| Gastric cancer is the second most common cancer in the world. Incidence and mortality rates are higher in countries such as Japan, China, South America, Portugal, and Italy. In Zhejiang province of China, the incidence and mortality rates are higher than their average rates of whole country. The geographical differences in gastric cancer frequency may be due to molecular as well as environmental differences.Now, carcinogenesis is widely regarded as a multistep process involving an accumulation of genetic alterations. Multiple genetic alterations including activation of oncogenes such as k-ras mutation,c-erbB2 amplification, inactivation of tumor suppressor genes such as APC gene and TP53 gene mutation, chromosomal loss of heterozygosity, genetic instability, telomerase activation, CpG methylation and alterations of cell-cycle regulators have been detected. Multistep tumor development included precancerous lesion, early stage, advanced stage (invasion and metastasis), and so on.However, many study results are different, even some are contradictory. It may be correlation with two below reasons. First, the molecular mechanisms of gastric adenocarcinoma probably have differences among different human races or different regions. Second, each experiment groups select different experiment materials, methods and markers. Especially to experiment materials as templates, tumor tissue blocks are so extensively used, that the percent of tumor cells in the blocks becomes a direct important factor to affect experiment results. In addition, the studies of multisteps are focused on primary tumor lesions at different stages, but not on invasive and metastatic lesions. Compared with primary tumor, few studies of invasive and metastatic lesions could be seen in gastric adenocarcinoma progression.Therefore, we selected two important molecular events, microsatellite instability and TP53 gene mutation, and investigated their alterations during the progression of gastric adenocarcinoma. Applying laser microdissection after established LMD-PCR and IHC-LMD-PCR technology, we separately microdissected and collected primary tumor, intravasal tumor emboli and/or lymph node metastasis and/or distal organ metastasis and/or distal disseminated tumor in the same gastric adenocarcinoma. Then, analysis of microsatellite instability and TP53 gene mutation, comparison of relationship between TP53 gene mutation and TP53 protein expression, and exploration of their changes as well as biological significance in invasive and metastasis lesion, were earned out. And it would provide the important experimental basis for the molecular-pathological diagnosis of gastric adenocarcinoma later.Part 1:Establishing LMD-PCR Technology from Paraffin-Embedded TissueSectionsWe established the LMD-PCR technology from six aspects as below.(1)Establishing the stable technology of LMD-PCR from formalin-fixed and paraffin-embedding tissue(PET) sections. To find the fittest DNA extraction method of LMD-PCR technology for following experiments, we compared four different DNA extraction-PCR methods(direct PCR, phenol-chloroform DNA extraction-PCR, WGA-PCR, proteinase K digestion-PCR) by amplifying B -globin, after microdissecting the same tumor cell number from 3 PET sections. (2)Effect of PET archived time: To evaluate the longest archived time of PET which can get satisfied amplification, we microdissected the archival PET section from 1986 to 1991 years (5 gastric adenocarcinoma cases per year), respectively. Then fragment 138bp and 270bp were amplified by PCR to confirm archival PET tissue from which year could remain to successfully get specified fragment.〦ffect of amplification fragment lengths: To observe the longest length fragment which can get satisfied amplification, we respectively amplified five different length fragments (110bp, 268bp, 536bp, 989bp, 1327bp) by PCR after laser microdissection and DNA extraction from 6 advanced gastric adenocarcinoma cases.〦ffect of cell number as template: To determine the least cell numbers as template w...
|
PDF Full Text Request