摘要
目的:探讨骨碎补总黄酮(osteopractic total flavone,OTF)对纳米羟基磷灰石-胶原(nano-hydroxyapatite collagen,nHAC)复合材料与成骨细胞-血管内皮细胞共培养体系中血管内皮细胞增殖的影响。方法:分别以浓度为500 μg·mL^-1 、250 μg·mL ^-1 、100 μg·mL^-1 、50 μg·mL ^-1 及0 μg·mL^-1 的OTF溶液干预生长在nHAC复合材料上的hFOB1.19人成骨细胞,干预24 h后收集5种上清液。再分别以5种上清液和人脐静脉血管内皮细胞(human umbilical vein endothelial cells,HUVEC)培养基混合培养HUVEC,培养24 h和48 h后进行划痕实验,计算细胞迁移率,确定OTF溶液最佳干预浓度和时间。在4个培养皿中铺满nHAC复合材料,加入DMEM/F12。A培养皿中不添加其他材料;B、D培养皿中接种hFOB1.19人成骨细胞,预培养24 h使细胞贴壁;C、D培养皿中加入OTF溶液,根据上一步划痕实验确定的最佳干预浓度和时间进行干预。干预结束后收集各培养皿中的上清液进行后续实验。接种HUVEC于HUVEC培养基上,分为空白组和A、B、C、D共5组;空白组不添加其他材料,A、B、C、D组分别加入A、B、C、D培养皿上清液;以CCK-8法测定细胞增殖率,HE染色后观察HUVEC形态,以ELISA法检测血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)和成纤维细胞生长因子-2(fibroblast growth factor-2,FGF-2)含量。结果:①OTF溶液最佳干预浓度与时间检测结果。5种浓度OTF溶液干预后,0~24 h细胞迁移率的差异有统计学意义[(16.46±4.01)%,(21.71± 1.30)%,(24.94±3.47)%,(22.08±2.46)%,(21.34±2.28)%, F=4.564,P =0.013],其中100 μg·mL ^-1 OTF溶液干预后的细胞迁移率高于其余4组( P=0.001,P=0.020,P=0.014,P =0.029);24~48 h细胞迁移率的差异无统计学意义[(6.06± 1.22)%,(5.37±1.85)%,(5.58±1.88)%,(6.14±1.52)%,(4.99±1.25)%, F=0.374,P =0.823]。提示OTF溶液最佳干预浓度为100 μg·mL ^-1 ,最佳干预时间为24 h。②HUVEC细胞活性检测结果。时间因素与分组因素存在交互效应( F=14.039, P =0.000 );5组HUVEC细胞增殖率总体比较,差异有统计学意义,即存在分组效应( F=83.285,P =0.000);干预开始后不同时点之间HUVEC细胞增殖率的差异有统计学意义,即存在时间效应( F=1 968.467,P =0.000);5组HUVEC细胞增殖率随时间变化均呈升高趋势,各组的变化趋势不完全相同(1.05±0.06,1.81±0.09,2.53±0.05,6.10±0.17,11.29±1.21, F=527.347,P=0.000;0.99±0.11,2.37±0.40,2.92±0.19,7.35±0.19,14.41±1.35,F=913.030,P=0.000;0.98±0.20,3.50±0.18,4.35±0.16,8.53±0.99,15.42±0.73,F=1 637.304,P=0.000;1.02±0.10,2.10±0.13,2.59±0.15,4.70±0.25,11.41±1.21,F=494.876,P=0.000;1.01±0.06,2.81±0.37,3.31±0.08,5.95±0.24,12.74±2.03,F=878.982,P =0.000);干预开始后0 d时,各组细胞增殖率比较,差异无统计学意义;干预开始后1 d、2 d时,B组HUVEC细胞增殖率最高、D组次之,其余3组增殖率较为接近;干预开始后3 d、4 d时,B组HUVEC细胞增殖率最高、A组次之,其余3组增殖率较为接近。③HUVEC形态观察结果。细胞形态观察结果显示,干预开始后0 d、1 d、2 d、3 d、4 d时各组HUVEC的形态均无明显差异。④VEGF和FGF-2含量检测。A、B、C、D 4组VEGF含量比较,差异无统计学意义(0.059±0.012,0.057±0.016,0.059±0.018,0.057±0.014, F=0.060,P = 0.980 )。4组FGF-2含量比较,差异有统计学意义[(215.83±19.56)pg·mL ^-1 ,(565.83±47.14)pg·mL^-1 ,(228.33± 17.67)pg·mL^-1 ,(445.00±17.69)pg·mL^-1 , F=317.377,P =0.000];B组的FGF-2含量高于其余3组( P=0.009,P=0.010,P= 0.025),D组的FGF-2含量高于A组和C组( P=0.013,P =0.017),A组和C组FGF-2含量的差异无统计学意义( P = 0.050 )。结论:人成骨细胞和HUVEC在nHAC复合材料上共培养,均可以良好贴附、生长和增殖;OTF不能促进nHAC复合材料与人成骨细胞-HUVEC共培养体系中HUVEC增殖;HUVEC的增殖可能与成骨细胞分泌FGF-2有关。
Objective: To explore the effects of osteopractic total flavone(OTF)on proliferation of vascular endothelial cells(VECs)of osteoblasts(OBs)-VECs co-culture system in nano-hydroxyapatite collagen(nHAC)composite. Methods: The hFOB1.19 human OBs were cultured in nHAC composite and were intervened by OTF solution with concentration of 500,250,100,50 and 0 μg/mL respectively for 24 hrs,and then 5 kinds of supernatants were collected.The HUVECs were cultured in human umbilical vein endothelial cells(HUVEC)medium supplemented with the 5 kinds of supernatants respectively.The scratch tests were performed after 24- and 48-hour cell cultivation to calculate the cell migration rate and determine the optimal intervention concentration and duration of OTF solution.Four petri dishes were overspread with nHAC composites and were added with DMEM/F12 medium respectively.No other mediums was added into petri dish A,while petri dish B and D were inoculated with hFOB1.19 human OBs and the OBs were precultured