甲状腺乳头状癌基因谱表达的初步研究

1、甲状腺乳头状癌基因谱表达的初步研究         11-02-13 09:31:00     作者：刘伟松,项丞,田冶    编辑：studa20【摘要】  目的：利用基因芯片筛选与甲状腺乳头状癌发生、发展和与恶性度相关的基因。方法：分别选取高、低分化甲状腺乳头状癌组织标本，抽提、纯化mRNA，同法再纯化同患者的正常甲状腺组织的mRNA；用Cy5荧光染料对甲状腺乳头状癌组织mRNA作探针标记，以Cy3荧光染料对正常黏膜组织的

2、mRNA作探针标记，一同和可检测4 096个基因片段的BiostarH40 s型基因芯片进行杂交得到的双色荧光标记芯片，将之分别扫描入计算机，进行结果分析。结果：高、低分化2种芯片上均有大量基因片段出现了的高/低表达的情况，高分化芯片上异常表达片段数量为低表达351条，高表达417条，低分化芯片上异常表达者为低表达410条，高表达415条。在2种芯片中均有相同表达差异趋势的基因片段中，共计有90条基因片段出现低表达，其中随恶性度增高而降低的基因有19条；有78条片段呈现高表达状态，其中随恶性度增高而表达增加的有25条。表达异常的片段功能较分散；有大量的不明功能的片段发现。结论：甲状腺乳头状癌的

3、发生、发展不能用单一的基因变异解释，而是由多基因共同作用的结果，并且变异具有明显的个体性差异；甲状腺乳头状癌的恶性度与多个基因片段异常表达相关。很多新发现的片段的功能不明了，对于确定甲状腺乳头状癌的发生发展内在机制仍有待研究。 【关键词】  基因芯片；基因表达；甲状腺癌ABSTRACTObjective: To screen genes that were correlative with carcinogenesis, development and malignance of papillary thyroid carcinoma using cDNA microarray.&#

4、160; Method: Two pieces of papillary thyroid carcinoma tissue with different malignance degree and samples from normal thyroid tissue were collected for mRNA extraction and purification. Marked the purified mRNA from carcinoma tissue with Cy5 deoxycytidine triphosphate for probes labeling and mRNA f

5、rom normal tissue with Cy3dCTP separately, mix these two kinds of mRNA probes together and then hybridize to the cDNA microarray. If Cy3 signal is stronger, the spot on the microarray would be green, means lowexpressed gene. While stronger Cy5 signal will lead to a red spot, which means overexpresse

6、d gene. Experiment gets 2 pieces of doublecolor cDNA microarrays and then they were scanned into computer and analyzed. Result: Differences were found between carcinoma tissue and normal thyoid tissue in their gene fragments' expression. In the welldifferentiated chip, there are 351 lowexpressed

7、 and 417 overexpressed gene fragments presented. To the poorly differentiated chip, the correspondence number was 410 and 415. 90 lowexpressed and 78 overexpressed genes fragments were individually presented in both chip segments with similar expression trend, of which, expression of 25 seemed corre

8、lated to malignant degree. Large quantity of genes with unknown functions were found. Conclusion: The carcinogenesis, development and malignance degree of papillary thyroid carcinoma is resulted from different kinds of abnormality of polygenes not a single gene. Since functions of many gene fragment

9、s has been unknown to us yet, further study still needs to be done.      KEY WORDS Gene chip; Gene expression; Thyroid carcinoma    甲状腺癌是头颈部最常见的恶性肿瘤，病理分型上分为4种：乳头状癌、滤泡型癌、髓样癌、未分化癌，其中又以乳头状癌(papillary thyroid carcinoma， PTC)所占比例最大，约为70%左右，故而获得了更多的研究关注。在以往的文献中，对于乳头状癌异常基

10、因片段表达情况的研究多为某一片段，难于产生较为全面的认识。本实验即利用基因芯片对甲状腺乳头状癌的基因进行大规模片段的研究，对其基因谱表达作一初步分析。1  材料与方法1.1  材料所有病例均来自哈尔滨医科大学附属肿瘤医院。术前选择单侧甲状腺发生肿瘤病例，手术中切取肿瘤组织和对侧腺体正常组织，分别进行液氮冷冻保存。术后根据病理诊断，确定为单侧甲状腺乳头状癌者标本留用，否则筛除。最终样本皆为女性。上海博星公司产BiostarH40 s基因芯片。均由上海博星公司对外试验室提供，包括TRIzol，异丙醇,氯仿，RNeasy Midi kit 75142，Easy Exteact，75%乙醇(RNasefree)，MilliQ水(RNasefree)，无水乙醇,杂交试剂盒等。其中TRIzol购自LifeTechnologies公司，试剂盒中RNAlater，DEPC，RNAsecure TM Reagent购自Qiagen公司。采用GenePix Pro 3.