【作者】 陆文总；

【导师】 刘福柱； 李青旺；

【作者基本信息】 西北农林科技大学 ， 动物学， 2009， 博士

【摘要】 宫颈癌是严重威胁女性健康的的恶性疾病,位居女性恶性肿瘤第二。目前使用化学合成药物治疗肿瘤的同时对机体产生毒害作用。因此,寻找安全有效的天然药物成为生物医药领域的一个研究热点。天然抗癌植物性多糖以其毒副作用小、生物活性强和不产生耐药性等优点引起了人们的高度重视。异叶败酱草作为我国传统的中草药,具有清热燥湿、止血、止带的功效,对治疗子宫糜烂、早期宫颈癌等效果显著。本研究旨在评估异叶败酱草多糖级分1 （PHB-P1）抗宫颈癌的作用效果,探讨其作用机制,为开发抗宫颈癌新药提供科学理论依据。试验一,异叶败酱草多糖的分离纯化和活性鉴定。本研究通过水提醇沉法提取了异叶败酱草粗多糖,并经过离子交换柱分离纯化得到分子量均一的异叶败酱草多糖级分1 （PHB-P1）,该多糖级分含有酮糖、葡萄糖糖醛酸等,提取率为1.57%。采用MTT法初步检测PHB-P1对人宫颈癌HeLa细胞和正常人外周血单核淋巴细胞的毒性作用,研究发现PHB-P1对HeLa细胞具有显著的毒性作用,其IC₅₀为52.26μg/mL,而对正常的人外周血单核淋巴细胞没有明显的毒性作用。试验二,异叶败酱草多糖（PHB-P1）抗HeLa细胞增殖机制的研究。本研究作为体外试验以HeLa细胞为研究对象,PHB-P1（50μg/mL）处理HeLa细胞24 h和48 h,通过各种荧光染色法、原位末端标记技术（TUNEL）、DNA片段化分析、细胞周期分析、反转录-聚合酶链式反应（RT-PCR）等技术,检测HeLa细胞的形态和生化特征的变化,以及细胞中caspase-3、明胶酶、端粒酶活性和相关基因表达的变化。研究结果表明PHB-P1（50μg/mL）能诱导HeLa细胞凋亡;能干扰HeLa细胞的细胞周期分布,使细胞在G0/G1期显著增加,伴随G2/M期细胞明显减少;能显著增强了HeLa细胞caspase-3的活性、显著抑制HeLa细胞分泌明胶酶、抑制HeLa细胞端粒酶的活性。而且,PHB-P1（50μg/mL）能够显著上调HeLa细胞中p53、Bax、p14ARF的mRNA表达量,显著下调Bcl-2的mRNA表达量。试验三,异叶败酱草多糖（PHB-P1）对荷瘤小鼠抗氧化力及免疫调节作用的影响。本研究以U14荷瘤小鼠为研究对象,生理盐水处理为阴性对照、环磷酰胺（CTX）为阳性对照药物,连续处理15 d,采用比色法等方法,从PHB-P1影响小鼠机体抗氧化能力和免疫调节方面分析了PHB-P1抗肿瘤的可能机制。研究发现PHB-P1高、低剂量（80、40 mg/kg b.w.）和CTX均能明显减缓肿瘤体积增大而抑制肿瘤生长,能有效地延长U14荷瘤小鼠的生命延长率,生命延长率分别为73.28%、40.46%和54.20%,对肝肾脏没有明显的毒害作用。PHB-P1高、低剂量能显著提高荷瘤小鼠血清中T-AOC活性（P<0.01, P<0.01）和显著降低MDA活性（P<0.05, P<0.05）,还能明显增强SOD活性（P<0.01, P<0.05）。而且,PHB-P1高剂量对肝组织SOD活性有显著的增强效果;PHB-P1高、低剂量均能明显提高荷瘤小鼠肝组织中T-AOC的水平,MDA水平显著减少。同时,PHB-P1高、低剂量处理均能提高荷瘤小鼠肾组织中T-AOC的水平,分别提高了79.65%和59.75%,但对肾组织中的MDA影响效果不明显,只有PHB-P1高剂量提高肾组织中SOD的水平（P<0.05）。PHB-P1高、低剂量（80、40 mg/kg b.w.）灌胃给药后,使荷瘤小鼠的胸腺指数从阴性组的0.79提高到1.37和1.22,脾脏指数而阴性组的11.71提高到13.58和11.88。CTX处理显著抑制小鼠胸腺指数。PHB-P1高、低剂量处理均可以显著提高荷瘤小鼠IFN-γ活性（P<0.01, P<0.05）,分别提高了41.22%和30.22%;高剂量PHB-P1能显著抑制TNF-α的活性（P<0.05）,低剂量PHB-P1的效果不明显;PHB-P1处理也能部分提高IL-2活性,但是效果不明显。试验四,异叶败酱草多糖（PHB-P1）抑制肿瘤生长作用机制的研究。本研究以U14荷瘤小鼠为研究对象,生理盐水处理为阴性对照、CTX为阳性对照药物,连续处理15 d,采用比色法、免疫组织化学技术、流式细胞术等方法,从PHB-P1影响小鼠肿瘤细胞周期和细胞凋亡方面综合分析了PHB-P1抗肿瘤的可能机制。研究结果表明异叶败酱草多糖PHB-P1高、低剂量（80、40mg/kg b.w.）和CTX处理可以有效地抑制U14实体瘤小鼠肿瘤的生长,肿瘤重量抑制率分别达42.68%、31.30%和54.06%,能诱导U14荷瘤小鼠肿瘤细胞发生凋亡,肿瘤细胞凋亡的百分率分别为20.96%、27.85%和31.03%。PHB-P1高、低剂量处理后小鼠血清乳酸脱氢酶（LDH）水平分别下降了23.94%和18.92% （P<0.01、P<0.01）,而PHB-P1高、低剂量处理均未能明显影响碱性磷酸酶（AKP）的水平。研究发现PHB-P1能干扰U14实体瘤小鼠肿瘤的细胞周期分布,使细胞在G0/G1期显著增加,伴随S期细胞明显减少;低剂量PHB-P1可以提高p19^ARF蛋白的表达量,但是效果不明显,而高剂量PHB-P1能显著增强p19^ARF蛋白的表达水平。而且PHB-P1能显著上调Bax蛋白的表达量,显著下调突变型p53和Bcl-2蛋白的表达量。本研究通过分离纯化得到异叶败酱草多糖PHB-P1,并通过体外培养的HeLa细胞和U14荷瘤小鼠模型试验,研究分析了PHB-P1抗宫颈癌的作用机制和调控途径,为PHB-P1作为天然抗癌药物的进一步研究和临床应用提供理论基础。更多还原

【Abstract】 Cervical cancer is one of the most common gynecologic, which is the second most common cancer in women worldwide and threatens the health of women. At present, chemical anticancer drugs are used to heal the patients efficiently, also result in serious side effects. So, many researchers pay attention to exploitation of natural drugs, which is effective and safe. Many botanical polysaccharides have been investigated with a view to their application for