低频超声联合微泡增强宫颈癌Hela细胞对顺铂敏感性的实验研究

Low-frequency ultrasound combined with microbubble agent enhances cytotoxicity of cisplatin to Hela cells

目的研究低频率、低声压超声激励微泡增强顺铂对宫颈癌Hela细胞的杀伤作用。方法实验分为对照组(C组),顺铂组(P组),顺铂联合超声组(PU组),超声微泡组(UM组),超声微泡联合顺铂组(PUM组)。采用频率1MHz、声压400 k Pa的非聚焦超声联合脂质微泡及顺铂,按照分组处理对数期生长的宫颈癌Hela细胞。Hoechst33342染色后通过荧光显微镜观察并计算各组细胞的凋亡率;采用Cell Counting Kit-8(CCK-8)法检测处理后各组细胞的增殖活性;对各组细胞行Annexin V-FITC/PI双染色后,运用流式细胞仪检测各组处理后24 h细胞凋亡情况。结果测定Hela细胞对顺铂的敏感性,顺铂的IC50为4.882μg/m L。通过流式细胞仪检测到超声微泡顺铂组(PUM)的细胞凋亡率为58.1%,较顺铂联合超声组(PU)增高40%,明显高于其余各组,组间差异有统计学意义(P<0.05);CCK-8检测结果显示PUM组较其余各组细胞生长明显受到抑制;Hoechst33342染色后,PUM组典型凋亡细胞明显多于其余各组。结论低频率低声压超声联合微泡可以增强顺铂对宫颈癌Hela细胞的杀伤作用,明显抑制Hela细胞生长,促进其凋亡。

Objective To investigate the antitumor effect of ultrasound-mediated chemotherapy, i. e. combination of microbubble agent and cisplatin exposing to low-intensity/low-frequency ultrasound, on human cervical cancer Hela cells. Methods Hela cells were divided into 5 groups： control group （C） , cisplatin group （P）, cisplatin combined ultrasound group （PU）, microbubble enhanced ultrasound （UM）, and cisplatin combined microbubble ultrasound group （PUM）. Nonfocused ultrasound with 1MHz frequency and 400 kPa acoustic pressure was applied to treat the cells in log-phrase of growth. Cell apoptosis was measured by Hoechst33342 staining. Cell proliferation rate was examined by CCK-8 assay. The proportion of cell apoptosis was determined by flow cytometry after cell were treated for 24 h and double-stained with Annexin V-FITC/PI, Results The IC50 of cisplatin was 4. 882 μg/mL in Hela cells. Flow cytometry analysis showed that apoptosis of PUM group was significantly higher than other groups （P 〈 0.05 ）. The apoptotic rate of PUM group was 58.1% , which was 40% higher than that in PU group. Cell growth was markedly inhibited in PUM group compared with other group detected by CCK-8 assay. Also, the apoptotic cells had typical morphological changes observed with inverted fluorescence microscope after Hoechst33342 staining. Conclusion Low- intensity and low-frequency ultrasound combined with microbubble agent can enhance the cytotoxicity of cisplatin to Hela cells by inhibiting proliferation and promoting apoptosis.