【作者】 邓鹏；

【导师】 唐安洲；

【作者基本信息】 广西医科大学 ， 耳鼻咽喉科学， 2010， 博士

【摘要】 目的目前治疗鼻咽癌的主要方法放射治疗和化学治疗虽取得了较高的有效率,但均存在较强的毒副反应,而且远处转移率较高,远期效果仍不理想,5年生存率仍徘徊于70％～80％左右。因此有必要寻找高效而低毒的抗鼻咽癌的药物。铁皮石斛属多年生附生草本植物的全草,是国家重点保护药物,含丰富的多糖、生物碱、多种人体必需的微量元素和氨基酸等。铁皮石斛提取物已经证实能显著抑制荷瘤小鼠骨肉瘤和肝癌细胞生长、抗肿瘤多药耐药性等,但在抗鼻咽癌方面国内外仍未见有报道。本课题从铁皮石斛药用有效部位茎部分离提取有效成分,拟系统研究铁皮石斛对人鼻咽癌细胞CNE1、CNE2的体内外抑瘤作用并探讨其作用机制,为从中草药成分中寻找确切有效的抗鼻咽癌药物,提高鼻咽癌治疗水平提供实验依据。研究内容：第一部分铁皮石斛及其含药血清的体外抗鼻咽癌作用方法分别采用MTT、光学显微镜、流式细胞仪与Western blot检测新鲜铁皮石斛提取物及含石斛大鼠血清抑制CNE1和CNE2细胞增殖与诱导凋亡作用及Bcl-xL, mcl-1、caspase-3、caspase-8和caspase-9蛋白的表达,了解石斛及其含药血清对鼻咽癌细胞的作用及机理。结果经MTT测试不同数目的CNE1及CNE2生长曲线,发现96孔板每孔加6000个CNE1及CNE2细胞时效果最好。经MTT法检测显示铁皮石斛提取物及含铁皮石斛大鼠血清对CNE1及CNE2均有明显抑制作用,以铁皮石斛提取物抑制率较高。经流式细胞仪检测显示,与对照组相比石斛提取物及其含药血清对CNE1和CNE2细胞株均有明显促进细胞凋亡作用(P<0.01),而且有明显的时间依赖性及浓度依赖性。用流式细胞仪分析显示32mg/L,64mg/L,128mg/L,256mg/L的石斛的周期阻滞均发生在G0／G1期,表现为该期细胞比例明显高于对照组(P<0.01),而G2／M期明显低于对照组(P<0.01)。四个浓度梯度的石斛血清分别对两个鼻咽癌细胞株的周期改变的影响。对于细胞系CNE1,除10％浓度组外,其他组的周期阻滞均发生在G0／G1期。对于细胞系CNE2,到除15％组外,其他组的周期阻滞均发生在G0／G1期,表现为该期细胞比例明显高于对照组(P<0.01),而G2／M期明显低于对照组(P<0.01)。Western blot结果显示：Bcl-xL和Mcl-1表达均下降,caspase-8、caspase-9有明显的表达；而caspase-3也有表达。提示石斛能抑制Bcl-xL和Mcl-1的表达,并可能与线粒体通路的细胞凋亡及死亡通路的细胞凋亡有关。第二部分铁皮石斛对鼻咽癌裸鼠移植瘤模型的作用方法将CNE1及CNE2细胞分别接种于裸鼠,待肿瘤长至一定体积后设对照组及药物组并用药物干预。测量裸鼠的体重、瘤体体积等,15天后取出瘤体并测量瘤重。观察肿瘤的显微结构及超微结构变化,用原位组织化学法(TUNEL法)检测肿瘤组织细胞凋亡、免疫组织化学法检测肿瘤组织内Bcl-xL, mcl-1和caspase-3的表达情况。结果随着浓度的增加可以明显的发现肿瘤的体积增长幅度、瘤重均是越来越轻。显微观察可见用药组的肿瘤细胞减少,纤维组织增多。超微结构下可见凋亡小体,免疫组化提示Bcl-xL, mcl-1阳性率减低,但可见caspase-3阳性率明显增高。Tunnel法检测提示用药组的凋亡细胞明显增多。结论铁皮石斛具有抑制人鼻咽癌CNE1和CNE2细胞增殖和诱导凋亡的作用,能抑制鼻咽癌裸鼠CNE1和CNE2移植瘤的成长。其机制可能与Bcl-xL, Mcl-1蛋白下调、促进Caspase 3的活化有关等有关,并可能与死亡受体通路的细胞凋亡和线粒体通路的细胞凋亡均有关。更多还原

