WO2011147086A1 - 载有干扰核糖核酸的细胞微粒子、其制备方法及其应用 - Google Patents

载有干扰核糖核酸的细胞微粒子、其制备方法及其应用 Download PDF

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WO2011147086A1
WO2011147086A1 PCT/CN2010/073262 CN2010073262W WO2011147086A1 WO 2011147086 A1 WO2011147086 A1 WO 2011147086A1 CN 2010073262 W CN2010073262 W CN 2010073262W WO 2011147086 A1 WO2011147086 A1 WO 2011147086A1
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cell
microparticles
sirna
cells
diseases
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PCT/CN2010/073262
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English (en)
French (fr)
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张辰宇
曾科
刘丹青
张玉婧
顾宏伟
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江苏命码生物科技有限公司
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Priority to PCT/CN2010/073262 priority Critical patent/WO2011147086A1/zh
Priority to US13/700,067 priority patent/US9376679B2/en
Priority to CN201080066281.0A priority patent/CN102858375B/zh
Priority to US13/700,062 priority patent/US9421167B2/en
Priority to EP10852044.6A priority patent/EP2578236B1/en
Priority to PCT/CN2010/079602 priority patent/WO2011147175A1/zh
Publication of WO2011147086A1 publication Critical patent/WO2011147086A1/zh

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Definitions

  • the present invention relates to cell microparticles carrying interfering ribonucleic acid, a preparation method and use thereof.
  • the present invention relates to a method of providing cell microparticles carrying interfering ribonucleic acid, supporting interfering ribonucleic acid on cell microparticles, and the role of this method in biomedical experimental technique improvement and disease prevention/treatment. Background technique
  • Microvesicles are a type of biofoam structure with a lipid bilayer membrane between 10 and 500 nm in size. It was reported as early as 1967, when it was called "platelet dust", which was derived from platelets and contained sputum, which promoted coagulation. In vitro studies have found that endothelial cells, vascular smooth muscle cells, platelets, white blood cells, lymphocytes, red blood cells, etc. can release microparticles. Depending on the way the microparticles are produced, the microparticles can be distinguished as exosomes and shedding vesicles.
  • Exosomes are cells that are stimulated to vomit into cells by multivesicular bodies (MVBs), while shedding vesicles are secreted directly from the cell surface by budding.
  • MVBs multivesicular bodies
  • shedding vesicles secreted by different cells are named differently, such as ectosomes secreted by neutrophils and monocytes, and microparticles secreted by platelets.
  • the membrane component of the microparticles depends on the cells from which they are derived, mainly composed of lipids and proteins, but the internal components of the microparticles are not known.
  • the plasma membrane of the cell microparticles contains the characteristics of the source cells, namely the molecular markers specific to the surface of the cell and the cell receptor/ligand. The clear physiological functions of cell microparticles have not been studied so far.
  • RNA interference Small interfering RNA
  • siRNA is a kind of double-stranded RNA molecule composed of more than 20 nucleotides, which can silence gene expression by using a message RNA (mRNA) that specifically degrades the target gene. Effect. This process is called RNA interference (RNAi).
  • RNA interference is a way of post-transcriptional silencing of genes and is one of the highly conserved phenomena of ancient and evolutionary biology. Specific and highly efficient inhibition of gene expression by siRNA-mediated recognition and targeting of cleavage of homologous target mRNA. RNA interference is characterized by biocatalytic reactions, which require the involvement of multiple protein factors and ATP. In recent years, breakthroughs have been made in RNA interference research, which was named one of the top ten scientific advances in 2001 by Science magazine and ranked first in the top ten scientific advances in 2002. 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 gene therapy in various diseases.
  • RNA interference can utilize siRNA or siRNA expression vectors quickly, economically, simply, and with high sequence specificity, it can specifically silence specific genes, gain functional loss or reduce the specificity of mutant sequences to eliminate target gene expression, so now It has become an important research tool for exploring gene function.
  • mutations in specific genes need to be lost or reduced to determine their function, so RNA interference can be used as a powerful research tool for functional genomic research.
  • the establishment of siRNA expression library construction method makes high-throughput selection of RNA interference technology possible, and it is of great significance to clarify the signal transduction pathway and discover new drug targets.
  • RNA interference technology is also widely used in the field of treatment of diseases.
  • RNA interference technology it was found that selecting a sequence in the viral genome that has no homology with the human genome as an inhibitory sequence can inhibit the replication of the virus while avoiding The toxic side effects of normal tissues.
  • the selection of the inhibitory sequence at a specific site can induce apoptosis induction in some malignant tumor cells with a clear gene mutation.
  • siRNA or shRNA expression against certain oncogenes or anti-apoptotic molecules can be guided by using a tumor-specific promoter to achieve specific killing of tumor cells.
  • RNA interference is aimed at gene silencing in the post-transcriptional stage, the whole process design is simpler, more rapid, and effective than the traditional gene therapy knockout. This opens up new avenues for gene therapy.
  • the general idea is to control the abnormal protein synthesis process or the replication and expression of exogenous pathogenic nucleic acids in the disease by strengthening the RNA interference mechanism of key genes, especially for nucleic acid viruses that cause serious harm to human health.
  • siRNA can effectively inhibit the replication of HIV virus in cultured cells in vitro.
  • siRNA can prevent HIV infection by inhibiting the HIV virus's own genes (such as pie, gag, rev, tat and env) or host genes (such as HIV's main receptor CD4).
  • HIV virus's own genes such as pie, gag, rev, tat and env
  • host genes such as HIV's main receptor CD4
  • RNA interference to silence the P53 gene can inhibit the transformation of tumor cells from benign to malignant.
  • RNA interference has been widely used in all aspects of biomedical research, there are still some problems that are currently difficult to solve. For example, with existing lipofection methods, the efficiency of siRNA into certain cells, such as immune cells, is very low. This affects its further application in this area.
  • siRNA can be injected directly into the animal, the uncoated siRNA has a very short half-life and is not satisfactory.
  • the carriers for siRNA drug delivery mainly include liposomes, Nanocapsules/Nanoparticles, ⁇ -cyclodextrinin inhibitor ( ⁇ -cyclodextrin compound), etc., although the drug can be prolonged.
  • the retention time in the body and the absorption rate of siRNA drugs are increased to some extent, but the targeting and high efficiency of the drug delivery are still insufficient. How to effectively administer humans, to ensure the effective release of the target organ target tissue, and to be highly safe, needs further study.
  • siRNA As an important means of biological and medical research and potential drugs, siRNA still has some unresolved problems.
  • the specificity (targeting), poor stability and low efficiency of delivery of siRNA are the main reasons hindering its application.
  • cell microparticles are a biofoam vector that is highly efficient and specific in transport in vivo. These cell microparticles vary in size and are distributed in the 10-500 nm range. In principle, the membrane components (including specific surface receptors and membrane lipid structures) of the microparticles released by different cells are identical to the plasma membrane components of the corresponding cells. Therefore, the cell microparticles have a receptor protein or membrane lipid structure specific to the surface of the source cell, and have high affinity with the corresponding target cell. If cell microparticles are used as a carrier for transporting siRNA, siRNA can be efficiently and selectively delivered to target cells/tissues, thereby greatly enhancing the function of siRNA to regulate cell function.
  • cell microparticles including all membrane lipid raft structures with cell microparticle characteristics, such as exosome and sheshed vesicles and shedding vesicles secreted by various cells
  • siRNA contained also has strong targeting, stability and high efficiency, and it has great application prospects in the research and treatment of disease mechanisms.
  • the inventors found that: transporting interfering ribonucleic acid with cell microparticles as a carrier (microRAN) Entering the target cell, because the cell microparticle is a substance secreted by the cell itself, it has biological affinity and does not cause damage to the organism itself.
  • the cell microparticle surface carries the surface cell-specific surface molecule, its target The cells have high affinity and thus can enter target cells efficiently and characteristically.
  • the interfering ribonucleic acid that enters the target cell can function to block the translation process of the target gene protein by binding to the specific sequence of the target gene messenger ribonucleic acid, thereby specifically blocking the gene expression.
  • cell microparticles are derived from cells and are present in the organism itself, thereby overcoming the toxicity of the currently synthesized drug carrier to the cells and damage to the body;
  • the various techniques used in the process of entering the cell microparticles are easy to implement, and the encapsulation efficiency is high, which increases the potential of its practical application to a certain extent; more importantly, the cell microparticles have a bilayer plasma membrane.
  • the structural vesicle structure, the outer membrane and the cytoplasmic membrane structure are similar, and can enter the cell by fusion with cell membrane and endocytosis.
  • the surface of the cell microparticle membrane carries molecular markers on the surface of the plasma membrane of the source cells, such as surface proteins and various receptors/ligands, it can efficiently enter the target cells through specific recognition.
  • the application of siRNA to cells secreted by primary cultures of the patient's own tissues or cells can also reduce immune rejection and further increase the efficiency of cell microparticles carrying siRNA into the organism. Based on the above advantages, the delivery of siRNA as a drug by cell microparticles as a carrier will play an important role in drug development and prevention and treatment of clinical diseases. Summary of invention
  • the present invention provides microparticles of cells containing interfering ribonucleic acids.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising cell microparticles containing interfering ribonucleic acid and a pharmaceutically usable carrier.
  • the present invention further provides a kit comprising a cell microparticle containing interfering ribonucleic acid or a pharmaceutical composition containing cell microparticles containing interfering ribonucleic acid, and instructions for use.
  • the invention further provides a method of preparing microparticles containing interfering ribonucleic acid, comprising the steps of:
  • siRNA and its control sequence into a donor cell, preferably by transfection or viral vector method; isolating cell microparticles containing interfering ribonucleic acid;
  • microparticles containing interfering ribonucleic acid are added to a receptor, preferably a recipient cell; the effect of the microparticles containing interfering ribonucleic acid into the recipient cell is studied.
