摘要:

Ras原致癌基因表現之蛋白質是一群結合GTP的蛋白。有很多的酪胺酸激素接受器(tyrosine kinase receptor)，如表皮生長因子(epidermal growth factor)、胰島素(insulin)和神經生長因子(nerve growth factor)都經由RAS蛋白來做信息傳遞。RAS蛋白有三種，分別為H-ras、K-ras和N-ras，位於細胞膜上，與細胞的生長分化有極大的關係。RAS蛋白以兩種狀態存在，一是靜止狀態，此時會結合GDP，屬於不活化狀態。另一種狀態則結合GTP，導致酵素活化和訊號傳遞。所以，RAS蛋白是一種重要的細胞訊號傳遞蛋白，如果發生突變，細胞的生長分化即受影響。最常發生在Ras基因的突變是鹼基的取代，突變發生在第12、13個密碼時，會加強GTP的結合。突變發生在第61個密碼時，GTPase水解的能力會減少。因此，基因的突變會經由GTP親和力增加，或GTPase活性減少，使得GTP無法正常回復到GDP，結果RAS蛋白持續發出訊號，進而破壞細胞正常的生長調控，因此Ras基因的突變與腫瘤的發生有密切的相關性。本論文擬探討在各種甲狀腺腫瘤中Ras基因突變的情形，我們收集了8例濾泡癌(follicular carcinoma)、42例乳突癌(papillary carcinoma)、2例未分化癌(anaplastic carcinoma)、5 例Hurthle氏細胞腺瘤、12例濾泡腺瘤(follicular adenoma)、20例結節性甲狀腺腫(nodular goiter)，先萃取各檢體的DNA，以PCR方法增幅Ras基因之突變點所在的片段，利用自動核酸序列分析方法，檢測不同的腫瘤中Ras基因突變的情況。結果發現89例甲狀腺腫瘤檢體中，有四例檢體具Ras基因突變，其中3例為濾泡癌，分別在H-ras胺基酸密碼第61位置、K-ras密碼第61及N-ras密碼第61位置；另1例為濾泡腺瘤，突變點位在N-ras密碼第61位置。另外，有33.7% (30/89)的檢體在H-ras密碼第27位置具有多型性(polymorphism)的情形，為中性突變，不影響其胺基酸種類。整體來說，Ras基因在甲狀腺腫瘤的突變盛行率為4.5%，其中濾泡癌突變盛行率為37.5%，濾泡腺瘤為8.3%，其他腫瘤則為零，推測Ras基因在甲狀腺的濾泡癌中突變盛行率最高。而Ras基因在甲狀腺良性腫瘤的突變盛行率為2.7%，惡性腫瘤為5.8%。Ras基因在甲狀腺腫瘤的突變盛行率不高，顯示甲狀腺腫瘤致癌過程可能牽涉到環境因子或其他基因的機轉，值得我們進一步研究。另一方面，偵測方法之敏感度亦可能造成本分析中盛行率低的原因，因此以變性梯度膠體電泳方法(Denaturing Gradient Gel Electrophoresis，DGGE)提高偵測之敏感度，目前找到四例核酸序列所沒有偵測到的突變的病例，顯示DGGE確為較敏感檢測單一鹼基突變之方法，可以幫助我們印證自動核酸序列分析之結果。

Abstrate:

Ras proto-oncogenes are members of the superfamily of GTP-binding proteins. Many tyrosine kinase receptors, including those for epidermal growth factor, insulin, and nerve growth factor, signal through RAS proteins. The product of members of this oncogene family (H-, K-, N-ras) is a 21 kD protein with nucleotide binding activity, involved in the transduction of information from the cell surface to the nucleus. The three RAS proteins exit in two states: a resting state in which they are bound to GDP and an active state in which they bind GTP. The most common form of mutational activation of Ras oncogenes in human tumors is through single base substations affecting either the GTP-binding of main (codons 12 and 13) or the GTPase domain (codon 61) of the protein. Thus, mutant RAS proteins result in constitutive activation of the downstream signaling cascade because their affinity for GTP is increased or their GTPase activity is decreased, so that the protein cannot return to the resting state. To investigate we have screened 89 thyroid tumor specimens, which include 8 follicular carcinomas (FC), 42 papillary carcinoma (PTC), 2 anaplastic carcinoma (AC),5 Hurthle cell adenoma (HA), 12 follicular adenoma (FA) and 20 nodular goiter (NG), for mutation in three Ras genes using PCR and automatic sequencing. Four tumors contained Ras gene mutation. Of these, three were identified among FC (37.5%), which mutation were in the codon 61 of each Ras genes. One mutation were at codon 61 of N-ras in FA specimens (8.3%). In addition, 33.7% (30/89) of specimens contain H-ras codon 27 polymorphism. In conclusion, our data indicated that the prevalence rates of Ras gene mutation were 5.8% and 2.7% in thyroid carcinoma and thyroid benign adenoma, respectively. Other environmental or genetic factors might also involved in the thyroid tumorigenesis and worth further investigation. The data were further confirmed using the combination of the PCR and denaturing gradient gel electrophoresis (DGGE). Four more cases of possible Ras mutation were detected which did not revealed by automatic sequencing , indicating that DGGE is a more sensitive method in detecting single nucleotide mutation. DGGE analysis should increase the detection rate of Ras gene mutation in our analysis.

中文摘要----------------1
英文摘要----------------3
背景介紹----------------4
實驗材料----------------8
實驗方法----------------13
結果--------------------18
討論--------------------21
參考資料----------------25
附表--------------------31
附圖--------------------39

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