WO2016066086A1 - 胎盘通透性小rna及其应用 - Google Patents

胎盘通透性小rna及其应用 Download PDF

Info

Publication number
WO2016066086A1
WO2016066086A1 PCT/CN2015/092974 CN2015092974W WO2016066086A1 WO 2016066086 A1 WO2016066086 A1 WO 2016066086A1 CN 2015092974 W CN2015092974 W CN 2015092974W WO 2016066086 A1 WO2016066086 A1 WO 2016066086A1
Authority
WO
WIPO (PCT)
Prior art keywords
fetus
small rna
sirna
small
rna
Prior art date
Application number
PCT/CN2015/092974
Other languages
English (en)
French (fr)
Inventor
张辰宇
李菁
张玉婧
曾科
Original Assignee
江苏命码生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 江苏命码生物科技有限公司 filed Critical 江苏命码生物科技有限公司
Priority to US15/523,323 priority Critical patent/US20170247696A1/en
Publication of WO2016066086A1 publication Critical patent/WO2016066086A1/zh

Links

Images

Classifications

    • 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
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • 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
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
    • 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
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
    • C12N15/1133—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses against herpetoviridae, e.g. HSV
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs
    • 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
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • the present invention relates to the use of placental permeability small RNA, and in particular to the direct administration of small RNA or small RNA containing substances to the mother by different methods from the placenta into the fetus to produce the desired effect on the fetus.
  • the placenta is an important organ formed during pregnancy. It is an interface formed between the maternal and fetal circulatory systems. Through this interface, the fetus can obtain nutrients and oxygen from the mother, and at the same time can discharge its own metabolic waste.
  • the placenta also acts as a barrier for selective material exchange.
  • the barrier separating the maternal and fetal circulatory systems consists of four parts: endothelial cells of the fetal blood vessels, trophoblast, cytotrophoblast, and syncytiotrophoclast. The substance enters the circulatory system of the fetus through the mother, and passes through the barrier formed by the above four layers of cells.
  • the mother After pregnancy, especially in the first trimester of pregnancy, the mother will undergo a series of physiological changes, one of which is low immune response, reduced resistance to various microorganisms, increased susceptibility to pathogen infection, and may also cause The virus lurking in the body is reactivated. In this infection, the pregnant woman has no self-conscious symptoms and only shows viremia.
  • intrauterine infections caused by various viruses and pathogens may cause permanent damage, including embryonic death, miscarriage, stillbirth, and various congenital malformations.
  • Toxoplasma, herpes simplex virus, cytomegalovirus, rubella virus, etc. can cause the fetus during pregnancy Pathogenic microorganisms of intrauterine infection.
  • the fetal immune response is not expressed until about 23 weeks of pregnancy, if the mother is infected with a teratogenic pathogen, it is necessary to wait until 23 weeks after gestation and after the cord blood test for virus-related antibodies or DNA, to determine whether the fetus is infected. At this time, if the infection is clear, it is very likely that an induction of labor will be required, which will cause greater damage to the pregnant woman.
  • the current treatment of intrauterine infections are: acyclovir, aggrecan or adenosine treatment of herpes simplex virus infection, but there are certain side effects; penicillin treatment for congenital syphilis If penicillin allergy can be changed to cephalosporin, but lack of specificity; treatment of sulfadiazine, pyrimethamine or spiramycin for toxoplasmosis, but there is a certain teratogenic effect; intramuscular injection of rubella virus Immunoglobulin, but the validity remains to be confirmed. And the risk of teratogenicity is inevitable during pregnancy.
  • small RNA small RNA
  • Another object of the invention is to provide a method of administering a small RNA to a fetus.
  • Another object of the present invention is to provide a kit for detecting small RNA.
  • a small RNA for the preparation of a medicament
  • the small RNA comprises: a small ribonucleic acid (miRNA), a small interfering ribonucleic acid (s iRNA) or a combination thereof
  • the medicament contains the small RNA as an active substance, wherein the drug is directly administered to a mother, and the small RNA enters the fetus through the placenta and acts on the fetus.
  • the administration comprises oral, intravenous, subcutaneous injection.
  • the small RNA comprises a miRNA, an siRNA or a plasmid carrying a miRNA/s iRNA.
  • the small RNA comprises a single-stranded nucleic acid sequence or a complement thereof.
  • the microRNA comprises a miRNA from a plant, an animal, a microorganism, or a synthetic miRNA, or a combination thereof.
  • the miRNA is a plant-derived miRNA.
  • the miRNA comprises a miRNA that inhibits viral infection or replication.
  • the miRNA comprises miRNA2911 from honeysuckle.
  • the small interference RNA (hereinafter referred to as siRNA) is derived from a plant, animal, microbial siRNA, or synthetic siRNA, or a combination thereof.
  • the medicament is for preventing and treating fetal diseases, or for regulating normal growth and development of the fetus.
  • the medicament contains 0.1-99.99% by weight of a pharmaceutically acceptable carrier and 0.01-99.9% by weight of the small RNA.
  • the small RNA comprises a modified or unmodified miRNA, a modified or unmodified siRNA, or a combination thereof.
  • the fetal disease includes congenital diseases (such as heart, liver, metabolic and other diseases), intrauterine infectious diseases during pregnancy (such as rubella virus, herpes virus or other diseases caused by microbial infections). ) and fetal distress caused by intrauterine distress.
  • congenital diseases such as heart, liver, metabolic and other diseases
  • intrauterine infectious diseases during pregnancy such as rubella virus, herpes virus or other diseases caused by microbial infections.
  • fetal distress caused by intrauterine distress.
  • the infectious disease includes infections caused by the following pathogens: virus, bacteria, mycoplasma, and chlamydia.
  • the virus comprises: a rubella virus and a herpes virus.
  • a second aspect of the invention provides a method of administering a small RNA to a fetus, the method comprising the steps of:
  • small RNA small ribonucleic acid
  • the small RNA comprises: microRNA (miRNA), small interfering ribonucleic acid (siRNA) or a combination thereof.
  • the administration described in step (a) comprises oral, inhalation, gavage, blood injection, intramuscular injection or a combination thereof.
  • entering the fetus refers to the entry of small RNA into the blood, plasma, serum, body fluids, cells, tissues, organs, or a combination thereof of the fetus.
  • the tissue comprises liver tissue of a fetus.
  • the method further includes the steps of:
  • the tissue comprises liver tissue of a fetus.
  • a third aspect of the invention provides a kit for administering a small RNA, the kit comprising:
  • the instructions describe the mode of administration of administering the drug to the mother and allowing the small RNA to enter the fetus through the placenta and act on the fetus.
  • the instructions further recite the use of the medicament for the prevention and treatment of fetal disease, or for regulating the normal growth and development of the fetus.
  • Figure 1 shows the results of deep sequencing of plant-derived microRNAs in human cord blood and amniotic fluid.
  • Figure 2 shows the level of miR2911 in the serum of pregnant mice in the honeysuckle extract (A: fold change; B: concentration).
  • Figure 2A is a fold change of miR2911 in the serum of pregnant mice in the honeysuckle extract;
  • Figure 3 shows the level of miR2911 in the honeysuckle extract in fetal liver.
  • Figure 4 shows the levels of synthetic siRNA in pregnant rat serum (A: fold change; B: concentration).
  • Figure 4A is a fold change of synthetic siRNA in pregnant rat serum;
  • Figure 4B shows the concentration of synthetic siRNA in pregnant rat serum.
  • Figure 5 shows the levels of synthetic siRNA in fetal rat liver.
  • Figure 6 shows the mRNA levels of alpha-fetoprotein in the liver of fetal rats.