  • the present invention also provides a method of preventing and/or treating a disease comprising: transferring a microparticle of a cell containing interfering ribonucleic acid into a recipient.
  • the invention also provides the use of cellular microparticles containing interfering ribonucleic acids for the transport of interfering ribonucleic acids.
  • Figure 1 shows a transmission electron micrograph of normal human serum/plasma cell microparticles.
  • Figure 3-A shows flow cytometry for the detection of cell microparticles
  • Figure 3-B shows flow cytometry for detection of microparticles containing siRNA
  • Figure 3-C shows that siRNA enters target cells with cell microparticles as a vector
  • Figure 4-A shows that siRNA specifically down-regulates target protein expression in target cells.
  • Figure 4-B shows the effect of microparticles without siRNA on the migration ability of target cells.
  • Figure 4-C Effect of microparticles containing siRNA on the migration ability of target cells
  • Figure 4-D shows statistical results of the effect of siRNA-containing cell microparticles on target cell migration ability
  • Cellular microparticles include natural bio-vesicles with a lipid bilayer membrane secreted by cells ranging from 10-500 nm in size, including through multivesicular bodies.
  • MVBs Exosome secreted by cells; shedding vesicles secreted by the cells and needles A special name for shedding vesicles secreted by various fine packets.
  • Cell microparticles include cell microparticles produced by various cells derived from humans or animals, in particular, cells of normal or diseased human or animal, and may be primary cultures, subcultures (cell lines), such as endothelial cells. , vascular smooth muscle cells, platelets, white blood cells, lymphocytes and red blood cells.
  • siRNA includes all siRNA sequences designed for receptor genes that specifically degrade target genes by the principle of RNA interference.
  • the invention also provides pharmaceutical compositions and kits useful for the treatment of diseases.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising cell microparticles containing interfering ribonucleic acid and a pharmaceutically usable carrier.
  • the carrier includes, for example, physiological saline, serum, cell culture fluid, phosphate buffered saline (PBS), and the like.
  • kits comprising a cell microparticle containing interfering ribonucleic acid or a drug composition containing cell microparticles containing interfering ribonucleic acid, and instructions for use.
  • Diseases which can be prevented and/or treated using cell microparticles containing interfering ribonucleic acid or a pharmaceutical composition or kit containing the interfering ribonucleic acid-containing cell microparticles include various tumors; various acute and chronic infectious diseases such as viral influenza, Viral diseases such as viral hepatitis, AIDS, SARS, such as bacterial diseases such as tuberculosis and bacterial pneumonia, and acute and chronic infectious diseases caused by various other pathogenic microorganisms; other acute and chronic diseases, such as respiratory diseases, immune system diseases, Blood and hematopoietic diseases, circulatory diseases, endocrine system metabolic diseases, systemic diseases, systemic diseases, urinary system diseases, reproductive system diseases and sports system diseases.
  • various acute and chronic infectious diseases such as viral influenza, Viral diseases such as viral hepatitis, AIDS, SARS, such as bacterial diseases such as tuberculosis and bacterial pneumonia, and acute and chronic infectious diseases caused by various other pathogenic microorganisms
  • the invention further provides a method of preparing microparticles containing interfering ribonucleic acid, comprising the steps of:
  • Cell microparticles containing interfering ribonucleic acid are isolated.
  • Methods of preparing cell microparticles include, for example, differential centrifugation, immunoadsorption, and ultrafiltration.
  • the method for preparing cell microparticles by differential centrifugation comprises, for example, the following steps: first, body fluid, Blood, cells, tissues, cells or tissues cultured in vitro are centrifuged to remove various types of cells and debris, and then the supernatant is ultracentrifuged, and the precipitate is a cell microparticle.
  • the preparation of the cell microparticles by immunoadsorption comprises, for example, the following steps:
  • an ultrafiltration method comprising, for example, the following steps:
  • the present invention provides a method of preparing siRNA-loaded cell microparticles which can efficiently and specifically enter a recipient cell or organism, and interfere with protein expression of its target gene by siRNA.
  • the method comprises:
  • siRNA-containing cell microparticles into a receptor, such as a recipient cell, preferably into a living organism.
  • a method of preparing cell microparticles containing interfering ribonucleic acid comprises:
  • the design of the target gene siRNA sequence follows the following principles:
  • siRNA fragment satisfies AAN19TT, NAN19N, NARN17Y and NAN 17Y (N stands for any base, and R and Y represent purine and pyrimidine, respectively).
  • the designed siRNA sequence was BLAST searched in the US NCBI (National Center for Biotechnology Infermation) EST or Unigene database to ensure the specificity of the siRNA sequence to the target gene.
  • the method of constructing the siRNA expression vector comprises: inserting a DNA molecule having a specific stem loop and a termination signal of about 70 bp in length into a specific vector.
  • the method of transfecting siRNA is carried out using a liposome (Invitrogen Lipofectamine 2000) method.
  • the method of preparing the microparticles of the cells is selected from one or more of a differential centrifugation method, an immunoadsorption method, and an ultrafiltration method.
  • the method for detecting the siRNA loading efficiency in the cell microparticles is selected from one or more of RT-PCR, Real time-PCR, Northern blot, immunofluorescence, and flow cytometry.
  • the RT-PCR method includes the following steps:
  • the Real-time PCR method includes the following steps:
  • the Northern blotting method includes the following steps: (1) collecting serum/plasma and cell and tissue samples;
  • Isotope signal detection such as phosphor screen scanning test results.
  • the immunofluorescence method includes the following steps:
  • Receptor cells include all existing cell lines, cell lines, and primary cultures of tissue/packages of normal human or disease patients.
  • Organisms into which cell microparticles can enter include humans, various animals, and various pathogenic microorganisms.
  • Animals include cartilage fish, bony fish, amphibians, reptiles, birds and mammals
  • pathogenic microorganisms include bacteria, spirochetes, mycoplasma, rickettsia, chlamydia and actinomycetes.
  • the pathogenic microorganism includes various DNA and RNA viruses such as hepatitis B virus, variola virus, HIV, SARS virus, influenza virus and the like.
  • siRNA and its control sequence are introduced into the donor cell by transfection
  • siRNA-containing cell microparticles Adding the siRNA-containing cell microparticles to the recipient cell
  • siRNA-containing cell microparticles into the recipient cells was investigated.
  • methods for performing gene function studies using siRNA-loaded cell microparticles include:
  • Methods for studying the effect of siRNA-containing cell microparticles on the function of the recipient cell after entry into the receptor include one or more of confocal fluorescence microscopy, Western blotting, and cell migration methods.
  • the Western blot method includes the following steps:
  • the present invention also provides a method of preventing and/or treating a disease comprising: transferring a microparticle of a cell containing interfering ribonucleic acid into a recipient.
  • a method for preventing/treating a disease using a cytoplasmic microparticle carrying siRNA includes:
  • Cell microparticles carry siRNA into the recipient cell or patient tissue, causing a change in the protein content of the target gene by interfering with the expression of its target gene.
  • Diseases include various tumors; various acute and chronic infectious diseases, such as viral influenza, viral hepatitis, AIDS, SARS and other viral diseases, such as tuberculosis, bacterial pneumonia and other bacterial diseases, and other acute causes caused by various pathogenic microorganisms Chronic infectious diseases; other acute and chronic diseases, such as respiratory diseases, immune system diseases, blood and hematopoietic diseases, circulatory diseases, endocrine system metabolic diseases, digestive diseases, nervous system diseases, urinary system diseases, reproductive system diseases and Sports system diseases.
  • infectious diseases such as viral influenza, viral hepatitis, AIDS, SARS and other viral diseases, such as tuberculosis, bacterial pneumonia and other bacterial diseases, and other acute causes caused by various pathogenic microorganisms Chronic infectious diseases
  • other acute and chronic diseases such as respiratory diseases, immune system diseases, blood and hematopoietic diseases, circulatory diseases, endocrine system metabolic diseases, digestive diseases, nervous system diseases, urinary system diseases, reproductive system diseases and Sports system diseases.
  • siRNA includes all siRNA sequences designed for pathogenic genes that specifically degrade target genes by the principle of RNA interference;
  • Genes include all gene fragments that can be transcribed into functional molecules, such as protein genes, mciroRNA gene, etc.
  • Pathogenic genes include humans, various animals, including cartilage fish, bony fish, amphibians, reptiles, birds and mammals, and other genes involved in the development of diseases, as well as various pathogenic microbial genes.
  • the above pathogenic microorganisms include bacteria, spirochetes, mycoplasma, rickettsia, chlamydia and actinomycetes.
  • the pathogenic microorganisms include various DNA and RNA viruses such as hepatitis B virus, variola virus, HIV, SARS virus, influenza virus and the like.
  • the invention also provides the use of cellular microparticles containing interfering ribonucleic acids for the transport of interfering ribonucleic acids.
  • the serum/plasma or cultured cells are centrifuged at 300 g for 5 minutes, and the supernatant is taken; (2) the supernatant is centrifuged at 1500 g for 20 minutes, and the supernatant is taken; (3) the supernatant is centrifuged at l OOOg for 30 minutes. Take the supernatant. (4) The supernatant was centrifuged at l l OOOOg for 70 minutes, and the precipitate was taken as a microparticle.
  • the isolated cell microparticles were observed under transmission electron microscopy, including: Cell microparticle precipitation was fixed overnight at 4 ° C with a 2.5% glutaraldehyde fixative, and rinsed three times with PBS for 10 minutes each time. Thereafter, it was fixed at room temperature for 60 minutes with 1% osmium tetroxide. The fixed sample was embedded in 10% gelatin and then fixed at a temperature of 4 °C with glutaraldehyde, and the sample was cut into small pieces (volume less than 1 mm 3 ). The samples were dehydrated with increasing concentrations of ethanol solution (30%, 50%, 70%, 90%, 95% and 100% ⁇ 3). After embedding with epoxy resin, it was sliced with a Leica UC6 ultramicrotome and finally observed with a FEI Tecnai T20 transmission electron microscope at 120 kV.
