【作者】 李卫；

【导师】 何援利；

【作者基本信息】 南方医科大学 ， 妇产科（专业学位）， 2013， 博士

【摘要】 研究背景上皮性卵巢癌是妇科恶性肿瘤中最致命的肿瘤,死亡率位居妇科肿瘤第一位。尽管手术治疗对早期卵巢癌的疗效良好,但绝大多数患者病情会出现晚期进展。手术和化学药物治疗是卵巢癌治疗中的两个重要手段,由于晚期卵巢癌的根治性手术不能达到理想和满意程度,故化学药物治疗对手术后的治疗就显得更为重要。但由于晚期卵巢癌极易产生对化学药物的耐药和全身毒副反应,应用现有的化学药物治疗仍然有很高的复发率和未控率,因此研发新型的高效低毒的抗肿瘤药物是目前卵巢癌治疗的研究热点。目前国内外研究的新型抗肿瘤药物种类如下：1.以细胞信号分子为靶点：包括蛋白络氨酸激酶抑制剂、各种信号转导通路抑制剂、法尼基转移酶抑制剂、细胞周期调控剂、蛋白酶体抑制剂、组蛋白去乙酰化酶抑制剂等。2.血管生成抑制剂。3.抗癌细胞脱落、粘附和基底膜降解：抗转移药。4.以端粒酶为靶点：端粒酶抑制剂。5针对肿瘤细胞耐药：耐药逆转剂。6.促进肿瘤细胞向成熟分化：分化诱导剂。7.特异性杀伤癌细胞：(抗体或毒素)导向治疗。8.增强放疗和化疗的疗效：放化疗增敏剂。9.提高或调节机体免疫功能生物反应调节剂。10.针对癌基因和抑癌基因的基因治疗：导入野生型抑癌基因、自杀基因、抗耐药基因及反义寡核苷酸、肿瘤疫苗、RNA干扰、MicroRNA等。11.靶向cox-2途径：cox-2抑制剂等。12.天然动植物药物：如原花青素、儿茶素等。13.化学合成的抗肿瘤药物：通过计算机辅助设计模拟靶向肿瘤特异性靶点的抗肿瘤药物,如化学合成的小分子抗肿瘤药物等。近年来化学合成的小分子抗肿瘤药物成为新型抗肿瘤药物的研究热点。本实验所用化合物即为模拟锰超氧化物歧化酶(MnSOD)结构所设计的化合物。人体正常的新陈代谢都会产生自由基,但在特殊情况下会产生大量自由基,这些大量的自由基通过氧化作用使人体的细胞和组织受到损伤,包括自由基使酶失活、自由基损害细胞膜、自由基损伤导致基因突变和癌变,故自由基是许多疾病发生发展的病理学基础。超氧化物歧化酶是细胞内重要的酶性自由基清除剂,可催化O-2歧化为H2O2和O2。人体内超氧化物歧化酶中最重要的就是锰超氧化物歧化酶(MnSOD)。MnSOD基因的表达和调控对机体自由基-自由基清除剂的平衡体系至关重要,是抗氧化系统的最重要的酶之一。目前,大量研究已证实MnSOD与肿瘤细胞生长、分化、侵袭及耐药等多种生物学行为关系密切。但MnSOD在肿瘤细胞中的表达报道不一。普遍认为,MnSOD在大多数肿瘤细胞中活性减弱,但其在卵巢癌细胞中活性增强。卵巢癌中,高表达的MnSOD对肿瘤细胞有保护作用,并可促进肿瘤分化进展,但是,产生对卵巢癌保护性MnSOD量的这一氧化还原平衡被打破后,MnSOD表达进一步增高,引起H202过量,反过来又会抑制肿瘤细胞增殖,并可导致肿瘤细胞凋亡。由此推测MnSOD超过卵巢癌保护性表达量的话,MnSOD反而可能会抑制卵巢癌发展。近年来运用MnSOD化学模拟物来替代天然MnSOD成为研究热点。由于天然MnSOD分子量大、体内半衰期短、不易透过细胞膜、稳定性差等原因,使其药用价值受到了极大的限制。为此,生物化学界的科研人员用化学手段合成与表征具有相关结构的铜、锰、铁等金属离子的小分子配合物来模拟超氧化物歧化酶以克服天然超氧化物歧化酶的缺点,从而使模拟化合物的小分子配合物有运用于临床的可能。兰州大学采用水溶性较好的柔性脂肪胺和具有良好生物相容性的邻香草醛合成了水溶性和脂溶性较好的MnSOD配合物,并已研究证实其具有较高的活性。该化合物具有水脂兼溶、活性高、稳定性好、分子量小、易跨膜、成本低廉、纯度高、产率高等特点,可见其很好的克服了天然MnSOD的局限性,从而有运用于临床的潜在可能性。MnSODm通过非细胞毒性作用抑制肿瘤,没有化学药物的毒副作用,具有高效低毒的特点,使得MnSODm成为有临床应用前景的抗肿瘤药物。任何药物运用于临床的前提是其作用机制必须明确。而目前国内外甚少有MnSODm抗肿瘤作用的研究,而对其抗肿瘤的机制更是研究甚少。国外有文献报道,锰超氧化物歧化酶模拟化合物(MnSODm)联合庆大霉素治疗,可以减低耳毒性,MnSODm与顺铂协同作用降低顺铂造成的耳毒性,干预MnSOD表达,增强顺铂化疗作用；而MnSODm对肿瘤的治疗,国内仅见安娴、范临兰等研究了MnSODm对白血病K562细胞的增殖抑制作用及诱导细胞凋亡作用。本实验应用MnSODm同时干预体内外肿瘤细胞增长,以观察其抗肿瘤作用。本课题以人上皮性卵巢癌SKOV3细胞及其裸鼠移植瘤为研究对象,体内实验通过移植瘤组织HE染色,电子显微镜观察药物治疗后肿瘤组织的病理学变化,并借助于称瘤重及测量瘤体大小计算抑瘤率,查血常规、肝肾功能变化,检测药物毒性反应；免疫组化法检测瘤组织相关癌基因；体外实验通过CCK-8法检测细胞增殖与活性,PI单染和倒置显微镜观察细胞凋亡,电镜观察凋亡细胞超微结构的变化,流式细胞术观察细胞凋亡周期,PCR、蛋白印迹法研究核因子(NFKB)、bax和bcl-2等基因表达变化,体内体外实验相结合,从而研究MnSODm在体内外对SKOV3细胞系及其裸鼠移植瘤生长的抑制作用并探讨分子机理,为卵巢癌治疗提供新思路,新方法。第一章MnSODm抑制人上皮性卵巢癌SKOV3细胞增殖并诱导其凋亡目的：观察MnSODm对人上皮性卵巢癌SKOV3细胞生长的抑制作用。方法：1.取对数生长期的SKOV3细胞,用不同浓度的MnSODm (0μg/ml,10u g/ml,50μg/ml)处理Oh、24h、48h、72h、CCK-8法检测MnSODm处理前后细胞增殖与活性的变化,顺铂对照组顺铂浓度为10μg/ml。滴加CCK-8试剂后,在450nm波长处检测各孔吸光度(OD)值,记录结果。2.取对数生长期的SKOV3细胞,用不同浓度的MnSODm (0μg/ml,10μ g/ml,50μg/ml)处理72h,倒置显微镜观察MnSODm处理前后细胞形态学变化。3.取对数生长期的SKOV3细胞,用不同浓度的MnSODm (Oμg/ml,10μg/ml,50μg/ml)处理72h,PI单染法观察MnSODm处理前后细胞发生凋亡的形态学变化。4.取对数生长期的SKOV3细胞,用不同浓度的MnSODm (0μg/ml,10μg/ml,50μg/ml)处理72h,透射电镜观察MnSODm处理前后SKOV3细胞超微结构的改变。5.取对数生长期的SKOV3细胞,用不同浓度的MnSODm (0μg/ml,10μg/ml,50μg/ml)处理72h,加入Annexin V和PI染色,流式细胞术检测MnSODm处理前后细胞凋亡率的变化。6.统计学分析：采用SPSS17.0统计软件分析。计量资料以均数±标准差表示(x±s),采用单因素方差分析(One-way ANOVA),组间比较采用多重比较LSD法。P<0.05为差异有统计学意义。结果：1.与对照组比较,MnSODm处理后SKOV3细胞的增殖及活性明显受到抑制(P<0.05),呈时间依赖性和浓度依赖性。与顺铂相比,MnSODm浓度为10μg/ml时,其24、48、72h对细胞的抑制作用均小于顺铂(P<0.05),而MnSODm浓度为50μg/ml时,其24h对细胞的抑制作用小于顺铂(P<0.05),但48、72小时对细胞的抑制作用则强于顺铂(P<0.05)。有统计学意义。2.与对照组比较,倒置显微镜下SKOV3细胞经MnSODm作用72h后,细胞变圆、皱缩、破裂,呈不规则形,出现漂浮状态生长的细胞数量明显增加,细胞内部结构受到破坏,存活的细胞明显减少。3. MnSODm作用后,PI染色显示SKOV3凋亡细胞明显增多。4. MnSODm处理72h后,透射电镜下SKOV3细胞出现细胞体积缩小,胞膜破损直至消失,核染色质高度浓缩、电子密度增高并边集于核膜下,有的核染色质呈半月形,有的出现核固缩和核碎裂,胞质出现空泡变性等明显的凋亡细胞的超微结构改变。5. MnSODm处理72h后,应用流式细胞术显示SKOV3细胞凋亡率明显增高,与对照组比较(P<0.05),有统计学意义。结论MnSODm可抑制人上皮性卵巢癌SKOV3细胞增殖,其抑制肿瘤细胞的增殖作用呈时间依赖性和浓度依赖性,MnSODm可诱导SKOV3细胞凋亡。第二章MnSODm抑制人上皮性卵巢癌SKOV3细胞增殖及诱导其凋亡的可能机制的研究目的：探讨MnSODm抑制人上皮性卵巢癌SKOV3细胞增殖及诱导其凋亡的可能机理的研究。方法：1.取对数生长期的SKOV3细胞,用不同浓度的MnSODm (0μg/ml,10μ g/ml,50μg/ml)处理72h,流式细胞术检测MnSODm处理前后SKOV3细胞周期分布的变化。2.取对数生长期的SKOV3细胞,用不同浓度的MnSODm (0μg/ml,10μg/ml,50μg/ml)处理72h,荧光发光法检测MnSODm处理前后SKOV3细胞中凋亡相关因子Caspase-3/7的活性。3.取对数生长期的SKOV3细胞,用不同浓度的MnSODm (0μg/ml,10μ g/ml,50μg/ml)处理72h,RT-PCR法检测MnSODm处理前后SKOV3细胞中核因子-κ B/p65、细胞凋亡相关因子bcl-2、bax mRNA表达的变化。4.取对数生长期的SKOV3细胞,用不同浓度的MnSODm (0μg/ml,10μ g/ml,50μg/ml)处理72h,Western印迹法检测MnSODm处理前后SKOV3细胞中核因子-κB/p65、细胞凋亡相关因子bcl-2、bax蛋白表达的变化。5.统计学分析：采用SPSS17.0统计软件处理,计量资料以均数±标准差(x±s)表示。采用单因素方差分析(One-way ANOVA),组间比较采用多重比较LSD法。P<0.05为差异有统计学意义。结果：1.与对照组相比,MnSODm处理后,SKOV3细胞周期分布发生明显改变,表现为G0/G1期细胞增多,S期细胞减少,细胞周期阻滞于G0/G1期(P<0.05),差异有统计学意义。2.不同浓度的MnSODm处理SKOV3细胞72h后,SKOV3细胞中凋亡相关因子Caspase-3/7活性无明显改变(P>0.05),差异没有统计学意义。