76, SEQ NO.77, SEQ NO.78, SEQ NO.83.
  • sRNAs also have the functions of (1) increasing the expression level or activity of alkaline phosphatase gene, or promoting osteoblast differentiation, and (2) reducing the expression level or activity of tartrate-resistant acid phosphatase, or inhibiting osteoclast differentiation.
  • the metabolic bone disease is osteoporosis, including postmenopausal osteoporosis and senile osteoporosis.
  • the "subject" is an animal, such as: a mammal, including primates (such as: humans, non-human primates, such as: monkeys and chimpanzees), non-primates (such as: cattle, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, horses and whales), or birds (e.g. chickens, ducks or geese).
  • the subject is human.
  • single-stranded sRNA and double-stranded RNA can be synthesized using standard methods known in the art, non-limiting examples include chemical synthesis using phosphotriester, phosphite, or phosphoramidite chemistry and solid phase techniques , such as the solid-phase phosphoramidite triester method.
  • the synthesized oligonucleotides were separated and purified to obtain the target product.
  • G", “C”, “A”, “T” and “U” respectively generally represent nucleotides, which respectively contain the bases of guanine, cytosine, adenine, thymidine and uracil.
  • ribonucleotide or “nucleotide” also refers to modified nucleotides.
  • guanine, cytosine, adenine, and uracil can be used without substantially changing the base pairing properties of the oligonucleotide comprising nucleotides with these substituted moieties. Other partial body replacements.
  • sRNA is a non-coding sRNA molecule present in an organism. If there is no special description, "sRNA” herein includes unmodified sRNA, modified sRNA, 5' end and/or 3' end truncated sRNA, homology greater than or equal to 80%, 85%, 90%, 95% of sRNAs, fusion sRNAs, complementary or hybridized sRNAs, fully complementary double-stranded RNAs or partially complementary double-stranded RNAs, sRNAs containing hairpin structures, or sRNAs represented by formula (I).
  • each strand of a double-stranded sRNA molecule are ribonucleotides, and each strand or both strands may also include one or more non-ribonucleotides, such as: deoxyribonucleotides and/or modified Nucleotides.
  • sRNA used in this specification may include chemically modified ribonucleotides; sRNA may be modified on multiple nucleotides.
  • modified refers to nucleotides each independently having a sugar moiety group, a modified internucleotide linkage, and/or a modified nucleobase.
  • modified sRNA includes substitutions, additions, or exclusions such as functional groups or atoms at internucleotide linkages, sugar moiety groups, or nucleobases.
  • Modifications suitable for use in the formulations of the invention include all modifications disclosed herein or known in the art.
  • sRNA includes any such modifications for sRNA-type molecules.
  • fusion sRNA means that after a base is fused to the 3' end of the sRNA sequence, its degradation rate will slow down and its stability will increase; After function remains unchanged.
  • alkaline phosphatase is one of the phenotypic markers of osteoblasts, which can directly reflect the activity or function of osteoblasts.
  • An increase in alkaline phosphatase indicates that the cells are differentiated into osteoblasts.
  • the higher the alkaline phosphatase level the more obvious the osteogenic differentiation of cells.
  • the level of alkaline phosphatase can be detected by MC3T3-E1 cells (related experiments described in L.Darryl Quarles, et, al. Distinct proliferative and differentiated stages of murine MC3T3-E1 cells in culture: an in vitro model of osteoblast development. Journal of Bone and Mineral Research, 7(6).1992.683-692).
  • “Tartrate-resistant acid phosphatase” can be released into the blood by osteoclasts, which is a blood index indicating the body's osteoclast activity.
  • osteoclasts which is a blood index indicating the body's osteoclast activity.
  • the level of tartrate-resistant acid phosphatase can be detected by the RAW264.7 cell line (related experiments are described in Patricia Collin-Osdoby.et, al. (2003).
  • the same or related biological effect means that it has the effect of preventing, treating or improving the subject's bone metabolism disease; increasing the expression level or activity of alkaline phosphatase gene, and/or reducing the expression level or activity of tartrate-resistant acid phosphatase; Promote osteoblast differentiation, and/or inhibit osteoclast differentiation; or prevent, treat or improve osteoporosis symptoms; or prevent, treat or improve postmenopausal osteoporosis or senile osteoporosis.
  • Figures 1-3 show the relative alkaline phosphatase activity of the OP1-OP84 group.
  • Figures 4-6 show the relative activity of tartrate-resistant acid phosphatase in the OP1-OP84 group.
  • kits Materials and reagents whose sources are not emphasized in the present invention are commonly used materials and reagents in the art.
  • the kit that mentions the source of the kit, refer to the instructions of the kit for operation. If there is no mention of the operation method, operate according to the instruction manual of the commercially available instrument, or operate according to the routine operation method of those skilled in the art.
  • Embodiment one the preparation of Chinese medicine composition and single flavor Chinese medicine decoction
  • the crude drug of each dose of Chinese medicine composition contains the Chinese medicine combination obtained by boiling Epimedium 24-30g, Dipsacus 12-15g, Anemarrhena 12-15g, Salvia miltiorrhiza 12-15g, Psoralen 12-15g, and Rehmannia glutinosa 12-15g. Thing (soup), the specific cooking steps are as follows:
  • Example 2 Obtaining sRNA in single-flavored traditional Chinese medicine soup
  • the first step use the improved CTAB method to independently extract total RNA from the single-flavored traditional Chinese medicine soup, the specific steps are as follows:
  • the second step obtain the sRNA sequence from the total RNA of the single Chinese medicine decoction obtained in the first step, the specific method is as follows:
  • Data quality control remove low-quality sequences with a ratio of N>10% (the number of bases with a quality value Q ⁇ 3 accounts for more than 50% of the entire sequence);
  • sRNA sequencing data preprocessing The sRNAs in 6 kinds of single traditional Chinese medicine decoctions were obtained, and the data were combined to form a Chinese medicine sRNA group (sRNA group 1).
  • Example 3 Obtaining sRNA sequences from total RNA in human blood after taking medicine and analyzing the sequencing data
  • Step 1 Preparation of human blood total RNA:
  • the second step use the human whole blood total RNA after drinking the traditional Chinese medicine composition (decoction) in the first step to obtain sRNA and perform sequencing:
  • Data quality control remove low-quality sequences with a ratio of N>10% (the number of bases with a quality value Q ⁇ 3 accounts for more than 50% of the entire sequence);
  • sRNA sequencing data preprocessing Obtain the sRNA group (sRNA group 2) in the blood of the first volunteer taking the medicine 0d, the sRNA group (sRNA group 3) in the blood of taking the medicine 3d, and the sRNA group (sRNA group 4) in the blood of taking the medicine 7d; the second volunteer sRNA group (sRNA group 5) in the blood after taking the medicine for 0 days, sRNA group in the blood for 3 days after taking the medicine (sRNA group 6), and sRNA group in the blood for 7 days after taking the medicine (sRNA group 7).
  • Step 3 Sequence screening and expression calculation
  • Use bowtie2 to compare the RNA sequencing data, and classify the sequences in sRNA group 3 that overlap with the sequences in sRNA group 1 as sRNA group 8; calculate the abundance ratio of the corresponding sRNA sequences in sRNA group 8 and sRNA group 2 to be greater than or equal to 1.5 sRNA (the calculation method is: sRNA for a specific sequence, the number of sequencing sequences in sRNA group 8/(the number of sequencing sequences in sRNA group 2+0.1), the same below), to obtain sRNA group 9; The intersecting sequences in sRNA group 1 were classified as sRNA group 10; the sRNAs whose abundance ratio of corresponding sRNA sequences in sRNA group 10 and sRNA group 2 were greater than or equal to 1.5 were calculated, and sRNA group 11 was obtained.
  • the sequence in sRNA group 6 that overlaps with the sequence in sRNA group 1 is classified as sRNA group 12; the sRNA whose abundance ratio between sRNA group 12 and the corresponding sRNA sequence in sRNA group 5 is greater than or equal to 1.5 is calculated, and sRNA group 13 is obtained;
  • the sequences in sRNA group 7 that overlap with the sequences in sRNA group 1 were classified as sRNA group 14; the sRNAs whose abundance ratio of the corresponding sRNA sequences in sRNA group 14 and sRNA group 5 were greater than or equal to 1.5 were calculated, and sRNA group 15 was obtained.
  • the combined sRNA group of sRNA group 9, sRNA group 11, sRNA group 13 and sRNA group 15 is regarded as plant-derived sRNA enriched in the subject's blood after taking the medicine, see Table 1.
  • Example 4 Osteogenesis test of double-stranded RNA corresponding to sRNA derived from medicinal plants in MC3T3-E1 cell line
  • MC3T3-E1 cell line On the MC3T3-E1 cell line, add appropriate amount of ascorbic acid, sodium glycerophosphate and dexamethasone to induce osteogenic differentiation, and detect the effect of sRNA on osteogenic differentiation by detecting the content of alkaline phosphatase.
  • MC3T3-E1 cells reached the logarithmic growth phase, they were divided into 12-well plates (1 mL medium/well), incubated overnight at 37°C, and subsequent experiments were performed after the cells adhered to the wall.
  • Double-stranded nonsense sequence artificially synthesized double-stranded unmodified RNA with flush ends, and the specific sequence of one strand is: 5'-UUCUCCGAACGUGUCACGU (SEQ ID NO.85).
  • Plant homologous double-stranded RNA For the 84 sRNAs shown in Table 1, the same RNA and complementary RNA were synthesized to form double-stranded unmodified RNA with flush ends.
  • Double-stranded nonsense sequence and double-stranded unmodified RNA were artificially synthesized by CRO company.
  • Blank group MC3T3-E1 cells were cultured in ⁇ -MEM medium (non-inducing medium 1) without osteogenic inducer.
  • Model group MC3T3-E1 cells were cultured in ⁇ -MEM medium (induction medium 1) containing osteogenic inducers.
  • MC3T3-E1 cells were cultured with ⁇ -MEM medium (induction medium 1) containing an osteogenic inducer, and lovastatin (Lovastain) was added at a final concentration of 0.04 ⁇ mol/L.
