Preliminary Study Of The Eca109Cells Biological Effects Under Different Dose Fractionation Mode Irradiation

Objective:In hope to provide some theoretical basis for unconventionalradiotherapy, We observe and compare radiation damage effects andexpression of apoptosis related genes （TNF and SODD）between groups bysimulating different dose fractionation on esophageal cancer cells Eca109.Method:(1) Culture Eca109cells and establish the Department of Eca109cells inthe exponential growth phase.(2) Establish groups of different fractionated dose irradiation program:the blank control group(0), the hyperfractionated group (1), the conventionalfractionation group (2), the hypofractionated group (3), the hypofractionatedgroup (7).(3) Observe morphological changes of irradiated cells.(4) Detect TNF mRNA and SODD mRNA，expression in each group withRT-PCR on the first day after simulation irradiation(5) Detect TNF protein and SODD proteins，expression in each groupwith Western Blot on the first day after simulation irradiation.(6) Detect the absorbance values (OD) of cells in each group on the1th,4th,7th,10th days after simulation irradiation with CCK-8and draw thesurvival curve.Results:(1) Morphological changes of each group，s cell were observed in Hoechst33258staining:The hyperfractionated group (1)54.9%of the irradiated cells were in thestate of―apoptoticnecrosis‖. Conventional fractionation group(2)50.5%ofthe irradiated cells were in the state of―oncosticnecrosis‖;30.3%of theirradiated cells were in the state of―apoptoticnecrosis‖. The hypofractionatedgroup (3)72.1%of the irradiated cells were in the state of―oncosisnecrosis‖,19.2%were in the state of―apoptotic necrosis‖. The hypofractionated group (7)53.6%of the irradiated cells were in the state of―apoptoticnecrosis‖,7.1%were in the state of―oncosisnecrosis‖.(2) Expression quantity of TNF mRNA and SODD mRNA was detectedwith RT-PCR.Mean differences of the TNF mRNA，s expression quantity in each groupwere compared: blank control group (0) with hypofractionated group (3)0.10(P=0.01); blank control group (0) with hypofractionated group (7)0.14(P=0.00); hyperfractionation group (1) with hypofractionated group (3)0.13(P=0.00); hyperfractionation group (1) with the hypofractionated group (7)0.18(P=0.00); conventional fractionation group (2) with the hypofractionatedgroup (3)0.09(P=0.03); conventional fractionation (2) with thehypofractionated group (7)0.13(P=0.00). all were statistically significant.Mean differences of the SODD mRNA，s expression quantity in eachgroup were compared: blank control group (0) and hyperfractionated group (1)0.39(P=0.00); blank control group (0) and hypofractionated group (3)0.14(P=0.03); the hyperfractionated group (1) and conventional fractionationgroup (2)0.33(P=0.00); the hyperfractionated group (1) and hypofractionatedgroup (3)0.25(P=0.00); the hyperfractionated group (1)and thehypofractionated group(7)0.13(P=0.04); conventional fractionation group (2) and the hypofractionated group (7)0.13(P=0.00).all were statisticallysignificant.(3) The TNF protein and SODD protein’s expression quantity in eachgroup was detected with Western Blot.The mean differences of TNF protein’s expression quantity of each groupwere compared: blank control group (0) and hyperfractionated group (1)3.30(P=0.00); blank control group (0) and conventional fractionation (2)4.98(P=0.00); blank control group(0) and hypofractionated group (3)5.39(P=0.00); blank control group (0) and hypofractionated group (7)4.61(P=0.00);all were statistically significant.The mean differences of SODD protein’s expression quantity in eachgroup were compared: blank control group (0) and hyperfractionated group(1)0.67(P=0.00); blank control group (0) with conventional fractionationgroup(2)0.68(P=0.00); blank control group(0) and hypofractionated group(3)0.76(P=0.00); the hyperfractionated group (1) hypofractionated group (7)0.80(P=0.00); conventional fractionation group (2) with thehypofractionated group (7)0.82(P=0.00); the hypofractionated group (3)and the hypofractionated group (7)0.90(P=0.00); all were statisticallysignificant.(4) Absorbance values (OD) of cells in each group were detected withCCK-8, and the survival curve was drawn.By comparing between Conventional fractionation group (2),hypofractionated group (3) and hypofractionated group (7) on the1th,4th,7th,10th days after the simulation irradiation, the absorbance values of cells werenot significantly different (P>0.05). On the1th,4th,10th day, thehyperfractionated Group (1)’s absorbance values were significantly lower than other groups (P <0.05). Absorbance values of each simulated irradiationgroup had no significant differences on the7th day (P>0.05).Conclusion:(1) The hyperfractionated irradiation induced most of Eca109cells toperform apoptoticnecrosis, but the hypofractionated irradiation induced mostof them to perform oncosticnecrosis. The results of CCK-8suggested that thehyperfractionated radiotherapy would have better tumor control rate thanother modes of dose fractionation in the treatment of esophageal cancer.(2) Among the simulation irradiation groups, the hyperfractionated group(1)’s TNF mRNA’s expression was the highest, so was SODD mRNA. Itsuggests that a fight should exist between promoting apoptotic genes andanti-apoptotic gene in the process of induced tumor cells’dying.(3) In the analog irradiation groups, both TNF protein and SODD proteinsuffered massive reduction. It suggests that injury of other organelles exceptDNA can not be ignored, because their functional status may affect thesynthesis of protein, while protein is the executor of the life behavior.