【作者】 陈文娟；

【导师】 熊正爱；

【作者基本信息】 重庆医科大学 ， 妇产科学， 2013， 博士

【摘要】 近年来，脉冲电场(Pulsed Electric Fields, PEF)在生物医学领域的运用越来越受到国内外学者的关注。前期的研究工作中发现微秒和纳秒级脉冲电场作用于肿瘤细胞会产生明显的杀伤效应，为脉冲电场用于肿瘤的治疗奠定了一定的基础。然而，微秒和纳秒脉冲电场必须以电极针为电场载体引入体内，以穿刺的方式引导至肿瘤组织，这在一定程度上限制了该方法的临床应用及适应症。能够无创进入体内，达到肿瘤表面的皮秒脉冲电场应运而生。匹配超宽带冲激脉冲辐射天线(IRA)的皮秒脉冲电场以其优异的方向性和精细的操控性以及靶向聚焦特性，成为具有巨大发展潜力的生物医学无创治疗的新能源。IRA可以在某一特定的范围内进行能量传输，将脉冲电磁波汇聚于指定区域，甚至摧毁目标，辐射能量在时间和空间上都很集中。根据这一特点，通过IRA将皮秒脉冲电场输送入肿瘤组织，便可实现肿瘤的无创治疗。目前，对于皮秒脉冲电场用于肿瘤治疗的研究还鲜有报道。根据脉冲电场对细胞作用的“窗口效应”及微秒脉冲的电穿孔效应和纳秒脉冲的细胞内处理效应，从理论上讲，皮秒级脉冲电场对细胞的主要作用应该发生在细胞器。另外，我们课题组前期实验发现皮秒脉冲电场会诱发肿瘤细胞线粒体膜电位的变化。因此，在本研究中我们将进一步深入探讨皮秒脉冲电场对另外一种细胞器-内质网功能的影响。宫颈癌是女性生殖系统常见的恶性肿瘤之一，现已严重威胁着全球妇女的身心健康。目前宫颈癌的发病逐渐呈现年轻化趋势，传统的手术治疗就意味着失去生育能力，这对于年轻患者是很难接受的，因此，非侵袭性的保留生育功能的治疗手段渐渐成为医患双方的共同需求。宫颈尽管不在体表，但可以通过简单的方法充分暴露，这也为皮秒脉冲电场的应用提供了便利的条件。基于此，本课题选取了人宫颈癌HeLa细胞作为体外实验的研究对象，以不同强度的皮秒脉冲电场为研究手段，应用流式细胞计数法(flowcytometry，FCM)、Hoechest染色及透射电子显微镜检测皮秒脉冲电场处理后对细胞凋亡及增殖的影响，运用激光共聚焦显微镜观察胞内钙离子浓度的变化，采用RT-PCR、Real-time PCR、Western Blot的方法在分子水平上探讨皮秒脉冲电场作用下内质网应激相关分子mRNA及蛋白表达水平的变化，寻找皮秒脉冲电场诱导HeLa细胞凋亡可能的分子机制，以期为皮秒脉冲电场用于宫颈癌的治疗提供理论依据，也为宫颈癌患者寻求一种无创的保留生育功能的新的治疗手段。第一部分皮秒脉冲电场诱导HeLa细胞凋亡效应的研究第一节皮秒脉冲电场诱导HeLa细胞凋亡现象的观察目的：观察皮秒脉冲电场处理HeLa细胞后，细胞凋亡现象的发生。方法：将HeLa细胞分为四个组，即：对照组和三个不同电场强度作用的实验组(200kV/cm组,400kV/cm组,600kV/cm组)。实验组皮秒脉冲电场的其它参数如下：固定脉宽800ps，频率3Hz，脉冲个数2000个。脉冲电场处理后细胞培养时间为12h。采用Hoechst33258染色观察特征性的细胞凋亡变化；流式细胞仪对细胞凋亡率和细胞周期的变化进行检测；透射电子显微镜分别对对照组和实验组细胞的超微结构进行观察。结果：皮秒脉冲电场处理HeLa细胞后，通过Hoechst33258染色，在倒置荧光显微镜下观察发现，实验组HeLa细胞核产生强烈的蓝色荧光，表明细胞在脉冲电场作用下发生了凋亡；流式细胞仪检测到实验组细胞的凋亡率较对照组明显增加，且随着电场强度的增大，发生凋亡的细胞增多，在细胞分裂周期中停留在G2/M期的细胞数逐渐增多；透射电镜下观察到实验组细胞呈现典型的凋亡形态以及线粒体，内质网的肿胀。结论：皮秒脉冲电场诱导了HeLa细胞的凋亡，并将细胞周期阻滞在G2/M期。且随着电场强度的增大，细胞的凋亡率逐渐升高，细胞周期中被阻滞在G2/M期的细胞数也逐渐增多。第二节皮秒脉冲电场对HeLa细胞中Caspase系列分子表达的影响目的：探讨皮秒脉冲电场作用后，HeLa细胞中凋亡相关分子Caspase-12，Caspase-9以及Caspase-3在转录及翻译水平的变化。方法：实验分组同前。皮秒脉冲电场处理HeLa细胞后培养12h，提取细胞内总的RNA，RT-PCR的方法检测caspase-12，caspase-9以及caspase-3在转录水平mRNA的相对含量。提取细胞内总蛋白，WesternBlot测定pro-caspase-12，pro-caspase-9，pro-caspase-3及PARP的蛋白水平。结果：与对照组相比，皮秒脉冲电场处理过的HeLa细胞中，caspase-12，caspase-9以及caspase-3的转录上调，且随着场强的增大，caspase相对mRNA水平逐渐升高。蛋白水平上，实验组HeLa细胞中无活性的pro-caspase-12，pro-caspase-9和pro-caspase-3的表达量随场强的增大而减少。此外，具有活性的PARP(cleaved-PARP，89kDa)含量随场强的增大明显增加。结论：皮秒脉冲电场诱导HeLa细胞凋亡时启动了caspase级联事件，导致了caspase的活性片段在转录和翻译水平表达量的增加，HeLa细胞在皮秒脉冲电场作用下发生的凋亡是caspase依赖性的。第二部分皮秒脉冲电场经内质网途径诱导HeLa细胞凋亡机制的研究目的：探讨皮秒脉冲电场作用于HeLa细胞后，内质网应激凋亡的发生及其机制。方法：皮秒脉冲电场处理HeLa细胞后，继续培养12h，利用钙离子荧光探针标记，流式细胞仪和激光共聚焦显微镜的方法检测胞内Ca2+浓度的变化；Western blot检测内质网上PERK和eIF2α的磷酸化水平以及ATF6的活化；Real time-PCR和Western blot从转录和翻译水平检测分子伴侣GRP78和GRP94的表达，以及促凋亡转录因子CHOP的表达。结果：皮秒脉冲电场作用后，HeLa细胞胞浆中Ca2+浓度较对照组显著升高。内质网上PERK和eIF2α的磷酸化水平增加以及ATF6被活化。分子伴侣GRP78和GRP94以及促凋亡转录因子CHOP在转录和翻译水平均表达上调。结论：皮秒脉冲电场作用于HeLa细胞后，观察到明显的细胞内处理效应，对内质网产生了显著的影响。启动了内质网应激相关的HeLa细胞凋亡。第三部分皮秒脉冲电场对内质网应激相关的线粒体凋亡通路分子表达的影响目的：探讨皮秒脉冲电场作用下，HeLa细胞中与内质网应激密切相关的线粒体凋亡途径中有关分子的表达。方法：采用RT-PCR，Western blot的方法检测的Bcl-2和Bax转录和翻译水平的变化。Western blot方法检测线粒体Cytc的释放。结果：与对照组相比，皮秒脉冲电场处理过的HeLa细胞中，抗凋亡因子Bcl-2在转录和翻译水平上表达下调，促凋亡因子Bax在转录和翻译水平上表达上调，胞浆中Cyt c的含量增加，且三者的含量变化都与皮秒脉冲电场的强度大小呈正相关。结论：结合前期实验的结果，可以得出：线粒体途径在皮秒脉冲电场诱导的HeLa细胞凋亡中发挥着重要的作用。通过Bcl-2可将线粒体和内质网两条凋亡通路联系起来，它们之间的促凋亡作用可能存在一定的协同关系。更多还原

