WO2020211841A1 - 细胞去服务化状态的分子标记物检测及其调控方法 - Google Patents

细胞去服务化状态的分子标记物检测及其调控方法 Download PDF

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WO2020211841A1
WO2020211841A1 PCT/CN2020/085364 CN2020085364W WO2020211841A1 WO 2020211841 A1 WO2020211841 A1 WO 2020211841A1 CN 2020085364 W CN2020085364 W CN 2020085364W WO 2020211841 A1 WO2020211841 A1 WO 2020211841A1
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cells
snorna
tissue
service state
gland
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French (fr)
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张辰宇
周祯
胡秀婷
李菁
周心妍
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南京大学
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Priority to EP20792239.4A priority Critical patent/EP3943609A4/en
Priority to JP2021562082A priority patent/JP2022529484A/ja
Priority to US17/604,707 priority patent/US20220195525A1/en
Publication of WO2020211841A1 publication Critical patent/WO2020211841A1/zh
Priority to IL287347A priority patent/IL287347A/en
Priority to JP2023197126A priority patent/JP2024028724A/ja

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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the invention belongs to the field of biotechnology, and specifically relates to the separation, qualitative and quantitative analysis of nucleolar ribonucleic acid molecules in human and animal tissues and cells that are used to distinguish the de-service state of cells. It also relates to the regulation of human and animal tissues and cells.
  • the nucleolar ribonucleic acid is used to change the application method of the cell to serve.
  • Small nucleolar RNAs are a class of medium-length non-coding small RNAs, their lengths range from 60-300 nt, which can combine with nucleolar ribonucleoprotein to form a snoRNP complex.
  • genes encoding small nucleolar RNAs mainly exist in the intron regions of protein coding genes or non-protein coding genes, and undergo further post-transcriptional processing to form mature small nucleolar RNAs.
  • the post-transcriptional modification of RNA, the stability of mRNA and the control of translation are important components of gene expression regulation.
  • rRNA ribosomal RNA
  • snRNA small nuclear RNA
  • the present inventors selected small nucleolar ribonucleic acid as the research object, hoping to distinguish this phenomenon by detecting the expression changes of small nucleolar ribonucleic acid in the cell, and also hoped that the expression of small nucleolar ribonucleic acid in the cell could be further reduced from The de-serving state is changed to the serving state, which can play a role in clinical treatment.
  • the object of the present invention is to provide a detection marker for the de-service state of cells, the marker includes any one or more of the following detectable snoRNA in animal and plant cells, preferably any two One or more than two kinds: See http://snoopy.med.miyazaki-u.ac.jp/ for snoRNA sequence.
  • Another object of the present invention is to provide a snoRNA marker detection method for detecting the de-service state of the above-mentioned cells, by which the state of the cells can be further evaluated.
  • Another object of the present invention is to provide a method for regulating snoRNA in organisms, by regulating the snoRNA to switch between the service state and the de-service state of the cell.
  • small nucleolar RNA small nucleolar RNA
  • detection reagent for preparing reagents or kits for detecting the service status of cells.
  • the servicing state includes an in-service state, an out-of-service state, or a transition thereof.
  • the snoRNA is derived from mammals, rodents, and primates.
  • the snoRNA is derived from human and mouse.
  • the snoRNA is organ, tissue, and cell specific snoRNA.
  • the organ is selected from the following group: joints, ligaments, tongue, salivary glands, parotid glands, mandibular glands, sublingual glands, pharynx, esophagus, stomach, small intestine, duodenum, jejunum, ileum , Large intestine, liver, gallbladder, mesenteric, pancreas, nasal cavity, pharynx, larynx, trachea, bronchus, lung, diaphragm, kidney, ureter, bladder, urethra, ovary, fallopian tube, uterus, vagina, placenta, testis, epididymis, Vas deferens, seminal vesicles, prostate, bulbar urethral glands, penis, scrotum, pituitary gland, pineal gland, thyroid, parathyroid, adrenal gland, pancreas, heart, arteries, veins, capillaries, lymphatic vessels, lymph
  • the tissue is selected from the following group: epithelial tissue (single-layer epithelial tissue, single-layer columnar epithelial tissue, pseudo-stratified ciliated epithelial tissue, stratified cubic epithelial tissue), connective tissue (fat tissue, Loose connective tissue, fibrous connective tissue, hyaline cartilage tissue, elastic cartilage tissue, fibrocartilage tissue, bone tissue, blood tissue), nerve tissue and muscle tissue (heart muscle, skeletal muscle, smooth muscle).
  • epithelial tissue single-layer epithelial tissue, single-layer columnar epithelial tissue, pseudo-stratified ciliated epithelial tissue, stratified cubic epithelial tissue
  • connective tissue fat tissue, Loose connective tissue, fibrous connective tissue, hyaline cartilage tissue, elastic cartilage tissue, fibrocartilage tissue, bone tissue, blood tissue
  • nerve tissue and muscle tissue herein, skeletal muscle, smooth muscle.
  • the cells include cells derived from endoderm, including exocrine epithelial cells, barrier cells, and hormone secreting cells.
  • Exocrine epithelial cells include, horuner cells in the duodenum, goblet cells in the respiratory and digestive tract, pit cells in the stomach, principal cells, parietal cells, pancreatic acinar cells, small intestinal Paneth cells, and lung II Type alveolar cells, rod-shaped cells in the lungs.
  • Barrier cells include type I lung cells, gallbladder epithelial cells, vesicle heart cells, intercalary tube cells, and intestinal brush border cells.
  • hormone secreting cells enteroendocrine cells, thyroid cells, parathyroid cells, and pancreatic islet cells.
  • Enteroendocrine cells include K cells, L cells, I cells, G cells, enterochromaffin cells, enterochromaffin-like cells, N cells, S cells, D cells, and Mo cells; thyroid cells include thyroid epithelial cells and parafollicular cells Cells; parathyroid cells include parathyroid main cells, eosinophils; pancreatic islet cells include Alpha cells, Beta cells, Delta cells, Epsilon cells, and pp cells.
  • the cells derived from the ectoderm mainly include exocrine epithelial cells, hormone secreting cells, epithelial cells, and nervous system cells.
  • exocrine cells include salivary gland mucin cells, salivary gland plasma cells, tongue Fengaibone gland cells, breast cells, lacrimal gland cells, ear wax gland cells, exocrine sweat gland dark cells, exocrine sweat gland bright cells, apocrine sweat gland cells, and eyelid ink Glandular cells, adipocytes, horuna cells of the duodenum.
  • Hormone secreting cells include corticotropin cells, gonadotropin cells, prolactin cells, melanocytes, growth hormone cells, thyroid stimulating cells in the anterior and middle lobe of the pituitary; large cell neurosecretory cells, small cell neurosecretory cells, Chromium cells.
  • Epithelial cells include keratinocytes, epidermal basal cells, melanocytes, medullary hair cells, cortical hair axon cells, epidermal hair axon cells, Huxley layer hair root sheath cells, outer root sheath hair cells, surface epithelial cells, base cells , Intercalated tube cells, striated tube cells, lactiferous duct cells, ameloblasts.
  • Cells in the nervous system are divided into five categories, sensor cells, autonomic nerve cells, sensory organs and peripheral neuron supporting cells, central nervous system neurons and glial cells, and lens cells.
  • Sensor cells include cortical auditory inner hair cells, cortical auditory outer hair cells, olfactory epithelial basal cells, cold-sensitive primary sensory neurons, heat-sensitive primary sensory neurons, epidermal Merkel cells, olfactory receptor neurons, and pain-sensitive primary Sensory neurons, proprioceptive primary sensory neurons, tactile sensitive primary sensory neurons, carotid somatic chemical receptor bulb cells, outer hair cells of the vestibular system of the ear, inner hair cells of the vestibular system of the ear, taste-receiving cells of taste buds , Retina photoreceptor cells, retinal photoreceptor cells can be subdivided into photoreceptor rod cells, photoreceptor blue-sensitive cone cells, photoreceptor green-sensitive cone cells, and photoreceptor red-sensitive cone cells.
  • Autonomic nerve cells include cholinergic nerve cells, adrenergic nerve cells, and polypeptide nerve cells.
  • Sensory organs and peripheral neuron supporting cells include cortical inner pillar cells, outer cortical pillar cells, inner cortical finger cells, outer cortical finger cells, cortical border cells, cortical Hensen cells, vestibular supporting cells, taste bud supporting cells, olfactory epithelial supporting cells, Schwann cells, satellite glial cells, enteric glial cells.
  • Central nervous system neurons and glial cells include neuronal cells, astrocytes, oligodendrocytes, ependymal cells, and pituitary cells. Neuronal cells can be divided into interneurons and main cells.
  • Neurons include basket cells, wheel cells, stellate cells, Golgi cells, granular cells, Lugaro cells, unipolar brush cells, Martinotti cells, chandelier cells, Cajal–Retzius cells, Double-bouquet cells, neuroglia Plasma cells, horizontal retinal cells, amacrine cells, spinal interneurons, runshock cells; main cells include spindle neurons, fork neurons, pyramidal cells, stellate cells, border cells, hairy cells, Purkinje cells , Medium-sized spiny neurons, pyramidal cells include location cells, positioning cells, velocity cells, direction recognition cells, and giant pyramidal cells. Lens cells include lens epithelial cells and lens fiber cells containing crystallin.
  • Cells derived from mesoderm mainly include metabolism and storage cells, secretory cells, barrier cells, extracellular matrix cells, contractile cells, blood and immune system cells, germ cells, trophoblast cells, and mesenchymal tissue cells.