  • Nonsense sequence group use ⁇ -MEM medium (induction medium 1) containing osteogenic inducer to culture MC3T3-E1 cells, and use RNAimax transfection reagent to transfect double-stranded nonsense sequence into cells, double-stranded nonsense The final concentration of the sequence was 5OnM.
  • Nucleic acid drug group use ⁇ -MEM medium (induction medium) containing osteogenic inducer to culture MC3T3-E1 cells, and use RNAimax to transfect 84 plant homologous double-stranded RNAs into the cells, plant homologous double-stranded The final concentration of RNA was 50nM.
  • the cells were washed with PBS, and the alkaline phosphatase content was detected using an alkaline phosphatase detection kit.
  • the treated cells were stained for alkaline phosphatase using the alkaline phosphatase staining solution kit.
  • the foregoing 31 double-stranded sRNAs can improve the degree of osteogenic differentiation of induced MC3T3-E1 cells, and can treat or slow down osteoporosis.
  • Example 5 Test of the effect of modified RNA of sRNA derived from medicinal plants in inhibiting osteoclast formation in RAW264.7 cell line
  • RAW264.7 cells were cultured to the logarithmic growth phase, they were divided into 12-well plates (1 mL medium/well), incubated overnight at 37°C, and subsequent experiments were performed after the cells adhered to the wall.
  • Blank group RAW264.7 cells were cultured in DMEM (low glucose) medium (non-inducing medium 2) without osteoclast inducer.
  • Model group RAW264.7 cells were cultured in DMEM (low glucose) medium (induction medium 2) containing osteoclast inducer.
  • Nonsense sequence group use DMEM (low glucose) medium (induction medium 2) containing osteoclast inducer to culture RAW264.7 cells, and use RNAimax transfection reagent to transfect the aforementioned double-stranded nonsense sequence into the cells, double-stranded
  • DMEM low glucose
  • induction medium 2 induction medium 2
  • RNAimax transfection reagent to transfect the aforementioned double-stranded nonsense sequence into the cells, double-stranded
  • the final concentration of nonsense sequence was 50 nM.
  • Nucleic acid drug group RAW264.7 cells were cultured in DMEM (low sugar) medium (induction medium 2) containing osteoclast inducer, and RNAimax was used to transfect the aforementioned 84 plant homologous double-stranded RNA cells, and plant homologous The final concentration of double-stranded RNA was 50nM.
  • the relative activity of the anti-tartrate acid phosphatase of the representative model group (Con) and the nonsense sequence group (NC) is significantly greater than that of the blank group (Blank), indicating that the cell osteoclast model constructed by the present invention is Effectively, compared to the blank cells in the model, they can effectively differentiate towards osteoclasts.
  • nucleic acid drug group of OP83 (21 groups in total) can not only reduce the activity of tartrate-resistant acid phosphatase, but also increase the activity of alkaline phosphatase.
  • Embodiment 6 The effect of modified single-stranded sRNA derived from medicinal plants on osteoporosis model mice
  • mice 16-20g C57BL/6 female mice of 6-8 weeks were used in the experiment. Mice were ovariectomized by dorsal incision, and bilateral ovariectomized mice simulated postmenopausal osteoporosis. As the mouse osteoporosis model of the present invention, they were used to detect the effect of drug intervention on osteoporosis mice.
  • RNA Artificially synthesized sRNA, the specific sequence is: 5'-UUCUCCGAACGUGUCACGU (SEQ ID NO.85).
  • the ribonucleoside at the 3' end is modified with 2'-oxygen-methylation, and the modified RNA can make the RNA more stable and slow down its degradation rate.
  • RNAs shown in OP5, OP20, OP30, OP58, and OP83 in Table 1 were synthesized respectively, and their 3' ribonucleosides were modified with 2'-oxygen-methylation.
  • the sRNA modified with 2'-oxygen-methylation at the 3' terminal ribonucleoside was artificially synthesized by CRO company.
  • Xianling Gubao capsules were taken out and dissolved in distilled water to form a 20 mg/mL drug suspension, which was used as a control group.
  • Sham operation group (sham group): Compared with the model in step 1, only the adipose tissue around the ovary was removed, the ovary was preserved, the fallopian tube was not ligated, sutured, and distilled water was given by intragastric administration once a day for 5 weeks.
  • This sham-operated mouse simulates trauma in order to evaluate the effect of surgical operation and stress on the experimental results, and this model does not cause osteoporosis in mice.
  • Model group (OVX group): For the model mice in step 1, distilled water was given by intragastric administration once a day for 5 weeks.
  • Nonsense sequence group (NC group): For the model mice in step 1, the nonsense sequence liposome complex was administered intragastrically, at a dose of 10 nmol/mouse, once a day for 5 weeks.
  • Nucleic acid drug group For the model mice in step 1, 2'-oxygen-methylation-modified OP5 (SEQ ID NO.87), OP20 (SEQ ID NO.88), OP30 (SEQ ID NO. 89), OP58 (SEQ ID NO.90), OP83 (SEQ ID NO.91) nucleic acid drug liposome complex, the dosage of nucleic acid drug is 10nmol/one, once a day, for 5 weeks.
  • mice were sacrificed by plucking the eyeballs to collect blood and dissecting the cervical spine. Stripped the femur, removed the connective tissue and muscle tissue on the surface of the femur, fixed the femur with 4% paraformaldehyde aqueous solution for 24-48 hours, washed it three times with PBS, stored it in 75% ethanol, and used a microCT instrument. (manufacturer SCANCO Medical AG, model ⁇ CT-100) for microCT scanning.
  • CT images show that the bone density of the model group is significantly lower than that of the sham operation group, and the reduction of bone density in the model group can be improved by the compound Chinese medicine group.
