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Small nucleic acid drug for treating acute lung injury caused by sepsis

A technology for acute lung injury and small nucleic acid drugs, applied in the field of biomedical materials, can solve problems such as designing siRNA, achieve low cost, simple production and synthesis process, and low immunogenicity

Inactive Publication Date: 2014-03-26
NANJING UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, no siRNA targeting HuR has been designed for the treatment of acute lung injury in sepsis

Method used

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  • Small nucleic acid drug for treating acute lung injury caused by sepsis
  • Small nucleic acid drug for treating acute lung injury caused by sepsis
  • Small nucleic acid drug for treating acute lung injury caused by sepsis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: HuR siRNA destabilizes TNF-α mRNA

[0032] In order to determine that HuR is involved in the regulation of TNF-α mRNA stability, we screened and synthesized multiple siRNAs against HuR, and selected a fragment with the best interference effect, which was 5'-AACAUUCAACGCUGUCGGUGAGU-3' (SEQ ID NO.27). This siRNA was transfected into A549 cells with lipofectamine2000, and the protein expression was verified by western blot. It was found that the RNAi almost completely inhibited the expression of HuR ( figure 1 ). In this case, the effect of HuR siRNA on the degradation rate of TNF-α mRNA was studied by reverse transcription PCR method, adding Act D (mRNA synthesis inhibitor) to cells transfected with HuR siRNA or control siRNA, and then collecting Act D treatment After 1 and 2 hours, the total RNA was extracted from the cells, and PCR amplification was carried out after reverse transcription. The experimental results showed that after HuR was interfered, the ha...

Embodiment 2

[0033] Example 2: HuR can bind TNF-α mRNA 3'UTR to play a stabilizing role

[0034] TNF-α3'UTR plays a key role in mRNA stability. In order to determine the minimum sensitive response element on TNF-α3'UTR, the present invention adopts PCR and molecular cloning methods to construct human TNF3'-UTR full length and 4 truncated variants. The luciferase reporter plasmids of the body were named T789, T430, T360, T142 and T55 ( Figure 4 ). The PCR primers involved in its construction are as follows:

[0035]

[0036] These reporter plasmids and renillia plasmids (purchased by Invitrogen) were co-transfected into cells using lipofectamine2000, and the luciferase activity was determined. The results showed that TNF-α 3'UTR full-length (T789) had obvious destabilizing effect, and T55 could Destabilization of the full-length TNF-α 3'UTR by partial substitution ( Figure 4 ). In order to further study the function of T55, we transfected the cells with the above variants and then ...

Embodiment 3

[0042] Example 3: In vivo knock-down HuR inhibits acute lung injury

[0043] 3.1 Experimental method

[0044] 3.1.1 Modeling of LPS-induced acute lung injury in mice

[0045] The experiment was divided into PBS negative control group and LPS treatment group. The PBS and LPS (500ug / ml) solution was stimulated by ultrasonic atomization for 30 minutes, and the mice were inhaled. After an interval of 1 hour, they were atomized again for 30 minutes. The mice were killed at 8 hours, and the lungs were taken. A part of the lung tissue was fixed in 4% neutral paraformaldehyde and stained with H&E, and the remaining part of the tissue was homogenized for MPO activity determination. 3.1.2 In vivo detection of HuR siRNA fragments with Cy5 fluorescence

[0046] Mice were injected with Cy5-labeled HuR siRNA into the tail vein. After 24 hours, the lung tissue was taken out and placed on a fluorescence imager to observe the distribution of HuR siRNA in the lung tissue. Take out a small pi...

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Abstract

The present invention discloses a use of cholesterol-modified HuR interference siRNA in preparation of drugs for treating acute lung injury caused by sepsis. According to the present invention, the related HuR interference siRNA is an artificially-synthesized and cholesterol-modified small nucleic acid drug; with intravenous administration, results show that the HuR siRNA can be richly distributed in lung tissue, enter lung cells, inhibit a plurality of 3'-UTR of mRNA of inflammatory factors having HuR binding sites, and promote TNFalpha RNA degradation so as to inhibit acute lung injury induced by bacterial lipopolysaccharide (LPS), wherein TNFalpha expression can be inhibited; and the small nucleic acid drug provides significant release effects for lung inflammation, is applicable for intravenous administration, inhalation administration or intratracheal administration, and is the small nucleic acid drug having application values and prospects and provided for treating acute lung injury under the premise of lack of effective and related acute lung injury treatment nucleic acid drugs in the prior art.

Description

technical field [0001] The invention belongs to the technical field of biomedical materials, and relates to the use of a small molecule RNA, that is, HuR siRNA, in preparing a medicine for treating sepsis acute lung injury or pulmonary inflammatory disease. Background technique [0002] Acute lung injury (ALI) is a systemic inflammatory response syndrome characterized by refractory hypoxemia and respiratory distress caused by various pathogenic factors inside and outside the lung. Common causes of acute lung injury include sepsis, trauma, and shock. Sepsis is its most common cause. [0003] At present, the main pathogenesis of acute lung injury in sepsis includes the following: (1) cellular mechanism: the main cells involved in inflammation include neutrophils, alveolar macrophages, pulmonary vascular endothelial cells, alveolar epithelial cells, lymphocytes, etc. . Studies by Lea F et al. have shown that neutrophil apoptosis is inversely proportional to the severity of s...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61P11/00
Inventor 殷武曹丹卞金俊华子春
Owner NANJING UNIV
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