  • Figure 7 shows the inhibition of HCMV virus by siRNA in vitro.
  • Figure 8 shows that siRNA enters fetuses through the mouse maternal and inhibits the rubella virus in the body.
  • the inventors have conducted extensive and in-depth research to apply exogenous small RNA from different sources to the mother. It is first discovered that small RNA can enter the fetus through the placenta and regulate fetal gene expression, affecting fetal growth and disease status.
  • the present invention has been completed on the basis.
  • Small RNA small RNA
  • small ribonucleic acid refers to a small fragment of RNA of a length of twenty-five nucleotides; according to the widely accepted classification method proposed by Steven Buckingham in May 2003, small RNA (small RNAs) are non-coding RNAs other than transcribed RNA (including ribosomal RNA and transfer RNA), including microRNAs, short interfering RNAs (siRNA), small nucleolar RNA ( snoRNA) and small nuclear RNA (snRNA).
  • small RNA small ribonucleic acid
  • small RNA are non-coding RNAs other than transcribed RNA (including ribosomal RNA and transfer RNA), including microRNAs, short interfering RNAs (siRNA), small nucleolar RNA ( snoRNA) and small nuclear RNA (snRNA).
  • microRNAs are a class of single-stranded small RNA molecules of about 19-23 nucleotides in length, located in the non-coding region of the genome, which are highly conserved in evolution and can inhibit the translation process of target genes.
  • Gene expression is regulated and closely related to many normal physiological activities of animals, such as biological individual development, tissue differentiation, apoptosis and energy metabolism, and is also closely related to the occurrence and development of many diseases.
  • Existing studies have also confirmed that plant miRNAs can also enter the animal through food intake and participate in regulatory activities.
  • RNA interference is a way of post-transcriptional regulation of genes.
  • siRNA can specifically recognize its target gene and recruit a protein complex called RNA induced silencing complex (RISC).
  • RISC contains ribonuclease and the like, and can specifically and efficiently inhibit gene expression by targeting cleavage of homologous mRNA. Since RNA interference technology can specifically eliminate or turn off the expression of specific genes, this technology has been widely used in biomedical experimental research and treatment of various diseases.
  • the invention also provides a kit for administering a small RNA, the kit comprising:
  • the instructions describe the mode of administration of administering the drug to the mother and allowing the small RNA to enter the fetus through the placenta and act on the fetus.
  • the instructions further recite the use of the medicament for the prevention and treatment of fetal disease, or for regulating the normal growth and development of the fetus.
  • the small RNA reaches the fetus through the placenta and has a direct effect, and the degree of fetal damage is minimized.
  • Example 1 Plant-derived microribonucleic acid through the mother placenta
  • Trizol was purchased from Invitrogen; reverse transcriptase and Taq enzyme were purchased from Dalian Baocheng Bioengineering Co., Ltd.; microRNA probe was purchased from Invitrogen.
  • Amniotic fluid is collected and mixed into a group of 10 samples to extract RNA for deep sequencing.
  • the cord blood was collected, centrifuged at 3000 rpm for 15 minutes, the upper serum was collected, and each 10 samples were mixed into a group, and RNA was extracted for deep sequencing.
  • Figure 1 shows the results of deep sequencing of plant-derived microRNAs in human cord blood and amniotic fluid.
  • the plants tested Source microRNAs include rice-derived osa-miR156a, osa-miR-168a, osa-miR167a, osa-miR166a, osa-miR-172a, osa-miR-164a.
  • the plant's microRNA osa-miR156a, osa-miR-168a, osa-miR167a, osa-miR166a, osa-miR-172a, osa-miR can be detected in human cord blood and amniotic fluid.
  • the presence of -164a indicates that plant-derived microRNAs can pass through the maternal placenta.
  • the sequences of the above miRNAs can be obtained from public database miRBase queries.
  • Example 2 Plant-derived microribonucleic acid enters the fetus through the maternal placenta
  • Honeysuckle was purchased from Xiansheng Pharmaceutical; Trizol was purchased from Invitrogen; reverse transcriptase and Taq enzyme were purchased from Dalian Baocheng Bioengineering Co., Ltd.; microRNA probe was purchased from invitrogen
  • C57/BL6J mice were purchased from the Model Animal Center of Nanjing University and belonged to SPF mice, and were raised according to the standard. One male and two female mice were mated together, and the female rats were examined for the next day. If they were seen, the mating was successful. The test was performed after the placenta matured 14 days after pregnancy.
  • the honeysuckle extract was intragastrically administered at a dose of 1 ml of honeysuckle extract per pregnant mouse, and the pregnant mice were treated 3 hours later. Blood was taken from the eyeball, serum was collected by centrifugation at 3000 rpm, and the fetal rat was isolated. After careful washing, the liver tissue of the fetal rat was isolated and stored in liquid nitrogen.
  • the control group was intragastrically administered with an equal volume of physiological saline. 3 hours to treat pregnant mice. Blood was taken from the eyeball, serum was collected by centrifugation at 3000 rpm, and the fetal rat was isolated. After careful washing, the liver tissue of the fetal rat was isolated and stored in liquid nitrogen.
  • Figure 2 shows the level of miR2911 in the serum of pregnant mice in the honeysuckle extract.
  • Figure 2A is a fold change of miR2911 in the serum of pregnant mice in the honeysuckle extract;
  • the expression level of miR2911 in the serum of the pregnant rats in the experimental group was significantly increased as compared with the control group. It is indicated that miR2911 in the honeysuckle extract passed through the pregnant mother's intestine and entered the circulatory system of the pregnant mother.
  • the expression level of miR2911 in the liver of the experimental group was significantly increased as compared with the control group. It is indicated that miR2911 in the maternal serum passes through the placenta and enters the liver tissue of the fetus.
  • Example 3 Artificially synthesized small interfering nucleic acid (siRNA) of alpha-fetoprotein, which is highly expressed in the fetal phase, can pass through the placenta and regulate the expression of alpha-fetoprotein mRNA.
  • siRNA small interfering nucleic acid
  • siRNA was purchased from Invitrogen; alpha-fetoprotein mRNA was purchased from Invitrogen, microRNA probe was purchased from invitrogen; Trizol was purchased from invitrogen; reverse transcriptase and Taq enzyme were purchased from Dalian Baocheng Bioengineering Co., Ltd.
  • This example demonstrates the use of artificially synthesized small interfering nucleic acid (siRNA) traces of alpha-fetoprotein (AFP), which is highly expressed in the fetal phase, demonstrating that small interfering nucleic acids can pass through the placenta and regulate the expression of alpha-fetoprotein mRNA.
  • siRNA small interfering nucleic acid
  • AFP alpha-fetoprotein
  • mice The pre-experimental treatment of experimental mice was as described in Example 4. The experiment was performed after the placenta matured 14 days after pregnancy. The experimental group was intragastrically administered with 2 nmol/mouse and 5 nmol/mouse, while the control group was intragastrically administered with an equal volume of physiological saline. The mice were treated 3 hours later, blood was collected from the eyeballs, and fetal rats were taken, and liver tissues were isolated.
  • Figure 4 shows the levels of synthetic siRNA in pregnant rat serum.
  • Figure 4A is a fold change of synthetic siRNA in pregnant rat serum;
  • Figure 4B shows the concentration of synthetic siRNA in pregnant rat serum.
  • the expression level of the synthesized siRNA in the serum of the pregnant rats was significantly increased when the intragastric dose was 5 nmol/mouse.
  • the synthetic siRNA showed no significant change in serum expression levels in pregnant mice. This indicates that exogenous siRNA can be absorbed by the intestine into the circulatory system of the pregnant mother.
  • Figure 5 shows the levels of synthetic siRNA in fetal rat liver.
