血清血浆对成骨细胞增殖和分化的影响

Effects of Rat RP and RS on Cell Proliferation and Osteoblastic Differentiation of Rat Osteoblasts

目的观察大鼠血清和血浆对大鼠成骨细胞的增殖和分化的影响。方法取出生24h SD大鼠头颅骨,Ⅰ型胶原酶多次消化后获得成骨细胞,DMEM加10%FBS进行常规培养,同时细胞免疫荧光鉴定Col1a1的表达,传代后分别用10%血清(rat serum,RS)和10%血浆(rat plasma,RP)进行培养,分别命名为血清组和血浆组。培养24h,镜下观察细胞形态变化及检测增殖蛋白PCNA的表达; CCK8方法检测成骨细胞增殖。成骨诱导刺激1周后,检测(alkaline phosphatase,ALP)染色、ALP活性,同时定量PCR检测ALP、Runx2、Col1a1和OCN基因的表达。分化3周后,进行矿化结节染色。成骨细胞经过血清和血浆处理6h后,标记钙离子探针检测钙离子内流。结果几乎所有的细胞均表达Ⅰ型胶原。血清组成骨细胞明显变得细长,失去成骨细胞原有的特征;大鼠血浆组的PCNA表达水平低于大鼠血清组,CCK8检测表明血清组细胞增殖速度高于血浆组细胞;大鼠血浆组碱性磷酸酶活性及染色显著强于大鼠血清组;成骨性基因ALP、Runx2、OCN和Col1a1在大鼠血浆刺激下,表达明显高于血清组。大鼠血浆组的钙离子内流和矿化结节明显优于大鼠血清组。结论大鼠血清刺激大鼠成骨细胞的增殖,在促进成骨分化方面的作用弱于大鼠血浆的作用。

Objective To investigate the effects of rat plasma and rat serum on the proliferation and differentiation of osteoblasts. Methods rat osteoblasts were isolated from craniums of newly born (within 24 hours)SD rats,and digested with type I collagenase for many times.Osteoblasts were routinely cultured with DMEM plus 10% FBS and identified by detecting the in situ expression of Collal. Osteoblasts were cultured with 10% rat plasma (RP group)and 10% rat serum (RS group)in DMEM for 24 hours after trypsin dispersing.Cell morphology was investigated under inverted microscope.Osteoblasts'proliferation was detected by CCK8 method and the expression levels of PCNA.After osteoblasts were differentiated for one week,ALP staining and ALP activities were detected,and the mRNA levels of ALP,Runx2,Collal and OCN were analyzed by quantitative PCR (qPCR).Mineralized nodules from differentiated osteoblasts were stained by Alizarin red S after three weeks'differentiation.The osteoblasts were incubated with Ca^2+-sensitive dye fluo4-AM molecular probes for 60min after osteoblasts were treated with RS or RP for 6 hours,and labeled cells were investigated under inverted fluorescence microscope.Results All the osteoblast express Collal.The cell morphology was changed from polygon to fibroblastic cells after rat osteoblasts were cultured with medium containing RS.The cell proliferation was accelerated in RS group by cell counting and detecting the expression levels of PCNA.The ALP activities of osteoblasts in RP group were higher than that in RS group.The expression levels of osteogenic genes ALP,Runx2,Collal and OCN in RP group were much higher than that in RS group.Both the mineralized nodules and the intracellular calcium were more obvious in RP group than RS group.Conclusion RS promoted the osteoblasts proliferation,and the effects of osteogenic differentiation by RS were weaker than RP.