^(99)Tc^m标记反义miR208b寡核苷酸及其转染离体肥大心肌细胞的实验研究

Transfection of hypertrophic cardiac myocytes in vitro with ^(99)Tc^m-labeled antisense mi R208b oligonucleotide

目的探索用放射性核素99Tcm标记反义mi R208b寡核苷酸,并转染离体早期肥大心肌细胞的实验过程及方法。方法合成针对mi R208b的反义mi R寡核苷酸(AMO),LNA(带锁核酸)修饰AMO,将双功能螯合剂NHS-MAG3(N-羟基琥珀酰亚胺-巯基乙酰基三甘氨酸)与LNA-AMO偶联后,用99Tcm标记,然后用Sep-Pak C18反相层析法对NHS-MAG3-LNA-AMO及其标记物进行洗脱纯化,前者同时经Gene Quant DNA/RNA calculator测定在260 nm波长处的吸光度;不同检测方法测定纯化后标记物的标记率、放化纯度、稳定性以及与靶基因杂交的活性;建立由血管紧张素Ⅱ(AngⅡ)诱导的离体心肌肥大细胞模型,检验早期肥大心肌细胞内mi RNA208b的相对表达量以及经脂质体包裹的99Tcm-NHS-MAG3-LNA-AMO在早期肥大心肌细胞内的滞留率。结果纯化后的标记物标记率>84%,放化纯度>86%,标记物分别在新鲜人血清、生理盐水中孵育12 h后,放化纯度均大于80%,并具有与靶向基因杂合的能力。离体心肌肥大模型成功建立,测得早期心肌肥大细胞内mi RNA208b表达升高,且最终标记物转染细胞后6 h滞留率大于20%。结论在双功能螯合剂NHS-MAG3以及LNA的介导下,成功将放射性核素99Tcm标记于NHSMAG3-LNA-AMO,最后将反义探针顺利转染早期离体肥大心肌细胞,这为后续的在体心肌肥大核素显像提供重要实验基础。

Objective To test the efficiency of transfecting99Tcm- labeled anti- mi R208 b oligonucleotide into early hypertrophic cardiac myocytes in vitro. Methods The anti- oligonucleotide targeting mi R208b（AMO） was synthesized and modified with LNA followed by conjugation with N-hydroxysuccinimidyl S-acetyl-meraptoacetyl triglycine（NHS-MAG3） and radiolabeling with99 Tcm. NHS- MAG3- LNA- AMO and labeled AMO were purified with Sep- Pak C18 column chromatography, and the former was examined for UV absorption at the 260 nm using Gene Quant DNA/RNA calculator. The labeling efficiency,radiochemical purity, stability and molecular hybridization activity were analyzed. An angiotensin II- induced cell model of hypertrophic cardiac myocytes was transfected with99Tcm-NHS-MAG3-LNA-AMO via liposome, and the relative expression of mi RNA208 b and retention ratio of the labeled AMO in early hypertrophic cells were determined. Results The labeling efficiency and radiochemical purity of the labeled AMO after purification exceeded 84% and 86%, respectively. The radiochemical purities of the labeled AMO incubated in serum and normal saline for 12 h were both higher than 80%, and the labeled AMO showed a capacity to hybridize with the target gene. In the hypertrophic model of cardiac myocytes, the retention ratio of labeled AMO at 6 h was higher than 20%. Conclusion The99Tcm- labeled antisense probe can be efficiently transfected into hypertrophic cardiac myocytes in vitro, which provides an experimental basis for subsequent radionuclide imaging studies.