  • fluorescently labeled siRNA was transfected into cells using the following procedure, and the efficiency of transfection was examined.
  • siRNA sequence was first designed for different sites of the human c-myb gene sequence:
  • siRNA designed above was commercially synthesized, and the siRNA against the c-myb gene was labeled with the green fluorescent dye FITC.
  • Lipofectamine 2000 (Invitrogen, USA) siRNA was transfected into human monocyte/macrophage cell line THP-1 cells (Shanghai Cell Bank of Chinese Academy of Sciences) by the following methods:
  • THP-1 cell culture was cultured in RPMI 1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA) at 5% C0 2 at 37 °C.
  • Mixture C and mixture D are mixed to form a mixture F, which is allowed to stand for 20 minutes.
  • ReDetecting c-myb gene messenger RNA levels by Real time-PCR method to detect interference effects Rate including:
  • the reverse transcription reaction system includes 4 ⁇ 1 5 AMV buffer, 2 ⁇ l lOmM each dNTP mixture ( Takara, ⁇ ), 0.5 ⁇ 1 RNase Inhibitor ( Takara, Japan), 2 ⁇ 1 AMV ( Takara, Japan), and 0.5 lOligodT ( Takara, Japan) .
  • the reaction step is 15 minutes at 16 ° C, 1 hour at 42 ° C, and 5 minutes at 85 ° C;
  • the data processing method is the AC T method, and the AC T is set to the number of cycles when the reaction reaches the domain value.
  • the flow cytometry method includes the following steps: collecting the interfered THP-1 cells, adjusting the cell concentration to 10 6 /ml; measuring the fluorescence intensity of the cells by flow cytometry (BD FACS, Calibur), and detecting the forward direction
  • the lateral voltage amplification mode is lin
  • the fluorescence intensity is detected by FL1-H
  • the voltage amplification mode of the FL-1H channel is log.
  • fluorescence microscopy can also be used to detect the efficiency of siRNA entry into recipient cells. Specific steps include: placing THP-1 cells transfected with c-myb siRNA on a fluorescence inverted microscope (OLYMPUS) stage, excitation wavelength 488nm detection. As a result, as shown in Fig. 2-C, the transfected cells showed green fluorescence (as shown by the bright spots in Fig. 2-C), indicating that the siRNA enters the cells with high efficiency.
  • Example 3 Cell microparticles carry siRNA into recipient cells
  • the efficiency of loading siRNA was measured by collecting the granules of THP-1 cells transfected with c-myb siRNA, and added to the target cells to measure the efficiency of entry into the target cells.
  • THP-1 The human monocyte/macrophage cell line THP-1, which plays an important role in the inflammatory response, was selected as a research object.
  • THP-1 cells were cultured in RPMI 1640 medium (Gibco supplemented with 10% fetal bovine serum (Gibco, USA). , US), 5% C0 2 , cultured at 37 °C.
  • RPMI 1640 medium Gibco supplemented with 10% fetal bovine serum (Gibco, USA). , US)
  • C0 2 fetal bovine serum
  • THP-1 cell microparticles transfected with c-myb siRNA were isolated.
  • the efficiency of the loaded cytoplasmic particles loaded with siRNA was measured by flow cytometry.
  • FIG. 3-A The forward lateral voltage amplification mode is log when detecting, and the fluorescence intensity is detected by FL1-H, and the FL-1 channel voltage amplification mode is log.
  • a part of the cell microparticles secreted by THP-1 (the right part of the vertical line in Fig. 3-B) was labeled with a fluorescent label. Since c-myb siRNA is fluorescently labeled, the fluorescence intensity of microparticles can be detected by flow cytometry to reflect the efficiency of microparticle loading of siRNA.
  • HMCE-1 HMEC-1 human venular endothelial cell HMEC-1 (USA Center for Disease Control).
  • HMCE-1 HMEC-1 was cultured in MCDB supplemented with 10 ng/mL epidermal growth factor (Becton-Dickinson, USA), 10 ng/mL hydrocortisone (Sigma), and 10% fetal bovine serum (Gibco, USA).
  • 5% C0 2 was cultured at 37 °C.
  • siRNA which is a drug can be highly affinity-specifically and specifically delivered to target cells by microparticles, and the purpose of drug prevention/treatment can be achieved by affecting the function of target cells involved in disease onset.
  • siRNA-bearing cell microparticles to study gene function
  • the siRNA targeting the target gene is efficiently and specifically transferred into the recipient cell by using the microparticles to specifically reduce the expression of the target gene in the recipient cell, mimic the pathological condition or function as a gene knockout. Study the physiological functions of target genes in cells.
  • the c-myb gene was selected as a research object, and the c-myb gene encodes a cellular transcription factor, which plays an important role in cell differentiation, proliferation, migration, and blood cell survival.
  • C-myb has been shown to be an important proto-oncogene and is closely related to the development of many cancers.
  • Example 2 Using the method for isolating cell microparticles in Example 1, isolating THP-1 cell microparticles transfected with c-myb siRNA
  • c-myb and GAPDH were labeled with a monoclonal antibody against c-myb (Santa Cluz) and a monoclonal antibody against GAPDH (Santa Cluz), respectively.
  • siRNA entry into recipient cells can be detected by Western blot techniques that detect specific protein expression levels.
  • this example also examined the effect of microparticle-borne siRNA on the migration ability of HMEC-1 cells in its target cells.
  • the c-myb gene As an important transcription factor, the c-myb gene has an important influence on cell growth, migration and differentiation. Although studies have demonstrated that c-myb has a significant regulatory effect on the migration of a variety of cells, this example specifically reduces the c- in endothelial cell line HMEC-1 cells by using siRNA contained in cell microparticles as an experimental method. The myb protein is expressed to detect the migration function of the cells in this case, thereby examining whether the c-myb gene has an effect on the migration function of endothelial cells.
  • Cell migration assays include: Polycarbonate membrane (8- ⁇ pore size) at the bottom of the upper chamber of Transwell Boyden Chamber (6.5 mm, Costar, Cambridge, MA, USA) covered with 0.1% gelatin; used for HMEC-1 cells The serum-free medium is suspended and the concentration is controlled at
  • Fig. 4-B The migration ability of HMCE-1 cells (Fig. 4-C) treated with cell microparticles carrying c-myb siRNA was significantly enhanced. The number of cells in 5 random fields was counted, and the results are shown in Fig. 4-D. The number of cells migrated by HMCE-1 cells treated with cell microparticles carrying c-myb siRNA was significantly increased compared with the control.
  • c-myb gene inhibits the migration of endothelial cells.
  • this example demonstrates that the preparation of siRNA-loaded cell microparticles and related methods can be used as a medical biological research method to study the function of a gene by specifically reducing the expression of a certain gene in the cell.
  • Example 5 Prevention/Treatment of Disease Using Cell Microparticles Loaded with siRNA
  • cell microparticles containing siRNA against HIV (HIV) gene were used to inhibit the survival and reproduction of HIV in its host cells.
  • HIV HIV
  • the results are shown in Figure 5.
  • the ordinate shows the amount of HIV virus in the host cells. If a blank cell with no virus at all is used as a control (column represented by horizontal axis 1), set its value to 1; then cells that are added to the virus without taking any treatment (columns represented by horizontal axis 2) The virus content will be more than 16 times that of the control cells. However, if cell microparticles loaded with viral siRNA are used as a treatment, the amount of virus in the host cell is greatly reduced. As a result, it was found that after the siRNA contained in the cell microparticles (the column represented by the horizontal axis 5, 6), the virus in the host cell was reduced to about 40% (the column represented by the horizontal axis 6).
  • the HIV virus in the host cell can be completely inhibited, and the HIV virus content can be reduced to and without the virus group (horizontal axis 1). Representing the pillars) the same level.
  • siRNA-loaded cell microparticles due to the vector itself, rather than siRNA
  • the column represented by the horizontal axis 3 As can be seen from the results, only the empty vector could not inhibit the HIV virus, and it was also proved that the virus inhibition was not the vector but the siRNA itself.
  • Example 6 Characterization of a pharmaceutical composition consisting of cell microparticles and siRNA loaded thereon This example examined the presence of a pharmaceutical composition consisting of cell microparticles and siRNA loaded thereon by a series of methods.
  • Example 3 According to the method of Example 3, whether the siRNA, i.e., whether the cytoplasmic granules and the siRNA complex, were composed of the particles isolated and purified and identified as the microparticles by flow cytometry was examined, and the results are shown in Fig. 3-B. Since the siRNA is fluorescently labeled, if the cell microparticles contain siRNA, the cell microparticles must also be fluorescently labeled. Therefore, we used flow cytometry to detect the fluorescence of cell microparticles. As can be seen from Figure 3-B, a large number of cell microparticles carry fluorescence (Fig.
  • the invention provides that: (1) cell microparticles carrying interfering ribonucleic acid.
  • siRNA contained in cell microparticles to various clinical diseases (including various tumors; various acute and chronic infectious diseases, and acute and chronic infectious diseases caused by various other pathogenic microorganisms; other acute and chronic diseases, such as respiratory diseases, Immune system diseases, diseases of the blood and hematopoietic diseases, such as circulatory diseases of cardiovascular and cerebrovascular diseases, diseases of endocrine and metabolic diseases, diseases of the digestive system, diseases of the nervous system, diseases of the urinary system, diseases of the reproductive system and diseases of the motor system; 3) Application of cell microparticles to efficiently and specifically deliver interference with the ribonucleoside, as an experimental means to study specific gene functions.
