加热联合吲哚美辛对人肝癌MHCC97L细胞增殖和侵袭能力的影响

Effects of thermotherapy combined with indomethacin on proliferation and invasiveness of hepato- cellular carcinoma cell line MHCC97L

目的观察水浴加热联合吲哚美辛对人肝癌MHCC97L细胞增殖和侵袭能力的影响。方法42℃水浴加热前2h加入0．2mmol／L吲哚美辛，加热组不加药，对照组不加热、不加药，其他处理相同并在同一时间点观察。在不同的时间观察细胞增殖、生长曲线、克隆形成率、流式细胞术（FCM）分析细胞周期、侵袭、运动、血管内皮生长因子（VEGF）和基质金属蛋白酶（MMP）-2蛋白表达[酶联免疫吸附试验（ELISA）法]。结果与对照组比较，加热＋吲哚美辛明显抑制MHCC97L细胞增殖（P〈0．01），其最大抑制效应为加热后48h（53．6％），细胞倍增时间延长了2．07倍；加热＋吲哚美辛组48、96h的细胞集落形成率均明显降低（P〈0．01）；FCM显示，吲哚美辛能逆转加热对细胞周期的影响，加热＋吲哚美辛组48h和96h的G，期细胞比例均明显升高，S＋G2期细胞比例均明显降低（P〈0．05）。加热＋吲哚美辛组48h和96h相同数量的细胞穿过人工基底膜到达Transwell小室膜背面的细胞平均数（侵袭实验）和穿过Transwell小室膜到达背面的MHCC97L细胞平均数（运动实验）均明显低于加热组（P〈0．01）；ELISA法检测发现，加热＋吲哚美辛组48、96h的分泌量均明显低于加热组（P〈0．05）。结论吲哚美辛抑制体外加热后肝癌细胞的增殖，其作用和抑制细胞进入DNA合成期和分裂期有关；进一步抑制其侵袭运动能力，其作用和MMP-2、VEGF表达降低有关。

Objective To investigate the effects of thermotherapy combined with indomethacin on proliferation and invasiveness of hepatocellular carcinoma cell line MHCC97L. Methods Indomethacin （0. 2 retool/L） was added 2 h before thermotherapy. Chemicals were omitted for the thermotherapy group. Chemicals and thermotherapy were omitted for the control group. Proliferation, growth curve and plate effi- ciency were observed. Flow cytometry was used to analyze the cell cycle. Transwell invasion assay and cell motility assay were done. The expression ov vascular endothelial growth factor （VEGF） and matrix metallo- proteinase-2 （MMP-2） protein in MHCC97L cells was detected by using enzyme-linked immunosorbent as- say. Results Compared to the control group, the proliferation of MHCC97L cells was significantly de- creased after thermotherapy combined with indomethacin （ P 〈 0. 01 ）. The inhibition ratio reached the maximum at 48 h after thermotherapy up to 53.6%, and the doubling time increased 2. 07 times. The plate efficiency of the same number of cells 48 and 96 h after thermotherapy combined with indomethacin was de- creased （P 〈 0.01 ）. Flow cytometry revealed that the ratio of cells in G1 phase was increased and that in S ＋ G2 phase decreased 48 and 96 h after thermotherapy combined with indomethacin, respectively （ P 〈 0. 01, P 〈 0. 05 ）. The average amount of invading cells per field in cell invasion assay and motility assay of the same number of cells 48 and 96 h after thermotherapy combined with indomethacin was significantly less than solitary thermotherapy group, respectively （ P 〈 0. 01 ）. Compared to solitary thermotherapy group, the expression of MMP-2 and VEGF 48 and 96 h after thermotherapy combined with indomethacin was de-creased, respectively （P 〈 0. 05 ）. Conclusion Indomethacin suppressed thermotherapy-augmented prolif- eration of MHCC97L cells, which was in part mediated by inhibiting DNA synthesis and mitosis of the cells. Indomethacin further inhibited the invasiveness of the cells, which was associated with the reduced VEGF and MMP-2 protein expression.