| Esophageal cancer (EC) is one of the six most common malignant diseases in the world with remarkable geographical distribution. The prognosis of esophageal cancer is very poor; five-year survival rate is only about 10 percent for the patients at late or advanced stage. Linzhou city (formerly Linxian) and nearby counties in Henan province have been well-recognized as the high incidence area in the world. In the past decades, studies by scientists from China and abroad in this area have indicated that esophageal carcinogenesis was a multistep progressive process involved by multiple genetic changes (accumulation and overlap). The early characteristic for the subjects predisposed to EC is the abnormal proliferaion in epithelial cells, morphologically manifested as basal cell hyperplasia (BCH), dysplasia (DYS) and carcinoma in situ (CIS), which could be considered as precancerous lesions of EC. The precancerous lesions are unstale, i. e, it can develop to cancer, or to stay couple of years without any changes, even return to normal. What is the most important factor to decide the precancerous lesions developing in different directions, especially in those precancerous lesions with similar morphological changes? What is the key point to drive the mild precancerous lesions to more severe ones? Our hypothesis is that there may be different molecular changes under the precancerous lesions with similar morphology. To further characterize the molecular changes in EC carcinogenesis will shed light on elucidating the mechanisms of EC and establishing the biomarkers for early diagnosis and high-risk population screening.Recent studies indicate that the alterations of microsatellite DNA maybe one of the important markers to induce normal cell to immortal or neoplastic transformation. The alterations of microsatellite DNA are divided into two parts. One is microsatellite instability (MSI), which could be defined as the length differences of microsatellite DNA between normal and tumor cell at the same individual characterized by the increase or decrease of repeat units. The other is loss ofheterozygosity (LOH), which could be defined as one allele loss in tumor compared with two alleles in normal tissue. It has been recognized that the changes of microsatellite DNA may be one of important markers for tumor diagnosis.The present studies were undertaken to characterize the changes of microsatellite DNA in esophageal carcinogenesis, loss of heterozygosity (LOH) in specific loci was thus analyzed using microsatellite polymorphic markers spanning frequent loss regions based on the results of cytogenetic studies. Allelic deletions were examined using 18 polymorphic markers on chromosomes 3p, 5q, 6p, 9p, 13q, 16q, 17, and 18q.III. 1,2. 1 Materials and methodsIII. 1.2.1.1 Precancerous and carcerous tissuesThirty-two surgically resected SCC specimens were collected from Linzhou, the high-incidence area for EC in Henan. Of the EC patients, there were 18 males and 14 females with an average of 59 years (range: 44-73 years). All the SCC patients were not treated by either chemotherapy or radiotherapy before operation. Surgically resected specimens were divided into two parts. One fixed with 85% ethanol and paraffin embedded for histopathological diagnosis. The other stored in the liquid nitrogen and then transferred in a -80℃ freezer until use. 10/32 cases were taken from cancers and adjacent regions. Various tissues from cancer and adjacent parts were frozen and serially sectioned into 5μm and 30-60 slides were collected for DNA extracting and LOH analysis. HE staining was performed every five slides for histopathological diagnosis. According to cell morphologic changes, the esophageal epithelia were divided into BCH, DYS, CIS and SCC.III. 1.2.1.2 Laser capture microdissection (LCM)According to the situation of cells by HE, we dropped little glycerol in matched spots, separated precancer, career and normal cells by 5ml syringe in anatomy microscope, put them to 180μl lysis buffer, added 20μl proteinase K, ov...
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