| ObjectivesGlioma is one of the most common types of central nervous tumors, which accounts for about 40%-60% of the primary intercranical tumor. Due to its invasion characteristic and the trend from lower-level to high-level, surgical treatment is difficult to cure the glioma and the therapeutic effect was poor even though combining the radiotherapy and chemotherapy. The postoperative recurrence rate of glioma is high; the prognosis of the patients is poor; mortality rate is high, too. The oncogene and tumor suppressor gene play an important role in the occurrence and development process of tumor. The activation of proto-oncogenes and the inactivation of tumor-suppressor genes make the regulation of cell cycle and cell apoptosis out of control. The wild type p53 gene is a common tumor suppressor gene, the main biological function of which is through a series of signal transduction pathways regulating cell cycle and cell apoptosis to maintain the stability of the cellular genome, thus inhibiting the occurrence of tumor. This study aims to enhance wtp53 gene transfected to the murie glioma C6 cells by ultrasound-targeted microbubble destruction technology. In addition, the feasibility of ultrasound-mediated SonoVue microbubble irradiation enhancing pEGFP-C1-wtp53 plasmid transfected into C6 cells was explored and the parameters of microbubble concentration and plasmid concentration was optimized. MethodThe cultured murine glioma C6 cells suspension in vitro could be divided into five groups: the blank control group, the plasmid + ultrasound group, the plasmid + microbubble group, the ultrasound + microbubble group, the plasmid + ultrasound + microbubble group. According to the microbubble concentration, the plasmid + ultrasound + microbubble group is divided into 5%, 10%, 20%, 30%, 40% groups. And plasmid concentration could be divided into 5ug/ml, 10ug/ml, 15ug/ml, 20ug/ml, 30ug/ml. Upon exposure to 1 MHz pulsed ultrasound beam(0.8 W/cm2, 20 % duty cycle) for 50 s, the wtp53 gene was transfected to C6 cells. After 24h- 48 h of incubation, fluorescence microscope cell observed transfection rate; Trypan blue staining method detected cell mortality; RT-PCR detected the wtp53 mRNA expression in the transfection cells; and flow cytometry instrument analyzed the apoptosis rate and the cell cycle distribution. Result1. The gene delivery efficiency increased significantly at 20% and 30% concentrations and reached the highest level. However, the cell survival rate significantly reduced as SonoVue microbubble concentration >30%(P<0.05).The gene delivery efficiency increased significantly with DNA concentrations, peaked at DNA concentrations of 15?g/ml(P<0.05); However, the cell survival rate reduced(P<0.05). The cell survival rate significant decreased at DNA concentrations of 20?g/ml or 30?g/ml(P<0.01).Taking into account the efficient gene delivery and cell viability, the optimal exposure parameters were determined to microbubble concentration of 20% and the wtp53 concentration of 15?g/ml in this study.2. UTMD as a safe and effective method not only facilitated gene delivery but successfully made the gene expressing stably in the cell further.Under the optimized ultrasound irradiation conditions, no significant difference in cell viability was observed among the A, B, C and D groups(P>0.05).In comparison with other groups, the transfection efficiency was higher in the wtp53 + microbubble + ultrasound group: wtp53 mRNA expression increased. What's more, tumor cells apoptosis, count of cell cycle G0/G1 phase and cell mortality rate increased statistically(P<0.05). Compared with the control group, the cell mortality rate of ultrasound + microbubble group increased, but the difference between two groups was not statistically significant(P>0.05). ConclusionUltrasound-targeted microbubble destruction as a security and efficient method of gene therapy, could enhance the wtp53 gene transfection in glioma C6 cell, and the wtp53 gene could be stably expressed. |
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