  • the synthetic siRNA was significantly increased in the liver tissue of the fetus when the intragastric dose was 5 nmol/mouse; however, when the intragastric dose was 2 nmol/mouse, There was no significant change in the expression level of the synthesized siRNA in fetal liver tissue. This indicates that the pregnant mother's siRNA can cross the placenta to reach the liver tissue of the fetus, but low doses may not be detected in the fetus, and when the intragastric dose is increased, the siRNA entering the fetal rat liver tissue is sufficient. Detected.
  • Figure 6 shows the mRNA levels of alpha-fetoprotein in the liver of fetal rats.
  • the mRNA level of alpha-fetoprotein was significantly decreased when the intragastric dose was 5 nmol/mouse compared with the control group. However, there was no significant change in alpha-fetoprotein mRNA levels when the gavage dose was 2 nmol/mouse. This indicates that siRNA enters mouse liver tissue and exerts its biological function, which reduces the level of alpha-fetoprotein mRNA in fetal liver and affects the expression of genes in the fetus, thus further affecting biological processes such as liver development.
  • Example 4 Inhibition of human cytomegalovirus (HCMV) by siRNA
  • the main content of this example is an in vitro experiment to verify that siRNA can effectively inhibit human cytomegalovirus.
  • HCMV AD169 virus strain was purchased from CGMCC. Human embryonic lung fibroblasts were infected with HCMV AD169 virus at an effective infectious dose for subsequent experiments.
  • the 122 bp downstream of the initiation codon was selected as the target sequence.
  • Target sequence of siRNA GGCTTGAGGGAAGGCACATAACT (SEQ ID NO. 7)
  • Antisense strand 5'UUAUGUGCCUUCCCUCAAGUU-3' (SEQ ID NO. 9)
  • Infected cells were collected 48 hours after virus infestation, infected cells were treated with siRNA, control group was set up, and samples were taken at regular intervals to detect virus titer values.
  • RNA was designed for the key gene E1 antigen gene fragment of Rubella virus, which was administered to mice by injection of a plasmid carrying siRNA, and finally it was proved that siRNA can cross the placenta to reach the fetus. Antiviral effect.
  • RNA was designed for the non-coding region of the key gene SL4 of Rubella virus. It was found that inhibiting the non-coding region of the 3'-end SL4 of rubella virus can effectively inhibit the virus activity, and design a small interference for the non-coding region of SL4.
  • the cDNA sequence of RNA (Gene ID: M74327.1, searched in the GENEBANK database of the National Library of the United States), selected 482 bp downstream of the initiation password:
  • Target sequence of siRNA GACGACCATTATCGTTCAGATAA (SEQ ID NO. 10)
  • siRNA-RV The designed siRNA sequence, here named siRNA-RV:
  • Antisense strand 5'-AUCUGAACGAUAAUGGUCGUU-3' (SEQ ID NO. 12)
  • Trizol was purchased from Invitrogen; reverse transcriptase and Taq enzyme were purchased from Dalian Baocheng Bioengineering Co., Ltd.; siRNA was purchased from Invitrogen; microRNA probe was purchased from Invitrogen; experimental animal C57/BL6J mice Purchased from the Model Animal Center of Nanjing University; the rubella virus strain M15240 was purchased from CGMCC.
  • C57/BL6J mice were purchased from the Model Animal Center of Nanjing University and belonged to SPF mice, and were raised according to the standard. One male and two female mice were mated together, and the female rats were examined for the next day. If they were seen, the mating was successful. The test was performed after the placenta matured 14 days after pregnancy. Forty pregnant rats were divided into four groups, 10 in each group. Four groups of mice were injected with rubella virus at 14 days of pregnancy, and the follow-up test was started 48 hours after the disease.
  • siRNA-RV fragment was ligated to the pAdI-cut pAdTrack-CMV vector by restriction enzyme digestion and enzyme digestion, and then identified by sequencing.
  • the correct clone was named: pCMV-siRNA-RV.
  • an empty vector plasmid pCMV-control control plasmid was prepared as a negative control.
  • mice One group of mice was injected with pCMV-siRNA-RV by tail vein injection at a concentration of 20 nmol/mL, an injection volume of 100 ⁇ L to a final concentration of 2 nmol/mouse, and two groups of mice were injected with pCMV-siRNA-RV in the tail vein.
  • the pregnant rats were killed. Take the blood of pregnant mice and separate the fetal rats. After careful washing, the fetal rat blood is taken and stored in liquid nitrogen.
  • Fig. 8 The virus titer in the serum of the pregnant mice and the fetal rats obtained in the step 5.3 was examined, and the results are shown in Fig. 8.
  • specific siRNA can effectively reduce the viral value in pregnant mice and pass it to the fetus through the placental barrier, and the concentration gradient test results show that the antiviral effect of siRNA is concentration dependent.
  • the present invention provides an effective method for combating rubella virus during pregnancy.
  • Example 4 and Example 5 further demonstrate that microRNA can enter the fetus through the placenta.
  • exogenous microRNAs in the maternal diet can enter the fetus and regulate the expression of genes in the fetus, affecting the growth and development of the fetus. It shows that the maternal diet plays an important role in regulating the epigenetics of the fetus.
  • the present invention provides a new standard for the evaluation of dietary quality of pregnant women.
  • the present invention provides endogenous small RNA and food and microbial small RNA of a pregnant mother through the placenta
  • the maternal small RNA of the present invention passes through the placenta, affecting not only the growth and development of the fetus, but also the possible diseases, and may also affect the development and diseases after birth; the present invention provides a new method for treating intrauterine fetal diseases. .
  • MicroRNAs are a class of small molecule nucleic acids that have received widespread attention in the last decade. Studies have found that tiny ribonucleic acid molecules can be stably present in serum and are species-popular. Previous studies by the present inventors have confirmed that humans and animals can ingest microRNAs in plants through diet. Exogenous plant microribonucleic acid enters the intestine and is absorbed by the intestine. It is encapsulated in microvesicles in the intestinal epithelial cells. These tiny microvesicles carrying exogenous plant micronuclei are secreted into the circulatory system. In turn, exogenous microRNAs are introduced into tissues. In tissues, exogenous microRNAs regulate the expression of target genes and regulate the biological functions of humans and animals. On this basis, the concept of miRNA is a new class of nutrients.
  • the present invention uses biological means to detect that during pregnancy, the endogenous small RNA of the pregnant mother and the exogenous foods and microorganisms are small.
  • RNA can enter the fetus through the placenta and affect the growth and development of the fetus by regulating the genes of the fetus.
  • These small RNAs can affect not only the growth and development of the fetus, but also the possible diseases, and may also affect the development and disease after birth.
  • the present invention provides a novel method of treating intrauterine infections in fetal diseases by providing drugs and methods that specifically target and modulate these small RNAs.
  • the present invention provides a new standard for the evaluation of dietary quality in pregnant women.
  • the diet of pregnant women is also crucial for fetal growth and development.
  • the Dietary Balance Index (DBI) evaluation system is used to measure whether the dietary intake of pregnant women is reasonable. If dietary intake does not maintain a balanced nutritional intake, supplements can be made with nutritional supplements.
  • DBI Dietary Balance Index
  • this evaluation system is flawed because it does not take miRNAs into account. For example, some plant foods are rich in miRNAs that are detrimental to fetal growth and development, but in this system, these plants are only a source of vitamins and cellulose, so pregnant women are not advised to avoid ingestion.
  • the invention also provides for the use of small RNAs that can pass through the placenta as a nutritional supplement for pregnant women.
  • the present invention biologically studies the endogenous small RNA of the pregnant mother and the food and microbial small RNA can enter the fetus through the placenta and have biological functions. Based on this, the present invention solves the following technical problems:

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Virology (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Communicable Diseases (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Oncology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

提供了一种小核糖核酸(小RNA)在制备药物中的用途，该药物含有所述小RNA作为活性物质，其中该药物直接施用于母体，而所述小RNA经胎盘进入胎儿并作用于胎儿。试验表明，通过口服和灌胃等不同途径对母体直接施用所述小RNA时，所述小RNA可以通过胎盘进入胎儿从而对胎儿产生直接作用。

Description

胎盘通透性小RNA及其应用 技术领域
本发明涉及胎盘通透性小RNA的用途，具体地涉及使用不同方法对母体直接施用小RNA或含有小RNA的物质由经胎盘进入胎儿从而对胎儿产生预期作用。
背景技术
胎盘是在妊娠期间形成的一个重要器官，它是在母体和胎儿的循环系统之间形成的一个交界面，通过这个交界面，胎儿可以从母体获得营养物质和氧气，同时可以将自身代谢废物排出；胎盘也起到一种屏障作用，进行选择性的物质交换。具体来说，分开母体和胎儿循环系统的屏障由4部分组成，它们分别是：胎儿血管的内皮细胞，滋养层(trophoblast),细胞滋养层(cytotrophoblast)和合胞体滋养层(syncytiotrophoclast).一般认为一种物质要通过母体进入胎儿的循环系统，要穿过上述4层细胞构成的屏障。研究表明，胎盘在物质输送过程中，氧、二氧化碳和许多小分子依靠扩散与渗透；大分子如蛋白质、抗体、激素等则依靠主动转运和吞饮转运；葡萄糖是胎儿热能的主要来源，以易化扩散方式通过胎盘；胎血内氨基酸浓度高于母血，以主动转运方式运出胎盘；母体中自由脂肪酸以简单扩散方式通过胎盘；一些金属离子如钠、钾(Na、K)，维生素类及代谢抑制剂可按主动转运的方式通过胎盘；一些更大的分子(如Rh阳性抗原等)一般不能转运，只有在缺氧、创伤、特殊分娩的情况下，可能发生窜流而混杂。
有研究表明，不仅母体的营养物质可以通过胎盘进入胎儿，母体摄取的一些有害物质也可以通过胎盘影响胎儿的生长发育。许多胎儿先天性疾病的发生跟胎儿在母体中受到的外界环境有着密切的关系。其中，宫内感染，即母体的病原体通过胎盘组织后进入胎儿，是造成胎儿畸形、流产或死胎的一个重要因素。妊娠后，尤其在妊娠的头三个月，母体会发生一系列生理变化，其中之一是免疫反应功能低下，对各种微生物的抵抗力降低，对病原体感染的易感性增加，也可能使本来潜伏在体内的病毒再活化。这种感染时，孕妇本人多无自觉症状而仅表现为病毒血症。但对胎儿而言，多种病毒及病原体引起的宫内感染则有可能造成永久性的损害，包括胚胎死亡、流产、死产、各种先天畸形等。弓形虫、单纯疱疹病毒、巨细胞病毒、风疹病毒等都是在怀孕期能够引起胎儿 宫内感染的病原微生物。
由于胎儿的免疫反应要到妊娠约23周才有所表达，所以，若母体感染了致畸病原，要等到妊娠23周及以后抽取脐血检测相关病毒抗体或DNA，才可以明确胎儿是否感染。此时若明确感染，很有可能需进行引产，这将对孕妇身心造成较大伤害。
若不进行引产，目前宫内感染的治疗方法有：对单纯疱疹病毒感染的无环鸟苷、丙氧鸟苷或阿糖腺苷治疗，但有一定的毒副作用；对先天性梅毒的青霉素治疗，若青霉素过敏可改用先锋霉素，但缺乏特异性；对弓形虫病的磺胺嘧啶、乙胺嘧啶或螺旋霉素治疗，但存在一定的致畸作用；对风疹病毒的肌肉注射人多克隆免疫球蛋白，但有效性仍待证实。且孕期用药都不可避免致畸的风险。
因此，治疗宫内感染及其造成的发育缺陷的有效、安全的方法亟待出现。
发明内容
本发明的目的是提供一种用于制备药物的小核糖核酸(small RNA,以下称为小RNA)用途。
本发明的另一目的是提供一种对胎儿施用小RNA的方法。
本发明的另一目的是提供一种检测小RNA的试剂盒。
本发明的第一方面，提供了一种用于制备药物的小RNA用途，其中，所述的小RNA包含：微小核糖核酸(miRNA)，小干扰核糖核酸(s iRNA)或其组合，所述药物含有所述小RNA作为活性物质，其中所述药物直接施用于母体，而所述小RNA经胎盘进入胎儿并作用于胎儿。
在另一优选例中，所述的施用包括口服、静脉注射、皮下注射。
在另一优选例中，所述的小RNA包括miRNA、siRNA或载有miRNA/s iRNA的质粒。
在另一优选例中，所述的小RNA包括单链核酸序列或其互补序列。
在另一优选例中，所述的微小核糖核酸(mircoRNA,以下称为miRNA)包括来自植物、动物、微生物的miRNA、或人工合成的miRNA、或其组合。
在另一优选例中，所述的miRNA为来自植物的miRNA。
在另一优选例中，所述的miRNA包括能抑制病毒感染或复制的miRNA。
在另一优选例中，所述的miRNA包括来自金银花的miRNA2911。
在另一优选例中，所述的小干扰核糖核酸(small interference RNA以下称为siRNA)来自植物、动物、微生物的siRNA、或人工合成的siRNA、或其组合。
在另一优选例中，所述药物用于预防和治疗胎儿疾病，或用于调节胎儿的正常生长发育。
在另一优选例中，所述的药物含有0.1-99.99wt％药学上可接受的载体和0.01-99.9wt％的所述小RNA。
在另一优选例中，所述的小RNA包括修饰的或未修饰的miRNA，修饰的或未修饰的siRNA或其组合。
在另一优选例中，所述的胎儿疾病包括先天性疾病(如心脏，肝脏，代谢性和其它疾病)，妊娠期宫内感染性疾病(如风疹病毒，疱疹病毒或其它微生物感染引起的疾病)和宫内窘迫引起的胎儿窘迫症。
在另一优选例中，所述的感染性疾病包括以下病原体引起的感染：病毒、细菌、支原体和衣原体。
在另一优选例中，所述的病毒包括：风疹病毒和疱疹病毒。
本发明的第二方面提供了一种对胎儿施用小RNA的方法，所述的方法包含以下步骤：
(a)对母体施用小核糖核酸(小RNA)，使得所述小RNA由母体经胎盘进入胎儿并作用于胎儿，其中，所述的小RNA包含：微小核糖核酸(miRNA)，小干扰核糖核酸(siRNA)或其组合。
在另一优选例中，步骤(a)中所述的施用包括：口服、吸入、灌胃、血液注射、肌肉注射或其组合。
在另一优选例中，在步骤(a)中所述的“进入胎儿”指小RNA进入胎儿的血液、血浆、血清、体液、细胞、组织、器官、或其组合。
在另一优选例中，所述的组织包括胎儿的肝脏组织。
在另一优选例中，所述方法还包括步骤:
(b)检测来自所述母体和/或胎儿的样本中的所述小RNA存在与否与含量，其中所述样本中包括：血液、血浆、血清、体液、细胞、组织、器官、或其组合。
在另一优选例中，所述的组织包括胎儿的肝脏组织。
本发明的第三方面提供了一种用于施用小RNA的试剂盒，所述试剂盒包含：
(a)一容器以及位于所述容器内的药物，所述药物含有所述小RNA作为活 性物质，其中当所述药物直接施用于母体时所述小RNA经胎盘进入胎儿并作用于胎儿；
(b)使用说明书。
在另一优选例中，所述说明书记载了以下施用方式：将所述药物施用于母体，并使得所述小RNA经胎盘进入胎儿并作用于胎儿。
在另一优选例中，所述的说明书还记载了所述药物用于预防和治疗胎儿疾病，或用于调节胎儿的正常生长发育。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
下列附图用于说明本发明的具体实施方案，而不用于限定由权利要求书所界定的本发明范围。
图1显示植物来源的微小核糖核酸在人的脐带血和羊水中的深度测序结果。