  • various clinical diseases including various tumors; various acute and chronic infectious diseases, and acute and chronic infectious diseases caused by various other pathogenic microorganisms; other acute and chronic diseases, such as respiratory diseases, Immune system diseases, diseases of the blood and hematopoietic diseases, such as circulatory diseases of cardiovascular and cerebrovascular diseases, diseases of endocrine and metabolic diseases, diseases of the
  • the present invention provides a method for preparing cell microparticles carrying interfering ribonucleic acid, which has high targeting, stability and high efficiency.
  • siRNA can stably, efficiently, and specifically enter cell cells by acting on its target gene, thereby causing a fix to the function of the target cell. Therefore, siRNA-loaded cell microparticles can not only function as a biomedical research tool in gene function research, but also act as a drug, efficiently and specifically enter the organism, and change gene expression and affect cell function. , thereby treating the role of prevention/disease.

Description

载有干扰核糖核酸的细胞微粒子、 其制备方法及其应用 发明领域
本发明涉及载有干扰核糖核酸的细胞微粒子、 制备方法及其应用。 具 体地， 本发明涉及提供载有干扰核糖核酸的细胞微粒子， 将干扰核糖核酸 负载于细胞微粒子的方法， 以及此方法在生物医学实验技术改良及疾病预 防 /治疗中的作用。 背景技术
细胞微粒子 (microvesicles)是一类大小在 10-500nm之间， 具有脂质双 层膜的生物嚢泡结构。 早在 1967年就有报道， 当时被称为" platelet dust", 其来源于血小板， 包含嚢泡， 具有促进凝结的作用。 体外研究发现内皮细 胞、 血管平滑肌细胞、 血小板、 白细胞、 淋巴细胞、 红细胞等都能释放微 粒子。 根据微粒子产生的途径， 又可以将微粒子区分为 exosomes 和 shedding vesicles。 Exosomes是细胞在被刺激的情况下, 通过多嚢泡小体 ( Multivesicular bodies, MVBs )胞吐到细胞夕卜的， 而 shedding vesicles则 是直接从细胞表面以出芽的方式分泌出来的。 目前， 不同细胞分泌的 shedding vesicles被命名为不同的名称，比如中性粒细胞和单核细胞分泌的 被称为 ectosomes, 而血小板分泌的则被称为 microparticles。
细胞微粒子的膜成分取决于其来源的细胞， 主要由脂质和蛋白质组 成， 但细胞微粒子的内部成分尚不清楚。 细胞微粒子的质膜含有来源细胞 的特征， 即来源细胞表面特异性的分子标记以及细胞受体 /配体。 细胞微粒 子明确的生理功能至今尚未研究清楚。
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 ( messager 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干扰是基因转录后沉默的一种方式，是生物界古老而且进化的高 度保守的现象之一。通过 siRNA介导的识别，并靶向切割同源性靶 mRNA 的方式， 特异性高效抑制基因表达。 RNA干扰具有生物催化反应特征， 反 应中需要多种蛋白因子以及 ATP参与。 近几年来 RNA干扰研究取得了突破性进展， 被《Science》 杂志评为 2001年的十大科学进展之一， 并名列 2002年十大科学进展之首。 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基因治疗领域。
在 RNA干扰技术出现以前， 基因敲除 (gene knockout) 是主要的反向 遗传学 （reverse genetics )研究手段， 但其技术难度高， 操作复杂， 周期 长。 由于 RNA干扰可以利用 siRNA或 siRNA表达载体快速、 经济、 简便 的同时又具有高度的序列专一性， 可以特异地使特定基因沉默， 获得功能 丧失或减低突变序列特异方式剔除目的基因表达， 所以现在已经成为探索 基因功能的重要研究手段。 在功能基因组研究中， 需要对特定基因功能丧 失或减低突变， 以确定其功能， 因此 RNA干扰可以作为一种强有力的研 究工具， 用于功能基因组的研究。 同时 siRNA表达文库构建方法的建立， 使得 RNA干扰技术进行高通量 选成为可能， 对阐明信号转导通路， 发 现新的药物作用靶点有重要意义。
RNA干扰技术也被广泛应用与治疗疾病领域。 在利用 RNA干扰技术 对 HIV-1、 乙型肝炎、 丙型肝炎等进行基因治疗研究中发现， 选择病毒基 因组中与人类基因组无同源性的序列作为抑制序列可在抑制病毒复制的 同时避免对正常组织的毒副作用。 同时将抑制序列选择在特定的位点， 可 对部分有明确基因突变的恶性肿瘤细胞产生凋亡诱导作用。 此外尚可通过 使用肿瘤特异性启动子， 引导针对某些癌基因或抗凋亡分子的 siRNA或 shRNA表达， 从而达到特异性杀伤肿瘤细胞的目的。
由于 RNA干扰是针对转录后阶段的基因沉默， 相对于传统基因治疗 对基因水平上的敲除， 整个流程设计更简便， 且作用迅速， 效果明显， 为 基因治疗开辟了新的途径。 其总体思路是通过加强关键基因的 RNA干扰 机制， 控制疾病中出现异常的蛋白合成进程或外源致病核酸的复制及表 达， 尤其针对引起一些对人类健康严重危害的核酸病毒。
目前，研究已经证明， siRNA能有效地抑制 HIV病毒在体外培养细胞 中的复制。 siRNA可以通过抑制 HIV病毒自身基因 （如 pie, gag, rev, tat 和 env )或宿主基因（如 HIV的主要受体 CD4 )达到阻止 HIV感染的目的。 同时研究发现， 在两种自身免疫性肝炎的小鼠模型中静脉注射抑制 Fas的 siRNA进入小鼠， 肝细胞中的 Fas mRNA及蛋白质水平均降低， 故保护了 肝细胞免受自身免疫肝炎引起的细胞凋亡的伤害。 更有研究发现： 利用
RNA干扰去沉默 P53基因， 可以抑制肿瘤细胞从良性到恶性的转化。
虽然 RNA干扰已广泛应用于生物医学研究各个方面， 但目前该技术 仍有一些难以解决的问题。 例如， 应用现有的脂质体转染方法， siRNA进 入某些细胞， 如免疫细胞的效率非常低。 这就影响其在这一领域的进一步 应用。
同时， 尽管 siRNA药物的研究开发取得了不少成果， 但要将其作为药 物真正用于医疗还面临很多问题。 虽然可以直接将 siRNA 注射到动物体 内， 但是这种没有经过包裹的 siRNA的半衰期极短， 治疗效果差强人意。 目 前， siRNA 药物运送的载体主要有脂质体、 毫微型胶裏 ( Nanocapsules/Nanoparticles ). β 环糊精包含物 ( β-cyclodextrininclusion Compound或称 β环糊精胶裏） 等， 虽然可以延长药物在体内的存留时间 并一定程度上提高 siRNA药物的吸收率，但是其传送药物的靶向性和高效 性仍然不足。 如何对人进行有效的给药， 既能确保药效在靶器官靶组织有 效释放， 还要具有高度安全性等等问题都尚需进一步研究。
