Study On Anti-glioma Effect Of Radiation By Per1、Per2Genes In Vivo

Objective: To explore the expression and at different time periods radiotherapyregulation of circadian clock genes Period1and Period2(Per1,Per2) in gliomasand normal brain tissue. Observation the effect of Per1、 Per2on the C6gliomaand normal tissue cells on the apoptosis rate and effects of proliferation capacityby radiation.Methods: The C6glioma cells were cultured and inoculated into the caudatenucleus in the right side of the SD rats and establishment the glioma model of rat.The rats were housed under a standard light/dark cycle of12h:12h. In this study,times are reported as Zeitgeber time (ZT), or hours after light onset. Thus, ZT0indicates when lights were turned on and ZT12when lights were turned off. Theanimals were adapted to the12h:12h light/dark cycle for3weeks before theexperiments.Tumor-bearing rats were killed at ZT4, ZT8, ZT12, ZT16, ZT20, and ZT24(ZT0). Tumor tissue and contralateral normal tissue were rapidly removed andfrozen in liquid nitrogen. Tissue was then processed for quantification of Per1andPer2mRNA by real-time RT-PCR.Tumor-bearing rats were administered a single15-Gy dose of x-rays (Varian2100C/D, USA) at each of the following time points: ZT4, ZT8, ZT12, ZT16,ZT20, ZT24. We measured cell proliferation and apoptosis in glioma and normaltissue by determining levels of proliferating cell nuclear antigen (PCNA) andterminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotinnick end-labeling (TUNEL) to observe the different of radiosensitivity. Results: We measured the expression of Per1mRNA over a period of24h in bothnormal and glioma tissue from rats. In normal tissues, the Per1mRNA levelpeaked at ZT8, after which it gradually decreased and had a nadir at ZT20-24. Incontrast, in the glioma tissues, the Per1mRNA level peaked at ZT4, it was low atZT12, then it hit a second peak around ZT16and a second trough around ZT24. Innormal tissue, Per2mRNA expression levels began to rise immediately after ZT0,reached a peak at ZT12, and then gradually fell to a nadir at ZT24. In contrast, inglioma tissue, the level of Per2mRNA peaked at ZT24(ZT0), fell to a low at ZT8,then rose to a second peak around ZT12, followed by a second trough aroundZT20.We measured cell proliferation in glioma and normal tissues at ZTs whenPer1and Per2mRNA levels were high and low, following irradiation with a singledose of x-radiation. In glioma tissues, the proportion of proliferating cells at ZT4,when Per1expression was at its peak, was not significantly different from theproportion at ZT12, when Per1expression was at a nadir. Similar results wereobtained in normal tissue, where the proportion of proliferating cells was notsignificantly different between ZT4and ZT12. Proliferation in glioma tissuecorrelated with the level of Per2expression. The proportion of proliferating cellsat ZT0, when Per2expression was maximal, was significantly lower than at ZT8,when Per2expression was at a nadir. In contrast, in normal tissue, the proportionof proliferating cells was not significantly different between ZT0and ZT8.We detected apoptosis in glioma and normal tissue after irradiating animals at timeswhen Per1and Per2mRNA levels were high and low. In glioma tissue, the proportion ofapoptotic cells was not significantly different than Per1expression was at a nadir. Similarly,in normal tissue, the proportion of apoptotic cells was not significantly different betweenZT4and ZT12. Apoptosis in glioma tissue correlated with the level of Per2expression. When Per2expression was maximal the proportion of apoptotic cells was significantlyhigher than the nadir. In contrast, in normal tissue, the proportion of apoptotic cells was notsignificantly different.Conclusion: This study confirmed that of Per1, Per2exhibited a gene expressionrhythms in the rat glioma cells in vivo expression of rhythm for12hours, normaltissue for24hours. Ionizing radiation can induce tumors of Per1, Per2exhibited athe increased expression and inhibition of tumor cell value-added, and promote theapoptosis of the tumor tissue. Preliminary exploration of the glioma-ray sensitiverhythm, provides a theoretical basis for the treatment of gliomas time inradiotherapy, a new comprehensive treatment of gliomas.