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制备纯化短葶山麦冬皂苷C(DT-13)单克隆抗体,选用人脐静脉内皮细胞(HUVEC)及人宫颈癌细胞(HeLa)为研究对象,通过MTT法筛选给药浓度,以细胞免疫荧光定位法探索DT-13在细胞内的作用分布和蓄积情况及其特点。结果显示,在对细胞无显著毒性作用的DT-13浓度下,DT-13能迅速透过细胞膜进入胞浆,逐渐进入细胞核并长时间蓄积。DT-13(5.0μmol/L)在给药30 min前主要分布于人脐静脉内皮细胞(HUVEC)细胞的胞浆,给药12 h后主要蓄积于细胞核内且持续时间可达24 h。而对人宫颈癌细胞(HeLa),与HUVEC细胞相比,DT-13(2.5μmol/L)能更快进入并蓄积于细胞核。提示DT-13可能通过作用于细胞核而发挥其主要的生物活性。该方法能直观、可靠、便捷地观察DT-13在细胞内的定位及动态变化,为中药皂苷类活性成分的药效及作用机理研究提供新的技术方法。
The purified monoclonal antibody of Ophiopogon japonicus Cynanchum C (DT-13) was prepared and the human umbilical vein endothelial cells (HUVECs) and human cervical cancer cells (HeLa) were selected as the research objects. The drug concentration was screened by MTT method, Fluorescent localization method to explore the role of DT-13 in the cell distribution and accumulation of the characteristics and characteristics. The results showed that DT-13 could rapidly penetrate the cell membrane into the cytoplasm and gradually enter the nucleus and accumulate for a long time under the concentration of DT-13 which has no significant toxic effect on the cells. DT-13 (5.0μmol / L) was mainly distributed in the cytoplasm of human umbilical vein endothelial cells (HUVECs) 30 min before administration, and mainly accumulated in the nucleus after 12 h of administration and lasted for 24 h. However, for human cervical carcinoma cells (HeLa), DT-13 (2.5 μmol / L) could enter and accumulate in the nucleus faster than HUVEC cells. Tip DT-13 may play its main biological activity by acting on the nucleus. The method can directly, reliably and conveniently observe the localization and the dynamic change of DT-13 in cells and provide a new technical method for studying the pharmacodynamics and the action mechanism of the traditional Chinese medicine saponin active component.