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-外科学(骨外科)专业论文.docx

华中科技大学硕士学位论文 华 中 科 技 大 学 硕 士 学 位 论 文 PAGE 4 PAGE 4 Abstract Objective This study was conducted to discuss the effects of low-intensity pulsed ultrasound （ LIPU ） on the cell proliferation and extracellular matrix secretion of precartilagious stem cells. To learn about the related molecular mechanism of this phenomenon. Method Isolated precartilagious stem cells from the neonatal SD rat’s metaphysics and purified with magnetic activated cell sorting, identified by immunocytochemistry with the specific marker (FGFR-3). Take the third generation of cells which in good conditions. Digest them by trypsin then use contain serum medium terminate digestion and suspend cells. Counting with a microscope then seed cells in 96-well culture plates with the density of 1.6X103/ml. Divid them into 4 groups（control、5min/day、12min/day and 20min/day）. 4 holes are includede in each group. At 37 ℃, saturated humidity, 5% C02 incubator cultured 24h, Intervene cells with low-intensity pulsed ultrasound （ intensity ， 100mW/cm2 ；frequency，1MHz；interval，20%）after them adhere to the bottum.Smear coupling agent evenly in the ultrasonic probe then put it under the culture plate. The cells were detected with CCK-8 to assess cell proliferation at 1day, 3day, 5day. Then seed cells in Petri dish 5 mm in diameter with the density of 2X105/ml.The groups are the same with the former.The cells were collected for RT-PCR detection of Sox-9, Col2α1, aggrecan, TGF-β1 gene expression 30min after the intervention finished. . Result LIPU can promote the proliferation of SD rats，s PCSCs, compared with the control group on the first day only the group of 20 minutes / day shows more obvious proliferation than the other groups, the difference was statistically significant; and in three days and five days after continuous stimulation the discrepancy between experimental and control groups were significantly different, but the differences between experimental groups was not sign