【Abstract】 In recent years, the application of pulsed electric field (PEF) inbiomedical fields was given more and more attention from scholars athome and abroad. Previous studies have showed that microsecond andnanosecond pulsed electric fields produced significant killing effects intumor cells. However, the application of ms, μs and ns PEF still needs touse the invasive or minimally invasive needle or plate electrodes, to guidethe puncture of tumor tissue, which to some extent limit the clinicalapplication of this method. Therefore, picosecond pulsed electric fieldscame into being, which can be transferred to target deep tissuenon-invasively and reach the surface of tumor precisely.Matching the impulse radiating antenna (IRA), picosecond pulsedelectric fields with excellent directivity and fine handling, would become anew method in the field of the non-invasive treatment in tumors. IRA canbe carried out energy within the particular scope. It can converge theelectromagnetic pulse on a designated area, or even destroy the target.Radiation energy is concentrated both in time and in space. According tothis feature, picosecond pulsed electric field was transported into the tumortissue by IRA. And the non-invasive treatment of tumors can come true.But the research of the biological effects of psPEF on cells is limited.According to the window effect of pulses electric fields on cells, the electroporation effect of microsecond pulses and the intracellularelectromanipulations effect of nanosecond pulses, the main role ofpicosecond pulsed electric fields should be in the organelles. In addition,previous experiments of our group found that the picosecond pulsedelectric fields cause changes in mitochondrial membrane potential of tumorcells. So, in this study we will further investigate the influence of thepicosecond pulsed electric field on the function of another organelle-endoplasmic reticulum.Cervical carcinoma, the most common malignant tumor of femalereproductive system, is a serious threat to global women’s physical andmental health. Its incidence is gradually getting younger and younger inrecent years. The traditional surgical treatment means the loss of fertilityand affects patients’ sexual function, which is very difficult to accept foryoung patients. Therefore, non-invasive treatment with preserved fertilityhas increasingly become the expectation for both doctors and patients.Although the cervical is not in the surface, it can be fully exposed by asimple method, which provides convenient conditions for the application ofpicosecond pulsed electric field (psPEF).Based on this, human cervical carcinoma HeLa cells were exposed topsPEF to investigate the underlying mechanisms of apoptosis in this study.Treatment with psPEF led to marked cell apoptosis and cell cycle arrest atG2/M phase. In addition, psPEF impacted the phosphorylation levels ofendoplasmic reticulum sensors and up-regulated the expression ofGlucose-regulated protein78(GRP78), Glucose-regulated protein94(GRP94) and C/EBP homologous protein (CHOP). These changes wereaccompanied by the elevation of Ca2+concentrations