  • the osteoporosis induced by this model can be alleviated by appropriate drugs.
  • This model is suitable for Screening of anti-osteoporotic drugs.
  • the osteoporosis of the nonsense sequence group was comparable to that of the model group, the bone mineral density of the Chinese medicine compound group and the nucleic acid drug group was basically the same as or slightly higher than that of the sham operation group, and the bone mineral density of the Chinese medicine compound group and the nucleic acid drug group was significantly higher than that of the model group and nonsense sequence groups.
  • the nucleic acid drugs tested in the examples can effectively treat or relieve osteoporosis.
  • the software system supporting the instrument is used to perform the following statistical analysis on the Micro CT results:
  • Tb.Th trabecular bone width
  • the data of the test nucleic acid drug group is better than that of the Xianling Gubao Capsule group, which can be used to treat or relieve osteoporosis.
  • the model group is very close to the nonsense sequence group, which shows that in this test, the nonsense sequence has no obvious impact on each index, and the change in the actual results is caused by traditional Chinese medicine and nucleic acid drug liposome complex.

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Abstract

提供了一种来源于药用植物的sRNA，包含SEQ ID NO.1至SEQ ID NO.84任意一种未经修饰sRNA、经修饰的sRNA、5'端和/或3'端截短的sRNA、同源性大于80％sRNA、融合sRNA、互补或杂交的sRNA、完全互补的双链RNA或局部互补的双链RNA，含有发夹结构的sRNA或其组合，能够用于治疗或预防骨代谢疾病。

Description

来源于药用植物的sRNA及其应用 技术领域
本发明属于药用小核酸分子技术领域，特别涉及一种来源于药用植物组合物中分离的sRNA及其应用。
背景技术
骨质疏松症(osteoporosis，OP)是最常见的骨骼疾病，是一种以骨量低，骨组织微结构损坏，导致骨脆性增加，易发生骨折为特征的全身性骨病。2001年美国国立卫生研究院将其定义为以骨强度下降和骨折风险增加为特征的骨骼疾病，提示骨量降低是骨质疏松性骨折的主要危险因素，但还存在其他危险因素。OP可发生于任何年龄，但多见于绝经后女性和老年男性。
随着人口老龄化进程的加速，骨质疏松症的发病率紧随心血管病、糖尿病，已跃居至慢性疾病的第3位，成为世界的常见病和多发病。严重影响人们的身体健康和生活质量，并给社会带来巨大的经济负担。据2015年相关预测，我国2015、2035和2050年主要用于骨质疏松性骨折(腕部、椎体和髋部)的医疗费用将分别高达720亿元、1320亿元和1630亿元。对于骨质疏松症的防治，现阶段主要的治疗药物有钙剂、维生素D和骨吸收抑制药(包括雌激素、选择性雌激素受体调节剂、双膦酸盐等)等三大类。然而这些药物的长期使用会增加妇科癌病、心血管病、血栓的发生率，且存在肾脏损伤、疗程长、费用高、疗效不确切等诸多问题，使得患者在治疗过程中因无法耐受不良反应及承担高昂的治疗费用被迫终止治疗。因此，寻找新型的对抗骨质疏松症的治疗措施仍然是十分必要的。
sRNA(small RNA，小RNA)是一类长度小于200nt的核糖核酸片段，大部分的sRNA为非编码RNA(non-coding RNA,ncRNA)，包括：微sRNA(microRNA，miRNA)，核仁sRNA(small nucleolar RNA，snoRNA)，piwi蛋白相互作用RNA(piwi-interacting RNA，piRNA)，核sRNA(small nuclear RNA，U-RNA)，小干扰RNA(small interfering RNA，siRNA)等。sRNA广 泛存在于动物、植物及微生物中，并通过诱导基因沉默的方式参与调控细胞增殖、分化、代谢和死亡等关键生物学过程。
药用植物是指医学上可用于预防或治疗疾病的植物，我国是药用植物资源最为丰富、使用历史最为悠久、历史记载最为详实的国家之一。目前已知的药用植物有效成分为化学小分子、生物小分子(糖苷、生物碱等)及生物大分子(蛋白质等)，但仍有很大部分药用植物有效成分不明，且已知有效成分难以替代植物整体的作用。最近研究发现，稻米来源的miR168a1、金银花来源的miR29112、红景天来源的HJT-sRNA-m73、草莓来源的FvmiR168、拟南芥来源的miR1594等药用植物sRNA均可以跨物种调控基因表达和机体生理病理学过程。这些研究成果表明，sRNA可能是药用植物一种新的有效成分，为药用植物的机制研究开启了全新的领域。
由淫羊藿、续断、丹参、知母、补骨脂、地黄六味中药组成的中药复方，用于治疗肝肾不足、瘀血阻络所致的骨质疏松症。现有技术中该中药复方多采用水提取或乙醇提取，没有对其植物来源的sRNA进行系统的研究。
发明内容
本发明的目的在于提供一种来源于药用植物的sRNA。具体而言，是从淫羊藿、续断、丹参、知母、补骨脂、地黄组成的复方中药组合物中分离获得的sRNA。
本发明的另一目的在于提供来源于上述药用植物sRNA的用途。所述用途包括预防、治疗或改善受试者骨代谢疾病的用途，及其制备药物组合物的用途。
本发明的另一目的在于提供用于制备上述来源于药用植物的sRNA的基因工程表达载体，以及包含该基因工程表达载体的宿主细胞。
为了实现上述目的，本发明采用了以下技术方案：
一方面，本发明提供了一种sRNA，所述sRNA包含如下任一序列所示的sRNA或其组合：
SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11、SEQ ID NO.12、SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15、SEQ ID  NO.16、SEQ ID NO.17、SEQ ID NO.18、SEQ ID NO.19、SEQ ID NO.20、SEQ ID NO.21、SEQ ID NO.22、SEQ ID NO.23、SEQ ID NO.24、SEQ ID NO.25、SEQ ID NO.26、SEQ ID NO.27、SEQ ID NO.28、SEQ ID NO.29、SEQ ID NO.30、SEQ ID NO.31、SEQ ID NO.32、SEQ ID NO.34、SEQ ID NO.35、SEQ ID NO.36、SEQ ID NO.37、SEQ ID NO.38、SEQ ID NO.39、SEQ ID NO.42、SEQ ID NO.44、SEQ ID NO.45、SEQ ID NO.46、SEQ ID NO.47、SEQ ID NO.48、SEQ ID NO.49、SEQ ID NO.50、SEQ ID NO.51、SEQ ID NO.53、SEQ ID NO.54、SEQ ID NO.55、SEQ ID NO.56、SEQ ID NO.57、SEQ ID NO.58、SEQ ID NO.59、SEQ ID NO.60、SEQ ID NO.61、SEQ ID NO.62、SEQ ID NO.63、SEQ ID NO.64、SEQ ID NO.65、SEQ ID NO.66、SEQ ID NO.67、SEQ ID NO.68、SEQ ID NO.69、SEQ ID NO.70、SEQ ID NO.71、SEQ ID NO.72、SEQ ID NO.73、SEQ ID NO.75、SEQ ID NO.76、SEQ ID NO.77、SEQ ID NO.78、SEQ ID NO.80、SEQ ID NO.81、SEQ ID NO.82、SEQ ID NO.83、SEQ ID NO.84。在另一些实施方案中，sRNA具备SEQ ID NO.1至SEQ ID NO.84中任一序列所示的RNA或其组合。
在一些实施方案中，sRNA包含SEQ ID NO.1至SEQ ID NO.84中任一序列所示的sRNA的5’端和/或3’端截短1-5个或1-3个核苷酸的sRNA或其组合。在另一些实施方案中，sRNA具备SEQ ID NO.1至SEQ ID NO.84中任一序列所示的sRNA的5’端和/或3’端截短1-5个或1-3个核苷酸的RNA或其组合。所述截短sRNA与SEQ ID NO.1至SEQ ID NO.84对应序列具备相同或关联的生物效果。
在一些实施方案中，sRNA包含与SEQ ID NO.1至SEQ ID NO.84中任一序列所示的sRNA的同源性大于等于80％、85％、90％、95％的sRNA或其组合。在一个另实施方案中，sRNA具备与SEQ ID NO.1至SEQ ID NO.84中任一序列所示的sRNA的同源性大于等于80％、85％、90％、95％的sRNA或其组合。所述同源sRNA与SEQ ID NO.1至SEQ ID NO.84对应序列具备相同或关联的生物效果。
在一些实施方案中，sRNA包含SEQ ID NO.1至SEQ ID NO.84中的任一sRNA序列的融合RNA或其组合。所述融合sRNA与SEQ ID NO.1至SEQ ID NO.84对应序列具备相同或关联的生物效果。
在一些实施方案中，sRNA包含SEQ ID NO.1至SEQ ID NO.84RNA中的任一sRNA互补或杂交的sRNA或其组合。所述互补或杂交sRNA与SEQ ID NO.1至SEQ ID NO.84对应序列具备相同或关联的生物效果。
在一些实施方案中，sRNA包含SEQ ID NO.1至SEQ ID NO.84中的任一sRNA和能够与该sRNA杂交的核酸序列杂交所形成的，完全互补的双链sRNA或局部互补的双链RNA或其组合。在另一些实施方案中，sRNA具备SEQ ID NO.1至SEQ ID NO.84中的任一sRNA和能够与该sRNA杂交的核酸序列杂交所形成的，完全互补的双链sRNA或局部互补的双链RNA或其组合。所述完全互补或局部互补的双链sRNA与SEQ ID NO.1至SEQ ID NO.84中的对应序列具备相同或关联的生物效果。
在一些实施方案中，sRNA是SEQ ID NO.1至SEQ ID NO.84中的任一sRNA且含有发夹结构的sRNA或其组合。在另一个实施方案中，sRNA具备SEQ ID NO.1至SEQ ID NO.84中的任一sRNA且含有发夹结构的sRNA或其组合。
在一些实施方案中，sRNA是SEQ ID NO.1至SEQ ID NO.84中的任一sRNA并且含有2'-氟修饰的核苷酸、2’-甲氧基乙基修饰的核苷酸、2'-脱氧-修饰的核苷酸、2’-氨基-修饰的核苷酸、2’-O-烯丙基-修饰的核苷酸、2’-C-烷基-修饰的核苷酸、2’-羟基-修饰的核苷酸、2’-O-烷基-修饰的核苷酸或其组合；优选2'-O-甲基修饰的核苷酸，更优选RNA的3’端为2'-O-甲基修饰的核苷酸。使用上述修饰核苷酸的sRNA与SEQ ID NO.1至SEQ ID NO.84中的对应序列具备相同或关联的生物效果。
在一些实施方案中，sRNA优选包含以下任一序列所示的sRNA或其组合：SEQ NO.2、SEQ NO.5、SEQ NO.9、SEQ NO.11、SEQ NO.13、SEQ NO.14、SEQ NO.18、SEQ NO.20、SEQ NO.30、SEQ NO.34、SEQ NO.38、SEQ NO.39、SEQ NO.45、SEQ NO.46、SEQ NO.51、SEQ NO.58、SEQ NO.64、SEQ NO.67、SEQ NO.69、SEQ NO.70、SEQ NO.71、SEQ NO.73、SEQ NO.75、SEQ NO.76、SEQ NO.77、SEQ NO.78、SEQ NO.80、SEQ NO.81、SEQ NO.82、SEQ NO.83、SEQ NO.84。
