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Chitosan-Math1 gene nanoparticle and application thereof in treatment of deafness

A nanoparticle and chitosan technology, applied in the field of nanomaterials, can solve unseen problems and achieve the effects of easy control, uniform size and control

Inactive Publication Date: 2010-12-01
GENERAL HOSPITAL OF PLA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0013] However, there have been no reports on the preparation of chitosan-Math1 gene nanoparticles and transfection of in vitro cultured cells with chitosan-Math1 gene nanoparticles and the expression of Math1 gene

Method used

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  • Chitosan-Math1 gene nanoparticle and application thereof in treatment of deafness
  • Chitosan-Math1 gene nanoparticle and application thereof in treatment of deafness
  • Chitosan-Math1 gene nanoparticle and application thereof in treatment of deafness

Examples

Experimental program
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Effect test

Embodiment 1

[0050] Embodiment 1: Construction of PRK5-Math1 plasmid

[0051] The total RNA of the 16-day brain tissue of embryonic mice was extracted by the Trizol method, cDNA was synthesized by reverse transcription, and the Math1 gene containing the E box was synthesized by the PCR method, and ECOR1 and BamH1 restriction sites were added to the 5' and 3' ends. The PCR amplified product was digested with ECOR1 and BamH1, and after purification, it was ligated with the PRK5 plasmid (Clontech Company) digested with ECOR1 and BamH1 to construct the PRK5-Math1 plasmid. The Math1 gene has Figure 5 sequence shown.

[0052] Amplification primers are as follows:

[0053] F: 5'-GGAATTAAAATAGTTGGGGGACC-3',

[0054] R: 5'-TGGACAGCTTCTTGTTGGCTT-3',

[0055] Amplification conditions were: 94°C for 5 min; 94°C for 1 min; 58°C for 40 sec; 72°C for 40 sec; 35 cycles, 72°C for 5 min.

Embodiment 2

[0056] Embodiment 2: Construction of PRK5-Math1-EGFP plasmid

[0057] The plasmid pEGFP-C2 (Invitrogen Company) containing the EGFP gene and the PRK5-Math1 plasmid of Example 1 were double-digested with Hpa1 and Xbal1 enzymes, respectively, purified and recovered, and connected by T4 ligase to construct the PRK5-Math1-EGFP plasmid.

Embodiment 3

[0058] Example 3: Amplification and purification of PRK5-Math1-EGFP

[0059] Take 5 μl of plasmid PRK5-Math1-EGFP, add 100 μl of competent Escherichia coli DH5α bacteria, mix well, ice-bath for 30 minutes, heat shock at 42°C for 1 minute, ice-bath for 2 minutes, add 800 μl of LB medium, and incubate at 37°C for 1 hour. Take 100 μl of the bacterial solution and spread it on a plate containing ampicillin, and incubate it upside down at 37°C for 16 hours. Pick a single colony from the plate, inoculate it in 5 ml of LB liquid medium containing ampicillin, shake at a constant temperature of 37°C overnight, and grow the bacteria to late logarithmic phase. Plasmids were extracted according to the instructions of the plasmid extraction kit (QIAGEN).

[0060] Take 0.5-1 μg of the plasmid, add 5U of endonuclease (no more than 1 / 10 of the total reaction volume), the reaction volume is 20 μl, bathe in a warm water for 2 hours, and take a small amount of samples for agarose gel electropho...

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Abstract

The invention discloses a chitosan and / or chitosan derivative-Math1 gene nanoparticle as well as a preparation method and application thereof. The gene nanoparticle is prepared by carrying out complex coacervation on chitosan and plasmid containing Math1 gene. The gene nanoparticle has controllable particle diameter and uniform size, is beneficial to surface modification, capable of improving theexpression and delivery capacities of the Math1 gene and being used for treating sensorineural deafness resulted from hair cell loss caused by noise, medicine poisoning, and the like.

Description

technical field [0001] The invention belongs to the technical field of nanometer materials, and in particular relates to chitosan-Math1 gene nanoparticle, its preparation method and its application in the treatment of deafness. Background technique [0002] Gene delivery usually requires a carrier. An ideal gene carrier can deliver the target gene safely and efficiently to specific diseased cells or tissues, so as to achieve the purpose of treatment. Currently, the commonly used gene vectors mainly include viruses and liposomes. Viral vectors such as retrovirus, adenovirus (Ad), adeno-associated virus (AAV), lentivirus and herpes simplex virus (HSV) have high expression levels, but they have no specific selectivity and many side effects, such as carcinogenicity, wild-type in vivo Type virus production, cell pathological changes, etc., and the length of the gene that can be loaded is limited. Liposome carrier can carry a larger number of genes, but its molecular weight is l...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/36A61K47/48A61P27/16C12N15/85A61K47/61
Inventor 杨仕明吴雁韩东翟所强高维强任丽丽郭维维胡吟燕李兴启孙建和赵立东韩东一杨伟炎
Owner GENERAL HOSPITAL OF PLA
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