短葶山麦冬皂苷C的细胞内分布研究

Study on the Distribution in Cells by Ophiopogonin C

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。

To investigate the effect and its feature of location and accumulation of Ophiopogonin C（DT-13） in HUVEC /HeLa cells moreover,a new method for the study of efficacy and mechanism of saponins of Traditional Chinese medicine（TCM） is provided.We obtaind and purified enough DT-13 monoclonal antibodies with the pre-obtained DT-13 hybridoma cell line 1B12,its activities with icELISA are also detectad.The effect of proliferation on HUVEC / HeLa cells after DT-13 with different series of concentrations by MTT assay is investigated.The effect and its feature of location and accumulation of DT-13 in HUVEC / HeLa cells by immunflouresence（FITC DAPI staining） method and studied.The results showed that under the no-cytotoxicitiy concentration of DT-13,it can be carried thraugh in to cells＇ cytoplasm quickly and then got into nucleus gradually and accumulated for a long time.When in HUVEC cells,DT-13（5.0 μmol / L） is in cytoplasm 30 min before,12 h later it is basically all into the nucleus accumulating for at least 24 h.While in HeLa cells,DT-13（2.5 μmol / L） is faster to get into nucleus than in HUVEC cells,slao accumulate for at least 24 h.The resluts suggest that DT-13＇ pharmacological activities may be related to the substances in nucleus mainly,and DT-13＇ main mechanism remains to be further studied.In summary,DT-13＇ localization and dynamic process in cells can be detected by this method with the advantage of direct-viewing,reliability and convenience.Also,this method can be referred when studying on other saponins of TCM or their analogues.