【作者】 代佑果；

【导师】 崔进；

【作者基本信息】 昆明医科大学 ， 外科学， 2014， 博士

【摘要】 背景：取自海平面下200m的深层海水,经过处理后成为“生理性深层海水”(Physiological deep-sea water, PDSW),富含多种微量元素和矿物质,尤其含有硒、锶、铜和锌等可以预防癌症的元素。热疗是继手术、化疗、放疗和生物治疗之外,治疗癌症很有前途的一种方法。我们假设全身热疗结合PDSW能够治疗肝细胞癌,并能增强荷瘤裸鼠的耐受力,延长生存时间。方法：第一部分实验研究拟探讨“生理性深层海水(PDSW)”对45℃高温环境动物耐受性影响及机制。将取自我国海南省海域的深层海水进行制备,制成“生理性深层海水(PDSW)",检测所含有的部分元素,并以昆明地区自来水(生活饮用水)相对应检测的数据作为对照。20只昆明小鼠,雌雄各半,随机分为对照组和实验组,对照组小鼠饮用自来水,实验组饮用深层海水,连续15天,然后把各组小鼠放入45℃高温环境下饲养,记录每只鼠死亡时间,直至各组小鼠全部死亡为止。并取每只鼠脑、肺、心、肝和肾做病理检查。Western-blot检测各组肝组织(Heat shock proteins-72)HSP72表达。第二部分实验研究拟探讨热疗结合PDSW的体外和体内抗癌作用。在体外实验中,体外培养的正常肝细胞和人肝癌QGY-7703细胞被随机分为PDSW组和生理盐水组,PDSW组加入PDSW,生理盐水(normal saline,NS)组给予等量生理盐水,24小时后,每天分别接受40℃热疗6h及43℃热疗1h,在热疗后的24、48和72h,用MTT法检测热疗结合PDSW对正常肝细胞及人肝癌QGY-7703细胞的抑制率。同时检测3PDSW及NS在40℃6h连续10天状态下对人肝癌QGY-7703细胞克隆形成率的影响。在体内实验中,荷人肝癌QGY-7703细胞的Balb/c裸鼠被随机分为两组(n=6),对照组给予自来水,实验组给予硬度3000ppm的PDSW,喂养30天,隔天测量肿瘤体积,评估PDSW对肿瘤生长的作用。然后两组动物每3天给予40℃全身热疗6h,直到荷瘤小鼠全部死亡,热疗中隔天测量肿瘤的体积,并评估PDSW联合全身热疗对肿瘤的抑制作用以及荷瘤裸鼠的生存时间,获取小鼠肿瘤做病理检测和电子显微镜检测。结果：在第一部分实验中,45℃高温环境热耐受实验结果显示,PDSW组小鼠的生存时间比自来水组长,差异显著(P<0.01)。该结果结合组织学结果表明,PDSW能增加实验动物对高温的耐受性,延长实验小鼠的生存时间。Western-blot检测发现PDSW组肝组织HSP72表达较TW组强,差异显著(P<0.05)。在第二部分实验中,MTT检测结果显示肿瘤抑制率在两组均呈时间和浓度依赖性。PDSW组的肿瘤抑制率明显比生理盐水组高,差异显著(P<0.05)。结果表明在体外PDSW能够增加热疗的抗癌敏感性。而正常肝细胞组,PDSW对正常肝细胞的生长抑制率明显低于NS组,结果表明PDSW具有保护正常肝细胞的作用。此外,PDSW组的克隆形成率较NS组低,差异显著(P<0.05)。给予PDSW喂养的荷人肝癌QGY-7703细胞的Balb/c裸鼠,其肿瘤体积与用自来水喂养的荷瘤裸鼠比较没有差异(P>0.05)。结果表明PDSW没有促进荷瘤裸鼠肿瘤生长的作用。在热疗21天后,PDSW组荷瘤小鼠的肿瘤明显比对照组小,差异显著(P<0.05)。结果表明PDSW能够增强全身热疗的抗癌作用,且PDSW组的热效应比对照组提前约4天。全身热疗后,PDSW组荷瘤小鼠的生存时间比自来水组长,差异显著(P<0.05)。该结果表明,PDSW能够提高荷瘤小鼠的热耐受性,并能延长荷瘤小鼠的生存时间。热疗21天后,组织学结果显示自来水组肿瘤仅有小片坏死,而PDSW组有大片肿瘤坏死。两组坏死面积百分率比较差异显著(P<0.01)。透射电镜结果显示PDSW组主要以坏死为主,而自来水组则以细胞凋亡为主,结果表明PDSW联合热疗提前了人肝癌QGY-7703细胞的凋亡时间,推测PDSW能够降低该细胞的临界温度。结论：在第一部分实验中,“生理性深层海水(PDSW)"可以增强小鼠对45℃高温环境的耐受性,其机制考虑与PDSW促进HSP72表达有关。第二部分实验研究结果表明PDSW能够提高正常肝细胞、实验动物对热的耐受性。PDSW没有促进人肝癌QGY-7703细胞生长的作用,当联合全身热疗使用时,可明显改善荷瘤实验动物的生存时间,并能增强全身热疗的抗癌作用。研究结果可望为肝细胞癌提供一种新的治疗策略,相关的机制有待进一步研究。更多还原

