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LncRNA targeting Notch1 gene and use of LncRNA

A gene-targeted technology, applied in LncRNA and its application fields, can solve problems such as large side effects and ineffective primary progressive MS, and achieve the effects of less toxic side effects, inhibition of central nervous system inflammation and demyelination

Active Publication Date: 2021-04-09
XUZHOU MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0003] At present, large doses of glucocorticoids, immunoglobulins and interferon β-1 are commonly used in the clinical treatment of central nervous system inflammatory demyelinating diseases, but long-term use has serious side effects, such as obesity, insulin resistance, abnormal blood lipids and blood pressure, digestive Ulcer, osteoporosis complicated with osteonecrosis, rash, acute renal failure, etc., and almost ineffective for primary progressive MS

Method used

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  • LncRNA targeting Notch1 gene and use of LncRNA
  • LncRNA targeting Notch1 gene and use of LncRNA
  • LncRNA targeting Notch1 gene and use of LncRNA

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Experimental program
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Effect test

Embodiment 1

[0032] experimental method:

[0033] EAE mouse model induction: 300 μg of myelin oligodendrocyte glycoprotein antigen (Myelinoligodendrocyte glycoprotein amino acids 35–55, MOG 35-55 ) was dissolved in 0.1ml of sterile PBS, 0.5mg of inactivated Mycobacterium tuberculosis was dissolved in 0.1ml of complete Freund's adjuvant, and the two were repeatedly sucked and blown through a three-way tube until a water-in-oil mixture was formed. State, make EAE antigen-induced emulsion, and inject it into mice through subcutaneous multi-point on the back; at the same time, dissolve 200ng pertussis in 0.2ml sterile PBS, on the 0th day and the 2nd day after EAE mouse antigen induction 200 ng of pertussis toxin was injected intraperitoneally. Daily observation, double-blind method for scoring the neurological function of the mice. The criteria are as follows: 0 points, no clinical manifestations; 1 point, decreased tail tone or paralysis of mice; 2 points, partial hindlimb paralysis; 3 poin...

Embodiment 2

[0037] Cultivation of primary cells: take C57BL / 6 suckling mice born within 24 hours, soak them in 75% ethanol for 3-5 minutes under the ultra-clean workbench to disinfect the skin, decapitate, carefully cut the skull with ophthalmic scissors under aseptic conditions, and take On both sides of the brain, the meninges were peeled off under a stereoscope, and the cerebral cortex was taken out and placed in a sterile petri dish filled with pre-cooled D-Hanks solution. Then, rinse twice with pre-cooled D-Hanks solution, cut up the cerebral cortex to make mince, digest with 0.25% trypsin solution at 37°C for 5-10min, and culture completely with DMEM / F12 containing 10% fetal bovine serum The digestion was terminated by pipetting gently, centrifuged at 1200r / min for 5min, washed twice with pre-cooled DMEM / F12 complete medium, filtered with stainless steel mesh (ф=200μm), and transferred to a poly-L-lysine-uncoated Add an appropriate amount of DMEM / F12 complete medium to a glass cultu...

Embodiment 4

[0041] Primary mouse astrocytes were incubated overnight in serum-free medium, then stimulated with IL-9, and total cellular RNA was collected at different time points. Real-time PCR detection of Gm13568 and Notch1 levels in astrocytes, see the results Figure 8 .

[0042] Figure 8 The expression levels of Gm13568 and Notch1 in astrocytes after IL-9 stimulation at different times, it can be seen that the expression levels of Gm13568 and Notch1 mRNA increase or decrease synchronously, and the changes are consistent, which indicates that Gm13568 may be a positive regulator of Notch1 .

[0043] The sequence of Gm13568 was amplified and cloned into a lentiviral transfer vector carrying the GFAP promoter of astrocytes, named LV-Gm13568. Since the sequence of Gm13568 was completely complementary to the sense strand at the 9075-9497 site of the Notch1 gene, we adopted The method of expressing the sequence of its binding region with Notch1 uses competitive inhibition to down-regul...

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Abstract

The invention discloses a long-chain non-coding RNA (LncRNA) Gm13568 targeting a Notch1 gene. A sequence of the LncRNA Gm13568 is shown as SEQ ID NO:1. When inventors study a disease process of mice of a multiple sclerosis model, interleukin 9 (IL-9) is found to be significantly up-regulated, astrocytes are activated and proliferated, and meanwhile, Notch1 signals are activated and secretion of proinflammatory factors is increased. Researches prove that the IL-9 activates a Notch1 signal pathway of the astrocytes and promotes a generation of a large number of the inflammatory factors; An in-vivo inhibition of the Gm13568 remarkably reduces the activation of the Notch1 signals and the generation of the inflammatory factors, relieves demyelination, only specifically targets the astrocytes in the central nervous system, and does not cause damage to other tissue cells. Therefore, a Gm13568 inhibitor can be used as a new medicine for treating inflammatory demyelinating diseases of the central nervous system.

Description

technical field [0001] The invention relates to a LncRNA targeting Notch1 gene and application thereof, belonging to the technical fields of genetic engineering and biomedicine. Background technique [0002] Inflammatory demyelinating diseases of the central nervous system are a very common type of autoimmune diseases of the central nervous system mediated by T cells, mainly including multiple sclerosis (MS), acute disseminated encephalomyelitis (ADEM), Subacute neuromyelitis optica (SMON), concentric sclerosis, central allergic diseases induced by various causes (such as after infection with various biological pathogens, after vaccination, heterosexual protein allergy, and drug or chemical allergic reaction) (such as acute demyelinating leukoencephalopathy and acute demyelinating myelitis), the pathogenesis has not been fully elucidated. [0003] At present, large doses of glucocorticoids, immunoglobulins and interferon β-1 are commonly used in the clinical treatment of ce...

Claims

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Application Information

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IPC IPC(8): C12N15/113A61K45/00A61P25/00A61P25/28
CPCA61K45/00A61P25/00A61P25/28C12N15/113C12N2310/00
Inventor 刘晓梅尤红娟张清秀周峰陈国芳汤仁仙郑葵阳李向阳华慧王卫晓于倩
Owner XUZHOU MEDICAL UNIV
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