Intraabdominelle pH-Verschiebungen unter den Bedingungen der CO₂-Laparoskopie führen zu einer Verminderung der intraperitonealen fibrinolytischen Kompetenz

Abstract

Background: The local oncological effects of CO₂ pneumoperitoneum are unclear, especially in advanced colorectal tumours. Both in vitro and in vivo studies show an influence on the growth rate of colon carcinoma cells under conditions of CO₂ pneumoperitoneum. Alterations in the fibrinolytic activity of tumour and mesothelial cells are under discussion as a possible cause of this effect. To simulate a pneumoperitoneum, we have developed a chamber in which cells can be cultivated under variable pH values and gas and pressure conditions. In this study, the effects of CO₂ pressure and the pH value on the fibrinolytic activity of colon carcinoma cells and mesothelial cells were investigated. Method: The rectangular Plexiglas chamber (26 × 26 × 7 cm) constructed in our laboratory can be closed by means of a lid which provides an air-tight seal. The chamber has 3 apertures, through which the gas can be fed and the gas pressure and gas concentration measured (by means of a barometer and gas analyser, respectively). Human mesothelial cells and colon carcinoma cell lines (WIDR, Colo201, Colo205, T84, HT29, SW48, HCT116) were cultivated in the chamber (37 °C) in gelatin-coated Petri dishes at a pH of 6.1 (a value which, according to the literature, corresponds to the intraperitoneal pH value under conditions of CO₂ pneumoperitoneum) and gas-equilibrated medium. After 1/2, 1, 2 and 5 h with and without CO₂ pressure (10 mm Hg) 2 Petri dishes in each case were removed and the total RNA of the cells isolated before a quantitative RNA measurement of plasminogen activators tPA and uPA and of the plasminogen activator inhibitor PAI was performed by mean of real-time one-step RT-PCR in an I-cycler. Cells in the incubator which were cultivated and prepared at a physiological pH value of 7.4 and without CO₂ pressure served as controls. Results: Both with and without CO₂ gassing at a pH of 6.1, the mesothelial cells within 5 h showed a decrease in tPA expression (mean ± SEM amol RNA/ 100 ng total RNA) compared with the control group (pH 6.1: 0.68 ± 0.06; pH 6.1 + CO₂: 0.62 ± 0.06, control: 1.12 ±0.16). By contrast a significant increase in PAI expression was seen in the tumour cell lines SW48 (pH6.1: 0.01 ±0.002; pH 6,l+CO₂: 0.014 ±0.003, control: 0.005 ± 0.0007), HCT116 (pH 6.1: 14.66 ± 4.1; pH 6.1 + CO₂: 8.03 ± 0.16, control: 0.95 ± 0.07) and WiDr (pH 6.1: 3.35 ± 0.15; pH 6.1 + CO₂: 1.23 ± 0.1, control: 0.36 ± 0.02) (p < 0.05, after multivariance analysis (ANOVA)). Conclusion: The decrease in tPA expression in the mesothelial cells and the increase in PAI expression in the tumour cell lines shows, that the use of CO₂ pneumoperitoneum can lead to an attenuation of the intraperitoneal fibrinolytic response. The decrease in the intraabdominal pH value appears to be of particular relevance here. This observation may be of fundamental importance for understanding interactions between tumour cells and mesothelial cells under conditions of CO₂ laparoscopy. Inert gases such as helium or nitrogen, which hardly lead to any shifts in intra-abdominal pH, may therefore be a useful alternative to CO₂ laparoscopy from an oncological point of view.