超声辐照联合双自杀基因慢病毒载体微泡对宫颈癌细胞的杀伤效应

目的： 探讨超声辐照联合双自杀基因慢病毒载体微泡对宫颈癌细胞的体外杀伤效应，为宫颈癌的靶向基因治疗奠定基础。方法：构建双自杀基因慢病毒载体pLenti6-KDRP-CD/TK-EGFP，转染293T细胞并计算病毒滴度，与声诺维微泡混悬液混匀孵育，测定其包封率和载药量。运用载药微泡对宫颈癌HeLa细胞进行干预，试验设空白对照组、慢病毒组、慢病毒载体微泡组、慢病毒载体微泡联合超声辐照组，分别使用4种超声声强 (0.25、0.5、1.0、2.0 W/cm2) 300 kHz，辐照30 s。应用四甲基偶氮唑盐 (MTT)比色实验和流式细胞仪检测各组干预方法对癌细胞增殖和凋亡率的影响。结果：荧光显微镜下可观察到EGFP的表达，说明载体构建成功，转染后获得病毒滴度为3.5×1012 pfu/L，包封率为90.6％±3.1％，载药量为29.2％±0.9％。MTT检测发现与空白对照组比较，慢病毒组、慢病毒载体微泡组、不同超声声强+慢病毒载体微泡组对细胞增殖均有明显抑制作用 (P<0.05或P<0.01)，随着超声辐照声强的增加，癌细胞的抑制率逐步增高 (P<0.05)，但2.0 W/cm2组与1.0W/cm2组相比抑制率无明显差异 (P>0.05)；各组抑制作用于24 h后均有所下降 (P均<0.05)，48 h组与24 h组比较抑制作用无明显变化 (P>0.05)。通过流式细胞术检测发现与空白对照组比较，各干预处理组对HeLa细胞的凋亡作用均显著升高 (P均<0.05)；运用超声辐照后，其凋亡率升高趋势更为显著 (P<0.01)。结论：双自杀基因慢病毒载体对宫颈癌HeLa细胞具有显著的杀伤效应；联合超声辐照其载药微泡的杀伤效应可明显增强，具有协同作用。

Lethal effects on cervical cancer cells by ultrasonic irradiation combined with lentivirus vector microbubble of double suicide gene

OBJECTIVE: To verify the lethal effects on cervical cancer cells by ultrasonic irradiation combined with lentivirus vector microbubble of double suicide gene，to lay the foundation of targeted gene therapy in cervical cancer. METHODS：After constructing lentivirus vector carrying the double suicide gene system (pLenti6-KDRP-CD/TK-EGFP)，transfecting it into the 293T cells，computing virus titer and mingling with microbubble (sonovue)，we then examined the encapsulating rate and drug loading capacity. The cervical cancer cells (HeLa) were divided to four groups：the blank group；the lentivirus vector group；the lentivirus vector microbubble group；ultrasonic irradiation+lentivirus vectormicrobubblegroup：(radiated 30 s by 0.25，0.5，1.0，2.0 W/cm2) 300 kHz.We examined the effect on proliferation of cervical cancer cells by MTT assay and apoptosis by flow cytometry. RESULTS：The virus titer，encapsulating rate and drug loadings capacity were assessed and the expression of EGFP were examined under the fluorescence microscope. MTT assay showed that proliferation of cervical cancer cells was obviously inhibited in the lentivirus vector group，the lentivirus vector microbubblegroup and 0.25 W/cm2+ the lentivirus vector microbubblegroup (P<0.05). When the intensity of ultrasound was higher than 0.5 W/cm2，the inhibition effect was more significant (P<0.01). With progressively lighter intensity of ultrasound，inhibition rates of cervical cancer cells were gradually increased until 2.0 W/cm2，when the inhibition rates tended to be steady. After 24 h，the inhibition effect decreased obviously (P<0.05).After 48 h，there was no significant change for this effect (P>0.05).The apoptosis rates of cervical cancer cells were obviously increased in the lentivirus vector group and lentivirus vector microbubble group (P<0.05). When combining with ultrasonic irradiation，the apoptosis rate of cervical cancer cells was more significant (P<0.01). CONCLUSION： Microbubble carrying lentivirus vector of double suicide gene could kill cervical cancer cells (HeLa). The lethal effect on cervical cancer cells was obviously increased by combining with ultrasonic irradiation.