  • Metabolism and storage cells include white fat cells, brown fat cells and liver fat cells.
  • Secretory cells include adrenal cortex globular zone cells to produce mineralocorticoids, adrenal cortex fascicular zone cells to produce glucocorticoids, adrenal cortex reticular zone cells to produce androgens, three types of adrenal cortex cells, as well as ovarian follicular inner membrane cells, granular lutein cells , Luteal cells, testicular stromal cells, seminal vesicle cells, prostate cells, globular gland cells, Pap cells, gland cells around the urethra or urethra, endometrial cells, paraglomerular cells, renal dense plaque cells, pericytes, Mesangial cells of kidney. Barrier cells can be divided into three types.
  • the urinary system includes podocytes, proximal tubule brush border cells, Henry's ring segment cells, distal renal tubule cells, master cells in renal collecting duct cells, intercalated cells, and transitional epithelium.
  • Cells; the reproductive system includes duct cells, efferent duct cells, epididymal primary cells, epididymal basal cells; endothelial cells in the circulatory system.
  • Extracellular matrix cells include ear vestibular semicircular canal epithelial cells, cortical interdental epithelial cells, loose connective tissue fibroblasts, corneal fibroblasts, tendon fibroblasts, bone marrow reticular tissue fibroblasts, other non-epithelial fibroblasts, Pericytes, such as: hepatic stellate cells, intervertebral disc nucleus pulposus cells, hyaline chondrocytes, fibrochondrocytes, elastic chondrocytes, osteoblasts, osteoprogenitor cells, ocular vitreous clear cells, extra-auricular lymphatic space stellate cells, pancreatic stars Shaped cells.
  • Contractile cells include red skeletal muscle cells, white skeletal muscle cells, intermediate skeletal muscle cells, muscle spindle nuclear pocket cells, muscle spindle nuclear chain cells, muscle satellite cells, six types of skeletal muscle cells, cardiomyocytes, sinoatrial node cells, Purkinje Fibroblasts include three types of cardiomyocytes, as well as smooth muscle cells, iris myoepithelial cells, and exocrine adenomyotic myoepithelial cells.
  • Blood and immune system cells include red blood cells, megakaryocytes, platelets, monocytes, macrophages in connective tissue, Langerhans cells in the epidermis, osteoclasts, dendritic cells, microglia, and neutral Granulocytes, eosinophils, basophils, hybridoma cells, mast cells, helper T cells, suppressor T cells, cytotoxic T cells, natural killer T cells, B cells, natural killer cells, reticulocytes, Stem and progenitor cells of the blood and immune system.
  • Germ cells include oocytes/oocytes, sperm cells, spermatogonia, spermatocytes, and sperm.
  • Trophoblasts include granular cells in the ovary, supporting cells in the testis, and epithelial reticular cells. Almost tissue cells include almost tissue kidney cells.
  • the snoRNA is one or more of those shown in Table 1, or a combination thereof.
  • the snoRNA is selected from the group consisting of SNORA41, SNORD85, SNORD115, SNORD116, SNORD14c, SNORD14d, SNORD55, SNORD15b, SNORD23, SNORD96a, SNORD123, SNORD34, SNORD82.
  • a method for determining molecular markers of servicing status which includes the steps:
  • the first service status is in service status.
  • the second service state is an unserved state.
  • the snoRNA is derived from mammals, rodents, and primates.
  • the snoRNA is derived from human and mouse.
  • the snoRNA is organ, tissue, and cell specific snoRNA.
  • the organs include joints, ligaments, tongue, salivary glands, parotid glands, mandibular glands, sublingual glands, pharynx, esophagus, stomach, small intestine, duodenum, jejunum, ileum, large intestine, liver , Gallbladder, mesenteric, pancreas, nasal cavity, pharynx, larynx, trachea, bronchus, lung, diaphragm, kidney, ureter, bladder, urethra, ovary, fallopian tube, uterus, vagina, placenta, testis, epididymis, vas deferens, seminal vesicle, Prostate, bulbar urethral gland, penis, scrotum, pituitary gland, pineal gland, thyroid, parathyroid, adrenal gland, pancreas, heart, arteries, veins, capillaries, lymphatic vessels, lymph nodes, bone
  • the tissues include epithelial tissues (single-layer epithelial tissue, single-layer columnar epithelial tissue, pseudo-stratified ciliated epithelial tissue, stratified cubic epithelial tissue), connective tissue (fat tissue, loose connective tissue, Fibrous connective tissue, hyaline cartilage tissue, elastic cartilage tissue, fibrocartilage tissue, bone tissue, blood tissue), nerve tissue and muscle tissue (myocardium, skeletal muscle, smooth muscle).
  • epithelial tissues single-layer epithelial tissue, single-layer columnar epithelial tissue, pseudo-stratified ciliated epithelial tissue, stratified cubic epithelial tissue
  • connective tissue fat tissue, loose connective tissue, Fibrous connective tissue, hyaline cartilage tissue, elastic cartilage tissue, fibrocartilage tissue, bone tissue, blood tissue
  • nerve tissue and muscle tissue myocardium, skeletal muscle, smooth muscle.
  • the cells include cells derived from endoderm, including exocrine epithelial cells, barrier cells, and hormone secreting cells.
  • Exocrine epithelial cells include, horuner cells in the duodenum, goblet cells in the respiratory and digestive tract, pit cells in the stomach, principal cells, parietal cells, pancreatic acinar cells, small intestinal Paneth cells, and lung II Type alveolar cells, rod-shaped cells in the lungs.
  • Barrier cells include type I lung cells, gallbladder epithelial cells, vesicle heart cells, intercalary tube cells, and intestinal brush border cells.
  • hormone secreting cells enteroendocrine cells, thyroid cells, parathyroid cells, and pancreatic islet cells.
  • Enteroendocrine cells include K cells, L cells, I cells, G cells, enterochromaffin cells, enterochromaffin-like cells, N cells, S cells, D cells, and Mo cells; thyroid cells include thyroid epithelial cells and parafollicular cells Cells; parathyroid cells include parathyroid main cells, eosinophils; pancreatic islet cells include Alpha cells, Beta cells, Delta cells, Epsilon cells, and pp cells.
  • the cells derived from the ectoderm mainly include exocrine epithelial cells, hormone secreting cells, epithelial cells, and nervous system cells.
  • exocrine cells include salivary gland mucin cells, salivary gland plasma cells, tongue Fengaibone gland cells, breast cells, lacrimal gland cells, ear wax gland cells, exocrine sweat gland dark cells, exocrine sweat gland bright cells, apocrine sweat gland cells, and eyelid ink Glandular cells, adipocytes, horuna cells of the duodenum.
  • Hormone secreting cells include corticotropin cells, gonadotropin cells, prolactin cells, melanocytes, growth hormone cells, thyroid stimulating cells in the anterior and middle lobe of the pituitary; large cell neurosecretory cells, small cell neurosecretory cells, Chromium cells.
  • Epithelial cells include keratinocytes, epidermal basal cells, melanocytes, medullary hair cells, cortical hair axon cells, epidermal hair axon cells, Huxley layer hair root sheath cells, outer root sheath hair cells, surface epithelial cells, base cells , Intercalated tube cells, striated tube cells, lactiferous duct cells, ameloblasts.
  • Cells in the nervous system are divided into five categories, sensor cells, autonomic nerve cells, sensory organs and peripheral neuron supporting cells, central nervous system neurons and glial cells, and lens cells.
  • Sensor cells include cortical auditory inner hair cells, cortical auditory outer hair cells, olfactory epithelial basal cells, cold-sensitive primary sensory neurons, heat-sensitive primary sensory neurons, epidermal Merkel cells, olfactory receptor neurons, and pain-sensitive primary Sensory neurons, proprioceptive primary sensory neurons, tactile sensitive primary sensory neurons, carotid somatic chemical receptor bulb cells, outer hair cells of the vestibular system of the ear, inner hair cells of the vestibular system of the ear, taste-receiving cells of taste buds , Retina photoreceptor cells, retinal photoreceptor cells can be subdivided into photoreceptor rod cells, photoreceptor blue-sensitive cone cells, photoreceptor green-sensitive cone cells, and photoreceptor red-sensitive cone cells.
  • Autonomic nerve cells include cholinergic nerve cells, adrenergic nerve cells, and polypeptide nerve cells.
  • Sensory organs and peripheral neuron supporting cells include cortical inner pillar cells, outer cortical pillar cells, inner cortical finger cells, outer cortical finger cells, cortical border cells, cortical Hensen cells, vestibular supporting cells, taste bud supporting cells, olfactory epithelial supporting cells, Schwann cells, satellite glial cells, enteric glial cells.
  • Central nervous system neurons and glial cells include neuronal cells, astrocytes, oligodendrocytes, ependymal cells, and pituitary cells. Neuronal cells can be divided into interneurons and main cells.
  • Neurons include basket cells, wheel cells, stellate cells, Golgi cells, granular cells, Lugaro cells, unipolar brush cells, Martinotti cells, chandelier cells, Cajal–Retzius cells, Double-bouquet cells, neuroglia Plasma cells, horizontal retinal cells, amacrine cells, spinal interneurons, runshock cells; main cells include spindle neurons, fork neurons, pyramidal cells, stellate cells, border cells, hairy cells, Purkinje cells , Medium-sized spiny neurons, pyramidal cells include location cells, positioning cells, velocity cells, direction recognition cells, and giant pyramidal cells. Lens cells include lens epithelial cells and lens fiber cells containing crystallin.
