【作者】 汤昊；

【导师】 孙颖浩； 王林辉； 侯建国； 许传亮；

【作者基本信息】 第二军医大学 ， 外科学， 2008， 博士

【摘要】 一、研究背景目前随着我国人口老龄化、人民生活水平的提高以及饮食结构的变化,前列腺癌发病率正呈逐年上升趋势,对老年男性健康的影响正逐渐凸现出来。以上海地区为例,前列腺癌发病率在1985年是2.6/10万男性人口,2000年增长到7.7/10万男性人口,2005年已达到10/10万男性人口。高压氧（hyperbaric oxygen,HBO）是指在海平面位置2-3倍标准大气压的环境下吸入100%的纯氧,它可以使动脉血氧分压超过2000mmHg,组织氧分压接近400mmHg。HBO引起的组织氧分压升高以及氧输送增加不依赖于血红蛋白,如此高浓度的氧可以产生一系列生物化学、细胞学以及生理学方面有益的影响。HBO现已被广泛应用于临床之中,对于恶性肿瘤理论上讲HBO能够消除肿瘤自身缺氧所产生对于放疗、化疗等治疗的抵抗性,从而提高相关治疗的疗效。但是自1966年Johnson和Lauehlan首先报道HBO可能具有促进肿瘤细胞生长的作用之后,陆续有类似的个案报道出现,更加剧了人们的担心,此后很长一段时期恶性肿瘤及其病史被视为HBO治疗的禁忌症。目前HBO对于恶性肿瘤自然进程的确切影响仍不清楚,对于肿瘤细胞的生长是否具有促进生长作用仍存有争论。多数学者认为HBO并不促进肿瘤细胞的生长,但是仍有相当多的研究结果不支持此观点;于此同时,目前普遍认为HBO本身对于肿瘤细胞并没有明确的杀伤作用,单独使用HBO治疗恶性肿瘤并不具有疗效,其治疗作用往往是通过与其它治疗方法的联合应用而得以实现。通过复习与本研究相关的文献资料发现,目前缺乏HBO对于前列腺癌细胞生长影响的实验性研究;对于HBO在前列腺癌治疗中的应用价值暂无实验研究方面的依据。针对上述现状,设计本课题拟研究以下两方面问题:首先,HBO对于前列腺癌细胞的生长是否具有促进作用,临床上应用HBO治疗前列腺癌患者是否安全?其次,HBO对于前列腺癌细胞是否具有增强其对放疗、化疗等治疗敏感性的实验依据,临床中是否可以将HBO作为前列腺癌的辅助治疗?二、研究目的1、研究高压氧对前列腺癌LNCaP细胞株（激素依赖型）、PC-3细胞株（激素非依赖型）生长的影响;2、探讨高压氧在前列腺癌治疗中的应用价值。三、研究方法1、将LNCaP细胞株、PC-3细胞株随机分为对照组常压常氧条件下培养,实验组每日进行1次0.25 MPa（1个标准大气压=0.1 MPa）高压氧暴露,持续70分钟,连续5日;2、分别应用光学显微镜及透射电子显微镜、CCK—8细胞计数试剂盒、流式细胞仪以及Transwell迁移小室,对所有两组细胞的形态学、增殖能力、细胞周期分布以及迁移能力进行观察比较;3、构建PC—3细胞株对照组及实验组SCID小鼠移植瘤模型,观察并对比两组移植瘤在生长体积变化、瘤体微血管密度（MVD）、以及瘤细胞增殖（Ki—67蛋白）与凋亡（p53、p27蛋白）方面的差异。四、结果1、不同处理组的两类细胞大体及超微形态结构对比均未发现明显改变,细胞增殖及迁移能力均不存在显著性差异（p＞0.05）;2、流式细胞仪检测显示高压氧导致LNCaP、PC—3细胞株在G₂/M期的蓄积;3、成功建立两组PC-3细胞株移植瘤动物模型,致瘤率为100%,肿瘤接种后第28天,对照组移植瘤体积为（433.6±12.8）mm³,实验组为（425.8±13.9）mm³,两组移植瘤生长体积变化差异无显著性（p＞0.05）;4、对照组移植瘤微血管密度及Ki—67、p53、p27蛋白表达的阳性细胞率分别为20.1±2.31、（50.6±7.3）%、（30.5±4.7）%、（85.3±6.4）%,实验组分别为22.4±3.19、（55.2±6.7）%、（31.2±5.3）%、（80.4±5.7）%,病理学特征均无显著性差异（p＞0.05）。五、结论1、高压氧不促进前列腺癌细胞的增殖,对前列腺癌中新生血管也无明显影响,高压氧可能不会促进潜伏性前列腺癌的进展,即前列腺癌患者接受高压氧疗可能是安全的;2、高压氧促使两类前列腺癌细胞在G₂/M期的蓄积,形成细胞周期同步化,可能会增强前列腺癌对其它治疗方法如放疗、化疗等的敏感性,提高治疗效果;3、由于条件限制和认识不足,目前高压氧在前列腺癌治疗的临床应用尚较少,应用价值有待进一步验证,其合理的病例选择以及最佳的联合应用方案尚需进一步研究。更多还原

