Application of p65 in preparation of medicament for up-regulating SIRT1 expression

A kind of medicine and the technology of use, which is applied in the field of use of p65 in the preparation of medicines that up-regulate SIRT1 expression

Inactive Publication Date: 2011-05-11
THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although SIRT1 can maintain gene stability, it also inhibits the activity of tumor suppressor genes, which makes SIRT1 have potential tumorigenic propertie

Method used

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  • Application of p65 in preparation of medicament for up-regulating SIRT1 expression
  • Application of p65 in preparation of medicament for up-regulating SIRT1 expression
  • Application of p65 in preparation of medicament for up-regulating SIRT1 expression

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1 Experimental material

[0032] 1. Strains and plasmids

[0033] Escherichia coli DH5α (genetic type supE44 hsdR17 recA1 endA1 gyrA96) (purchased from Beijing Tiangen Company, catalog number is CB101-03). The pTASirt-promoter (2825bp) plasmid is obtained by ligating the 2825bp promoter region of human SIRT1 (ref Gene_NM_012238 range=chr10: 69311608-69314485) into the pTA-Luc plasmid with MluI and XholI as restriction sites. For the NF-κB luciferase reporter plasmid, four κB response elements GGGACTTTCC were inserted in series into pGL3basic, with KpnI and BglII as restriction sites. The MMP-9 luciferase reporter plasmid is to clone the human MMP-9 gene promoter (-711~+19bp, refGene_NM_004994 range=chr20:44070243-44070972) into the Mlu I and BglII sites of the pGL-3basic vector. The pcDNA3.1-p65 and pcDNA3.1-p50 expression plasmids are human-derived p65 and p50 cDNAs (the p65 gene cDNA number is NM_021975.3, and the p50 gene cDNA number is NM_003998.3) sequ...

Embodiment 2

[0044] Embodiment 2 experimental method

[0045] 1. Bacterial manipulation and transformation of plasmids

[0046] 1.1 Preparation of Competent Escherichia coli DH5a

[0047] Inoculate a single colony of host bacteria into 5ml LB medium, shake overnight at 37°C, inoculate 1 / 50 volume of the overnight bacterial solution into 50-100ml LB medium the next day, shake at 37°C, and wait for the OD of the bacterial solution 600 When it is close to 0.3-0.4, centrifuge at 4°C and 4000 rpm for 10 minutes, collect the bacteria, and take 1 / 5 volume of the original bacteria solution with pre-cooled 0.1mol / LCaCl 2 Gently add the solution to the cells, gently shake the centrifuge tube on the ice bath to slowly disperse the cells, let stand in the ice bath for 20 minutes, centrifuge at 4°C, and discard the supernatant. Repeat the previous operation, after collecting the cells by centrifugation under the same conditions, add 1 / 50 volume of pre-cooled CaCl to the cells 2 , mix well, and stand...

Embodiment 3

[0124] Embodiment 3 experimental result

[0125] 1. p65-induced up-regulation of SIRT1 expression

[0126] NF-κB consists of two subunits, p65 and p50, in which p65 can bind to DNA and has transcriptional activity, while p50 has only DNA binding domain. In the study, we focused on the role of NF-κBp65 subunit with transcriptional activation function in inducing SIRT1 expression. The transcription factor NF-κB plays a role in the nucleus. In the resting state, IκB combines with NF-κB to form a trimer, which covers the nuclear localization signal of NF-κB and exists stably in the cytoplasm of the cell. In the TNF-κB Under the stimulation of α, IL-6, oxidative stress, UV, etc., the phosphorylation of IκB dissociates from the trimer, and NF-κB translocates from the cytoplasm to the nucleus. After treating the vascular smooth muscle cell line A7r5 with TNF-α for 30 minutes, the cells were fixed in situ, and immunofluorescence detection was performed. It was found that after treat...

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Abstract

The invention relates to application of p65 in preparation of a medicament for up-regulating silence information regulator 1 (SIRT1) expression. Particularly, the medicament is a purified protein type medicament, and the formulation of the medicament is the one suitable for protein medicament application; and particularly, the medicament is a recombinant nucleic acid construct for expressing p65 protein. The invention also relates to application of p65 in preparation of a medicament for treating diseases of mammals comprising people benefiting from SIRT1 expression up-regulation, wherein the diseases of mammals comprising people benefiting from SIRT1 expression up-regulation are selected from metabolic syndromes, tumors, neurodegenerative diseases, cardiovascular diseases, inflammations or mitochondria related diseases.

Description

technical field [0001] The present invention relates to the use of p65 in the preparation of medicines for up-regulating the expression of SIRT1 in mammals, including humans. The invention also relates to the use of p65 in the preparation of medicines for the treatment of diseases in mammals including humans that are benefited from the increased expression of SIRT1. Background technique [0002] The function of SIRT1 [0003] The Sir2 (silence information regulator) gene family is a conserved NAD+-dependent histone / non-histone deacetylase that exists from archaea to mammals. In yeast, Sir2, together with several proteins it interacts with, plays an indispensable role in gene silencing, genome stability, cell lifespan, and metabolic regulation. There are seven Sir2 homologous genes in mammals, named SIRT1 to SIRT7, among which SIRT1 and Sir2 have the highest homology (Frye, 1999), and the research is the most in-depth. SIRT1 is located on q21.3 of chromosome 10, the gene le...

Claims

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Application Information

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IPC IPC(8): A61K38/17A61K48/00A61P3/10A61P3/04A61P3/00A61P35/00A61P25/28A61P25/16A61P9/00A61P9/04A61P9/10A61P29/00A61P43/00
Inventor 张慧娜刘德培高鹏李莉陈厚早
Owner THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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