纤维蛋白凝胶对成骨细胞增殖与分化的影响

EFFECTS OF FIBRIN GELS ON CELL PROLIFERATION AND DIFFERENTIATION IN MC3T3E1 CELL LINE

目的分析MC3T3E1细胞在纤维蛋白凝胶(fibrin gel,FG)内的细胞形态学、细胞增殖和成骨细胞分化作用,为将FG应用于组织工程奠定基础。方法将MC3T3E1细胞接种于3种不同浓度(20、10、5mg/mL)的FG内,分别设为实验组A、B、C组,对照组(D组)采用无凝胶正常培养基培养。于不同时间点通过倒置相差显微镜和激光扫描共聚焦显微镜观察细胞在凝胶内的形态学变化,荧光分光光度计测定细胞增殖状态,酶标仪和von Kossa染色分析细胞ALP活性和钙盐沉积;培养14d和21d行RT-PCR检测ALP和骨涎蛋白(bone sialoprotein,BSP)mRNA的表达。结果MC3T3E1细胞在A、B、C组FG内培养至21d,细胞具有较长突起且连接成网,而D组细胞呈纺锤形或立方形。D组细胞随培养时间延长荧光强度逐渐增高,至14d达最高,28d降至最低;A、B、C组细胞培养至28d时荧光强度达最高,与D组比较差异有统计学意义(P<0.05)。D组细胞随培养时间延长ALP活性逐渐升高,至28d达最高;A、B组培养21d时ALP活性最强,与D组比较差异有统计学意义(P<0.05),其余各组各时间点与D组比较差异均无统计学意义(P>0.05)。von Kossa染色示培养21、28d,A组在细胞附近的凝胶区出现矿化结节,D组至28d仍未见矿化结节形成。RT-PCR检测示培养后21d,A组的ALPmRNA和BSP mRNA表达均较D组增强(P<0.05)。结论MC3T3E1细胞在FG内培养21d后成骨细胞分化最明显,细胞增殖维持活跃状态。

Objective To analyze MC3T3E1 cell morphology, proliferation, and osteogenic differentiation in fibrin gel （FG） so as to lay a fundament for use of FG in tissue engneering. Methods MC3T3E1 cells were incubated in three concentrations （20, 10 and 5 mg/mL）of FG as the experimental groups （groups A, B and C） and in the common medium culture as the control group （group D）. The cell morphology and distribution in FG were observed by inverted phase contrast microscope and confocal laser scanning microscope at different time. The cell proliferation was assessed by fluorospectrophotometer. The alkaline phosphatase （ALP） activity was detected by automatic biochemistry analyses and yon Kossa staining was used to analyze calcium salts mineralization. RT-PCR was used to analyze the ALP and bone sialoprotein （BSP） mRNA expression at 14 and 21 days. Results In groups A, B and C, the MC3T3E1 cells had long processes which connected each other and formed network; but fusiform or cube cells were observed in group D at 21 days. The fluorescence intensity was increased gradually with time, was the highest at 14 days and the lowest at 28 days in group D; it was highest in groups A, B and C at 28 days, there were statistically significant differences when compared with group D （P 〈 0.05）. The ALP activity was increased gradually with time, and it was the highest at 28 days in group D and at 21 days in groups A and B, there were significant differences （P 〈 0.05）, no statistically significant differences compared with group D at other time points （P 〉 0.05）. The mineralization nodus were seen at 21 and 28 days in group A, but no mineralization nodus was seen in group D at 28 days. The RT-PCR results showed the mRNA expressions of ALP and BSP were enhanced in group A when compared with group D （P 〈 0.05）. Conclusion The osteogenic differentiation was most obvious and cell proliferation was most active after 21 days of incubation in FG.