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Specific long-acting siRNA and application thereof to treatment of renal fibrosis

A renal fibrosis and specific technology, applied in the field of RNA interference, can solve problems such as ignorance, achieve the effect of alleviating the degree of fibrosis, good drug effect, and inhibiting the process of fibrosis

Inactive Publication Date: 2018-05-22
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It has been reported that Kindlin-2 can participate in renal fibrosis by activating the TGF signaling pathway. However, the regulation of Kindlin-2 on other signaling pathways can be used as a target for inhibiting renal fibrosis. The effect is not known

Method used

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  • Specific long-acting siRNA and application thereof to treatment of renal fibrosis
  • Specific long-acting siRNA and application thereof to treatment of renal fibrosis
  • Specific long-acting siRNA and application thereof to treatment of renal fibrosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] A specific long-acting siRNA (Kindlin-2 siRNA) and negative control siRNA:

[0025] Kindlin-2 siRNA, sense: 5′-CAGCGAGAAUCUUGGAGGC-3′,

[0026] Negative Control siRNA, sense: 5′-UUCUCCGAACGUGUCACGUTT-3′,

[0027] Synthesized by Guangzhou Ruibo Company:

[0028]

[0029] In Kindlin-2 gene half-knockout animals (FERMT2+ / -) C57 mice and WT C57 mice were made UUO models, and then compared the Hippo signaling pathway, EMT indicators, fibrosis indicators, Changes in the Wnt signaling pathway, etc., and compare the degree of Hippo inhibition and the degree of fibrosis in the level of Kindlin-2 expression.

[0030] Western Blot detection

[0031] 1) Polyacrylamide gel electrophoresis (SDS-PAGE)

[0032] ① Glue preparation

[0033] Prepare the lower layer separating gel with the required concentration, pour the gel after mixing well, and carefully add distilled water on the surface of the gel, pour out the uncondensed distilled water after the gel naturally condenses, po...

Embodiment 2

[0059] In this experiment, 8 cases of renal fibrosis were collected from the Third Hospital of Peking University. The expression levels of Kindlin-2, YAP, p-YAP, and MOB were detected by immunohistochemistry, and the specific detection methods are as follows.

[0060] The tissue detection effectiveness and specificity of the antibody obtained by immunohistochemistry detection, the specific steps are as follows:

[0061] 1) Immunohistochemical staining of paraffin sections (Envision two-step method)

[0062] ① Dewaxing and hydration of paraffin sections: xylene Ⅰ and Ⅱ for 10 minutes each; 100% alcohol Ⅰ and Ⅱ for 5 minutes each; 95% alcohol Ⅰ and Ⅱ for 5 minutes each; 90% alcohol for 5 minutes; 80% alcohol for 5 minutes and 70% alcohol for 5 minutes;

[0063] ② 3%H 2 o 2 Block endogenous peroxidase, at room temperature, 10min;

[0064] ③ Rinse with tap water for 2 minutes and distilled water for 2 times;

[0065] ④ Antigen restoration (antigen restoration solution is divi...

Embodiment 3

[0074] Twelve C57 male mice were randomly divided into 6 groups, named Group1-6, so the animals underwent right kidney UUO surgery on the first day. The first group G1 was injected with 5nmol Kindlin-2 long-acting siRNA on the first day, the third day, and the fifth day respectively; the G2 group was treated with the same dose of Kindlin-2 long-acting siRNA on the second day and the fourth day; Group G2 was treated on the third day and the fifth day; Group G4 and G5 were treated once on the fourth and fifth day respectively; Group G6 was the experimental control group, and the treatment was given on the first day, the third day and the end of the fifth day respectively. Intravenous injection of 5nmol Control long-acting siRNA, see specific dosage regimen Figure 5 a. All animals were sacrificed on the 7th day and bilateral kidneys were fixed and protein was extracted.

[0075] The results of the experiment showed that the degree of water accumulation in the UUO kidneys of th...

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Abstract

The invention discloses specific long-acting siRNA and application thereof to treatment of renal fibrosis. The specific long-acting siRNA specifically targets integrin-interacting protein Kindlin-2, can effectively reduce the expression level of the renal Kidlin-2 protein, and can effectively activate TGFbeta and Wnt signaling pathways in kidney and inhibit a Hippo signaling pathway so as to treatthe renal fibrosis and delay occurrence thereof; furthermore, animal experiments verify that tail vein injection of the specific long-acting siRNA after occurrence of UUO-induced fibrosis can effectively inhibit the progression of the fibrosis and alleviate the degree of the fibrosis and the effect of early medication is good.

Description

technical field [0001] The invention belongs to the technical field of RNA interference, in particular to a specific long-acting siRNA and its application in treating renal fibrosis. Background technique [0002] Renal fibrosis is a pathophysiological change, which is a gradual process in which the function of the kidney changes from healthy to damaged, then damaged, and finally lost. Renal fibrosis is a complex pathological process involving multiple signaling pathways, and so far there is no effective treatment. Finding effective therapeutic targets to block multiple pathogenic signaling pathways and block or delay the fibrosis process will be good news for patients with acute and chronic kidney diseases. It has been reported that Kindlin-2 can participate in renal fibrosis by activating the TGF signaling pathway. However, whether the regulation of Kindlin-2 on other signaling pathways can be used as a target for inhibiting renal fibrosis, and targeted therapy after fibro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K31/7088A61P13/12
CPCA61K31/7088C12N15/113C12N2310/141
Inventor 张宏权战军宋佳桂
Owner PEKING UNIV
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