补肾祛瘀前癌消对前列腺癌LNCaP细胞生物学行为的影响

【作者】 李子文；

【导师】 郝钰；

【作者基本信息】 北京中医药大学 ， 中医学， 2011， 硕士

【摘要】 研究背景前列腺癌是全球第五大常见癌症,亚洲地区是公认的前列腺癌低发地区。但是,随着生活条件的改善和诊断水平的不断提高,在我国,尤其是大城市,前列腺癌的发病率逐年升高。激素疗法是早期前列腺癌患者有效的治疗手段,可明显延缓前列腺癌进展、提高患者生存率。然而,大多数患者在经过中位时间12-18个月激素治疗后,几乎所有患者都进展为激素非依赖性前列腺癌,进入此阶段后,激素治疗失效,病情进展迅速,最终走向死亡。中药治疗与现代治疗方法相比,副作用较小,作用层次和靶点多,越来越多地被应用于肿瘤疾病的治疗。本项目采用的补肾祛瘀中药复方拟称前癌消,是临床治疗前列腺癌的经验方。本实验室裸鼠体内研究结果提示,对于经过去势治疗的雄激素依赖性前列腺癌,该方有一定的增强去势疗效的作用,并可延缓去势后前列腺癌由雄激素依赖向雄激素非依赖转化；对于未经去势治疗的雄激素依赖性前列腺癌,该方有一定的促肿瘤生长作用,应该慎用。本实验选取前期体内实验所使用的也是研究中经常使用的雄激素敏感性LNCaP(?)田胞作为体外实验研究对象,以期通过本实验进一步研究前癌消对前列腺癌细胞的作用,为补肾祛瘀法在临床上治疗前列腺癌提供实验依据。目的：以人前列腺LNCaP细胞为研究模型,观察前癌消对该细胞的生物学行为产生的影响,从而为阐明其作用机制提供实验研究基础。方法：采用雄性Wistar大鼠双侧睾丸摘除术和前癌消水煎剂灌胃方法,获得4组不同的含药血清：正常对照组、单纯给药组、单纯去势组和去势给药组。用各组大鼠血清对人雄激素依赖性前列腺癌LNCaP细胞实施干预,采用MTT法检测前癌消对LNCaP细胞增殖的影响；采用PI单染法和PI/Annexin V双染法结合流式细胞术,检测前癌消对LNCaP细胞周期和凋亡的影响；采用Hoechst 34580荧光染色观察LNCaP细胞凋亡的细胞核形态学改变；采用Fluo-4 AM荧光染色结合激光共聚焦显微镜观察并测定延时钙离子荧光强度和瞬时钙离子荧光强度的变化；采用划痕法结合活细胞工作站长时间连续观察前癌消对LNCaP细胞迁移的影响。结果1、前癌消单纯给药组血清对LNCaP细胞增殖具有一定的促进作用。单纯去势组血清对LNCaP细胞增殖具有一定的抑制作用。去势给药组血清与单纯去势组血清相比,有轻度的促进细胞增殖的作用。2、单纯去势组血清能够导致LNCaP细胞出现GO/G1期周期阻滞。去势给药组血清与单纯去势组血清相比,对LNCaP细胞周期的阻滞作用更明显。3、单纯给药组血清、单纯去势组血清和去势给药组血清干预24和48小时后,都具有明显的促进LNCaP(?)田胞凋亡的作用,其中,单纯去势组血清促凋亡作用最强,去势给药组血清次之,单纯给药组血清最弱。激光共聚焦显微镜下可见相应作用时间和处理组出现典型的凋亡细胞核改变。4、除正常对照组外,其他各组血清处理24小时后,LNCaP细胞胞浆中的钙离子荧光强度均有升高,其中,单纯去势组LNCaP细胞胞浆钙离子荧光强度最强,单纯给药组次之,去势给药组最弱。血清作用48小时后,去势给药组LNCaP细胞胞浆钙离子荧光强度最强,单纯去势组次之,单纯给药组最弱。5、各组血清处理LNCaP(?)细胞,可见胞浆中瞬时钙离子的变化模式不同。除单纯去势组外,其他各组钙离子荧光强度均在观察期内开始降低。6、去势给药组血清处理的LNCaP细胞在迁移实验研究的观察期内,迁移速度始终最快,其次是单纯去势组、单纯给药组,最慢的是正常对照组。但这种差异不具有统计学意义。结论：单纯使用前癌消含药血清,可轻度促进雄激素依赖性前列腺癌LNCaP细胞增殖,但有一定的阻滞细胞周期和促进细胞凋亡的作用。单纯去势组血清,能够明显抑制LNCaP细胞增殖,导致细胞周期阻滞,并促进该细胞凋亡,促凋亡作用可能与细胞内钙离子超载有关。去势给药组血清的周期阻滞和促凋亡作用较单纯去势组血清更强,促凋亡作用可能与细胞内钙离子超载有关。单纯给药组、单纯去势组和去势给药组血清都能使LNCaP细胞迁移速度加快,但鉴于体外培养条件单一,无法模拟体内环境中其他因素的影响,故有待进一步研究。更多还原