in intracellular.Furthermore, activation of caspase-12, caspase-9, caspase-3, increasing therelease of cytochrome c and up-regulation of Bax, and down-regulation of Bcl-2were observed in HeLa cells. Taken together, our findings suggestthat psPEF is an efficient apoptosis-inducing agent for HeLa cells, whichexerts its effects, at least partially, via the endoplasmic reticulum stress andcaspase-dependent signaling pathways.Part1Picosecond pulsed electric fields induced apoptosis in HeLa cellsChapter1The apoptosis phenomena of HeLa cells under Picosecondpulsed electric fieldsObjective: To observe the occurrence of apoptosis of HeLa cells aftertreated with picosecond pulsed electric fields.Methods: HeLa cells were divided into control group and fourexperimental groups (different amplitudes of picosecond pulsed electricfields treated groups). The parameters of experimental groups are asfollows: Fixed pulse width800ps, frequency3Hz, the number of pulses2000, three different electric field strength (200kV/cm,400kV/cm,600kV/cm). After treated with pulsed electric fields, the cell was culturedfor12h. Flow cytometry was used to detect changes in the rate of apoptosisand cell cycle; Hoechst33258staining was used to observe thecharacteristic changes in apoptosis; The ultrastructures of cells of thecontrol and experimental groups were observed by transmission electronmicroscopy.Results: After treated with psPEF, the apoptosis rate of theexperimental groups was significantly increased than the control group.The cell cycle distribution was assessed by monitoring intensity of PI fluorescence. Results indicated that psPEF blocked cells cleavage in phaseG2/M of the cell cycle. After treated with different amplitudes of psPEFand cultured for12h, typical apoptotic morphological changes of HeLacells were detected by Hoechst33258staining. The quantity of positivecells of the experimental groups increased significantly compared to thecontrol group. The experimental group cells showed typical apoptoticmorphology and the mitochondria and endoplasmic reticulum swollenunder effect of psPEF.Conclusion: The picosecond pulsed electric fields induced apoptosisof HeLa cells, and the cell cycle was arrested at the G2/M phase.Chapter2Caspase activity in HeLa cells exposed to picosecond pulsedelectric fieldsObjective: To explore the activity of apoptosis-related factorscaspase-12, caspase-9and caspase-3after exposed to picosecond pulsedelectric fields.Methods: After treated with different amplitudes of psPEF andcultured for12h, the total RNA and proteins of HeLa cells was extracted.The level of mRNA of caspase-12, caspase-9and caspase-3was detectedby RT-PCR. And the level of proteins of pro-caspase-12, pro-caspase-9,pro-caspase-3and PARP was detected by Western Blot.Results: Compared with the control group, the level of transcriptionof caspase-12, caspase-9and caspase-3was upregulated in theexperimental groups. And as the field strength increases, the relativemRNA levels of caspase gradually increased. In the levels of protein, theexpression of inactive