The Establishment Of Animal Models With Pancreatic Cancer And Initial Drug Trial In Nude Mice

Objective: To develop an animal model of pancreatic cancer that accurately mimics the human condition,we restricted carcinogetic exposure to the pancreas.Two different doses of dimethylbenzanthracine (DMBA) were implanted directly into the pancreas,by comparision, determined a better approach to the development of pancreatic cancer in experimental models; Dynamically investigated the expression of K-ras, p53 and Smad4 in removed pancreatic tissue by immunohistochemistry, further study the molecular mechanisms underlying pathogenesis of pancreatic cancer. To study the model of tumor induction through the direct injection of human pancreatic cancer cells into the subcutaneous dorsa of mice in the proximal midline.Human pancreatic cancers overexpress epidermal growth factor receptor (EGFR), The progressive growth and metastasis of pancreatic cancer depend in part on EGFR, We wanted to determine the effect of anti-EGFR monoclonal antibody (MMAb-2) therapy on the growth of human pancreatic cancer in nude mice model. Methods: Male Sprague-Dawley rats weighing 150-200 g were divided into control group and administered DMBA groups.Two groups of administered DMBA rats were exposed to two doses of DMBA(6mg,9mg) through the direct implantation of carcinogen crystals into the pancreas.DMBA crystals were implanted into the pancreas by sharply incising the pancreatic membrane,placing a previously measured amount of DMBA into the pancreas with interrupted 6-0 Prolene sutures.The rats were housed in cages before and after all surgical procedures, were allowed a standard rat food and water.They were killed at 1,2,3,4, 5,6, and 7 months.At death both treatment and control animals underwent abdominal exploration with inspection of the pancreas.The pancreas was serially sectioned after formalin fixation and embedded in paraffin,and 4um hematoxylin-eosin stained sections were prepared.The pancreas was removed from each rat and evaluated histologically.Detcted the expression of K-ras, p53 and Smad4 in removed pancreatic tissue by immunohistochemistry(LSAB). The mice were housed and maintained in laminar flow cabinets under specific pathogen-free conditions.A tumor cell suspension (SW1990) of 4*106 cells in 0.2 mlRPMI1640 was injected into the subcutaneous dorsa of mice in the proximal midline. The mice were weighed, and tumors were measured each week in two diameters with a dial caliper.When the tumor volumes were approximately 150 mm3, mice were randomized into 8 groups of 6 animals each.The treatment groups received intraperitoneal injections of MMAb-2 or 5-Fu or gemcitabine or MMAb-2, 5-Fu and gemcitabine in combination respectively. The control group only received intraperitoneal injection of saline.After 4 weeks for treatment,the mice were killed. Tumors were excised and weighed. The size and weight of the pancreatic tumors were recorded. Histopathology confirmed the identity of the disease. The blood routine was done..Tumor tissues were harvested and fixed in 10% neutral buffered formalin . All tissues were paraffin-embedded. Sections (5um) were first stained with hematoxylineosin(H&E)to evaluate tissue viability and quality. Investigated the expression of bax and bcl-2 in removed pancreatic tissue by immunohistochemistry.Tumor tissues were examined immunofluorescence for cells apoptosis. Results: The pancreatic masses were observed after 4 months.The masses varied in size from 0.5-1.5cm in diameter and appeared firm and white. Histologically,cribriforming epithelial growth can be seen in conjunction with nuclear changes ofmalignancy-large nuclei with nucleoli. By comparision,cancerigenic rate in the 6mg group was higher.the death rate was lower. Dynamically detected the expression of K-ras, p53 and Smad4 in removed pancreatic tissue, the K-ras mutation occurred during the early stage of pancreatic carcinogenesis, p53 and Smad4 Smad4 mutation occurred during the relatively late stages.Compare with adenoma and hyperplasia, the expression of K-ras, p53 and Smad4 in carcinoma tissue was higher,there were significant difference among them. The pancreatic masses were present in 90% nude mice after injection of human pancreatic cancer cells into the subcutaneous dorsa of mice in the proximal midline. The masses varied in size and were usually firm and white.The treatment groups produced a reduction in the volume of pancreatic tumors in mice, respectively.Tumors from mice treated exhibited necrotic zones and contained a large number of infiltrating , cells apoptosis was clear. Immunohistochemical analysis revealed increased degrees of apoptosis in treated tumors compared to untreated tumors.Conclusitions: These results demonstrated that pancreatic ductal adenocarcinoma may be easily be induced in the rats with 6 mg DMBA implantation. The animal model may provide a basis for further study the molecular mechanisms underlying pathogenesis of pancreatic cancer. High frequency mutations of K-ras, p53 and Smad4 associated with pancreatic adenocarcinomas. Pancreatic cancer can easierly be induced in the nude mice through the direct injection of human pancreatic cancer cells. Tumor suppression may be due to increased tumor cell apoptosis. These data showed that human pancreatic cancer can be inhibited in a mouse model with MMAb-2. Anti-EGFR therapy can be combined with conventional chemotherapy by showing better. Anti-EGFR therapy may provide potential for future treatment of pancreatic cancer.