【作者】 董丹；

【导师】 李玉；

【作者基本信息】 大连医科大学 ， 肿瘤学， 2007， 硕士

【摘要】 研究背景原发性肝癌是我国常见的恶性肿瘤,其死亡率位居各类肿瘤第二位。现代精确放疗是治疗肝癌的常用手段之一。然而,在给予足以杀灭肿瘤组织放射剂量的同时常会损伤周围重要组织。而许多肿瘤由于其本身对射线抵抗或肿瘤内存在大量对放射线不敏感的乏氧细胞,即使给予足够的射线剂量仍无法有效控制肿瘤。因此,有关放射增敏剂的研究具有重要临床意义。多西紫杉醇是一种新型半合成紫杉醇类衍生物,体外研究表明,多西紫杉醇可诱导多种肿瘤细胞凋亡,影响细胞周期分布[1]。临床研究证实多西紫杉醇对多种肿瘤细胞有放射增敏作用且不加重正常组织的放射损伤,但有关多西紫杉醇对肝癌细胞的放射增敏鲜见报道。研究目的本实验研究旨在探索多西紫杉醇对人肝癌细胞株SMMC-7721的细胞毒性及放射增敏作用,为有效提高肝癌的放疗效果提供试验依据。实验方法(1)将复苏的人肝癌细胞系SMMC-7721进行传代培养,显微镜观察细胞形态变化,定期换液及传代,待细胞生长良好,进入对数生长期后进行试验。(2)MTT法测定多西紫杉醇对SMMC-7721细胞的生长抑制作用。(3)荧光倒置相差显微镜观察浓度为IC50的多西紫杉醇不同处理时间后的细胞形态学变化。(4)MTT法测定多西紫杉醇对SMMC-7721细胞的放射增敏作用。( 5)流式细胞分析方法检测多西紫杉醇联合放射线对细胞周期的影响。(6)流式细胞分析方法检测多西紫杉醇联合放射线对细胞周期影响的时效性。实验结果(1)人肝癌细胞系SMMC-7721传代培养3天后,细胞生长良好,进入对数生长期。(2)各浓度的多西紫杉醇分别作用3 h,6 h,9 h,12 h,24 h,48 h后,细胞抑制率随着药物浓度的增加而升高,随着药物作用时间的延长而升高,且药物浓度和处理时间之间存在协同作用。析因方差分析结果表明不同药物浓度处理组中除200μg/ml,400μg/ml外,其他各浓度处理组间两两比较均有显著统计学差异( F=15723.685,P =0.0000 ) ,不同处理时间组间细胞抑制率有显著统计学差异(F=11740.167,P=0.0000),不同浓度和时间协同组间细胞抑制率有显著统计学差异(F=739.916,P=0.0000)。多西紫杉醇对SMMC-7721细胞的IC50为21.69μg/ml,95%的可信区间为19.12μg/ml ~24.60μg/ml。确定随后的增敏实验选择浓度为:4μg/ml。(3)荧光倒置相差显微镜(200×)观察显示:正常肿瘤细胞呈团块状排列,团块内细胞数量不等,细胞间排列紧密;细胞大小不一,异形性明显,呈梭形或多角形;胞膜、胞浆等细胞内各结构清晰。胞核卵圆形,核膜清晰,核仁明显。药物作用于细胞后3 h:肿瘤细胞体积增大,胞膜、胞浆、核膜等细胞内结构显示不清,核仁尚清;6 h:多数细胞间出现融合现象,呈团块状,团块内细胞各结构均显示不清,细胞表面可见大小不等、分布均匀的空泡;9 h:细胞内空泡开始融合变大,细胞团内各结构显示更不清楚;12 h:细胞数目减少,细胞表面可见黑色颗粒并均匀分布,细胞团内各结构无法分辨,部分细胞固缩、变圆;24 h:细胞排列松散,细胞间隙增大,仅见模糊的细胞大体轮廓,细胞内各结构无法分辨,胞膜仍可见。细胞表面散在空泡,胞浆缺失。细胞开始固缩、溶解,并可见典型的核固缩、核碎裂及细胞变性坏死现象;48 h:细胞排列更为松散,胞膜不完整,表现为变性或溶解样坏死。(4)多西紫杉醇预处理3 h、6 h、9 h、12 h、24 h、48 h后照射,不同预处理时间组间SER有显著性差异( F = 32.43935,P = 0.0000),不同预处理时间组间两两比较均具显著性差异( P < 0.05)。各组SER分别为: 1.21、1.33、1.50、1.68、1.72、1.83,随预处理时间延长,SER逐渐增加,在预处理3 h～12 h期间,SER增高速率最快,此后随孵育时间延长,SER呈较为平缓增长趋势。(5)单纯4μg/ml多西紫杉醇处理和2 Gy照射均引起明显的细胞周期G2/M期阻滞,且多西紫杉醇与照射有协同作用。(6)随着多西紫杉醇预处理时间的延长,G2/M期细胞所占比例逐渐增加,多西紫杉醇预处理12 h、24 h、48 h,其G2/M期细胞比例分别为32.23%,50.23%,60.13%。结论多西紫杉醇对人肝癌细胞系SMMC-7721有明显毒性作用和放射增敏作用,其IC50为21.69μg/ml,95%的可信区间为19.12μg/ml~24.60μg/ml,放射增敏作用的机制可能与引起G2/M期阻滞,影响细胞周期的重分布有关。更多还原

