低强度脉冲场超声促进人脂肪间充质干细胞的增殖和黏附  

Low-intensity pulsed ultrasound promotes the proliferation and adhesion of human adipose-derived mesenchymal stem cells

作　　者：陈扬[1] 黄邓高[1] 高元慧[1] 王顺兰[1] 曹卉[1] 郑琳麟 何浩伟 罗思琴 肖敬川[1] 张应爱[1] 张淑芳[1] Chen Yang;Huang Denggao;Gao Yuanhui;Wang Shunlan;Cao Hui;Zheng Linlin;He Haowei;Luo Siqin;Xiao Jingchuan;Zhang Yingai;Zhang Shufang(Affiliated Haikou Hospital of Xiangya Medicine College,Central South University,Haikou 570208,Hainan Province,China)

机构地区：[1]中南大学湘雅医学院附属海口医院,海南省海口市570208

出　　处：《中国组织工程研究》2021年第25期3949-3955,共7页Chinese Journal of Tissue Engineering Research

基　　金：海南省自然科学基金(819QN386),项目负责人:黄邓高;海南省自然科学基金(819QN390),项目负责人:高元慧,海南省自然科学基金(18A200137),项目负责人:张应爱;海南省重大科技项目(ZDZX2013003,ZDKJ2017007),项目负责人:张淑芳。

摘　　要：背景:间充质干细胞是目前组织工程和再生医学研究的热点之一,然而如何在体外进行干细胞的有效扩增以满足临床需求仍是研究者们面临的难题。目的:探讨低强度脉冲场超声对人脂肪间充质干细胞增殖和黏附的影响。方法:取第3代人脂肪间充质干细胞,在人脂肪间充质干细胞培养24,48,72 h时以0,10,30,50 mW/cm~2低强度脉冲场超声强度进行第1,2,3次刺激,每次刺激时间为5 min,刺激结束后将细胞置于培养箱继续培养24 h,然后采用CCK-8实验筛选最佳刺激条件。选用30 mW/cm~2强度刺激2次后的细胞为实验组进行后续实验,0 mW/cm~2刺激为对照组。流式细胞仪术检测细胞表面标志物;免疫组化染色观察细胞增殖相关抗原Ki-67和黏着斑激酶FAK的表达;RT-PCR检测细胞增殖通路相关基因Cyclin D1和c-myc的表达;转录组测序检测转录水平基因的变化。结果与结论:①30 mW/cm~2强度连续刺激2次是促进人脂肪间充质干细胞增殖的最佳条件;②与对照组相比,30 mW/cm~2低强度脉冲场超声刺激后细胞表面标志物未改变,增殖相关基因CyclinD1、c-myc和黏着斑激酶FAK表达上调;③转录组测序结果显示有27个基因出现显著性差异,其中15个基因上调,12个基因下调;④结果表明,低强度脉冲场超声通过上调CyclinD1、c-myc基因和FAK的表达,伴随转录组多个基因的调控,在细胞未分化的状态下,促进人脂肪间充质干细胞增殖。BACKGROUND:Mesenchymal stem cells are one of the hot topics in tissue engineering and regenerative medicine.However,how to effectively expand the stem cells in vitro to meet the clinical needs is still a challenge for researchers.OBJECTIVE:To investigate the effect of low-intensity pulsed ultrasound on the proliferation and adhesion of human adipose-derived mesenchymal stromal cells.METHODS:The human adipose-derived mesenchymal stromal cells of the passage 3 were cultured for 24,48 and 72 hours.The first,second and third times of stimulation were performed with 0,10,30,and 50 mW/cm2 low-intensity pulsed ultrasound intensity,and each stimulation time was 5 minutes.After the stimulation,the cells were placed in the incubator for 24 hours.CCK-8 test was used to screen the best stimulation conditions.The cells stimulated twice with 30 mW/cm2 were selected as the experimental group for follow-up experiments,and 0 mW/cm2 for the control group.Flow cytometry was used to detect cell surface markers.Immunohistochemistry was used to observe the expression of proliferation related antigen Ki-67 and focal adhesion kinase FAK.RT-PCR was used to detect the expression of Cyclin D1 and c-myc.Transcriptome sequencing was used to detect the changes of transcription genes.RESULTS AND CONCLUSION:(1)30 mW/cm2 continuous stimulation twice was the best condition to promote the proliferation of human adipose-derived mesenchymal stem cells.(2)Compared with the control group,cell surface markers remained unchanged after 30 mW/cm2 low-intensity pulsed ultrasound stimulation,and the expression of proliferation-related genes CyclinD1,c-myc and focal adhesion kinase FAK were up-regulated.(3)Transcriptome sequencing results showed that there were significant differences in 27 genes,among which 15 genes were up-regulated and 12 genes were down-regulated.(4)It is concluded that low-intensity pulsed ultrasound promotes human adipose-derived mesenchymal stromal cells proliferation in undifferentiated cells by upregulating the expression of CyclinD

关 键 词：干细胞 脂肪间充质干细胞 超声 低强度脉冲场超声 细胞增殖 细胞黏附 基因 

分 类 号：R459.9[医药卫生—治疗学] R394.2[医药卫生—临床医学]