机械牵张应力对人根尖牙乳头干细胞增殖分化的影响

Effects of mechanical stretch stress on the proliferation and differentiation of human stem cells from the apical papilla

目的根尖牙乳头干细胞(SCAP)被看作是根尖周组织再生的种子细胞并被用于以干细胞为基础的根尖周组织再生工程中。文章探讨不同力值的机械牵张应力对人SCAP增殖、分化潜能的影响。方法利用组织块酶消化法及有限稀释法分离、培养人SCAP并加以鉴定。根据对细胞加载机械牵张应力大小不同将实验分为150、200、250 g组,另设空白对照组(未做加力处理)。利用MTT法检测不同大小静态机械牵张应力刺激对SCAP增殖活性的影响。利用Western blot检测机械牵张应力作用下SCAP成骨/成牙分化相关蛋白(ALP、OSX、DSP)表达的变化以及不同大小静态机械牵张应力作用下SCAP内质网应激分子伴侣GRP78的表达情况。结果 SCAP细胞经过3周的成骨诱导后进行茜素红染色,镜下可见块状矿化结节的出现,SCAP细胞经过2周的成脂诱导后进行油红O染色,镜下可见红染的脂滴出现。SCAP细胞表面抗原表型通过流式细胞仪检测分析显示:SCAP对CD31(2.46%,内皮细胞标记物)和CD45(0.07%,造血干细胞标记物)呈阴性表达,对CD90(99.89%,间质细胞标记)和STRO-1(15.21%,干细胞标记)呈阳性表达。与150 g组比较,200 g加力组的加载机械牵张应力第2天,SCAP增殖能力明显下降(P<0.05);250 g加力组的加载机械牵张应力的第2、3、4、5天,SCAP增殖能力显著下降(P<0.05)。与200 g组比较,250 g加力组的加载机械牵张应力的第2、3、4、5天,SCAP增殖能力显著下降(P<0.05)。Western blot检测结果显示:加载牵张应力的第5天,与对照组相比,150 g组、200 g组、250 g组成骨分化相关蛋白ALP和OSX和DSP表达均显著升高(P<0.05)。与150 g组相比,200g组ALP、OSX表达差异无统计学意义(P>0.05),DSP表达显著升高(P<0.05);250 g组ALP、OSX、DSP表达均显著降低(P<0.05)。与200 g组相比,250 g组ALP、OSX、DSP表达均显著降低(P<0.05)。Western blot检测显示:加载牵张应力的第5天,与对照组GRP78表达(0.279±0.085)相比,150、200、250 g组GRP78表达(1.085±0.128、1.289±0.076、0.810±0.067)均显著升高(P<0.05)。结论机械牵张应力对人根尖牙乳头干细胞的增殖和成骨/成牙本质分化具有调控作用。内质网应激参与了机械牵张应力作用下的SCAP成骨/成牙分化过程,并促进了SCAP成骨/成牙分化。

Objective SCAP are seen as seed cells of peri-apical tissue regeneration and used in periapical tissue regeneration project based on stem cells .In this study, we aim to explore the ef-fectdifferent mechanical stretch stress on the proliferation and differen-tiation potential of human stem cells from the apical papilla （ SCAP ） ,and to clarify the mechanism of how mechanical stretch stress regulate human SCAP ,which will provide theoretical guidance for ortho-dontic treatment . Methods Human SCAP was isolated , cultured and identified by combined explants method and enzymatic separa-tion method and limited dilution .MTT assay was used to detect the effect different static mechanical stretch stress stimulation have on the proliferation of SCAP .Western blot was used to detect the expression changes of SCAP osteogenesis /odontoblast differentiation-re-lated protein （ALP, OSX,DSP） under mechanical stretch stressand to detect the expression of SCAP endoplasmic reticulum stress mo -lecular chaperone GRP 78 under different static mechanical stretch stress . Results SCAP were successfully isolated and cultured , and we induced SCAP to differentiate into osteoblasts and adipocytes successfully by osteogenic medium and adipogenic medium .Flow cytometry was performed in accordance with SCAP immunophenotype .Compared with the control group , 150g mechanical stretch stress stimulation promoted SCAP proliferation first and then inhibited SCAP proliferation [（0.481±0.226）,（1.375±0.104）,（1.425± 0.136）,（1.556±0.268）,（0.589±0.29）,P〈0.05].It was same in the 200g group.250g mechanical stretch stress stimulation signifi-cantly inhibited SCAP proliferation [（0.373±0.146）,（0.545±0.069）,（0.745±0.273）,（0.967±0.278）,（1.060±0.362）,P〈0.05]. The expression levels of ALP , OSX and DSP protein in each group were higher than those in the control group （P〈0.05）.Compared with the control group, the expression of GRP78 protein was up-regulated （P〈0.05）. Conclusion Mechanical stretch stress could regulate the SCAP proliferation and osteogenesis/odontoblast differentiation .What′s more,endoplasmic reticulum stress played a role in osteogenesis/odontoblast differentiation under mechanical stretch stress and promoted SCAP osteogenesis /odontoblast differentiation .