【作者】 聂斌；

【导师】 周菊英；

【作者基本信息】 苏州大学 ， 肿瘤学， 2010， 硕士

【摘要】 目的:观察塞来昔布的辐射增敏效应,探讨自噬和自噬性死亡在塞来昔布对脑胶质瘤细胞SHG-44辐射增敏效应中的作用。方法:选择脑胶质瘤细胞株SHG-44作为研究对象,以不同浓度塞来昔布(0、30、50、100μM)设立药物组(D组),以不同剂量6MV-X线照射(0、1、2、4、6、8Gy)设立辐射组(R组),两者交互设立联合组(D+R组)。采用四甲基偶氮唑盐(MTT)比色实验检测塞来昔布对SHG-44细胞增殖抑制作用;集落形成法检测塞来昔布对SHG-44辐射增敏作用;流式细胞仪检测细胞周期分布和凋亡;吖啶橙染色、绿色荧光FITC- LC3-Ⅱ抗体和电镜共同检测细胞内自噬体的分布和数量。结果:1. MTT检测显示:塞来昔布抑制SHG-44细胞增殖,与浓度和时间相关,随药物浓度增加以及作用时间延长细胞活性越低(P<0.05)。克隆形成实验结果和细胞存活曲线相关参数显示:塞来昔布增加SHG-44细胞的辐射敏感性,放射增敏比SERD0=1.20。2.流式细胞仪对细胞周期和凋亡的检测显示:塞来昔布30μM诱导肿瘤细胞G2/M期阻滞,并延长辐射8Gy诱导的细胞G2/M期阻滞时间(C、D、R和D+R组的G2/M分别为:9.87±1.34%,20.14±2.52%,29.15±1.99%,34.26±2.20%,P<0.05),而非促进细胞凋亡(R:8.24±0.93%,D+R:9.71±1.24%,P=0.158)。随着辐射剂量增加,G2/M期细胞比例增多(2Gy、6Gy和8Gy G2/M:14.06±1.37%、18.72±3.77%、29.15±1.99%,P<0.05)。3.细胞自噬的检测:①细胞自噬形态学的电镜观察发现塞来昔布组、辐射组和联合组的细胞胞浆内可见不同阶段自噬小体,而无明显的核浓缩和核碎片;②细胞吖啶橙染色发现塞来昔布30μM联合辐射8Gy组细胞胞质内染成红色的酸性囊泡结构数量高于对照组、塞来昔布30μM组和辐射8Gy组(6.19±0.56 vs. 1.08±0.21、1.94±0.06、2.71±0.14,P<0.05);③通过FITC -LC3-Ⅱ标记自噬小体特异性膜蛋白在细胞胞质形成绿色小点,联合组阳性细胞比例高于对照组、药物组和辐射组(43.51±3.26% vs. 9.34±1.05%、18.46±2.67 %、24.81±2.04%, P<0.05)。4.随着辐射剂量的递增G2/M期细胞比例增多,同时细胞自噬水平也相应增加,统计软件对细胞自噬水平和细胞G2/M期比例行相关性分析,显示二者呈线性相关(相关系数r=0.97,P<0.05)。5.随着细胞自噬水平增加,细胞的克隆源性存活呈指数性递减。统计软件分析,显示细胞存活分数与细胞自噬水平成指数性负相关(相关指数R=0.98 P<0.05)。结论:1.塞来昔布在体外实验中提高脑胶质瘤细胞株SHG-44的辐射敏感性。2.塞来昔布增强辐射诱导SHG-44细胞G2/M期阻滞,促进细胞自噬。诱导细胞自噬性死亡,可能是塞来昔布增强脑胶质瘤细胞辐射敏感性机制之一。3.细胞自噬和自噬性死亡可能与细胞G2/M期阻滞有关。更多还原

【Abstract】 Objective: To evaluate Celecoxib’s radiosensitizing effect and to analyze the effect of autophagy and autophagic cell death on Celecoxib radiosensitizing human glioma SHG-44 cell lines.Methods: The human glioma cell SHG-44 was managed in vitro. Group of drug (Group D) was divided according to the different concentration of Celecoxib (0μM, 30μM, 50μM and 100μM);and the group irradiation (Group R) divided according to the different dose of 6-MV X-ray(0Gy, 1Gy, 2Gy, 4Gy, 6Gy and 8Gy), the other group was formed by Celecoxib combined with irradiation (Group D+R). MTT assay was performed to determine the effects of Celecoxib on SHG-44 cell growth; Celecoxib’s radiosensitization was measured by clongenic assay; the cell cycle redistribution and apoptosis was analyzed by Flow-cytometric analysis. Meanwhile, acridine orange (AO), FITC-LC3-Ⅱand transmission electron microscope were used to detect autophagy.Results:1. The outcome of MTT assay suggested that Celecoxib may produce a concentration- dependent and time-dependent inhibition of SHG-44 cell proliferation(P<0.05). Celecoxib showed radiosensitizing effect to a certain extent in SHG-44 cells by colony forming assays, the sensitizating enhancement ratio SERD0=1.20.2. Flow-cytometric analysis showed that Celecoxib 30μM induced G2/M arrest and enhanced prolongation of G2/M arrest that induced by irradiation 8Gy, (C,D,R and D+R G2/M: 9.87±1.34%, 20.14±2.52%, 29.15±1.99%, 34.26±2.20%, respectively; P<0.05),however, apoptotic cell death was not apparent (R: 8.2±0.93%, D+R: 9.7±1.24%, P=0.158). Furthermore prolongation of G2/M arrest was demonstrated dose-dependent manner (2, 6 and 8Gy G2/M: 14.06±1.37%, 18.72±3.77%, 29.15±1.99%, respectively; P<0.05).3. To investigate autophagy, first we performed TEM, SHG-44 cells treated with irradiation and / or Celecoxib showed prominent formation of autophagic vesicles with lamellar structures or residual digested material, without the nucleus condensation or segmentation. Next, cells incubated by acridine orange (AO) after irradiation 8Gy and/or Celecoxib 30μM, which emitted red fluorescein in acidic vesicular organelles (C, D, R and D+R: 1.08±0.21, 1.94±0.06, 2.71±0.14, 6.19±0.56; P < 0.05), and FITC-MAP1- LC3-Ⅱantibody was used to label LC3-Ⅱ, which was bounded on the membrane of autophagosomes. The level of autophagy of the group combination cells showed more than the R group or the Celecoxib group(C, D, R and D+R: 9.34±1.05%, 18.46±2.67%, 24.81±2.04%, 43.51±3.26%, P<0.05).4. As the dose of radiation increased, G2/M phase was prolonged, while the level of autophagy increased in a corresponding. Statistical software for correlation analysis showed that there is a linear correlation between the prolongation of G2/M arrest and the level of cellular autophagy (correlation coefficient r=0.97,P<0.05). 5. As the level of autophagy increased, the clongenic survival decreased exponentially. Statistical analysis showed that there is a negative correlation between the logarithm of cell survival fraction and the level of cellular autophagy (correlation index R=0.98,P<0.05).Conclusions:1. Celecoxib enhances the radiosensitivity of glioma cells SHG-44 in vitro.2. Celecoxib enhances SHG-44 cells G2/M phase arrest by radiation-induced and promoted cell autophagy. To induce autophagic cell death may be one of the mechanisms of Celecoxib radiosensiting glioma cells SHG-44.3. Induction of autophagy and autophagic cell death may be in association with the cells G2/M phase arrest.更多还原