  • Cells derived from mesoderm mainly include metabolism and storage cells, secretory cells, barrier cells, extracellular matrix cells, contractile cells, blood and immune system cells, germ cells, trophoblast cells, and mesenchymal tissue cells.
  • Metabolism and storage cells include white fat cells, brown fat cells and liver fat cells.
  • Secretory cells include adrenal cortex globular zone cells to produce mineralocorticoids, adrenal cortex fascicular zone cells to produce glucocorticoids, adrenal cortex reticular zone cells to produce androgens, three types of adrenal cortex cells, as well as ovarian follicular inner membrane cells, granular lutein cells , Luteal cells, testicular stromal cells, seminal vesicle cells, prostate cells, globular gland cells, Pap cells, gland cells around the urethra or urethra, endometrial cells, paraglomerular cells, renal dense plaque cells, pericytes, Mesangial cells of kidney. Barrier cells can be divided into three types.
  • the urinary system includes podocytes, proximal tubule brush border cells, Henry's ring segment cells, distal renal tubule cells, master cells in renal collecting duct cells, intercalated cells, and transitional epithelium.
  • Cells; the reproductive system includes duct cells, efferent duct cells, epididymal primary cells, epididymal basal cells; endothelial cells in the circulatory system.
  • Extracellular matrix cells include ear vestibular semicircular canal epithelial cells, cortical interdental epithelial cells, loose connective tissue fibroblasts, corneal fibroblasts, tendon fibroblasts, bone marrow reticular tissue fibroblasts, other non-epithelial fibroblasts, Pericytes, such as: hepatic stellate cells, intervertebral disc nucleus pulposus cells, hyaline chondrocytes, fibrochondrocytes, elastic chondrocytes, osteoblasts, osteoprogenitor cells, ocular vitreous clear cells, extra-auricular lymphatic space stellate cells, pancreatic stars Shaped cells.
  • Contractile cells include red skeletal muscle cells, white skeletal muscle cells, intermediate skeletal muscle cells, muscle spindle nuclear pocket cells, muscle spindle nuclear chain cells, muscle satellite cells, six types of skeletal muscle cells, cardiomyocytes, sinoatrial node cells, Purkinje Fibroblasts include three types of cardiomyocytes, as well as smooth muscle cells, iris myoepithelial cells, and exocrine adenomyotic myoepithelial cells.
  • Blood and immune system cells include red blood cells, megakaryocytes, platelets, monocytes, macrophages in connective tissue, Langerhans cells in the epidermis, osteoclasts, dendritic cells, microglia, and neutral Granulocytes, eosinophils, basophils, hybridoma cells, mast cells, helper T cells, suppressor T cells, cytotoxic T cells, natural killer T cells, B cells, natural killer cells, reticulocytes, Stem and progenitor cells of the blood and immune system.
  • Germ cells include oocytes/oocytes, sperm cells, spermatogonia, spermatocytes, and sperm.
  • Trophoblasts include granular cells in the ovary, supporting cells in the testis, and epithelial reticular cells. Almost tissue cells include almost tissue kidney cells.
  • the snoRNA is selected from the group consisting of SNORA41, SNORD85, SNORD115, SNORD116, SNORD14c, SNORD14d, SNORD55, SNORD15b, SNORD23, SNORD96a, SNORD123, SNORD34, SNORD82.
  • the method is non-diagnostic and non-therapeutic.
  • snoRNA or its promoter or antagonist for the preparation of drugs or preparations for regulating the service status of cell tissues or organs.
  • the servicing state includes an in-service state, an out-of-service state, or a transition thereof.
  • the snoRNA is one or more of those shown in Table 1, or a combination thereof.
  • the snoRNA is selected from the group consisting of SNORA41, SNORD85, SNORD115, SNORD116, SNORD14c, SNORD14d, SNORD55, SNORD15b, SNORD23, SNORD96a, SNORD123, SNORD34, SNORD82.
  • Figure 1 shows the RT-PCR results of some snoRNAs detected in various tissues of normal mice.
  • Figure 2 shows the real-time PCR results of some snoRNAs detected in various tissues of normal mice.
  • Figure 3 shows the expression changes of leptin gene at different time points after adipocyte culture in vitro.
  • Figure 4 shows the snoRNA expression profiles of adipocytes in different states detected by high-throughput sequencing.
  • SnoRNA a class of molecular markers used to diagnose cell state.
  • it studies the changes in the cell state after regulating the expression of snoRNA, and provides a method for transforming the service state and de-service state of cells for biological experiments and clinical applications. The present invention has been completed on this basis.
  • differentiated cells In the concept of classical cell biology, all mature cells with specific functions are called differentiated cells. If differentiated cells lack specific functions for regulating homeostasis or serving the internal environment, they are now simply called For "de-differentiation", it is neither different from real undifferentiated cells such as precursor cells, nor distinguished by specific molecular markers.
  • the present invention has discovered that differentiated cells have two states: one is enmunting (with the service function of regulating homeostasis) and the other is the demunting state (demunting, without the service function of regulating homeostasis). Both of these states are differentiated states, so the survival and metabolism of cells (related gene expression and protein synthesis) are the same, and there is a difference in service functions.
  • demunting cells and precursor cells are that they have different survival metabolic states (precursor cells have not yet differentiated), but the same state of lack of specific service functions.
  • the increase of tissue-specific SnoRNA is a biomarker for identifying demunting cells; increasing SnoRNA can change the enmunting state of the cell to the demunting state; decreasing SnoRNA can change the demunting state of the cell to the enmunting state. All tissue cells have these two states at the same time. Under most pathological conditions, the proportion of demunting cells will increase, leading to organ failure. Reducing the pathologically increased demunting cells will have a therapeutic effect on all histopathological failures such as the arousal of patients with cerebral death, the growth of functional nerves in patients with paraplegia, and the increase of the proportion of activated functional neurons in the brain.
  • the present invention discovers two states of differentiated cells (in serving state and de-serving state), distinguishing between de-serving state cells and undifferentiated cells, and the study of these two states can explain the original biological and medical research Many unexplainable phenomena in
  • the present invention can quickly and simply identify the in-service state and the un-served state of cells, avoiding the need for complex studies on cell function, state, transcriptome and proteome changes to identify this Two cell states;
  • the present invention can effectively transform the in-service state and the out-of-service state of cells, and has huge application prospects in biological research and clinical application;
  • the present invention further discovers the important regulatory function of snoRNA in organisms, and provides a new direction for biological theoretical research and application research.
  • the detection method for detecting the snoRNA marker in the de-service state of cells is selected from the group consisting of reverse transcription polymerase chain reaction (RT-PCR), real-time fluorescent quantitative reverse transcription polymerase chain reaction (Real-time RT-PCR), Northern blot hybridization method (Northern blotting), high-throughput sequencing technology or biochip method.
  • RT-PCR reverse transcription polymerase chain reaction
  • Real-time RT-PCR real-time fluorescent quantitative reverse transcription polymerase chain reaction
  • Northern blot hybridization method Northern blotting
  • high-throughput sequencing technology biochip method.
  • the RT-PCR method is a preferred method and includes the following steps:
  • the Real-time PCR method is another preferred method and includes the following steps:
  • the Nortern blotting method includes the following steps:
  • the high-throughput sequencing method includes the following steps:
  • the biochip method includes the following steps:
  • the control method is selected from interference RNA control method and CRISPR/Cas9 control method.
  • the preferred method for interfering RNA regulation includes the following steps:
  • siRNA sequence Design interfering RNA (siRNA sequence) and shRNA sequence capable of expressing interfering RNA (see excel for siRNA sequence and shRNA sequence);
  • the CRISPR/Cas9 regulation method is another preferred method and includes the following steps:
  • RT-PCR technology is used to detect the snoRNA content in human and animal cells. The specific steps are as follows:
  • the reverse transcription reaction system includes 2 ⁇ l 5 ⁇ AMV buffer, 1 ⁇ l dNTP mixture, 0.5 ⁇ l RNAase Inhibitor, 0.5 ⁇ l AMV enzyme, 1 ⁇ l gene-specific reverse primer, 1 ⁇ g total RNA and supplemental RNAase free water to a total volume of 10 ⁇ l.
  • the reaction steps include incubating at 16°C for 15 minutes, reacting at 42°C for 1 hour, and incubating at 85°C for 5 minutes.
  • Figure 1 The results are shown in Figure 1, the results of RT-PCR by extracting total RNA from multiple tissues of normal mice.
  • the inventors selected more than 900 snoRNAs from mice for PCR reaction.
  • Figure 1 shows four snoRNAs (SNORA41, SNORD85, SNORD115 and SNORD116). It can be seen from the results that these four snoRNAs can be detected in the heart, liver, spleen, lung, kidney, brain, and muscle.
  • real-time PCR technology is used to detect the snoRNA content in human and animal cells.
  • Real-time PCR experiment principle and experiment procedure are the same as RT-PCR, the only difference is that the fluorescent dye Eva Green is added during PCR.
  • the instrument used was ABI Prism7300 fluorescent quantitative PCR instrument, and the reaction conditions were 95°C, 5 minutes for one cycle ⁇ 95°C, 15 seconds, 60°C, for 40 cycles in 1 minute.
  • the data processing method is the ⁇ CT method, and CT is set as the number of cycles when the reaction reaches the threshold.
  • FIG. 2 The result is shown in Figure 2.
  • the inventors used Real-time PCR to detect the expression levels of all mouse snoRNA in various tissues.
