低强度脉冲超声对雪旺细胞增生及NT-3基因表达的影响

目的研究培养的雪旺细胞在低强度脉冲超声(low-intensity pulsed ultrasound,LIPUS)直接作用下,对细胞增生、NT-3及脑源性神经生长因子(BDNF)mRNA表达的影响。方法取新生1-3dWistar大鼠坐骨神经,经过酶消化分离雪旺细胞,培养在6孔板上。每天给予超声频率1MHz及声强(SATA)0.1W/cm25min连续刺激,持续14d。对照组除不给予超声刺激外,其他条件与实验组相同。采用5-溴-2-脱氧尿嘧啶实验(BrdU摄入实验)检测细胞增生率的变化,免疫组化及RT-PCR实验检测细胞因子的变化。结果免疫组化结果显示,分离的雪旺细胞标记物S-100鉴定98%以上阳性。BrdU摄入实验发现受超声刺激的细胞在培养第4,7,10,14天细胞增生率较对照组增高,其中前3个时间点差异具有统计学意义。在刺激第14天,实验组NT-3mRNA水平较对照组有显著上调,NT-3/β-actin比值分别为0.56±0.13和0.41±0.09(n=6,P<0.05)。而BDNF水平实验组较对照组显著下调,BDNF/β-actin比值分别是0.51±0.05和0.60±0.08(n=6,P<0.05)。免疫细胞化学证实2组雪旺细胞NT-3及BDNF均有超过98%的阳性率,但表达强度不同。结论给予低强度脉冲超声刺激可促进培养的雪旺细胞增生及神经生长因子表达的改变,本研究为低强度脉冲超声的应用提供了理论依据。

Effect of Low-intensity Pulsed Ultrasound on Schwann Cell Proliferation and Expression of Neurotrophin-3 Protein in Vitro

Objective It is generally known that low-intensity pulsed ultrasound(LIPUS) accelerates peripheral nerve tissue regeneration. However, the precise cellular mechanism is still unclear. The purpose of this study was to determine how the Schwann cells respond directly to LIPUS stimuli. Methods Schwann cells were enzymatically isolated from postnatal 1-3 day rat sciatic nerve tissue and cultured in a 6-well plate. The ultrasound was applied at a frequency of 1 MHz and an intensity of 100mW/cm² spatial average temporal average(ISATA) for 5 min/day. The control group was cultured in the same way but without an administration of ultrasound. We investigated the effect of LIPUS on cell proliferation, neurotrophin-3(NT-3) and brain-derived neurotrophic factor(BDNF) mRNA expression by the methods of BrdU uptake, immunohistochemistry and RT-PCR. Results Immunohistochemistry demonstrated that more than 98% of the cultured cells were positive in S-100, NT-3 and BDNF. Stimulated cells also revealed an increased rate of cell proliferation by using 5-bromo-2'-deoxyuridine(BrdU) assay. The mRNA expression of NT-3 by RT-PCR assays was significantly up-regulated at day 14 after the LIPUS stimulation, whereas, the mRNA expression of BDNF was significantly down-regulated. Conclusion These results suggested that the application of LIPUS can promote cell proliferation and NT-3 gene expression, and down-regulates BDNF gene expression in Schwann cells. The signal-regulated mechanisms involved in this process remains unclear and requires further research.