3. MnSODm处理后,SKOV3细胞中核因子-κB/p65、细胞凋亡相关因子bcl-2mRNA表达明显下调(P<0.05),而bax mRNA表达明显上调(P<0.05),差异有统计学意义。4. MnSODm作用SKOV3细胞72h后,Western检测结果显示：SKOV3细胞中NF-κB/p65蛋白与对照组(0μg/ml)比较,表达明显降低,而其表达降低相应调控其下游凋亡抑制因子bcl-2蛋白表达逐渐降低,而凋亡促进因子bax蛋白表达明显升高,差异有统计学意义(P<0.05)。结论：MnSODm可能是通过影响细胞周期分布,更重要的是通过核因子-κB/p56途径来调控凋亡相关因子bcl-2、bax从而实现抑制人卵巢癌SKOV3细胞的增殖并诱导其凋亡作用的,而其抑制卵巢癌细胞增殖与Caspase-3/7因子无关。第三章MnSODm对卵巢癌细胞SKOV3裸鼠异体移植瘤生长的抑制作用目的：建立SKOV3裸鼠异体移植瘤模型,观察阳性对照药物顺铂和不同剂量的MnSODm对移植瘤生长的抑制作用。方法：1.细胞培养：将SKOV3细胞用含10%新生小牛血清的RPMI1640培养基加青霉素和链霉素,于37℃、5%CO2混合气体95%饱和湿度的孵育箱内培养。每2-3d换液1次。2.种植瘤体：取对数生长期的细胞,胰酶消化后配成2.5×106个细胞/ml,取细胞悬液按每鼠0.2ml接种于荷瘤小鼠背部皮下,肿瘤稳定生长后,选择肿瘤生长旺盛且无破溃的荷瘤小鼠,颈椎脱位处死,切开皮肤,剥离肿瘤。3.异体移植：将瘤组织剪切成1.5mm×1.5mmx1.5mm左右的小块儿,用套管针接种于SPF级别BALB/c-nu裸小鼠(4-6周龄,雌性,体重20-22g)背部皮下。用游离标尺测量移植瘤直径。以皮下结节直径大于0.5cm为成瘤标准。4.分组：裸小鼠移植瘤模型建成后,随机分组为：阴性对照组即生理盐水对照组、化疗药阳性对照组即顺铂组、MnSODm高剂量组、MnSODm中剂量组和MnSODm低剂量组,各药物腹腔注射治疗7天。5.给药7天后,次日停药,使用电子天平称重,将各组裸鼠眼眶取血后处死,使用内装有肝素抗凝剂收集血液,取300μ1血液使用血细胞分析仪检测白细胞及血小板数目,取0.5ml血液静置30min后,离心取血清,检测各组裸鼠血清中谷丙转氨酶(ALT),尿素氮(BUN),血肌酐(Ccr)的变化。6.处死裸小鼠后,剥除肿瘤,称瘤重,测量瘤体积；计算肿瘤抑制率(抑制率大于30%为有效)和T/C值,T/C值的计算方法：T/C=(TRTV/CRTV)×100%其中TRTV为治疗组RTV, CRTV为阴性对照组RTV, RTV=VT/V0,VT为给药7天后瘤的体积,V0为给药时测量瘤的体积；结果T/C小于40%为有效。7.处死裸小鼠后,取各组肿瘤组织用10%甲醛固定,常规脱水、石蜡包埋,HE染色后光镜下进行组织学观察；并留组织行免疫组织化学法染色。8.采用SPSS17.0统计软件处理,计量资料以均数±标准差表示(x±s),进行正态性及方差齐性检验,两组间均数使用独立样本t检验,两组用药前后比较使用配对t检验,多组比较采用单因素方差分析(One-way ANOVA),组间比较采用多重比较LSD法,以a=0.05为检验水准,当P<0.05时,差异有统计学意义；当P>0.05时,差异无统计学意义。结果：1.实验动物一般情况观察：在对瘤体移植后7天内的观察得出,异体移植瘤生长的一般情况较好,无样本丢失；并观察了分组后各干预组裸鼠及其移植瘤的表现,得出各治疗组均能够明显改善裸鼠精神、活动度、反应,瘤体生长等一般情况。2.肿瘤体积的变化：各治疗组肿瘤体积与对照组比较,肿瘤体积明显缩小(P<0.05),有统计学意义。各治疗组治疗前后肿瘤体积比较(P<0.05),有统计学意义。T/C比值结果：顺铂组T/C比值是36%、MnSODm高剂量组是33%、中剂量组是41%、低剂量组是45%。T/C小于40%为治疗有效,。3.各组移植瘤的肿瘤重量和治疗后裸鼠体质量的变化：杀死裸鼠后剥取瘤体称瘤重,计算肿瘤抑制率,各治疗组与对照组比较,肿瘤重量明显减低,除MnSODm低剂量组外,其余各治疗组与对照组比较(P<0.05)有统计学意义,MnSODm高剂量组肿瘤抑制率66.30%,中剂量组肿瘤抑制率54.02%,低量组肿瘤抑制率32.98%。肿瘤抑制率>30%为有效。在处死前对各组裸鼠进行称重后,发现各组裸鼠在药物干预下体质量未发生明显改变,各治疗组与对照组比较(P>0.05)无统计学意义。4.组织形态学观察：生理盐水对照组表现低分化腺癌特点,肿瘤呈实性团块儿状,癌细胞密集,细胞有明显异型性,核大深染,染色质呈团块状,有明显核仁,胞浆丰富,可见较多核分裂像,间质极少。MnSODm高剂量组HE染色表现：细胞排列较稀疏,可见大片红染无结构的坏死区,瘤细胞核深染、固缩；MnSODm中剂量组坏死面积较大；MnSODm低剂量组变化不明显,与生理盐水组染片相似。顺铂治疗组图片表现与高剂量组相似。5.对血细胞及肝肾功能的影响：使用LSD法统计后结果提示,不同剂量的MnSODm治疗组与生理盐水组比较(P>0.05),无统计学意义；顺铂治疗组与生理盐水组比较(P<0.05),差异有统计学意义,与MnSODm高、中剂量组比较(P<0.05)有统计学意义,与MnSODm低剂量组比较(P>0.05)无统计学意义；MnSODm各治疗组相互比较(P>0.05)无统计学意义。结论：1.成功建立裸鼠异体移植瘤动物模型,为进一步研究MnSODm对人上皮性卵巢癌细胞SKOV3裸鼠异体移植瘤奠定基础。2.体内实验证实MnSODm对人上皮性卵巢癌细胞SKOV3裸鼠异体移植瘤生长具有抑制作用,且具有低毒的优点。第四章MnSODm对人上皮性卵巢癌SKOV3细胞裸鼠异体移植瘤作用前后bcl-2、bax表达的影响目的：采用免疫组织化学方法检测MnSODm对人上皮性卵巢癌SKOV3细胞裸鼠移植瘤bcl-2及bax蛋白的影响,探讨MnSODm对人上皮性卵巢癌SKOV3细胞裸鼠移植瘤生长抑制作用可能机理的研究方法：1.裸鼠移植瘤模型的建立；2.随机分4个组：阴性对照组即生理盐水对照组、MnSODm高剂量组、MnSODm中剂量组和MnSODm低剂量组,腹腔注射上述药物治疗7天。3.停药次日终止观察,处死裸鼠,将瘤体标本应用免疫组化(SABC)法对进行组织切片染色,使用bcl-2及bax两种免疫组化实验试剂；4.完成染片后,使用高清晰数码显微镜摄片,采集图片,使用Motic Images Advanced312分析系统处理数据,导出数据进行统计学处理,检测bcl-2及bax蛋白在人上皮性卵巢癌SKOV3细胞裸鼠移植瘤组织中及MnSODm各剂量组干预后的强弱表达结果。5.统计学分析：SPSS17.0统计软件处理,计量资料以均数±标准差表示(x±s)。采用单因素方差分析(One-way ANOVA),组间比较采用多重比较LSD法,P<0.05为有差异,结果具有统计学意义。结果：1.药物治疗后裸鼠肿瘤细胞bcl-2蛋白的表达：阴性对照组免疫组化结果提示,人上皮性卵巢癌SKOV3细胞裸鼠移植瘤组织中,bcl-2蛋白的表达定位于细胞浆与核膜处,胞浆或是核膜中着色数量较多,呈现棕褐色深染,说明bcl-2蛋白在人上皮性卵巢癌SKOV3细胞裸鼠移植瘤组织中的表达水平较高；MnSODm三个不同梯度给药组,着色的胞浆范围逐渐减少,着色深度亦逐渐变浅,说明bcl-2蛋白的表达与梯度呈非直线相关性。在MnSODm高剂量组中仅见极少数着色,主要定位于细胞浆,核膜较少见,说明bcl-2蛋白在MnSODm高剂量干预下以低表达显示。2.药物治疗后裸鼠肿瘤细胞bax蛋白的表达：bax蛋白表达,其定位于胞浆；bax在人上皮性卵巢癌SKOV3细胞裸鼠移植瘤组织中表达较弱,胞浆中呈现棕色浅染,着色范围小,说明bax蛋白在其中表达很少,几乎不表达；MnSODm三个不同梯度给药组,胞浆及核膜着色范围逐渐增大,细胞浆着色增强,显示bax蛋白的表达呈梯度上升的趋势。其中MnSODm高剂量组中见染色呈棕黄色,说明bcl-2蛋白在MnSODm高剂量干预下以高表达呈现。3. MnSODm各治疗组bcl-2、bax表达与阴性对照组(生理盐水组)之间比较(P<0.05),有统计学意义。可见MnSODm不同浓度干预组均能够下调bcl-2的表达,上调bax蛋白的表达。4. MnSODm高、中、低三个浓度组bcl-2、bax表达比较(P<0.05),有统计学意义。随着MnSODm剂量的增加,bcl-2的表达减弱,bax的表达增强。结论：1.bcl-2及bax参与了上皮性卵巢癌SKOV3移植瘤组织细胞生长抑制的过程；2.证实了MnSODm可能通过上调bax蛋白,下调bcl-2蛋白来抑制人上皮性卵巢癌细胞SKOV3裸鼠异体移植瘤之生长。全文小结1.成功建立人上皮性卵巢癌SKOV3细胞裸鼠异体移植瘤动物模型,为进一步研究MnSODm对人上皮性卵巢癌细胞SKOV3裸鼠异体移植瘤治疗奠定了基础。本模型系二次传代后,肿瘤在体内成长,成瘤率稳定,试验周期短,易于实施干预措施,样本具有均一性和可重复性,能真实反映肿瘤在体内的演变过程。2.观察荷瘤鼠体重、一般状况变化,检测小鼠用药物治疗前后血白细胞、血小板、转氨酶、肌酐和尿素氮的变化,真实反映了MnSODm在体内毒性低的特点。3.首次将MnSODm应用于体内试验,为MnSODm有可能应用于临床提供了可靠的临床前实验室研究依据。4. MnSODm可抑制SKOV3细胞增殖,呈时间依赖性和浓度依赖性,MnSODm可诱导SKOV3细胞凋亡。5.MnSODm高剂量组肿瘤抑制率最高,作用于肿瘤细胞72h的细胞凋亡率最高,体内外实验均证实MnSODm对SKOV3的作用呈时间依赖性和浓度依赖性,但是毒性没有相应增加。6.MnSODm作用于体内试验,采取腹腔内注射,局部皮肤组织没有出现破溃、红、肿、热、痛等现象,也没有出现腹泻腹胀等消化道不适症状,说明该种化合物不仅体内代谢造成的毒性反应小,直接组织刺激性亦很小,值得进一步研发。7. MnSODm可能是通过影响细胞周期分布,主要将肿瘤细胞调控在Go/G1期；更重要的是通过核因子-κB/p65途径来调控凋亡相关因子bcl-2、bax从而实现抑制人卵巢癌SKOV3细胞的增殖并诱导其凋亡作用的,而其抑制卵巢癌细胞增殖与Caspase-3/7因子无关。更多还原