  • Figure 2 only shows the expression levels of SNORA41, SNORD85, SNORD115 and SNORD116 in the heart, liver, spleen, lung, kidney, brain and muscle, respectively.
  • U6 was used as an internal reference in the experiment. It can be known from the test results that SNORA41 is highly expressed in the liver, SNORD85 is highly expressed in the lung, spleen, muscle and kidney, and SNORD115 and SNORD116 are specifically highly expressed in brain tissue. From Figure 2, there are tissue-specific snoRNAs in various tissues, and the differential expression of these snoRNAs may be related to the differences in the functions performed by different tissues in the body.
  • the inventors used a high-throughput sequencing method to detect the snoRNA expression profile of adipocytes at different time points after in vitro culture.
  • the specific experimental steps are as follows:
  • mice Collect white adipose tissue of mice in different states. After being reduced into small pieces, adipocytes are collected at 0 hour, 3 hours, 6 hours, 9 hours, 12 hours and 24 hours, and total RNA and total protein are extracted.
  • RNA Isolate small RNA and build a library.
  • the total RNA was cut into gel using PAGE electrophoresis to separate the 18-30nt RNA.
  • Add digestive enzymes to remove the 3'linker.
  • Use RT primers for reverse transcription extension Synthesize one-strand cDNA.
  • Use high-sensitivity polymerase to amplify cDNA, enrich the cDNA with 3'linker and 5'linker at the same time, amplify the library yield.
  • Use PAGE electrophoresis to separate the 100-120bp range PCR product, effectively remove the primer By-products such as dimers. Perform quality inspection on the constructed library, and sequence the library qualified by the quality inspection.
  • FIG. 3 The results are shown in Figure 3.
  • the present inventors detected the expression levels of adipocyte-specific service genes such as leptin in adipocytes at different time points.
  • Figure 2 only shows the changes in the expression level of the leptin gene.
  • the results showed that as the time of in vitro culture increased, both the mRNA level and the protein level of leptin gene decreased until no longer expressed. It shows that the adipocytes changed from the serving state to the unserved state after being isolated.
  • the present inventors used restriction endonucleases to process the control plasmid, after recovering the linear vector, ligated the RVG-SNORD115 shRNA combined fragment with the vector using T4 ligase; the resulting ligation product was subjected to transformation experiments and spread on the resistant plate ; On the second day, a single clone was picked and sequenced to confirm the correctness of the plasmid sequence.
  • the control plasmid and SNORD115 siRNA/RVG plasmid were injected into the tail vein of normal mice at a dose of 10 mg/kg; 12 hours later, the mice were sacrificed, and the liver tissue and brain tissue of the mice were respectively subjected to in situ hybridization experiments.
  • a green fluorescent modification was added to the sequence that is completely complementary to the SNORD115 siRNA sequence as a detection probe to indicate the distribution of siRNA in tissue sections; the results show that there are a large number of siRNA in the liver tissue of mice Distribution, and a small amount of siRNA can also be detected in brain tissue, proving that SNORD115 siRNA/RVG plasmid does have the effect of brain targeting.
  • the present inventors also injected the control plasmid and SNORD115 siRNA/RVG plasmid into the tail vein of mice at a dose of 10 mg/kg, once every two days. A total of 7 injections, the mice were sacrificed 24 hours after the last injection, and the brain tissues of the mice were taken for Real-time PCR experiments. The results show that plasmid molecules can effectively reduce the expression level of SNORD115 in brain tissue in vivo.

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Abstract

