中国组织工程研究

中国组织工程研究 ›› 2019, Vol. 23 ›› Issue (2): 251-256.doi: 10.3969/j.issn.2095-4344.0608

• 材料力学及表面改性 material mechanics and surface modification • 上一篇    下一篇

运用液体流动装置模拟的流体剪切应力下骨髓瘤细胞对骨细胞、破骨细胞和骨细胞表达RANKL的影响

王晓桃1，田 申1，何玉婵1，吴春叶1，张俊艳1，阳少芳1，王航飞1，You Lidan2   

  1. 1桂林医学院第二附属医院血液内科，广西壮族自治区桂林市 541000；2加拿大多伦多大学，工程生物学实验室，加拿大多伦多市
  • 收稿日期:2018-08-06 出版日期:2019-01-18 发布日期:2019-01-18
  • 通讯作者: Wang Xiaotao, Department of Hematology, the Second Affiliated Hospital of Guilin Medical University, Guilin 541000, Guangxi Zhuang Autonomous Region, China
  • 作者简介:王晓桃，女，1975年生，湖南省永州市人，2016年广西医科大学毕业，博士，教授，主要从事多发性骨髓瘤骨病的临床与基础研究。
  • 基金资助:

    国家自然科学基金资助项目(81460038，项目负责人：王晓桃)；广西自然科学基金面上项目(2017GXNSFAA198178)；桂林市科学研究与技术开发项目(20170109-49)

Effect of fluid shear force from a fluid flow device simulating mechanical motion on RANKL expression in myeloma cells, osteocytes and osteoclasts

Wang Xiaotao1, Tian Shen1, He Yuchan1, Wu Chunye1, Zhang Junyan1, Yang Shaofang1, Wang Hangfei1,  You Lidan2   

  1.  王晓桃，博士，教授，桂林医学院第二附属医院血液内科，广西壮族自治区桂林市  541000
  • Received:2018-08-06 Online:2019-01-18 Published:2019-01-18
  • About author:Wang Xiaotao, MD, Professor, Department of Hematology, the Second Affiliated Hospital of Guilin Medical University, Guilin 541000, Guangxi Zhuang Autonomous Region, China
  • Supported by:

    the National Natural Science Foundation of China, No. 81460038 (to WXT); the Natural Science Foundation of Guangxi Zhuang Autonomous Region (General Program), No. 2017GXNSFAA198178; the Scientific Research and Technology Development of Guilin, No. 20170109-49

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文题释义：
骨髓瘤细胞：具有无限分裂能力，由于该细胞并非高度分化细胞，因此分裂能力较强，而其他肿瘤细胞分裂能力较弱，因此多在制备单克隆抗体时与B淋巴细胞融合，使单克隆抗体迅速大量增殖。
体外低强度震动流体室装置(flow fluid chamber)：模拟骨髓瘤骨病患者轻微运动或机械运动时产生的流体剪切力在骨髓瘤环境下对骨细胞的影响。
 
 
背景：骨髓瘤骨病因骨骼疼痛、溶骨性破坏导致患者可能懒动而导致骨质丢失增加、骨病加重。
目的：探讨运用液体流动装置模拟人机械运动时产生的流体剪切力在骨髓瘤环境下，瘤细胞(U266细胞)对骨细胞(Y4细胞)、破骨细胞的作用，及其对骨细胞分泌的RANKL表达的影响。
方法：①建立液体流动装置，实验组为U266细胞培养液，对照组为Y4标准培养液，每组均设流动和不流动模式；体外细胞传代培养骨髓瘤细胞系U266细胞和小鼠骨细胞系Y4细胞；②再取新的Y4细胞，按实验分组再培养48 h，显微镜下观察骨细胞、破骨细胞的形态并计数；③ELISA定量检测RANKL的水平；Western Blotting检测RANKL蛋白；④建立RANKL+实验样品+标准培养液的体系，取RAW264.7细胞体外培养，用RANKL干预诱导，TRAP染色观察RAW264.7细胞株分化的破骨细胞数量及状态。
结果与结论：①与对照组相比，U266细胞培养上清下的Y4细胞计数显著下降，形态变化明显；TRAP阳性细胞数增加和RANKL蛋白表达显著升高(P均< 0.05)；②动模式较非流量模式，Y4细胞数量明显增多，TRAP阳性细胞数明显减少，骨细胞表达的RANKL蛋白降低(P均< 0.05)；③结果说明，骨髓瘤细胞可以抑制正常骨细胞的生长和促进RANKL蛋白和破骨细胞的增殖。与静态相比，流体切应力可促进骨细胞的增殖，RANKL蛋白表达受抑制和抑制破骨细胞的增殖。因此，推测机械运动可以防止骨髓瘤骨病进展。 

关键词: 骨髓瘤骨病, 流动液体室装置, 骨细胞, 破骨细胞, 核转录因子κB配体, RANKL, 组织构建

Abstract:

BACKGROUND: Myeloma patients have less movement because of bone pain and osteolytic destruction, which result in bone loss and disease aggravation.

OBJECTIVE: To explore the effect of myeloma cells (U266 cells) on osteocytes (Y4 cells), osteoblasts and osteoclasts as well as RANKL secreted by osteocytes under fluid shear stress simulating human mechanical movement by a fluid flow devise.
METHODS: A flow and no-flow model was established. U266 cell culture fluid was used in the experimental group and Y4 standard culture fluid was used in the flow and control groups. Myeloma cell line U266 and mouse osteocytes cell line Y4 were cultured and passaged in vitro. Y4 cells were allotted and cultured for 48 hours, and the morphology of osteocytes and osteoclasts was observed and counted under microscope. The level of RANKL was quantitatively determined by ELISA and western blot assay. The RANKL, sample and standard culture system was established, RAW264.7 cells were cultured in vitro, and induced by RANKL. Number and morphology of RAW264.7 cells differentiating into osteoclasts were observed by TRAP staining.
RESULTS AND CONCLUSION: Compared with the control group, Y4 cells in U266 culture supernatant exhibited morphological changes and their number was significantly decreased. The number of TRAP-positive osteoclasts was increased and the expression of RANKL protein in Y4 cells was significantly up-regulated (P < 0.05). Comparison between the flow model and the no-flow model showed that the number of Y4 cells was significantly increased, the expression of RANKL protein decreased and the number of TRAP-positive osteoclasts was significantly reduced (P < 0.05). Our study indicates that myeloma cells can inhibit the growth of normal osteocyte and might promote the expression of RANKL protein and the growth of osteoclasts. Compared with static state, fluid shear stress may promote the proliferation of osteocytes and might inhibit the expression of RANKL protein and the growth of osteoclasts. So, we hypothesize that mild exercise may prevent the progression of myeloma bone disease. 

Key words: Multiple Myeloma, Osteoclasts, RANK Ligand, Tissue Engineering

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