甲狀腺乳頭狀癌基因譜表達(dá)的初步研究

1、甲狀腺乳頭狀癌基因譜表達(dá)的初步研究         11-02-13 09:31:00     作者：劉偉松,項(xiàng)丞,田冶    編輯：studa20【摘要】  目的：利用基因芯片篩選與甲狀腺乳頭狀癌發(fā)生、發(fā)展和與惡性度相關(guān)的基因。方法：分別選取高、低分化甲狀腺乳頭狀癌組織標(biāo)本，抽提、純化mRNA，同法再純化同患者的正常甲狀腺組織的mRNA；用Cy5熒光染料對(duì)甲狀腺乳頭狀癌組織mRNA作探針標(biāo)記，以Cy3熒光染料對(duì)正常黏膜組織的

2、mRNA作探針標(biāo)記，一同和可檢測(cè)4 096個(gè)基因片段的BiostarH40 s型基因芯片進(jìn)行雜交得到的雙色熒光標(biāo)記芯片，將之分別掃描入計(jì)算機(jī)，進(jìn)行結(jié)果分析。結(jié)果：高、低分化2種芯片上均有大量基因片段出現(xiàn)了的高/低表達(dá)的情況，高分化芯片上異常表達(dá)片段數(shù)量為低表達(dá)351條，高表達(dá)417條，低分化芯片上異常表達(dá)者為低表達(dá)410條，高表達(dá)415條。在2種芯片中均有相同表達(dá)差異趨勢(shì)的基因片段中，共計(jì)有90條基因片段出現(xiàn)低表達(dá)，其中隨惡性度增高而降低的基因有19條；有78條片段呈現(xiàn)高表達(dá)狀態(tài)，其中隨惡性度增高而表達(dá)增加的有25條。表達(dá)異常的片段功能較分散；有大量的不明功能的片段發(fā)現(xiàn)。結(jié)論：甲狀腺乳頭狀癌的

3、發(fā)生、發(fā)展不能用單一的基因變異解釋，而是由多基因共同作用的結(jié)果，并且變異具有明顯的個(gè)體性差異；甲狀腺乳頭狀癌的惡性度與多個(gè)基因片段異常表達(dá)相關(guān)。很多新發(fā)現(xiàn)的片段的功能不明了，對(duì)于確定甲狀腺乳頭狀癌的發(fā)生發(fā)展內(nèi)在機(jī)制仍有待研究。 【關(guān)鍵詞】  基因芯片；基因表達(dá)；甲狀腺癌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    甲狀腺癌是頭頸部最常見的惡性腫瘤，病理分型上分為4種：乳頭狀癌、濾泡型癌、髓樣癌、未分化癌，其中又以乳頭狀癌(papillary thyroid carcinoma， PTC)所占比例最大，約為70%左右，故而獲得了更多的研究關(guān)注。在以往的文獻(xiàn)中，對(duì)于乳頭狀癌異常基

10、因片段表達(dá)情況的研究多為某一片段，難于產(chǎn)生較為全面的認(rèn)識(shí)。本實(shí)驗(yàn)即利用基因芯片對(duì)甲狀腺乳頭狀癌的基因進(jìn)行大規(guī)模片段的研究，對(duì)其基因譜表達(dá)作一初步分析。1  材料與方法1.1  材料1.1.1  標(biāo)本制備 所有病例均來(lái)自哈爾濱醫(yī)科大學(xué)附屬腫瘤醫(yī)院。術(shù)前選擇單側(cè)甲狀腺發(fā)生腫瘤病例，手術(shù)中切取腫瘤組織和對(duì)側(cè)腺體正常組織，分別進(jìn)行液氮冷凍保存。術(shù)后根據(jù)病理診斷，確定為單側(cè)甲狀腺乳頭狀癌者標(biāo)本留用，否則篩除。最終樣本皆為女性。1.1.2  基因芯片 上海博星公司產(chǎn)BiostarH40 s基因芯片。1.1.3  實(shí)驗(yàn)試劑 均由

11、上海博星公司對(duì)外試驗(yàn)室提供，包括TRIzol，異丙醇,氯仿，RNeasy Midi kit 75142，Easy Exteact，75%乙醇(RNasefree)，MilliQ水(RNasefree)，無(wú)水乙醇,雜交試劑盒等。其中TRIzol購(gòu)自LifeTechnologies公司，試劑盒中RNAlater，DEPC，RNAsecure TM Reagent購(gòu)自Qiagen公司。1.1.4  儀器與設(shè)備 S200純化柱購(gòu)自Amersham Pharmacia Biotech Inc，CT0250型真空濃縮儀購(gòu)自CHRIST Ltd.，GenePix 4 000B型芯片掃描儀購(gòu)自AXON Ltd，核酸分析儀產(chǎn)自Biochrom Ltd. Cambridge CB4 OFJ England，MilliQ水生成儀購(gòu)自MILIPORE Ltd.，DY891型電動(dòng)玻璃勻漿機(jī)產(chǎn)自寧波新芝科器研究所，F(xiàn)R280型電泳儀和FR200型凝膠成像儀產(chǎn)自上海復(fù)日科技，QL901型漩渦混合器購(gòu)自海門市麒麟醫(yī)用儀器廠，DND9052型雜交箱產(chǎn)自上海精宏實(shí)驗(yàn)設(shè)備有限公司。 1.1.5  計(jì)算機(jī)處理軟件 采用GenePix Pro 3.0計(jì)算機(jī)