【Abstract】 Backgrounds Primary hepatocarcinoma is a common cancer, its mortality rate ranks secondly in various tumors. Modern precise radiation therapy is a common means of treating liver cancer. However, enough dose radiation to kill the tumor may damage important tissue around. Due to a lot of hypoxic cells insensitive to radiation in many tumors, even adequate dose radiation is still unable to control the tumor effectively, therefore, it has important clinical significance to study the radiation sensitizer. Docetaxel is a new semi-synthetic derivatives of paclitaxel, vitro studies showed that docetaxel can induce apoptosis, influence distribution of cell cycle. Clinical research had shown that docetaxel could increase radiation sensitivity of tumor cells without increasing the radiation injury of normal tissue. However, there were rare reports that docetaxel could increase radiation sensitivity of hepatoma cells.Objectives This study aims to explore the radiosensitizing effect and toxicity of docetaxel on human hepatocellular carcinoma cell line SMMC-7721, which provides experimental basis for enhancing the therapeutic effect of liver cancer.Methods (1)Recovered, Passaged and Cultured SMMC-7721. Observed cell morphology by inverted microscope. Liquided and passaged regularly and experimented when cell entered the logarithmic growth phase. (2) Inhibition effect of docetaxel on the growth of SMMC-7721 was determined by MTT assay. (3) The differences of morphology changes between docetaxel of different time was observed by inverted fluorescence microscope. (4) Radiation sensitizing effect of docetaxel on SMMC-7721 cell was determined by MTT assay. (5) The effect of docetaxel combiningwith radiation on the cell cycle was detected by flow cytometry. (6) Timeliness effects of docetaxel plus radiation on the cell cycle was detected by flow cytometry.Results (1) After cultured for three days, SMMC-7721 growed well and entered the logarithmic growth phase. (2) After treated by docetaxel of different concentration for 3 h, 6 h, 9 h, 12 h, 24 h, 48 h respectively, inhibition rate increased with the increase of concentration and treating time. Results of analysis of variance showed that there were significant differences of inhibition rate among differentent treating time groups ( F=11740.167, P=0.0000), different concentration groups except 200μg/ml group, 400μg/ml group, showed a significant difference ( F=15723.685, P=0.0000), IC50 was 21.69μg/ml, 95% confidence interval was 19.12μg/ml~24.60μg/ml. There were significant differences of inhibition rate among cooperative groups of different concerntration and time(F=739.916, P=0.0000).(3) Observed by inverted fluorescence microscope (200×): normal tumor cells was blob-like arrangement, the cell number in blob is different, cell ranged closely, size was different, profile was obvious, cell were shuttle or polygon; structures within the cell such as membrane, cytoplasm and so on were clear. So were oval nucleus, nuclear membrane and nucleus. After treated by docetaxel for 3 h: Volume of tumor cell augmented, the membrane, cytoplasm, the nuclear membrane and other structures were unclear, but nucleus was still clear; 6 h : Most cells were fused and arranged like blob, structures within the cell of blob was unclear, there were uniform cavitation on the surface of cells; 9 h: Intracellular vacuole began fusing and increasing, structure within the cell revealed even more dim; 12 h: The number of cells decreased, there were black uniform particles on the surface of cells and structures within the cell could not be resolved, some cells shrank, rounded; 24 h: Cells ranged loose and intercell space increased, a vague outline of the whole cell can be seen only, the cell structure could not be resolved, the membrane was still visible. Cell surface scattered vacuoles, there was no cytoplasmic. Cell began to shrink and dissolve and there were typical nuclear pyknosis, nuclear fragmentation and cell necrosis; 48 h: Cells ranged more loose, no whole cell membrane can be seen, cells presented degeneration or dissolved necrosis. (4) Pretreated bydocetaxel for 3 h, 6 h, 12 h, 24 h, 48 h before exposure, it presented significant differences in SER among groups (F = 32.43935, P = 0.0000), it presented significant differences in SER between groups (P<0.05). SER was 1.21,1.33,1.50,1.68,1.72 and 1.83 respectively. With the increase in time of pretreatment, SER gradually increase, pretreatment of 3 h~12 h, SER increase fastest, SER subsequently growed slowly. (5)Only treatment of 4μg/ml docetaxel or radiation of 2 Gy caused significant G2/M block, it presented a synergistic effect of docetaxel and irradiation. (6) With the extension of pretreatment time, percentage of G2/M cell increased gradually, pretreatment of 12 h, 24 h, 48 h, percentage of G2/M was 32.23%,50.23% and 60.13% respectively.Conclusion Docetaxel had significant radiosensitizing effect and toxicity on SMMC-7721, IC50 was 21.6μg/ml, 95% confidence interval was 19.12μg/ml ~24.60μg/ml, mechanism of radiosensitizing effect may be related to the induction of G2/M block and redistribution of cell cycle.更多还原