本发明提供了一种核仁小RNA(snoRNA)或其检测试剂的用途，用于检测细胞服务化状态，并建立了snoRNA的检测和调控方法。

Description

细胞去服务化状态的分子标记物检测及其调控方法 技术领域
本发明属于生物技术领域，具体涉及人和动物组织和细胞中用于区分细胞去服务状态的核仁小核糖核酸分子的分离、定性和定量分析，同时也涉及到调控人和动物组织和细胞中的核仁小核糖核酸用于改变细胞去服务状态的应用方法。
背景技术
核仁小RNA(snoRNA)是一类中等长度的非编码小RNA,它们的长度在60-300nt不等，能与核仁核糖核蛋白结合形成snoRNP复合物。在脊椎动物中编码核仁小RNA的基因主要存在于蛋白编码基因或非蛋白编码基因的内含子区域，并且经过进一步的转录后加工处理形成成熟的核仁小RNA。在人类细胞中的，RNA的转录后修饰、mRNA的稳定性和翻译的控制是基因表达调控的重要组成部分。snoRNA和它们产生的功能短片段RNA在这些过程中发挥着重要作用：它们指导核糖体RNA(rRNA)和核小RNA(snRNA)的修饰、影响其互补前体mRNA的剪接以及控制mRNA的翻译和稳定性。外界因素以及细胞内部的信号级联反应能够导致snoRNA表达水平的变化，进而引起细胞水平的生理变化、器官功能障碍以及各种疾病。通过对snoRNA的检测与调控，可以有效的确定并改变细胞的服务化与去服务化状态。
由于关于细胞不同服务状态的研究才刚刚开始，目前尚无具体有效界定细胞服务化状态和去服务化状态的有效分子标志物。
因此，本领域迫切需要建立有效的snoRNA的检测和调控方法。
发明内容
目前，在医学临床过程中，有很多生理现象无法解释，如肝炎患者不能过度疲劳、肾肿瘤病人尽管肿瘤病灶很小但仍会因肾衰而死、植物人虽然没有正常反射但是生理状态正常等等一系列的现象。而通过对细胞去服务化状态的研究，可以进一步帮助更好解释这些问题。而这一系列的研究前提就是需要寻找到一种能够标识这种状态的标志物。
本发明人选取小核仁核糖核酸作为研究对象，希望通过检测细胞中小核仁核糖核酸的表达变化来区分这一现象，同时更希望通过对细胞中小核仁核糖核酸的表达进行调控进一步将细胞从去服务化状态改变为服务化状态，从而能在临床上起 治疗的作用。
本发明的目的首先是提供一种细胞去服务化状态的检测标记物，所述标记物包括以下在动物、植物细胞中存在且可检测的snoRNA中的任意一种或一种以上，优选任意两种或两种以上：snoRNA序列见http://snoopy.med.miyazaki-u.ac.jp/。
本发明的另一个目的是提供检测上述细胞去服务状态的snoRNA标记物检测方法，通过该方法可以进一步评价细胞的状态。
本发明的另一个目的是提供调控生物体内snoRNA的方法，通过对snoRNA进行调控对细胞的服务状态和去服务状态进行转换。
在本发明的第一方面，提供了一种核仁小RNA(snoRNA)或其检测试剂的用途，用于制备检测细胞服务化状态的试剂或试剂盒。
在另一优选例中，所述的服务化状态包括在服状态、去服状态、或其转换。
在另一优选例中，所述的snoRNA来源于哺乳动物、啮齿动物、灵长类动物。
在另一优选例中，所述的snoRNA来源于人、小鼠。
在另一优选例中，所述的snoRNA为器官、组织、细胞特异性的snoRNA。
在另一优选例中，所述的器官选自下组：关节、韧带、舌、唾腺、腮腺、下颌腺、舌下腺、咽、食道、胃、小肠、十二指肠、空肠、回肠、大肠、肝脏、胆囊、肠系膜、胰脏、鼻腔、咽、喉、气管、支气管、肺、横膈膜、肾、输尿管、膀胱、尿道、卵巢、输卵管、子宫、阴道、胎盘、睾丸、附睾、输精管、精囊、前列腺、尿道球腺、阴茎、阴囊、脑下垂体、松果体、甲状腺、副甲状腺、肾上腺、胰腺、心脏、动脉、静脉、微血管、淋巴管、淋巴结、骨髓、胸腺、脾脏、肠相关淋巴组织、扁桃腺、大脑、间脑、脑干、中脑、桥脑、延髓、小脑、脊髓、脑室、脉络丛、脑神经、脊神经、神经节、肠神经系统、角膜、虹膜、睫状体、晶状体、视网膜、耳垂、鼓膜、听小骨、耳蜗、耳前庭、半规管、舌、皮肤等。
在另一优选例中，所述的组织选自下组：上皮组织(单层上皮组织、单层柱状上皮组织、假复层纤毛上皮组织、复层立方上皮组织)、结缔组织(脂肪组织、疏松结缔组织、纤维结缔组织、透明软骨组织、弹性软骨组织、纤维软骨组织、骨组织、血液组织)、神经组织和肌肉组织(心肌、骨骼肌、平滑肌)。
在另一优选例中，所述的细胞包括来源于内胚层的细胞包括外分泌上皮细胞，屏障细胞，激素分泌细胞。外分泌上皮细胞包括，十二指肠的歩茹呐氏细胞，呼吸道、消化道杯状细胞，胃部的小凹细胞，主细胞，壁细胞，胰腺腺泡细胞，小肠帕内特细胞，肺Ⅱ型肺泡细胞，肺部棒状细胞。屏障细胞包括I型肺细胞，胆囊上皮细胞，泡 心细胞，闰管细胞，肠刷状缘细胞。激素分泌细胞有肠内分泌细胞，甲状腺细胞，甲状旁腺细胞，胰岛细胞四类。肠内分泌细胞包括K细胞，L细胞，I细胞，G细胞，肠嗜铬细胞，肠嗜铬细胞样细胞，N细胞，S细胞，D细胞，Mo细胞；甲状腺细胞包括甲状腺上皮细胞，滤泡旁细胞；甲状旁腺细胞包括甲状旁腺主细胞，嗜酸细胞；胰岛细胞包括Alpha细胞，Beta细胞，Delta细胞，Epsilon细胞，pp细胞。来源于外胚层的细胞主要有外分泌上皮细胞，激素分泌细胞，上皮细胞，神经系统细胞等。其中外分泌细胞包括唾液腺黏液细胞，唾液腺浆液细胞，舌部的冯哎波讷腺细胞，乳腺细胞，泪腺细胞，耳的耳垢腺细胞，外分泌汗腺暗细胞，外分泌汗腺亮细胞，顶泌汗腺细胞，眼睑的墨氏腺细胞，脂肪腺细胞，十二指肠的歩茹呐氏细胞。激素分泌细胞包括垂体前部及中叶的促皮质激素细胞、促性腺细胞、催乳素细胞、促黑激素细胞、生长激素细胞、促甲状腺细胞；大细胞性神经分泌细胞，小细胞神经分泌细胞，嗜铬细胞。上皮细胞包括角质形成细胞，表皮基底细胞，黑素细胞，髓质毛细胞，皮质毛轴突细胞，表皮毛轴突细胞，Huxley层毛根鞘细胞，外根鞘毛细胞，表面上皮细胞，基地细胞，闰管细胞，纹状管细胞，输乳管细胞，成釉细胞。神经系统中的细胞分为五类，传感器细胞，自主神经细胞，感觉器官和外周神经元支持细胞，中枢神经系统神经元和神经胶质细胞，晶状体细胞。传感器细胞包括皮质听觉内毛细胞，皮质听觉外毛细胞，嗅上皮基底细胞，冷敏感的初级感觉神经元，热敏感的初级感觉神经元，表皮Merkel细胞，嗅觉受体神经元，疼痛敏感的初级感觉神经元，本体感觉初级感觉神经元，触感敏感的初级感觉神经元，颈动脉体细胞化学受体球细胞，耳前庭系统的外毛细胞，耳前庭系统的内毛细胞，味蕾的味觉感受细胞，视网膜感光细胞，视网膜感光细胞可细分为感光器杆细胞，光感受器蓝色敏感的视锥细胞，光感受器绿色敏感的视锥细胞，光感受器红色敏感的视锥细胞。自主神经细胞包括胆碱能神经细胞，肾上腺素能神经细胞，多肽神经细胞。感觉器官和外周神经元支持细胞包括皮质内柱细胞，皮质外柱细胞，皮质内指细胞，皮质外指细胞，皮质边缘细胞，皮质Hensen细胞，前庭支持细胞，味蕾支持细胞，嗅上皮支持细胞，施万细胞，卫星神经胶质细胞，肠神经胶质细胞。中枢神经系统神经元和神经胶质细胞包括神经元细胞，星形胶质细胞，少突胶质细胞，室管膜细胞，垂体细胞，其中神经元细胞可分为中间神经元和主细胞，中间神经元包括篮状细胞，车轮状细胞，星状细胞，高尔基细胞，颗粒细胞，Lugaro细胞，单极刷细胞，Martinotti细胞，枝形吊灯细胞，Cajal–Retzius细胞，Double-bouquet细胞，神经胶质细胞，视网膜水平细胞，无长突细胞，脊髓中间神经元，闰绍细胞；主细胞包括主轴神经元，叉神经元，锥体细胞，星状细胞，边界细胞，多毛细胞，浦肯野细胞，中型多刺神经元，锥体细胞包括位置细胞，定位细胞，速度 细胞，方向辨识细胞，巨大锥体细胞。晶状体细胞包括晶状体上皮细胞和含结晶素的晶状体纤维细胞。来源于中胚层的细胞主要包括代谢与储存细胞，分泌细胞，屏障细胞，细胞外基质细胞，收缩细胞，血液和免疫系统细胞，生殖细胞，滋养细胞，间质组织细胞九类。代谢与储存细胞包括白色脂肪细胞，褐色脂肪细胞和肝脏脂肪细胞。分泌细胞包括肾上腺皮质球状带细胞产生盐皮质激素，肾上腺皮质束状带细胞产生糖皮质激素，肾上腺皮质网状带细胞产生雄激素三种肾上腺皮质细胞以及卵巢卵泡内膜细胞，颗粒叶黄素细胞，黄体细胞，睾丸间质细胞，精囊细胞，前列腺细胞，球腺细胞，巴氏腺细胞，尿道或尿道周围腺体细胞，子宫内膜细胞，肾球旁细胞，肾致密斑细胞，肾极周细胞，肾系膜细胞。屏障细胞可分为三类，泌尿系统中包括足细胞，近端小管刷状缘细胞，亨利氏环细段细胞，肾远端小管细胞，肾集合管细胞中的主细胞以及闰细胞，移行上皮细胞；生殖系统中包括导管细胞，传出导管细胞，附睾主细胞，附睾基底细胞；循环系统中的内皮细胞。细胞外基质细胞包括耳前庭半规管上皮细胞，皮质齿间上皮细胞，松散的结缔组织成纤维细胞，角膜成纤维细胞，肌腱成纤维细胞，骨髓网状组织成纤维细胞，其他非上皮成纤维细胞，周细胞，如：肝星状细胞，椎间盘髓核细胞，透明软骨细胞，纤维软骨细胞，弹性软骨细胞，成骨细胞，骨祖细胞，眼玻璃体透明细胞，耳外淋巴间隙星状细胞，胰腺星状细胞。收缩细胞包括红色骨骼肌细胞，白色骨骼肌细胞，中间骨骼肌细胞，肌梭核袋细胞，肌梭核链细胞，肌卫星细胞六种骨骼肌细胞，心肌细胞，窦房结细胞，蒲肯野纤维细胞三种心肌细胞，以及平滑肌细胞，虹膜肌上皮细胞，外分泌腺肌上皮细胞。血液和免疫系统细胞包括红细胞，巨核细胞，血小板，单核细胞，结缔组织中的巨噬细胞，表皮中的朗格汉斯细胞，破骨细胞，树突状细胞，小胶质细胞，中性粒细胞，嗜酸性粒细胞，嗜碱性粒细胞，杂交瘤细胞，肥大细胞，辅助T细胞，抑制T细胞，细胞毒T细胞，自然杀伤T细胞，B细胞，自然杀伤细胞，网状细胞，血液及免疫系统的干细胞和祖细胞。生殖细胞包括卵原细胞/卵母细胞，精细胞，精原细胞，精母细胞，精子。滋养细胞包括卵巢中的颗粒细胞，睾丸中的支持细胞，上皮网状细胞。简直组织细胞包括简直组织肾细胞。