inhibiting tumor growth because they possess strong bioactivity and do not result in drug resistance. Patrinia heterophylla Bunge is being used as a traditional medicine against earlier period cervical cancer, typhoid, leucorrhea, metrorrhagia and metrostaxis. The aim of our study was to assess the effects of polysaccharides fraction 1 of Patrinia heterophylla Bunge （PHB-P1） on cervical cancer and to explain its mechanism, which might provide theoretical supports for developing new anti-cervical cancer.Extraction and activity identification of polysaccharides of Patrinia heterophylla Bunge were studied in the first experiment. Crude polysaccharides of Patrinia heterophylla Bunge was got by water-extraction and alcohol precipitation, and a polysaccharides fraction 1 （PHB-P1） was isolated through further purification, which was uniform in molecular weight and contained ketose, uronic acids and so on. Extraction percentage of PHB-P1 was 1.57%. Cytotoxicity to HeLa was found significantly by treatment of PHB-P1 and the IC50 was 52.26μg/mL, and it failed to inhibit significantly the growth of human peripheral blood mononuclear cells by MTT assay.The inhibitory effects of PHB-P1 on HeLa cells proliferation were performed in the second experiment in vitro. HeLa cells were treated with PHB-P1 （50μg/mL） for 24 h and 48 h. The methods were used in the experiment such as fluorescent stainings, analysis of DNA fragmentation, Tdt-mediated dUTP nick end labeling （TUNEL） assay, cell cycle analysis and reverse transcription-polymerase chain reaction （RT-PCR）, which assessed the change of cell morphous and biochemistry feature, and detected the change of activities of gelatinase and telomerase, and examined the expression of some genes in HeLa cells. Our results showed that PHB-P1 could induce HeLa cells apoptosis and inhibit significantly the activities of gelatinase and telomerase. Furthermore, cell cycle analysis showed the accumulation of tumor cells in the G0/G1 phase and a relative decrease of the G2/M phase. And the caspase-3 acticity was increased significantly. Meanwhile, PHB-P1 showed the up-regulation of p53, p14ARF and Bax; also PHB-P1 significantly inhibited the expression of Bcl-2 in tumor tissues.The effects of PHB-P1 on antioxidant capability, immunomodulation cell cycle distribution and induced-apoptosis of U14 tumor-bearing mice were investigated in the third experiment. In the study, cyclophosphamide was considered as positive control drug and physiologic saline was used for negative control group. The U14 tumor-bearing mice models were established, and all the animals were treated the drugs for 15 d. The colorimetric method was used in the experiment. Our results showed that the growth of solid tumor was inhibited significantly after administrated U14-bearing mice model with PHB-P1 （80, 40 mg/kg b.w.） and CTX for 15 days, and the life span increasing ratio was 