不同压力下CO_2气腹处理对结肠癌HT-29细胞增殖及侵袭能力的影响

The effect of CO_2 pneumoperitoneum on the proliferation and invasion of HT-29 colon cancer cells under different pressures

目的观察不同压力CO_2气腹处理对结肠癌HT-29细胞体内外增殖与侵袭能力的影响。方法以HT-29细胞暴露于5 mm Hg、10 mm Hg及15 mm Hg CO_2压力下1h建立体外模拟气腹模型为三个实验组,另以常规培养HT-29细胞为对照组。处理1 h后,立即取各组细胞培养液检测pH值。常规培养24 h后将各组细胞接种裸鼠腹腔建立体内模型(每组对应6只裸鼠),同时体外以四唑盐比色法(methyl thiazolyl tetrazolim,MTT)检测各组细胞粘附率,免疫细胞化学法检测E-钙粘蛋白的表达。继续常规培养5 d,绘制细胞生长曲线。3 w后开腹检查裸鼠腹腔肿瘤种植转移情况并测量肿瘤平均质量,以免疫组化法检测肿瘤组织中E-钙粘蛋白的表达。结果气腹处理1 h后,实验组细胞培养液即时p H值显著低于对照组(均P<0.05)。10 mm Hg组和15mm Hg组与对照组相比,细胞粘附率下降、裸鼠腹腔内肿瘤平均质量减小、E-钙粘蛋白表达水平降低(均P<0.05),5 mm Hg组上述指标与对照组比较,差异均无统计学意义(均P>0.05)。各实验组细胞增殖速度随气腹处理压力的升高呈下降趋势。结论模拟10 mm Hg及15 mm Hg CO_2气腹处理,可降低结肠癌HT-29细胞的体内外的增殖能力,不增加在腹腔的侵袭能力。5 mm Hg CO_2气腹处理对HT-29细胞没有显著影响。

Objective To explore the effect of CO2 pneumoperitoneum on the proliferation and invasion of HT-29 colon cancer cells under different pressures. Methods With an in vitro artificial pneumoperitoneum model, HT-29 cells were exposed to CO2-insufflation of three pressure groups（5, 10 and 15 mm Hg） for 1h. The HT-29 cells in the control group were conventionally cultured. The p H value of cell culture medium was measured immediately after the intervention. After 24 h of routine culture, HT-29 cells were inoculated in nude mice to establish in vivo model（6 mice in each group）. In vitro, the cell adhesion rate and immunocytochemistry assay of Ecadherin expression were detected by using Methylthiazolyl tetrazolim（MTT）. After 5 days of conventional culture, the growth curves of the cells in each group were drown. After 3 weeks, the metastasis of peritoneal tumor in nude mice was examined by laparotomy and the average mass of tumor was measured. The expression of E-cadherin in tumor sections was detected by immunohistochemistry. Results After 1 hour of the intervention, the p H value of the cell culture fluid in the three observation groups was significantly lower than the control group（P 0.05）. Compared with the control group, the cell adhesion rate, the growth status of tumor in peritoneal cavity,and the expression level of E-cadherin in the 10 mm Hg group and the 15 mm Hg group were significantly lower（P 0.05）, while they were not significantly different between the 5 mm Hg group and the control group（P 〈 0.05）. With the increasing of the pressure of the pneumoperitoneum, there was a decending trend. Conclusion The treatment of 10 mm Hg and 15 mm Hg CO2 pneumoperitoneum in vitro can decrease the proliferation ability of HT-29 colon cancer cells, and without influencing the invasion ability. While 5 mm Hg CO2 pneumoperitoneum has no significant effect on HT-29 cells.