体外培养人骨髓间充质干细胞向成骨细胞分化的流体剪切应力参数

Parameters of fluid shear stress in the differentiation of cultured human mesenchymal stem cells in vitro into osteoblasts

目的:流体剪切力是骨骼组织中骨骼细胞所感受到的主要应力刺激,可促进骨骼细胞的增殖、分化及保持细胞正常的生理功能。应用流体剪切力刺激体外培养的人骨髓间充质干细胞,观察其向成骨细胞分化的可行性。方法:实验于2004-04/12在第三军医大学西南医院完成,骨髓来源于正常志愿献髓者。进行人骨髓间充质干细胞的分离培养,实验组应用平行平板流动腔对自愿捐献的人骨髓间充质干细胞给予强度为0.3Pa的流体剪切力刺激30min,对照组不加此干预,两组物理环境相同。应用流式细胞仪检测细胞周期,应用透射电镜观察细胞超微结构,检测细胞内碱性磷酸酶含量。评估其向成骨细胞分化和具备功能的特征。放免法检测培养上清液骨钙素含量。评估其是否向成骨细胞成熟晚期分化。并与静态培养对照组作对比。结果:①实验组经流体剪切力作用后,细胞胞体变大,突起较多,多角形细胞较对照组增多;透射电镜观察可见细胞胞浆丰富、核浆比例小,有较多的内质网,高尔基体发达,核仁明显,呈成熟细胞表现。②实验组细胞S期(DNA合成期)比例较对照组明显增高[(13.53±1.47)%,(7.56±2.54)%,P<0.01]。③实验组细胞内碱性磷酸酶含量第9天开始增高,且随时间延长,增高速度加快。④第12天及第24天检测的上清液骨钙素含量两组无明显差别(t=0.263,-0.406,P>0.05)。结论:本实验发现强度为0.3Pa作用30min的流体剪切力可以使人骨髓间充质干细胞早期的细胞内碱性磷酸酶含量增高,细胞开始向成骨细胞分化,但未能促使晚期的骨钙素表达升高,说明此强度的流体剪切力不能成功促进人骨髓间充质干细胞最终实现向成骨方向分化。需要继续探索能够促进人骨髓间充质干细胞向成骨方向分化的流体剪切力的更有效的参数。

AIM:Fluid shear stress(FSS),the major stress that stimulates the cells in ske letal tissues,can accelerate the proliferation of skeletal cells,promote their d ifferentiation and keep their physiological function normal.This study was to st imulate the human mesenchymal stem cells(hMSCs) cultured in vitro by FSS,and pro be into the feasibility of differentiation of hMSCs into osteoblasts. METHODS:The experiment was carried out in Southwest Hospital, Third Military Medical University between April to December 2004,and bone marrow was derived f rom normal volunteers.Having been cultured in vitro to differentiate,the hMSCs w ere stimulated by FSS at 0.3 Pa for 30 minutes using parallel- plate flow chamb er in experimental group,but added with nothing in control group in separate phy sical environment.The cell cycle of hMSCs was measured by flow cytometry,ultrast ructure of hMSCs by transmission electric microscopy(TEM),and meanwhile the leve l of alkaline phosphatase(ALP) was detected so as to evaluate the characters of the differentiation and function of the hMSCs.The concentration of osteocalcin o f the suspension in the culture medium was detected by radioimmunoassay in order to judge whether they were in the late stage of differentiation into osteoblast s,which were then compared with the controls cultured statically. RESULTS:① After the stimulation of FSS,the cell body of hMSCs became bigger, more cell processes and more polygonal cells could be seen in the experiment gro up than in the control group.Shown by TEM,more cytoplasm,lower ratio of nuclei t o plasm,more endoplasmic reticulums,more Golgi bodies,more obvious nucleoli and maturer cells were found in the experiment group,as compared with the control gr oup.② The cell ratio in S period in the experiment group was higher than that i n the control group significantly[(13.53± 1.47)% ,(7.56± 2.54)% ,P< 0.01].③ The level of ALP began to increase from the 9th day,and the increase speed was accelerated over time in the experiment group.④ The osteocalcin concentration o f the culture medium had no significant difference between the two groups on the 12th and 24th days(t=0.263,- 0.406,P >0.05). CONCLUSION:FSS(0.3 Pa,30 minutes) can increase the level of ALP of hMSCs in e arly stage,induce the hMSCs to differentiate into osteoblasts,but not increase t he expression of osteocalcin in early stage,which indicates that FSS at 0.3 Pa c an not successfully enhance the differentiation of hMSCs into osteoblasts in the end.It is necessary to investigate more contributing parameters of FSS that can induce hMSCs to differentiate into osteoblasts.