siRNA作为生物、 医学研究的重要手段以及潜在的药物， 目前还存在 一些未解决的问题， 递送 siRNA的特异性 (靶向性)差、 稳定性差、 效率低 是妨碍其应用的主要原因， 因此， 迫切需要一种更加稳定、 高效、 特异的 运送 siRNA的方式， 高效地、 特异性地传送 siRNA。
申请人意外地发现： 细胞微粒子是一种在生物体内转运效率高， 特异 性强的生物嚢泡载体。这些细胞微粒子大小不一，分布在 10-500纳米范围。 原则上不同细胞释放的微粒子的膜表成分（包括特异性表面受体及膜脂结 构）与对应细胞的质膜成分相同。 因此， 细胞微粒子带有来源细胞表面特 有的受体蛋白或膜脂结构， 与对应的靶细胞有高亲和性。 如果釆用细胞微 粒子作为运送 siRNA的载体， 就可以高效率、 选择性地递送 siRNA到靶 细胞 /组织， 从而大大提高 siRNA调控细胞功能的作用。 显然， 由于细胞 微粒子（包括所有具有细胞微粒子特征的膜脂嚢泡结构， 比如 exosome和 shedding vesicles以及针对各种细胞分泌的 shedding vesicles的特称 ) 本身 具有和特定组织及细胞结合的特异性， 其所载 siRNA也呈现较强的靶向 性、 稳定性及高效率， 它在疾病机制的研究及治疗方面有重大应用前景。
发明人研究发现： 以细胞微粒子作为载体运送干扰核糖核酸 ( microRAN ) 进入靶细胞 , 由于细胞微粒子是细胞自身分泌的物质， 具 有生物亲和性， 不会对生物体本身造成伤害； 同时， 由于细胞微粒子表面 携带了来源细胞特异的表面分子， 对其靶细胞具有高亲和性， 因此可以高 效、 特性地进入靶细胞。 进入靶细胞的干扰核糖核酸可以发挥其功能， 通 过与靶基因信使核糖核酸特定序列的结合， 阻断靶基因蛋白质翻译过程， 从而起到特异性阻断基因表达的作用。
应用细胞微粒子作为载体运送 siRNA的优势在于： 首先， 细胞微粒子 来源于细胞， 是生物体本身存在的， 从而可以克服目前人工合成的药物载 体对细胞的毒性以及对身体的损害； 其次， 将 siRNA包裹进入细胞微粒子 的过程中所应用的各种技术手段都很容易实现， 同时包裹效率很高， 这在 一定程度上加大了其实际应用的潜力； 更重要的是细胞微粒子是具有双层 质膜结构的嚢泡结构，外膜和细胞质膜结构类似，可以通过和细胞膜融合、 胞吞作用进入细胞。 同时， 由于细胞微粒子膜表面携带有来源细胞的质膜 表面的分子标记物， 如表面蛋白、 各种受体 /配体等， 可以通过特异性的识 别， 高效地进入靶细胞。 如果应用病人自身组织或细胞的原代培养物分泌 的细胞微粒子包裹 siRNA还可减少免疫排斥并进一步提高细胞微粒子携 带 siRNA进入生物体的效率。 基于以上优势， 细胞微粒子作为载体运送 siRNA作为药物将会在药物开发及临床疾病的预防和治疗中起到重要的 作用。 发明概述
本发明提供了含有干扰核糖核酸的细胞微粒子。
本发明还提供一种药物组合物， 包括含有干扰核糖核酸的细胞微粒子和 药物上可用的载体。
本发明进一步提供了一种试剂盒， 其中包括含有干扰核糖核酸的细胞微 粒子或者含有含有干扰核糖核酸的细胞微粒子的药物组合物， 以及使用说 明。
本发明此外还提供了一种制备含有干扰核糖核酸的细胞微粒子的方法， 包括下列步骤：
应用细胞转染技术将干扰核糖核酸转入细胞； 或病毒载体法将干扰核糖 核酸转入细胞； 分离含有干扰核糖核酸的细胞微粒子。
本发明还提供了一种研究方法， 包括：
将 siRNA及其对照序列导入供体细胞内， 优选通过转染或病毒载体法； 分离含有干扰核糖核酸的细胞微粒子；
将该含有干扰核糖核酸的细胞微粒子加入受体， 优选受体细胞； 研究该含有干扰核糖核酸的细胞微粒子进入受体细胞后的影响。
本发明还提供了一种预防和 /或治疗疾病的方法， 包括：将含有干扰核糖 核酸的细胞微粒子转入受体。
本发明还提供了含有干扰核糖核酸的细胞微粒子在输送干扰核糖核酸 中的用途。 附图说明
图 1显示正常人血清 /血浆细胞微粒子的透射电镜图。
图 2-A Real time-PCR方法检测 c-myb基因 siRNA干扰效率
图 2-B流式细胞仪检测转染效率的结果
图 2-C荧光显微镜检测转染效率的结果
图 3-A显示流式细胞法检测细胞微粒子
图 3-B显示流式细胞法检测含有 siRNA的细胞微粒子
图 3-C 显示 siRNA以细胞微粒子作为载体进入靶细胞
图 4-A显示 siRNA在靶细胞特异性将靶蛋白表达量下调
图 4-B 显示未含有 siRNA的细胞微粒子对靶细胞迁移能力的影响 图 4-C 含有 siRNA的细胞微粒子对靶细胞迁移能力的影响
图 4-D显示含有 siRNA的细胞微粒子对靶细胞迁移能力影响的统计结 果
图 5 含有 siRNA的细胞微粒子对 HIV病毒的抑制作用 具体实施方式
含有干扰核糖核酸的细胞微粒子
细胞微粒子包括各种大小在 10-500nm之间， 由细胞分泌的具有脂质 双层膜的天然生物嚢泡， 包括通过多嚢泡小体 ( Multivesicular bodies ,
MVBs )分泌的 Exosome; 细胞以出芽方式分泌的 shedding vesicles以及针 对各种细包分泌的 shedding vesicles的特称。
细胞微粒子包括来自人或动物的各种细胞产生的细胞微粒子， 特别地， 包括正常的或者患病的人或动物的细胞，可以是原代培养物、传代培养物（细 胞系）， 例如内皮细胞、 血管平滑肌细胞、 血小板、 白细胞、 淋巴细胞和红 细胞。
siRNA包括所有针对受体基因设计的通过 RNA干扰原理特异性降解靶 基因的 siRNA序列。
本发明还提供了可用于疾病治疗的药物组合物及试剂盒。
根据本发明的一个实施方案， 本发明提供了一种药物组合物， 包括含有 干扰核糖核酸的细胞微粒子以及药物上可用的载体。 载体例如包括生理盐 水、 血清、 细胞培养液、 磷酸盐緩冲液（PBS )等。
根据本发明的一个实施方案， 本发明提供了一种试剂盒， 其中包括含有 干扰核糖核酸的细胞微粒子或者含有含有干扰核糖核酸的细胞微粒子的药 物组合物， 以及使用说明。
使用含有干扰核糖核酸的细胞微粒子或者含有该含有干扰核糖核酸的 细胞微粒子的药物组合物或试剂盒能够预防和 /或治疗的疾病包括各种肿 瘤； 各种急慢性传染病， 例如病毒性流感、 病毒性肝炎、 艾滋病、 SARS 等病毒性疾病， 例如结核、 细菌性肺炎等细菌性疾病， 以及其它各种病原 微生物导致的急慢性传染病； 其它急慢性疾病， 例如呼吸系统疾病， 免疫 系统疾病，血液与造血系统疾病，循环系统疾病， 内分泌系统代谢性疾病， 消 4匕系统疾病， 申经系统疾病， 泌尿系统疾病， 生殖系统疾病和运动系统 疾病。 方法
本发明此外还提供了一种制备含有干扰核糖核酸的细胞微粒子的方法， 包括下列步骤：
应用细胞转染技术将干扰核糖核酸转入细胞； 或病毒载体法将干扰核糖 核酸转入细胞；
分离含有干扰核糖核酸的细胞微粒子。
制备细胞微粒子的方法包括，例如差速离心法、免疫吸附法和超滤法。 优选釆用差速离心法制备细胞微粒子，例如包括下述步骤：先将体液、 血液、 细胞、 组织、 体外培养的细胞或组织离心， 除去各类细胞及碎片， 然后将上清超速离心， 取沉淀便是细胞微粒子。
或者优选地， 使用免疫吸附法制备细胞微粒子， 例如包括以下步骤：
( 1 )先将体液、 血液、 细胞、 组织、 体外培养的细胞或组织离心， 去除各 种细胞及碎片， 取上清； （2 )将吸附在组织培养皿上的细胞特异性的抗体 或免疫磁珠 ( Invitrogen , 美国）与上清温育 （例如 30~60分钟）， 得到被 吸附的细胞微粒子。
或者优选地， 釆用例如包括以下步骤的超滤法：
( 1 )先将体液、 血液、 细胞、 组织、 体外培养的细胞或组织离心去除 各种细胞及碎片， 取上清； （2 )将上清放入带有 100KD 滤膜的浓缩离心 管 （Millipore, 美国）， 于 4000转 /分钟进行离心， 浓缩即得细胞微粒子。
优选地， 本发明提供了一种制备负载有 siRNA的细胞微粒子的方法， 该微粒子可以高效、特异进入受体细胞或生物体， 通过 siRNA干扰其靶基 因的蛋白表达。
根据本发明的一个实施方案， 该方法包括：
1 ) 将 siRNA包裹进入供体细胞微粒子；
2 ) 分离供体细胞分泌的细胞微粒子；
3 ) 检测细胞微粒子中 siRNA的装载效率；
4 ) 将含有 siRNA的细胞微粒子引入受体，例如受体细胞，优选注 (射) 入生物体。
根据本发明的另一个实施方案， 制备含有干扰核糖核酸的细胞微粒子 的方法包括：
1 ) 设计针对靶基因的 siRNA序列；
2 ) 化学合成成熟 siRNA或构建 siRNA表达载体；
3 ) 应用细胞转染技术将 siRNA转入细胞或将 siRNA表达载体转入 细胞。
优选， 靶基因 siRNA序列的设计遵守以下原则：
( 1 ) siRNA片段满足 AAN19TT, NAN19N , NARN17Y 和 NAN 17Y ( N代表任何碱基， R和 Y分别代表嘌呤和嘧啶）。