【Abstract】 BackgroundEpithelial ovarian cancer is the most fatal tumor in gynecological malignant tumor, mortality ranks first in gynecologic oncology. Although surgical treatment has a good effect on early-stage ovarian cancer, yet the vast majority of patients’ condition will advance in late stage.It is difficult to treat advanced ovarian cancer with existing chemotherapy drug, because advanced ovarian cancer is susceptible to have drug resistance and systemic toxic and side reactions to chemotherapy drugs, however, the advanced ovarian cancer has lost its conditions and chance for operation, therefore, chemotherapy still remains the most important treatment method for advanced ovarian cancer, that is to say, chemotherapy and surgery occupy the equal important position in the treatment of ovarian cancer.Thus it can be seen, It is very necessary to develop new type of antitumor drugs with high efficiency and low toxicity, the research focus of the new way to treat ovarian cancer is to seek new antitumor drugs.The research of new types of anticancer drugs are as follows:researchers favor target cell signaling molecules, such as protein tyrosine kinase inhibitors, proteasome inhibitors, histone deacetylase inhibitors, angiogenesis inhibitors antimetastatic drugs, telomerase inhibitors and so on. In recent years, researchers incline toward computer-aided design simulation anticancer drugs which target tumor-specific targets, such as chemical synthesis of small molecule anticancer drugs, a research focus of the new anticancer drugs. The compound used in this experiment is the analog compound designed by the structure of the manganese superoxide dismutase (MnSOD).The body’s normal metabolism will generate free radicals, but will produce a large number of free radicals in special circumstances, which through oxidation damage the body of the cell and tissue, including inactivating the enzyme, damaging cell membranes and leading to mutations and cancer. So the free radicals is the basis of the development of the pathology of many diseases. Intracellular superoxide dismutase (SOD) is an important enzymatic radical scavenger, which can be catalyzed O-2disproportionation of H2O2and O2. Superoxide dismutase in the human body is the most important manganese superoxide dismutase (MnSOD). Balance system between MnSOD gene expression and regulation of the body’s free radicals-radical scavenger is essential, and it is one of the most important enzymes in the antioxidant system.Currently, a large number of studies have confirmed that the MnSOD and growth, differentiation, invasion and resistance of tumor cell and other biological behaviors are closely related. MnSOD Generally speaking, MnSOD activity decreased in most tumor cells, but its activity increased in ovarian cancer. In ovarian cancer, high expression of MnSOD has a protective effect on tumor cells, and may contribute to tumor differentiation progress. However, when production of amount of protective MnSOD of oxidation in ovarian cancer restoration balance is broken, MnSOD expression further increased, causing the excess of H2O2which will inhibit the proliferation of tumor cells, and can lead to apoptosis of tumor cells. It is easily inferred that if MnSOD is more than the protective expression level of ovarian cancer, MnSOD but may inhibit the development of ovarian cancer.In recent years, the use of the MnSOD chemical stimulants to replace natural MnSOD becomes a research hotspot. Due to the large molecular weight of the natural MnSOD, short vivo half-life, easy access to the cell membrane and poor stability reasons, its medicinal value is greatly limited. Biochemistry sector researchers, with the help of synthesis and characterization of chemical, synthesize small molecules, of copper, manganese, iron and other metal ions, which have related structures complex to simulate the SOD in order to overcome the disadvantages of natural SOD, so that the analog compounds of small molecule could be applied clinically. Lanzhou University uses flexible aliphatic amines and preferably water-soluble biocompatible to synthesize water-soluble and fat-soluble preferably MnSOD complexes of o-vanillin, which has a higher activity that has been confirmed in study. The compound has a water grease and