131I标记适配子的优化研究及其稳定性的(1)
【摘要】 目的 探讨用氯氨T法和Iodogen法进行适配子的131I标记的可行性及标记产物在不同条件下的稳定性。方法 用氯氨T法和Iodogen法分别对适配子进行131I的标记，用纸层析法测定2种方法不同反应时间的标记率，测定标记产物在不同条件下的稳定性。结果 2种方法在反应时间为1 min时的标记率分别为(94.11±0.30) %和(94.01±0.07) %，比较差异无统计学意义(P > 0.05);置于4℃与室温24 h后，标记产物在磷酸盐缓冲液(PBS)、无血清培养基、含10%血清培养基、纯血清中的放射性化学纯度分别为(97.46±0.77) %、(94.76±1.57) %、(85.18±2.27) %、(67.96±1.91) %与(95.78±1.98) %、(56.11±4.06) %、(23.96±3.76) %、(9.45±1.75) %，除PBE介质外，标记产物在4℃条件与室温条件下的放射性化学纯度均有差异(P均 < 0.001);2种温度条件下标记产物在无血清培养基与含10%血清培养基中的放射性化学纯度均有差异(P均 < 0.001)。结论 氯氨T法和Iodogen法均可高效标记适配子，标记产物在低温及无血清条件中稳定性更好。
【关键词】 适配子;氯氨T法;Iodogen法;131I;稳定性
Optimization and evaluation of the stability of 131I labeling aptamer Xu Jiehua， Zhang Guixiong， Yang Min， Huang Wenshan. Department of Nuclear Medicine， the Third Affiliated Hospital of Sun Yat-sen University， Guangzhou 510630， China
Corresponding author， Xu Jiehua， E-mail： xujhgz3@163.com
【Abstract】 Objective To investigate the feasibility of labeling aptamer with 131I using chloramine-T or Iodogen methods and evaluate its stability under different conditions. Methods Aptamer was labeled with 131I using the chloramine-T and Iodogen methods. The labeling rate of different reaction times were measured by paper chromatography. The stability of labeled products under different conditions was determined. Results The labeling rates of two methods at 1 min of reaction time were (94.11±0.30) % and (94.01±0.07) %， respectively， there was no significant difference between them (P > 0.05). The radiochemical purity of labeled products in phosphate buffer(PBS)， serum-free medium， 10% serum medium and serum were (97.46±0.77) %， (94.76±1.57) %， (85.18±2.27) % and (67.96±1.91) % at 4℃for 24 h， (95.78 ±1.98) %， (56.11±4.06) %， (23.96±3.76) % and (9.45±1.75) % at room temperature for 24 h， respectively. Statistical significance was observed under two temperature conditions in serum-free medium， 10% serum medium and serum (all P < 0.001). At 44℃ and room temperature， the radiochemical purity of labeled products in the serum-free medium and 10% serum medium significantly differed (both P < 0.001). Conclusion Both chloramine-T and Iodogen methods can efficiently label the aptamer， and the labeling products have better stability under low temperature and serum-free conditions.