【Abstract】 BackgroundProstate cancer is the fifth most common cancer throughout the world. It is supposed t hat Asia enjoys a low incidence of prostate cancer, however, the incidence of prostate ca ncer there has been increasing year by year with the improvement of living conditions, t he changes in lifestyle and the progress in diagnosis. In China, the incidence of prostate cancer increases gradually especially in big cities. Hormone therapy is an effective treatme nt in th early stage of prostate cancer. Short or long term of hormone deprivation thera py can help to improve survival and delay the progress of the disease. However, after a medium period of 12 to 18 months, nearly all patients under the treatment of hormone deprivation therapies are progressing into the hormone independent stage of prostate ca ncer. Hormone therapy introduce no therapeutic actions in this stage, neither other thera pies. A rapid progression can be observed in this stage and most patients stride to the e nd of the life.Traditional Chinese medicine performs well in the clinical practice in treating different ki nds of cancers and it is more and more employed in cancer treatment these years for th e advantages of little side effect, multi-targets and multi functioning levels, focusing on c omprehensive regulation of the body compared to the modern therapies. The QAX presc ription used in our program is an effective QAX created by Prof. Pei Wang on the basi s of his experiences in treating cancers, which is called the QAX with the function of n ourishing the kidney and promoting blood circulation. The previous research in our labor atory indicated that this QAX should be used with cautions during the stage of androge n-dependent prostate cancer and it will give good performance on treating the disease du ring the androgen-independent stage. Besides, it can delay the disease progression from a ndrogen-dependent stage into the androgen-independent. LNCaP cells, the classic androge n-dependent prostate cancer cells, are used in our program to test the effects of the QA X above in vitro, so as to point out the proper moment to use the QAX with the func tions of nourishing the kidney and promoting the blood circulation in clinical practice.ObjectiveHuman prostate cancer LNCaP cells are employed in our program as the research model to observe the effects of the QAX with the function of nourishing the kidney and pro moting blood circulatioin on the biological behaviors of LNCaP cells in order to supply the research basis for expanding its clinical application. MethodsThe methods of intragastric administration of equivalent dose decoction and surgical rem oval of the testicles were employed to prepare 4 groups of different rat serums, the Nor mal Control group, the QAX group, the castration group and the Castration+QAX grou p. The serums described above are used to interfere the human androgen-dependent pros tate cancer LNCaP cells and MTT-asssav was used to measure the effects of the serums on cell proliferation; PI staining and PI/Annexin V double-staining were employed to te st the effects on cell cycle and apoptosis; Hoechst 34580 fluorescent staining was practic ed to observe the nuclear morphological changes of apoptosis cells caused by the serums; Fluo-4 AM fluorescent staining was performed to observe the time-lapse and instant cha nges of fluorescence indensity of cytosolic calcium caused by the serums with the help o f Laser Scanning Confocal Microscope; improved wounding assay was tried here to carry out continuous observation of the effects of the serums on the migration of LNCaP ce lls with the help of living cell station.Results1 Serums containing the QAX with the function of nourishing the kidney and promoting the blood circulation can accelerate the proliferation of LNCaP c ells, while as the castration group serum can suppress it. Compared to cast ration group, the Castration+QAX group can mildly accelerate the LNCaP prol iferation.2 Castration group serum can cause the G0/G1 cell cycle arrest in LNCaP cel ls. Compared to castration group, the Castration+QAX group can cause more si gnificant cycle arrest in the cells.3 After an interfereing period of 24 and 48 hrs, all groups performed signi ficant effects of promoting apoptosis in LNCaP cells aparting from the Norm al Control group, within which the castration group produced the strongest effect followed by the Castration+QAX group and at last the QAX group. The correspondingly typical nuclear morphological changes of apoptosis cells were observ ed by Laser Scanning Confocal Microscope.4 After 24 hrs of serums treatment, the cytosolic calcium indensity of LNCa P cells were observed to be increased. The castration group showed the high est indensity and then the QAX group and the Castration+QAX group the lowes t. After 48 hrs of serums treatment, the Castration+QAX group showed the hi ghest indensity followed by the castration group and the QAX group at last.5 The instant changing patterns of cytosolic calcium fluorescence indensity were different in different groups. The fluorescence indensity of calcium started to decrease in all groups exept the castration group.6 During the observing period of experiment, the migration speed of LNCaP c ells in Castration+QAX group was the fastest and then the castration group and the QAX group, the Normal Control group cells were the slowest, however, with no statistical significance.ConclusionThe QAX with the function of nourishing the kidnedy and promoting blood cir culation can mildly accelerate the proliferation of human prostate cancer L NCaP cells and can cause cell cycle arrest and apoptosis in the cells. Cast ration can significantly suppress the proliferation of the cells and cause cell cyecle arrest and accelerate cell apoptosis, which can be related to t he calcium overload. The QAX, castration or the combination of coumpound an d castration can accelerate the migration of LNCaP cells, however, consider ing the simple cultural conditions which can not simulate the enviroment in vivo, we should further our research on the effects on migration of the ce lls.更多还原