在另一优选例中，所述的snoRNA为表1所示的一种或多种、或其组合。
在另一优选例中，所述的snoRNA选自下组：SNORA41、SNORD85、SNORD115、SNORD116、SNORD14c、SNORD14d、SNORD55、SNORD15b、SNORD23、SNORD96a、SNORD123、SNORD34、SNORD82。
在本发明的第二方面，提供了一种确定服务化状态分子标志物的方法，包括步骤：
(1)提供第一服务状态下的细胞，检测所述第一服务状态下的snoRNA的种类和数量，从而获得第一数据集；
(2)提供第二服务状态下的细胞，检测所述第二服务状态下的snoRNA的种类和数量，从而获得第二数据集；
(3)将第一数据集和第二数据集进行比较，确定在第一服务状态和第二服务状态下特有的snoRNA的种类和数量信息，从而鉴别在第一服务状态和第二服务状态下特有的分子标志物。
在另一优选例中，所述的第一服务状态为在服状态。
在另一优选例中，所述的第二服务状态为去服状态。
在另一优选例中，所述的snoRNA来源于哺乳动物、啮齿动物、灵长类动物。
在另一优选例中，所述的snoRNA来源于人、小鼠。
在另一优选例中，所述的snoRNA为器官、组织、细胞特异性的snoRNA。
在另一优选例中，所述的器官包括关节、韧带、舌、唾腺、腮腺、下颌腺、舌下腺、咽、食道、胃、小肠、十二指肠、空肠、回肠、大肠、肝脏、胆囊、肠系膜、胰脏、鼻腔、咽、喉、气管、支气管、肺、横膈膜、肾、输尿管、膀胱、尿道、卵巢、输卵管、子宫、阴道、胎盘、睾丸、附睾、输精管、精囊、前列腺、尿道球腺、阴茎、阴囊、脑下垂体、松果体、甲状腺、副甲状腺、肾上腺、胰腺、心脏、动脉、静脉、微血管、淋巴管、淋巴结、骨髓、胸腺、脾脏、肠相关淋巴组织、扁桃腺、大脑、间脑、脑干、中脑、桥脑、延髓、小脑、脊髓、脑室、脉络丛、脑神经、脊神经、神经节、肠神经系统、角膜、虹膜、睫状体、晶状体、视网膜、耳垂、鼓膜、听小骨、耳蜗、耳前庭、半规管、舌、皮肤等。
在另一优选例中，所述的组织包括上皮组织(单层上皮组织、单层柱状上皮组织、假复层纤毛上皮组织、复层立方上皮组织)、结缔组织(脂肪组织、疏松结缔组织、纤维结缔组织、透明软骨组织、弹性软骨组织、纤维软骨组织、骨组织、血液组织)、神经组织和肌肉组织(心肌、骨骼肌、平滑肌)。
在另一优选例中，所述的细胞包括来源于内胚层的细胞包括外分泌上皮细胞，屏障细胞，激素分泌细胞。外分泌上皮细胞包括，十二指肠的歩茹呐氏细胞，呼吸道、消化道杯状细胞，胃部的小凹细胞，主细胞，壁细胞，胰腺腺泡细胞，小肠帕内特细胞，肺Ⅱ型肺泡细胞，肺部棒状细胞。屏障细胞包括I型肺细胞，胆囊上皮细胞，泡心细胞，闰管细胞，肠刷状缘细胞。激素分泌细胞有肠内分泌细胞，甲状腺细胞，甲状旁腺细胞，胰岛细胞四类。肠内分泌细胞包括K细胞，L细胞，I细胞，G细胞，肠嗜铬细胞，肠嗜铬细胞样细胞，N细胞，S细胞，D细 胞，Mo细胞；甲状腺细胞包括甲状腺上皮细胞，滤泡旁细胞；甲状旁腺细胞包括甲状旁腺主细胞，嗜酸细胞；胰岛细胞包括Alpha细胞，Beta细胞，Delta细胞，Epsilon细胞，pp细胞。来源于外胚层的细胞主要有外分泌上皮细胞，激素分泌细胞，上皮细胞，神经系统细胞等。其中外分泌细胞包括唾液腺黏液细胞，唾液腺浆液细胞，舌部的冯哎波讷腺细胞，乳腺细胞，泪腺细胞，耳的耳垢腺细胞，外分泌汗腺暗细胞，外分泌汗腺亮细胞，顶泌汗腺细胞，眼睑的墨氏腺细胞，脂肪腺细胞，十二指肠的歩茹呐氏细胞。激素分泌细胞包括垂体前部及中叶的促皮质激素细胞、促性腺细胞、催乳素细胞、促黑激素细胞、生长激素细胞、促甲状腺细胞；大细胞性神经分泌细胞，小细胞神经分泌细胞，嗜铬细胞。上皮细胞包括角质形成细胞，表皮基底细胞，黑素细胞，髓质毛细胞，皮质毛轴突细胞，表皮毛轴突细胞，Huxley层毛根鞘细胞，外根鞘毛细胞，表面上皮细胞，基地细胞，闰管细胞，纹状管细胞，输乳管细胞，成釉细胞。神经系统中的细胞分为五类，传感器细胞，自主神经细胞，感觉器官和外周神经元支持细胞，中枢神经系统神经元和神经胶质细胞，晶状体细胞。传感器细胞包括皮质听觉内毛细胞，皮质听觉外毛细胞，嗅上皮基底细胞，冷敏感的初级感觉神经元，热敏感的初级感觉神经元，表皮Merkel细胞，嗅觉受体神经元，疼痛敏感的初级感觉神经元，本体感觉初级感觉神经元，触感敏感的初级感觉神经元，颈动脉体细胞化学受体球细胞，耳前庭系统的外毛细胞，耳前庭系统的内毛细胞，味蕾的味觉感受细胞，视网膜感光细胞，视网膜感光细胞可细分为感光器杆细胞，光感受器蓝色敏感的视锥细胞，光感受器绿色敏感的视锥细胞，光感受器红色敏感的视锥细胞。自主神经细胞包括胆碱能神经细胞，肾上腺素能神经细胞，多肽神经细胞。感觉器官和外周神经元支持细胞包括皮质内柱细胞，皮质外柱细胞，皮质内指细胞，皮质外指细胞，皮质边缘细胞，皮质Hensen细胞，前庭支持细胞，味蕾支持细胞，嗅上皮支持细胞，施万细胞，卫星神经胶质细胞，肠神经胶质细胞。中枢神经系统神经元和神经胶质细胞包括神经元细胞，星形胶质细胞，少突胶质细胞，室管膜细胞，垂体细胞，其中神经元细胞可分为中间神经元和主细胞，中间神经元包括篮状细胞，车轮状细胞，星状细胞，高尔基细胞，颗粒细胞，Lugaro细胞，单极刷细胞，Martinotti细胞，枝形吊灯细胞，Cajal–Retzius细胞，Double-bouquet细胞，神经胶质细胞，视网膜水平细胞，无长突细胞，脊髓中间神经元，闰绍细胞；主细胞包括主轴神经元，叉神经元，锥体细胞，星状细胞，边界细胞，多毛细胞，浦肯野细胞，中型多刺神经元，锥体细胞包括位置细胞，定位细胞，速度细胞，方向辨识细胞，巨大锥体细胞。晶状体细胞包括晶状体上皮细胞和含结晶素的晶状体 纤维细胞。来源于中胚层的细胞主要包括代谢与储存细胞，分泌细胞，屏障细胞，细胞外基质细胞，收缩细胞，血液和免疫系统细胞，生殖细胞，滋养细胞，间质组织细胞九类。代谢与储存细胞包括白色脂肪细胞，褐色脂肪细胞和肝脏脂肪细胞。分泌细胞包括肾上腺皮质球状带细胞产生盐皮质激素，肾上腺皮质束状带细胞产生糖皮质激素，肾上腺皮质网状带细胞产生雄激素三种肾上腺皮质细胞以及卵巢卵泡内膜细胞，颗粒叶黄素细胞，黄体细胞，睾丸间质细胞，精囊细胞，前列腺细胞，球腺细胞，巴氏腺细胞，尿道或尿道周围腺体细胞，子宫内膜细胞，肾球旁细胞，肾致密斑细胞，肾极周细胞，肾系膜细胞。屏障细胞可分为三类，泌尿系统中包括足细胞，近端小管刷状缘细胞，亨利氏环细段细胞，肾远端小管细胞，肾集合管细胞中的主细胞以及闰细胞，移行上皮细胞；生殖系统中包括导管细胞，传出导管细胞，附睾主细胞，附睾基底细胞；循环系统中的内皮细胞。细胞外基质细胞包括耳前庭半规管上皮细胞，皮质齿间上皮细胞，松散的结缔组织成纤维细胞，角膜成纤维细胞，肌腱成纤维细胞，骨髓网状组织成纤维细胞，其他非上皮成纤维细胞，周细胞，如：肝星状细胞，椎间盘髓核细胞，透明软骨细胞，纤维软骨细胞，弹性软骨细胞，成骨细胞，骨祖细胞，眼玻璃体透明细胞，耳外淋巴间隙星状细胞，胰腺星状细胞。收缩细胞包括红色骨骼肌细胞，白色骨骼肌细胞，中间骨骼肌细胞，肌梭核袋细胞，肌梭核链细胞，肌卫星细胞六种骨骼肌细胞，心肌细胞，窦房结细胞，蒲肯野纤维细胞三种心肌细胞，以及平滑肌细胞，虹膜肌上皮细胞，外分泌腺肌上皮细胞。血液和免疫系统细胞包括红细胞，巨核细胞，血小板，单核细胞，结缔组织中的巨噬细胞，表皮中的朗格汉斯细胞，破骨细胞，树突状细胞，小胶质细胞，中性粒细胞，嗜酸性粒细胞，嗜碱性粒细胞，杂交瘤细胞，肥大细胞，辅助T细胞，抑制T细胞，细胞毒T细胞，自然杀伤T细胞，B细胞，自然杀伤细胞，网状细胞，血液及免疫系统的干细胞和祖细胞。生殖细胞包括卵原细胞/卵母细胞，精细胞，精原细胞，精母细胞，精子。滋养细胞包括卵巢中的颗粒细胞，睾丸中的支持细胞，上皮网状细胞。简直组织细胞包括简直组织肾细胞等。
在另一优选例中，所述的snoRNA选自下组：SNORA41、SNORD85、SNORD115、SNORD116、SNORD14c、SNORD14d、SNORD55、SNORD15b、SNORD23、SNORD96a、SNORD123、SNORD34、SNORD82。
在另一优选例中，所述的方法为非诊断性和非治疗性的。
在本发明的第三方面，提供了一种snoRNA或其促进剂或其拮抗剂的用途，用于 制备药物或制剂，所述药物或制剂用于调控细胞组织或器官的服务状态。
在另一优选例中，所述的服务化状态包括在服状态、去服状态、或其转换。
在另一优选例中，所述的snoRNA为表1所示的一种或多种、或其组合。
在另一优选例中，所述的snoRNA选自下组：SNORA41、SNORD85、SNORD115、SNORD116、SNORD14c、SNORD14d、SNORD55、SNORD15b、SNORD23、SNORD96a、SNORD123、SNORD34、SNORD82。
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
图1显示了正常小鼠各个组织中检测得到的部分snoRNA的RT-PCR结果。
图2显示了正常小鼠各个组织中检测得到的部分snoRNA的real-time PCR结果。