chief dissolved, high activity and good stability, with small molecular weight, easy transmembrane, low-cost, high purity, high yield and other characteristics, which can be seen to overcome the limitations of natural MnSOD, in order to apply the possibility of the clinical potential.MnSODm Inhibits the tumor, under the help of non-cytotoxic effect, and has no toxic side effects of chemotherapy drugs with high efficiency and low toxicity characteristics, making itself potential clinical application of anticancer drugs. Any drug used in clinical premise should be sure that its mechanism of action must be clear. The research about anti-tumor effect of MnSODm at home and abroad is rare, and research of its anti-tumor mechanism is still unmanned. The foreign reference figures that manganese superoxide dismutase mimetic compounds (MnSODm) combined with gentamicin treatment, can reduce ototoxicity, and tumor treatment of MnSODm, only could be found at home in An Xia、n Fan Pro Lan which study inhibition of MnSODm on the proliferation of leukemia K562cells and induction of cell apoptosis.The subject chooses MnSODm, a new research focus and research gaps of anticancer drugs. Considering human epithelial ovarian carcinoma cell line SKOV3and its xenograft tumor in transplanted tumor tissue as the research object, the experiment uses HE staining, electron microscopy drugs for the observation of pathological changes of the tumor tissue after treatment, with the help of the calculation of tumor weight inhibitory rate and measurement of tumor size, as well as the immunohistochemical assay tumor tissue cancer gene. The experiment observes expression changes of cell proliferation and activity of CCK-8assay, PI single stain, inverted microscope apoptosis, flow cytometry apoptosis cycle, PCR, NF-KB, BAX and BCL-2gene, the combination of in vitro and in vivo experiments, the experiment studies growth inhibition of MnSODm on SKOV3cell lines and xenograft tumor in vitro and in vivo, andexplores the molecular mechanism to provide new ideas and new methods for the treatment of ovarian cancer.Chapter1Inhibition on proliferation OF MnSODm on human epithelial ovarian cancer SKOV3cell and induction of apoptosisObjective:The observation of growth inhibition.of MnSODm on human epithelial ovarian cancer SKOV3cellMethod:1. By means of CCK-8, take the logarithmic growth phase SKOV3cells with different concentrations of MnSODm (Oug/ml, lOug/ml,50ug/ml) to process cell proliferation and activity changes in the control group whose cisplatin concentration is10ug/ml in Oh,24h,48h,72h,.before and after the treatment of MnSODm,2.Take the logarithmic phase of SKOV3cells with different concentrations MnSODm (Oug/ml,10ug/ml,50ug/ml) to process for72h, and observe the morphological changes inverted the microscope MnSODm treated cells.3. Take the logarithmic phase of SKOV3cells, for72h with different concentrations MnSODm (Oug/ml,10ug/ml,50ug/ml) to observe the morphological changes of cell apoptosis PI single staining before and after the process of MnSODm.4.Take logarithmic growth phase SKOV3cells with different concentrations of MnSODm (Oug/ml,10ug/ml,50ug/ml) to process for72h, and use transmission electron microscope to observe the ultrastructural changes SKOV3cells before and after MnSODm treatment.5.Take SKOV3cells in logarithmic growth phase, with of different concentrations MnSODm (0ug/ml,10ug/ml,50ug/ml) to process for72h, flow cytometry the apoptosis rate changes before and after MnSODm treatment.6.Statistical analysis:use SPSS17.0statistical analysis software to analyze measurement data which were expressed as mean±standard deviation, and use single-factor analysis of variance (One-way ANOVA) to compare the groups with the help of multiple comparison LSD method. P<0.05was considered statistically significant.Results:1.Compared with the control group, after MnSODm treatment, SKOV3cell proliferation activity was significantly inhibited in a time-dependent and concentration-dependent. Compared with cisplatin the MnSODm concentration10ug/ml for24,48,72h, the inhibition of the cells were less than cisplatin, and the MnSODm concentration50ug/ml24h cell inhibition is less than smooth platinum, but at48and72hours, cell inhibition is stronger than cisplatin.2.After Treatment of