低强度超声波对人骨髓间充质干细胞增殖的影响  

Study on proliferation effects of low intensity pulsed ultrasound on human bone marrow mesenchymal stem cells in vitro

作　　者：李志[1] 谢树才 张剑权[1] 符国珍[1] 吴红芬 吕明[1] 符真[1] 周帅[1] Li Zhi;Xie Shucai;Zhang Jianquan;Fu Guozhen;Wu Hongfen;Lyu Ming;Fu Zhen;Zhou Shuai(Department of Hepatobiliary Surgery,Affiliated Haikou Hospital of Central South University Xiangya School of Medicine,Haikou 570208,Chin;Department of Gastroenterology,the Affiliated Haikou Hospital of Central South University Xiangya School of Medicine,Haikou 570208,China)

机构地区：[1]中南大学湘雅医学院附属海口医院肝胆外科,海口570208 [2]中南大学湘雅医学院附属海口医院消化内科,海口570208

出　　处：《中华实验外科杂志》2018年第11期2006-2009,共4页Chinese Journal of Experimental Surgery

基　　金：海南省科技资助项目（ZDXM2015082）;海南省自然科学基金（817382）

摘　　要：目的探讨低强度脉冲式超声波（LIPUS）对人骨髓间充质干细胞（hBMSCs）体外培养增殖的影响及细胞周期蛋白（Cyclin）D1在LIPUS促进hBMSCs增殖中的作用机制。方法以细胞浓度为2.0×10^4／ml接种于12孔培养板，分别采用辐射强度为0、30、40、50、60、80mW／cm^2的LIPUS每天刺激5min，0、24、72、96h后观察细胞形态学变化，用Scepter 2.0手持细胞计数器进行细胞计数，应用流式细胞仪检测细胞周期和增殖指数，细胞增殖-毒性检测试剂盒（CCK-8）检测细胞增殖活力，Western blot检测各强度组Cyclin D1表达水平，反转录-聚合酶链反应（RT-PCR）检测各强度下Cyclin D1 mRNA的表达水平。结果细胞计数发现，除30mW／cm^2（P=0.871）外，40W／cm^2（P=0.006）、50W／cm^2（P=0.001）、60W／cm^2（P=0.001）、80W／cm^2（P=0.004）组细胞数均高于对照组；细胞增殖活力检测发现，30mW／cm^2（P=0.042）、40W／cm^2（P=0.012）、50W／cm^2（P=0.001）、60W／cm^2（P=0.001）、80W／cm^2（P=0.046）组细胞增殖活力较对照组明显增高；细胞周期检测表明，LIPUS组细胞增殖指数显著高于对照组（P=0.001）；Western blot和RT-PCR检测发现，与对照组比较，除30mW／cm^2（P=0.149）外，40W／cm^2（P=0.002）、50W／cm^2（P=0.004）、60W／cm^2（P=0.023）、80W／cm^2（P=0.012）组Cyclin D1的表达均上调；除30mW／cm^2（P=0.075）外LIPUS组Cyclin D1 mRNA表达上调，差异有统计学意义（P=0.001）。结论LIPUS能显著促进hBMSCs增殖，通过上调Cyclin D1的表达促进hBMSCs从G1期进入S期，从而促进其增殖。Objective To investigate the effect of low intensity pulsed ultrasound （LIPUS） on the proliferation of human bone marrow mesenchymal stem cells （hBMSCs） and the effect of Cyelin D1 on the proliferation of hBMSCs induced by LIPUS. Methods The cells were inoculated on the a 12-well culture culture plate at a cell concentration of 2.0×10^4 cells/ml, LIPUS with radiation intensity of 0, 30, 40, 50, 60 and 80 mW/cm^2 was used to stimulate for 5 rain, the morphological changes of the cells were observed at 0, 24, 72 and 96 h, cell counting was performed with a Scepter 2.0 handheld cell counter, the cell cycle and value-added index were measured by flow cytometly, the cell proliferation was detected by cell counting kit-8 （CCK-8）. The expression of Cyclin D1 was detected by Western blotting. The expression of Cyclin D1 mRNA was detected by reverse transcriptase-polymerase chain reaction （ RT -PCR）. Results Cell counts were found except for the 30 mW/cm^2 （ P = 0. 871 ） group that the number of cells in 40W/cm^2 （P=0.006）, 50W/cm^2 （P= 0.001）, 60W/cm^2 （P= 0.001）, 80 W/cm^2 （ P = 0.004） group was significantly higher than that in control group. Cell proliferation assay was found that the proliferation of 30 mW/cm^2 （P=0.042）, 40 W/cm^2 （P=0.012）, 50 W/cm^2 （P= 0. 001 ）, 60 W/cm^2 （ P = 0. 001 ）, 80 W/cm^2 （ P = 0.046） cells was significantly higher than that of the control group. Flow cytometry detection found that the cell proliferation index of LIPUS group was significantly higher than that of control group （P =0.001 ）. Western blotting and RT-PCR showed that there was no significant difference between the 30 mW/cm^2 group and the control group. The expression of Cyclin D1 was significantly increased in 40 W/cm^2 （ P=0.002 ）, 50 W/cm^2 （ P = 0.004 ）, 60 W/cm^2 （ P = 0.023 ）, 80 W/cm^2 （P = 0.012） groups. The expression of Cycling D1 mRNA was significantly different from that of control group （P = 0. 001 ）. Conclusion LIPUS can significantl

关 键 词：低强度脉冲式超声 骨髓间充质干细胞 增殖 细胞周期蛋白D1 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]