( 2 ) 选择无重复序列的互补 DNA外显子序列以及具有均衡 A、G、 C、 T含量 （或 GC含量在 30% ~ 70% ) 的反义链。 ( 3 ) 避开序列中有成串的单一碱基， 尤其是 G碱基。
( 4 ) 避开 3'和 5'端未翻译的区域（ 5'-UTR, 3'-UTR ), 通常这些位 点是 mRNA结合蛋白的结合位点。
( 5 ) 避开起始密码子或者外显子与外显子的交界区域（ exon-exon boundaries )。
将设计的 siRNA序列在美国 NCBI ( National Center for Biotechnology Infermation ) 的 EST或 Unigene数据库进行 BLAST检索， 以保证 siRNA 序列对靶基因的特异性。
设计 4个以上 siRNA序列， 进行化学合成后， 通过实验 选出沉默效 果最好的 siRNA序列， 进行下一步的基因功能研究。 优选， 构建 siRNA表达载体的方法包括： 将长约 70bp的含有特定茎 环以及终止信号的 DNA分子插入到特定载体中。
优选， 转染 siRNA的方法釆用脂质体 （Invitrogen公司 Lipofectamine 2000 ) 法。
细胞微粒子的制备方法选自差速离心法、 免疫吸附法和超滤法中的一 种或多种。
检测细胞微粒子中 siRNA 装载效率的方法选自 RT-PCR、 Real time-PCR、 Northern blot、 免疫荧光、 流式细胞法中的一种或多种。
RT-PCR方法， 例如包括以下步骤：
( 1 )提取 RNA 干扰后的细胞 /组织总 RNA, 通过逆转录反应得到 cDNA样品；
( 2 )应用靶基因特异性引物进行 PCR反应；
( 3 ) 进行 PCR产物的琼脂糖凝胶电泳；
( 4 ) EB染色后在紫外灯下观察结果。
Real-time PCR方法， 例如包括以下步骤：
( 1 )提取 RNA干扰后的细胞 /组织 ,通过 RNA逆转录反应得到 cDNA 样品；
( 2 )设计靶基因特异性引物；
( 3 )加入荧光探针进行 PCR反应；
Northern blotting方法， 例如包括以下步骤： ( 1 ) 收集血清 /血浆及细胞、 组织样本；
( 2 ) 通过 Trizol试剂提取总 RNA;
( 3 ) 进行变性 PAGE电泳和膜转移实验；
( 4 ) 制备同位素标记靶基因探针；
( 5 ) 进行膜杂交反应；
( 6 ) 同位素信号检测如磷屏扫描检测结果。
免疫荧光方法， 例如包括以下步骤：
( 1 ) 将细胞附着在支持物上；
( 2 ) 应用细胞固定剂如多聚曱醛等， 将细胞固定；
( 3 ) 应用脱脂牛奶或牛血清白蛋白对细胞进行封闭；
( 4 ) 应用荧光标记抗体标记特异性靶基因蛋白；
( 5 ) 荧光显微镜下观测细胞荧光强度。
受体细胞包括现有所有细胞系、 细胞株、 以及正常人或疾病患者自身 组织 /细包的原代培养物。
细胞微粒子能够进入的生物体包括人类、 各种动物及各种致病微生 物。 动物包括软骨鱼、 硬骨鱼、 两栖动物、 爬行动物、 鸟类及哺乳类， 致 病微生物包括细菌、 螺旋体、 支原体、 立克次体、 衣原体和放线菌。 特别 的， 所述致病微生物包括各 DNA及 RNA病毒， 如乙肝病毒、 天花病毒、 艾滋病病毒、 SARS 病毒、 流感病毒等。
本发明还提供了一种研究方法， 包括：
通过转染将 siRNA及其对照序列导入供体细胞内；
分离含有 siRNA的细胞微粒子；
将该含有 siRNA的细胞微粒子加入受体细胞；
研究该含有 siRNA的细胞微粒子进入受体细胞后的影响。
根据本发明的一个实施方案，应用携带 siRNA的细胞微粒子进行基因 功能研究的方法包括：
( 1 )通过转染将 siRNA及其对照序列导入供体细胞内；
( 2 )分离制备含有 siRNA的供体细胞微粒子；
( 3 )将细胞微粒子加入受体细胞；
( 4 )研究细胞微粒子携带 siRNA进入受体细胞后对受体细胞功能的影 响， 从而研究其靶基因对细胞功能的影响； 研究含有 siRNA的细胞微粒子进入受体后对受体细胞功能的影响的方 法包括共聚焦荧光显微镜法、 Western bloting方法、 细胞迁移方法的一种 或几种。
例如， Western blot方法包括以下步骤：
( 1 ) 应用蛋白质裂解液提取细胞或组织总蛋白；
( 2 ) 进行 SDS-PAGE电泳和膜转移实验；
( 3 ) 应用脱脂牛奶或牛血清白蛋白对膜进行封闭；
( 4 ) 应用 HRP标记过的特异性抗体，对膜上的特异性靶基因蛋白 进行标记；
( 5 ) 加 HRP底物， 产生发光反应；
( 6 ) 放射自显影。
本发明还提供了一种预防和 /或治疗疾病的方法， 包括：将含有干扰核糖 核酸的细胞微粒子转入受体。
根据本发明的一个实施方案， 应用携带 siRNA的细胞微粒子预防 /治 疗疾病的方法包括：
1)通过转染将 siRNA导入供体细胞内；
2)分离制备含有 siRNA的供体细胞微粒子；
3)将细胞微粒子加入受体细胞或注入病人体内。
4)细胞微粒子携带 siRNA进入受体细胞或病人组织， 通过干扰其靶基 因表达， 引起靶基因蛋白质含量的改变。
5)通过改变细胞内蛋白质影响细胞功能， 从而起到预防 /治疗疾病的作 用。
疾病包括各种肿瘤； 各种急慢性传染病， 例如病毒性流感、 病毒性肝 炎、 艾滋病、 SARS等病毒性疾病， 例如结核、 细菌性肺炎等细菌性疾病， 以及其它各种病原微生物导致的急慢性传染病； 其它急慢性疾病， 例如呼 吸系统疾病， 免疫系统疾病， 血液与造血系统疾病， 循环系统疾病， 内分 泌系统代谢性疾病， 消化系统疾病， 神经系统疾病， 泌尿系统疾病， 生殖 系统疾病和运动系统疾病。
siRNA包括所有针对致病基因设计的通过 RNA干扰原理特异性降解靶 基因的 siRNA序列；
基因包括所有能转录成有功能分子的基因片段， 例如蛋白质基因、 mciroRNA基因等。 致病基因包括人类， 各种动物， 包括软骨鱼、 硬骨鱼、 两栖动物、 爬行动物、 鸟类及哺乳动物等生物自身参与疾病发生发展的各 种基因， 以及各种致病微生物基因。
上述致病微生物包括细菌、 螺旋体、 支原体、 立克次体、 衣原体和放 线菌。特别的，所述致病微生物包括各种 DNA及 RNA病毒，如乙肝病毒、 天花病毒、 艾滋病病毒、 SARS 病毒、 流感病毒等。
本发明还提供了含有干扰核糖核酸的细胞微粒子在输送干扰核糖核酸 中的用途。 实施例
可以理解的是， 在此描述的特定实施方式通过举例的方式来表示， 其并 不作为对本发明的限制。 在不偏离于本发明范围的情况下， 本发明的主要特 征可以用于各种实施方式。 本领域的技术人员将会意识到或能够确认， 使用 常规实验， 许多等同物都能应用于本文所描述的特定步骤中。 这些等同物被 认为处在本发明的范围之内， 并且被权利要求所覆盖。 实施例 1 血清 /血浆和细胞培养液中细胞微粒子的分离及检测 本实施例分别釆用差速离心法方法分离出血清 /血浆和细胞培养液中 细胞微粒子：
具体地， 先将血清 /血浆或培养细胞于 300g离心 5分钟， 取上清； （2 ) 将上清于 1500g离心 20分钟， 取上清； （3 )将上清于 l OOOOg离心 30分 钟， 取上清。 （4 )将上清于 l l OOOOg离心 70分钟， 取沉淀即为细胞微粒 子。
将分离得到的细胞微粒子在透射电镜下观察， 包括： 细胞微粒子沉淀 用 2.5%戊二醛固定液在 4 °C固定过夜， PBS漂洗三遍， 每遍 10分钟。 之 后用 1 %的四氧化锇室温固定 60分钟。 固定后的样品用 10%的明胶包埋， 然后在 4 °C用戊二醛再固定后， 将样品切成小块（体积小于 1立方毫米）。 样品用依次增高浓度的乙醇溶液脱水 （30%, 50%, 70%, 90%, 95% and 100%χ3 )。 用环氧树脂浸透包埋后， 用 Leica UC6 超薄切片机切片， 最后 用 FEI Tecnai T20 透射电子显微镜在 120 kV条件下观察。
釆用差速离心法分离得到细胞微粒子的透射电镜图如图 1所示， 显示 从正常人血清 /血浆中分离到的细胞微粒子大小不一，分布在 10-500 nm范 围。 实施例 2 siRNA转染进入供体细胞
本实施例釆用以下步骤将荧光标记的 siRNA转染进入细胞，并检测转 染效率。
首先针对人 c-myb基因序列的不同位点设计 siRNA序列：
(正义链 +环 +反义链）： 5'-GGTGGAACAGAATGGAACA TTGAAGAAG TGTTCCATTCTGTTCCACC TT-3';
同时设计一条随机序列作为阴性对照：
(正义链 +环 +反义链）5'-GACTTCATAAGGCGCATGC TTGAAGAAG GCATGCGCCTTATGAAGTC TT-3' .