MnSODm, SKOV3cell survival cells were significantly reduced under inverted microscope.3.After Treatment of MnSODm, PI staining showed that the SKOV3apoptotic cells increased.4.After Treatment of MnSODm, transmission electron microscopy under SKOV3cells appear obvious ultrastructural changes of apoptotic cells.5.After Treatment of MnSODm, flow cytometry displays that the SKOV3cell apoptosis was significantly higher.Conclusion:MnSODm can inhibit the proliferation of SKOV3cell in human epithelial ovarian cance.Its effect on inhibition of tumor cell proliferation is dependent on the time and concentration and also MnSODm can induce apoptosis of SKOV3cells. Chapter2Study on the mechanism of inhibition of MnSODm on proliferation of epithelial ovarian cancer SKOV3cell and induction of apoptosisObjective:Explore the mechanism of inhibition of MnSODm on proliferation of epithelial ovarian cancer SKOV3cell and induction of apoptosis.Method:l.Take SKOV3cells in logarithmic growth phase, with of different concentrations MnSODm (0ug/ml,10ug/ml,50ug/ml) to process for72h, using flow cytometry to detect changes in the SKOV3cell cycle distribution before and after MnSODm treatment.2.Take the logarithmic growth phase SKOV3cells with different concentrations of MnSODm (Oug/ml, lOug/ml,50ug/ml) to process for72h, using fluorescent chemiluminescence to detect SKOV3cells of apoptosis-related factor Caspase-3/7activity before and after MnSODm treatment.3.Take the logarithmic growth phase SKOV3cells with different concentrations of MnSODm (Oug/ml,10ug/ml,50ug/ml) to process for72h, using RT-PCR assay to detect the expression changes of nuclear factor-KB/p65、apoptosis-related factors bcl-2,and bax mRNA in SKOV3cells in cells before and after MnSODm treatment.4.Take the logarithmic growth phase SKOV3cells with different concentrations of MnSODm (Oug/ml,10ug/ml,50ug/ml) to process for72h, using Western blotting MnSODm to detect the change of bax protein expression.of SKOV3cells of nuclear factor-κB/p65, cells wither apoptosis associated factor bcl-2,.5.Statistical analysis:use SPSS17.0statistical software to analyze data expressed as mean±standard deviation. Measurement data are compared, using independent samples t test, and P<0.05was considered statistically significant.Results: 1. Compared with the control group, after MnSODm treatment, the SKOV3cell cycle distribution changed significantly, the performance increase in cells in G0/G1phase, and S phase cells decreased cell cycle arrest in G0/G1phase.2.After MnSODm treatment, SKOV3cell apoptosis-related factors Caspase-3/7activity did not change significantly.3After MnSODm treatment, SKOV3cells nuclear factor-κB/p56, apoptosis-related factor bcl-2mRNA expression was significantly lowered, and bax mRNA expression was significantly increased.4.After MnSODm treatment, SKOV3cells nuclear factor-κB/p56, apoptosis-related factor bcl-2protein expression was significantly reduced, while bax protein expression was significantly increased.Conclusion:MnSODm, through affecting cell cycle distribution, and more importantly, through nuclear factor-κB/p56way, regulates apoptosis-related factors bcl-2and bax in order to achieve inhibition on proliferation of human ovarian cancer SKOV3cell and induction of apoptosis. Its inhibition on proliferation of ovarian cancer cell has nothing to do with factor of Caspase-3/7.Chapter3MnSODm’s inhibition on growth of transplanted allogeneic Tumor cells in SKOV3nude ratObjective:Set up the model of transplanted tumor cells in SKOV3nude rat, to observe inhibition of positive drugs, cisplatin and different doses of MnSODm on growth of transplanted Tumor cells.Method:1. Cell cultivation:SKOV3cells were cultivated in incubation box containing10%fetal calf serum, the RPMI1640culture radical penicillin and streptomycin, at 37℃,5%CO2gas mixture as well as95%humidification. Medium was changed every2-3days.2. Plantation of tumor:Take cells with the logarithmic growth phase, trypsinize dubbed2.5X106cells/ml, and take the cell suspension0.2ml per mice from tumor-bearing rat subcutaneously. After stable growth of the tumor, the tumor-bearing rats with strong tumor growth and ulceration were sacrificed and dislocate cervical, the skin and the tumors.3. Allograft:Tumor tissue was cut into small pieces (about1.5mm3trocar, subcutaneously) and then was inoculated in into nude rats of SPF level BALB/c-nu (4-6weeks old, female, weight20-22g). Use a ruler to measure the diameter of the transplanted tumor free. Set tumor subcutaneous nodules greater than0.5mm as the standard.4. Grouping:After the completion of xenograft model of the nude rats, randomized experimental group was divided into saline Control group, the chemotherapy drug positive control group (Cisplation), high dose group (MnSODm high), medium dose group (MnSODm medium) and low dose group (MnSODm low). And all groups were given saline10mg/kg, cisplatin10mg/kg, MnSODm high dose of20mg/kg, medium doses of10mg/kg and low dose of5mg/kg. Drug treatment lasted for seven days.5. With7days’dosing, drug treatment withdrew the next day and the rats were weighed. Nude rats were killed and the author collected orbital blood with anticoagulant, took300ul to check the number of blood cells and platelet in the blood analyzer leukocyte, then took0.5ml blood stand for30min, centrifuged to obtain serum, and detected changes in serum alanine aminotransferase (ALT), blood urea nitrogen (BUN), and serum creatinine (Ccr) in each group.6. Nude mice were sacrificed, and the author removed the tumor, measured tumor volume and calculated tumor inhibition rate (greater than30%inhibition rate to be valid) with the formula:T/C=(TRTC/CRTV)×100%detection, in which TRTV stands for the treatment group, RTV, CRTV for the negative control group RTV, RTV=VT/V0.VT is the tumor volume after administration of7days. V0is the measurement of tumor volume when administered and results of less than40%T/C is valid.7. Nude rats were sacrificed, and the author took the vehicle control group (saline), the chemotherapy drug positive control group and the experimental drug with MnSODm dose group fixed with10%formalin, dehydrated, embedded in paraffin and HE staining. After all these operations, the above tumors were observed under light microscope and organizations were stained immunochemically.(see chapter Ⅱ for details).8. The author used SPSS17.0statistical software to process measurement data which are presented as mean±standard deviation expressed, normality and homogeneity of variance test, and the mean between the two groups used independent samples t-test, and the two groups before and after treatment compared with the help of paired t test, One-way ANOVA is used in comparison among groups while LSD is used in comparison between different groups,with a=0.05level of inspection. The difference was statistically significant when P<0.05, while the difference was not statistically significant when P>0.05.Results:1. General observation of experimental animal:7days after tumor transplantation, growth of xenograft tumor in the general situation is better, with no sample loss. After observation of each intervention group of nude rats with transplanted tumor, each treatment group were able to significantly improve the nude rats’ spirit, activity, response, tumor growth.2Change of volume of different treatment groups with the SKOV3xenograft tumor:with comparison of tumor volume the SKOV3xenograft in nude rats found in each group before and after treatment, the tumor volume difference was significant (P <0.05).7days after treatment at different drug concentrations, the author measured of tumor length diameter, and calculated the volume change of the volume and the group. The results suggested that there are statistically significant (P<0.05). T/C ratio results indicated that cisplatin group T/C ratio was36%, MnSODm high dose group was33%, middle dose group was41%, and the low dose group was45%. T/C less than40%of the treatment is effective, and the cisplatin MnSODm high-dose group less than40%is effective. Naked eye stripped transplanted tumors tumor found in the different concentrations MnSODm treatment group and negative control group, significantly reducing the volume and the cisplatin group which differed obviously from volume of MnSODm high dose group.3. Difference in weight and volume of tumor of tumor xenografts and nude body:nude rats were killed after stripping tumor and weighed tumor, and calculated the rate of tumor suppressor. Between each treatment group and the control group, tumor weight was significantly reduced, except in low dose