| Objective:This study aims to test radiation-sensitivitizing effect induced by mitochondria transplantation on human glioma cells(U87)and its mechanism.Methods:The mitochondria of human astrocytes(HA)were extracted and co-cultured with human glioma cells(U87),U87 cells were exposured to X-Ray.Western Blot was used to detect the expression of Cyto-C,Bax and Bcl-2 proteins in U87 cells;the Annexin V/PI double staining method was used to detect the apoptosis;the RT-CES system was used to evaluate viability of U87 cells;the clonogenic assay was performed to compare the proliferation status of each group;Western Blot was used to detect the expression of Fascin-1and MMP-2 proteins in U87 cells;The expression of Fascin and MMP-2 in U87 cells was detected by Western Blot,and the in vitro transfer ability of U87 cells was tested by scratch test and in vitro invasion test.and the distribution of filopodia in cells was observed using phalloidin staining.Results:Firstly,the mitochondria were still active after being extracted from HA cells.The free mitochondria labeled with mito-tracker red could enter U87 cells by co-culture and amplify nd1 gene by Polymerase Chain Reaction(PCR).The results of detection of Fis1 and Drp1 proteins by Western Blot showed that the free mitochondria could survive in U87 cells and replicate in large quantities.Secondly,when the fluorescence intensity in the cell was quantitatively analyzed,the fluorescence intensity of the single cell increased from 0.08 to 1.83 when the free mitochondria and U87 cells were co-cultured for 12 h,indicating that the free mitochondria can enter the U87 cells by co-culture.The results showed that the expression of Cyto-C and Bax increased from 179.5%and 198.5%to 251.72%and256.10%,respectively,and the expression of Bcl-2 decreased from 57.17%to 22.23%,respectively.The growth curve of U87 cells in the combined group was increased to19.2%and 43.8%compared with that of the alone irradiation group using the Annexin V/PI double staining method.The results of RT-CES showed that the growth curve of U87 cells in the combined group was inhibited in 96 h,and the formation rate of the combined group decreased from 53%to 29.3%in the alone irradiation group.Thirdly,mtDNA-deficient?~0cells were constructed.Single cell electrophoresis showed that after 4Gy X-ray irradiation,comet trailing rate in?~0+Mito group was significantly higher than that in?~0 group,?-H2AX immunofluorescence assay revealed a large number of DNA double bond breaks in?~0+Mito group.At the same time,the colony formation rate of?~0 Mito group was significantly lower than that of?~0 group after irradiation.Forth,Western Blot showed that the content of the Fascin and MMP-2 protein in the cells decreased to 53.39%and 55.17%,respectively,while the contents of the Fascin and MMP-2 protein in the cells decreased to 48.19%and 44.23%,respectively,when the mitochondria were transplanted for 12 h.It was found that the filiform pseudo-foot of the mitochondrial transplantation group was significantly reduced as compared with the control group.The results of the scratch test show that the combined group can further inhibit the migration function of U87 cells.In vitro,the results of in vitro invasion test showed that the number of invasion in the treatment group decreased to 57.2%in the control group,the invasion quantity of the X-ray alone treatment group was 30.8%,and the invasion number of the combined group was significantly reduced to 14%.Conclusion:Overall,This study has found that,The free mitochondria can enter the U87 cells of the glioma by co-culture,and the U87 cells have a radiosensitization effect.After the co-culture for 12 h,4 Gy X-ray irradiation can be carried out,and the U87 cells can be used for inducing apoptosis,inhibiting proliferation and anti-tumor metastasis and the like.The mechanism of action and X-ray irradiation activate the mitochondrial apoptosis pathway and reduce the degree of malignancy of the tumor cells. |
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