图3显示了脂肪细胞离体培养后不同时间点leptin基因的表达变化。
图4显示了高通量测序检测到的不同状态脂肪细胞的snoRNA的表达谱。
具体实施方式
本发明人经过广泛而深入的研究，首次发现了通过研究各种组织和细胞在服务状态和去服务状态下的snoRNA的特异性变化，可以得到一类在细胞两种状态下差异表达程度较大的snoRNA，应用于诊断细胞状态的一类分子标注物。同时，研究调控snoRNA表达量后细胞状态的变化，提供一种用于转换细胞服务化状态和去服务化状态的方法，用于生物实验和临床应用。在此基础上完成了本发明。
细胞服务状态
在服：enmunte(动词)；enmunting(动名词)；enmuntion(名词)；enmuntive(形容词)；enmuntively(副词)
去服：demunte(动词)；demunting(动名词)；demuntion(名词)；demuntive(形容词)；demuntively(副词)
在经典细胞生物学的概念中，所有成熟并有特定功能的细胞被称之为已分化细胞，而如果分化细胞缺失了特定的为调控内稳态或内环境服务的功能，现在只简单地称为"去分化"，与前体细胞等真正尚未分化细胞既无区别，也没有特定分子 标志物区分。
本发明发现了分化细胞存在着两个状态：一个是在服状态(enmunting，具有调控内稳态的服务功能)，另一个是去服状态(demunting，没有调控内稳态的服务功能)。这两种状态都是分化状态，因此细胞的生存代谢(相关基因表达和蛋白合成)都是一样的，服务功能有无的区别。demunting细胞与前体细胞的区别是：生存代谢状态不同(前体细胞还未分化)，但特定服务功能缺失的状态相同。
组织特异性SnoRNA的升高是鉴定demunting细胞的生物标志物；增高SnoRNA可以使细胞的enmunting状态变为demunting状态；减低SnoRNA，可以把细胞的demunting状态转变为enmunting状态。所有组织细胞都同时存在这两种状态。在大多数病理条件下，demunting细胞比例会增高，而导致器官衰竭。减低病理性增高的demunting细胞，会对脑死患者的唤醒，截瘫患者的功能性神经生长，增加大脑中激活的功能性神经元的比例等所有组织病理性功能衰竭都有治疗作用。
本发明的主要优点包括：
1)本发明发现了分化细胞的两种状态(在服状态和去服状态)，区分了去服状态细胞和未分化细胞，通过对这两种状态的研究能够解释原有生物学、医学研究中很多没法解释的现象；
2)本发明通过建立有效的snoRNA的检测方法，能够快速简单的鉴定细胞的在服状态和去服状态，避免了需通过对细胞功能、状态、转录组和蛋白组变化的复杂研究来鉴定这两种细胞状态；
3)本发明通过建立有效的snoRNA的调控方法，能够有效的转化细胞的在服状态和去服状态，在生物研究和临床应用上有巨大的应用前景；
4)本发明进一步发现了snoRNA在生物体内的重要调控功能，为生物学理论研究和应用研究提供了新的方向。
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，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)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，否则百分比和份数是重量百分比和重量份数。
通用方法
检测细胞去服务状态的snoRNA标记物的检测方法选自反转录聚合酶链式反应方法(RT-PCR)、实时荧光定量反转录聚合酶链式反应方法(Real-time RT-PCR)、Northern印记杂交方法(Northern blotting)、高通量测序技术或生物芯片方法。
RT-PCR方法为优选方法，包括以下步骤：
1)提取受试细胞样品的总RNA(例如使用Trizol试剂提取)，通过RNA逆转录反应得到cDNA样品；或者以受试细胞样品的裂解液作为缓冲液进行逆转录反应制备cDNA样品；
2)用snoRNA设计引物进行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方法为另一优选方法，包括以下步骤：
1)提取受试细胞样品的总RNA(例如使用Trizol试剂提取)，通过RNA逆转录反应得到cDNA样品；或者以受试细胞样品的裂解液作为缓冲液进行逆转录反应制备cDNA样品；
2)用snoRNA设计引物和荧光染料进行PCR反应(snoRNA的PCR引物见表格)；
3)检测并比较受试细胞样本相对于正常状态细胞样本中snoRNA的量的变化。
Nortern blotting方法包括以下步骤：
1)提取受试细胞样品的总RNA(例如使用Trizol试剂提取)；
2)进行变形PAGE电泳和膜转移实验；
3)制备同位素标记或者地高辛标记核酸探针；
4)进行膜杂交反应；
5)同位素信号或者地高辛信号检测。
高通量测序方法包括如下步骤：
1)提取受试细胞样品的总RNA(例如使用Trizol试剂提取)；
2)进行变形PAGE电泳回收50nt以下RNA分子；
3)将adapter primer酶联在RNA分子的3'端和5'端；
4)进行RT-PCR反应并进行测序；
5)数据分析和处理。
生物芯片方法包括如下步骤：
1)将人中全部七百多种snoRNA库点阵并制备生物芯片；
2)提取受试细胞样品的总RNA(例如使用Trizol试剂提取)；
3)通过柱分离来分离小核糖核酸；
4)利用T4RNA连接酶进行微小核糖核酸荧光标记；
5)与生物芯片进行杂交反应。
调控方法选自干扰RNA调控法和CRISPR/Cas9调控法。
干扰RNA调控方法为优选方法包括如下步骤：
1)设计干扰RNA(siRNA序列)和能够表达干扰RNA的shRNA序列(siRNA序列和shRNA序列见excel)；
2)制备干扰RNA或能表达相应干扰RNA的shRNA的质粒/病毒；
3)使用转染方法将干扰RNA或表达干扰RNA的质粒转进细胞或使用病毒感染的方法感染细胞；
4)收集细胞检测细胞状态。
CRISPR/Cas9调控法为另一优选方法包括如下步骤：
1)设计sgRNA；
2)构建sgRNA质粒载体同时构建表达sgRNA和Cas9蛋白的aav病毒；
3)使用转染方法转染sgRNA质粒和Cas9质粒到靶细胞中；或将构建好的aav病毒与细胞一同孵育；
4)收集细胞检测细胞状态。
实施例1.
细胞中snoRNA的RT-PCR实验
为了检测不同细胞服务状态下的snoRNA的特异变化，使用RT-PCR技术检测人和动物细胞中的snoRNA含量，具体步骤为：
(1)收集人或其他动物的细胞；
(2)制备cDNA样品。使用TRIzol试剂先提取细胞总RNA，然后通过RNA逆 转录反应得到cDNA。逆转录的反应体系包括2μl 5×AMV buffer、1μl dNTP mixture、0.5μl RNAase Inhibitor、0.5μl AMV酶、1μl基因特异性反向引物、1μg总RNA和补RNAase free水至总体积10μl。反应步骤为16℃孵育15分钟，42℃反应1小时，85℃孵育5分钟。
(3)PCR电泳及观察。取1μl上一步反应得到的cDNA，加入0.3μl Taq酶、0.4μl dNTP mixture、1.2μl 25mM MgCl2、2μl 10×PCR Buffer、0.5μl特异正向引物、0.5μl特异反向引物和14.1μl水，20μl体系进行PCR。PCR的反应条件是：95℃、5分钟进行一个循环→95℃、15秒，60摄氏度、1分钟进行40个循环。PCR产物取10μl进行3％琼脂糖凝胶电泳，EB染色后紫外灯下观察。
结果如图1所示，以正常小鼠多个组织为研究对象，通过提取总RNA，进行RT-PCR的结果。本发明人选用了小鼠全部九百多个snoRNA进行PCR反应，图1为其中的4种snoRNA(SNORA41、SNORD85、SNORD115和SNORD116)。从结果可以看出这四种snoRNA在心脏、肝、脾脏、肺、肾、脑和肌肉中都能够检测到。
实施例2.
细胞中snoRNA的real-time PCR实验
为了检测不同细胞服务状态下的snoRNA的特异变化，使用real-time PCR技术检测人和动物细胞中的snoRNA含量。Real-time PCR实验原理及实验步骤同RT-PCR一样，唯一不同是在PCR的时候加入了荧光染料Eva Green。仪器使用的是ABI Prism7300荧光定量PCR仪，反应条件为95℃、5分钟进行一个循环→95℃、15秒，60摄氏度、1分钟进行40个循环。数据处理方法为ΔΔCT法，CT设为反应达到阈值时的循环数，则每个snoRNA相对于标准内参的表达量可以用方程2 -ΔCT表示，其中ΔCT＝CT 样品-CT 内参。
结果如图2所示。本发明人使用了Real-time PCR分别检测了小鼠全部snoRNA在各个组织中的表达量。图2仅显示出SNORA41、SNORD85、SNORD115和SNORD116分别在心脏、肝、脾脏、肺、肾、脑和肌肉中的表达量。实验中使用U6作为内参。由检测结果可以知道，SNORA41在肝中高表达，SNORD85在肺、脾、肌肉和肾中高表达，而SNORD115和SNORD116特异性在脑组织中高表达。从图2可以，各个组织中存在组织特异性表达的snoRNA，这些snoRNA差异表达可能与不同组织在机体中行使的功能差异相关。
实施例3.