在一些实施方案中，sRNA优选包含以下任一序列所示的sRNA或其组合：SEQ NO.1、SEQ NO.2、SEQ NO.3、SEQ NO.4、SEQ NO.5、SEQ NO.7、 SEQ NO.8、SEQ NO.10、SEQ NO.12、SEQ NO.13、SEQ NO.14、SEQ NO.15、SEQ NO.16、SEQ NO.17、SEQ NO.18、SEQ NO.19、SEQ NO.20、SEQ NO.21、SEQ NO.22、SEQ NO.23、SEQ NO.24、SEQ NO.25、SEQ NO.26、SEQ NO.27、SEQ NO.28、SEQ NO.29、SEQ NO.30、SEQ NO.31、SEQ NO.32、SEQ NO.34、SEQ NO.35、SEQ NO.36、SEQ NO.37、SEQ NO.38、SEQ NO.39、SEQ NO.42、SEQ NO.44、SEQ NO.46、SEQ NO.47、SEQ NO.48、SEQ NO.49、SEQ NO.50、SEQ NO.53、SEQ NO.54、SEQ NO.55、SEQ NO.56、SEQ NO.57、SEQ NO.58、SEQ NO.59、SEQ NO.60、SEQ NO.61、SEQ NO.62、SEQ NO.63、SEQ NO.64、SEQ NO.65、SEQ NO.66、SEQ NO.67、SEQ NO.68、SEQ NO.69、SEQ NO.71、SEQ NO.72、SEQ NO.73、SEQ NO.76、SEQ NO.77、SEQ NO.78、SEQ NO.83。
在一些实施方案中，sRNA优选包含以下任一序列所示的sRNA或其组合：SEQ NO.2、SEQ NO.5、SEQ NO.13、SEQ NO.14、SEQ NO.18、SEQ NO.20、SEQ NO.30、SEQ NO.34、SEQ NO.38、SEQ NO.39、SEQ NO.46、SEQ NO.58、SEQ NO.64、SEQ NO.67、SEQ NO.69、SEQ NO.71、SEQ NO.73、SEQ NO.76、SEQ NO.77、SEQ NO.78、SEQ NO.83。
一方面，本发明提供一种式(Ⅰ)所述的sRNA，
(X 1) n-UCGGUCGGGCUGGGG-(X 2) m  (Ⅰ)
其中，
X 1中的任意一个独立的选自G-、UGGG-、CUGGG-、GCUGGG-、GGCUGGG-、GGGCUGGG-、AGGGCUGGG-或其组合；
X 2中的任意一个独立的选自-C、-CG、-CGC、-CGCG或其组合；
m或n＝0、1、2、或3；
其中UCGGUCGGGCUGGGG的相同部分sRNA序列为SEQ ID NO.86。
在一个实施方案中，所述sRNA具有与式(Ⅰ)结构sRNA同源性大于等于80％、85％、90％或95％的sRNA；
在一个实施方案中，所述sRNA是式(Ⅰ)结构sRNA的互补序列。
在一些实施方案中，在式(Ⅰ)sRNA的5’端和/或3’端进一步融合1-5个核苷酸，优选1-3个核苷酸。
在一些实施方案中，式(Ⅰ)sRNA进一步包含，能够与该sRNA杂交的核酸序列杂交所形成的，完全互补的双链sRNA或局部互补的双链sRNA或其 组合。
在一些实施方案中，式(Ⅰ)sRNA含有发夹结构。
在一些实施方案中，式(Ⅰ)sRNA含有2'-氟修饰的核苷酸、2’-甲氧基乙基修饰的核苷酸、2'-脱氧-修饰的核苷酸、2’-氨基-修饰的核苷酸、2’-O-烯丙基-修饰的核苷酸、2’-C-烷基-修饰的核苷酸、2’-羟基-修饰的核苷酸、2’-O-烷基-修饰的核苷酸，优选2'-O-甲基修饰的核苷酸，优选sRNA的3’端为2'-O-甲基修饰的核苷酸。
在一些实施方案中，式(Ⅰ)sRNA包括SEQ NO.2、4-18、30、33-36、42-46所示的sRNA或其组合。在另一些实施方案中，式(Ⅰ)sRNA具有SEQ NO.2、4-18、30、33-36、42-46所示的sRNA或其组合。
一方面，本发明提供一种sRNA，具备SEQ ID NO.42所示的sRNA的5’端截短1-9个核苷酸，和/或3’端截短1-4个核苷酸。具体的截短数量选择1个、2个、3个、4个、5个、6个、7个、8个或9个。
另一方面，本发明提供一种基因工程表达载体，所述基因工程表达载体能够转录出本发明所述的sRNA。sRNA为前述任意一种未经修饰sRNA，经修饰的sRNA，5’端和/或3’端截短的sRNA,同源性大于等于80％、85％、90％、95％的sRNA，融合sRNA，互补或杂交的sRNA，完全互补的双链RNA或局部互补的双链RNA，含有发夹结构的sRNA，式(Ⅰ)所示的sRNA。基因工程表达载体在受试者表达相应的RNA，发挥相应的药理作用并实现基因治疗。
另一方面，本发明提供一种宿主细胞，所述宿主细胞包含基因工程表达载体。
在一个实施方案中，基因工程表达载体在受试者(比如，骨代谢疾病或骨质疏松症患者)表达相应的sRNA，进而发挥相应的药理作用，实现基因治疗。在另一实施方案中，基因工程表达载体在体外转录出相应的sRNA，可以采用宿主细胞或无细胞体系来转录，经提取(纯化)后，施加给受试者。
在一些实施方案中，所述药物组合物是适用于灌胃、口服、静脉内、皮下、经皮、肌肉内、动脉内、腹膜内、肺内、脑脊髓内、关节内、滑膜内、鞘内、心室内、或吸入途径施用的药物剂型。
在一些实施方案中，提供了一种药物递送复合物，所述药物递送复合物 中含有药物递送载体和本发明的sRNA、重组表达载体、宿主细胞。在一些实施方式中，所述药物递送载体为脂质体。
另一方面，本发明提供一种所述sRNA、其基因工程表达载体、其宿主细胞或其药物组合物在制备预防、治疗或改善受试者骨代谢疾病药物中的用途。
另一方面，本发明提供一种所述sRNA、其基因工程表达载体、其宿主细胞或其药物组合物在预防、治疗或改善受试者骨代谢疾病药物中的用途。
在一些实施方案中，所述骨代谢疾病通过提高碱性磷酸酶基因表达量或活性，和/或者降低抗酒石酸酸性磷酸酶表达量或活性进行预防、治疗或改善。
在一些实施方案中，所述骨代谢疾病通过促进成骨细胞分化，和/或者抑制破骨细胞分化进行预防、治疗或改善。
在一些实施方案中，提高碱性磷酸酶基因表达量或活性，或者促进成骨细胞分化的sRNA选自：SEQ NO.2、SEQ NO.5、SEQ NO.9、SEQ NO.11、SEQ NO.13、SEQ NO.14、SEQ NO.18、SEQ NO.20、SEQ NO.30、SEQ NO.34、SEQ NO.38、SEQ NO.39、SEQ NO.45、SEQ NO.46、SEQ NO.51、SEQ NO.58、SEQ NO.64、SEQ NO.67、SEQ NO.69、SEQ NO.70、SEQ NO.71、SEQ NO.73、SEQ NO.75、SEQ NO.76、SEQ NO.77、SEQ NO.78、SEQ NO.80、SEQ NO.81、SEQ NO.82、SEQ NO.83、SEQ NO.84。
在一些实施方案中，降低抗酒石酸酸性磷酸酶表达量或活性，或者抑制破骨细胞分化的sRNA选自：SEQ NO.1、SEQ NO.2、SEQ NO.3、SEQ NO.4、SEQ NO.5、SEQ NO.7、SEQ NO.8、SEQ NO.10、SEQ NO.12、SEQ NO.13、SEQ NO.14、SEQ NO.15、SEQ NO.16、SEQ NO.17、SEQ NO.18、SEQ NO.19、SEQ NO.20、SEQ NO.21、SEQ NO.22、SEQ NO.23、SEQ NO.24、SEQ NO.25、SEQ NO.26、SEQ NO.27、SEQ NO.28、SEQ NO.29、SEQ NO.30、SEQ NO.31、SEQ NO.32、SEQ NO.34、SEQ NO.35、SEQ NO.36、SEQ NO.37、SEQ NO.38、SEQ NO.39、SEQ NO.42、SEQ NO.44、SEQ NO.46、SEQ NO.47、SEQ NO.48、SEQ NO.49、SEQ NO.50、SEQ NO.53、SEQ NO.54、SEQ NO.55、SEQ NO.56、SEQ NO.57、SEQ NO.58、SEQ NO.59、SEQ NO.60、SEQ NO.61、SEQ NO.62、SEQ NO.63、SEQ NO.64、SEQ NO.65、SEQ NO.66、SEQ NO.67、SEQ NO.68、SEQ NO.69、SEQ NO.71、SEQ NO.72、SEQ NO.73、SEQ NO.76、SEQ NO.77、 SEQ NO.78、SEQ NO.83。
在一些实施方案中，所述sRNA优选SEQ NO.2、SEQ NO.5、SEQ NO.13、SEQ NO.14、SEQ NO.18、SEQ NO.20、SEQ NO.30、SEQ NO.34、SEQ NO.38、SEQ NO.39、SEQ NO.46、SEQ NO.58、SEQ NO.64、SEQ NO.67、SEQ NO.69、SEQ NO.71、SEQ NO.73、SEQ NO.76、SEQ NO.77、SEQ NO.78、SEQ NO.83。这些sRNA同时具备(1)提高碱性磷酸酶基因表达量或活性，或者促进成骨细胞分化，和(2)降低抗酒石酸酸性磷酸酶表达量或活性或者抑制破骨细胞分化的作用。
在一些实施方案中，所述骨代谢疾病为骨质疏松症，包括绝经后骨质疏松症和老年性骨质疏松症。
在一些实施方案中，所述“受试者”为动物，如：哺乳动物，包括灵长类(如：人类、非人类灵长类，例如：猴子与黑猩猩)、非灵长类(如：牛、猪、骆驼、大羊驼、马、山羊、兔子、绵羊、仓鼠、天竺鼠、猫、狗、大鼠、小鼠、马与鲸鱼)，或鸟类(例如：鸡、鸭或鹅)。在一个实施方案中，该受试者为人类。
在一些实施方案中，单链sRNA和双链RNA可采用本领域中已知标准方法进行合成，非限制性实例包括使用磷酸三酯、亚磷酸酯或亚磷酰胺化学和固相技术进行化学合成，例如固相亚磷酰胺三酯法。合成的寡核苷酸经过分离纯化，得到目标产物。
定义
为了更容易了解本发明，首先定义某些术语。此外应注意，不论何时出示的参数数值或数值范围，其均指示该出示数值及该范围之间的数值与范围亦为本发明一部分。
本文所采用术语“包括”意指词组“包括(但不限于)”，并可与其交换使用。本文所采用术语“包含”意指词组“包含(但不限于)”，并可与其交换使用。
本文所采用术语“或”意指“和/或”，并可与其交换使用，除非另有说明。
“G”、“C”、“A”、“T”与“U”分别一般代表核苷酸，其分别包含鸟嘌呤、胞嘧啶、腺嘌呤、胸苷与尿嘧啶的碱基。然而，应了解，术语“核糖核苷酸”或“核苷酸”也指经修饰的核苷酸。本领域普通技术人员应了解，鸟嘌呤、胞嘧啶、腺嘌呤与尿嘧啶可在不会实质上改变该包含带有这些置换部分体的核苷酸的寡核苷酸碱基配对性质下改用其他部分体置换。
“sRNA”是一种存在于生物体内的非编码sRNA分子。如果没有特殊的说明，本文中“sRNA”包括未经修饰sRNA，经修饰的sRNA，5’端和/或3’端截短的sRNA，同源性大于等于80％、85％、90％、95％的sRNA，融合sRNA，互补或杂交的sRNA，完全互补的双链RNA或局部互补的双链RNA，含有发夹结构的sRNA，或式(Ⅰ)所示的sRNA。
双链sRNA分子中各链的大多数核苷酸为核糖核苷酸，各链或两链还可包括一个或多个非核糖核苷酸，例如：脱氧核糖核苷酸和/或经修饰的核苷酸。此外，本说明书所采用sRNA可能包括经化学修饰的核糖核苷酸；sRNA可能在多个核苷酸上进行修饰。
本文所采用术语“经修饰”是指分别独立具有糖部分基团、经修饰的核苷酸间键联、和/或经修饰的核碱基的核苷酸。因此，术语“经修饰的sRNA”包括在核苷酸间键联、糖部分基团、或核碱基上的取代、加成、或排除例如：官能基或原子。适用于本发明制剂的修饰法包括本文所揭示或本领域中已知的所有修饰形式。针对本说明书与权利要求目的，“sRNA”包括用于sRNA型分子的任何这些修饰。
术语“融合sRNA”是指在sRNA序列的3’端融合一段碱基之后，其降解速度会变慢，稳定性增加；其与RNA互补或形成复合物所需构象维持序列是不变的，融合后功能不变。
术语“碱性磷酸酶”是成骨细胞的表型标志物之一，它可直接反映成骨细胞的活性或功能状况。碱性磷酸酶升高，代表细胞向成骨分化。碱性磷酸酶水平越高，细胞向成骨分化越明显。可以通过MC3T3-E1细胞检测碱性磷酸酶的水平(相关实验描述于L.Darryl Quarles,et,al.Distinct proliferative and differentiated stages of murine MC3T3-E1 cells in culture:an in vitro model of osteoblast development.Journal of Bone and Mineral Research, 7(6).1992.683-692)。
“抗酒石酸酸性磷酸酶”可以被破骨细胞释放到血液中，是指示机体破骨活性的血液指标。通过检测抗酒石酸酸性磷酸酶的活性，可以观测细胞向破骨分化的程度，从而观测sRNA对抑制细胞向破骨细胞分化的影响。抗酒石酸酸性磷酸酶活性越高，细胞向破骨分化越明显。可以通过RAW264.7细胞系检测抗酒石酸酸性磷酸酶的水平(相关实验描述于Patricia Collin-Osdoby.et,al.(2003).Rankl-mediated osteoclast formation from murine raw 264.7cells.Methods Mol Biol,80,187-202；和William J.Boyle.et,al.(2003).Osteoclast differentiation and activation.Nature,423(6937),337-342.)
