大鼠注射型富血小板纤维蛋白诱导干细胞成神经分化  

Neuronal differentiation of bone marrow mesenchymal stem cells induced by injectable platelet-rich fibrin in rat

作　　者：高海明 黄陈翼 李秀军 刘宗超 吴佳奇 GAO Hai-ming;HUANG Chen-yi;LI Xiu-jun;LIU Zong-chao;WU Jia-qi(Department of Orthopedic Surgery,The Affiliated Hospital of Traditional Chinese Medicine,Southwest Medical University,Luzhou 646000,China)

机构地区：[1]西南医科大学附属中医医院骨科,四川泸州646000

出　　处：《中国矫形外科杂志》2020年第6期537-541,共5页Orthopedic Journal of China

基　　金：四川省教育厅自然科学重点项目(编号:17ZA0432)。

摘　　要：[目的]探讨注射型富血小板纤维蛋白(i-PRF)体外诱导骨髓间充质干细胞(BMSCs)成神经细胞分化的能力。[方法]自10~15 d龄SD乳鼠胫骨骨髓分离培养BMSCs至第4代。自SD乳鼠静脉血采用改良低速离心法备制i-PRF。将第4代BMSCs随机分为空白组、150μl组和300μl组,分别加入i-PRF 0、150、300μl,并加入12%FBS进行培养。于第7、14、21 d观察各组细胞形态改变并以免疫组化法及Western blot检测其神经分化情况。[结果]BMSCs经i-PRF诱导7 d后细胞形态开始改变,21 d后突触生长,出现类神经元细胞形态;而空白组细胞形态无改变。细胞免疫组化显示150、300μl组Nestin蛋白表达阳性,而空白组为阴性。免疫荧光染色结果显示150、300μl组S-100β蛋白表达阳性率显著高于空白组。Western blot检测结果表明Nestin、N-cadherin蛋白相对表达量依次为150μl组>300μl组>空白组,差异具有统计学意义(P<0.05)。[结论]i-PRF能有效诱导BMSc成神经细胞分化且受i-PRF剂量的影响。[Objective] To investigate the capacity of injectable platelet-rich fibrin(i-PRF) inducing neuronal differentiation of bone marrow mesenchymal stem cells(BMSCs) in vitro in rats. [Methods] The BMSCs were isolated from the tibial bone marrow of the Sprague-Dawley neonatal rats aged 10~15 days, and cultured until the 4 thgeneration. The i-PRF was prepared with modified low-speed centrifugation by using the venous blood of the rates. After that, the BMSCs of 4 thgeneration were randomly divided into the blank group, 150 μl group and 300 μl group, and furtherly cultured with 0 ul 150 μl and 300 μl of iPRF combined with 12% FBS respectively. The morphological changes of cells were observed on the7 th, 14 thand 21 stdays and the neuronal differentiation were detected by immunohistochemistry and Western blot. [Results] The morphology of BMSCs began to change after 7 days of i-PRF induction, and synaptic growth and neuron-like cell morphology appeared 21 days later.Cell immunohistochemistry showed positive Nestin protein expression in the 150 μl and 300 μl groups, whereas negative expression in the blank group. In addition, the immunofluorescence staining revealed positive S-100 β protein expression in the150 μl group and 300 μl group, while negative in the blank group. Furthermore, the western blot assessment showed that the relative expression of Nestin and N-cadherin protein ranked top-down in a sequence of the 150 μl group > the 300 μl group > the blank group, which proved statistically significant(P<0.05). [Conclusion] The i-PRF does have capacity of inducing neuronal differentiation of BMSCs in this experiment, which might be regulated by the i-PRF concentration.

关 键 词：注射型富血小板纤维蛋白 骨髓间充质干细胞 诱导分化 神经组织工程 

分 类 号：R318[医药卫生—生物医学工程]