图2显示金银花提取物中的miR2911在孕鼠血清中的水平(A：倍数变化；B：浓度)。图2A是金银花提取物中的miR2911在孕鼠血清中倍数变化；图2B金银花提取物中的miR2911在孕鼠血清中浓度。
图3显示金银花提取物中的miR2911在胎鼠肝脏中的水平。
图4显示合成siRNA在孕鼠血清中的水平(A：倍数变化；B：浓度)。图4A是合成siRNA在孕鼠血清中的倍数变化；图4B合成siRNA在孕鼠血清中的浓度。
图5显示合成siRNA在胎鼠肝脏中的水平。
图6显示胎鼠肝脏中甲胎蛋白的mRNA水平。
图7显示siRNA在体外对HCMV病毒的抑制作用。
图8显示siRNA通过小鼠母体进入胎鼠，并对其体内风疹病毒产生抑制作用。
具体实施方式
本发明人经过广泛而深入的研究，对母体施用不同来源的外源性小RNA，首次发现小RNA可以通过胎盘进入胎儿，并对胎儿基因表达进行调控，影响胎儿生长发育及疾病状态，在此基础上完成了本发明。
小核糖核酸(小RNA)
在本发明中，术语“小核糖核酸(小RNA)”指长度大小为二十几个核苷酸的小片段RNA；根据Steven Buckingham于2003年5月提出的被广泛认同的分类方法，小RNA(small RNAs)是指非编码RNA中除转录RNA(包含核糖体RNA和转移RNA)外的部分，包括微小RNA(microRNAs)、小的干涉RNA(short interference RNAs,siRNA)、小核仁RNA(snoRNA)和小核RNA(snRNA)等。
其中，微小核糖核酸(microRNA,miRNA)是一类长约19-23个核苷酸的单链小核糖核酸分子，位于基因组非编码区，进化上高度保守，可以通过抑制靶基因的翻译过程对基因表达进行调节，并与动物的许多正常生理活动，如生物个体发育、组织分化、细胞凋亡以及能量代谢等密切相关，同时也与许多疾病的发生及发展存在着紧密的联系。现有研究还证实，植物性miRNA还可以通过摄食进入动物体内，并参与调控活动。
小干扰核糖核酸(small interfering RNA，siRNA)是一类由20多个核苷酸组成的双链RNA分子，可以通过特异性降解靶基因的信使核糖核酸(messenger RNA，mRNA)起到沉默基因表达的作用。这一过程被称为RNA干扰(RNA interference，RNAi)。RNA干扰是基因转录后调控的一种方式。siRNA可以对其靶基因进行特异性识别，并能招募被称为沉默复合体(RNA induced silencing complex，RISC)的蛋白质复合体。RISC包含核糖核酸酶等，可以通过靶向切割同源性mRNA的方式，特异、高效地抑制基因的表达。由于使用RNA干扰技术可以特异性剔除或关闭特定基因的表达，所以该技术已被广泛用于生物医学实验研究及各种疾病的治疗领域。
试剂盒
本发明还提供了一种用于施用小RNA的试剂盒，所述试剂盒包含：
(a)一容器以及位于所述容器内的药物，所述药物含有所述小RNA作为活性物质，其中当所述药物直接施用于母体时所述小RNA经胎盘进入胎儿并作用于胎儿；
(b)使用说明书。
在另一优选例中，所述说明书记载了以下施用方式：将所述药物施用于母体，并使得所述小RNA经胎盘进入胎儿并作用于胎儿。
在另一优选例中，所述的说明书还记载了所述药物用于预防和治疗胎儿疾病，或用于调节胎儿的正常生长发育。
本发明的主要优点在于：
(1)首次揭示了小RNA可以通过胎盘进入胎儿。
(2)提供了一种通过对母体施用小RNA进入胎儿，来调控胎儿中基因的表达，对胎儿的生长发育产生影响的方法。
(3)通过对母体施用小RNA，如注射载有小RNA的质粒等方式，所述小RNA经胎盘到达胎儿并产生直接作用，对胎儿伤害度降低到最小程度
(4)提供了一种研发治疗宫内胎儿疾病的新药方法。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)或植物分子生物学-实验手册(Plant Molecular Biology-A Laboratory Mannual,Melody S.Clark编,Springer-verlag Berl in Heidelberg,1997)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例1：植物来源的微小核糖核酸通过母体胎盘
1.1脐带血及羊水的收集和检测。
试剂：Trizol购自invitrogen公司；逆转录酶和Taq酶购自大连宝成生物工程有限公司；微小核糖核酸探针购自invitrogen公司。
收取羊水，每10个样本混合成一组，提取RNA，进行深度测序。收集脐带血，3000rpm离心15分钟，收集上层血清，每10个样本混合成一组，提取RNA，进行深度测序。
具体结果如图1所述。图1显示了植物来源的微小核糖核酸在人的脐带血和羊水中的深度测序结果。以总的动物微小核糖核酸的拷贝数作为校正，检测的植物来 源的微小核糖核酸包括水稻来源的osa-miR156a,osa-miR-168a,osa-miR167a,osa-miR166a,osa-miR-172a,osa-miR-164a。从图1可以看出，在人的脐带血和羊水中可以检测到植物的微小核糖核酸osa-miR156a,osa-miR-168a,osa-miR167a,osa-miR166a,osa-miR-172a,osa-miR-164a的存在，说明植物来源的微小核糖核酸可通过母体胎盘。上述miRNA的序列可从公共数据库miRBase查询获得。
miRNA名称 SEQ ID NO.: 序列(5’-3‘)
osa-MIR156a 1 UGACAGAAGAGAGUGAGCAC
osa-MIR168a 2 UCGCUUGGUGCAGAUCGGGAC
osa-MIR167a 3 UGAAGCUGCCAGCAUGAUCUA
osa-MIR166a 4 GGAAUGUUGUCUGGUUCAAGG
osa-MIR172a 5 AGAAUCUUGAUGAUGCUGCAU
osa-MIR164a 6 UGGAGAAGCAGGGCACGUGCA
实施例2：植物来源的微小核糖核酸通过母体胎盘进入胎儿体内
金银花购自先声药业；Trizol购自invitrogen公司；逆转录酶和Taq酶购自大连宝成生物工程有限公司；微小核糖核酸探针购自invitrogen公司
2.1金银花水提物的制备：按照每1克金银花加25毫升水，文火煎煮30分钟，收集汤汁浓缩至1克金银花提取物/毫升。
2.2动物实验
C57/BL6J小鼠均购自南京大学模式动物中心，属于SPF级小鼠，按照标准饲养。将1只雄鼠与两只雌鼠合笼交配，次日清晨检查雌鼠阴栓，若见栓则证明交配成功，待到怀孕14天胎盘成熟后进行实验。金银花提取物按照每只孕鼠1ml金银花提取物的剂量的灌胃，3小时后处理孕鼠。眼球取血，3000rpm离心收集血清，分离胎鼠，仔细冲洗后分离胎鼠肝脏组织，液氮中保存。对照组灌胃等体积的生理盐水。3小时候处理孕鼠。眼球取血，3000rpm离心收集血清，分离胎鼠，仔细冲洗后分离胎鼠肝脏组织，液氮中保存。
2.3血清的RNA提取和Real-time PCR检测。