“相同或关联的生物效果”是指具有预防、治疗或改善受试者骨代谢疾病的作用；提高碱性磷酸酶基因表达量或活性，和/或者降低抗酒石酸酸性磷酸酶表达量或活性；促进成骨细胞分化，和/或者抑制破骨细胞分化；或预防、治疗或改善骨质疏松症状；或者预防、治疗或改善绝经后骨质疏松症或老年性骨质疏松症。
附图说明
图1-图3显示了OP1-OP84组的碱性磷酸酶相对活性。
图4-图6显示了OP1-OP84组的抗酒石酸酸性磷酸酶相对活性。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将对本发明实施方式作进一步地详细描述。
本发明没有强调来源的材料和试剂均为本领域常用材料和试剂。提及试剂盒来源的试剂盒的应用，参照试剂盒的说明书进行操作。没有提及操作方法的，按照商购仪器的说明书进行操作，或者按照本领域技术人员的常规操作方法进行操作。
实施例一：中药组合物和单味中药汤汁的制备
每剂中药组合物的生药含有由淫羊藿24-30g、续断12-15g、知母12-15g、丹参12-15g、补骨脂12-15g、地黄12-15g熬煮得到的中药组合物(汤汁)， 具体熬煮步骤如下：
取中药生药淫羊藿24-30g、续断12-15g、知母12-15g、丹参12-15g、补骨脂12-15g、地黄12-15g，加500mL水，冷水浸泡25-35min，加热沸腾后改小火30-35min后改大火10-15min，开盖冷却至室温，倾倒出上面的液体得到中药组合物。
单味中药淫羊藿、续断、知母、丹参、补骨脂、地黄分别独立地取100g，加500mL水，冷水浸泡25-35min，加热沸腾后改小火30-35min后改大火10-15min，开盖冷却至室温，倾倒出上面的液体分别得到六种单味中药汤汁。
实施例二：获取单味中药汤汁中的sRNA
第一步：用改良的CTAB法分别独立地从单味中药汤汁中提取总RNA，具体步骤如下：
(1)取实施例1所制备的单味中药汤汁。
(2)向单味中药汤汁中加入β-巯基乙醇和CTAB裂解液(65℃预热)，65℃放置30min；
(3)低温、高速离心8-15min；
(4)取上清，加入等体积的异丙醇，充分混匀后于-40℃静置20min；
(5)低温、高速离心8-15min后弃去上清；
(6)用DPEC处理的水溶解沉淀，加入β-巯基乙醇和CTAB裂解液(65℃预热)，65℃放置30min；
(7)加入等体积的氯仿，4℃静置15min；
(8)低温、高速离心15-20min后取上清；
(9)向上清中加入0.6倍体积的异丙醇(-40℃预冷)，充分混匀后于-40℃静置30min；
(10)低温、高速离心8-15min后弃去上清；
(12)用75％乙醇清洗，烘干得到单味中药汤汁的总RNA沉淀。
第二步：从第一步中得到的单味中药汤汁的总RNA获取sRNA序列，具体方法如下：
采用Illumina公司的Nextseq CN500测序仪，1×75bp模式，对前述总RNA进行测序。方法如下所示：
1)样本质量检测：Agilent 2100对样本进行检测，当合格后采用标准的Illumina文库构建流程建立sRNA文库；
2)sRNA文库构建：将构建完成的文库用Applied Biosystems公司采用StepOnePlus QPCR进行质检，当文库检测为单峰且浓度高于3nM，体积大于25μL即为质量合格；
3)cDNA簇的制备：按照Illumina标准cDNA簇生成，混合测定和定量方法进行样本文库混合，制备cDNA簇；
4)数据质量控制：去除N的比例＞10％的低质量(质量值Q≤3的碱基数目占整个序列的50％以上)序列；
5)sRNA测序数据预处理。得到6种单味中药汤汁中的sRNA，将数据合并，共同组成中药sRNA组(sRNA组1)。
实施例三：从服药后人血总RNA中获取sRNA序列并对测序数据分析
第一步：受试者人血总RNA的制备：
两位志愿者分别连续7天服用中药药方，每剂为实施例1所制备的中药组合物1份，每日2剂。
分别于服药后的0d(指服药前)，3d，7d抽全血，用Trizol-LS法提取总RNA，提取步骤如下：
(1)向250μl全血中加入Trizol LS，充分震荡裂解，加入氯仿，4℃静置15min；
(2)低温、高速离心15-20min后取上清；
(3)向上清中加入0.6倍异丙醇(-40℃预冷)，充分混匀后于-40℃静置30min；
(4)低温、高速离心15-20min后弃去上清；
(5)用75％乙醇清洗，烘干得到各份RNA沉淀。
第二步：利用第一步饮用中药组合物(汤剂)后的人全血总RNA获取sRNA并进行测序：
采用Illumina公司的Nextseq CN500测序仪，1×75bp模式，对前述各份总RNA分别进行测序。方法如下所示：
1)样本质量检测：Agilent 2100对样本进行检测，当合格后采用标准 的Illumina文库构建流程建立sRNA文库；
2)sRNA文库构建：将构建完成的文库用Applied Biosystems公司采用StepOnePlus QPCR进行质检，当文库检测为单峰且浓度高于3nM，体积大于25μL即为质量合格；
3)cDNA簇的制备：按照Illumina标准cDNA簇生成，混合测定和定量方法进行样本文库混合，制备cDNA簇；
4)数据质量控制：去除N的比例＞10％的低质量(质量值Q≤3的碱基数目占整个序列的50％以上)序列；
5)sRNA测序数据预处理。得到第一位志愿者服药0d血液中的sRNA组(sRNA组2)、服药3d血液中的sRNA组(sRNA组3)、服药7d血液中的sRNA组(sRNA组4)；第二位志愿者服药0d血液中的sRNA组(sRNA组5)、服药3d血液中的sRNA组(sRNA组6)、服药7d血液中的sRNA组(sRNA组7)。
第三步：序列筛选及表达量计算
使用bowtie2进行RNA测序数据比对，将sRNA组3中与sRNA组1中序列有交集的序列归为sRNA组8；计算sRNA组8中与sRNA组2中相应sRNA序列的丰度比值大于等于1.5的sRNA(计算方法为：针对特定序列的sRNA，sRNA组8中的测序序列数/(sRNA组2中的测序序列数+0.1)，下同)，得sRNA组9；将sRNA组4中与sRNA组1中序列有交集的序列归为sRNA组10；计算sRNA组10中与sRNA组2中相应sRNA序列的丰度比值大于等于1.5的sRNA，得sRNA组11。
将sRNA组6中与sRNA组1中序列有交集的序列归为sRNA组12；计算sRNA组12中与sRNA组5中相应sRNA序列的丰度比值大于等于1.5的sRNA，得sRNA组13；将sRNA组7中与sRNA组1中序列有交集的序列归为sRNA组14；计算sRNA组14中与sRNA组5中相应sRNA序列的丰度比值大于等于1.5的sRNA，得sRNA组15。
将sRNA组9、sRNA组11、sRNA组13和sRNA组15合并后的sRNA组，视为服药后在受试者血液中富集的植物来源的sRNA，参见表1。
表1服药后在受试者血液中富集的植物来源的sRNA
编号 代号 序列(5’-3’)
SEQ ID NO.1 OP1 UGGAAUGUAGAGAAGUGUGUGU
SEQ ID NO.2 OP2 CUGGGUCGGUCGGGCUGGGG
SEQ ID NO.3 OP3 AAGGAUUGGCUCUAAGGGCUGGG
SEQ ID NO.4 OP4 UGGGUCGGUCGGGCUGGGGC
SEQ ID NO.5 OP5 CUGGGUCGGUCGGGCUGGGGC
SEQ ID NO.6 OP6 CUGGGUCGGUCGGGCUGGGGCG
SEQ ID NO.7 OP7 UGGGUCGGUCGGGCUGGGGCG
SEQ ID NO.8 OP8 GCUGGGUCGGUCGGGCUGGGG
SEQ ID NO.9 OP9 GCUGGGUCGGUCGGGCUGGGGC
SEQ ID NO.10 OP10 AGGGCUGGGUCGGUCGGGCUGGGG
SEQ ID NO.11 OP11 GGCUGGGUCGGUCGGGCUGGGG
SEQ ID NO.12 OP12 AGGGCUGGGUCGGUCGGGCUGGGGC
SEQ ID NO.13 OP13 GGCUGGGUCGGUCGGGCUGGGGC
SEQ ID NO.14 OP14 GGGCUGGGUCGGUCGGGCUGGGGC
SEQ ID NO.15 OP15 GCUGGGUCGGUCGGGCUGGGGCG
SEQ ID NO.16 OP16 AGGGCUGGGUCGGUCGGGCUGGGGCG
SEQ ID NO.17 OP17 GGGCUGGGUCGGUCGGGCUGGGGCG
SEQ ID NO.18 OP18 GGCUGGGUCGGUCGGGCUGGGGCG
SEQ ID NO.19 OP19 GCAUUGUGGUUCAGUGGUAGAAUUCUCGC
SEQ ID NO.20 OP20 GCAUUGUGGUUCAGUGGUAGAAUU
SEQ ID NO.21 OP21 GCAUUGUGGUUCAGUGGUAGAAUUCUCG
SEQ ID NO.22 OP22 GCAUUGUGGUUCAGUGGUAGAAUUCU
SEQ ID NO.23 OP23 AGCGCUGAGAAGACGGUCGAAC
SEQ ID NO.24 OP24 AGCGCUGAGAAGACGGUCGAACU
SEQ ID NO.25 OP25 AGCGCUGAGAAGACGGUCGAACUUG
SEQ ID NO.26 OP26 GCGCUGAGAAGACGGUCGAACUUGAC
SEQ ID NO.27 OP27 AGCGCUGAGAAGACGGUCGAACUUGAC
SEQ ID NO.28 OP28 AGCGCUGAGAAGACGGUCGAACUUGA
SEQ ID NO.29 OP29 AAAAAGUUACCAUUACUGAGU
