目的：探讨99mTc标记针对靶向趋化因子受体4（CXCR4）mRNA的小干扰RNA（Small interference RNA，siRNA）分子探针用于乳腺癌荷瘤裸鼠基因显像的价值。方法：以双功能螯合剂HYNIC为螯合物对以CXCR4 mRNA某段序列为靶点的siRNA及人无关基因序列为靶点的对照siRNA进行99mTc标记，对其标记率、放化纯及稳定性进行分析。30只乳腺癌荷瘤裸鼠，建立的人乳腺癌MDA-MB-231裸鼠肿瘤模型，随机分成3组（每组10只），分别进行99mTcO4-、99mTc-HYNIC-siRNA（干扰组）和99mTc-HYNIC-siRNA（对照组）肿瘤显像。测量并计算每个时间点的（T/M）的比值。所得测量数据使用方差分析和两样本t检验进行统计分析。结果：干扰组的99mTc标记率为（61.26±2.47）%，对照组的99mTc标记率为（60.85±2.76）%。干扰组和对照组经纯化后，放化纯均达90%以上。干扰组和对照组分别溶于37℃的PBS和正常人新鲜血清中30 min和120 min后，性质保持稳定。注射不同探针后1 h，4 h及10 h时，干扰组T/M比值分别为3.486±0.145、4.574±0.222和6.608±0.366；对照组分别为1.286±0.189、1.348±0.204和1.354±0.238；游离99mTcO4-组分别为1.317±0.164、1.322±0.159和1.401±0.145，在每个成像时间点，3组之间出现的T/M比值差异有统计学意义。干扰组在1 h、4 h、10 h各时间点的T/M比值与对照组及游离99mTcO4-组之间相比有差异，并且差异均有统计学意义（t对照=29.20、33.84、38.07， t游离=31.29、37.61、41.86，P均<0.01），而对照组各时间点的T/M比值与游离99mTcO4-组差异均无统计学意义（t=0.392、0.318、0.533，P均>0.05）。结论：以99mTc标记的siRNA探针可特异性聚集于乳腺癌荷裸鼠肿瘤模型的肿瘤组织，为特异性诊断乳腺癌提供了一种新的方法。
Abstract
Objective: To investigate the possibility of using small interference RNA(siRNA) targeting human chemokine receptor 4(CXCR4) radiolabeled with 99mTc for diagnosing human breast cancer. Methods: Small interference RNA targeted to CXCR4 mRNA and negative control siRNA were synthesized and radiolabeled by 99mTc with the bifunctional chelator HYNIC. The labeling efficiency, radiochemical purity and stability were investigated. Animal models of nude mice bearing human breast cancer MDA-MB-231 were established, divided them into three groups, 10 mice in each group. 99mTcO4-, 99mTc-HYNIC- siRNA(interference group), 99mTc-HYNIC-siRNA(negative control group) were separately injected through tail vein to the three groups. After 99mTcO4-, 99mTc-HYNIC-siRNA(interference group), 99mTc-HYNIC-siRNA(negative control group) were introduced into these animal models, tumor images were acquired with SPECT, the ratio of T/M was calculated. The data were analyzed by two-sample t test and analysis of variance. Results: The labeling efficiency of siRNA(interference group) and siRNA(negative control group) were (61.26±2.47)% and (60.85±2.76)% respectively. The radiochemical purities were both above 90%. Incubated in fresh human serum and PBS for 30 min and 120 min at 37℃, respectively, probes’ properties remained stable in 2 hours. At 1, 4 and 10 h after injection of different probes, the T/M ratios of 99mTc-HYNIC-siRNA(interference group) were 3.486±0.145, 4.574±0.222 and 6.608±0.366, and that of 99mTc-HYNIC-siRNA(negative control group) were 1.286±0.189, 1.348±0.204 and 1.354±0.238, with 99mTcO4- group 1.317±0.164, 1.322±0.159 and 1.401±0.145 respectively, and there were statistically significant differences in different groups at every time spots. The ratios of T/M in 99mTcO4- group and 99mTc-HYNIC-siRNA(negative control group) were significantly lower than those in 99mTc-HYNIC-siRNA(interference group) at 1, 4, 10 h respectively(tcontrol=29.20, 33.84 and 38.07, 99mTcO4-=31.29, 37.61 and 41.86, all P<0.01), and the T/M ratios at 1 h, 4 h, 10 h of 99mTc-HYNIC-siRNA(negative control group) and 99mTcO4- groups were not significantly different(t=0.392, 0.318 and 0.533, all P>0.05). Conclusions: The 99mTc-HYNIC-siRNA(interference group) could specificity accumulated in breast cancer tissue. The imaging with 99mTc-HYNIC-siRNA may be a promising method for diagnosis of breast cancer.
关键词
乳腺肿瘤 /
放射性核素显像
Key words
Breast neoplasms /
Radionuclide imaging
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基金
黑龙江省自然科学基金（面上项目）（H2015066）。
