高强度聚焦超声介导脂质体pEGFP-N1质粒转染胰腺癌细胞的实验研究

Enhancement of high intensity focused ultrasound-mediated transfection of pEGFP-N1 plasmid DNA in pancreatic carcinoma cells in vitro

目的通过观察高强度聚焦超声(HIFU)介导脂质体增强型绿色荧光蛋白质粒pEGFP-N1转染辐照后残余活细胞的效率,探讨HIFU联合基因治疗胰腺癌的可行性。方法 HIFU辐照胰腺癌PANC-1、CFPAC-1细胞,热电偶测温仪记录温度变化,锥虫蓝染色试验检测辐照后即刻杀伤效应,CCK-8试剂盒测定存活细胞生长曲线。辐照后立即加入脂质体pEGFP-N1质粒转染48h,荧光显微镜观察存活细胞中GFP瞬间转染阳性率。比较在不同发射功率及时间作用下温度、细胞活力与转染效率变化特征。结果辐照功率30、50、70W/cm2的平台期温度及达到平台期时间分别为47℃、61s,62℃、44s,82℃、33s。在辐照功率为70W/cm2、辐照10s时温度为56.51℃。胰腺癌PANC-1、CFPAC-1细胞即刻活力分别为(85.91±2.23)%、(84.35±1.65)%;辐照后48h存活细胞的增殖抑制率分别为56.47%、44.60%;存活细胞中脂质体pEGFP-N1质粒瞬间转染率分别为(68.31±2.61)%、(65.85±6.70)%,较对照组分别增加3.7倍和4.3倍。两株细胞在温度变化、细胞破坏、细胞增殖及抑制、质粒转染效率等方面没有统计学差异。结论 HIFU在灭活胰腺癌细胞的同时,可促进辐照区域存活细胞的基因转染,HIFU联合基因治疗具有可行性。

Objective To determine whether high intensity focused ultrasound （HIFU） enhances in vitro transfection of liposome encapsulating plasmids contain enhanced green fluorescent protein N1 （ pEGFP- N1 ） cDNA in pancreatic cancer cells, to explore the feasibility of using a combined HIFU and genetic thera- py for treating pancreatic cancers, and to evaluate the effect of HIFU on cell destruction. Methods Pancre- atic cancer cell models PANC-1 and CFPAC-1 were used. The effect of HIFU treatment on cancer cells was monitored with real-time temperature measurement by thermocouple. The effect of cell destruction effect by HIFU was evaluated with Trypan blue dye exclusion test. Surviving cell growth curve of cancer cells after exposure to HIFU was accessed regularly with cell count kit-8. Liposome encapsulating pEGFP-N1 was trans- fected into cancer cells, and fluorescent protein expression in surviving cells was detected with fluorescence microscope 48 h later. The temperature, the cell activity, and transfection efficiency of cell samples exposed to HIFU of different exposure factors were compared. Results The plateau temperature and time required to reach the temperature were （47℃ ,61 s）, （62℃ ,44 s）, and （82℃ ,33 s） at exposure power of 30, 50, and 70 W/cm2, respectively. The plateau temperature was 56.51℃ after exposure of 10 s at 70 W/cm2, when cell activities of PANC-1 and CFPAC-1 cells were （85.91 ± 2.23） % and （84.35 ±1.65 ） % respectively. Under the same condition, the inhibition rate of growth were 56.47% and 44.60% for the two cell types, and the GFP positive cell of surviving cell expression were （ 68.31± 2.61 ） % and （ 65.85± 6.70 ） % respectively 48 h after treatment. The GFP positive cells in exposure groups increased by 3.7-fold and 4.3- fold compared with the control group for the two cell types. There was no significant difference in temperature, cell destruction, surviving cell growth curve, and transfection efficiency between the two cell types. Conclusion The HIFU enhances the transfection of gene in surviving cells exposed to HIFU by causing cellular destruction in target region, which indicates that a combined of HIF15 and genetic therapy is feasible in treatment of pancreatic cancer.