SEQ ID NO.30 OP30 UCGGUCGGGCUGGGGCGC
SEQ ID NO.31 OP31 GCGCUGAGAAGACGGUCGAACUUGACU
SEQ ID NO.32 OP32 AGCGCUGAGAAGACGGUCGAACUUGACU
SEQ ID NO.33 OP33 AGGGCUGGGUCGGUCGGGCUGGGGCGC
SEQ ID NO.34 OP34 UCGGUCGGGCUGGGGCGCG
SEQ ID NO.35 OP35 CUGGGUCGGUCGGGCUGGGGCGC
SEQ ID NO.36 OP36 GGCUGGGUCGGUCGGGCUGGGGCGC
SEQ ID NO.37 OP37 CGGCGACUCUGGACGCGAGCU
SEQ ID NO.38 OP38 CGGCGGCGACUCUGGACGCGAGCU
SEQ ID NO.39 OP39 GGCGGCGACUCUGGACGCGAGCU
SEQ ID NO.40 OP40 GCGGCGACUCUGGACGCGAGCU
SEQ ID NO.41 OP41 UACAGUACUGUGAUAACUGAUU
SEQ ID NO.42 OP42 AGGGCUGGGUCGGUCGGGCUGGGGCGCG
SEQ ID NO.43 OP43 CUGGGUCGGUCGGGCUGGGGCGCG
SEQ ID NO.44 OP44 GUCGGUCGGGCUGGGGCGCG
SEQ ID NO.45 OP45 GGCUGGGUCGGUCGGGCUGGGGCGCG
SEQ ID NO.46 OP46 GCUGGGUCGGUCGGGCUGGGGCGCG
SEQ ID NO.47 OP47 UACAGUACUGUGAUAACUGACU
SEQ ID NO.48 OP48 ACAGUACUGUGAUAACUGACU
SEQ ID NO.49 OP49 UCCGACUGUUUAAUUAAAACA
SEQ ID NO.50 OP50 UCUAGUCCGACUUUGUGAAAUGA
SEQ ID NO.51 OP51 AUCCGACUGUUUAAUUAAAAC
SEQ ID NO.52 OP52 AUCCGACUGUUUAAUUAAAACA
SEQ ID NO.53 OP53 GGGAAUCCGACUGUUUAAUUAAAACA
SEQ ID NO.54 OP54 CCGAAAGAUGGUGAACUAUGCC
SEQ ID NO.55 OP55 UCGUAACAAGGUUUCCGUA
SEQ ID NO.56 OP56 ACCCGAAAGAUGGUGAACUAUGCC
SEQ ID NO.57 OP57 UGUACACACCGCCCGUCGCU
SEQ ID NO.58 OP58 UAACAAGGUUUCCGUAGG
SEQ ID NO.59 OP59 UUUGUACACACCGCCCGUCGCU
SEQ ID NO.60 OP60 CGGGAUAAGGAUUGGCUC
SEQ ID NO.61 OP61 UCGGGAUAAGGAUUGGCUC
SEQ ID NO.62 OP62 GGGAUAAGGAUUGGCUCUA
SEQ ID NO.63 OP63 UUCGGGAUAAGGAUUGGCUC
SEQ ID NO.64 OP64 AUAGGGGCGAAAGACUAAUCGAACCA
SEQ ID NO.65 OP65 CGAAAGAUGGUGAACUAUGCU
SEQ ID NO.66 OP66 UGUACACACCGCCCGUCGCUACU
SEQ ID NO.67 OP67 AGGGAACGUGAGCUGGGUUUAGACCG
SEQ ID NO.68 OP68 GUAACUUCGGGAUAAGGAUUGGC
SEQ ID NO.69 OP69 CACGACUCUCGGCAACGGAUA
SEQ ID NO.70 OP70 GGGUCUGUAGCUCAGCUGGUAGAGCAC
SEQ ID NO.71 OP71 CGUAACUUCGGGAUAAGGAUUGGC
SEQ ID NO.72 OP72 CGUAACUUCGGGAUAAGGAUUGGCU
SEQ ID NO.73 OP73 CGUAACUUCGGGAUAAGGAUUGGCUC
SEQ ID NO.74 OP74 CCGUAACUUCGGGAUAAGGAUUGGC
SEQ ID NO.75 OP75 CCGUAACUUCGGGAUAAGGAUUGGCU
SEQ ID NO.76 OP76 CCGUAACUUCGGGAUAAGGAUUGGCUC
SEQ ID NO.77 OP77 GGGUCUGUAGCUCAGCUGGUUAGAGCACC
SEQ ID NO.78 OP78 ACAAUGUAGGUAAGGGAAGUCG
SEQ ID NO.79 OP79 AGGGAACGUGAGCUGGGAUUAGACCG
SEQ ID NO.80 OP80 CACACGACUCUCGGCAACG
SEQ ID NO.81 OP81 CGCGGGCUCUGCCCGUUGCUCUGA
SEQ ID NO.82 OP82 CGCGUCGCACGGAUUCGU
SEQ ID NO.83 OP83 CCGCGUCGCACGGAUUCGU
SEQ ID NO.84 OP84 ACAAUGUAGGUAAGGGAAGUCGG
实施例四：药用植物来源的sRNA对应的双链RNA在MC3T3-E1细胞系中的促成骨作用测试
在MC3T3-E1细胞系上，加入适量的抗坏血酸、甘油磷酸钠和地塞米松进行成骨诱导分化，通过检测碱性磷酸酶的含量并检测sRNA对成骨分化的影响。
将MC3T3-E1细胞至对数生长期时，分至12孔板(1mL培养基/孔)，37℃孵育过夜，待细胞贴壁后进行后续实验。
双链无义序列：人工合成末端齐平的双链无修饰RNA，其中一条链的具体序列为：5’-UUCUCCGAACGUGUCACGU(SEQ ID NO.85)。
植物同源双链RNA：针对表1所示84个sRNA，分别合成相同的RNA和互补的RNA，组成末端齐平的双链无修饰RNA。
双链无义序列和双链无修饰RNA由CRO公司进行人工合成。
空白组：使用不含有成骨诱导剂的α-MEM培养基(非诱导培养基1)培养MC3T3-E1细胞。
模型组：使用含有成骨诱导剂的α-MEM培养基(诱导培养基1)培养MC3T3-E1细胞。
阳性药物组：使用含有成骨诱导剂的α-MEM培养基(诱导培养基1)培养MC3T3-E1细胞，并加入终浓度为0.04μmol/L的洛伐他汀(Lovastain)。
无义序列组：使用含有成骨诱导剂的α-MEM培养基(诱导培养基1)培养MC3T3-E1细胞，并使用RNAimax转染试剂转染双链无义序列到细胞中，双链无义序列终浓度为50nM。
核酸药物组：使用含有成骨诱导剂的α-MEM培养基(诱导培养基)培养MC3T3-E1细胞，并分别使用RNAimax转染84个植物同源双链RNA到细胞中，植物同源双链RNA终浓度均为50nM。
将不同成分与细胞共同孵育3d后，分别用PBS清洗细胞，使用碱性磷酸酶检测试剂盒检测碱性磷酸酶含量。
碱性磷酸酶检测结果参见图1-3，其中Con代表模型组，NC代表无义序列组，洛伐他汀代表阳性药物组，OPi组表示其中使用的植物同源双链RNA中一条链与表1中OPi所示序列相同，i为1到84之间的自然数。
将处理的细胞使用碱性磷酸酶染色液试剂盒进行碱性磷酸酶染色。
从图1-3的结果可见，代表模型组和无义序列组的碱性磷酸酶的活性相差不明显，而核酸药物组中OP2、OP5、OP9、OP11、OP13、OP14、OP18、 OP20、OP30、OP34、OP38、OP39、OP45、OP46、OP51、OP58、OP64、OP67、OP69、OP70、OP71、OP73、OP75、OP76、OP77、OP78、OP80、OP81、OP82、OP83、OP84(31个组)对应的双链RNA处理后碱性磷酸酶的活性明显高于模型组。特别地，OP5、OP9、OP13、OP20、OP34、OP51、OP64、OP67、OP70、OP73、OP75、OP77、OP78、OP80、OP81、OP82、OP83，比阳性药物洛伐他汀组碱性磷酸酶的活性更高。
前述31个双链sRNA能够针对诱导的MC3T3-E1细胞提高成骨分化程度，能够治疗或减缓骨质疏松症。
实施例五：药用植物来源的sRNA的修饰RNA在RAW264.7细胞系中的抑制破骨细胞形成的作用测试
在RAW264.7细胞系上，加入适量的RANKL诱导细胞破骨分化，并检测sRNA对破骨分化的影响。
将RAW264.7细胞培养至对数生长期时，分至12孔板(1mL培养基/孔)，37℃孵育过夜，待细胞贴壁后进行后续实验。
空白组：使用不含有破骨诱导剂的DMEM(低糖)培养基(非诱导培养基2)培养RAW264.7细胞。
模型组：使用含有破骨诱导剂的DMEM(低糖)培养基(诱导培养基2)培养RAW264.7细胞。
无义序列组：使用含有破骨诱导剂的DMEM(低糖)培养基(诱导培养基2)培养RAW264.7细胞，并使用RNAimax转染试剂转染前述双链无义序列到细胞中，双链无义序列终浓度为50nM。
核酸药物组：使用含有破骨诱导剂的DMEM(低糖)培养基(诱导培养基2)培养RAW264.7细胞，并分别使用RNAimax转染前述84个植物同源双链RNA细胞中，植物同源双链RNA终浓度均为50nM。
将不同成分细胞组共同孵育4d后，用PBS清洗细胞，使用抗酒石酸酸性磷酸酶检测试剂盒检测抗酒石酸酸性磷酸酶含量。检测结果参见图4-6，其中Blank代表空白组，Con代表模型组，NC代表无义序列组，OPi组表示其中使用的植物同源双链RNA中一条链与表1中OPi所示序列相同，i为1到84之间的自然数。
