【摘要】 目的探讨以靶向趋化因子受体4（CXCR4）为靶点的^99mTc标记小干扰RNA（small interference RNA,siRNA）对人乳腺癌MDA-MB-231细胞目的基因表达的影响,为后续的乳腺癌CXCR4-siRNA基因显像奠定基础。方法人工设计合成三段针对CXCR4 mRNA的siRNA,同时设计出阴性对照序列,质体分别包裹三段干扰序列,转染MDA-MB-231细胞,24 h后进行PCR实验,检测CXCR4 mRNA的表达情况,在48 h后进行Western blot实验,检测CXCR4蛋白的表达,综合分析,选出最佳干扰序列。使用螯合剂HYNIC对干扰效果最好siRNA序列及siRNA（阴性对照）进行放射性^99mTc标记,分别检测其标记率、放射性化学纯度、稳定性及干扰活性。多组间计量资料比较用方差分析进行比较,两组间计量资料比较用LSD-t检验进行比较。结果三段不同序列siRNA作用于MDA-MB-231细胞后,CXCR4 siRNA1作用后的CXCR4 mRNA及蛋白表达量减少的情况与其它两条干扰序列差异有统计学意义。^99mTc-HYNICCXCR4 siRNA1的标记率为（61. 26±2. 47）%（n=5）,阴性对照^99mTc-HYNIC-CXCR4 siRNA的标记率为（60. 85±2. 76）%（n=5）。^99mTc-HYNIC-CXCR4 siRNA1的稳定性好,放射性化学纯度均能达到90%。^99mTc标记前后CXCR4 siRNA干扰活性无差别。结论 CXCR4 siRNA探针能与CXCR4 mRNA上的目的序列结合,抑制目的基因的表达。合成的探针分子稳定性好,具备活体乳腺癌显像的基础条件,有望为乳腺癌精准诊疗提供一条新路径。更多还原

【Abstract】 Objective To investigate the effect of human chemokine receptor 4（ CXCR4） small interference RNA（ siRNA） labeled with^99mTc on the target gene expression of human breast cancer MDA-MB-231 cells,and to lay the foundation for the subsequent development of CXCR4-siRNA gene in breast cancer. Methods Three siRNAs targeted to CXCR4 mRNA were designed and synthesized,and negative control sequences were designed. The plastics werewrapped in three sections of the interference sequence,its carrier transfection breast cancer cell line MDA-MB-231. The mRNA and protein expression of the target gene with RT-PCR after 24 h transfection was investigated and Western blot was used after 48 h to filter out the target sequence which interference effects is best. The HYNIC as its chelating,radiolabeled the CXCR4 siRNA sequence that interference effects was best and siRNA（ negative control） radioactive with^99mTc. The labeling efficiency,stability,radiochemical purity,the activity of molecular hybridi-zation and the interference activity after radiolabeled were analysed. Multivariate metering data were compared using the variance analysis,and the comparison between the two groups was compared with the LSD-t test. Results Three different sequences of siRNA were administered to MDA-MB-231 cells,CXCR4 siRNA1 after the role of CXCR4 mRNA and protein expression decreased with the other two interference sequence difference was statistically significant. The labeling rate of^99mTc-HYNIC-CXCR4 siRNA1 was（ 61. 26 ± 2. 47） %（ n =5）,and the labeling rate of the negative control^99mTc-HYNIC-CXCR4 siRNA was（ 60. 85 ±2. 76） %（ n = 5）.^99mTc-HYNIC-CXCR4 siRNA1 had good stability. Radioactive chemical purity reached 90%. There was no difference in CXCR4 siRNA interference activity before and after^99mTc labeling. Conclusion The CXCR4 siRNA probe binds to the target sequence on CXCR4 mRNA,inhibiting the expression of the gene of interest. Synthetic probe molecular stability,with the basic conditions of live breast cancer imaging,is expected to provide a new path for the diagnosis and treatment of breast cancer.更多还原