group MnSODm. The rest of the treatment group and the control group (P<0.05) have significant difference:66.30%tumor inhibition rate for MnSODm high-dose group,54.02%tumor inhibition rate for middle dose group, and32.98%tumor inhibition rate for the low-dose group. Weighing nude rats before execution, the author found that under drug intervention the weight of unde rats did not change significantly and each treatment group, compared with the control group, was statistically significant (P>0.05).4Observation of tissue morphology:as for control group, the performance had characteristics of poorly differentiated adenocarcinoma. The tumors were solid group pieces like intensive cancer cells, cellular atypia, and a large deeply stained chromatin lumps with obvious nucleoli, abundant cytoplasm, showing more mitotic little interstitial. The performance of HE staining in MnSODm high dose group was: sparsely arranged cells, visible necrotic area of large tracts of red dye structure, deeply stained tumor nuclei, pyknosis. the medium dose group of MnSODm had a larger necrotic area while low-dose group of MnSODm changed significantly, with the physiological stained piece similar to the control group.5. Effect on Blood cells and function of Liver and kidney:as statistical results suggest with the help of LSD, the difference between treatment group with different dose of MnSODm and saline group (P>0.05) was not statistically significant; the comparison between the treatment group with cisplatin and the saline group (P< 0.05), has significant differences.The comparison between the treatment group with cisplatin and high dose group of MnSODm as well as medium dose group of MnSODm has no significant differences. The comparison among high dose group of MnSODm, medium dose group of MnSODm and low dose group of MnSODm has no statistical difference.Conclusion:1. Successfully established animal model of epithelial ovarian cancer SKOV3nude rats xenograft and laid the foundation for further research MnSODm.2.Experiments confirmed growth inhibition of MnSODm on epithelial ovarian cancer SKOV3nude mice xenograft, and has the advantages to lower the impact on kidney functions.Chapter4The study of the mechanism of the inhibitory effects of MnSODm on human epithelial ovarian cancer SKOV3cells in nude rats xenograftObjective:Using immunohistochemistry, the author detect effects of MnSODm on human epithelial ovarian cancer SKOV3cells in nude rats with transplanted tumor of bcl-2and bax protein, and explored the mechanism of growth inhibition of MnSODm on human epithelial ovarian carcinoma cell line SKOV3xenograft tumor.Method:1. Set up the transplantation model of nude rats;2. Randomized grouping as Chapter One, excluded the positive control group of chemotherapy drugs (Cisplation Group), and other treatment groups were measured like the first chapter;3. Terminated to observe next day after withdrawal, and the mice were sacrificed, and the tumor specimens by immunohistochemistry (SABC) were sectioned and stained,with bcl-2and bax immunohistochemistry reagents;4. After completion of the stained piece, with high-definition digital microscope radiography, the author collected pictures and analyzed system for processing data, exported data for statistical analysis, and detected bcl-2and bax protein in human epithelial ovarian cancer SKOV3cells in nude rats with transplanted tumor tissue and strength of expression in each dose group after the intervention MnSODm.5. Statistical analysis:with SPSS17.0statistical software, the measurement data are presented as mean±standard deviation expressed. Single-factor analysis of variance (One-way ANOVA) groups were compared using multiple comparison LSD method, P<0.05, and results had statistically significant difference.Results:1.Expression of nude rats’tumor cells bcl-2protein after drug treatment: immunohistochemistry results of control group suggested that the expression of Bcl-2protein in human epithelial ovarian cancer SKOV3cells in nude rats with transplanted tumor tissue were located in the cytoplasm nuclear membrane at more positive control group cytoplasmic or nuclear membrane in coloring the number, showing deeply stained brown,and indicating higher levels of bcl-2protein expression in human epithelial ovarian cancer SKOV3cells