从图4-6的结果可见，代表模型组(Con)和无义序列组(NC)的抗酒石酸酸性磷酸酶相对活性明显大于空白组(Blank)，说明本发明所构建的细胞破骨模型是有效的，模型中相对于空白的细胞能够有效地向破骨方向分化。
由此可见，OP1、OP2、OP3、OP4、OP5、OP7、OP8、OP10、OP12、OP13、OP14、OP15、OP16、OP17、OP18、OP19、OP20、OP21、OP22、OP23、OP24、OP25、OP26、OP27、OP28、OP29、OP30、OP31、OP32、OP34、OP35、OP36、OP37、OP38、OP39、OP42、OP44、OP46、OP47、OP48、OP49、OP50、OP53、OP54、OP55、OP56、OP57、OP58、OP59、OP60、OP61、OP62、OP63、OP64、OP65、OP66、OP67、OP68、OP69、OP71、OP72、OP73、OP76、OP77、OP78、OP83的核酸药物组(共66组)处理后，抗酒石酸酸性磷酸酶相对活性明显低于模型组和无义序列组，能够有效地抑制细胞朝向破骨方向分化，能够治疗或减缓骨质疏松症。
结合图1-3，前述66组中OP2、OP5、OP13、OP14、OP18、OP20、OP30、OP34、OP38、OP39、OP46、OP58、OP64、OP67、OP69、OP71、OP73、OP76、OP77、OP78、OP83的核酸药物组(共21组)除了能够降低抗酒石酸酸性磷酸酶活性之外，还能够提高碱性磷酸酶的活性。
实施例六：修饰的药用植物来源的单链sRNA在骨质疏松模型小鼠上的作用
1.双侧卵巢切除骨质疏松小鼠模型的建立
实验所用6-8周16-20g C57BL/6雌性小鼠。小鼠采用背部切口进行卵巢切除，双侧卵巢切除小鼠模拟绝经后骨质疏松，作为本发明的小鼠骨质疏松模型，用于检测药物干预对于骨质疏松症小鼠的影响。
2.实验药物准备
无义序列对照药物的制备：
人工合成的sRNA，具体序列为：5’-UUCUCCGAACGUGUCACGU(SEQ ID NO.85)。将其3’端核糖核苷用2’-氧-甲基化修饰，修饰后的RNA能够更使RNA更稳定，减缓其降解速度。
核酸药物的制备：
人工合成的sRNA，分别合成表1中OP5、OP20、OP30、OP58、OP83 中所示的RNA，并将其3’端核糖核苷用2’-氧-甲基化修饰。
3’端核糖核苷用2’-氧-甲基化修饰的sRNA由CRO公司进行人工合成。
将20nmol的2’-氧-甲基化修饰的OP5、OP20、OP30、OP58、OP83或无义序列对照药物)分别加入DEPC水溶解后再加入脂质，形成无义序列NC脂质体复合物、2’-氧-甲基化修饰的核酸药物OP5、OP20、OP30、OP58、OP83脂质体复合物。
将仙灵骨葆胶囊内容物取出，用蒸馏水溶解成20mg/mL的药物悬浊液，作为对照组。
3.实验分组
假手术组(sham组)：相对于步骤1的模型，只切除卵巢周围的脂肪组织，保留卵巢，不结扎输卵管，做缝合处理，灌胃给予蒸馏水，每日一次，持续5周。该假手术小鼠模拟创伤，以便评估手术操作与应激对实验结果的影响，该模型不会导致小鼠骨质疏松症。
模型组(OVX组)：针对步骤1的模型小鼠，灌胃给予蒸馏水，每日一次，持续5周。
无义序列组(NC组)：针对步骤1的模型小鼠，灌胃给予无义序列脂质体复合物，用量为10nmol/只，每日一次，持续5周。
核酸药物组：针对步骤1的模型小鼠，灌胃分别给予2’-氧-甲基化修饰的OP5(SEQ ID NO.87)、OP20(SEQ ID NO.88)、OP30(SEQ ID NO.89)、OP58(SEQ ID NO.90)、OP83(SEQ ID NO.91)核酸药物脂质体复合物，核酸药物用量为10nmol/只，每日一次，持续5周。
中药复方组(仙灵骨葆胶囊组)：针对步骤1的模型小鼠，灌胃给予前述药物悬浊液，仙灵骨葆胶囊内容物用量为236mg/kg小鼠体重，每日一次，持续5周。
前述各组均在手术12天后开始给药，饲养管理环境相同。
4.MicroCT试验
小鼠摘眼球取血后离断颈椎处死，剥离股骨，剔除股骨表面结缔组织与肌肉组织，4％多聚甲醛水溶液固定股骨24-48h后PBS洗3次，保存于75％乙醇，采用microCT仪(制造商为SCANCO Medical AG，型号为μCT-100)进行microCT扫描。
分别获取股骨远端纵向图像，股骨远端横向图像，另一角度股骨远端横向图像。
CT图像显示，模型组比假手术组骨密度明显更低，模型组的骨密度降低的情况能被中药复方组改善，该模型诱导的骨质疏松疾病能够通过合适的药物缓解，该模型适用于抗骨质疏松药物的筛选。
无义序列组的骨质疏松情况与模型组相当，中药复方组与核酸药物组的骨密度与假手术组基本相当或略高，中药复方组与核酸药物组的骨密度显著性高于模型组和无义序列组。实施例测试的核酸药物，能够有效地治疗或缓解骨质疏松症。
采用仪器配套的软件系统，对Micro CT结果进行如下统计分析：
(1)BMD(骨密度)结果显示，测试核酸药物组的骨密度均略高于假手术组，相当于模型组的1.12-1.34倍，相当于无义序列组的1.11-1.33倍。
(2)Tb.Th(骨小梁宽度)结果显示测试核酸药物组的骨小梁宽度高于假手术组，相当于模型组的1.39-1.68倍，相当于无义序列组的1.42-1.70倍。
(3)Ct.Th(皮质骨厚度)结果显示测试核酸药物组的皮质骨厚度接近于假手术组，相当于模型组的1.18-1.42倍，相当于无义序列组的1.18-1.42倍。
测试核酸药物组的数据优于仙灵骨葆胶囊组，能够用于治疗或缓解骨质疏松症的作用。模型组与无义序列组均十分接近，这说明本项测试中，无义序列对各个指标没有明显影响，实际结果的变化是中药以及核酸药物脂质体复合物引起的。
由技术常识可知，本发明可以通过其它的不脱离其精神实质或必要特征的实施方案来实现。因此，上述公开的实施方案，就各方面而言，都只是举例说明，并不是仅有的。所有在本发明范围内或在等同于本发明的范围内的改变均被本发明包含。

Claims (24)

  1. 一种sRNA，包含SEQ ID NO.1至SEQ ID NO.84中任一序列所示的sRNA或其组合。
  2. 权利要求1所述的sRNA，所述sRNA选自
    包含SEQ ID NO.1至SEQ ID NO.84中任一序列所示的sRNA的5’端和/或3’端截短1-5个或1-3个核苷酸的sRNA或其组合；或者包含与SEQ ID NO.1至SEQ ID NO.84中任一序列所示的sRNA的同源性大于等于80％、85％、90％、95％的sRNA或其组合；或者
    包含SEQ ID NO.1至SEQ ID NO.84中的任一sRNA的序列的融合sRNA或其组合；或者
    包含SEQ ID NO.1至SEQ ID NO.84 RNA中的任一sRNA互补或杂交的sRNA或其组合；或者
    包含SEQ ID NO.1至SEQ ID NO.84中的任一sRNA和能够与该sRNA杂交的核酸序列杂交所形成的，完全互补的双链sRNA或局部互补的双链sRNA或其组合；或者
    包含SEQ ID NO.1至SEQ ID NO.84中的任一sRNA且含有发夹结构的sRNA或其组合；或者
    SEQ ID NO.1至SEQ ID NO.84中的任一sRNA含有2'-氟修饰的核苷酸、2’-甲氧基乙基修饰的核苷酸、2'-脱氧-修饰的核苷酸、2’-氨基-修饰的核苷酸、2’-O-烯丙基-修饰的核苷酸、2’-C-烷基-修饰的核苷酸、2’-羟基-修饰的核苷酸、2’-O-烷基-修饰的核苷酸，优选2'-O-甲基修饰的核苷酸，优选sRNA的3’端为2'-O-甲基修饰的核苷酸。
  3. 权利要求1或2中所述的sRNA，其中的sRNA包含以下任一序列所示的sRNA或其组合，
    SEQ NO.2、SEQ NO.5、SEQ NO.9、SEQ NO.11、SEQ NO.13、SEQ NO.14、SEQ NO.18、SEQ NO.20、SEQ NO.30、SEQ NO.34、SEQ NO.38、SEQ NO.39、SEQ NO.45、SEQ NO.46、SEQ NO.51、SEQ NO.58、SEQ NO.64、SEQ NO.67、SEQ NO.69、SEQ NO.70、SEQ NO.71、SEQ NO.73、SEQ NO.75、SEQ NO.76、 SEQ NO.77、SEQ NO.78、SEQ NO.80、SEQ NO.81、SEQ NO.82、SEQ NO.83、SEQ NO.84。
  4. 权利要求1或2中所述的sRNA，其中的sRNA包含以下任一序列所示的sRNA或其组合，
    SEQ NO.1、SEQ NO.2、SEQ NO.3、SEQ NO.4、SEQ NO.5、SEQ NO.7、SEQ NO.8、SEQ NO.10、SEQ NO.12、SEQ NO.13、SEQ NO.14、SEQ NO.15、SEQ NO.16、SEQ NO.17、SEQ NO.18、SEQ NO.19、SEQ NO.20、SEQ NO.21、SEQ NO.22、SEQ NO.23、SEQ NO.24、SEQ NO.25、SEQ NO.26、SEQ NO.27、SEQ NO.28、SEQ NO.29、SEQ NO.30、SEQ NO.31、SEQ NO.32、SEQ NO.34、SEQ NO.35、SEQ NO.36、SEQ NO.37、SEQ NO.38、SEQ NO.39、SEQ NO.42、SEQ NO.44、SEQ NO.46、SEQ NO.47、SEQ NO.48、SEQ NO.49、SEQ NO.50、SEQ NO.53、SEQ NO.54、SEQ NO.55、SEQ NO.56、SEQ NO.57、SEQ NO.58、SEQ NO.59、SEQ NO.60、SEQ NO.61、SEQ NO.62、SEQ NO.63、SEQ NO.64、SEQ NO.65、SEQ NO.66、SEQ NO.67、SEQ NO.68、SEQ NO.69、SEQ NO.71、SEQ NO.72、SEQ NO.73、SEQ NO.76、SEQ NO.77、SEQ NO.78、SEQ NO.83。
