【作者】 陈少雅；

【导师】 陈崇宏；

【作者基本信息】 福建医科大学 ， 内科学， 2007， 博士

【摘要】 褪黑激素(melatonin,MT)是生物体内普遍存在的一种吲哚类激素,在人体内主要由松果体合成和分泌。MT具有提高机体免疫功能、抗氧化作用及抗肿瘤活性等重要的生理及药理作用,且相对安全。因此,MT的功能研究是目前国内外研究热点之一。MT的肿瘤化学预防、抗肿瘤活性及其应用前景,已引起人们的关注。中、晚期宫颈癌的死亡率仍居高不下。宫颈癌手术治疗之后必须进行化疗。研究表明,宫颈癌患者血浆中MT水平较正常人低。因此,我们推测MT可能有助于宫颈癌的治疗。本工作采用体外培养的肿瘤细胞系HeLa细胞等及动物移植性肿瘤模型,观察MT对肿瘤的抑制情况,以进一步明确MT的抗肿瘤活性。运用Western Blotting法观察MT对HeLa细胞内NF-κB的含量变化及其调控蛋白的调控情况,以证实NF-κB的含量及活性变化与MT抗肿瘤活性的关系。进一步分析MT对HeLa细胞细胞周期的影响,MT对HeLa细胞形态的影响,HeLa细胞运用MT后胞内抗氧化能力的变化以及MT1及MT2褪黑激素膜受体在HeLa细胞上的表达。通过上述研究,有助于评价MT的抗肿瘤活性及其相关作用,为在肿瘤的综合治疗中提供一种具多种治疗效应且毒性相对小的肿瘤治疗或辅助治疗用药,提供重要的理论基础和实验依据。1.褪黑激素的体内外抗肿瘤活性研究从HL60、B16、HeLa及MCF-7的生长曲线中可见,随着MT剂量的增加,作用时间的延长,它对肿瘤细胞的抑制增殖活性明显增强,生长曲线也随之下移。MT作用72小时,HeLa的IC50为61.71μg / mL ,与其余三种细胞系比较,HeLa细胞的IC50最小。结果进一步证实MT可抑制体外培养肿瘤细胞的增殖。MT组、MT+CTX组及MT+5-FU组较对照组、CTX组及5-FU组在实验结束时体重均值有所增加,结果显示,MT没有明显的毒性反应。单用MT 80mg / kg在不产生明显毒性反应基础上,对小鼠实体瘤U14的生长有一定的抑制作用,最大抑瘤率为30.9 %。MT+CTX联用(q = 1.072)及MT+5-FU联用(q = 1.093)均可产生单纯相加作用。结果提示,MT可在不产生毒性反应的基础上抑制小鼠实体瘤U14的生长,与CTX联用、5-FU联用均可增强抑瘤作用。2.褪黑激素对HeLa细胞NFκB及其相关蛋白IκKα/β、IκB-α及p-IκB-α水平的影响Western blotting分析显示,MT 0.2μg /μL作用1d、2d及3d,MT作用2d后对HeLa细胞NFκB水平的抑制作用呈时间依赖性下调,IκB-α水平呈时间依赖性上调,p-IκB-α水平则呈时间依赖性下调,而IκKα/β水平也呈时间依赖性下调。MT 0.008μg /μL, 0.04μg /μL及0.2μg /μL作用3d,对HeLA细胞NFκB的抑制作用呈明显的浓度依赖性;p-IκB-α水平呈浓度依赖性下调,从MT 0.04μg /μL开始,IκKα/β水平呈浓度依赖性下调而IκB-α水平呈浓度依赖性上调。结果提示,MT对HeLA细胞NFκB蛋白的抑制作用呈浓度依赖性及时间依赖性。结果还表明,MT可能通过直接或间接抑制IκKα/β而使IκB-α蛋白降解减少,水平上调,p-IκB-α蛋白水平下调,可能导致IκB-α对NFκB抑制增加,使NFκB活性降低。说明MT在产生抗增殖活性剂量下能下调NFκB水平;而且NFκB活性可能也因调控蛋白IκKα/β水平下调,IκB-α水平上调及p-IκB-α水平下调而受到抑制。3褪黑激素对HeLa细胞抑制增殖活性的其余相关作用荧光显微镜下,未经MT处理的经胰酶消化的HeLa细胞呈圆形;MT 1μg /μL作用24h、48h及72h,胞膜溶解的坏死细胞随作用时间的延长而逐渐增多,凋亡细胞少见。由此可见,MT主要致HeLa细胞坏死而少致凋亡。电镜下,未经MT处理的对照组细胞,异染色质少,常染色体多;MT组0.2μg /μL作用72h,可见胞膜不完整的坏死细胞及少量胞膜完整、染色质聚集在核膜的周边的凋亡细胞。流式细胞仪检测显示,MT 0.008μg /μL、0.04μg /μL及0.2μg /μL作用72h后,S期细胞比例随剂量增加而逐渐降低。结果提示,MT可能抑制HeLa细胞的S期。HeLa细胞的总抗氧化能力及总SOD、GSH的水平测定显示:MT作用于HeLa细胞1d、2d及3d,低剂量MT组、中剂量MT组、高剂量MT组及极高剂量MT组的T-AOC均数分别与对照组比较,均有增加。随着MT浓度的增加,作用时间的延长,T-AOC逐渐增强,以1μg /μL MT作用3d为最明显;T-SOD均数均有所增加,GSH含量均数也有所增加,但均不存在明显的时间与剂量依赖性。Western blotting法及免疫组织化学法结果提示,HeLa细胞上未能检测到MT1及MT2受体,MT抑制HeLa细胞的增殖活性可能与激动MT膜受体无关,但核受体ROR/RZR的激动作用是否存在,有待证实。这些结果提示,MT在抑制HeLa细胞增殖的剂量下引起细胞形态的改变主要以坏死为主,还能抑制细胞周期的S期,并且提高HeLa细胞的T-AOC,其中T-SOD及GSH水平提高。综上所述,本工作提示:1.褪黑激素对体外培养的肿瘤细胞HeLa、MCF-7、HL60及B16均有抑制增殖活性,其中对HeLa细胞的抑制作用最显著,抑制浓度范围在0.008μg /μL～1μg /μL。MT对HeLa细胞的IC50为0.617μg /μL。2.褪黑激素对小鼠移植性肿瘤U14有一定的抑制作用,MT 80mg / kg对U14的抑瘤率达30.9 %;与5-FU、CTX合用可能有协同抑制增殖作用。3.首次证实褪黑激素对HeLa细胞的抑制增殖活性可能与下调NFκB水平有关,而且存在一定的剂量依赖性与时间依赖性。这可能与MT的抗增殖机制有关。4.首次证实褪黑激素可能通过直接或间接抑制IκKα/β而使IκB-α蛋白降解减少,含量增多,p-IκB-α蛋白因而水平降低,从而可能导致IκB-α对NFκB抑制增加,使NFκB活性降低。这可能与MT的抗增殖机制有关。5.褪黑激素抑制HeLa细胞增殖活性可能与增强HeLa细胞的抗氧化能力有关。MT 0.008μg /μL～1μg /μL可剂量依赖性及时间依赖性增强HeLa细胞的T-AOC,其中T-SOD及GSH的水平提高。这可能与MT的抗增殖活性有关。6.褪黑激素抑制HeLa细胞增殖活性可能与抑制细胞周期的S期有关。MT 0.008μg /μL～0.2μg /μL作用72h后,S期细胞比例随剂量增加而逐渐降低。这可能与MT的抗增殖活性也有关。更多还原

【Abstract】 Melatonin(MT) is the indole hormone widely existing in organism. It possesses some important physiological and pharmacologic properties, such as enhancing body immune function, elevating the antioxidative activities and antitumor activities. Furthermore, melatonin has very low toxicity. Therefore, the study on function of melatonin has been one of the most interesting research fields.It is one of the focuses that MT can be a chemopreventive drug and antineoplastic activities and its prospect of application. The death