结直肠粘膜隐窝异常病灶K-ras、APC基因突变的研究

目的　分析隐窝异常病灶（aberrantcrypt foci,ACF）的基因水平变化特点及其作为结直肠癌最早期形态变化的分子依据,探讨ACF与腺瘤之间的关系。方法　经微解剖分离提取15例正常粘膜腺体、34例ACF、15例腺瘤和35例结直肠癌组织进行DNA测序,检测K-ras第12、13、61密码子和APC第15外显子“突变密集区”的突变。结果　正常粘膜无K-ras和APC基因突变,ACF、腺瘤和癌组织K-ras突变率分别为17.6%（6/34）、13.3%（2/15）和14.3%（5/35）,3者突变率相近。4例ACF、腺瘤和癌组织的突变位于12密码子的第2碱基（GGT→GAT）,2例ACF突变发生在13密码子的第2碱基（GGC→GAC）。ACF中K-ras的突变特点与同来源的癌组织保持一致,即患者年龄较大,大体以隆起型为主,所有组织未发现有61密码子的突变。癌和腺瘤APC基因突变率相近,癌为22.9%（8/35）,腺瘤为26.7%（4/15）明显高于ACF的2.9%（1/34,P<0.05),癌组织APC与患者的年龄、肿瘤位置、大体类型、组织分化程度无关。结论　ACF可能是结直肠癌最早期的形态改变,ACF的形态学变化、部分基因的改变均有别于腺瘤,提示（1）ACF有可能是腺瘤前的形态改变;（2）结直肠癌的发生可能存在“正常上皮→ACF→癌变”这一途径。

Mutations of APC and K-ras gene in aberrant crypt foci from human colon

OBJECTIVE: To analyze the mutations of K-ras and APC gene in normal colorectal mucosa, aberrant crypt foci (ACF), adenoma and colorectal carcinoma (CRC); To study the genetic alteration in ACF and its possibility of being a molecular marker of very early colon cancer and to explore the relationship of ACF and colorectal adenoma. METHODS: DNA from 35 CRC, 15 adenomas, 34 ACF and 15 cases of normal mucosa with mucous glands were isolated by micro-dissection. Direct gene sequencing of k-ras gene including codon 12, 13 and 61 as well as the mutation cluster region (MCR) of APC gene. RESULTS: Mutation frequency of k-ras gene in ACF, adenoma and carcinoma was 17.6% (6/34), 13.3% (2/15), and 14.3% (5/35) respectively, showing no difference between the three pathologic changes. The ras gene mutation sites in adenomas, carcinomas and 4 ACF were limited to codon 12 (GGT-->GAT), but in 2 ACF, they were located in codon 13 (GGC-->GAC). K-ras gene mutation in ACF was found more frequently in old patients and in patients with polypoid cancers. No mutations were found in codon 61 in three types of tissue. Mutation rate of APC genes in adenomas and carcinomas was 22.9% (8/35) and 26.7% (4/15) respectively, which was higher than that of ACF 2.9%, (P < 0.05). APC gene mutation in carcinomas was not correlated with age, location, size and differentiation. CONCLUSION: The morphological changes and gene mutation status are different in ACF and adenomas. ACF is a very early morphological lesion in the carcinogenesis of colorectal carcinoma. Therefore, ACF is considered to be the putative "microadenoma" where the development of colorectal carcinomas may be from "normal epithelium to ACF and then to carcinomas".