Chinese Journal of Tissue Engineering Research ›› 2021, Vol. 25 ›› Issue (25): 3949-3955.doi: 10.12307/2021.003
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Chen Yang, Huang Denggao, Gao Yuanhui, Wang Shunlan, Cao Hui, Zheng Linlin, He Haowei, Luo Siqin, Xiao Jingchuan, Zhang Yingai, Zhang Shufang
Received:
2020-06-20
Revised:
2020-06-28
Accepted:
2020-07-23
Online:
2021-09-08
Published:
2021-03-24
Contact:
Huang Denggao, Associate researcher, Affiliated Haikou Hospital of Xiangya Medicine College, Central South University, Haikou 570208, Hainan Province, China
Zhang Shufang, Researcher, Professor, Affiliated Haikou Hospital of Xiangya Medicine College, Central South University, Haikou 570208, Hainan Province, China
About author:
Chen Yang, Associate chief technician, Affiliated Haikou Hospital of Xiangya Medicine College, Central South University, Haikou 570208, Hainan Province, China
Supported by:
CLC Number:
Chen Yang, Huang Denggao, Gao Yuanhui, Wang Shunlan, Cao Hui, Zheng Linlin, He Haowei, Luo Siqin, Xiao Jingchuan, Zhang Yingai, Zhang Shufang. Low-intensity pulsed ultrasound promotes the proliferation and adhesion of human adipose-derived mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2021, 25(25): 3949-3955.
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2.1 筛选最佳刺激条件 在人脂肪间充质干细胞培养24,48,72 h时以0,10,30,50 mW/cm2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检测细胞增殖活性,结果发现刺激2次后,各组细胞增殖活性均高于对照组(P < 0.05),其中30 mW/cm2组增殖活性最大,在第3次刺激后,10,30 mW/cm2组增殖能力高于对照组(P < 0.05),同样30 mW/cm2组增殖活性最大。因此选用 30 mW/cm2强度刺激2次后的细胞进行后续实验。将 30 mW/cm2组设为实验组,0 mW/cm2组设为对照组。镜下观察30 mW/cm2组的细胞数量增多,而细胞形态不变,见图1。"
2.5 转录组实验结果 2.5.1 差异分析 基因的差异表达分析使用edgeR软件进行。首先根据基因的原始reads数生成相对表达量,其单位为CPM(每百万计数);然后根据分组情况计算差异表达基因。差异表达倍数通过log2转换。显著差异表达基因的筛选阈值为P值< 0.05且|log2(FC)|≥ 1。图5A为基因散点图,图5B为显著差异表达基因热图。与对照组相比,低强度脉冲场超声刺激组有15个基因上调和12基因下调,差异有显著性意义。 2.5.2 差异基因GO通路注释及富集 使用分组差异基因比较中显著(q值 < 0.05)结果进行GO的注释与富集。注释和富集分析的方法为R语言clusterProfiler扩展包,同时使用FDR方法进行多重假阳性矫正。GO显著富集(P值 < 0.05)的主要通路进行注释。结果显示差异基因主要富集的前30类生物学组分为分子功能(序列特异性DNA结合和核酸绑定转录因子活性等)、生物学进程(RNA合成和代谢进程、核酸模板转录调节和染色体结构等)和细胞组分(包括细胞核内腔、核浆和蛋白复合体等),见图6A。 2.5.3 差异基因KEGG通路注释及富集 使用分组差异基因比较显著(P < 0.05)的结果进行KEGG的注释与富集。注释和富集分析的方法为R语言clusterProfiler 扩展包,同时使用FDR方法进行多重假阳性矫正。对KEGG显著富集(P < 0.05)的主要通路进行注释并绘制柱状图。实验结果显示,相关基因主要富集到微环境信号转导、氨基酸代谢、细胞群体和细胞运动性等功能,见图6B。"
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