雌激素受体核酸适配体在乳腺癌免疫组织化学检测中的应用

The Application of Estrogen Receptor Aptamer in Immunohistochemical Detection of Breast Cancer

目的筛选出能应用于乳腺癌免疫组织化学检测的雌激素受体(ER)核酸。方法制备ER蛋白,用指数富集的配体系统进化(SELEX)技术筛选出对ER具有高亲和力和特异性的核酸适配体。采用配体-受体相互反应实验来测定合成的ER适配体复合物的亲和力,用生物素化ER适配体和ER单克隆抗体分别作为一抗对105例乳腺癌组织进行免疫组化染色,以检测合成的ER适配体在癌组织中的特异性表达。用Kappa检验进行一致性检验。结果线性回归法计算获得生物素化适配体与ER复合物的解离常数Kd值为(0.34±0.05) nmol/L (n=3,r=0.989),最大结合力(B_(max))为769.23 fmol/(mg prot·nmol^(-1)·L^(-1))。实验结果表明,分别用生物素化ER适配体和ER单克隆抗体分别作为一抗进行免疫组化染色的结果具有高度一致性。其中ER阳性和阴性样本n=105,kappa值=0.943,95%可信区间=0.879～1.006,P<0.05;而ER弱阳性和中度/强表达样本n=75,kappa值=0.805,95%可信区间=0.642～0.967,P<0.05。进一步观察发现,ER适配体和ER抗体能使乳腺癌组织同一部位出现阳性表达,而阴性对照组均无表达。2例子宫内膜癌用生物素化ER适配体检测,癌细胞ER表达均为阳性。结论我们合成的ER适配体在体外能高度结合ER,ER适配体和ER抗体都能检测乳腺癌组织中ER的特异性表达。

Objective To synthesize and select an estrogen receptors(ER)aptamer that can be used in immunostaining of breast cancer tissues.Methods ER protein was purified.ER aptamer that showed a high affinity and specificity for ER was synthesized and selected and by SELEX.Ligand-receptor interactions assay was adopted to measure the affinity of the aptamer-ER complex.Both the biotinylated aptamer and the anti-ER monoclonal antibody were tested for immunohistochemical staining of ER status on 105breast cancer samples.Agreement on the detection of ER expression was determined by Kappastatistics.Results The dissociation contant(Kd)of the biotinylated aptamer-ER complex,as calculated by a linear regression analysis,was determined to be(0.34±0.05)nmol/L(n=3;r=0.989).The binding capacity(Bmax)was 769.23fmol/mg prot·nmol^-1·L^-1).The ER aptamer and the anti-ER antibody both exhibited identical specificity to ER-expressing breast cancer cells.There was a high agreement between the two methods(n=105,Kappavalue=0.943,95%confident interval=0.879-1.006,P<0.05for the ER positive and negtive samples;n=75,Kappavalue=0.805,95%confident interval=0.642-0.967,P<0.05for the ER weak and moderate/strong expression samples).Both the anti-ER antibody and the ER aptamer can also recognized breast cancer cells at the same sites.There was no expression in the negative controls.There were also positive expressions in the 2endometrial cancer tissues by using biotinylated aptamer.Conclusion Our results indicated that the synthesized ER aptamer has a high affinity to bind ER.ER aptamer and the anti-ER antibody can both be used for immunohistochemical staining of ER status in breast cancer tissue.