富血小板纤维蛋白提取液对MC3T3-E1细胞影响的实验研究

Effects of platelet-rich fibrin extract on MC3T3-E1 cell

目的探讨富血小板纤维蛋白提取液（platelet—rich fibrin extract，PRFe）对成骨细胞增殖、分化及细胞骨架的影响，以期为富血小板纤维蛋白在临床的应用提供理论基础。方法实验组使用含PRFe的α-最低必需培养基（α-minimum essential medium，α-MEM）培养液（10％胎牛血清）培养细胞，对照组使用不含PRFe的Ot—MEM培养液（10％胎牛血清）。甲基噻唑基四唑（methylthiazolyltetrazolium，MTT）法检测培养1、3、5d细胞增殖的A值；以碱性磷酸酶（alkalinephosphatase，ALP）活性检测1、3、5、7d的细胞分化情况；茜素红染色观察14、21d细胞分泌钙结节的情况；激光共聚焦显微镜观察培养后3、6、9及12h的细胞骨架形态；荧光实时定量PCR检测核心结合因子α1（core—bindingfactord1，CbfM）和骨钙蛋白基因分别在培养3、7d的表达。结果MTT检测结果显示，随培养时间延长，细胞数量逐渐增加，在培养1、3、5d实验组细胞A值（分别为0．336±0．011、0．571±0．039、0．787±0．050）均显著高于对照组（分别为0．300±0．021、0．387±0．040、0．527±0．034）（P〈0．05）。两组ALP活性均随培养时间延长而增加，实验组的增加更为显著，在培养1、3、5、7d实验组A值（分别为0．146±0．014、0．199±0．017、0．390±0．020及0．492±0．019）均显著大于对照组（分别为0．115±0．014、0．145±0．015、0．190±0．015及0．230±0．026）（P〈0．05）。茜素红染色结果显示，两组随培养时间延长细胞分泌的钙结节均逐渐增多，培养14、21d实验组细胞分泌的钙结节积分吸光度值（分别为22．119±3．694、31．528±3．162）均较对照组（分别为8．498±2．041、15．162±2．526）显著增加（P〈0．05）。在各时间点，实验组细胞骨架均较对照组更加伸展；两组细胞的基因表达量也随时间逐渐增加，实验组细胞CbfM和骨钙蛋白基因表达量均显著高于对照组（P〈0.05）。结论PRFe能有效促进成骨细胞的增殖和分化，并对细胞骨架早期的排列和伸展有明显的促进作用。

Objective To evaluate the effect of platelet-rich fibrin extract （PRFe） on proliferation and differentiation and F-actin cytoskeleton of osteoblasts. Methods The experimental group used the （x-minimum essential medium（α-MEM） containing PRFe（ 10% fetal bovine serum）, and the control group used the α-MEM （10% fetal bovine serum）.The number of the osteoblasts at 1st,3rd,5th d was detected by methyl thiazolyl tetrazolium（MTT） assay, and the differentiation of osteoblast at 1 st,3rd,5th,7 th d detected by the activity of alkaline phosphatase（ALP）. The alizarin red dye was used to observe the number of calcium nodus at 14th ,21st d. The F-actin cytoskeleton was evaluated by confocal laser scanning microscope （CLSM） at 3rd,6th, 9th, 12th h. The level of osteogenetic biomarkers osteoczlcin（OCN） and core-binding factor cd （Cbfcd） at 3rd,7th d were quantified by real-time PCR. Results A significant increase of absorbance at lst,3rd,5th d was showed in experimental group （0. 336 ±0. 011,0. 571 ± 0. 039,0. 787 ± 0. 050） compared to control group （0. 300 ± 0. 021, 0. 387 ±0. 040,0. 527 ±0. 034） （P 〈0. 05）. The absorbance of experimental group at 1st ,3rd,5th ,7th d（0. 146 ± 0. 014,0. 199±0. 017,0. 390±0. 020,0. 492±0. 019） was significantly higher than that of control group（0. 115 ± 0. 014,0. 145 ±0. 015,0. 190 ±0. 015,0. 230 ±0. 026） （P 〈0. 05）. The integrated absorbance of the calcium nodusin experimental group at 14th,21st d（22. 119 ± 3. 694,31.528± 3. 162） was significantly higher than in control group（8. 498±2.041,15. 162 ±2.526） （P 〈0.05）. The Cbfal and OCN gene expression in experimental group was higher than in control group （ P 〈 0. 05 ） . Conclusions PRFe could enhance the proliferation and differentiation of osteoblasts and promote the spread of F-actin eytoskeleton.