大鼠雪旺细胞对两种来源的成骨细胞增殖分化影响的研究

目的 研究应用雪旺细胞(SCs)对两种来源的成骨细胞(OB)的增殖与分化的影响,为构建神经化组织工程骨提供体外实验理论依据.方法 通过采用股骨、胫骨髓腔冲洗法获得SD大鼠骨髓基质干细胞(BMSCs),采用胰蛋白酶消化新生鼠颅骨获得OB及采用消化后组织块法获得SCs.将BMSCs在成骨诱导液作用3周,鉴定BMSCs向OB分化.采用96孔共培养板将第2代SCs种植于上室,颅骨来源OB种植下室,共培养为实验组,无SCs干预共培养为对照组.将诱导分化的OB种植于下室,第2代SCs种植于上室,共培养为实验组,无SCs干预共培养为对照组,分别于共培养后1、3、5、7、9d采用甲基噻唑基四唑(MTT)进行增殖比较.采用6孔共培养板,实验组上室种植SCs,下室分别种植颅骨来源及BMSCs来源的OB,不加SCs的两种来源的OB作为对照组,分别共培养后于3、7 d检测两种来源OB的碱性磷酸酶、骨钙素、骨桥蛋白、骨形态发生蛋白-2 mRNA表达.结果 SCs对颅骨来源的OB在共培养3、5、7、9 d 4个时间点均有明显促增殖作用,在碱性磷酸酶、骨桥蛋白、骨形态发生蛋白-2mRNA在各个时间段均起到抑制作用.SCs对大鼠BMSCs来源的OB在共培养1、3 d无明显促增殖作用,在5、7、9 d有明显促增殖作用,在成骨培养基的环境里,SCs可促进BMSCs诱导的OB分化.结论 选择BMSCs来源的OB 与 SCs共培养更适合用于构建神经化组织工程骨.

Proliferation and differentiation of osteoblasts from two sources co-cultured with rat Schwann cells

Objective To explore the proliferation and differentiation of osteoblasts from 2 sources co-cultured with SD rat Schwann cells(SCs) . Methods Bone marrow stromal cells (BMSCs) were obtained by washing the femoral and tibial bone marrow cavities in SD rats. Osteoblast differentiation of the third passage of BMSCs was induced by incubation in osteogenic medium. Primary rat calvarial osteoblasts were obtained by digestion of the calvarial bone in one day old SD rats. The cells were cultured in DMEM supplemented with 10% fetal bovine serum(FBS) . SCs of passage 2 were obtained by digestion of sciatic nerve. The SCs were identified by S-100. The proliferation of 2 kinds of osteoblasts co-cultured with SCs was tested using 96 co-culture plate by methyl thiazdyl tetrazolium(MTF). Real-time PCR was used to test the osteoblast differentiation through co-culturing with SCs in 3 d and 7 d. The osteoblasts were implanted in the subtus chamber. The SCs were implanted in the superior chamber. Results SCs enhanced significantly the proliferation of calvarial osteoblasts at 7 time points. The expression levels of OPN mRNA, OCN mRNA, ALP mRNA, and BMP-2 mRNA of the osteoblasts were significantly lower in the experiment group than in the control group in 3 d and 7 d. SCs also enhanced significantly the proliferation of the induced osteoblasts in 5 d, 7 d and 9 d. The expression levels of OPN mRNA, OCN mRNA, ALP mRNA, and BMP-2 mRNA of the induced osteoblasts were significantly higher in the experiment group than in the control group in 3 d and 7 d, except the level of ALP mRNA in 7 d.Conclusions The BMSCs-induced osteoblasts cocultured with SCs may be used as seed cells to construct neurotized tissue engineered bone.