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Construction method of SOD (superoxide dismutase) gene modified mesenchymal stem cell and application thereof

A kind of stem cell and gene modification technology, applied in the field of stem cells, to achieve the effect of prolonging the survival period, broad application prospects, and enhancing the relative activity of SOD

Inactive Publication Date: 2012-09-12
SHENZHEN BEIKE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, experimental research on MSC gene modification has just emerged, and there is no relevant report on gene transfection of MSC in the treatment of ARS at home and abroad.

Method used

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  • Construction method of SOD (superoxide dismutase) gene modified mesenchymal stem cell and application thereof
  • Construction method of SOD (superoxide dismutase) gene modified mesenchymal stem cell and application thereof
  • Construction method of SOD (superoxide dismutase) gene modified mesenchymal stem cell and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Construction of SOD gene-modified mesenchymal stem cells.

[0026] Adenovirus pDC315 vector, pBHGloxΔE1 and 3Cre were purchased from Microbix Company.

[0027] Connect the SOD3 gene to the adenovirus pDC315 vector to obtain the adenovirus pDC315 recombinant vector; cultivate the 293 cell line, and use the Lipofectamine transfection reagent to co-transfect the adenovirus pDC315 recombinant vector, pBHGloxΔE1, and 3Cre plasmid vector into the 293 cell line. Cultivate for 11 days, until the 293 cell line completely develops pathological effects, collect the adenovirus strain, save the strain, confirm that the SOD3 gene has been successfully connected to the adenovirus vector; amplify the adenovirus strain in large quantities, and infect the 293 cell line , after observing the cell outtoxicity, the adenovirus supernatant was collected, purified and concentrated by ultrafiltration, and the titer of adenovirus in the supernatant was determined by the method of TCI...

Embodiment 2

[0028] Example 2 Identification of EC-SOD overexpression in MSCs.

[0029] After obtaining suitable adenovirus infection conditions, the MSC cell line overexpressing EC-SOD was obtained, and the EC-SOD protein content in MSC cells was detected by Western Blot, and the results were as follows Figure 5 Shown in -A; SOD relative activity in the cell culture fluid measured by enzyme labeling method, the result is as follows Figure 5 -B is shown. Figure 5 It shows that the EC-SOD gene carried by adenovirus is successfully expressed in MSC, the EC-SOD protein content in MSC cells increases, and the relative activity of SOD in cell culture medium increases.

Embodiment 3

[0030] Example 3 The therapeutic effect of SOD gene-modified mesenchymal stem cells on irradiated mice.

[0031] In the present invention, mice are irradiated with cesium 137 gamma rays as a radiation damage model. There are 10 mice in each group, and the mice are treated with SOD gene-modified mesenchymal stem cells through the tail vein 4 hours after irradiating with 5.5Gy rays. The survival of the mice, the results are as follows Figure 6 shown. Among them, UC: umbilical cord mesenchymal stem cells; UC+Null: umbilical cord mesenchymal stem cells infected with empty adenovirus; UC+SOD: umbilical cord mesenchymal stem cells infected with SOD3-adenovirus; BM: bone marrow-derived mesenchymal stem cells Stem cells, BM+Null: bone marrow mesenchymal stem cells infected with empty adenovirus; BM+SOD3: bone marrow mesenchymal stem cells infected with SOD3-adenovirus. Injection volume of SOD3 gene modified mesenchymal stem cells 5×10 5 , two stem cell injections, the injection ...

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Abstract

The invention provides a construction method of a SOD (superoxide dismutase) gene modified mesenchymal stem cell. The construction method comprises the following steps: (A) connecting an SOD3 gene to an adenovirus vector to obtain a recombinant adenovirus vector; B) co-transfecting the recombinant adenovirus vector and a packaging plasmid to a 293 cell line by utilizing a transfecting agent, culturing until the 293 cell line completely generates a cytopathic effect, collecting an adenovirus strain, storing the strain and confirming that the SOD3 gene is connected to the adenovirus vector; C) amplifying the adenovirus strain, infecting the 293 cell line, collecting the adenovirus supernatant, and determining the titer after ultrafiltration, purification and concentration; and D) infecting the mesenchymal stem cell in different conditions respectively by the adenovirus, and determining the best infection condition to obtain the SOD gene modified mesenchymal stem cell. The EC-SOD (extracellular superoxide dismutase) gene over-expressed SOD gene modified mesenchymal stem cell can be prepared by the method, and can be applied to treatment of mouse with radiation so as to prolong the survival period of the radiated mouse.

Description

technical field [0001] The invention belongs to the technical field of stem cells, in particular to a method for constructing SOD gene-modified mesenchymal stem cells and its application. Background technique [0002] Acute Radiation Syndrome (ARS) occurs in clinical tumor patients during radiotherapy, nuclear terrorism, and nuclear accidents. Tissues in the human body with fast cell renewal and rapid metabolism are particularly sensitive to radiation, such as hematopoietic tissue and intestinal tissue. After the Fukushima nuclear leak in Japan in 2010, all countries began to pay more attention to nuclear safety issues. Finding more effective treatments for treating and preventing ARS is an important task related to national security and national health. For the treatment of ARS, some supportive therapies are routinely used, including blood transfusion, fluid replacement, electrolytes, antibiotics, and antiviral drugs, but the therapeutic effect is not obvious. At present,...

Claims

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Application Information

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IPC IPC(8): C12N15/861C12N5/10A61K48/00A61K38/44A61P39/06
Inventor 李陶甘靖毅姜新根孟繁伟李婵刘宗才丁长才姜舒胡祥
Owner SHENZHEN BEIKE BIOTECH
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