Immunoliposome based on Nrdp1 gene RNAi and miR-494 and application thereof

A technology of mir-494 and immunoliposome, which is applied in the field of genetic engineering, can solve the problems of lack of therapeutic drugs, inflammatory reactions that do not cause clinicians, damage, etc., and achieve the effect of inhibiting the initiation process of inflammation, good application prospects, and increasing content

Inactive Publication Date: 2019-02-01
AFFILIATED YONGCHUAN HOSPITAL OF CHONGQING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although modern medical research has gradually recognized the role of vascular response after cerebral hemorrhage and the cascading inflammatory response in the destruction of the brain microenvironment and the pathological process of nerve cell damage, the inflammatory response has not attracted the attention of clinicians, and Clinically, there is also a lack of corresponding therapeutic drugs

Method used

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  • Immunoliposome based on Nrdp1 gene RNAi and miR-494 and application thereof
  • Immunoliposome based on Nrdp1 gene RNAi and miR-494 and application thereof
  • Immunoliposome based on Nrdp1 gene RNAi and miR-494 and application thereof

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Effect test

Embodiment 1

[0053] Example 1 Preparation of immunoliposome (RNAi of Anti-F4 / 80-Lip-miR-494 / Nrdp1 gene) based on RNAi of miR-494 / Nrdp1 gene

[0054] 1. Preparation and detection of RNAi recombinant eukaryotic expression vector of miR-494 / Nrdp1 gene

[0055] (1) Preparation of RNAi recombinant eukaryotic expression vector of miR-494 / Nrdp1 gene

[0056] The nucleotide sequence shown as SEQ ID NO:1 was synthesized. Two corresponding inverted repeat sequences were designed: as shown in SEQ ID NO: 2 and SEQ ID NO: 3, they were used as primers to clone and obtain the target DNA fragment.

[0057] The above DNA strands were annealed and ligated with the plasmid vector pGenesil-1. T4 ligase ligated the annealed nucleotide to pGenesil-1 and transformed it into Escherichia coli DH5α, screened positive clones with LB plates containing ampicillin, extracted plasmids for identification, and inserted NotI and XbaI sites of pGenesil-1 The positive cloning plasmid of the RNAi gene of the miR-494 / Nrdp1 ...

Embodiment 2

[0073] Anti-inflammatory effect research of embodiment 2 immunoliposome

[0074] 1. Detection of NRDP1 in microglial cells after red blood cell lysate treatment,

[0075]Add 10 μl of erythrocyte lysate to 0.1 mg of immunoliposome-incubated microglia or control microglia, at 37°C for 24 hours, and detect the expression of NRDP1 in microglial cells stimulated by erythrocyte lysate by WB method. Research results such as image 3 As shown, immunoliposome-incubated microglia expressed less NRDP1 than control microglia.

[0076] 2. Detection of inflammatory factors in microglial cells after red blood cell lysate treatment,

[0077] Add 10 μl of erythrocyte lysate to 0.1 mg of immunoliposome-incubated microglia or control microglia, 37°C, 24h, use ELISA to detect that erythrocyte lysate stimulates microglia to secrete IL-8 and TNF- Alpha capabilities. Research results such as Figure 4 As shown, the immunoliposome-incubated microglia secreted less IL-8 and TNF-α than the control...

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Abstract

The invention belongs to the field of genetic engineering, and in particular, relates to an immunoliposome based on Nrdp1 gene RNAi and miR-494 and an application thereof. The liposome is encapsulatedwith recombinant plasmids of miR-494 and the Nrdp1 gene RNAi, and is covalently bonded with an anti-F4 / 80 monoclonal antibody. A preparation method includes three steps: preparation of miR-494 and Nrdp1 gene RNAi expression vectors, liposome encapsulation of miR-494 and Nrdp1 gene RNAi recombinant eukaryotic expression vectors, and bonding of the liposome encapsulated with the miR-494 and the Nrdp1 gene RNAi with the anti-F4 / 80 monoclonal antibody. The immunoliposome can actively target to microglia cells, is released in cells and expresses the miR-494 and the Nrdp1 gene RNAi, inhibits the expression of Nrdp1, thereby playing an anti-inflammatory role; the immunoliposome has the advantages of high efficiency, low toxicity, and simple preparation method, can be used for preparing anti-inflammatory drugs, and has good application prospects in inhibition of a secondary inflammatory reaction of cerebral hemorrhage.

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular to an immunoliposome based on Nrdp1 gene RNAi and miR-494 and application thereof. Background technique [0002] Cerebral hemorrhage (cerebral hemorrhage) refers to the hemorrhage caused by the rupture of blood vessels in the non-traumatic brain parenchyma, accounting for 20% to 30% of all strokes, and the fatality rate in the acute phase is 30% to 40%. The cause of the occurrence is mainly related to cerebrovascular lesions, that is, closely related to hyperlipidemia, diabetes, high blood pressure, aging of blood vessels, and smoking. Patients with cerebral hemorrhage often develop sudden onset due to emotional agitation and exertion, and have a high early mortality rate. Most of the survivors have sequelae such as motor disorders, cognitive impairments, and speech and swallowing disorders to varying degrees. None of the current drugs for the treatment of cerebral hemorrhage has...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/70A61K9/127A61K31/7105A61K47/68A61P29/00A61P9/10A61P7/04
CPCA61K9/127A61K31/7105A61K47/6803A61P7/04A61P9/10A61P29/00C12N15/113C12N15/70C12N2310/141
Inventor 冯桂波
Owner AFFILIATED YONGCHUAN HOSPITAL OF CHONGQING MEDICAL UNIV
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