in nude rats with transplanted tumor tissue; in the different gradient administration group of MnSODm, coloring cytoplasm gradually reduced faded pigmentation, and the bcl-2protein expression gradient was non-linear correlation. Only a handful of coloring was seen in MnSODm high dose group, and was mainly located in the cytoplasm.The nuclear membrane is rare, indicating the low expression of bcl-2protein MnSODm high dose intervention.2. Expression of bax protein in nude rats’tumor cells after drug treatment:the expression of positive control group bax protein could be seen, which is located in the cytoplasm. The weak reflected in its expression in human epithelial ovarian cancer SKOV3cells in nude rats with transplanted tumor tissue with rendered brown light staining in the cytoplasm. The expression of bax protein is rare, in which almost no expression could be found with only a handful of coloring; in MnSODm different gradient administration group, cytoplasmic and nuclear membrane coloring gradually increased coloring of the deepened cytoplasm and Bax protein expression was gradient upward trend. MnSODm high dose group was stained brownish yellow, and presented with high expression of bcl-2protein in MnSODm high dose intervention.3. Comparison between the intervention group and Control group MnSODm different concentrations (P<0.05):the expression of down-regulated bcl-2in MnSODm different concentrations of the intervention group could be visible, expression of raised Bax protein could also be found;4.comparison between MnSODm Medium Group (10mg/kg) MnSODm Low Group (5mg/kg) and MnSODm High Group (20mg/kg)(P<0.05):results suggested that as MnSODm dose increased, expression of Bax protein bcl-2decreased. The expression of down-regulated bcl-2in MnSODm different concentrations of the intervention group could be visible, expression of raised Bax protein could also be found.Conclusion:1. bcl-2and bax were involved in the process of growth inhibition on the epithelial ovarian cancer SKOV3cell in tumor tissues;2. Confirmed that the MnSODm high dose group, by increasing bax protein and decreasing bcl-2protein, inhibited the growth of human epithelial ovarian cancer cells in SKOV3nude mouse xenograft.Summary1. Successfully establishing animal model of epithelial ovarian cancer SKOV3nude mice xenograft lays the foundation for further research MnSODm. After the second passage of the lines of the model, the tumor grows in vivo. The stable growth rate of tumor and the short experimental period make it easy to implement interventions. The sample has the homogeneity and reproducibility and it can truly reflect the evolution process of the tumor in vivo.2.Observing the weight of the tumor-bearing mice and general state of change, and detecting the changes in the white blood cells, platelets, transaminases, creatinine and urea nitrogen in mice after drug treatment can truly reflect the low toxicity characteristics of MnSODm in vivo.3.Firstly applied the MnSODm in vivo test providesa possible clinical application of MnSODm for a reliable basis of pre-clinical laboratory studies.4.MnSODm can inhibit the proliferation of SKOV3cell, which is dependent on time and concentration, and MnSODm can induce apoptosis in SKOV3cells.5.Observing from the vivo tests, the highest dose of MnSODm has the highest rate of tumor suppressor and the highest rate of apoptosis of tumor cells in72h. The in vitro and in vivo experiments confirmed that the role of MnSODm on SKOV3was time-dependent and concentration-dependent but no corresponding increase in toxicity.6.MnSODm acts on vivo test. Taking an intraperitineal injection that the local skin tissue does not appear ulceration, red, swelling, heat, pain, and other phenomena, diarrhea, bloating and other gastrointestinal symptoms did not appear. All of this show the compound in vivo metabolic cause toxicity directly organized the irritation is also very small, it is worthy of further research and development.7.MnSODm, through affecting cell cycle distribution, and, more importantly, through regulating nuclear factor-κB/p56way of apoptosis-related factors bcl-2and bax, can achieve inhibition on proliferation of human ovarian cancer SKOV3cell, induce apoptosis, and inhibit ovarian cancer cell proliferation factor of Caspase-3/7.更多还原