[Abstract]
Objective:This study was to construct the shRNA plasmid targeting the oncogene c-Myc and slience the expression of c-Myc gene in human glioblastoma cell lines IN500Δcells using RNA interference technique. Then, this study was also to approach the effects of c-Myc oncogene on glioma cells’proliferation and apoptosis both in vivo and in vitro. Meanwhile, this study detected the influence on the expression of VEGF in IN500Δcells after silencing the c-Myc oncogene and discussed the position of c-Myc gene in glioma and its role on the regulation of angiogenesis. Finally, significant experimental data was provided for the gene therapy and anti-angiogenesis therapy to gliomas.Methods:1 Construction of shRNA plasmids and cell transfection: Construct the shRNA interference plasmid neamed pCMYC targeting c-Myc gene and also construct the pHK plasmid as negative control. Transfect the pCMYC and pHK plasmids into the IN500Δcells respectively through liposome mediating and establish the IN500Δ-pCMYC and IN500Δ-pHK cell lines following the green fluorescent protein marking the plasmid after G418 screening.2 Detection of the shRNA’s silencing effects on the targeting gene: Seen the non-transfection IN500Δcells as blank control, detect the expressive change of c-Myc and VEGF both on mRNA and protein level by RT-PCR and immunocytochemistry method respectively among different cell groups.3 The influence of pCMYC plasmid on the cell cycle and cell apoptosis in IN500Δcells: Detect the change of cell cycle and cell apoptosis among IN500Δ, IN500Δ-pHK and IN500Δ-pCMYC cell groups through flow cytometry.4 The influence of pCMYC plasmid on cell proliferation in vivo through bearing cancer nude mice experiment: Inoculate the IN500Δ, IN500Δ-pHK and IN500Δ-pCMYC cells into nude mice subcutaneously. Observe the tumor formation rate and the tumor growing information and draw the tumor volume growth curve and tumor weight bar graph.5 The influence of pCMYC plasmid on the expression of c-Myc and VEGF on protein level in IN500Δcells in vivo: Decte the expression of c-Myc and VEGF on protein level in the three different xenograft tumors by immunohistochemistry.6 The influence of pCMYC plasmid on the cell apoptosis of IN500Δcells in vivo: Detect the cell apoptosis information in the three different xenograft tumors by TUNEL in situ cell apoptosis detecting method. 7 Statistical analysis: Using SPSS15.0 statistical software, analyse the experimental data scientifically. The comparison of interclass measurement data applied the one-way ANOVA method and the comparison of two classes applied LSD and SNK-T test. The comparison of enumeration data applied non-parameter test. P
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