富血小板血浆对人乳牙牙髓干细胞的增殖与向成骨分化的调控作用

Regulation of platelet-rich plasma on proliferation and osteogenic differentiation of human deciduous dental pulp stem cells

目的探讨富血小板血浆(mPRP)对人乳牙牙髓干细胞(SHED)的增殖与向成骨分化的调控中的作用。方法收集陕西省宝鸡市中医医院门诊6~8岁儿童拔除的乳牙,酶消化法培养SHED,将细胞随机分为:对照组、3μmol/L组和10μmol/L组,依照分组依次以0μmol/L、3μmol/L、10μmol/LmPRP作用于SHED。采用MTT法检测mPRP对SHED增殖能力的影响,采用碱性磷酸酶(ALP)试剂盘检测mPRP作用于SHED后ALP活性的改变,RT-PCR方法检测细胞内骨涎蛋白与骨钙素mRNA含量的改变。结果①采用mPRP培养的SHED生长形态良好,单细胞形式的成纤维细胞样形态,体积较小,大多数细胞为长梭型或多角型,有些细胞呈现出立方形,细胞胞质均匀,胞体丰满。每5~7天汇合可传代。②mPRP对SHED细胞具有促进其增殖的能力,3μmol/L组与10μmol/L组SHED细胞测得的OD值均高于对照组(P<0.05)。随着mPRP浓度的升高,测得的OD值增大(P<0.05)。③ALP活性方面,在3μmol/L组和10μmol/L组,测得的OD值均随着时间的变化,OD值增大,且差异具有统计学意义(P<0.05)。3μmol/L组与10μmol/L组测得的OD值在不同时间点上均高于对照组,且差异具有统计学意义(P<0.05)。④3μmol/L组及10μmol/L组SHED细胞中骨涎蛋白和骨钙素的mRNA含量均高于对照组,且随着mPRP的浓度升高,促进作用逐渐增强(P<0.05)。结论mPRP对SHED细胞的增殖和向成骨分化均具有一定的促进作用,随着浓度的增大和作用时间的延长,其促进增殖的能力也随之增强,可见mPRP促进SHED的增殖和向成骨分化具有一定效果。

Objective To investigate the role of platelet-rich plasma (mPRP) in regulating the proliferation and osteogenesis of human deciduous dental pulp stem cells (SHED). Methods SHED were cultured by enzymatic digestion method from the extracted deciduous teeth of 6~8 year-old children in Chinese Medicine Hospital of Baoji City. The cells were randomly divided into control group,3 μmol/L group and 10 μmol/L group. According to the grouping,0 μmol/L,3 μmol/L,10 μmol/L mPRP were applied to SHED. The effects of mPRP on the proliferation of SHED were detected by MTT method. Alkaline phosphatase (ALP) reagent disc was used to detect the alteration of ALP activity after mPRP acted on SHED. The changes of bone sialoprotein and osteocalcin in cells were detected by RT-PCR. Results ①SHED cultured with mPRP had good growth morphology,fibroblast-like morphology in single cell form and small size. Most of the cells were spindle-shaped or polygonal. Some cells showed cubic shape,homogeneous cytoplasm and plump cell body. Every 5~7 days confluence can be passed on.②mPRP could promote the proliferation of SHED cells. OD values of SHED cells in 3 μmol/Land 10 μmol/L groups were higher than those in control group ( P <0.05). With the increase of mPRP concentration,the measured OD value increased ( P <0.05).③For ALP activity,the OD values measured in 3 μmol/L and 10 μmol/L groups increased with time,and the difference was statistically significant ( P <0.05). The OD values measured in 3 μmol/L and 10 μmol/L groups were higher than those in control group at different time points,and the difference was statistically significant ( P <0.05).④The expression levels of BSP and osteocalcin in SHED cells of 3 μmol/L and 10 μmol/L groups were higher than those of control group,and the promotion effect was gradually enhanced with the increase of mPR P concentration ( P <0.05). Conclusion MPRP can promote the proliferation and osteogenic differentiation of SHED cells to a certain extent. With the increase of concentration and the prolongation of action time,its ability to promote proliferation is also enhanced. It can be seen that mPRP can promote the proliferation and osteogenic differentiation of SHED cells to a certain extent.