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Umbilical cord mesenchymal stem cell modified by Nrf2 gene as well as preparation method and application thereof

A technology of mesenchymal stem cells and gene modification, applied in the field of stem cells

Inactive Publication Date: 2020-01-24
李陶 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no report at home and abroad on the use of Nrf2 gene to modify umbilical cord mesenchymal stem cells to prepare anti-senile dementia drugs

Method used

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  • Umbilical cord mesenchymal stem cell modified by Nrf2 gene as well as preparation method and application thereof
  • Umbilical cord mesenchymal stem cell modified by Nrf2 gene as well as preparation method and application thereof
  • Umbilical cord mesenchymal stem cell modified by Nrf2 gene as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] The construction of embodiment one recombinant plasmid

[0064] 1. Collect 2ml of peripheral anticoagulant blood from 16 normal healthy people (28.3±10.5 years old), and mix all the blood evenly. Take 2ml of well-mixed peripheral blood, add 12ml of erythrocyte lysate within 4h, centrifuge at 1000rpm for 5min, discard the supernatant, resuspend and wash with PBS, add 1ml of Trizol lysate, and extract RNA. According to Takara RT Master Mix reverse transcription kit operating instructions, take 500ng of total RNA for reverse transcription, the reaction system is 1μl, the reaction conditions are 37°C for 15min, 85°C for 15s, and the reaction product is stored at -20°C for future use. The reaction system is as follows:

[0065] Template RNA 1μl

[0066] RT enzyme Master Mix 2μl

[0067] DEPC ddH 2 O 7μl

[0068] 2. Use primers Forward: 5'-cgcggatccgcgatggatttgat-3', Reverse: 5'-cgacgcgtcgagaaaaactag-3', and use the cDNA obtained in step 1 as a template for conventiona...

Embodiment 2

[0072] Example 2 Acquisition of Nrf2 Gene Modified Umbilical Cord Mesenchymal Stem Cells

[0073] 1. Take 5×10 293T cells in the logarithmic growth phase 6 Inoculate each in a 100mm cell culture dish. After 24 hours, when the cell confluence reaches 80%-90%, replace it with 6ml of fresh DMEM complete medium. After 4 hours, add 0.5ml of the target plasmid pWPXL-Nrf210μg, packaging plasmid pMD2.G 6.5 μg and envelope plasmid psPAX2 3.5μg mixture, and slowly add 50μl CaCl 2 , mixed gently, used for lentivirus packaging. The virus liquid after packaging for 48h and 72h was collected, concentrated and stored at -80°C for later use.

[0074] 2. Collect discarded umbilical cord tissue from the hospital, transfer the umbilical cord tissue to a sterile collection bottle, store it at 2-8°C, and separate Wharton's jelly within 12 hours. Separated Wharton's jelly with hMSC SFM culture medium adherent culture in T75 culture flask, placed at 37 degrees Celsius, 5% CO 2 cultured in an i...

Embodiment 3

[0078] Example 3 UCMSC-Nrf2 against nerve cell damage

[0079] When cells are damaged or die, intracellular lactate dehydrogenase (lactate dehydrogenase, LDH) will be released into the cell culture medium, so LDH is an important indicator for detecting cell damage or death. The hydrogen peroxide model of nerve cell PC12 and the Aβ25-35 model were used as nerve cell injury models. Take the nerve cell PC12 to 1×10 5 The density of / hole is inoculated in 6 hole plate, and adds the UCMSC (1 * 10 4 / hole). After 24 hours of culture, the cells were divided into A: control group, B: hydrogen peroxide group, C: UCMSC+H 2 o 2 Panel, D: UCMSC-Nrf2+H 2 o 2 Group, E: Aβ25-35 group, F: UCMSC+Aβ 25-35 Group, G: UCMSC-Nrf2+Aβ25-35 group. Add H to the holes corresponding to the group 2 o 2 and Aβ25-35, so that their final concentrations were 200 μM and 20 μM, respectively. After co-incubating for 12 hours, the cell supernatant was collected, and LDH was detected according to the in...

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Abstract

The invention relates to the technical field of stem cells, in particular to umbilical cord mesenchymal stem cell modified by a Nrf2 gene as well as a preparation method and an application thereof. The invention discloses the umbilical cord mesenchymal stem cell modified by the Nrf2 gene. The umbilical cord mesenchymal stem cell modified by the Nrf2 gene is the umbilical cord mesenchymal stem cellfor overexpression of the Nrf2. The resistance of the Nfr2 gene modified UCMSC to nerve injury is enhanced, the oxidative stress can be effectively antagonized, and the Nfr2 gene modified UCMSC can effectively promote the UCMSC to be converted into neural stem cells compared with the unmodified UCMSC. The UCMSC modified by the Nfr2 gene is transplanted into a dementia mouse body; the Nfr2 gene modified UCMSC can significantly improve learning and memory impairment of dementia mice, protect oxidative damage of brain tissues of the dementia mice, reduce activity of hippocampus AChE and NO in the brains of the dementia mice and inhibit release of inflammatory factors in the hippocampus tissues in the brains of the senile dementia mice. The technical support is provided for preparing a medicine for treating senile dementia by the stem cell.

Description

technical field [0001] The invention relates to the technical field of stem cells, in particular to a Nrf2 gene-modified umbilical cord mesenchymal stem cell and a preparation method and application thereof. Background technique [0002] Alzheimer's disease (Alzheimer's disease, AD) is a progressive neurodegenerative disease, clinically manifested as memory and cognitive dysfunction, loss of language and behavioral ability, and severe amnesia and motor function in the later stage of the patient weaken, and eventually lead to death. Due to the long course of AD and the lack of effective treatment methods, many AD patients have brought serious burdens to families and society. Therefore, finding effective AD treatment methods has become an urgent topic in the medical field. [0003] Due to the existence of the blood-cerebrospinal fluid barrier, it is difficult for many drugs to enter the nervous system tissue, which leads to great limitations in the treatment of AD with tradi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N5/0797A61K35/28A61P25/28
CPCA61K35/28A61P25/28C07K14/47C12N5/0623C12N5/0665C12N2510/00
Inventor 李陶
Owner 李陶
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