目的构建新型人卵巢癌HO8910细胞株靶向输送系统TAT-OSBP-EGFP ，并对其靶向输送特性和体外活性进行研究鉴定。方法采用PGEX-6P-3质粒分别构建TAT-EGFP、OSBP-EGFP 和TAT-OSBP-EGFP 表达载体，重组质粒转化BL21（ DE3）大肠杆菌，经IPTG诱导表达和GST SefinoseTM Resin柱亲和层析纯化后，采用SDS-PAGE 和Western blotting 鉴定。流式细胞术分析不同浓度（0、1、5、10μmol/L） TAT-EGFP、OSBP-EGFP和TAT-OSBP-EGFP融合蛋白处理HO8910细胞2h后的细胞穿膜率，细胞免疫荧光法检测3种融合蛋白对HO8910的输送特性（以人结肠癌LoVo细胞作对比），CCK-8法检测0、1、5、10、15、40、60、80、100μmol/L TAT-OSBP-EGFP处理HO8910细胞2h后的细胞活性。结果成功构建TAT-OSBP-EGFP原核表达载体，并获得可溶性融合蛋白。与TAT-EGFP相比，当浓度为10μmol/L时，TAT-OSBP-EGFP处理后HO8910细胞的穿膜率无明显升高（ P＞0．05）；但当浓度为1、5μmol/L时，TAT-OSBP-EGFP处理后HO8910细胞的穿膜率升高，差异有统计学意义（ P＜0．01）。经TAT-OSBP-EGFP蛋白处理，HO8910细胞内荧光强度高于LoVo细胞；经TAT-EGFP蛋白处理，HO8910细胞内绿色荧光强度与LoVo细胞相似；经OSBP-EGFP 蛋白处理，两种细胞内未见明显的绿色荧光。不同浓度 TAT-OSBP-EGEP 对HO8910细胞活性无影响（ P＞0．05）。结论成功构建针对人卵巢癌HO8910细胞株的靶向输送系统，为下一步肿瘤靶向药物输送载体的构建并发挥肿瘤的靶向杀灭作用打下了良好的基础。%Objective To construct the new targeting delivery system TAT-OSBP-EGFP of human ovarian cancer cell line HO8910, and perform targeted delivery of its properties and in vitro activity identification. Methods The expression vectors of TAT-OSBP-EGFP, TAT-EGFP and OSBP-EGFP via pGEX-6P-3 plasmid were constructed and transfected into E. coli BL21(DE3). The a-bove vectors were expressed with IPTG induction, purified with GST SefinoseTM Resin colum and subsequently identified by SDS-PAGE and Western blotting analysis. Flow cytometry was used to analyze the cell penetrating efficiency of HO8910 cells which was processed by TAT-EGFP, OSBP-EGFP and TAT-OSBP-EGFP in different concentrations(0, 1, 5, 10μmol/L). The targeted delivery character-istic of the three fusion proteins for HO8910 cells was observed by fluorescence microscopy in comparison with human colon cancer Lo-Vo cells. The CCK-8 kit was employed to detect the activity of HO8910 cells treated with 0, 1, 5, 10, 15, 40, 60, 80, 100μmol/L TAT-OSBP-EGFP for 2h. Results The TAT-OSBP-EGEP expression vector was successfully constructed and a soluble fusion protein was smoothly obtained. Compared with TAT-EGFP, significantly increase was observed in the cell-penetrating rate under 1 and 5μmol/L but not 10μmol/L TAT-OSBP-EGFP to HO8910 cells. The fluorescence intensity( FI) treated by TAT-OSBP-EGFP was higher in HO8910 cells versus LoVo cells. No significant difference was observed on FI between HO8910 cells and LoVo cells treated with TAT-EGFP , while no FI was found in cells treated with OSBP-EGFP . TAT-OSBP-EGEP of different concentrations showed no effect on cell activity( P>0. 05) . Conclusion The carrier system with good human ovarian cell line HO8910 targeting delivery performance is suc-cessfully constructed, providing a good foundation of the next tumor-targeting drug delivery vector and tumor targeting killing system.
展开▼