rate of metaphase or advanced cervical carcinoma has kept on high level. After surgical operation, the patients suffered from metaphase or advanced cervical carcinoma are necessary for the chemotherapy. Studies showed that the plasma MT concentration in patients with cervical cancer was lower than that of normals. Therefore, it is logical to assume that MT may be help to cervical carcinoma patients.In the present study, the antineoplastic effects of melatonin determined by cultured cell lines and animal transplanting tumor models in order to make clear further the antineoplastic effects of melatonin. The expression of NF-κB and its regulatory proteins by MT on HeLa cells were analysied primarily by Western blotting analysis. Then, the study was designed to evaluate the influence of melatonin on the cell division cycle of HeLa cells, to explore morphological alterations of HeLa cells by fluorescene staining and transmission electron micrograph. In addition, the T-AOC and the level of T-SOD and GSH on HeLa cells by MT would be measured. The paper would detect the MT membrane receptors MT1 and MT2 on HeLa cells by means of Western blotting analysis and immunohistochemical staining.These study contributed to a better understanding of the antineoplastic activities and its related effects which were undertaken to supply the experimental foundations for the potential clinical application of melatonin and its homologens as a kinds of antineoplastic drugs or adjuvants in treatment of neoplasms.1 Antineoplastic activities of MTThe cell growth curve of HL60, B16, HeLa and MCF-7 were determined by MTT assay. With the elevation of dose and accession of time, the antiproliferative activities induced by MT increased. All of the cell growth curve were significantly lower. The IC50 on HeLa cells which was the smallest one of the four cell lines by MT for 72hr was 61.71μg / mL. The above results have been confirmed further that MT had an antiproliferative activities on cultured tumor cell lines in vitro.It was shown that MT inhibited the murine U14 carcinoma in a dose-dependent manner with the maximum tumor growth inhibitory rate of 30.9% at a dose of MT 80mg / kg. It was also shown that the means of body weight either MT group or MT in combined with CTX or 5-FU were gain more than that of both control and CTX group. It confirmed that MT had no obvious toxicity. The results observed that both of MT in combined with CTX (q=1.072)and with 5-FU(q=1.093) were showed synergistic. These results suggested that melatonin possesses an antineoplastic activities in vivo.2 The influences of MT on the levels of NFκB and its related proteins IκB-α, p-IκB-αand IκKα/βon HeLa cellsIt was shown that MT could down-regulate NFκB protein level in a time- dependent and dose-dependent manner after treated with melatonin 0.2μg /μL for 1d, 2d and 3d and with melatonin 0.008μg /μL, 0.04μg /μL and 0.2μg /μL for 3 days by Western blotting analysis. It was observed that MT could down-regulate IκKα/βand p-IκB-αand up-regulate IκB-αafter treated with melatonin 0.2μg /μL for 1d, 2d and 3d in a time-dependent manner from the second day. The results revealed that MT could down-regulate IκKα/βand p-IκB-αand up-regulate IκB-αafter treated with melatonin 0.008μg /μL, 0.04μg /μL and 0.2μg /μL for 3 days in a dose-dependent manner from MT 0.04μg /μL. These results suggested that MT could down-regulate NFκB protein level while exerting the antiproliferative activities. Moreover, These results also suggested that MT regulated NFκB related proteins level, including down-regulate IκKα/βand p-IκB-αand up-regulate IκB-α. These may be result in inhibiting NF-κB activity.3 Other effect of MT related to its antiproliferation activitiesIt was observed that HeLa cells stained with AO/EB were damaged in a time-dependent manner by fluorescene staining. As the time go on, more and more necrocytosis were seen and less apoptosis by MT 1μg /μL for 24h, 48h and 72h.The results showed more somatic chromosome and less hetero-chromosome of the control group. It was observed that much more necrocytosis and less apoptosis presented on HeLa cells exposed to MT 0.2μg /μL for 72h by transmission electron micrograph.The results also showed that HeLa cells in phase S was reduced in a dose-dependent manner while exposed to melatonin at 0.008μg /μL、0.04μg /μL and 0.2μg /μL for 72h by flow cytometric analysis.It was shown that MT enhanced T-AOC in a time-dependent and dose-dependent manner on HeLa cells after exposed to melatonin at 0.008μg /μL , 0.04μg /μL, 0.2μg /μL and 1μg /μL for 1d, 2d and 3d by uv-vis spectrophotometer.MT elevated the level of T-SOD(dose and group as the same as above mentioned) and GSH(dose and group similar to above mentioned, not including the very high group MT) on HeLa cells. The results showed that MT1 and MT2 MR were not be detected on HeLa cells by Western blotting analysis and immunohistochemical staining. Whether the MT nucleus receptors ROR/RZR existed on HeLa cells and participated the antiproliferation activities of MT were still unknown and need to be further confirmed.These results above mentioned revealed that MT inhibited HeLa cells with necrosis and less apoptosis, along with reducing the cell numbers of phase S in the cell division cycle, enhancing the T-AOC and increasing the level of T-SOD and GSH. These rusults determined that MT1 and MT2 MR were not be detected on HeLa cells and MT nucleus receptors ROR/RZR existed or not were still unknown and need to be analysed.Summary1. It have been confirmed further that MT 0.008μg /μL-1μg /μL had an antiproliferative activities on HeLa cells in vitro. The IC50 of MT on HeLa cells was 0.617μg /μL.2. MT 80mg / kg possesses an antineoplastic activities on murine transplant carcinoma U14 and had no obvious toxicity in vivo. The maximum inhibitory rate on U14 was 30.9 %. Both MT in combined with CTX (q=1.072)and with 5-FU(q=1.093) were showed synergistic.3.MT could down-regulate NFκB protein level while exerting the antiproliferative activities, which may be one of mechanisms of the antiproliferative activity of it.4. MT regulated NFκB related proteins level, including down-regulate IκKα/βand p-IκB-αand up-regulate IκB-α. These may be result in inhibiting NF-κB activity, which may be contribute to the antiproliferative activity of it.5.MT 0.008μg /μL - 1μg /μL enhanced T-AOC in a time-dependent and dose-dependent manner on HeLa cells. MT elevated the level of T-SOD and GSH, which may be related to the antiproliferative activity of it. 6. HeLa cells in phase S was reduced by MT 0.008μg /μL - 0.2μg /μL for 72 hr, which may be also related to the antiproliferative activity of it.更多还原