富血小板血浆对三维培养中脐带间充质干细胞成骨分化的影响

Effect of platelet-rich plasma on osteogenic differentiation of umbilical cord mesenchymal stem cells in three-dimensional culture

目的探讨脐血来源的富血小板血浆(PRP)对三维培养体系中脐带间充质干细胞成骨分化的影响。方法收集足月健康剖宫产新生儿脐带标本,采用组织块培养法进行脐带间充质干细胞(h UC-MSCs)的分离培养,观察细胞形态学特征;采用二次离心法提取脐血富血小板血浆和乏血小板血浆(PPP)。将PRP作为h UC-MSCs培养添加剂,PPP凝胶作为h UC-MSCs包埋载体。实验分为A组:10%FBS(对照组);B组:10%FBS+PRP;C组:10%FBS+PPP凝胶;D组:10%FBS+PPP凝胶+PRP。培养7 d后添加成骨诱导培养基,于第7、14天收集各组细胞上清液,比色法测定各组细胞上清液的碱性磷酸酶(ALP)活性。14 d后收集各组h UC-MSCs,RT-PCR测定各组h UC-MSCs成骨相关基因Runx2、ALP和Osteocalcin(OC)的相对表达量。结果经分离纯化的脐带间充质干细胞接种7 d后,各组细胞培养上清中ALP活性的差异无统计学意义;加入成骨诱导培养基7 d后,C、D组中ALP活性高于A、B组,D组高于C组(均P<0.01)。与A组比较,C、D组中Runx2、ALP、OC基因表达均明显上调(P<0.01),其中D组表达最高;B组中Runx2、ALP基因表达提高(P<0.01),而OC基因表达差异无统计学意义(P>0.05)。结论 PPP凝胶可作为体外培养h UC-MSCs的三维载体;PRP可提高h UC-MSCs成骨分化能力。

Objective To investigate the effect of umbilical cord blood-derived platelet-rieh plasma （UCB-PRP） on osteo- genie differentiation of human umbilical cord mesenehymal stem cells （hUC-MSCs） in three-dimensional culture. Methods Umbilical cord and umbilical cord blood from full-term neonatus were collected. Tissue block culture method was used for isolation and culture of hUC-MSCs, and morphological characteristics of the cells were observed; UCB-PRP and UCB-PPP were extracted from umbilical cord blood after twice centrifugation. During the three-dimensional culture, UCB-PRP was used as additive and UCB-PPP gel as encapsulating material for hUC-MSCs. There were four groups divided： Group A： 10%FBS （Control Group）; Group B： 10%FBS＋ PRP; Group C： 10%FBS＋ PPP gel; Group D： 10%FBS＋ PPP gel＋ PRP. Culture medium of osteogenic induction was added, supernatant in each group was collected on Day 7 and 14, and eolorimetric assay was used to measure the activity of alkaline phosphatase （ALP） in supernatant in each group, hUC-MSCs in each group was collected on Day 14, and RT-PCR was used to measure relative expression of Runx2, ALP and Osteocalcin （OC）. Results There was no signifi- cant difference in ALP activity of supematant among all groups on Day 7 of inoculation; After addition of osteogenie induction medium, ALP activity in Group C and D was higher than that in Group A and B, and that in Group D was higher than Group C （P〈0.01）. Compared with Group A, Runx2, ALP and OC gene expression in Group C and D was significantly up-regulated （P〈 0.0]）, and Group D showed the highest expression; the expression of Runx2 and ALP gene in Group B was up-regulated （P〈 0.01）, but there was no statistical difference in the expression of OC gene （P〉0.05）. Conclusion UCB-PPP gel can be used as a three-dimensional carrier for in vitro culture of hUC-MSCs; and UCB-PRP can improve osteogenic differentiation of hUC-MSCs.