【Abstract】 BACKGROUNDWith the development of China and diet changed,the overall incidence of prostate cancer in men increased annually and became a more and more serious healthy problem to the elderly man.Between 1985 and 2005,the incidence of prostate cancer in men increased from 2.6 per 100,000 to 10 per 100,000 in ShangHai territory,an increase of approximately 300%.Hyperbaric oxygen（HBO）—100 percent oxygen at two to three times the atmospheric pressure at sea level—can result in arterial oxygen tension in excess of 2000 mmHg and oxygen tension in tissue of almost 400 mmHg.HBO increases oxygen tensions and oxygen delivery to tissues independent of hemoglobin.Such doses of oxygen have a number of beneficial biochemical,cellular,and physiologic effects. Over the past 50 years,HBO therapy has been recommended and used in a wide variety of medical conditions.In theory,HBO may remove the hypoxic stimulus;that drives tumour angiogenesis and other adaptations that facilitate tumour growth in a hypoxic environment,thus sensitizing them to radiotherapy,chemotherapy and other therapeutic treatment modalities.Although serious adverse events are rare,HBO cannot be regarded as an entirely benign intervention.In 1966 Johnson and Lauchlan first reported a tumor-stimulatory effect with HBO,demonstrating increased metastases in patients with cervical cancer.The effect of HBO administration on tumour growth has been inconclusive.HBO has been previously contraindicated in the treatment of malignancy due to concern that an increased oxygen pressure may stimulate tumour growth via re-oxygenation of hypoxic tumour cells and increased neovascularization.It has further been suggested that HBO may increase the incidence and/or rate of growth of local recurrence or remote metastatic disease in patients with a history of malignancy.Although most of the experimental and clinical studies suggest that HBO has no direct effect on tumors,there is a considerable amount of conflicting evidence to support the idea that HBO has an effect.The general consensus is that HBO could not kill tumor cells directly and remains ineffective as a stand-alone therapy.However,HBO may enhance the efficacy of certain therapies that are limited because of the hypoxic tumor microenvironment.But the exact effect of HBO on the natural history of malignancy is uncertain. Minimal evidence exists regarding the direct effect of HBO on prostate cancer cells. And there are no experimental studies showing treatment with HBO during other therapeutic treatment modalities is to improve the response of prostate cancer.The experimental studies could answer the following questions:1.Does HBO accelerate the growth of indolent prostate cancer cells（suggesting HBO administration is safe when it is used to manage patients treated by other therapies for prostate cancer）?2.HBO may improve the ability of other therapies to kill hypoxic prostate cancer cells. As an adjuvant therapy,HBO may result in a reduction in mortality and recurrence?OBJECTIVE1.To detect the effects of HBO on human prostate LNCaP and PC-3 cell lines.2.Assess HBO potential role in therapy for prostate cancer.METHODS1.Human prostate LNCaP and PC-3 cell lines were randomized to undergo 5 sessions of either HBO or normobaric air in standardized conditions.Microscope and transmission electron microscope were used to detect the cell morphosis.2.Detect LNCaP and PC-3 cell lines proliferation in response to HBO by the CCK-8 assay.3.Observe the effection of HBO on LNCaP and PC-3 cell cycle by FCM.4.Transwell system were used to assess HBO effect on LNCaP and PC-3 cell migration.5.Form prostate cancer PC-3 cell xenografts,and analysis of developed tumours’ grow up.At the end of the experiment detect xenografts’s microvessel density（MVD）,apoptosis markers（p53,p27 protein） and proliferative index（Ki-67 protein） by immunohistochemistry.RESULTS1.Exposure to HBO at 2.5 atm for 5 treatment sessions comprised a daily 70-min session had no effect on LNCaP and PC-3 cells’ proliferation,ultramicro morphosis and cell migration in vitro.2.HBO induced LNCaP and PC-3 cell lines to enter the cell cycle and accumulated them in G₂/M stage.3.No differences were observed in volume of prostate cancer PC-3 cell xenografts, tumour microvessel density,proliferative index and apoptosis markers compared to the control group（p＞0.05）.CONCLUSIONSHBO did not have a tumour stimulatory effect on prostate cancer and may potentially be used safely as an adjuvant therapy.HBO therapy may improve therapeutic efficacy of prostate cancer. Further research may be warranted pending outcomes of the clinical research in evaluating the adjunctive potential of HBO with other therapeutic treatment modalities.更多还原