高通量测序检测脂肪组织离体后不同培养时间后的snoRNA含量实验
为了检测snoRNA在不同的细胞状态下是否存在差异表达。本发明人使用了高通量测序的方法检测了离体培养后不同时间点脂肪细胞的snoRNA表达谱。具体的实验步骤如下：
(1)收集不同状态下的小鼠的白色脂肪组织。减成小碎块后分别在0小时、3小时、6小时、9小时、12小时和24小时的时候收取脂肪细胞，提取总RNA和总蛋白。
(2)分离小RNA并建库。将Total RNA使用PAGE电泳切胶分离18-30nt的RNA。使用5-adenylated、3-blocked的单链DNA接头连接到(RNA的3'端。加入消化酶，去除3'接头。5'接头连接至上步产物的5'端。以RT引物进行逆转录延伸，合成一链cDNA。使用高敏聚合酶对cDNA进行扩增，富集同时连接有3'接头和5'接头的cDNA，放大文库产量。使用PAGE电泳分离100-120bp范围PCR产物，有效去除了引物二聚体等副产物。对构建的文库进行质量检测，将质量检测合格的文库上机测序。
(3)使用Illumina Hiseq2500对建立的cDNA文库进行测序。
(4)测序数据生成及后续分析。将生成的测序图像文件使用CASAVA转换成序列文件后，分别去除测序质量较低的tag、有5'接头污染的tag、没有3'接头序列的tag、没有插入片段的tag、包含polyA的tag和小于18nt的tag。剩余的序列使用bowtie分别与已知的snoRNA进行比对注释。将所有样本数据汇总得到脂肪细胞在不同状态下的snoRNA表达谱数据。
(5)分别使用Real-time PCR方法和Western blotting方法检测步骤(1)中各个时间点脂肪细胞leptin的mRNA和蛋白含量。
结果如图3所示，本发明人检测了不同时间点脂肪细胞中的leptin等脂肪细胞特有的服务基因的表达含量。图2仅显示了leptin基因的表达含量的变化。结果显示，随着离体培养的时间增加，leptin基因无论是mRNA水平还是蛋白水平，均下降直至不再表达。显示了脂肪细胞离体后从在服状态转变为去服状态。
如图4所示，通过对不同时间点脂肪组织snoRNA的表达谱分析，本发明人发现SNORD14c/d、SNORD55、SNORD15b、SNORD23、SNORD96a、SNORD123、SNORD34和SNORD82等snoRNA的表达量随着时间的变化而变化。这九种snoRNA在离体培养3h后开始升高，在9h的时候表达量升至最高点，后在24h后下降。以上数据表明，脂肪细胞在离体培养后，随着外界条件改变逐渐转为去 服务状态的同时，snoRNA的表达量也跟随上升。上述结果提示上述几种snoRNA可以作为脂肪细胞去服务化状态的分子标志物。
表1 对应于SnoRNA的人工序列(Artificial Sequence)或cDNA序列
Figure PCTCN2020085364-appb-000001
实施例4.
使用质粒表达的siRNA在小鼠脑内抑制SNORD115的表达
本发明人利用限制性内切酶对对照质粒进行处理，回收线性载体后，利用T4连接酶将RVG-SNORD115 shRNA组合片段与载体进行连接；得到的连接产物进行转化实验并涂布于抗性平板；第二天挑取单克隆，测序确定质粒序列的正确性。
将对照质粒和SNORD115 siRNA/RVG质粒按照10mg/kg的剂量对正常小鼠进行尾静脉注射；12小时后，处死小鼠，取小鼠的肝组织和脑组织分别进行原位杂交实验。在本组实验中，将与SNORD115 siRNA序列完全互补配对的序列添加绿色荧光修饰作为检测探针，以指示siRNA在组织切片中的分布情况；结果表明，在小鼠的肝脏组织中有大量siRNA的分布，而在脑组织中也能检测到少量siRNA，证明SNORD115 siRNA/RVG质粒确实具有脑靶向的效果。
为了证明这些分布的siRNA是否能够在体内有效抑制SNORD115的表达情况，本发明人同样按照10mg/kg的剂量将对照质粒和SNORD115 siRNA/RVG质粒对小鼠进行尾静脉注射，每两天注射一次，共注射7次，最后一次注射24小时后处死小鼠，取小鼠的脑组织进行Real-time PCR实验。结果表明，质粒分子在体内可以有效降低脑组织中SNORD115的表达水平。
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 (11)

  1. 一种核仁小RNA(snoRNA)或其检测试剂的用途，其特征在于，用于制备检测细胞服务化状态的试剂或试剂盒。
  2. 如权利要求1所述的用途，其特征在于，所述的服务化状态包括在服状态、去服状态、或其转换。
  3. 如权利要求1所述的用途，其特征在于，所述的snoRNA来源于哺乳动物、啮齿动物、灵长类动物。
  4. 如权利要求1所述的用途，其特征在于，所述的snoRNA为器官、组织、细胞特异性的snoRNA。
  5. 如权利要求1所述的用途，其特征在于，所述的器官选自下组：关节、韧带、舌、唾腺、腮腺、下颌腺、舌下腺、咽、食道、胃、小肠、十二指肠、空肠、回肠、大肠、肝脏、胆囊、肠系膜、胰脏、鼻腔、咽、喉、气管、支气管、肺、横膈膜、肾、输尿管、膀胱、尿道、卵巢、输卵管、子宫、阴道、胎盘、睾丸、附睾、输精管、精囊、前列腺、尿道球腺、阴茎、阴囊、脑下垂体、松果体、甲状腺、副甲状腺、肾上腺、胰腺、心脏、动脉、静脉、微血管、淋巴管、淋巴结、骨髓、胸腺、脾脏、肠相关淋巴组织、扁桃腺、大脑、间脑、脑干、中脑、桥脑、延髓、小脑、脊髓、脑室、脉络丛、脑神经、脊神经、神经节、肠神经系统、角膜、虹膜、睫状体、晶状体、视网膜、耳垂、鼓膜、听小骨、耳蜗、耳前庭、半规管、舌、皮肤。
  6. 如权利要求1所述的用途，其特征在于，所述的组织选自下组：上皮组织、结缔组织、神经组织和肌肉组织。
  7. 如权利要求1所述的用途，其特征在于，所述的细胞包括来源于内胚层的细胞包括外分泌上皮细胞，屏障细胞，激素分泌细胞。
  8. 如权利要求1所述的用途，其特征在于，所述的snoRNA选自下组：SNORA41、SNORD85、SNORD115、SNORD116、SNORD14c、SNORD14d、SNORD55、SNORD15b、SNORD23、SNORD96a、SNORD123、SNORD34、SNORD82。
  9. 一种确定服务化状态分子标志物的方法，其特征在于，包括步骤：
    (1)提供第一服务状态下的细胞，检测所述第一服务状态下的snoRNA的种类和数量，从而获得第一数据集；
    (2)提供第二服务状态下的细胞，检测所述第二服务状态下的snoRNA的种类和数量，从而获得第二数据集；
    (3)将第一数据集和第二数据集进行比较，确定在第一服务状态和第二服务状态 下特有的snoRNA的种类和数量信息，从而鉴别在第一服务状态和第二服务状态下特有的分子标志物。
  10. 一种snoRNA或其促进剂或其拮抗剂的用途，其特征在于，用于制备药物或制剂，所述药物或制剂用于调控细胞组织或器官的服务状态。
  11. 如权利要求10所述的用途，其特征在于，所述snoRNA选自下组：SNORA41、SNORD85、SNORD115、SNORD116、SNORD14c、SNORD14d、SNORD55、SNORD15b、SNORD23、SNORD96a、SNORD123、SNORD34、SNORD82。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116672366A (zh) * 2023-05-11 2023-09-01 山东第一医科大学附属眼科研究所(山东省眼科研究所、山东第一医科大学附属青岛眼科医院) 脉络丛上皮细胞在替代角膜内皮细胞方面的应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011036091A1 (de) * 2009-09-23 2011-03-31 Sirs-Lab Gmbh Verfahren zur in vitro erfassung und unterscheidung von pathophysiologischen zuständen
CN104894259A (zh) * 2015-06-01 2015-09-09 北京泱深生物信息技术有限公司 Tex19基因在胆管癌诊断和治疗中的应用

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106434648A (zh) * 2010-07-19 2017-02-22 F·C·贝内特 肌强直性营养障碍蛋白激酶(dmpk)表达的调节
WO2014110230A2 (en) * 2013-01-09 2014-07-17 Health Research, Inc. Methods for diagnosing cancer based on small nucleolar rna hbii-52
FR3012739A1 (fr) * 2013-11-07 2015-05-08 Centre Nat Rech Scient Sno arn, compositions et utilisations
CN104561363A (zh) * 2015-02-04 2015-04-29 安徽省农业科学院水稻研究所 用于筛选水稻耐旱品种的snoRNA基因分子标记的引物
JP2016214239A (ja) * 2015-05-15 2016-12-22 国立大学法人高知大学 膵がんマーカー
CN106282321B (zh) * 2015-05-26 2019-12-03 中山大学 由组织snoRNA组成的肝癌复发风险预测标志物及试剂盒
JP6143233B2 (ja) * 2015-08-05 2017-06-07 国立大学法人京都大学 運動ニューロン疾患の検査方法及び治療剤のスクリーニング方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011036091A1 (de) * 2009-09-23 2011-03-31 Sirs-Lab Gmbh Verfahren zur in vitro erfassung und unterscheidung von pathophysiologischen zuständen
CN104894259A (zh) * 2015-06-01 2015-09-09 北京泱深生物信息技术有限公司 Tex19基因在胆管癌诊断和治疗中的应用

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CUOMOD ET AL.: "Transcriptional Landscape of Mouse-Aged Ovaries Reveals a Unique Set of Non-coding RNAs Associated with Physiological and Environmental Ovarian Dysfunctions", CELL DEATH DISCOVERY, vol. 4, 5 December 2018 (2018-12-05), XP055746051 *
CUOMOD ET AL.: "Transcriptional Landscape of Mouse-Aged Ovaries Reveals a Unique Set of Non-coding RNAs Associated with Physiological and Environmental Ovarian Dysfunctions", CELL DEATH DISCOVERY, vol. 4, no. 112, 5 December 2018 (2018-12-05), XP055746051 *
SAMBROOK ET AL.: "Molecular Cloning: Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
See also references of EP3943609A4
ZHANG, XIAOCHEN ET AL.: "Structure and Function of snoRNAs", CHINESE BULLETIN OF LIFE SCIENCES, vol. 20, no. 2, 30 April 2008 (2008-04-30), XP055746065 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116672366A (zh) * 2023-05-11 2023-09-01 山东第一医科大学附属眼科研究所(山东省眼科研究所、山东第一医科大学附属青岛眼科医院) 脉络丛上皮细胞在替代角膜内皮细胞方面的应用

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