Sequential analysis of K-ras mutations in aberrant crypt foci and colonic tumors induced by azoxymethane in Fischer-344 rats on high-risk diet.
Sequential analysis of K-ras mutations in aberrant crypt foci and colonic tumors induced by azoxymethane in Fischer-344 rats on high-risk diet.
复制标题
高风险饮食的 Fischer-344 大鼠中氧化偶氮甲烷诱导的异常隐窝病灶和结肠肿瘤中 K-ras 突变的序列分析。
作者：
N. Shivapurkar;Z. Tang;A. Ferreira;S. Nasim;C. Garett;O. Alabaster
40只Fischer-344雄性大鼠被给予高风险饮食（HRD），即高脂肪、低纤维和低钙。4周后，大鼠每周两次皮下给药。注射氧化偶氮甲烷（AOM，15 mg/kg体重），并保持相同的饮食直至死亡。在第12周和第20周分别处死8只大鼠，以显微解剖结肠中含有4个或更多隐窝/病灶的异常隐窝病灶（ACF）。其余24只大鼠在30周时处死以收获结肠肿瘤。应用聚合酶链反应（PCR）技术，从ACF和结肠肿瘤中扩增K-ras基因的特异性DNA片段。对PCR扩增的DNA进行测序以鉴定密码子12和13中的点突变。在ACF和结肠肿瘤中检测到的所有突变都是密码子12的第二位G到A的转换。12周和20周时，分别有2/8（25%）和3/8（37%）大鼠的ACF中存在这些突变。突变存在于7/24（29%）大鼠的结肠肿瘤中。这些结果为ACF中的K-ras突变（> 4个隐窝/病灶）作为暴露于AOM同时接受高风险西式饮食的F344大鼠结肠中恶性潜能的早期标志物的意义提供了重要证据。
Forty Fischer-344 male rats were given a high-risk diet (HRD) that was high in fat, low in fiber and low in calcium. After 4 weeks, the rats were given two weekly s.c. injections of azoxymethane (AOM, 15 mg/kg body wt), and remained on the same diet till death. Eight rats were killed at 12 weeks and again at 20 weeks in order to microdissect aberrant crypt foci (ACF) containing four or more crypts/focus from their colons. The remaining 24 rats were killed at 30 weeks to harvest colonic tumors. The polymerase chain reaction (PCR) was used to amplify specific DNA segments in the K-ras gene from ACF and colonic tumors. The PCR-amplified DNAs were sequenced to identify the point mutations in codons 12 and 13. All the mutations detected in the ACF and colonic tumors were G to A transitions in the second position of codon 12. These mutations were present in the ACF of 2/8 (25%) and 3/8 (37%) rats at 12 and 20 weeks respectively. The mutations were present in colonic tumors of 7/24 (29%) rats. These results provide important evidence for the significance of K-ras mutations in ACF (> 4 crypts/focus) as early markers of malignant potential in the colons of F344 rats exposed to AOM while receiving a high-risk western style diet.