取100微升血清，加入300微升DEPC水，混匀后加入200微升水饱和酚，剧烈震荡后加入200微升氯仿，充分震荡，16000g室温下离心15分钟；离心结束后 小心吸取上清(约400微升)，加入2倍体积异丙醇，在-20℃静置60分钟，之后16000g，4℃离心20分钟；离心结束后弃上清，加入75％乙醇，轻柔颠倒，16000g，4℃离心20分钟；离心结束后弃上清，加入20微升DEPC水溶解。取2微升RNA进行反转录，之后取1微升cDNA进行real-time PCR检测目标小核酸表达。具体结果如图2-图3所述。图2显示金银花提取物中的miR2911在孕鼠血清中的水平。图2A是金银花提取物中的miR2911在孕鼠血清中倍数变化；图2B金银花提取物中的miR2911在孕鼠血清中浓度。对照组：灌胃与实验组等体积的生理盐水(n＝8)；实验组：灌胃金银花提取液，剂量：1g金银花的提取物/只小鼠(n＝8)。从图2可以看出，与对照组相比，实验组孕鼠血清中miR2911的表达水平显著增加。说明金银花提取物中的miR2911通过了怀孕母体肠道进入了怀孕母体的循环系统中。图3显示金银花提取物中的miR2911在胎鼠肝脏中的水平。对照组：灌胃与实验组等体积的生理盐水(n＝16)；实验组：灌胃金银花提取液，剂量：1g金银花的提取物/只小鼠(n＝16)。从图3可以看出，与对照组相比，实验组胎鼠肝脏中miR2911的表达水平显著增加。说明怀孕母体血清中的miR2911通过了胎盘进入了胎儿的肝脏组织中。
实施例3:人工合成的胎儿期高表达的甲胎蛋白的小干扰核酸(siRNA)可以通过胎盘并且调节甲胎蛋白mRNA的表达
siRNA购自invitrogen公司；甲胎蛋白mRNA购自invitrogen公司，微小核糖核酸探针购自invitrogen公司；Trizol购自invitrogen公司；逆转录酶和Taq酶购自大连宝成生物工程有限公司
本实施例利用人工合成的胎儿期高表达的甲胎蛋白(alpha-fetoprotein,AFP)的小干扰核酸(siRNA)示踪，证明小干扰核酸可以通过胎盘并且调节甲胎蛋白mRNA的表达。
实验小鼠的实验前处理如实施例4所述。待到怀孕14天胎盘成熟后进行实验。实验组按照2nmol/只小鼠和5nmol/只小鼠灌胃，同时对照组灌胃等体积的生理盐水。3小时后处理小鼠，眼球取血收集血液，并取胎鼠，分离肝脏组织。
3.1组织的RNA提取及real-time PCR检测。
取适量组织，加入1ml Trizol，研磨棒研磨至组织分散，加入200微升氯仿，剧烈震荡，12000g，4℃离心15分钟；离心结束后小心吸取上清，加入等体积异丙醇，室温静置10分钟，之后12000g，4℃离心10分钟；离心结束后 弃上清，加入1毫升75％乙醇，12000g，4℃离心5分钟；离心结束后弃上清，加入适量DEPC水溶解。取1微克RNA进行反转录，之后取1微升cDNA进行real-time PCR检测目标小核酸表达和mRNA表达，具体结果如图4-图6所述。
图4显示合成siRNA在孕鼠血清中的水平。图4A是合成siRNA在孕鼠血清中的倍数变化；图4B合成siRNA在孕鼠血清中的浓度。对照组：灌胃与实验组等体积的生理盐水(n＝4)；实验组1：灌胃siRNA溶液，剂量：2nmol(n＝4)；实验组2：灌胃siRNA溶液，剂量：5nmol(n＝4)。
从图4可以看出，与对照组相比，灌胃剂量在5nmol/只小鼠时，合成的siRNA在孕鼠血清表达水平显著增加。但是，灌胃剂量在2nmol/只小鼠时，合成的siRNA在孕鼠血清表达水平没有显著变化。这说明外源性的siRNA可以被肠道吸收，进入怀孕母体的循环系统。
图5显示合成siRNA在胎鼠肝脏中的水平。对照组：灌胃与实验组等体积的生理盐水(n＝8)；实验组1：灌胃siRNA溶液，剂量：2nmol(n＝8)；实验组2：灌胃siRNA溶液，剂量：5nmol(n＝8)。
从图5可以看出，与对照组相比，灌胃剂量在5nmol/只小鼠时，合成的siRNA在胎鼠肝组织中显著升高；但是，灌胃剂量在2nmol/只小鼠时，合成的siRNA在胎鼠肝组织表达水平没有显著变化。这说明怀孕母体的siRNA可以穿过胎盘到达胎鼠的肝组织，但是低剂量灌胃后可能不足以在胎鼠中检测到，当灌胃剂量增到后，进入胎鼠肝组织的siRNA才足以被检测到。
图6显示胎鼠肝脏中甲胎蛋白的mRNA水平。对照组：灌胃与实验组等体积的生理盐水(n＝8)；实验组1：灌胃siRNA溶液，剂量：2nmol(n＝8)；实验组2：灌胃siRNA溶液，剂量：5nmol(n＝8)。
从图6可以看出，与对照组相比，灌胃剂量在5nmol/只小鼠时，甲胎蛋白的mRNA水平显著下降。但是，灌胃剂量在2nmol/只小鼠时，甲胎蛋白的mRNA水平没有显著变化。这说明siRNA进入小鼠肝组织后发挥其生物学功能，降低了胎鼠肝脏中甲胎蛋白mRNA水平，影响了胎儿中基因的表达，因而会进一步影响肝脏发育等生物学过程。
实施例4：利用siRNA抑制人巨细胞病毒(HCMV)
本实施例主要内容为体外实验，验证利用siRNA可有效抑制人巨细胞病毒。
具体实验步骤如下：
1)人胚胎肺成纤维细胞(进口IMR-90型，购自南京凯基生物科技发展有限公司)，37℃恒温培养于DMEM培养基，培养基中混入10％FBS和1％青霉素混匀。
2)HCMV AD169病毒株购自CGMCC。将人胚胎肺成纤维细胞在有效感染量下感染HCMV AD169病毒，以备后续实验使用。
3)siRNA的设计
针对HCMV主要的蛋白基因UL123(在美国国立图书馆的GENEBANK数据库检索的Gene ID:3077513)的cDNA序列，选择起始密码下游122bp处(SEQ ID NO.7:GGCTTGAGGGAAGGCACATAACT)作为靶序列。
siRNA的靶序列:GGCTTGAGGGAAGGCACATAACT(SEQ ID NO.7)
设计的siRNA序列：
正义链:5'-CUUGAGGGAAGGCACAUAAUU-3'(SEQ ID NO.8)
反义链:5’UUAUGUGCCUUCCCUCAAGUU-3’(SEQ ID NO.9)
4)病毒侵染48小时后收集感染细胞，用siRNA处理感染细胞，设置对照组，固定间隔时间取样，检测病毒滴度值。
实验结果如图7所示。由图7可以看出，特异性siRNA处理下的感染细胞中的病毒值随处理时间的逐渐降低，由此可知，特异性siRNA可有效降低HCMV病毒的侵染。本实施例证明本发明所提供特异性siRNA可特异性针对人巨细胞病毒发挥作用，在人类细胞系中起到抗病毒的作用。
实施例5：利用siRNA抑制风疹病毒
本实施例针对风疹病毒(Rubella virus)的关键基因E1抗原基因片段设计小干扰RNA，通过注射载有siRNA的质粒的方式向小鼠给药，最后证明siRNA可以穿过胎盘达到胎鼠，起到抗病毒作用。
5.1风疹病毒特异性siRNA的设计
针对风疹病毒(Rubella virus)的关键基因SL4的非编码区设计小干扰RNA，研究发现，抑制风疹病毒的3'端SL4的非编码区可有效抑制病毒活性，针对SL4的非编码区设计小干扰RNA(在美国国立图书馆的GENEBANK数据库检索的Gene ID:M74327.1)的cDNA序列，选择起始密码下游482bp处：
(SEQ ID NO.10:GACGACCATTATCGTTCAGATAA)作为靶序列。
siRNA的靶序列:GACGACCATTATCGTTCAGATAA(SEQ ID NO.10)
设计的siRNA序列，此处命名为siRNA-RV：
正义链:5'-CGACCAUUAUCGUUCAGAUUU-3'(SEQ ID NO.11)
反义链:5’-AUCUGAACGAUAAUGGUCGUU-3’(SEQ ID NO.12)
5.2孕鼠接种风疹病毒
试剂与实验动物：Trizol购自invitrogen公司；逆转录酶和Taq酶购自大连宝成生物工程有限公司；siRNA购自invitrogen公司；微小核糖核酸探针购自invitrogen公司；实验动物C57/BL6J小鼠购自南京大学模式动物中心；风疹病毒株M15240购自CGMCC。
C57/BL6J小鼠均购自南京大学模式动物中心，属于SPF级小鼠，按照标准饲养。将1只雄鼠与两只雌鼠合笼交配，次日清晨检查雌鼠阴栓，若见栓则证明交配成功，待到怀孕14天胎盘成熟后进行实验。取孕鼠40只，分四组，每组10只，四组小鼠均在怀孕14天时静脉注射风疹病毒，病程48小时起开始后续试验。
