导　　师： 马文丽

学科专业： 071010

授予学位： 硕士

作　　者： ;

机构地区： 中国人民解放军第一军医大学

摘　　要： 三氧化二砷/(As/_2O/_3/)，俗称砒霜，有剧毒，20世纪90年代初，我国学者孙鸿德等报道了用三氧化二砷治疗急性早幼粒细胞性白血病/(APL/)并获临床缓解。进一步研究发现，As/_2O/_3不仅对APL有效果，对其它类型的白血病以及许多的恶性肿瘤，如胃癌、肺癌、前列腺癌、乳腺癌等都有良好的治疗效果。作用机制主要是通过诱导肿瘤细胞部分分化、抑制肿瘤细胞生长和诱导肿瘤细胞凋亡，近年又发现三氧化二砷可以通过抑制肿瘤新生血管生成而发挥抗肿瘤的效果。 我们在实验中观察到As/_2O/_3作用人红白血病K562细胞后，细胞生长明显变缓，部分细胞出现皱缩、染色质浓聚及胞膜起泡现象，部分细胞胞膜破裂，在其周围有致密的凋亡小体出现，DNA电泳出现典型的凋亡“梯状”带，提示As/_2O/_3能有效抑制K562细胞生长，诱导K562细胞凋亡。 为了进一步阐述As/_2O/_3作用的分子生物学机制，我们首先应用RD-PCR技术，结合聚丙烯酰胺凝胶电泳和银染技术分离和显示了As/_2O/_3作用K562细胞前后的cDNA片段，发现了11个差异表达的基因片段。这些片段切胶回收后测序，结果提示它们与As/_2O/_3的作用密切相关。 进一步我们收集了K562细胞的cDNA片段，取其中的一部分制备了包含162个cDNA片段的K562细胞cDNA表达谱芯片。应用此自制的芯片，与As/_2O/_3作用K562细胞前后的标记cDNA杂交，以研究As/_2O/_3诱导K562细胞凋亡前后基因表达的改变。提取As/_2O/_3作用K562细胞前后的总RNA，用SuperScriptⅡ逆转录酶逆转录成cDNA第一链，并在逆转录的过程中，用Cy3／Cy5荧光染料分别标记对照组／处理组，与自制的K562细胞基因表达谱芯片杂交。发现K562细胞经As/_2O/_3作用后，162个基因片段的表达都有所降低，其中25个基因片段的表达降低较为显著/(Cy5／Cy3＜0.5/ Arsenic is a natural substance that has been used medicinally for over 2,400 years. In 1970s physicians in China began using arsenic trioxide to treat acute promyelocytic leukemia /(APL/) and achieved promising results,including clinical remission of several cases. After that,the studies aimed at the functions and mechanisms of arsenic trioxide carried out extensively. It showed that arsenic trioxide could be used not only in the treatment of APL,but also in other malignant tumors,such as gastric cancer,lung cancer,carcinoma of prostate,ect. The mechanisms of such treatment have been proposed as inhibition of proliferation and angiogenesis,as well as induction of differentiation and apoptosis,as has been tested by various in vivo and in vitro experiments. In our experiments,it has also been demonstrated that after the treatment of arsenic trioxide,the K562 cells has undergone major morphological changes,which included nuclear shrinkage,membrane bleb and scattered apoptotic bodies. DNA gel electrophoresis also discovered that the typical 'DNA ladder' phenomena in the treatment group,while the control group showed the regular genomic banding. All of these indicated that arsenic trioxide was a powerful chemotherapeutic agent and the cells in the treatment group were induced to apoptosis. To further understand the molecular mechanisms of arsenic trioxide treatment in the induced cellular apoptosis,we applied the Restriction Display-PCR /(RD-PCR/) technique combined with polyacrylamide gel electrophoresis and sliver staining techniques to separate the differentially expressed genes. We found 11 genes were differentially expressed in the treatment group,which the genes were sequenced and analyzed,showing that the isolated gene fragments were closely associated with the inhibition of cellular proliferation and the induction of apoptosis. Then,we applied the RD-PCR technique to isolated gene expression sequence tags /(EST/) from treated and controlled K562 cells. 852 ESTs were

关 键 词： 细胞 三氧化二砷 凋亡 芯片 微集阵列 聚丙烯酰胺凝胶电泳 作用机制

领　　域： [生物学]