Application of miR-486-3p to preparation of product for treating neuroinflammation caused by SAH

A technology of neuroinflammation and products, applied in the application field of neuroinflammation products, can solve problems such as difficult to achieve clinical effects and unable to cross the BBB

Active Publication Date: 2020-09-22
THE FIRST AFFILIATED HOSPITAL OF WANNAN MEDICAL COLLEGE YIJISHAN HOSPITAL OF WANNAN MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is estimated that 98% of drug molecules are difficult to achieve clinical effect because they cannot cross the BBB

Method used

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  • Application of miR-486-3p to preparation of product for treating neuroinflammation caused by SAH
  • Application of miR-486-3p to preparation of product for treating neuroinflammation caused by SAH
  • Application of miR-486-3p to preparation of product for treating neuroinflammation caused by SAH

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1, qRT-PCR verifies NGS data

[0035] 1. Extract the total RNA of exosomes by QIAzol-spin column method, measure the purity of RNA, and quantify the RNA. Using the corresponding solvent as the control (Blank), take 2 μL of the RNA solution and detect it in Merinton SMA4000, and observe A 260 / A 280 、A 260 / A 230 Ratio and continuous wavelength absorption peak, and calculate the concentration of RNA solution to judge the quality of RNA extraction: A 260 / A 280 >2.0 and <2.3, it can meet the requirements of subsequent RT-qPCR. Total RNA was extracted from exosomes or brain tissue using QIAzol Lysis reagent (Qiagen, Germany).

[0036] Table 1

[0037]

[0038]

[0039] 2. Quantitative PCR of miRNA: To analyze miRNA levels, total RNA was reverse-transcribed into cDNA using miRcute miRNA FirstStrand cDNA Synthesis Kit (Tiangen Biotechnology Company, China). Reverse transcription system preparation: total RNA 2μg, 2×miRNA RT Reaction Buffer 10μl, miRNA...

Embodiment 2

[0041] Example 2, RVG-LAMP2B-overexpression lentiviral vector construction

[0042] 1. Experimental method:

[0043] 1.1 Obtain the RVG-LAMP2B-mus sequence fragment by PCR method

[0044] 1.1.1 The whole gene synthesis of the vector was synthesized by Jierui Bioengineering Co., Ltd.

[0045] 1.1.2 Primers: Gene name: RVG-LAMP2B-mus; Cloning vector: pHBLV-CMV-MCS-3FLAG-EF1-ZsGreen-T2A-PURO, cloning vector such as figure 1 shown. Cloning strategy: BamHI+EcoⅠ. The upstream and downstream primers of the target gene were respectively added with homologous sequences on both sides of EcoI and BamHI on the PHBLV-CMV-MCS-3FLAG-EF1-ZSGREEN-T2A-PURO vector for subcloning of the vector. The primer sequences are as follows:

[0046] Primers were synthesized by Shanghai Hanbio Co., Ltd.

[0047] m-RVG-lamp2b-F: (SEQ ID NO.22)

[0048] agaggatcta tttccggtga attcgccacc atgtgcctct ctccggtta

[0049] m-RVG-lamp2b-R: (SEQ ID NO.23)

[0050] cacttaagct tggtaccgag gatcccagag tctgatatcc agc...

Embodiment 3

[0062] Example 3, RVG-LAMP2B-overexpression lentiviral packaging

[0063] 2. Experimental steps:

[0064] 2.1 Culture and transfection of 293T cells

[0065]When the 293T cells were cultured in a 10cm dish until 80-90% confluent, they were inoculated into a 15cm dish. Pour off the culture medium and wash the cells twice with 1mL D-Hank’s solution. Add 1mL Trypsin-EDTA solution, mix well, and place at 37°C for 2-3min.

[0066] Carefully suck off the trypsin solution, add 2 mL of DMEM culture solution containing 10% FBS, and pipette the cells to form a single-cell suspension. Inoculate the cell suspension into a 15cm culture dish, add 18mL DMEM culture solution containing 10% FBS, and mix well at 37°C in 5% CO 2 Incubate overnight.

[0067] Add 1.5mL serum-free DMEM to a sterile 5mL centrifuge tube, add shuttle plasmid V3120 and packaging plasmids (pGag / Pol, pRev, pVSV-G) in proportion, mix well, take another sterile 5mL centrifuge tube tube, add 1.5mL serum-free DMEM, the...

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Abstract

The invention discloses application of miR-486-3p to preparation of a product for treating neuroinflammation caused by SAH. According to the application of a miR-486-3p related biological material topreparation of the following (a1) or (a2) products, (a1) a product for treating SAH is prepared; (a2) a product for inhibiting neuroinflammation caused by SAH is prepared, wherein the related biological material of the miR-486-3p is the miR-486-3p, or an exosome loaded with the miR-486-3p, or a substance capable of promoting expression of the miR-486-3p. Experiments prove that miRNA can be more effectively transferred to the brain by fusing RVG into the exosome modified by membrane glycoprotein Lamp2b to load the miR-486-3p. The miR-486-3p plays an anti-inflammatory role by inhibiting expression of Sirtuin2 (Sirt2).

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of miR-486-3p in the preparation of products for treating neuroinflammation caused by SAH. Background technique [0002] Aneurysmal subarachnoid hemorrhage (SAH), usually caused by a ruptured aneurysm, is a clinical syndrome with an annual mortality rate of 45% and an incidence of approximately 6-16 per 100,000 people, usually young individual. SAH accounts for 5%-7% of the total incidence of stroke. The main prognostic determinants are early brain injury (EBI) and early cerebral vasospasm (CVS), and delayed cerebral ischemia (DCI) after SAH. A growing number of studies have shown that EBI after SAH may be the main factor leading to unfavorable outcomes after SAH. Several pathophysiological processes associated with EBI after SAH are associated with inflammatory cascades. The development of new therapeutic approaches is crucial to promote the recovery of EBI after...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/7105A61P25/00A61P29/00C12N15/113
CPCA61K31/7105A61P25/00A61P29/00C12N15/1136C12N2310/141
Inventor 赖年升姚阳李真保方兴根吴德刚袁金龙赵心同夏大勇
Owner THE FIRST AFFILIATED HOSPITAL OF WANNAN MEDICAL COLLEGE YIJISHAN HOSPITAL OF WANNAN MEDICAL COLLEGE
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