pro-caspase-12, pro-caspase-9and pro-caspase-3 was reduced with the increase of the field strength. In addition, the activityof PARP (Cleaved PARP,89kDa) significantly increased with the fieldstrength increasing.Conclusion: The caspase cascade of events was activated whenpsPEF induced the apoptosis of HeLa cells. The apoptosis of HeLa cellsunder psPEF is caspase-dependent.Part2Mechanism of apoptosis through the endoplasmic reticulumpathway in Hela cells induced by psPEFObjective: To investigate the mechanism of apoptosis in Hela cellsvia endoplasmic reticulum stress induced by intense psPEF.Methods: After treated with different amplitudes of psPEF andcultured for12h, cells were loaded with the calcium probe Fluo-3-AM andthen examined by immunofluorescence using confocal microscopy andFlow cytometry. The phosphorylation of PERK and eIF2α and theactivation of ATF6were analyzed by Western blot. The expression of themolecular chaperone GRP78and GRP94and the pro-apoptotictranscription factor CHOP was analyzed by Real Time-PCR and Westernblot from the transcriptional and translational levels.Results: The green fluorescence intensity increased after the pulses,which indicated the elevation of Ca2+concentrations. The expression ofGRP78, GRP94and CHOP had a significant increase12h after the pulses,regardless of the level of gene or protein, compared with the control group.p-PERK, p-eIF2α and ATF6fragments increased, whereas their normalforms did not change.Conclusion: After psPEF exposure, PERK and eIF2α were phosphorylated; ATF6was cleaved to the activated form ATF6fragmentation; CHOP has a significant expression up-regulation; thechaperone proteins GRP78and GRP94were up-regulated, which showedthat the apoptosis of HeLa cells under psPEF was related to endoplasmicreticulum stress.Part3Effects of psPEF on Bax, Bcl-2and Cyt c in HeLa cellsObjective: To investigate the effects of psPEF on Bax, Bcl-2and Cytc, which are associated with both endoplasmic reticulum stress andmitochondrial dysfunction.Methods: After treated with different amplitudes of psPEF andcultured for12h, the total RNA and proteins of HeLa cells was extracted.The level of mRNA of Bax and Bcl-2was detected by RT-PCR. And thelevel of proteins of Bax, Bcl-2and Cyt c was detected by Western Blot.Results: Treatment with psPEF resulted in an increase of cytosoliccytochrome c in a dose dependent manner in HeLa cells. RT-PCR andWestern blot analysis revealed that the expression of Bax was up-regulated.In contrast, the expression of Bcl-2was significantly down-regulated.Conclusion: psPEF induced the up-regulation of Bax expression,down-regulation of Bcl-2expression and the movement of Cyt c from themitochondria into the cytoplasm. Combined with the results of previousexperiments, it can be concluded: psPEF induced apoptosis of HeLa cells,which at least partially, by the induction of mitochondrial dysfunction. Twoapoptotic pathways could be linked by Bcl-2. And there may be part of thecollaborative relationship between the two pathway of the mitochondriadysfunction and endoplasmic reticulum stress.更多还原