  5. 权利要求1或2中所述的sRNA，其中的sRNA包含以下任一序列所示的sRNA或其组合，
    SEQ NO.2、SEQ NO.5、SEQ NO.13、SEQ NO.14、SEQ NO.18、SEQ NO.20、SEQ NO.30、SEQ NO.34、SEQ NO.38、SEQ NO.39、SEQ NO.46、SEQ NO.58、SEQ NO.64、SEQ NO.67、SEQ NO.69、SEQ NO.71、SEQ NO.73、SEQ NO.76、SEQ NO.77、SEQ NO.78、SEQ NO.83。
  6. 一种基因工程表达载体，所述基因工程表达载体能够转录出权利要求1-5中任一项所述的sRNA。
  7. 一种宿主细胞，所述宿主细胞包含权利要求4所述的重组表达载体。
  8. 一种药物组合物，包含权利要求1-5所述的sRNA、权利要求6所述的 重组表达载体或权利要求7所述的宿主细胞，以及药学上可接受的辅料，所述药物组合物是适用于灌胃、口服、静脉内、皮下、经皮、肌肉内、动脉内、腹膜内、肺内、脑脊髓内、关节内、滑膜内、鞘内、心室内、或吸入途径施用的药物剂型。
  9. 权利要求8所述的组合物，所述组合物为脂质体。
  10. 权利要求1-5任意一项所述的sRNA，权利要求6的基因工程表达载体，权利要求7的宿主细胞，或权利要求8-9任意一项所述的药物组合物在制备预防、治疗或改善受试者骨代谢疾病药物中的用途。
  11. 权利要求10所述的用途，所述骨代谢疾病通过提高碱性磷酸酶基因表达量或活性，和/或者降低抗酒石酸酸性磷酸酶表达量或活性进行预防、治疗或改善。
  12. 权利要求10所述的用途，所述骨代谢疾病通过促进成骨细胞分化，和/或者抑制破骨细胞分化进行预防、治疗或改善。
  13. 权利要求11或12所述的用途，其中提高碱性磷酸酶基因表达量或活性，或者促进成骨细胞分化的sRNA选自：
    SEQ NO.2、SEQ NO.5、SEQ NO.9、SEQ NO.11、SEQ NO.13、SEQ NO.14、SEQ NO.18、SEQ NO.20、SEQ NO.30、SEQ NO.34、SEQ NO.38、SEQ NO.39、SEQ NO.45、SEQ NO.46、SEQ NO.51、SEQ NO.58、SEQ NO.64、SEQ NO.67、SEQ NO.69、SEQ NO.70、SEQ NO.71、SEQ NO.73、SEQ NO.75、SEQ NO.76、SEQ NO.77、SEQ NO.78、SEQ NO.80、SEQ NO.81、SEQ NO.82、SEQ NO.83、SEQ NO.84。
  14. 权利要求11或12所述的用途，其中降低抗酒石酸酸性磷酸酶表达量或活性，或者抑制破骨细胞分化的sRNA选自：
    SEQ NO.1、SEQ NO.2、SEQ NO.3、SEQ NO.4、SEQ NO.5、SEQ NO.7、 SEQ NO.8、SEQ NO.10、SEQ NO.12、SEQ NO.13、SEQ NO.14、SEQ NO.15、SEQ NO.16、SEQ NO.17、SEQ NO.18、SEQ NO.19、SEQ NO.20、SEQ NO.21、SEQ NO.22、SEQ NO.23、SEQ NO.24、SEQ NO.25、SEQ NO.26、SEQ NO.27、SEQ NO.28、SEQ NO.29、SEQ NO.30、SEQ NO.31、SEQ NO.32、SEQ NO.34、SEQ NO.35、SEQ NO.36、SEQ NO.37、SEQ NO.38、SEQ NO.39、SEQ NO.42、SEQ NO.44、SEQ NO.46、SEQ NO.47、SEQ NO.48、SEQ NO.49、SEQ NO.50、SEQ NO.53、SEQ NO.54、SEQ NO.55、SEQ NO.56、SEQ NO.57、SEQ NO.58、SEQ NO.59、SEQ NO.60、SEQ NO.61、SEQ NO.62、SEQ NO.63、SEQ NO.64、SEQ NO.65、SEQ NO.66、SEQ NO.67、SEQ NO.68、SEQ NO.69、SEQ NO.71、SEQ NO.72、SEQ NO.73、SEQ NO.76、SEQ NO.77、SEQ NO.78、SEQ NO.83。
  15. 权利要求11或12所述的用途，其中所述sRNA选自：
    SEQ NO.2、SEQ NO.5、SEQ NO.13、SEQ NO.14、SEQ NO.18、SEQ NO.20、SEQ NO.30、SEQ NO.34、SEQ NO.38、SEQ NO.39、SEQ NO.46、SEQ NO.58、SEQ NO.64、SEQ NO.67、SEQ NO.69、SEQ NO.71、SEQ NO.73、SEQ NO.76、SEQ NO.77、SEQ NO.78、SEQ NO.83。
  16. 权利要求10-15任一项所述的用途，所述骨代谢疾病为骨质疏松症，优选绝经后骨质疏松症或老年性骨质疏松症。
  17. 权利要求1-5任意一项所述的sRNA或权利要求8-9任意一项所述的药物组合物在预防、治疗或改善受试者骨代谢疾病药物中的用途。
  18. 权利要求17所述的用途，所述骨代谢疾病通过提高碱性磷酸酶基因表达量或活性，和/或者降低抗酒石酸酸性磷酸酶表达量或活性进行预防、治疗或改善。
  19. 权利要求17所述的用途，所述骨代谢疾病通过促进成骨细胞分化，和/或者抑制破骨细胞分化进行预防、治疗或改善。
  20. 权利要求18或19所述的用途，其中提高碱性磷酸酶基因表达量或活性，或者促进成骨细胞分化的sRNA选自：
    SEQ NO.2、SEQ NO.5、SEQ NO.9、SEQ NO.11、SEQ NO.13、SEQ NO.14、SEQ NO.18、SEQ NO.20、SEQ NO.30、SEQ NO.34、SEQ NO.38、SEQ NO.39、SEQ NO.45、SEQ NO.46、SEQ NO.51、SEQ NO.58、SEQ NO.64、SEQ NO.67、SEQ NO.69、SEQ NO.70、SEQ NO.71、SEQ NO.73、SEQ NO.75、SEQ NO.76、SEQ NO.77、SEQ NO.78、SEQ NO.80、SEQ NO.81、SEQ NO.82、SEQ NO.83、SEQ NO.84。
  21. 权利要求18或19所述的用途，其中降低抗酒石酸酸性磷酸酶表达量或活性，或者抑制破骨细胞分化的sRNA选自：
    SEQ NO.1、SEQ NO.2、SEQ NO.3、SEQ NO.4、SEQ NO.5、SEQ NO.7、SEQ NO.8、SEQ NO.10、SEQ NO.12、SEQ NO.13、SEQ NO.14、SEQ NO.15、SEQ NO.16、SEQ NO.17、SEQ NO.18、SEQ NO.19、SEQ NO.20、SEQ NO.21、SEQ NO.22、SEQ NO.23、SEQ NO.24、SEQ NO.25、SEQ NO.26、SEQ NO.27、SEQ NO.28、SEQ NO.29、SEQ NO.30、SEQ NO.31、SEQ NO.32、SEQ NO.34、SEQ NO.35、SEQ NO.36、SEQ NO.37、SEQ NO.38、SEQ NO.39、SEQ NO.42、SEQ NO.44、SEQ NO.46、SEQ NO.47、SEQ NO.48、SEQ NO.49、SEQ NO.50、SEQ NO.53、SEQ NO.54、SEQ NO.55、SEQ NO.56、SEQ NO.57、SEQ NO.58、SEQ NO.59、SEQ NO.60、SEQ NO.61、SEQ NO.62、SEQ NO.63、SEQ NO.64、SEQ NO.65、SEQ NO.66、SEQ NO.67、SEQ NO.68、SEQ NO.69、SEQ NO.71、SEQ NO.72、SEQ NO.73、SEQ NO.76、SEQ NO.77、SEQ NO.78、SEQ NO.83。
  22. 权利要求18或19所述的用途，其中所述sRNA选自：
    SEQ NO.2、SEQ NO.5、SEQ NO.13、SEQ NO.14、SEQ NO.18、SEQ NO.20、SEQ NO.30、SEQ NO.34、SEQ NO.38、SEQ NO.39、SEQ NO.46、SEQ NO.58、SEQ NO.64、SEQ NO.67、SEQ NO.69、SEQ NO.71、SEQ NO.73、SEQ NO.76、SEQ NO.77、SEQ NO.78、SEQ NO.83。
  23. 权利要求17-22任一项所述的用途，所述骨代谢疾病为骨质疏松症， 优选绝经后骨质疏松症，或老年性骨质疏松症。
  24. 一种式(Ⅰ)所述的sRNA，
    (X 1) n-UCGGUCGGGCUGGGG-(X 2) m  (Ⅰ)
    其中，
    X 1中的任意一个独立的选自G-、UGGG-、CUGGG-、GCUGGG-、GGCUGGG-、GGGCUGGG-、AGGGCUGGG-或其组合；
    X 2中的任意一个独立的选自-C、-CG、-CGC、-CGCG或其组合；
    m或n＝0、1、2、或3。
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