Wei Xiao-Ling, Liu Dao-Yang, Pan Jie, Lu Pei-Jun, Zhao Jun
Shanghai Stomatological Disease Center, Shanghai Stomatological Disease Hospital. Shanghai 200001, China. E-mail:
Shanghai Kou Qiang Yi Xue. 2016 Oct;25(5):522-526.
To investigate the influence of lyophilization on the biological activity of recombinant lentiviral vectors of bone morphogenetic protein 2(BMP-2).
Recombinant lenti-BMP-2 was constructed. lenti-BMP-2 was transfected with rat bone marrow stromal cells (BMSCs) by multiplicity infection (MOI) of 10, 25, 50, 100, 200. The infection efficiency was observed by X-gal staining. Under suitable conditions, the lenti-BMP-2 and 10% trehalose ratio of lyophilized protective agent was mixed into the lyophilization form. Before and after lyophilization, the effect of lenti-BMP-2 on the proliferation of BMSCs was evaluated by MTS assay. The expression of BMP-2 protein in the cells of lyophilized lenti-BMP-2 was detected by ELISA method. The expression of Runx-2, OCN, Col1 and OPN in BMSCs was detected by real-time PCR after transfection of lyophilized lenti-BMP-2. SPSS13.0 software package was used for statistical analysis.
X-gal staining showed an MOI of 100 pfu/cell, and stable transfection efficiency. Before and after lyophilization, no significant change was observed in regard to the effect of lyophilized lenti-BMP-2 on BMSCs proliferation (P>0.05). ELISA method showed that BMSCs transfected by lyophilized lenti-BMP-2 could express BMP-2 protein continuously and stably at a high level. Before and after lyophilization, the result of real-time PCR showed that no significant difference in the expression of OPN,Col1,OCN and Runx-2 in BMSCs (P>0.05).
Lyophilized lenti-BMP-2 with trehalose can maintain high activity for a long time as an effective and reliable storage method.
探讨冻干处理对重组骨形态发生蛋白2（BMP-2）慢病毒载体生物活性的影响。
构建重组慢病毒-BMP-2。以10、25、50、100、200的感染复数（MOI）将慢病毒-BMP-2转染大鼠骨髓基质细胞（BMSCs）。通过X-gal染色观察感染效率。在合适条件下，将慢病毒-BMP-2与10%海藻糖比例的冻干保护剂混合制成冻干制剂。冻干前后，通过MTS法评估慢病毒-BMP-2对BMSCs增殖的影响。采用ELISA法检测冻干慢病毒-BMP-2细胞中BMP-2蛋白的表达。转染冻干慢病毒-BMP-2后，通过实时PCR检测BMSCs中Runx-2、OCN、Col1和OPN的表达。使用SPSS13.0软件包进行统计分析。
X-gal染色显示MOI为100 pfu/细胞时，转染效率稳定。冻干前后，冻干慢病毒-BMP-2对BMSCs增殖的影响无显著变化（P>0.05）。ELISA法显示，冻干慢病毒-BMP-2转染的BMSCs能够持续稳定地高水平表达BMP-2蛋白。冻干前后，实时PCR结果显示BMSCs中OPN、Col1、OCN和Runx-2的表达无显著差异（P>0.05）。
含海藻糖的冻干慢病毒-BMP-2作为一种有效可靠的保存方法，可长时间保持高活性。
