超声辐照微泡介导双自杀基因转染对乳腺癌杀伤作用的在体研究

Anti-tumor effect of suicide gene transfection mediated by microbubble-enhanced ultrasonic irradiation on breast cancer: an in vivo study

目的:探讨超声辐照微泡介导CD/TK双自杀基因质粒转染对在体乳腺癌的杀伤效应。方法:用人乳腺癌MCF-7细胞建立裸鼠移植瘤模型后,将荷瘤裸鼠随机分为对照组、质粒组、超声辐照组、超声辐照微泡组,每组5只。对照组仅给予CD与TK相应的前药(5-氟胞嘧啶与更昔洛韦);质粒组注射质粒(含CD/TK基因)与前药;超声辐照组注射质粒与前药,并予超声辐照;超声辐照微泡组注射靶向超声造影剂(质粒与微泡混合物)与前药,并予超声辐照。实验期间记录裸鼠肿瘤生长情况;处理结束后5 d,剥除肿瘤,计算各组的抑瘤率;用倒置荧光显微镜观察目的基因瘤内转染情况,计算基因转染效率;RT-PCR法检测目的基因的表达;免疫组化法计数肿瘤的微血管密度(MVD)。结果:与对照组比较,质粒组肿瘤的生长无统计学差异(P>0.05),而超声辐照组与超声辐照微泡组肿瘤生长被明显抑制(均P<0.05),质粒组、超声辐照组、超声辐照微泡组的抑瘤率分别为3.72%、21.40%、47.13%。质粒组、超声辐照组、超声辐照微泡组基因转染效率分别为0.78%、2.81%、23.87%,后者转染效率明显高于前两组(均P<0.05)。超声辐照组与超声辐照微泡组肿瘤组织中均出现CD/TK基因阳性片段,而对照组与质粒组的肿瘤组织中未见。超声辐照组与超声辐照微泡组肿瘤组织MVD计数均明显低于对照组,且超声辐照微泡组低于超声辐照组(均P<0.05),质粒组MVD计数与对照组无统计学差异(P>0.05)。结论:超声辐照微泡介导的双自杀基因系统能有效提高基因转染效率与表达,从而增强对肿瘤生长和肿瘤微血管的生成的抑制作用。

Objective：To investigate the anti-tumor effect of the transfection of plasmid containing CD/TK double suicide genes mediated by microbubble-enhanced ultrasound exposure on breast cancer in vivo.Methods：After the xenograft tumor model was created in nude mice using human breast cancer MCF-7 cells,the tumor-bearing mice were randomly divided into control group,plasmid group,ultrasonic irradiation group and ultrasonic irradiation plus microbubble agent group,with 5 mice in each group.Mice in control group were only given the corresponding CD and TK prodrugs（5-fluorocytosine and ganciclovir）,mice in plasmid group were injected with the plasmids（containing CD/TK gene） and prodrugs,mice in ultrasonic irradiation group were injected with plasmids and prodrugs combined with ultrasonic irradiation,and those in ultrasonic irradiation plus microbubble agent group were injected with targeted ultrasonic contrast agent（mixture of plasmids and microbubble agent） combined with ultrasonic irradiation.The growth of the tumors was recorded during the experiment.After the tumors were enucleated at post-treatment day 5,the tumor inhibition rate in each group was calculated,the transfection of the target genes in the tumors was observed by an inverted fluorescence microscope,and the transfection efficiency was also analyzed;the expression of the target genes was determined by RT-PCR method;the microvessel density（MVD） of the tumors was detected by immunohistochemical staining.Results：Compared with control group,the tumor growth in plasmid group showed no obvious difference（P〉0.05）,while in both ultrasonic irradiation group and ultrasonic irradiation with microbubble agent group it was significantly inhibited（both P〈0.05）,and tumor inhibition rate in plasmid group,ultrasonic irradiation group and ultrasonic irradiation plus microbubble agent group was 3.72%,21.40%and 47.13%,respectively.The gene transfection efficiency in plasmid group,ultrasonic irradiation group and ultrasonic irradiation plus microbubble agent group was 0.78%,2.81%and 23.87%,respectively,which in the latter group was significantly higher than that in the former two groups（both P〈0.05）.The positive DNA fragment of CD/TK gene was present in the tumors from the ultrasonic irradiation group or ultrasonic irradiation plus microbubble agent group,but was absent in those from control group and plasmid group.The MVD count in tumors from both ultrasonic irradiation group and ultrasonic irradiation plus microbubble agent group was significantly lower than that in control group（both P〈0.05）,which in ultrasonic irradiation with microbubble agent group was even lower than that in ultrasonic irradiation group（both P〈0.05）,but showed no statistical difference between plasmid group and control group（P〉0.05）.Conclusion：The double suicide gene system mediated by microbubble-enhanced ultrasonic irradiation can effectively increase the transfection efficiency and expression of the target gene,and thereby exert an enhanced inhibitory effect on tumor growth and angiogenesis.