Effects Of CO₂ Pneumoperitoneum On Gastric Cancer Cellular Biological Characteristic And Invasive Ability

Objective: Gastric cancer is one of the most common digestive tract malignant tumors in our country and the most popular one which could implant metastasis in peritoneal cavity. So far, resection is the only way of radical cures. On the dawn of 21 century, minimally invasive surgery came to be the new navigation light of surgery, in which the laparoscopic technology developed most quickly. Laparoscopic radical resection of colorectal cancer is widely practiced in the field of general surgery. And it has been proved to have at least same affects of cancer treatment comparing open surgery. Recently, laparoscopic radical gastrectomy is practiced extensively and show promised recent effects. However, its long term effects are still in studying. Under this bachground, we organized the center of microinvasive gastrointestinal surgery in Southwest Hospital in 2004. We have been studying the feasibility of technology and methods in laparoscopic radical gastrectomy. In our studies, we detected the positive rate of free gastric cancer cells in peritoneal cavity. At the same time, we observed the changes of peritoneum and serosa during laparoscopic radical gastrectomy. On the other hand, we established the mode of pneumoperitoneum in vitro. We observed the effects of different types and pressures artificial pneumoperitoneum on gastric cancer cells'proliferation, apoptosis, cell cycle, migration, cystoskeleton and transplantation tumor. Finally, we detected the changes of cell cycle proteins and apoptotic genes under different pressures CO₂ pneumoperitoneum to study the molecular mechanism.Methods: (1) Intraoperative peritoneal washings were collected from the patients of laparoscopic gastrectomy and open gastrectomy. HE staining and real time RT-PCR on CEA were used to examine the free gastric cancer cells. We observed the peritoneum and serosa of two groups by scanning electronic microscope. (2) Different groups of cultured human gastric cancer strain MNK-45 were exposed under in vitro pneumoperitoneum model. After expose, pH was test and MTT chronometry was used to detect cell growth. Cell cycles, index of proliferation and apoptotic ratio were detected by flowcytometry. Then we observed the migration numbers and cytoskeleton by Boyden millipore and laser scanning confocal microscope. (3) The nude mice tumor models were built with the exposed cancer cells by injecting subcutaneouly and were be observed extensively. (4) RT-PCR, Western-blot and immunoprecipitation were used to investigate the influence on Cyclin D1, CDK4, Rb and phosphorylated Rb of exposed gastric cancer cell. The expressions of Bcl-2/Bax were detected by immunohistochemistry.Results: (1) The positive rates of free gastric cancer cells detected by cytological and real time RT-PCR were no significant differences in the laparoscopic and open groups, respectively. The depth of tumor invasion, area of invaded serosa, regional lymph node involvement, and higher TNM staging were significantly associated with the presence of free cancer cells. The mesothelial cells were swelling and the junction between cells enlarged in the laparoscopic radical gastrectomy. There was no obvious difference in the open radical gastrectomy. (2) pH of CO₂ pneumoperitoneum group dropped obviously and related to pressure. There was no obvious difference in the mixed gas pneumoperitoneum group. (3) The proliferation activity, proliferation index of group under 15mmHg CO₂ pneumoperitoneum dropped significantly, while other groups show no significant changes. The apoptotic ratio in 15mmHg CO₂ pneumoperitoneum raised obviously, while there was no obvious difference in the other groups. (4) The migration ability of exposed gastric cancer cell strain MKN-45 under 0, 10, 12mmHg CO₂ and mixed gas pneumoperitoneum show no significant change, while that of 15mmHg CO₂ pneumoperitoneum group obviously dropped. The microfilament and microtubule in gastric cancer MKN-45 cell were ambiguity in 15 mmHg pressure of CO₂ group. (5) The protein, mRNA and binding ability of Cyclin D1 and CDK4 dropped obviously in the 15mmHg CO₂ pneumoperitoneum group. There were no changes of Rb in protein and mRNA levels, while the phosphorylated Rb dropped obviously. The expression of Bcl-2/Bax dropped obviously when it was exposed to 15mmHg CO₂ pneumoperitoneum. (6) In mice tumor model, the bulk, weight and Ki-67 expression of the transplantation tumor dropped obviously in 15mmHg CO₂ pneumoperitoneum group.Conclusion: (1) Laparoscopic radical gastrectomy doesn't raise the detective ratio of free gastric cancer cells in peritoneal cavity. It could injure the mesothelial cells in the wall peritoneum. (2) The CO₂ pneumoperitoneum pressure of 15 mmHg weaks the proliferation activity, proliferation index and increases the apoptotic ratio. The mechanism may due to the downregulation of Cyclin D1, CDK4, phosphorylated Rb, Bcl-2/Bax and decrease the binding ability of Cyclin D1 and CDK4. (3) The CO₂ pneumoperitoneum pressure of 0, 10 and 12mmHg do not promote gastric cancer MKN-45 cell mobility, while 15mmHg arrest MKN-45 migration. The mechanism may due to the ambiguity of microfilament and microtubule. (4) The CO₂ pneumoperitoneum pressure lower than 15mmHg do not promote gastric cancer growth in animal model, which may indicate usage in clinical laparoscopic radical gastrectomy applied on progressed cancer patients.