RESUMO
BACKGROUND AND AIMS: The aim of this study was to detect Helicobacter pylori (H. pylori) in colorectal cancer tissue specimens and relate the possible role of this microorganism in the etiology of colorectal cancer. PATIENTS AND METHODS: From February 2002 to April 2003 83 CRC patients (55 male, 28 female) and 40 control patients (19 male, 21 female) entered the prospective study. The biopsy samples of CRC tissue and normal mucosa were obtained during open surgery on CRC patients. In the control patients biopsy samples were taken during colonoscopy. Pathology confirmed adenocarcinoma in all the CRC patients. The existence of genetic material of H. pylori was determined by detection of the ureA gene by nested PCR. K-ras PCR was also performed on all patients. RESULTS: H. pylori PCR was positive in 1 case (1.2%) of CRC in the tumour tissue and in all 5 samples (6.0%) of the normal colonic mucosa in the cancer patients. The control patients were PCR positive to H. pylori in 13 samples (32.5%). According to Chi-square test, there is no statistical correlation between H. pylori infection and CRC (x2 = 2.9395; p > 0.05) but there is a significant prevalence of H. pylori infection in controls compared to CRC (x2 = 15.5625; p < 0.01). The K-ras PCR showed gene mutations in 19 tumour tissues of CRC (31.6%) and in 2 cases (3.4%) of normal colonic mucosa of CRC patients . In controls K-ras PCR showed one gene mutation (3.0%). There is a significant statistical correlation between K-ras mutation and CRC (x2 = 16.0694; p < 0.01). CONCLUSION: Our established PCR for H. pylori is feasible for CRC tissue as well. However, H. pylori is not considered to play an important role in the pathogenesis of CRC. The identification of K-ras mutations in routine PCR analysis correlates with the presence of CRC.
Assuntos
Neoplasias Colorretais/microbiologia , Helicobacter pylori/isolamento & purificação , Urease/genética , Colo/microbiologia , Neoplasias Colorretais/genética , Feminino , Genes ras , Helicobacter pylori/genética , Humanos , Mucosa Intestinal/microbiologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase/métodosRESUMO
Mutation of the K-ras gene is thought to be an early and important event in pancreatic carcinogenesis. In order to study the role of this molecular alteration in the transition from the normal to the neoplastic pancreatic cell, bovine pancreatic duct cells were first immortalized by SV40 large T antigen (Ag) complementary (c)DNA transfection and then transfected with a mutated K-ras gene. As did primary duct cells, the immortalized duct cells (more than 100 passages) expressed cytokeratins, carbonic anhydrase type-II, cystic fibrosis transmembrane conductance regulator (CFTR), and multidrug resistance (mdr). They grew as a single layer after transplantation under plastic domes and formed three-dimensional structures resembling ducts when grown on Matrigel. Cell growth was stimulated by insulin, epidermal growth factor (EGF), transforming growth factor (TGF)-alpha, but cells did not respond to gastrin and CCK-8. They did not form colonies in soft agar nor did they form tumors in nude mice. Immortalized cells transfected with mutated K-ras acquired the ability to form tumors after orthotopic injection into the nude mouse pancreas. It is concluded that SV 40 immortalized bovine pancreatic
Assuntos
Transformação Celular Neoplásica/patologia , Genes ras/genética , Mutação , Ductos Pancreáticos/patologia , Neoplasias Pancreáticas/patologia , Transfecção/métodos , Animais , Antígenos Transformantes de Poliomavirus/genética , Biomarcadores/análise , Bovinos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Transformada , Transformação Celular Neoplásica/química , Transformação Celular Neoplásica/genética , Células Clonais , DNA Complementar/genética , Fator de Crescimento Epidérmico/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Técnica Indireta de Fluorescência para Anticorpo , Insulina/farmacologia , Camundongos , Camundongos Nus , Ductos Pancreáticos/efeitos dos fármacos , Ductos Pancreáticos/metabolismo , Neoplasias Pancreáticas/química , Neoplasias Pancreáticas/genética , Reação em Cadeia da Polimerase , RNA Viral/biossíntese , Fator de Crescimento Transformador alfa/farmacologiaRESUMO
Preparation of pancreatic duct epithelial cells from adult organs is possible by limited digestion and outgrowth of cells. These primary cells are mitotically active for only a short period. Therefore transfection with SV40 large-T antigen is one method to obtain an immortalized cell clone. Because the transfection efficacy of primary cells with conventional vectors is comparatively low, our aim was to develop conditions with improved transfection rates. Best transfection rates (approximately 6% of the resting cells) were obtained by using the BES buffered saline (BBS) calcium phosphate (Ca-P) coprecipitation technique at low pH. By using these optimized transfection parameters, primary cultures of human pancreatic duct epithelial cells were successfully transfected with the plasmid pSV3neo, bearing the large- and small-T antigen of SV40. A G 418 resistant clone (E4) was maintained in culture for 14 months before reaching terminal crisis.