【Key words】 Aptamer;Chloramine-T;Iodogen;131I;Stability
寡核苷酸適配子(aptamer)是用指数富集配基的系统进化(SELEX)技术，通过体外筛选、扩增和富集获得能与多肽、蛋白质、细胞器、核酸以及细胞和组织等靶物质高亲和力、高特异性结合的单链DNA/RNA寡核苷酸[1-3]。本课题组在前期实验中利用基于细胞的SELEX技术(Cell-SELEX技术)筛选出多条对肝癌细胞HepG2细胞株具有高度靶向性及亲和力的适配子JHIT1 ～ JHIT7 (ssDNA)，其中JHIT2的亲和力最佳，然后对适配子JHIT2用Iodogen法进行131I的直接标记，但该法的标记率较低且稳定性较差[4-5]。在适配子筛选的过程中，我们对适配子进行了荧光标记[羧基荧光素(FAM)、FITC、Cy5等]用于流式细胞技术及体外荧光显像试验以探讨其靶向性与亲和力。适配子的荧光标记简单易行，可由公司直接合成及纯化。在这过程中，我们发现适配子连接上功能基团FAM后，FAM可提供多个131I标记的位点，为标记提供了结构基础，预计该法的标记效率会比直接标记法高。在本研究中，我们将在前期研究中筛选出的适配子JHIT2的末端连接上功能基团FAM，分别用氯氨T法及Iodogen法对其进行131I标记，比较2种标记法的标记效能，并研究其标记产物在不同条件下的稳定性，为适配子介导肝癌的靶向放射显像或靶向放射治疗奠定基础。, http://www.100md.com(许杰华?张桂雄?杨敏?黄文山)
【关键词】 适配子;氯氨T法;Iodogen法;131I;稳定性
Optimization and evaluation of the stability of 131I labeling aptamer Xu Jiehua， Zhang Guixiong， Yang Min， Huang Wenshan. Department of Nuclear Medicine， the Third Affiliated Hospital of Sun Yat-sen University， Guangzhou 510630， China
Corresponding author， Xu Jiehua， E-mail： xujhgz3@163.com
【Abstract】 Objective To investigate the feasibility of labeling aptamer with 131I using chloramine-T or Iodogen methods and evaluate its stability under different conditions. Methods Aptamer was labeled with 131I using the chloramine-T and Iodogen methods. The labeling rate of different reaction times were measured by paper chromatography. The stability of labeled products under different conditions was determined. Results The labeling rates of two methods at 1 min of reaction time were (94.11±0.30) % and (94.01±0.07) %， respectively， there was no significant difference between them (P > 0.05). The radiochemical purity of labeled products in phosphate buffer(PBS)， serum-free medium， 10% serum medium and serum were (97.46±0.77) %， (94.76±1.57) %， (85.18±2.27) % and (67.96±1.91) % at 4℃for 24 h， (95.78 ±1.98) %， (56.11±4.06) %， (23.96±3.76) % and (9.45±1.75) % at room temperature for 24 h， respectively. Statistical significance was observed under two temperature conditions in serum-free medium， 10% serum medium and serum (all P < 0.001). At 44℃ and room temperature， the radiochemical purity of labeled products in the serum-free medium and 10% serum medium significantly differed (both P < 0.001). Conclusion Both chloramine-T and Iodogen methods can efficiently label the aptamer， and the labeling products have better stability under low temperature and serum-free conditions.
【Key words】 Aptamer;Chloramine-T;Iodogen;131I;Stability
寡核苷酸適配子(aptamer)是用指数富集配基的系统进化(SELEX)技术，通过体外筛选、扩增和富集获得能与多肽、蛋白质、细胞器、核酸以及细胞和组织等靶物质高亲和力、高特异性结合的单链DNA/RNA寡核苷酸[1-3]。本课题组在前期实验中利用基于细胞的SELEX技术(Cell-SELEX技术)筛选出多条对肝癌细胞HepG2细胞株具有高度靶向性及亲和力的适配子JHIT1 ～ JHIT7 (ssDNA)，其中JHIT2的亲和力最佳，然后对适配子JHIT2用Iodogen法进行131I的直接标记，但该法的标记率较低且稳定性较差[4-5]。在适配子筛选的过程中，我们对适配子进行了荧光标记[羧基荧光素(FAM)、FITC、Cy5等]用于流式细胞技术及体外荧光显像试验以探讨其靶向性与亲和力。适配子的荧光标记简单易行，可由公司直接合成及纯化。在这过程中，我们发现适配子连接上功能基团FAM后，FAM可提供多个131I标记的位点，为标记提供了结构基础，预计该法的标记效率会比直接标记法高。在本研究中，我们将在前期研究中筛选出的适配子JHIT2的末端连接上功能基团FAM，分别用氯氨T法及Iodogen法对其进行131I标记，比较2种标记法的标记效能，并研究其标记产物在不同条件下的稳定性，为适配子介导肝癌的靶向放射显像或靶向放射治疗奠定基础。, http://www.100md.com(许杰华?张桂雄?杨敏?黄文山)