【作者】 王小强；

【导师】 曹建平；

【作者基本信息】 苏州大学 ， 放射医学， 2008， 硕士

【摘要】 目的研究咖啡酸苯乙酯(CAPE)对人宫颈癌Hela细胞放射增敏作用及机制。检测CAPE浓度及作用时间对人宫颈癌Hela细胞的抑制效应,确定用作放射增敏实验的药物浓度及时间;研究CAPE对人宫颈癌Hela细胞的放射增敏作用;探讨CAPE的放射增敏机理,为研究放射增敏剂的作用机制积累实验资料。方法(1)以人宫颈癌Hela细胞为研究对象,采用MTT技术,经浓度为0、6、10、20、30、40μg/ml的CAPE作用24小时,分析细胞抑制率与药物浓度的关系;浓度为20μg/ml的CAPE与细胞作用0、12、24、48、72小时,分析细胞抑制率与CAPE作用时间的关系;(2)细胞经0、2、4、6、8Gy剂量的60Coγ射线照射,比较单纯照射组和照射+CAPE组的克隆存活率,并求出增敏比(SER);细胞经0、1、2、3、4Gy 60Coγ射线照射后观察比较单纯照射组和照射+CAPE组之间细胞微核率及微核细胞率的差异;(3)利用流式细胞术检测细胞周期分布、western-blot技术检测周期蛋白CyclinB1的表达情况。结果(1)人宫颈癌Hela细胞经不同浓度的CAPE作用后,细胞抑制率呈浓度依赖性增高(P<0.01),细胞抑制率为20%的CAPE浓度为20μg/ml;(2)浓度为20μg/ml的CAPE与Hela细胞作用不同时间,细胞抑制率呈时间依赖性增加(P<0.01);(3)细胞经60Coγ射线照射后,Hela细胞克隆存活率随着照射剂量的增加而降低(P<0.01);相同剂量下,照射+CAPE组的Hela细胞克隆存活率低于单纯照射组(P<0.01);照射+CAPE组的Hela细胞的平均致死剂量(D0)(1.82Gy、1.45Gy)、准阈剂量(Dq)(3.21Gy、1.89Gy)较单纯照射组减小,SER为1.26>1;(4)相同60Coγ射线照射下,照射+CAPE组细胞微核率及微核细胞率均高于单纯照射组(P<0.01),两者细胞微核率及微核细胞率与照射剂量呈正相关(P<0.01),均可拟合成剂量效应直线方程y=a + bD(D为照射剂量);(5)流式细胞术检测发现,与对照组相比, CAPE组及照射组G2/M期的细胞比例升高(P<0.01),而在照射+CAPE组则降低(P<0.05);(6)western-blot分析,CAPE联合照射及单纯照射,CyclinB1蛋白表达均随着照射剂量增大而减少;相同照射剂量下,CAPE联合照射的Hela细胞CyclinB1蛋白表达较单纯照射减小。结论(1)CAPE对人宫颈癌Hela细胞的抑制率在研究的浓度范围内呈浓度依赖性;(2)CAPE对Hela细胞的抑制率在研究的时间范围内呈时间依赖性;(3)Hela细胞克隆存活率与照射剂量呈负相关;相同剂量下照射+CAPE组的克隆存活率低于单纯照射组;CAPE对Hela细胞的放射增敏作用可能与抑制细胞的亚致死性损伤修复能力有关;(4)Hela细胞微核率和微核细胞率与照射剂量成正相关;相同剂量下照射+CAPE组细胞微核率和微核细胞率高于单纯照射组,因此我们从微核实验可以得出咖啡酸苯乙酯对Hela细胞具有放射增敏作用;(5)CAPE对Hela细胞的放射增敏作用与G2/M期阻滞有关(;6)Hela细胞经CAPE联合照射CyclinB1蛋白表达降低,导致细胞G2/M期发生阻滞,从而发挥放射增敏作用。更多还原

【Abstract】 Objective This study was to investigate the effect of caffic acid phenethyl ester (CAPE), a component of propolis,on the radiosensitivity of Hela cells. The thesis contained: the drug’s influence on the proliferation of Hela cells, radiosensitizing effect and cell cycle of the cells and expression level of cell cycle regulatory protein cyclinB1 after the cells treated by 60Coγray. The data showed a primary investigation of CAPE radiosensitizing mechanisms.Methods (1) MTT assay was used to measure the inhibition effect of different concentration and time by caffic acid phenethyl ester (CAPE) on human Hela cells of cervical cancer. (2) Cloning survival assay and MNCF-T were used to study the radiosensitizing effect of CAPE acting on Hela cells (3) Flow cytometric analysis was adopted to detect the changes of cell cycle distribution induced by CAPE after 60Coγirradiation, and with western-blot to examine the CyclinB1 expression.Results (1) The inhibition of CAPE acting on human Hela cells of cervical cancer increased with concentrations(P<0.01), CAPE concentration with 20% of the inhibition rate is 20μg/ml.(2)The inhibition of CAPE acting on Hela cells was in a time-dependent manner(P<0.01). (3) Hela cells cloning survival decreased with the increase of radiation dose(P<0.01).At the same radiation dose, Human Hela cells cloning survival in combination group was less than in the sole irradiation group(P<0.01),The mean lethal dose(D0) (1.82Gy、1.45Gy)and the quasi-threshold dose(Dq) (3.21Gy、1.89Gy) of Hela cells in combination group decreased comparing with the sole irradiation group,SER was 1.26.(4) At the same radiation dose, MNF and MNCF in combination group was higher than in sole irradiation group(P <0.01), and MNF and MNCF of the two was positively correlated with the radiation dose, may be fitting the dose-effect of linear equation y = a + bD. (5) Compared with the control group, cells in G2/M phase of CAPE group and the sole irradiation group increased (P <0.01), while in the combination group decreased (P <0.05). (6) CyclinB1 protein expression reduced as the radiation dose increases between CAPE combination group and the sole irradiation one under the same radiation dose, CyclinB1 expression of Hela cells in combination group was less than the sole irradiation.Conclusion (1) The inhibition of CAPE acting on human Hela cells of cervical cancer increased with concentrations. (2) CAPE acting on Hela cells in a time-dependent inhibition. (3) Cloning survival negatively correlated with radiation dose. At the same radiation dose, Hela cells cloning survival was less in combination group than in sole irradiation one. Radiosensitizing effect by CAPE on cells may be related to the inhibition of the sub-lethal damage repair. (4) MNF and MNCF between combination group and the sole irradiation one were positively correlated with the radiation dose. At the same radiation dose,the former was higher than the latter. (5) CAPE acting on Hela cells may be correlation with G2/M arrest. (6) Compared with the sole irradiation group, cyclinB1 expression level decreased in combination group.更多还原