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目的研究纳米载体Ac-αCD携带的Bcl-xl反义寡核苷酸(antisense oligonucleotide,ASON)对大鼠肺动脉平滑肌细胞(rat pulmonary arterial smooth muscle cells,RPASMCs)增殖和凋亡作用。方法设计合成5’端标记Cy3的Bcl-xlASON,由纳米载体Ac-αCD携带。实验分3组:纳米载体携带的Bcl-xl ASON组(ASON-NPs组)、单纯纳米载体组(NPs组)和空白对照组,分别使用纳米载体Ac-αCD携带的Bcl-xl ASON、纳米载体Ac-αCD和培养液处理RPASMCs 48 h,激光共聚焦显微镜观察RPASMCs对纳米载体携带的Bcl-xl ASON的摄取情况;RT-PCR、Western blot检测Bcl-xl的mRNA和蛋白表达;MTT检测处理后细胞的增殖抑制率;流式细胞仪检测细胞凋亡率。结果激光共聚焦显微镜下可见ASON-NPs组细胞质内大量呈颗粒状均匀分布的红色荧光物质,空白对照组和NPs组细胞细胞质内未见红色荧光物质;ASON-NPs组处理的RPASMCs的Bcl-xl mRNA和蛋白表达显著低于空白对照组和NPs组(P<0.05);ASODN-NPs组、NPs组、空白对照组细胞抑制率分别为:(53.61±3.02)%、(6.30±1.90)%、(1.40±0.62)%,凋亡率分别为:(53.04±2.09)%、(10.98±2.03)%、(2.19±0.11)%、ASON-NPs组和NPs组细胞抑制率、凋亡率均显著高于空白对照组(P<0.01),ASON-NPs组均显著高于NPs组(P<0.01)。结论纳米载体Ac-αCD携带的Bcl-xl反义寡核苷酸能被RPASMCs有效摄取,从而抑制其增殖,促进凋亡。
Objective To investigate the effects of antisense oligonucleotide (ASON) carried by Nanocarrier Ac-αCD on the proliferation and apoptosis of rat pulmonary arterial smooth muscle cells (RPASMCs). Methods Bcl-xlASON was synthesized and labeled with Cy3 on the 5 ’end. It was carried by the nanocarrier Ac-αCD. The experiment was divided into three groups: Bcl-xl ASON group (ASON-NPs group), simple nanocarrier group (NPs group) and blank control group carried by nanocarrier, Ac-αCD and culture medium were used to treat RPASMCs for 48 h. The uptake of Bcl-xl ASON by RPASMCs was observed by laser scanning confocal microscopy. The mRNA and protein expression of Bcl-xl was detected by RT-PCR and Western blot. Cell proliferation inhibition rate; flow cytometry apoptosis rate. Results A large number of red fluorescence were found in the cytoplasm of ASON-NPs group under laser scanning confocal microscope. No red fluorescence was found in the cytoplasm of ASON-NPs group. Bcl-xl NPY group and blank control group were (53.61 ± 3.02)% and (6.30 ± 1.90)%, respectively, which were significantly lower than those in NPs group and blank control group (P <0.05) (1.40 ± 0.62)% and the apoptotic rates were (53.04 ± 2.09)%, (10.98 ± 2.03)% and (2.19 ± 0.11)%, respectively. The rates of cell inhibition and apoptosis were significantly higher in ASON-NPs and NPs groups (P <0.01), ASON-NPs group was significantly higher than NPs group (P <0.01). CONCLUSION: Bcl-xl antisense oligonucleotides carried by the nanocarrier Ac-αCD can be efficiently taken up by RPASMCs, thereby inhibiting their proliferation and promoting apoptosis.