5.3孕鼠接受siRNA治疗
主要实验材料：
戊巴比妥钠、注射器、人工构建的过表达如5.1中所述siRNA的质粒(pCMV-siRNA-RV)(南京大学生命科学学院提供)、淋巴细胞分离液(天津市灏洋生物制品科技有限公司)
实验方法及步骤：
5.3.1过表达siRNA-RV的质粒(pCMV-siRNA-RV)的构建
经酶切、酶连将siRNA-RV片段连接到经PacI酶切的pAdTrack-CMV载体上，随后经测序鉴定，将正确克隆命名为：pCMV-siRNA-RV。同时制备空载体质粒pCMV-control(control plasmid)作为阴性对照。
5.3.2动物实验：
取1组小鼠，每只小鼠通过尾静脉注射方式注射pCMV-siRNA-RV，浓度20nmol/mL,注射体积100μL至终浓度2nmol/只，2组小鼠尾静脉注射pCMV-siRNA-RV，浓度50nmol/mL,注射体积100μL至终浓度5nmol/只，3组小鼠尾静脉注射pCMV-control，浓度20nmol/mL,注射体积100μL至终浓度2nmol/只，4组小鼠尾静脉注射生理盐水，24小时候后处死孕鼠。取孕鼠血液，分离胎鼠， 仔细冲洗后取胎鼠血液，液氮中保存。
5.4孕鼠及小鼠病毒滴度值检测
检测步骤5.3中所获得孕鼠与胎鼠血清中的病毒滴度平均值，结果见图8。由图8可以看出，特异性siRNA可以有效降低孕鼠体内的病毒值，并通过胎盘屏障传递给胎鼠，并且浓度梯度实验结果来看，siRNA抗病毒效果呈浓度依赖。由此可见，本发明提供了一种有效的对抗孕期风疹病毒的方法。
实施例4和实施例5进一步验证说明微小核糖核酸可以通过胎盘进入胎儿。在妊娠期间，母体饮食中的外源性微小核糖核酸可以进入胎儿并调控胎儿中基因的表达，对胎儿的生长发育产生影响。说明母体的饮食对胎儿的表观遗传有着重要的调控作用。本发明为孕妇膳食质量评价提供了一种新标准。本发明提供了可通过胎盘的怀孕母体的内源性小RNA及食物和微生物小RNA作为
孕妇营养补充剂的应用。本发明的孕期母体小RNA通过胎盘，不仅影响胎儿宫内的生长发育及导致可能的疾病，也可能影响出生后的发育及疾病；本发明提供了一种可能的治疗宫内胎儿疾病的新方法。
讨论
微小核糖核酸(microRNA，miRNA)是近十年来广泛受到关注一类小分子核酸。研究发现，微小核糖核酸分子可以稳定的存在于血清中，并且具有物种普遍性。本发明人的前期研究证实，人类和动物可以通过饮食摄取植物中的微小核糖核酸。外源性植物微小核糖核酸进入肠道后由肠道吸收，在肠上皮细胞中被包裹进微小囊泡(microvesicle)中，这些携带外源性植物微小核糖核酸微小囊泡被分泌进入循环系统，进而携带外源性微小核糖核酸进入组织，在组织中，外源性微小核糖核酸调控其靶基因的表达，调节人类和动物的生物学功能。在此基础上，提出了miRNA是一类新的营养物质的概念。
鉴于微小核糖核酸在可以在生物体中跨界存在并且具有显著的生物学功能，本发明用生物学手段检测获知，在妊娠期间，怀孕母体的内源性小RNA及外源性食物和微生物小RNA可通过胎盘进入胎儿，并通过调控胎儿的基因进而影响胎儿的生长发育。这些小RNA不仅能影响胎儿宫内的生长发育及导致可能的疾病，也可能影响出生后的发育及疾病。
通过提供特异性靶向并调控这些小RNA的药物及方法，本发明提供了一种治疗宫内感染胎儿疾病的新方法。
此外，本发明还为孕妇膳食质量评价提供了一种新标准。
孕妇膳食对胎儿生长发育也至关重要。目前国内采用膳食平衡指数(DBI)评价系统来衡量孕妇的膳食摄入是否合理。若膳食的摄入不能维持营养摄入的平衡，可利用营养补充剂进行调节。但该评价系统存在缺陷，因其并未将miRNAs考虑在内。例如，一些植物性食物中富含对胎儿生长发育不利的miRNAs,但在该系统中，这些植物仅是维生素和纤维素的来源，故并不会建议孕妇避免摄入。
本发明还提供了可通过胎盘的小RNA作为孕妇营养补充剂的应用。
本发明通过生物学手段，研究怀孕母体的内源性小RNA及食物和微生物小RNA通过胎盘可以进入胎儿，并且具有生物学功能。在此基础上，本发明解决了以下技术问题：
1.胎儿宫内感染疾病治疗的缺陷；
2.孕妇膳食质量评价新标准缺失。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种小核糖核酸(small RNA,以下称为小RNA)的用途，其中，所述的小RNA包含：微小核糖核酸(miRNA)，小干扰核糖核酸(siRNA)或其组合，其特征在于，用于制备药物，所述药物含有所述小RNA作为活性物质，其中所述药物直接施用于母体，而所述小RNA经胎盘进入胎儿并作用于胎儿。
  2. 如权利要求1所述的用途，其特征在于，所述的微小核糖核酸(mircoRNA,以下称为miRNA)包括来自植物、动物、微生物的miRNA、或人工合成的miRNA、或其组合。
  3. 如权利要求1所述的用途，其特征在于，所述的小干扰核糖核酸(small interference RNA以下称为siRNA)来自植物、动物、微生物的siRNA、或人工合成的siRNA、或其组合。
  4. 如权利要求1所述的用途，其特征在于，所述药物用于预防和治疗胎儿疾病，或用于调节胎儿的正常生长发育。
  5. 如权利要求4所述的用途，其特征在于，所述的药物含有0.1-99.99wt％药学上可接受的载体和0.01-99.9wt％的所述小RNA。
  6. 如权利要求4所述的用途，其特征在于，所述的胎儿疾病包括先天性疾病(如心脏，肝脏，代谢性和其它疾病)，妊娠期宫内感染性疾病(如风疹病毒，疱疹病毒或其它微生物感染引起的疾病)和宫内窘迫引起的胎儿窘迫症。
  7. 一种对胎儿施用小RNA的方法，其特征在于，所述的方法包含以下步骤：
    (a)对母体施用小核糖核酸(小RNA)，使得所述小RNA由母体经胎盘进入胎儿并作用于胎儿，其中，所述的小RNA包含：微小核糖核酸(miRNA)，小干扰核糖核酸(siRNA)或其组合。
  8. 如权利要求7所述的方法，其特征在于，在步骤(a)中所述的“进入胎儿”指小RNA进入胎儿的血液、血浆、血清、体液、细胞、组织、器官、或其组合。
  9. 如权利要求7所述的方法，其特征在于，所述方法还包括步骤:
    (b)检测来自所述母体和/或胎儿的样本中的所述小RNA存在与否与含量，其中所述样本中包括：血液、血浆、血清、体液、细胞、组织、器官、或其组合。
  10. 一种用于施用小RNA的试剂盒，其特征在于，所述试剂盒包含：
    (a)一容器以及位于所述容器内的药物，所述药物含有所述小RNA作为活 性物质，其中当所述药物直接施用于母体时所述小RNA经胎盘进入胎儿并作用于胎儿；
    (b)使用说明书。
PCT/CN2015/092974 2014-10-28 2015-10-27 胎盘通透性小rna及其应用 WO2016066086A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US15/523,323 US20170247696A1 (en) 2014-10-28 2015-10-27 Microrna with placenta-permeability and uses thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201410591247.5 2014-10-28
CN201410591247.5A CN105561337B (zh) 2014-10-28 2014-10-28 胎盘通透性小rna及其应用