接下来， 商业合成上述设计的 siRNA, 同时釆用绿色荧光染料 FITC 标记针对 c-myb基因的 siRNA。
釆用脂质体 Lipofectamine 2000 (Invitrogen,美国） 将 siRNA转染进入 人单核 /巨噬细胞系 THP-1细胞（中国科学院上海细胞库），具体方法如下：
( 1 ) THP-1细胞培养在添加了 10%胎牛血清 （Gibco, 美国） 的 RPMI 1640培养基（Gibco, 美国） 中， 5% C02、 37 °C培养。
( 2 ) 将 30μ1 lipofectamine 2000和 600pmol 的阴性对照 siRNA分别 与 1ml OPTI-MEM ( Gibco, 美国 )混合， 形成混合物 A和混合物 B , 室温 下放置 5min。
( 3 ) 将 30μ1 lipofectamine 2000和 600pmol 的 c-myb siRNA分别 与 1ml OPTI-MEM ( Gibco, 美国 )混合， 形成混合物 C和混合物 D, 室温 下放置 5min。
( 4 ) 将混合物 A和混合物 B混合， 生成混合物 E , 放置 20min。
( 5 ) 将混合物 C和混合物 D混合， 生成混合物 F, 放置 20min。
( 6 ) 将混合物 E和 F分别加入对照组和实验组细胞中， 补足
OPTI-MEM到 15ml。 5 % C02、 37 °C培养。
( 7 ) 6小时后更换成正常培养液。
( 8 ) 24h~48h后转染结束， 可以收集样品。
釆用 Real time-PCR方法检测 c-myb基因信使 RNA水平以检测干扰效 率， 包括：
( 1 ) 收集转染后的 THP-1细胞
( 2 )制备 cDNA样品：釆用 Trizol试剂（Invitrogen,美国）提取总 RNA, 总 RNA逆转即得到 cDNA样品。逆转录的反应体系包括 4μ1 5 AMV緩冲 液、 2μ1 lOmM each dNTP mixture ( Takara, 曰本)、 0.5μ1 RNase Inhibitor ( Takara, 日本）、 2μ1 AMV ( Takara, 日本） 以及 0.5 lOligodT ( Takara, 日本）。反应步骤为 16°C孵育 15分钟， 42°C反应 1小时， 85°C孵育 5分钟；
( 3 ) Real-time PCR反应： 取 Ιμΐ cDNA, 加入 0.3μ1 Taq酶 ( Takara, 日本）， 0.5μ1 ΙΟμΜ正向和反向引物， 1.2μ1 25mM MgCl2, 1.6μ1 2.5mM each dNTP mixture ( Takara, 日本）， Ιμΐ 20 EVA GREEN , 2μ1 lO PCR緩冲液， 12.4μ1Η20, 20μ1体系进行 PCR。 仪器使用的是 ABI Prism 7300荧光定量 PCR仪， 反应条件为 95°C、 5分钟进行 1个循环→ 95°C、 15秒， 60°C、 1 分钟进行 40个循环；
( 4 )数据处理： 数据处理方法为 ACT法， ACT设为反应达到域值时 的循环数， 则两组样品干扰核糖核酸的比较可以用方程 2-ACT表示， 其中 △CT=CT groupl -CT group2。 组织和细胞数据处理方法是以 U6作为内标， 则两 组样品 mRNA表达量的比较可以用方程 2-ACT表示， 其中△Cτ= [CT mlRNA- Cτu6]groupl - [CT miR A - CTU6]group2。
结果如图 2-A所示， 与转染过随机序列的阴性对照（左柱）相比， 转染 过 c-mybsiRNA的细胞中 c-myb基因的 m RNA表达量显著降低， 说明该实 验中釆用的转染方法的可靠而高效的。
同时釆用流式细胞法检测 siRNA进入细胞的效率，结果如图 2-B所示。 流式细胞法包括下述步骤： 收集干扰后的 THP-1细胞， 将细胞浓度调整到 106个 /ml; 应用流式细胞仪检 ( BD FACS, Calibur ) 测细胞荧光强度， 检测时前向侧向电压放大模式均为 lin, 釆用 FL1-H检测荧光强度， FL-1H 通道电压放大模式为 log。 由结果可以看出， 与对照 （细线）相比， 转染 了 c-myb siRNA的实验组（粗线 )的荧光强度增强， 说明 siRNA的转染效 率高， 此种方法是一种检测 siRNA转染效率的直观高效的方法。
另外， 还可以釆用荧光显微镜方法检测 siRNA进入受体细胞的效率， 具体步骤包括： 将转染过 c-myb siRNA后的 THP-1细胞置于荧光倒置显 微镜 ( OLYMPUS ) 载物台， 激发波长 488nm检测。 结果如图 2-C所示， 转染过的细胞可显示绿色荧光（如图 2-C中亮点 所示）， 说明 siRNA进入细胞的效率很高。 实施例 3 细胞微粒子携带 siRNA进入受体细胞
本实施例通过收集转染过 c-myb siRNA的 THP-1细胞的细胞微粒子， 检测其装载 siRNA的效率， 并将其加入其靶细胞，检测其进入靶细胞的效 率。
选择在炎症反应中起重要作用的人单核细胞 /巨噬细胞系 THP-1 作为 研究对象， THP-1细胞培养在添加了 10%胎牛血清（Gibco,美国）的 RPMI 1640培养基（Gibco, 美国） 中， 5% C02、 37°C下培养。 首先， 应用实施 例 2中的 siRNA转染方法， 制备转染过 c-myb siRNA的 THP-1细胞。
接下来， 应用实施例 1中细胞微粒子的分离方法， 分离转染过 c-myb siRNA的 THP-1细胞微粒子。
再通过流式细胞法检测所分离的细胞微粒子装载 siRNA的效率。
结果如图 3-A 所示。 检测时前向侧向电压放大模式均为 log, 釆用 FL1-H检测荧光强度， FL-1通道电压放大模式为 log。 从结果可以看出， 在 THP-1分泌的细胞微粒子中， 有一部分（图 3-B竖线以右部分）被标记 荧光标记。 由于 c-myb siRNA被荧光标记， 因此， 应用流式细胞仪检测微 粒子的荧光强度， 就可以反映微粒子装载 siRNA的效率。
最后将携带有 c-myb siRNA的 THP-1细胞分泌的微粒子加入人微静脉 内皮细胞 HMEC-1 (美国佐治亚州疾控中心） 的细胞培养液中。 HMCE-1 HMEC-1培养在添加了 10 ng/mL表皮生长因子 （ Becton-Dickinson, 美 国）、 10 ng/mL氢化可的松 ( Sigma ) 和 10%胎牛血清 （ Gibco, 美国） 的 MCDB-131培养基中， 5% C02、 37°C下培养。
由于在生理状况下， 单核 /巨噬细胞可以和血管内皮细胞相互作用， 单 核细胞表面的分子可以特异性地和内皮细胞表面的配体 /受体结合，诱发一 系列信号传导及细胞生理活动。 因此， 应用单核 /巨噬细胞系 THP-1 和微 静脉内皮细胞系 HMEC-1就可以模拟体内这两种细胞的相互作用。
检测加入了携带 siRNA 的 THP-1微粒子进入 HMCE-1细胞的效率。 由于细胞微粒子被绿色荧光标记， 因此， 应用荧光显微镜检测 HMEC-1荧 光结果， 就可以反映微粒子进入 HMEC-1的效率。 结果如图 3-C所示。 由结果可以看出，携带 siRNA的细胞微粒子可以高效特异性的进入靶 细胞 HMEC-1 (图 3-C 中亮点）。 又由于所有细胞都能够像血细胞一样分 泌细胞微粒子； 并且所有细胞也可以像内皮细胞一样接受与其特异性作用 的细胞分泌的细胞微粒子。 因此， THP-1细胞微粒子进入 HMEC-1细胞的 作用方式也可模拟生物体上所有细胞微粒子进入其靶细胞的作用方式。
通过以上的实验结果不难发现， 细胞微粒子是理想的稳定、 高效、 特 异运载 siRNA的载体， 可以通过细胞微粒子将 siRNA传递给其受体细胞。 提示可以通过微粒子， 将作为药物的 siRNA高亲和性、特异性的递送到靶 细胞中， 通过影响参与疾病发病的靶细胞的功能， 达到药物预防 /治疗的目 的。 实施例 4 应用携带 siRNA的细胞微粒子研究基因功能
本实施列应用细胞微粒子将针对靶基因的 siRNA高效、特异性地转入 受体细胞中， 通过特异性降低受体细胞中靶基因的表达， 模拟病理状况或 起到基因敲除的作用， 以研究靶基因在细胞中的生理功能。
本实施例选择了 c-myb基因作为研究对象， c-myb基因编码一个细胞 转录因子， 在细胞的分化、 增殖、 迁移及血细胞的存活中发挥重要作用。 c-myb 已被证明是一个重要的原癌基因， 与多种癌症的发生发展有密切关 系。
为了研究 c-myb基因在内皮细胞中的功能， 设计了如下实验：
1 )应用实施例 2中的 siRNA转染方法， 制备转染过 c-myb siRNA的 THP-1细胞。
2 )应用实施例 1中细胞微粒子的分离方法， 分离转染过 c-myb siRNA 的 THP-1细胞微粒子
3 )将携带有 c-myb siRNA的细胞微粒子加入到 HMEC-1细胞的培养 液中， 6小时后收集细胞， 按照以下步骤进行 Western Blot 实验。 具体步 骤包括：
( 1 ) 应用蛋白质裂解液提取细胞总蛋白；
( 2 ) 在 90V恒压条件下进行 SDS-PAGE电泳；
( 3 ) 在 160mA恒流条件下进行膜转移实验；
( 4 ) 应用 5%脱脂牛奶对膜进行封闭； ( 5 ) 分别应用抗 c-myb 的单克隆抗体 （Santa Cluz 公司 ) 和抗 GAPDH ( Santa Cluz公司） 的单克隆抗体分别对 c-myb和 GAPDH进行 标记。
( 6 ) 加对应的 HRP标记二抗；
( 7 ) 加 HRP底物， 产生发光反应；
( 8 ) 放射自显影。
由于 siRNA通过与其靶基因 mRNA结合，招募细胞内的沉默复合物 将其靶基因 mRNA被降解，从而导致其靶基因蛋白表达水平的下降。因此， 通过检测特异性蛋白表达水平的 Western blot技术就可以检测 siRNA进入 受体细胞的效率。
