超声微泡介导肾小管上皮细胞β-半乳糖苷酶基因的转导

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/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。

Ultrasound-mediated microbubble destruction enchances β-galactosidase gene transfection and expression in HKCs

ObjectiveTo 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). Methodsβ-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.Resultsβ-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/cm2×60 s) of ultrasound exposure were higher than those of other groups, similar to those of Group 7.ConclusionUnder 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.