超声靶向破坏载氧微泡技术对缺氧微环境下胆管癌细胞生长影响

Effects of ultrasound-mediated destruction of oxygen loadedlipid microbubbles on the growth of human cholangiocarcinoma cell line under hypoxia

目的胆管癌细胞在缺氧微环境中能够快速增殖、侵袭和转移,超声靶向破坏微泡技术改变了肿瘤缺氧微环境。本研究旨在探讨超声靶向破坏载氧微泡技术对缺氧微环境下胆管癌细胞增殖、凋亡的影响。方法以DSPC+DPPE-MPEG 5000为外壳,通过机械振荡法制作载氧微泡,运用氯化钴(CoCl_2)模拟缺氧微环境。体外培养胆管癌细胞株QBC939和RBE,通过超声靶向破坏载氧微泡技术改变胆管癌生存的缺氧微环境,噻唑蓝(MTT)比色法检测各组细胞的生长情况,流式细胞仪检测细胞凋亡率和细胞周期。结果通过DSPC+DPPE5000的机械振荡法能够制作出平均粒径为1.91μm和载氧浓度为(2.57±0.82)×10^(-3) mg/mL的载氧微泡,QBC939和RBE细胞在CoCl_2模拟缺氧微环境中能够快速生长,QBC939细胞对照组和CoCl_2组A值分别为1.91±0.07和1.67±0.04,t=-7.855,P=0.001 4;RBE细胞对照组和CoCl_2组A值分别为2.81±0.04和1.84±0.04,t=-30.514,P=0.000 1,差异均有统计学意义。但相比于缺氧组细胞,实验组胆管癌细胞生长明显受到抑制(均P<0.05),凋亡率上升(均P<0.05),且具有剂量依赖性。细胞周期分析缺氧组S期细胞比例明显升高,增殖指数PI明显升高,差异具有统计学意义(均P<0.05),但缺氧组与微泡组间差异无统计学意义。结论胆管癌细胞能够在缺氧微环境下快速生长,而超声靶向破坏载氧微泡能够纠正细胞的缺氧微环境,促进细胞凋亡,抑制胆管癌细胞增殖,但该作用不依赖于细胞周期的调控。

OBJECTIVE The aim of this study was to investigate the effects of ultrasound-mediated destruction of oxygen-loaded lipid microbubbles on the proliferation and apoptosis of human cholangiocarcinoma cell lines under hypoxia microenvironment.METHODS A type of microbubbles carrying oxygen which used the DSPC and DPPE-MPEG 5000 as shell was producted by mechanic vibration.Cholangiocarcinoma cell lines（QBC939 and RBE）were cultured in vitro under chemical hypoxia induced by CoCl2（150μmol/L）,we changed hypoxic microenvironment of cholangiocarcinoma cells by ultrasound-mediated destruction of oxygen loaded lipid microbubbles.The cell growth of each group was detected by MTT,the apoptosis and cell cycle were detected by flow cytometry analysis.RESULTS The oxygen-loaded microbubbles appeared as a uniform size with a mean diameter of 1.91μm and oxygen concentration of（2.57±0.82）× 10^-3 mg/mL.Comparing with the control group,the proliferation of QBC939 and RBE in the hypoxic microenvironment which induced by CoCl2 was increased significantly,the two groups（control group,CoCl2 group）showed different A（1.91±0.07 and 1.67±0.04,t=-7.855,P=0.0014;2.81±0.04 and 1.84±0.04,t=-30.514,P=0.0001）,but the ultrasound-mediated destruction of oxygen loaded lipid microbubbles effectively inhibited the proliferation（P〈0.05）and significantly promoted apoptosis of QBC939 and RBE cells in time and dose dependent patterns（P〈0.05）.The results of cell cycle revealed that the number of cells in S-phase was increased significantly（P〈0.05）,however,there was no significant difference between the hypoxia group and microbubble group.CONCLUSIONS The hypoxia microenvironment can promote the growth of cholangiocarcinoma cell Lines,and ultrasound-mediated destruction of oxygen-loaded lipid microbubbles can inhibite the proliferation and promote apoptosis effectively by correcting the hypoxia microenvironment,which is not associated with the arrest of cell cycle.