结果如图 4-A所示。由结果可见， GAPDH作为内参，证明 Western blot 过程中加入的总蛋白量在所有条带中都是一样的。 同时， 与转染阴性对照 的细胞（条带 2 )相比， 转染过 c-myb siRNA的细胞（条带 3 ) 中 c-myb 蛋白的表达有明显下降。 由此， 本发明人证明： 细胞微粒子携带的 c-myb siRNA进入了 HMEC-1细胞， 并且对 HMEC-1细胞中 c-myb蛋白的表达 起到了干扰作用。 进一步证明了， 细胞微粒子可以将 siRNA高效、 特异性 的进入靶细胞， 以此作为一种实验手段， 研究基因在特定细胞中的功能。
同时本实施例还检测了微粒子携带的 siRNA对其靶细胞 HMEC-1 细 胞迁移能力的影响。
c-myb基因作为一个重要的转录因子， 对细胞的生长、 迁移和分化有 重要影响。 虽然研究已经证明 c-myb对多种细胞的迁移有明显调控作用， 因此本实施例通过应用细胞微粒子所载 siRNA作为一种实验方法，特 异性地降低内皮细胞系 HMEC-1细胞中的 c-myb蛋白表达，以检测在这种 情况下细胞的迁移功能， 以此研究 c-myb基因对内皮细胞的迁移功能是否 有作用。
具体实验步骤包括：
( 1 ) 应用实施例 2中的 siRNA转染方法，制备转染过 c-myb siRNA 的 THP-1细包。
( 2 ) 应用实施例 1 中细胞微粒子的分离方法， 分离转染过 c-myb siRNA的 THP-1细胞的细胞微粒子 ( 3 ) 携带有 c-myb siRNA的 THP-1细胞微粒子孵育 HMEC-1细 胞 2小时。
( 4 ) 检测 HMEC-1细胞迁移情况。
细胞迁移实验检测方法包括： 将 Transwell Boyden Chamber (6.5 mm, Costar, Cambridge, MA, 美国）的上面小室底部的聚碳酸酯膜 （8-μπι 孔径) 用 0.1%的明胶覆盖； HMEC-1 细胞用不含血清的培养基悬起， 浓度控制在
( 1-10 ) ΐθ5 cells/mL; 细胞用或者不用来源于 THP-1细胞的包含 siRNA的 细胞微粒子孵育 2小时， 然后将 HMCE- 1 细胞加入上面的小室， 同时在下 面的小室加入 0.5 mL的含有 10%胎牛血清的培养基； 5% C02的细胞培养 箱孵育 4 h ; 迁移到下层的细胞用 90%乙醇在室温下固定 15分钟； 洗涤； 用 0.1% 的结晶紫室温染色 15分钟； 将留在滤膜上的细胞小心地刮下； 拍 照 (Olympus, BX51 , Japan); 细胞计数。
迁移后的细胞显微图片如图 4-B\C\D所示。 由结果可见， 与阴性对照
(图 4-B )相比， 被携带 c-myb siRNA的细胞微粒子处理过的 HMCE-1细 胞（图 4-C ) 的迁移能力明显增强。 细胞计数 5个随机视野中的细胞数， 结果如图 4-D所示， 与对照相比， 被携带 c-myb siRNA的细胞微粒子处理 过的 HMCE-1细胞迁移过的细胞数显著能加。
由此可见，降低了表达量的 c-myb基因能够显著增强 HMEC-1细胞的 迁移能力， 因此， 相反的可以说明， c-myb基因对内皮细胞的迁移有抑制 作用。
因此， 本实施例证明， 可以将制备载有 siRNA的细胞微粒子及相关方 法作为一种医学生物学的研究手段， 通过特异性降低细胞内某种基因的表 达来研究该基因的功能。 实施例 5 应用载有 siRNA的细胞微粒子进行疾病的预防 /治疗 本实施例应用载有针对艾滋病病毒（ HIV )基因的 siRNA的细胞微粒 子抑制 HIV在其宿主细胞中生存及繁殖。
具体包括以下步骤：
1 ) 针对 HIV基因组序列设计 siRNA序列；
2 ) 将 siRNA序列插入到载体中；
3 ) 将携带有 HIV siRNA的载体转染进入供体细胞 293T细胞 中；
4 ) 收集供体细胞分泌的细胞微粒子；
5 ) 将供体细胞分泌的载有 HIV siRNA的细胞微粒子加入 HIV 宿主细包；
6 ) 检测宿主细胞中病毒含量。
结果如图 5所示， 纵坐标显示了 HIV病毒在宿主细胞中的含量。 如果 将完全不加病毒的空白细胞作为对照（横轴 1代表的柱子），将其值设为 1 ; 那么单加入病毒而不釆取任何治疗措施的细胞（横轴 2代表的柱子） 中的 病毒含量将会是对照细胞的 16倍多。 然而， 如果釆用载有病毒 siRNA的 细胞微粒子作为治疗手段， 宿主细胞内的病毒含量就会大量减少。 从结果 可见， 加入细胞微粒子所载的 siRNA (横轴 5、 6代表的柱子)后， 宿主细胞 内的病毒以降低到 40%左右 （横轴 6代表的柱子）。 更重要的是， 如果增 加微粒子所载 siRNA的用量（横轴 5代表的柱子）， 宿主细胞内的 HIV病 毒甚至可以被完全抑制， HIV病毒的含量可以下降到和不加病毒组（横轴 1代表的柱子）相同的水平。
另外， 为了排除载有 siRNA的细胞微粒子对 HIV的抑制作用是由于 载体本身， 而不是 siRNA造成的， 我们还向 HIV病毒感染的宿主细胞中 转入了不含 siRNA的空白载体作为另一个对照 （横轴 3代表的柱子）。 从 结果可以看出， 仅空载体无法对 HIV病毒产生抑制作用， 也证明了产生病 毒抑制作用不是载体而是 siRNA本身。
同时， 我们还加入了一种短肽类的抗艾滋病药物作为阳性对照 （横轴 4代表的柱子）。由结果可以看出，这种药物也只能将 HIV含量降低到 50% 左右。 因此， 可以看出载有 HIV siRNA的细胞微粒与常规治疗艾滋病的药 物相比， 其作用效率更高， 抑制病毒的效果更好。 也进一步说明应用载有 siRNA的细胞微粒子治疗疾病具有巨大的发展潜力。 实施例 6 细胞微粒子及其载有的 siRNA组成的药物组合物的检定 本实施例通过一系列方法检定细胞微粒子及其载有的 siRNA组成的 药物组合物的存在。
1 )通过实施例 2所述方法将荧光标记的 siRNA转染入供体细胞。 结果如图 2-C所示，在荧光显微镜下观察可见荧光标记的 siRNA已经转染 进入细胞。
2 )通过实施例 1所述方法分离鉴定转染过荧光标记的 siRNA的供体 细胞分泌的细胞微粒子。 结果如图 1所示， 可见分离得到的细胞微粒子从 形态、 大小、 膜结构等方面均符合细胞微粒子的特定。
3 )根据实施例 3 的方法， 通过流式细胞仪检测分离纯化并鉴定为细 胞微粒子的颗粒中是否包裹有 siRNA, 即是否组成了细胞微粒子和 siRNA 复合物， 结果如图 3-B所示。 由于 siRNA是被荧光标记的， 如果细胞微粒 子中含有 siRNA, 那么， 细胞微粒子也一定能被荧光标记。 因此， 我们釆 用流式细胞术检测细胞微粒子所带的荧光状况。 由图 3-B可见， 有大量细 胞微粒子携带有荧光（图 3-B 竖线以右部分）， 证明细胞微粒子中包裹有 siRNA, 也即证明了细胞微粒子 -siRNA药物复合物的存在。 本发明提供了包括： （ 1 )载有干扰核糖核酸的细胞微粒子。 （2 )应用 细胞微粒子所载 siRNA进行各种临床疾病 (包括各种肿瘤； 各种急慢性传 染病， 以及其它各种病原微生物导致的急慢性传染病； 其它急慢性疾病， 如呼吸系统疾病， 免疫系统疾病， 血液与造血系统疾病， 例如心脑血管疾 病的循环系统疾病， 内分泌代谢系统疾病， 消化系统疾病，神经系统疾病， 泌尿系统疾病， 生殖系统疾病和运动系统疾病 )治疗的研究； （ 3 )应用细 胞微粒子高效、 特异性递送干扰核糖核， 作为实验手段， 研究特定基因功 能。
通过上述一系列的研究， 很清楚本发明提供了一种制备载有干扰核糖 核酸的细胞微粒子的方法， 该方法具有较高靶向性、 稳定性及高效性。
根据上述方法，本发明人证明 siRNA可以通过细胞微粒子稳定、 高效、 特异性的进入靶细胞， 通过作用于其靶基因， 对靶细胞的功能产生一定影 响。由此，载有 siRNA的细胞微粒子不仅可以作为一种生物医学研究手段， 在基因功能研究中发挥作用； 同时还能作为药物， 高效特异性地进入生物 体， 起到改变基因表达， 影响细胞功能， 从而治疗预防 /疾病的作用。

Claims

权 利 要 求 书
1、 含有干扰核糖核酸的细胞微粒子。
2、 根据权利要求 1 的细胞微粒子， 其中所述细胞微粒子来自人或动物 的供体细胞。
3、 根据权利要求 2的细胞微粒子， 其中所述供体细胞包括： 细胞系、 原代细胞培养物。
4、 根据权利要求 1 的细胞微粒子， 其中干扰核糖核酸包裹于细胞微粒 子中。
5、根据权利要求 1的细胞微粒子，其中细胞微粒子的平均粒径为 10-500 纳米。
6、 根据权利要求 1 的细胞微粒子， 其中细胞微粒子包括 exosome 和 shedding vesicle以及其他来自细胞的生物嚢泡结构。
7、 一种试剂盒， 包括根据权利要求 1-6 中任一项所述的含有干扰核糖 核酸的细胞微粒子。
8、 一种药物组合物， 包括权利要求 1-6 中任一项所述的含有干扰核糖 核酸的细胞微粒子。
9、 制备权利要求 1-6 中任一项所述的细胞微粒子的方法， 包括下列步 骤：
将干扰核糖核酸转入细胞， 优选应用细胞转染技术或病毒载体法； 分离含有干扰核糖核酸的细胞微粒子。
10、 权利要求 9的方法， 其中通过选自差速离心法、 免疫吸附法和超滤 法中的一种或多种分离含有干扰核糖核酸的细胞微粒子。
11、 一种研究方法， 包括：
将权利要求 1-6中任一项的含有干扰核糖核酸的细胞微粒子引入受体； 研究该含有干扰核糖核酸的细胞微粒子进入受体后对受体功能的影响。
12、 一种预防和 /或治疗疾病的方法， 包括： 将权利要求 1-6中任一项所 述的含有干扰核糖核酸的细胞微粒子引入受体细胞内。
13、 根据权利要求 12的方法， 其中所述疾病包括
肿瘤； 急慢性传染病， 细菌性疾病， 以及其它各种病原微生物导致的急 慢性传染病； 呼吸系统疾病， 免疫系统疾病， 血液与造血系统疾病， 循环系 统疾病， 内分泌系统代谢性疾病， 消化系统疾病， 神经系统疾病， 泌尿系统 疾病， 生殖系统疾病和运动系统疾病。
14、 根据权利要求 13 的方法， 其中所述疾病包括病毒性流感、 病毒性 肝炎、 艾滋病、 SARS , 结核、 细菌性肺炎等细菌性疾病， 病原微生物导致 的急慢性传染病。
15、 权利要求 1-6中任一项权利要求中的含有干扰核糖核酸的细胞微粒 子在输送干扰核糖核酸中的用途。
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