【Abstract】 Purpose Now the main metheds curing with nasopharyngeal carcinoma include radiotherapy and chemotherapy,both of those have severe side effect, and the long distance diversion survival rate for five years is about 70%～80%,so seanching more metheds for NPC therapy is a hot topic. Officinal Dendrobium Stem belongs to the entire grass with perennial attachment herb,witch is full of polysaccharide、alkaloid,as well as necessary microelement and amino acids etc. the Extraction from officinal Dendrobium Stem can suppress liver cancer and Osteosarcoma in mice,and also can prevent the resistance of e multi-medicines in tumor. But until now, there are any report with the role of anti-nasopharyngeal carcinoma of officinal Dendrobium Stem.The purpose of our study is to research the effect of officinal Dendrobium Stem on CNE1 and CNE2 cells,tring to find its molecular mechanismsPart oneStudies on the Inhibiting nasopharyngealcarcinoma Effect of Officinal Dendrobium Stem and the serum including Officinal Dendrobium Stem in vitroMethods The cells of CNE1 and CNE2 are interfered by Officinal Dendrobium Stem and the serum including Officinal Dendrobium Stem. Then the growth inhibition and apoptosis of CNEl and CNE2 cells and the protein levels of Bcl-xL, mcl-1,caspase-3,caspase-8 and caspase-9 were examined by MTT assay, lightmicroscopy, flow cytometry and Western blot, respectively.Results MTT assay showed that 6000 CNE1 or CNE2 cells in one hole in 96 orifices were the best growing. It suggest that both of the extractive of fresh Officinal Dendrobium Stem and the serum with Officinal Dendrobium Stem, significantly inhibited proliferation of CNE1 and CNE2 cells Officinal Dendrobium Stem has the higher inhibition ratio.flow cytometry analysis showed:compared to the control group, both of the extractive of fresh Officinal Dendrobium Stem and the serum with Officinal Dendrobium Stem played an Obvious role in promoting apoptosis to CNE1 and CNE2 cells(P<0.01). The result was Closely Related with time and density.The detect of flow cytometry revealed:the cycle blockage of the four concentration gradient of Officinal Dendrobium Stem took place at the stage of G0/G1.The cell proportion of this period was higher than the control group(p<0.01),and obviously lower than control group at the stage of G2/M(p<0.01).The cyclomorphosis effect of serum with Officinal Dendrobium Stem of the four concentration gradient on the two NPC cell strain,.For the CNE1, all cycle blockage happened at the stage of G0/G1 excepting the concentration of 10%.For the CNE2,all cycle blockage happened at the stage of G0/G1 excepting the concentration of 15%. The cell proportion of this period was higher than the control group(p<0.01), and obviously lower than control group at the stage of G2/M(p<0.01).Western blot showed:both of the Bcl-xl and Mcl-1 expression were decreased. Caspase-8 and caspase-9 expression were expressed fully, specially for the group of 256mg/mL. The caspase-3 expression was also expressed. Part twoThe effect of Officinal Dendrobium Stem in Null Mice Grafted with Nasopharyngeal CarcinomaMethed The CNE1 and CNE2 cells were grafted in the null mice,when the tumor growed to a certain volume, the null mice were divided into control group and medicine group, were investigated by HE staining.Ultra micro-structured changes of Carcinoma cells were observed by transmission electron microscope(TEM).In TUNEL of histochemistry were used to detect the apoptosis of Carcinoma cells.Immunohistochemical method were used to detect the expression of Bcl-xL, mcl-1 and caspase-3.Results The volume and the the weigh of tumor wrer inhibited by Officinal Dendrobium Stem. The tumor cells were decreased and fibrous tissue was increased in the Medication group. Under ultramicrostructure apoptotic body can be seen. The rate of cell apoptosis for the Officinal Dendrobium Stem groups were higher than that of the control.The expression of Bcl-xL and mcl-1 and were lower and the expression of caspase-3 were higher than that of the control.Conclusion Officinal Dendrobium Stem was an effective Traditional Chinese medicine to cure of nasopharyngealcarcinoma witch Promoting to apoptotic and Suppressing the muLtiplication. It also could inhibite the growth the Nasopharyngeal Carcinoma witch is grafted in the null mice. This might relay with a diminution of the expression of the anti-apoptotic protein Mcl-land Bcl-xL.Also may be related with the way of caspase. This may indicatethat that both of the mitochondrial pathway and the death pathway were clearly involved.更多还原