丹皮酚联合^(125)I粒子内照射诱导A549肺癌凋亡的实验研究

Observation of apoptosis of A549 lung cancer in nude mice induced by paeonol combined with ^(125)I seed brachytherapy

目的:本研究旨在通过^(99)Tc^m-膜联蛋白V(Annexin V)显像及磁共振扩散加权成像(DWI)的表观弥散系数(ADC)值分析,活体内观察丹皮酚灌胃、^(125)I粒子植入及两者联合应用治疗裸鼠A549肺癌后肿瘤细胞的凋亡情况。方法:制作裸鼠A549肺癌模型52只,随机分成4组,每组13只,分别为A组:丹皮酚灌胃;B组:^(125)I粒子(14.8 MBq/粒)植入治疗;C组:丹皮酚灌胃+^(125)I粒子植入联合治疗;D组:"冷粒子"植入对照组。治疗10~14天后行DWI成像及ADC值分析,并同期行^(99)Tc^m-Annexin V显像,然后处死裸鼠,剥取瘤体称瘤重,测量残存肿瘤的体积并计算抑瘤率,取瘤体标本原位末端标记法(TUNEL)检测肿瘤细胞凋亡。结果:(1)采用丹皮酚灌胃、^(125)I粒子植入及联合治疗干预,均能明显抑制肿瘤的生长,肿瘤的质量和体积较对照组明显下降,其抑瘤率分别为35.6%、57.3%和74.4%,与对照组比较,均有显著性差异(均P<0.05);(2)A、B、C、D各组^(99)Tc^m-Annexin V细胞凋亡显像阳性率组间差异有统计学意义(χ~2=9.35,P=0.025),与D组相比,丹皮酚灌胃(χ~2=4.51,P=0.031)、^(125)I粒子植入(χ~2=7.96,P=0.004)和两者联合用药(χ~2=15.48,P<0.001)均能提高^(99)Tc^m-Annexin V显像的阳性率;A、B、C、D各组瘤体的^(99)Tc^m-Annexin V摄取比值(T/N)和ADC值组间差异有统计学意义(F=7.218、6.987,均P<0.05),且A、B、C三组的T/N和ADC值均较D组升高,以C组升高明显(均P<0.05);(3)TUNEL法细胞凋亡检测示联合治疗C组的凋亡细胞明显增多,细胞凋亡指数(AI)组间差异有统计学意义(F=5.123,P=0.038);(4)ADC、AI和T/N比值两两相关,ADC相关于AI和T/N的r值分别为0.748和0.782,P值分别为0.032和0.016,AI相关于T/N的r值=0.765,P=0.024。结论:丹皮酚灌胃与^(125)I粒子植入联用可诱导更多的A549细胞凋亡,两者联合应用有协同抗肿瘤作用,可提高治疗疗效;^(99)Tc^m-Annexin V显像联合ADC值分析可早期较全面的从活体水平监测细胞凋亡诱导的效果,从而有助于早期评价治疗疗效。

Objective： The aim of this study was to detect the apoptosis of A549 lung cancer cells after paeonol combined with 125I seed brachytherapy by 99Tcm-Annexin V imaging and analysis of apparent diffusion coefficient（ADC） values of the magnetic resonance diffusion-weighted imaging（DWI）. Methods： Fifty-two nude mice bearing human lung cancer cell line A549 were randomly divided into 4 groups（13 mice in each group）. Group A： paeonol treatment alone. Group B： 125I seed brachtherapy alone. Group C： combination of paeonol and 125I seed brachtherapy. Group D： cold seed implantation as the control group. 10~14 days after brachytherapy, all mice were performed DWI and 99Tcm-Annexin V imaging, and then all mice were sacrificed, the tumor weights and dimensions of survived mice were measured, tumor weight inhibition rate was calculated, tumor cell apoptosis was detected by terminal oxynucleotidy transferase mediated dUTP biotin nick end labeling（TUNEL） immunofluorescence. Results： After the intervention of paeonol, 125I seed brachtherapy and their combination therapy, the growth of tumor cells were significantly inhibited, tumor volume and quality decreased obviously compared with the control group, the tumor inhibitory rates were 35.6%, 57.3% and 74.4%, respectively. The difference in positive rate of 99Tcm-Annexin V imaging between group A, B, C and D were statistically significant（χ2=9.35, P=0.025）; Compared with group D, paeonol（χ2=4.51, P=0.031）, 125I seed brachtherapy（χ2=7.96, P=0.004） and the combination of both（χ2=15.48, P〈0.001） all could increased the positive rate of 99Tcm-Annexin V imaging. There were significant differences in the tumor uptake of 99Tcm-Annexin V（T/N） and the ADC values between groups（F=7.218, 6.987, both P〈0.05）, and the T/N and ADC values in group A, B and C were higher than that in group D, and the rise in group C was especially obvious（all P〈0.05）. The combination of paeonol and 125I seed brachtherapy enhanced cell apoptosis, and the apoptosis index（AI） between groups had statistically significant difference（F=5.123, P=0.038）. The ADC value was correlated with AI and T/N（r=0.748, 0.782, P=0.032, 0.016）, and AI was correlated with T/N（r=0.765, P=0.024）. Conclusion： Paeonol can enhance the anti-tumor effect combined with 125I seed brachytherapy, the mechanism may be related to induced cell apoptosis. 99Tcm-Annexin V imaging combined with the analysis of ADC value could early effectively detect the apoptosis of tumor cells, thus they as visual have the potential to predict efficacy in vivo.