CAJ | 학술논문

目的:以β-半乳糖苷酶基因作为报告基因,探讨超声微泡介导在人肾近端小管上皮细胞(HKCs)中的转染效率和安全性.方法:体外培养HKCs,分为单纯超声组、单纯微泡组、裸质粒组、超声+质粒组、微泡+质粒组、超声+微泡+质粒组以及VigoFect+质粒组.超声+微泡+质粒组应用微泡和Plenti6-LacZ质粒转导HKCs后,给予不同强度不同时间超声辐照.采用X-gal染色观察细胞基因转导效率,台盼蓝染色观察细胞存活率,Hochest染色观察细胞凋亡率.结果:超声+微泡+质粒组转染效率高于其他各组;与VigoFect+质粒组相比无统计学差异(P>0.05).随超声声强增加和辐照时间延长,HKCs存活率下降,凋亡率升高.声强为0.3 W/cm~2时,辐照时间为60 s时,细胞存活率和转导效率均较高,而细胞凋亡率较低.结论:在适当超声强度和辐照时间条件下,超声微泡可安全、有效地促进肾小管上皮细胞的基因转染,是一种较理想的基因转导方法,这为肾脏病的基因治疗提供了实验基础.gal染色观察细胞基因转导效率,台盼蓝染色观察细胞存活率,Hochest染色观察细胞凋亡率.结果:超声+微泡+质粒组转染效率高于其他各组;与VigoFet+质粒组相比无统计学差异(P>0.05).随超声声强增加和辐照时间延长,HKCs存活率下降,凋亡率升高.声强为0.3 W/cm2时,辐照时间为60 s时,细胞存活率和转导效率均较高,而细胞凋亡率较低.结论:在适当超声强度和辐照时间条件下,超声微泡可安全、有效地促进肾小管上皮细胞的基因转染,是一种较理想的基因转导方法,这为肾脏病的基因治疗提供了实验基础.gal染色观察细胞基因转导效率,台盼蓝染色观察细胞存活率,Hochest染色观察细胞凋亡率.结果:超声+微泡+质粒组转染效率
목적:이β-반유당감매기인작위보고기인,탐토초성미포개도재인신근단소관상피세포(HKCs)중적전염효솔화안전성.방법:체외배양HKCs,분위단순초성조、단순미포조、라질립조、초성+질립조、미포+질립조、초성+미포+질립조이급VigoFect+질립조.초성+미포+질립조응용미포화Plenti6-LacZ질립전도HKCs후,급여불동강도불동시간초성복조.채용X-gal염색관찰세포기인전도효솔,태반람염색관찰세포존활솔,Hochest염색관찰세포조망솔.결과:초성+미포+질립조전염효솔고우기타각조;여VigoFect+질립조상비무통계학차이(P>0.05).수초성성강증가화복조시간연장,HKCs존활솔하강,조망솔승고.성강위0.3 W/cm~2시,복조시간위60 s시,세포존활솔화전도효솔균교고,이세포조망솔교저.결론:재괄당초성강도화복조시간조건하,초성미포가안전、유효지촉진신소관상피세포적기인전염,시일충교이상적기인전도방법,저위신장병적기인치료제공료실험기출.gal염색관찰세포기인전도효솔,태반람염색관찰세포존활솔,Hochest염색관찰세포조망솔.결과:초성+미포+질립조전염효솔고우기타각조;여VigoFet+질립조상비무통계학차이(P>0.05).수초성성강증가화복조시간연장,HKCs존활솔하강,조망솔승고.성강위0.3 W/cm2시,복조시간위60 s시,세포존활솔화전도효솔균교고,이세포조망솔교저.결론:재괄당초성강도화복조시간조건하,초성미포가안전、유효지촉진신소관상피세포적기인전염,시일충교이상적기인전도방법,저위신장병적기인치료제공료실험기출.gal염색관찰세포기인전도효솔,태반람염색관찰세포존활솔,Hochest염색관찰세포조망솔.결과:초성+미포+질립조전염효솔
Objective To investigate the efficiency and safety of ultrasound-mediated microbubble destruction in enhancing β-galactosidase gene (β-gal gene) transfer into human proximal tubular cells(HKCs). Methods β-gal gene was transfected to HKCs as a mark gene with ultrasound-mediated microbubble destruction. Cultured HKCs were grouped to receive the following 7 treatments respectively: ultrasound alone; microbubble alone; naked plasmid; ultrasound and plasmid; microbubble and plasmid; ultrasound, microbubble, and plasmid; and VigoFect and plasmid. In Group 6, HKCs were exposed to ultrasound under different sound intensities and time. X-gal stainning, typan blue stainning, and Hochest stainning were used to detect the transfection efficiency, cell survival rate, and cell apoptosis rate, respectively.Results β-galactosidase expression could be observed in the ultrasound-mediated microbubble destruction groups. Along with the increasing of sound intensity and exposure time, the cell survival rate of HKCs decreased, and the cell apoptosis rate increased gradually. The transduction efficiency and survival rate in middle intensity (0.3 W/cm~2×60 s) of ultrasound exposure were higher than those of other groups, similar to those of Group 7.Conclusion Under optimum sound intensity and exposure time, ultrasound-mediated microbubble destruction can increase gene transfer into HKCs. This non-invasive gene transfer method may be a useful tool for clinical gene therapy.imil