Vogt Stephan, Ueblacker Peter, Geis Christopher, Wagner Bettina, Wexel Gabriele, Tischer Thomas, Krüger Achim, Plank Christian, Anton Martina, Martinek Vladimir, Imhoff Andreas B, Gansbacher Bernd
Department of Orthopaedic and Trauma Surgery, Technical University Munich, Munich, Germany.
Biomaterials. 2008 Mar;29(9):1242-9. doi: 10.1016/j.biomaterials.2007.11.013.
Since efficient transfer of foreign genes into primary articular chondrocytes (CC) is difficult, a VSV.G pseudotyped retroviral vector (Bullet) was developed for marker and growth factor gene transfer. Transduction efficiency was analysed by FACS. BMP2 production was determined by specific hBMP2-ELISA. BMP2 effect on cells regarding proteoglycan production was measured by alcian blue staining and dye quantification. Alkaline phosphatase activity was determined by enzymatic reaction with p-nitrophenyl phosphate at OD 405nm and proliferation rate was analysed by MTT-assay. ATDC5 cells (98.3+/-0.6%SD) were transduced to express the reporter gene eGFP. After 52 weeks 94.7+/-0.6%SD of cells were positive. Retroviral transduction efficiency for nlslacZ exceeded 92.3+/-6.1%SD in rabbit CC and expression remained high after 15 weeks (75.7+/-14.2%SD). ATDC5 cells and CC expressed the growth factor gene hBMP2 after retroviral transduction at different time-points. BMP2 led to an increase in proteoglycan and alkaline phosphatase production. Initially, the proliferation rate detected by MTT-assay increased in both the cell types; afterwards the proliferation rate was similar to controls. The described retroviral vector system achieved high initial transduction rates in ATDC5 cells and CC. Gene transfer was very stable over the time period analysed, rendering it a useful tool for future in vitro and in vivo studies on cartilage remodelling.
由于将外源基因有效导入原代关节软骨细胞（CC）存在困难，因此开发了一种VSV.G假型逆转录病毒载体（Bullet）用于标记基因和生长因子基因的转移。通过荧光激活细胞分选术（FACS）分析转导效率。通过特异性hBMP2-ELISA测定骨形态发生蛋白2（BMP2）的产生。通过阿尔辛蓝染色和染料定量来测量BMP2对细胞蛋白聚糖产生的影响。通过在405nm波长下与对硝基苯磷酸进行酶促反应来测定碱性磷酸酶活性，并通过MTT法分析增殖率。对ATDC5细胞（98.3±0.6%标准差）进行转导以表达报告基因增强型绿色荧光蛋白（eGFP）。52周后，94.7±0.6%标准差的细胞呈阳性。在兔CC中，nlslacZ的逆转录病毒转导效率超过92.3±6.1%标准差，并且在15周后表达仍保持较高水平（75.7±14.2%标准差）。在不同时间点进行逆转录病毒转导后，ATDC5细胞和CC表达生长因子基因hBMP2。BMP2导致蛋白聚糖和碱性磷酸酶产生增加。最初，通过MTT法检测到的两种细胞类型的增殖率均增加；之后增殖率与对照组相似。所描述的逆转录病毒载体系统在ATDC5细胞和CC中实现了较高的初始转导率。在所分析的时间段内基因转移非常稳定，使其成为未来软骨重塑体外和体内研究的有用工具。