73.28%, 40.46% and 54.20%, respectively. The poison effect of PHB-P1 on liver and kidney of the mice was not found. PHB-P1 （80, 40 mg/kg b.w.） treatment could elevate the level of serum T-AOC （P<0.01, P<0.01） and SOD （P<0.01, P<0.05）, but decrease the MDA level in the mice （P<0.05, P<0.05）. It also improved the T-AOC level and deducted the MDA level in liver of the mice. Only higher dose PHB-P1 could enhance significantly the level of SOD in liver of the mice. Meanwhile, the level of kidney tissues T-AOC was increased by 79.65% and 59.75% in the mice treated with PHB-P1 （80, 40 mg/kg b.w.）, but there was no obvious effect on MDA level. The higher dose PHB-P1 could enhance the level of SOD in the kidney tissue （P<0.05）. PHB-P1 （80, 40 mg/kg b.w.） treatment could increase the thymus index to 1.37 and 1.22 from 0.79 compared with the control group, and also the spleen index was increased from 11.71 to 13.58 and 11.88 respectively. However, CTX treatment inhibited significantly the thymus index. Moreover, the level of IFN-γwas increased by 41.22% and 30.22% in the mice treated with PHB-P1 （80, 40 mg/kg b.w.）, and higher dose PHB-P1 could inhibit significantly the activity of TNF-α（P<0.05） but the lower dose. Furthermore, the activity of IL-2 was elevated in some degree but the significant difference was not found.The effect mechanism of PHB-P1 on the tumor growth of U14 tumor-bearing mice was investigated in the fourth experiment. In the study, cyclophosphamide was considered as positive control drug and physiologic saline was used for negative control group. The U14 tumor-bearing mice models were established, and all the animals were treated the drugs for 15 d. The colorimetric method, flow cytometry, and immunohistochemistry and so on were used in the experiment. Our results showed that the solid tumor growth was significantly inhibited by treatment of PHB-P1 （80, 40 mg/kg b.w.） and CTX, and the tumor ratio was 42.68%, 31.10% and 54.06%, respectively. Moreover, PHB-P1 （80, 40 mg/kg b.w.） and CTX treatment could induce tumor cells apoptosis and the ratio was 27.85%, 20.96% and 31.03%, respectively. Furthermore, the level of serum LDH was decreased by 23.94% and 18.92% in the mice treatment of PHB-P1 （80, 40 mg/kg b.w.） but did not affect significantly the AKP activity. It showed the accumulation of tumor cells in the G0/G1 phase （P<0.01） and a relative decrease of the S phase （P<0.01） after PHB-P1 treatment. The solid tumor growth was inhibited by PHB-P1 treatment, which might result from the down-regulation of mutant p53 and Bcl-2 and the up-regulation of Bax and p19^ARF protein.The study was performed with HeLa cells in vitro and with U14-bearing mice models in vivo, we analyzed systematically the mechanism of anticancer and regulation pathway, which would provide theoretical supports for further development of PHB-P1 as a natural anti-cervical cancer drug.更多还原