Studies of genetic altarations in pcecancerous and background lesions by tissue microdissection.

首先，从福尔马林固定和显微切割的组织片段中提取的DNA的质量和数量进行了检查。从3 μ m厚的福尔马林固定材料上固定的200 ~ 400个细胞中可提取2 ~ 3 ng的DNA。理论上认为一个细胞核中含有12 ~ 15 pg的DNA，这一估计与前人的结果一致。本研究采用微切割技术提取的DNA进行了以下分析：（1）应用微卫星标记对40例早期肺腺癌和30例早期晚期肺腺癌进行了2 p、3 p、9 p、17 p、17 q位点的杂合性缺失（洛）检测。洛缺失率在早期腺癌中为19.8%，在早期进展期腺癌中为26.8%。这些结果表明，多个等位基因丢失是肺腺癌发生的极早期特征性异常之一。(2)用一个任意引物对同一个体的正常组织和肿瘤组织进行DNA指纹图谱的比较。41.7%的腺癌出现7号染色体缺失，84.6%的小细胞癌出现22号染色体缺失。40%以上的腺癌出现1、8、13号染色体序列的扩增，63.3%的鳞癌出现2号染色体序列的扩增。通过微卫星PCR分析证实了小细胞癌中22号染色体的洛杂合性缺失，表明染色体22 q13.3上的洛杂合性缺失在小细胞癌中非常常见。

First, the quality and quantity of the DNA extracted from formalin fixxed and microdissected tissue fragments were examined. Two to three ng DNA could be extracted from 200 to 400 cells which were scrached from 3 mum-thick formalin fixed materials. Theoretically, one nucleus is thought to contain 12 to 15 pg DNA,this estimation are consistent with the former results. Templates extracted from 10 to 20 cells could be successfully amplified by polymerase chain reaction.Using extracted DNAs by this tissue microdissection method, the following analyzes were done :(1) Small sized pulmonary adenocarcinomas, including 40 of early stage adenocarcinomas and 30 of early advanced adenocarcinomas were examined for loss of heterozygosity (LOH) on 2p, 3p, 9p, 17p, 17q using microsatellite markers. The frequencies of LOH were 19.8% in in early stage adenocar cinomas and 26.8% in early advanced adenocarcinomas. These results indicated that multiple allelic loss are one of the characteristic abnormalities of very early stage of pulmonary adenocarcinogenesis.(2) DNA fingerprints qenerated by a single arbitrary primer were compared between normal and tumor tissues of the same individuals which were fixed with methanol and microdissected. Loss of sequence of chromosome 7 was detected in 41.7% of adenocarcinomas and that of chromosome 22 in 84.6% of small cell carcinomas. Gains of sequences in chromosome 1,8,13 were detected in over 40% of adenocarcinomas and chromosome 2 in 63.3% of squamous cell carcinomas. LOH of chromosome 22 in small cell carcinomas were confirmed by microsatellite PCR analysis and suggested that LOH on chromosome 22q13.3 is very frequent event in small cell carcinoma.