低强度脉冲超声波对SD大鼠前软骨干细胞增殖及细胞外基质mRNA表达的影响

Effect of low-intensity pulsed ultrasound on cell proliferation and the mRNA expression of extracellular matrix of Sprague-Dawley rat precartilagious stem cells

背景:研究证明低强度脉冲超声波不仅可以促进骨折愈合,而且还对软骨细胞增殖和细胞外基质分泌具有一定的调节作用。目的:进一步验证低强度脉冲超声波对大鼠前软骨干细胞增殖及细胞外基质分泌的影响。方法:分离新生大鼠乳鼠的前软骨干细胞,鉴定后第3代细胞培养,分为空白对照组、5,12,20min/d组4组。以低强度脉冲超声波刺激后继续培养细胞24h。分别于刺激后第1,3,5天用CCK-8法检测细胞增殖。另以低强度脉冲超声波刺激后间隔30min提取总RNA,用RT-PCR法检测Ⅱ型胶原、Aggrecan、转化生长因子β1及Sox9基因的表达。结果与结论:低强度脉冲超声波对大鼠前软骨干细胞的增殖有促进作用,与对照组相比第1天时20min/d组增殖明显(P<0.05)。大鼠前软骨干细胞Ⅱ型胶原、Aggrecan、转化生长因子β1及Sox9基因mRAN的表达均有明显增加,刺激天数越多mRAN的表达量越多,以20min/d组增高最为明显。提示低强度脉冲超声波可以促进SD大鼠前软骨干细胞的增殖能力,并可以促进细胞外基质mRAN的表达,这一效应可能是通过转化生长因子β1及Sox9基因所介导的。

BACKGROUND： Studies show that low-intensity pulsed ultrasound can improve the healing of bone fracture and regulate the cell proliferation and extracellular matrix secretion of cartilage. OBJECTIVE： To identify the effect of low-intensity pulsed ultrasound on cell proliferation and extracellular matrix secretion of precartilagious stem cells （PCSCs） METHODS： The precartilagious stem cells were isolated from the neonatal Sprague-Dawley （SD） rats and purified with magnetic activated cell sorting, identified by immunocytochemistry with the specific marker （FGFR-3）. Passage 3 cells were divided into 4 groups： Blank control group, 5 minutes per day group, 12 minutes per day group and 20 minutes per day group. The cells were cultured for 24 hours continually after stimulated by LIPU. The cell proliferation was detected by CCK-8 method at the 1st, 3rd and 5th day. The total RNA was collected every 30 minutes after stimulated by low-intensity pulsed ultrasound, and the gene expression of collagen type Ⅱ, Aggrecan, transforming growth factor β1 and Sox9 was detected by real-time polymerase chain reaction. RESULTS AND CONCLUSION： Low-intensity pulsed ultrasound could promote the proliferation of SD rats PCSCs, and compared with the blank control group on the first day only the 20 minutes per day group showed more obvious proliferation than the other groups, the difference was statistically significant （P〈0.05）. Low-intensity pulsed ultrasound can also promote the gene expression of collagen type Ⅱ, Aggrecan, TGF-β1 and Sox9 on SD rats PCSCs significantly. Higher amount of mRNA expression resulted from more stimulation, and the most effective group is 20 minutes per day group. Low-intensity pulsed ultrasound can promote the cell proliferation and extracellular matrix secretion of rat PCSCs. This effect may be mediated by TGF-β1 and Sox9 gene.