A double-gene co-expression plasmid pires2-nrf2-dkk1 and its preparation method and application

A co-expression and dual-gene technology, which is applied to the dual-gene co-expression plasmid pIRES2-Nrf2-DKK1 and its preparation field, can solve the problems affecting the therapeutic effect of transplanted cells, increase the number of cells, and reduce the activity of stem cells, so as to improve the anti-stress effect. ability, maintain phenotype and function, and ensure the effect of continuous proliferation

Active Publication Date: 2021-08-27
THE THIRD PEOPLES HOSPITAL OF SHENZHEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is to overcome the defects and deficiencies such as the reduction of stem cell activity and the increase of the number of apoptotic cells in the existing stem cell treatment process, thereby affecting the therapeutic effect of transplanted cells on the damaged site, and providing a stem cell Highly expressed double-gene co-expression plasmid pIRES2-Nrf2-DKK1 that can significantly increase the antioxidant, anti-inflammatory, and anti-apoptotic abilities of mesenchymal stem cells

Method used

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  • A double-gene co-expression plasmid pires2-nrf2-dkk1 and its preparation method and application
  • A double-gene co-expression plasmid pires2-nrf2-dkk1 and its preparation method and application
  • A double-gene co-expression plasmid pires2-nrf2-dkk1 and its preparation method and application

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Experimental program
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Effect test

Embodiment 1

[0057] Example 1 Construction of double-gene co-expression plasmid pIRES2-Nrf2-DKK1

[0058] 1. According to the sequence information of Nrf2 (NM_006164) and DKK-1 (NM_012242) on NCBI, clone the CDS sequences of Nrf2 and DKK-1 genes, the CDS sequences of the Nrf2 and DKK-1 genes are shown as SEQ ID NO: 1~ 2; using the pIRES2-zsGreen vector as the backbone, replace the EGFP gene on the pIRES2-zsGreen vector with the DKK-1 CDS sequence, and then clone the Nrf2 CDS sequence between the SacI and BamHI restriction sites of the pIRES2-DKK1 vector;

[0059] 2. Results

[0060] The full length of the double-gene co-expression plasmid is 7151 bp, wherein the Nrf2 gene sequence is located at the 619th to 2436th position, and the DKK-1 gene sequence is at the 3016th to 3816th position.

Embodiment 2

[0062] When the third-generation adipose-derived mesenchymal stem cells (purchased from Guangzhou Saiye Company) and umbilical cord mesenchymal stem cells (the umbilical cord tissue was obtained from healthy puerpera cesarean section fetuses in the Department of Obstetrics and Gynecology, Shenzhen Third People's Hospital) reached 60% confluence Around the same time, the double-gene co-expression plasmid was transferred into the above two types of cells by Lipofectamine 3000 transfection method. The transfection procedure was carried out according to the manufacturer's instructions. Add 0.1 μg / mL of LPS and 200 μM H 2 o 2 Co-stimulated for 24 hours, in order to avoid external stimulation (LPS+H 2 o 2 ) and cells not transfected with double-gene co-expression plasmids were negative controls. Finally, the cells of each group were collected to detect the levels of indicators related to cell viability, cell apoptosis, and cell oxidation.

[0063] The cell viability was detected...

Embodiment 3

[0069] The mouse model of acute liver injury was established by intraperitoneal injection of Gal and LPS solution in PBS. In the experiment, NOD / SCID male mice were injected with 2x10 6 Pre-transfected hADMSCs and hUCMSCs were used for transplantation therapy. Three days later, mouse serum and liver tissues were collected for follow-up experiments.

[0070] The grouping situation is: Gal+LPS means that the mice were intraperitoneally injected with Gal and LPS at the same time.

[0071] Healthy is a healthy mouse without any treatment.

[0072] Gal+LPS+No stem cell refers to mice that are not injected with stem cells after intraperitoneal injection of Gal and LPS into mice;

[0073] Gal+LPS+Stem cell means that after the intraperitoneal injection of Gal and LPS into the mice, the untreated hADMSCs and hUCMSCs were injected into the tail vein;

[0074] Gal+LPS+Stem cell+Plasmid means that Gal and LPS are injected intraperitoneally into mice, and then hADMSCs and hUCMSCs tran...

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Abstract

The invention discloses a double-gene co-expression plasmid pIRES2-Nrf2-DKK1. The plasmid contains the original expression vector pIRES2-zsGreen, CDS sequences of Nrf2 and DKK-1 genes; the full length of the plasmid is 7151bp, and its sequence is shown in SEQ ID NO: 1, wherein the Nrf2 gene sequence is located at positions 619-2436, The sequence of DKK-1 gene is located at position 3016-3816. The double-gene co-expression plasmid can be highly expressed in human adipose-derived mesenchymal stem cells and human umbilical cord mesenchymal stem cells, and can significantly increase the ability of mesenchymal stem cells to resist oxidation, anti-inflammation, and anti-apoptosis; in vivo experiments prove that, Transplantation of the mesenchymal stem cells transfected with the plasmid can significantly improve the liver injury phenomenon of mice, help to improve the therapeutic effect of mesenchymal stem cell transplantation, and has great application prospects.

Description

technical field [0001] The invention belongs to the field of biotechnology. More specifically, it relates to a double-gene co-expression plasmid pIRES2-Nrf2-DKK1 and its preparation method and application. Background technique [0002] In recent years, the proportion of patients with liver injury caused by alcohol and drugs has been increasing. Epidemiological data show that about 3.8% of the world's liver disease deaths are caused by alcoholic cirrhosis caused by drinking. At the same time, about 10% of acute hepatitis cases each year are drug-induced liver injury. These patients with liver damage caused by alcohol or drugs are likely to progress to liver cancer, cirrhosis and liver failure. Currently, doctors mainly use drugs with hepatoprotective effect to conservatively treat patients with mild symptoms of liver damage. However, in critically ill patients, it is still possible to improve their condition only through liver transplantation. Although liver transplantati...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N5/10A61K35/28A61P1/16
CPCA61K35/28C12N5/0665C12N5/0667C12N15/85C12N2510/00
Inventor 肖佳刘映霞陈凤何留民
Owner THE THIRD PEOPLES HOSPITAL OF SHENZHEN
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