Publications (1)

Publication Number Publication Date
WO2016066086A1 true WO2016066086A1 (zh) 2016-05-06

Family

ID=55856612

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2015/092974 WO2016066086A1 (zh) 2014-10-28 2015-10-27 胎盘通透性小rna及其应用

Country Status (3)

Country Link
US (1) US20170247696A1 (zh)
CN (1) CN105561337B (zh)
WO (1) WO2016066086A1 (zh)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103589721A (zh) * 2012-08-15 2014-02-19 北京命码生科科技有限公司 植物微小核糖核酸的提取、制备及其应用
CN104080911A (zh) * 2011-11-30 2014-10-01 不来梅大学 miRNA在胎盘组织中的表达

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK0782862T3 (da) * 1994-10-14 2005-01-03 Daiichi Seiyaku Co Sammensætning indeholdende genetisk materiale
CN101812452B (zh) * 2010-04-21 2011-11-16 暨南大学 Rala基因的小干扰rna在制备抗白血病药物中的应用
CN102886050B (zh) * 2012-07-16 2014-04-02 中国科学院动物研究所 miRNA-489的用途及药物组合物
CN102888403A (zh) * 2012-07-19 2013-01-23 中国科学院动物研究所 miRNA-539及其反义核苷酸的用途和其药物组合物
CN102973953B (zh) * 2012-12-04 2014-08-06 中国科学院动物研究所 miRNA-874及其反义核苷酸的用途
CN103757024B (zh) * 2014-01-29 2015-10-28 中国人民解放军第二军医大学 一种干扰tlr4受体的小rna及其应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104080911A (zh) * 2011-11-30 2014-10-01 不来梅大学 miRNA在胎盘组织中的表达
CN103589721A (zh) * 2012-08-15 2014-02-19 北京命码生科科技有限公司 植物微小核糖核酸的提取、制备及其应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
JIN, XIN: "The Application of the Quantified Trans-placental RNAi Technology in Mouse Model s Establishment", CHINESE MASTER'S THESES FULL-TEXT DATABASE (MEDICINE AND HEALTH SCIENCES, 15 April 2014 (2014-04-15) *
LI, QINGZHI: "Identification of Plant miRNAs in Maternal-fetal Transport of Pigs", CHINESE MASTER'S THESES FULL-TEXT DATABASE (AGRICULTURE SCIENCE AND TECHNOLOGY, 15 March 2014 (2014-03-15) *
THERESA E. GRATSCH ET AL.: "RNA inhibition of BMP-4 gene expression in postimplantation mouse embryos", GENESIS, vol. 37, 31 December 2003 (2003-12-31), XP002330365, ISSN: 1526-968X *

Also Published As

Publication number Publication date
CN105561337B (zh) 2020-06-26
US20170247696A1 (en) 2017-08-31
CN105561337A (zh) 2016-05-11

Similar Documents

Publication Publication Date Title
CA2527109A1 (en) Oligo double-stranded rna inhibiting the expression of bcl-2 and medicinal composition containing the same
CN115161321B (zh) ssc-miR-30c-3p在制备抗PDCoV增殖药物中的应用
Neerukonda et al. Comparison of the transcriptomes and proteomes of serum exosomes from Marek’s disease virus-vaccinated and protected and lymphoma-bearing chickens
EP3222721A1 (en) Novel precursor mirna and application thereof in tumor treatment
CN102154293B (zh) 抑制猪瘟病毒复制感染的小干扰rna及制备方法和应用
CN101880677B (zh) 针对2009新甲型流感病毒多聚酶基因和核蛋白基因的siRNA序列及其应用
CN110772483B (zh) 硫化氢修饰的间充质干细胞外囊泡作为miRNA递送载体在缺氧缺血性脑损伤中的应用
CN102242080A (zh) miR-24用于治疗或诊断心衰或患心衰倾向或者改善心肌细胞功能的方法
CN100549179C (zh) 胃癌靶向AFP基因的siRNAs表达载体及其用途
CN115068611B (zh) ssc-miR-374b-3p在制备抗PDCoV增殖药物中的应用
WO2016066086A1 (zh) 胎盘通透性小rna及其应用
WO2015085903A1 (zh) 体内感染微生物、寄生微生物、共生微生物的非编码性rna及其鉴定和应用
Kumar et al. Control of HBV replication by antiviral microRNAs transferred by lentiviral vectors for potential cell and gene therapy approaches
Zhou et al. MicroRNA expression and analysis of immune-related putative target genes in ISKNV-infected spleen of mandarin fish (Siniperca chuatsi)
Zhang et al. Advances in the protection of intestinal mucosal barrier function by milk-derived miRNAs
CN107446952B (zh) 一种用于肠外营养相关性肝病药物的制备方法
CN109266684B (zh) 一种构建病原感染敏感性动物模型的方法
KR20220164524A (ko) 태반추출물 유래 마이크로 rna를 포함하는 항바이러스 조성물
CN107308178B (zh) 一种用于肠外营养相关性肝病药物
CN114177298B (zh) Adar1作为治疗肺动脉高压疾病药物的应用
CN104922697B (zh) 降低磷酸吡哆醇氧化酶治疗卵巢癌
WO2023245771A1 (zh) Helz2基因在肿瘤防治药物中的应用
US20210171949A1 (en) Targeting Mirnas for Exosomal Delivery or Cellular Retention
CN103656673B (zh) 人ywhaq基因的用途及其相关药物
CN108283646A (zh) hsa-miRNA-155-5p在制备抑制人肠道病毒71型药物中的应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15855022

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 15523323

Country of ref document: US

122 Ep: pct application non-entry in european phase

Ref document number: 15855022

Country of ref document: EP

Kind code of ref document: A1