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npm-targeting shRNA interference library and applications thereof

A targeting and drug technology, applied in the field of npm targeted shRNA interference library, can solve problems such as blanks, and achieve the effect of improving glucose tolerance, improving blood sugar control ability, and inhibiting insulin resistance

Active Publication Date: 2018-04-13
THE FIRST PEOPLES HOSPITAL OF NANTONG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current research on npm and diseases is mainly concentrated in the field of tumors, and the research on npm as a therapeutic target for diabetes is still blank

Method used

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  • npm-targeting shRNA interference library and applications thereof
  • npm-targeting shRNA interference library and applications thereof
  • npm-targeting shRNA interference library and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Twenty 4-week-old C57BL / 6J male mice were randomly divided into a normal diet group (NCD) of 10 and a high-fat diet group (HFD) of 10. The mice in the normal diet group were given common standard diet, in which the proportions of various ingredients in calories were: fat 10%, protein 22%, and carbohydrate 68%. The calorie ratios of various ingredients in a high-fat diet (HFD) are: 49% fat, 21% protein, and 30% carbohydrate. Body weights were recorded weekly. At the 20th week of feeding, a glucose tolerance (GTT) test was performed. GTT: After fasting for 16 hours, the mice were injected with 2g / kg of glucose through the peritoneum after the blood glucose level was measured, and the blood glucose levels at 30, 60, 90, and 120 minutes after the injection were measured respectively. Mice were anesthetized and sacrificed, and fresh liver tissue was obtained from mice. The tissue was lysed with an appropriate amount of protein homogenate, and Western blot analysis was perf...

Embodiment 2

[0030] Using shRNA interference to specifically inhibit the expression of the npm gene in the liver, the following four shRNAs were constructed into the Ad-Easy adenovirus vector, and mixed in equal amounts to make a shRNA interference library, and the adenovirus was packaged, as follows:

[0031] The adenovirus packaging vector was transfected into 293 cells with a fusion degree of 50-70%, and 6 μg of the plasmid was transfected per 10 cm culture dish. Eight hours after transfection, the medium containing the complex was discarded and replaced with new medium. 7-10 days after transfection, scrape the cells from the culture dish with a cell scraper and transfer to a 50ml centrifuge tube. After centrifugation, the supernatant was discarded, and the cells were resuspended in 2 ml of PBS solution. Quickly move the cells in liquid nitrogen, then dissolve them in a 37°C water bath and shake vigorously. Repeat this step 4 times. The above-mentioned virus-containing supernatant wa...

Embodiment 3

[0039] After mice were fed a normal diet (10% fat, 22% protein, 68% carbohydrate) or a high-fat diet (49% fat, 21% protein, 30% carbohydrate) for 12 weeks, the high-fat fed mice were randomized Divided into two groups, group 1 mice were injected with control shRNA adenovirus (1*10 8 PFU / only), another group of mice were injected with npm interfering adenovirus (1*10 8 PFU / piece). After the injection, continue to feed for 4 weeks, and detect the glucose tolerance (GTT) of the mice.

[0040] GTT: After the mice were fasted for 16 hours, after the blood glucose level was measured, the glucose solution of 2g / kg of glucose was injected peritoneally, and the blood glucose content at 30, 60, 90, and 120 minutes after the injection was detected by a blood glucose meter, and the blood glucose changes of the mice were made. curve.

[0041] The relative expression of nucleophosmin (npm) in the liver tissues of normal diet and high-fat-induced diabetic mice (high-fat diet), as figure ...

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Abstract

The present invention discloses an npm-targeting shRNA interference library and applications thereof, wherein the interference library comprises four shRNAs, and the specific sequences of the four shRNAs are 5'-TGACTGACCAGGAGGCTATTCAA-3',5'-TATGGAATGTTATGATAGGACA-3',5'-TGATGAAAATGAGCACCAG-3', and 5'-CTCCTGGAGGTGGTAACAAG-3'. According to the present invention, the results findsthat npm is significantly and highly expressed in insulin resistant liver tissues, and the high expression is mainly stimulated by saturated fatty acids, such that the expression of the npm gene in the liver can be specifically inhibited by using the shRNA interference method; and the mouse experiment results prove that the interference on the npm can significantly inhibit the insulin resistance induced by high-fat diet, and the glucose tolerance of mice is significantly improved, such that it is proved that the interference on the expression of the npm in the liver can inhibit the insulin resistance in mice, andcan significantly improve the blood glucose control ability of diabetic patients.

Description

technical field [0001] The invention belongs to the technical field of gene medicine, and in particular relates to an npm targeting shRNA interference library and its application. Background technique [0002] Nucleophosmin (npm) is a multifunctional protein in the human body, which is closely related to cell stress, chronic inflammation, metabolic regulation, cell survival and proliferation. npm is a widely expressed protein with high expression in various tissues such as liver, kidney, thymus, muscle, brain and fat. Studies have shown that npm is involved in the process of mitosis, and the loss of its expression can lead to the failure of cell mitosis and the stagnation of cell proliferation. Moreover, npm can promote the stability and transcriptional activity of p53 in the nucleus, thereby promoting the process of apoptosis. npm can also participate in the occurrence and process of chronic inflammation by regulating the key transcription factor of inflammation NF-kappaB...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K31/7088A61P3/10
CPCC12N15/113C12N2310/14C12N2310/531
Inventor 王晓华万春华黄华星王雪琴马宏仓晓敏
Owner THE FIRST PEOPLES HOSPITAL OF NANTONG
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