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
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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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