反义miR-21增加U251脑胶质瘤细胞对5-氟尿嘧啶化疗敏感性的研究

Antisense microRNA-21 enhances U251 human glioma cell chemo-sensitivity to 5-Fluorouracil

目的探讨as—miR-21增加U251脑胶质瘤细胞对5-FU化疗敏感性的效果。方法透析法制备5-FU／PAMAM载体，透射电镜观察形态，紫外分光光度计检测载药率和包封率；流式细胞仪检测转染效率并分析细胞凋亡比例；MTT法检测共转染后细胞生长抑制效果；免疫荧光检测Ki67和Bcl-2蛋白表达；Transwell检测细胞侵袭能力变化。结果纳米微粒形态规整；平均包封率为66．21％，平均载药量为31．77％；PAMAM转染效率为70．53％；共转染组细胞生长显著抑制（F=273．345，P=0．000）；凋亡比例升高（F=43．21，P=0．000），Ki67、Bcl-2表达均下调；细胞侵袭能力显著下降。结论PAMAM可以有效同载as—miR-21和5-FU，且更加有效地抑制U251脑胶质瘤细胞的体外生长并增加对5-FU的化疗敏感性。

Objective To study the effect of as -miR -21 enhance U251 human glioma cell chemo - sensitivity to 5 - FU. Method 5 - FU/PAMAM complex was prepared by dialysis method. As - miR - 21 was incubated with 5 - FU/PAMAM to form 5 - FU/PAMAM/as - miR - 21. Transmission electronic microscopy（TEM） was performed to observe the morphology of the nanoparticles. The drug loading efficiency and encapsulation efficiency was determined by ultraviolet spectroscopy （UV）. The transfection of PAMAM dendrimer was detected by flow cytometry assay. MTT assay was carried out to determine U251 cell growth survival rate. Cell apoptosis was analyzed by flow - cytometry assay, hnmunofluoresence staining was employed to determine Ki67 and Bcl - 2 expression in U251 cells after combination treatment. Transwell assay was performed to detect cell invasion ability. Results The morphology of the nanoparticle was sphere observed by TEM, and the diameter was less than 100 nm. The encapsulation efficiency and loading efficiency of drug were determined by ultraviolet spectroscopy to be 66. 21% and 31.77% , respectively. The results of flow cytometry assay showed that PAMAM dendrimer transfection efficiency was 70. 53%. The survival rate of U251 cells with as - miR - 21 and 5 - FU co - delivery treatment was significantly suppressed （ F =273. 345, P =0. 000）. The increased apoptosis nuclei percentage in co- delivery treated U251 cell was detected（ F =43.21, P =0. 000） by flow - cytometry assay and the expression of Ki67 and Bcl -2 were down regulated simultaneously analyzed by immunofluorescenee. Transwell result also demonstrated the cell invasion ability of U251 cells treated with 5 - FU combine with as - miR - 21 was decreased. Conclusions PAMAM dendrimer could effectively deliver as - miR - 21 and 5 - FU simultaneously;combination therapy can suppress U251 cell line growth effectively in vitro and can enhance the chemo - sensitivity of glioma cells to 5 - FU chemotherapy.