【Abstract】 Background. Recently, physiological deep-sea water (PDSW) from a depth of more than200m in depth is rich in trace metals, e.g., Se, Sr, and Zn which have been considered to be associated with prevention of carcinoma. Hyperthermia (HT) is a promising method for cancer treatment. We hypothesized that whole body hyperthermia would treat significantly hepatocellular carcinoma when combined with PDSW, and would improve heat tolerance and survival time in mice.Methods. In the first study, the effects of physiological deep-sea water on hyperthermal tolerance for Kunming (KM) mice in the45℃environment were exploredand. Deep-sea water (DSW) from the south Chinese sea was processed, and the metallic elements dissolved in the DSW were analysed. The animals were randomly divided into two groups with five animals:control group received tap water; the experimental group was treated with PDSW for15days. And then the animals fed in the45℃conditions. The survival time and histomorphometric analyses of the brain, lung, heart, liver and kidney were investigated.In the second study, the anti-cancer effects and enhanced resistance of PDSW in vitro and in vivo were investigated. In vitro, the cultured human hepatoma QGY-7703cells were randomly divided into tow groups:control group received saline;the experimental group received different concentrations of PDSW. Tow groups were heated respectively to6h of40℃or1h of43℃24,48,72hours after the administration of PDSW or saline, and QGY-7703cells proliferation capacity and toxicity were investigated by MTT assay. In vivo, the HCC-bearing Balb/c-nude mice were randomly divided into two groups with six animals:control group received tap water (TW); the experimental group was fed with PDSW of hardness3000for a further30days. And then tow groups of animal were heated to6h of40℃by the way of whole body hyperthermia every two day. Tumor volume and survival was evaluated. The histological appearance were measured by HE staining and transmission electron microscopy.Results. In the first study, the survival time in PDSW-fed group was significantly longer than in the control group (P<0.05).Moreover, histomorphometric analyses that PDSW could protect the brain, lung, heart, liver and kidney of KM mice from the45℃conditions. The results of western blot revealed that expression of HSP72of liver tissues for PDSW-fed group substantially increased, as compared to control mice (P<0.05).In the second study, the results of MTT assay showed that tumor inhibitory rate were time and concentration dependent in tow groups. Tumor inhibitory rate for PDSW group in different time were significantly higher than for the saline group (P<0.05), and the results revealed that PDSW could increased the anti-cancer sensitivity of HT in vitro. On the other hand,the inhibitory of hepatocyte for PDSW group in different time were significantly lower than for the saline group (P>0.05), and the results suggested that PDSW could protect the hepatocyte from HT in vitro.The change of tumor volume for the HCC-bearing Balb/c-nude mice fed with PDSW were not different compared with the TW (P>0.05), and the results showed the growth of tumor for the HCC-bearing Balb/c-nu mice weren’t enhanced by PDSW. But21days after HT, the tumor volume for the HCC-bearing Balb/c-nude mice fed with PDSW were smaller than with TW (P<0.05), and the results suggested PDSW could increased the anti-cancer effects of HT. Heating effect was advanced approximately by4days in the PDSW group compared with the TW group. The survival time of the PDSW group after HT were longer than of the TW group (P<0.05), and the results showed that PDSW could enhance heat tolerance and extend survival time for the HCC-bearing Balb/c-nude mice. A histological examination of the tumor revealed only a small necrotic area in the TW group as opposed to massive necrosis in the PDSW group. The percentage values of the necrotic area of the neo-graft tumor21days after HT were significantly larger for the PDSW group than for the TW group (P<0.01). The results of transmission electron microscopy suggested that necrosis was mainly event in the PDSW group, while apoptosis was mainly event in the TW group.Conclusions. Taken together, the results in the first study suggested that PDSW could improve hyperthermal tolerance of KM mice, which was considered the relation with HSP72expression resulted in PDSW.These results in the second study provided a possibility that continuous administration of PDSW can ameliorate tolerance in vitro or in vivo. PDSW significantly improved the survival time when combined with whole body hyperthermia, and could increased the anti-cancer effects of HT. This was supported by the histological findings and the results of MTT assay. These results might provide a novel and promising therapeutic option for HCC. The further mechanism would be explored in the future.更多还原