【作者】 李健；

【导师】 李青旺；

【作者基本信息】 燕山大学 ， 应用化学， 2009， 博士

【摘要】 宫颈癌是妇科易发恶性肿瘤,在发展中国家居妇科恶性肿瘤的首位。近20年来,宫颈癌发病逐渐趋于年轻化,且宫颈腺癌比例增加。因此,寻找科学、有效的途径来增强放、化疗疗效,改善预后成为宫颈癌治疗领域的一个研究热点。天然抗肿瘤药物以其毒副作用小和不产生耐药性等优点引起了高度关注,特别是在发展中国家得到了广泛的研究和利用。龙葵作为我国传统中药,具有抑菌、抗病毒和抗肿瘤等作用。本研究采用水提醇沉法从龙葵全草中提取龙葵多糖,并进一步分离、纯化获得质量稳定、分子量均一的龙葵多糖亚级分1a(SNL-P1a)。以小鼠荷U14宫颈癌模型为研究对象,环磷酰胺为阳性对照组,采用流式细胞术、免疫组织化学技术、酶联免疫吸附检测(ELISA)、透射电子显微镜技术(TEM)以及反转录-聚合酶链式反应(RT-PCR)等方法,对SNL-P1a可能的抗肿瘤作用及其机理,从机体免疫调节、机体抗氧化防御体系及肿瘤组织细胞周期和细胞凋亡干扰等方面进行了较深入的探讨,获得如下研究结果。荷宫颈癌U14小鼠模型经灌胃给予低、高剂量的SNL-P1a 13 d后,其实体瘤生长受到明显抑制(P<0.05),肿瘤重量抑制率分别为37.65和52.52%,体积抑制率分别为36.75和50.29%;而且,低、高剂量的SNL-P1a均可以显著抑制腹水性肿瘤细胞的生长(P<0.01),抑制率分别为40.00和53.68%;荷瘤小鼠的存活时间显著延长(P<0.05,P<0.01),生命延长率分别为43.95和76.43%。同时,SNL-P1a对小鼠宫颈癌细胞的肺转移也表现出显著的抑制作用,低、高剂量抑制率分别为38.92和53.89% ( P<0.05,P<0.01)。SNL-P1a低、高剂量处理可使荷瘤小鼠的胸腺重量显著增加(P<0.05),胸腺指数增长率分别为220.11和179.89%。同时,由于肿瘤侵袭,对荷瘤小鼠所造成的胸腺萎缩、结构破坏和细胞凋亡在SNL-P1a低、高剂量处理后都有显著改善作用,这种保护作用是通过上调Bcl-2/Bax比值、进而抑制细胞凋亡而调控;但SNL-P1a对荷瘤小鼠脾脏组织保护作用不显著(P>0.05)。SNL-P1a处理可使荷瘤小鼠外周血CD4+ T淋巴细胞亚群比例上升,CD8+ T淋巴细胞亚群比例显著降低(P<0.01),CD4+/CD8+倒置情况有显著改善。通过对血清相关细胞因子进行测定,荷瘤小鼠血清IFN-γ和IL-2水平均显著升高,IL-4水平显著降低(P<0.01,P<0.05),部分逆转Th1/Th2的Th2漂移;同时,SNL-P1a处理可降低血清TNF-α水平,使肿瘤细胞失去生长和转移的必要基质条件。SNL-P1a处理在体内可显著提高荷瘤小鼠血清总超氧化物歧化酶(T-SOD)和总抗氧化能力(T-AOC)水平(P<0.05,P<0.01),显著降低血清丙二醛(MDA)水平(P<0.05)。对其处理后荷瘤小鼠肝脏、肾脏进行分析,组织中T-SOD、T-AOC和谷胱甘肽还原酶(GR)水平都有所提高,但对肾脏GR水平无显著影响(P>0.05)。对荷瘤小鼠胸腺、脾脏组织进行测定,胸腺T-AOC、T-SOD和GR水平均显著提高(P<0.05,P<0.01),但对脾脏组织T-SOD水平无显著作用(P>0.05)。对小鼠肿瘤组织和血清进行分析,T-AOC、T-SOD、GR和乳酸脱氢酶(LDH)水平均显著降低(P<0.01),但对碱性磷酸酶(AKP)活性无显著影响(P>0.05);通过对相关自由基测定,SNL-P1a在体外对·OH自由基、O2·-自由基和DPPH自由基均有显著清除作用。SNL-P1a处理后,肿瘤组织细胞核增殖抗原(PCNA)、c-myc、突变型p53和cyclinD1蛋白的表达显著下调,p21蛋白的表达显著上调,从而使肿瘤组织出现G2/M期阻滞;同时,小鼠肿瘤组织细胞的Fas、FasL、Bax和Caspase-3蛋白表达显著上调,Bcl-2蛋白表达显著下调,进一步可使更多的肿瘤组织细胞通过死亡受体和线粒体两条途径同时发生凋亡。本论文对SNL-P1a抗小鼠宫颈癌和免疫调节作用进行了研究,对其作用机理和调控途径进行了较为深入的探讨,为SNL-P1a作为天然抗宫颈癌药物的进一步开发提供了理论依据。更多还原

