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Nlrp3 gene RNAi immunoliposome targeting microglial cells and its preparation method and application

A microglia and immunoliposome technology, applied in the field of biomedicine, can solve problems such as anti-inflammatory treatment of NLRP3 inflammasomes that have not been seen, and achieve the effect of good application prospects.

Inactive Publication Date: 2016-09-14
AFFILIATED YONGCHUAN HOSPITAL OF CHONGQING MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no report on the use of NLRP3 inflammasome for anti-inflammatory treatment

Method used

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  • Nlrp3 gene RNAi immunoliposome targeting microglial cells and its preparation method and application
  • Nlrp3 gene RNAi immunoliposome targeting microglial cells and its preparation method and application
  • Nlrp3 gene RNAi immunoliposome targeting microglial cells and its preparation method and application

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Experimental program
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Effect test

Embodiment 1

[0028] Embodiment 1, the preparation of the immunoliposome based on the RNAi of NLRP3 gene

[0029] 1. Construction of RNAi recombinant eukaryotic expression vector of NLRP3 gene

[0030] Design 2 inverted repeat sequences, as follows:

[0031] 5'-gatcggaagcagtttgaagaattattcaagacgtaattcttcaaactgcttctttttta-3' (SEQ ID NO. 1) and 3'-ccttcgtcaaacttcttaataagttctgcattaagaagtttgacgaagaaaaaattcga-5' (SEQ ID NO. 2). After the DNA chain is annealed, it is ligated between the NotI and XbaI restriction sites of the pGenesil-1 vector by T4 ligase to obtain the RNAi recombinant eukaryotic expression vector of the NLRP3 gene, which is called pGenesil-1-NLRP3 RNAi for short. Then, the pGenesil-1-NLRP3 RNAi recombinant plasmid was transformed into Escherichia coli DH5α competent cells, and positive clones were screened on LB plates containing ampicillin, and the positive clones were inoculated in LB liquid medium containing ampicillin for bacterial enrichment culture, and a large number of s...

Embodiment 2

[0040] Embodiment 2, research on the anti-inflammatory effect of immunoliposome

[0041] 1. Detection of NLRP3 in microglia after erythrocyte lysates were treated with immunoliposomes

[0042] Add 10 μL of erythrocyte lysate to 0.1 mg Anti-OX-42-Lip-NLRP3 RNAi-incubated microglia or unincubated control microglia, and then treat it at 37°C for 24 hours, and use Western blotting (WB ) method to detect the expression of NLRP3 in immunoliposome microglial cells stimulated by erythrocyte lysate, the results are as follows Figure 6 shown. The results showed that immunoliposome-incubated microglia expressed less NLRP3 than control microglia.

[0043] 2. Detection of inflammatory factors in microglial cells after erythrocyte lysates were treated with immunoliposomes

[0044] Add 10 μL of erythrocyte lysate to 0.1 mg of immunoliposome-incubated microglia or control microglia, and then treat it at 37°C for 24 hours, and use ELISA to detect that the erythrocyte lysate stimulates micr...

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Abstract

The invention discloses microglial cell-targeting NLRP3 gene RNAi immune liposome and a preparation method and application thereof. The microglial cell-targeting NLRP3 gene RNAi immune liposome is prepared by connecting an OX-42 monoclonal antibody on the surface of NLRP3 gene-encapsulated RNAi recombinant eukaryotic expression vector; the preparation method is simple; the obtained microglial cell-targeting NLRP3 gene RNAi immune liposome can actively target a microglial cell, release and express the RNAi sequence of an NLRP3 gene in the cell, and inhibit the expression of the NLRP3 so as to play an anti-inflammatory role. The preparation method thereof has the advantages of high efficiency, low toxicity, high simplicity and the like; the microglial cell-targeting NLRP3 gene RNAi immune liposome can be used for preparing anti-inflammatory medicines and has a good application prospect in inhibiting secondary inflammation of brain hemorrhage.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to NLRP3 gene RNAi immune liposome targeting microglial cells, and also relates to the preparation method and application of the liposome. Background technique [0002] Cerebral hemorrhage refers to non-traumatic cerebral parenchymal hemorrhage, which is mostly caused by blood vessel rupture caused by high blood pressure. With the improvement of people's living standards and the arrival of aging, the incidence of cerebral hemorrhage is also on the rise. According to the latest statistical data, the number of patients who die from cerebral hemorrhage every year is about 20% of the number of patients who die from all diseases. At present, the medical treatment of cerebral hemorrhage is mainly based on dehydration to reduce intracranial pressure, scavenging free radicals, nourishing nerves and symptomatic and supportive treatment. In recent years, there is no breakthrough treatm...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00A61K9/127A61K47/42A61P7/00A61P29/00A61P7/04A61P25/00
Inventor 杨曌
Owner AFFILIATED YONGCHUAN HOSPITAL OF CHONGQING MEDICAL UNIV
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