高能聚焦超声对人胰腺癌移植瘤凋亡相关基因表达的影响

Effects of high-intensity focused ultrasound on apoptosis.associated gene expression in xenografts with human pancreatic cancer

目的探讨高强度聚焦超声（HIFU）辐照对人胰腺癌移植瘤凋亡相关基因的影响。方法建立裸小鼠人胰腺癌YY－1细胞移植瘤模型并进行HIFU辐照，同时设置对照组，用原位末端转移酶标记（TUNEL）法检测肿瘤细胞凋亡率，Agilent人类基因表达谱芯片分析凋亡相关的差异性基因变化，反转录－聚合酶链反应（RT－PCR）、蛋白印迹法（Western blot）对筛选出的部分基因进行验证。结果治疗后第7、14天，辐照组肿瘤细胞凋亡率均高于对照组，凋亡率分别为[（63.6%±15.2%）比（19.0%±2.4%），P〈0.01]、[（41.4%±7.3%）比（18.0%±2.4%），P〈0.01]。表达谱芯片分析示与凋亡信号传导通路相关的差异表达基因共69个，其中上调基因44个，下调基因25个。选取其中4条基因进行RT－PCR验证，其表达结果与芯片结果一致；Western印迹结果示治疗后第7、14天，辐照组Bax蛋白相对表达量高于对照组，分别为[（0.39±0.11）比（0.20±0.09），P〈0.05]和[（0.46±0.12）比（0.24±0.10），P〈0.05]; Bcl－2蛋白相对表达量低于对照组，分别为[（0.68±0.14）比（1.56±0.21），P〈0.05]和[（0.51±0.16）比（1.57±0.22），P〈0.05]。结论HIFU辐照可引起多基因表达改变，通过调控多个信号通道促进肿瘤细胞凋亡，外源性凋亡通道可能发挥主要作用，结合进一步研究有助于阐释HIFU调控凋亡的机制。

Objective To identify the expression of apoptosis-associated genes of high- intensity focused ultrasound（HIFU） in xenograft with human pancreatic cancer.Methods Mice implated with human pancreatic cancer cells （YY-1） were divided into HIFU group or control group. Tumor cell apoptosis was verified by TUNEL. The expression of the apoptosis-associated genes was analyzed by Agilent Human Gene Expression. Selected genes was validated by quantitative real-time PCR（RT-PCR）and Western blot.Results The rate of tumor cell apoptosis in HIFU group was higher than that of control group at 7, 14 days after HIFU treatment （（63.6%±15.2%）vs （19.0%±2.4%）, P〈0.01）and （（41.4%±7.3%）vs（18.0%±2.4%）, P〈0.01）. Gene expression profiling revealed a total of 69 differentially expressed genes in related to apoptosis pathway, among which 44 genes were up-regulated, and 25 genes down-regulated. The RT-PCR results of selected 4 genes were consistent with those of gene expression profiling. The results of Western blot analysis at 7, 14 days after HIFU treatment showed that the expressions level of Bax protein in HIFU group was greater that of in control group （（0.39±0.11）vs （0.20±0.09）, P〈0.05）and （（0.46±0.12）vs（0.24±0.10）, P〈0.05）, while the expressions level of Bcl-2 protein in HIFU group was lower than that of in control group （（0.68±0.14）vs（1.56±0.21）, P〈0.05）and（（0.51±0.16）vs（1.57±0.22）, P〈0.05）.Conclusions HIFU could induce apoptosis and results in dramatic changes in gene expression, indicating that multiple pathways are involved. Although intrinsic pathway might be predominantly involved in HIFU-elicited apoptosis, further research is needed to clarify the detailed mechanisms.