【Abstract】 Uterine cervical carcinoma is one of the most common gynecologic malignant tumors in the world, and its occurrence ratio has taken the first place in the developing countries. Because it has the trend of rejuvenation in cervical cancer emergence, and the rate of cervical adenocarcinoma is increasing, find a feasible method to enhance the efficacy of radiotherapy and chemotherapy becomes increasingly attractive. Recently, natural-derived antitumor agents have attracted more attention for less side effects and no-drug-resistance.Solanum nigrum Linne is a kind of traditional Chinese medicine, and it has the effects of antibacteria, antivirus and antitumor. Polysaccharides of Solanum nigrum L. (SNL-P) was got by water-extraction and alcohol precipitation, and a sub-fraction 1a (SNL-P1a) was got through further purification which was homogeneous in molecular weight and stable in quality. After the cervical cancer-bearing mice models were established, the effects of SNL-P1a on body’s immune system, antioxidant defense system, cell cycle and apoptosis of tumor tissue cells were studied by methods of flow cytometry, immunohistochemistry, enzyme linked immunosorbent assay (ELISA), transmission electron microscopy (TEM) and reverse transcription-polymerase chain reaction (RT-PCR).After oral administrated tumor-bearing models with SNL-P1a for 13 days, the growth of solid tumor was inhibited significantly (P<0.05). The weight inhibition rate was 37.65 and 52.52%, and the volume inhibition rate was 36.75 and 50.19% for low and high dose treatment, respectively. SNL-P1a inhibited the growth of ascites tumor (P<0.01), and the inhibition rate was 40.00 and 53.68% for low and high dose treatment. It also prolonged the survival time of tumor–bearing mice (P<0.05, P<0.01), and the life span increasing rate was 43.95 and 76.43% respectively. Furthermore, it inhibited the lung metastasis of uterine cervical cancer (P<0.05, P<0.01), and the inhibition rate was 38.92 and 53.89%, respectively.SNL-P1a treatment enhanced the thymus weight of tumor-bearing mice (P<0.05), and the thymus index increasing rate was 220.11 and 179.89% for low and high dose respectively. SNL-P1a could ameliorate the atrophy, structure destruction and cell apoptosis of thymus tissue caused by tumor onslaught by elevating Bcl-2/Bax ratio and further repressing cell apoptosis. But the protection effect of SNL-P1a on spleen tissue was not significant.SNL-P1a treatment could increase the proportion of peripheral blood CD4+ T lymphocyte subpopulation and decrease the proportion of CD8+ T lymphocyte subpopulation significantly (P<0.01), so ameliorate the reversion status of CD4+/CD8+ of tumor-bearng mice. It could also up-regulate the levels of serum IFN-γand IL-2, but down-regulate the level of serum IL-4 (P<0.01, P<0.05), then reverse the shift of Th1/Th2 toward Th2 partially. Meanwhile, SNL-P1a treatment could also decrease the level of serum TNF-αsignificantly, which deprived the necessary conditions of the growth and metastasis of tumor cells.SNL-P1a treatment could increase the levels of serum T-SOD and T-AOC (P<0.05, P<0.01), but decrease the MDA level in tumor-bearing mice significantly (P<0.05). It also improved the T-SOD and T-AOC levels in liver and kidney of tumor-bearing mice, and improved the GR level in liver but did not influence the GR level in kidney (P>0.05). Furthermore, SNL-P1a could increase the T-AOC level in thymus and spleen tissue (P<0.05, P<0.01), increase T-SOD level in thymus (P<0.05), but not in spleen (P>0.05). It could also enhance GR level in thymus and spleen tissues. To tumor tissues, SNL-P1a treatment decreased T-AOC, T-SOD and GR levels significantly (P<0.01). Besides that, the serum LDH level of tumor-bearing mice was suppressed by the treatment of SNL-P1a markedly (P<0.01), but the serum AKP level remained unchanged (P>0.05). Experiments in vitro further showed that SNL-P1a had strong effects on scavenging·OH radical, O2·- radical and DPPH radical.The tumor cell cycle was arrested in G2/M phase by SNL-P1a treatment, which might cause by the down-regulation of proteins of proliferating cell nuclear antigen (PCNA), c-myc, mutant p53 and cyclinD1 and the up-regulation of protein p21. Meanwhile, SNL-P1a treatment also caused the up-regulation of proteins Fas, FasL, Bax and Caspase-3 and down-regulation of Bcl-2 protein in tumor tissue cells, which might lead to the apoptosis of tumor tissue cells.The studies of anti-cervical cancer and immunomodulating effects of SNL-P1a on tumor-bearing mice provided theoretical supports for further development of SNL-P1a as an anti-cervical cancer agent.更多还原