低强度脉冲超声对人脐带干细胞体外增殖和成软骨分化的影响

关键词：低强度脉冲超声  脐带间充质干细胞  组织工程  细胞增殖  成软骨分化

Keywords:low-intensity pulsed ultrasound  umbilical cord mesenchymal stem cells  tissue engineering  cell proliferation  chondrogenic differentiation

摘要：

      摘要 目的：研究低强度脉冲超声（LIPUS）对体外培养人脐带间充质干细胞（hUCMSCs）增殖和成软骨分化的影响。 方法：将P4代hUCMSCs随机分为对照组（C组）L30组（30mW/cm2）、L50组（50mW/cm2）、L80组（80mW/cm2），对照组予超声假照处理，各LIPUS刺激组分别刺激5、10、20min/d。LIPUS刺激后观察细胞生长情况，连续刺激1—5天后采用CCK-8检测细胞增殖活性；连续刺激并成软骨诱导培养2周后分别采用ELISA法检测软骨特异性胞外糖胺多糖（GAG）和Ⅱ型胶原蛋白（Col2）的浓度，以及细胞阿利新蓝染色和Col2、DAPI荧光染色观察上述两种软骨特异性蛋白的分布水平。 结果：①LIPUS刺激5d后观察刺激5min/d时L50组细胞集落更为密集，细胞增殖活性组内比较高于L30组和L80组（P<0.05），组间比较高于对照组（P<0.05），刺激10min/d、20min/d时各实验组随着刺激强度增加细胞增殖受到抑制，以L80组抑制增殖效果最显著（P<0.05）。②LIPUS刺激并成软骨诱导5min/d时L30组细胞更为扁平宽大且阿利新蓝染色面积及Col2荧光强度较L50组、L80组及对照组更大，进一步分析胞外分泌GAG和Col2蛋白的含量为实验组高于对照组，以L30最为显著（P<0.05），刺激10min/d、20min/d时各实验组之间以及与对照组组间无显著性差异。 结论：①适宜参数的LIPUS刺激促进细胞增殖，以（50mW/cm2、5min/d）最为显著，但随着刺激强度增大和刺激时间的延长，细胞的增殖能力下降；②在细胞诱导因子的协同作用下，在不影响细胞增殖率的情况下适宜参数（30mW/cm2、5min/d）的低强度脉冲超声可促进人脐带间充质干细胞成软骨分化。

Abstract:

      Abstract Objective：To investigate the effects of low intensity pulsed ultrasound (LIPUS) on proliferation and chondrogenic differentiation of human umbilical cord mesenchymal stem cells (hUCMSCs) in vitro. Method：P4 hUCMSCs were randomly divided into control group (group C， no LIPUS), LIPUS L30 group (30mW/cm2), L50 group (50mW/cm2), L80 group (80mW/cm2), Each LIPUS group was divided into 5, 10, 20 min/d subgroups. After 1-5 days continuous LIPUS stimulation, the cell proliferation activity was detected by CCK-8 After continuous stimulation and induction of cartilage for 2 weeks, the concentration of cartilage specific extracellular glycosaminoglycan(GAG) and type Ⅱ collagen(Col2) were detected by ELISA method, two kinds of cartilage were observed by cell Alicia blue staining and Col2 and DAPI fluorescence staining to assess the level of the distribution of specific proteins. Result：①After 5 min/d stimulation for 5 days, the cell colony of L50 group was more denser and the cell proliferation activity was more higher than that of L30 group， L80 group (P<0.05) and control group (PP< 0.05), The proliferation rate decreased more as the stimulation intensity or duration increased in 10 min/d and 20min/d groups, the L8o and 20min/d group is the most (P<0.05).②When LIPUS stimulation 5min/d and chondrogenic differentiation were used together, the cells in group L30 were more flat and larger, and the area of alcian blue staining and Col2 fluorescence intensity were greater than those in the L50 group, L80 group and the control group. The content of the exocrine GAG and Col2 protein in the experimental group was higher than that in the control group, and the most significant was L30 (P<0.05). There was no significant difference between the experimental group and the control group at 10min/d and 20min/d. Conclusion：①The LIPUS stimulation with appropriate parameters promotes cell proliferation. 50mW/cm2-5min/d is the most significant, but with the increase of stimulation intensity and stimulation time, the cell proliferation ability decreases. ②Under the synergy effect of cell inducible factors, the appropriate parameters (30mW/cm2, 5min/d) LIPUS stimulation can promote chondrogenesis of human umbilical cord mesenchymal stem cells.