for 24 hrs till cell adhesion.The petri dish C and D were supplemented with OTF solution and the optimal intervention concentration and duration were determined by above scratch tests.The supernatant in each petri dish was collected for the following experiments after the end of intervention.HUVEC were inoculated into HUVEC medium and were divided into blank group and group A,B,C and D.The blank group were not added with other mediums,and group A,B,C and D were added with supernatants that collected from petri dish A,B,C and D respectively.The HUVEC proliferation rate was detected by using CCK-8 method,and the morphology of HUVEC was observed through HE staining.The contents of vascular endothelial growth factor(VEGF)and fibroblast growth factor-2(FGF-2)were detected by using ELISA method. Results: After intervention by OTF solution with concentrations of 500,250,100,50 and 0 μg/mL,there was statistical difference in 0-24 hour migration rate of HUVEC between the 5 groups(16.46+/-4.01,21.71+/-1.30,24.94+/-3.47,22.08+/-2.46,21.34+/-2.28%, F=4.564,P =0.013),and the migration rate was higher in group that was intervented by OTF solution with concentration of 100 μg/mL compared to the other 4 groups( P=0.001,P=0.020,P=0.014,P =0.029).There was no statistical difference in 24-48 hour migration rate of HUVEC between the 5 groups(6.06+/-1.22,5.37+/-1.85,5.58+/-1.88,6.14+/-1.52,4.99+/-1.25%, F=0.374,P =0.823).Above results suggested that the optimal intervention concentration of OTF solution was 100 g/mL and the optimal intervention duration was 24 hrs.There was interaction between time factor and group factor in HUVEC activity( F=14.039,P =0.000).There was statistical difference in HUVEC proliferation rate between the 5 groups in general,in other words,there was group effect( F=83.285,P =0.000).There was statistical difference in HUVEC proliferation rate between different timepoints after the beginning of intervention,in other words,there was time effect( F=1 968.467,P= 0.000).The HUVEC proliferation rate presented a time-dependent increasing trend in the 5 groups,while the 5 groups were inconsistent with each other in the variation tendency(1.05+/-0.06,1.81+/-0.09,2.53+/-0.05,6.10+/-0.17, 11.29+/-1.21 , F=527.347,P =0.000;0.99+/-0.11,2.37+/-0.40,2.92+/-0.19,7.35+/-0.19,14.41+/-1.35, F=913.030, P =0.000;0.98+/-0.20,3.50+/-0.18,4.35+/-0.16,8.53+/-0.99,15.42+/-0.73, F=1 637.304,P =0.000;1.02+/-0.10, 2.10+/-0.13 ,2.59+/-0.15,4.70+/-0.25,11.41+/-1.21, F=494.876,P =0.000;1.01+/-0.06,2.81+/-0.37,3.31+/-0.08,5.95+/-0.24,12.74+/-2.03, F=878.982,P =0.000).There was no statistical difference in HUVEC proliferation rate between the 5 groups at the beginning of intervention.The HUVEC proliferation rate was highest in group B and second highest in group D,and was similar to each other in the other 3 groups at 1 and 2 days after the beginning of intervention.The HUVEC proliferation rate was highest in group B and second highest in group A,and was similar to each other in the other 3 groups at 3 and 4 days after the beginning of intervention.The results of observation on HUVEC morphology showed that there was no obvious difference in HUVEC morphology at the beginning of intervention and at 1,2,3 and 4 days after the beginning of intervention.There was no statistical difference in content of VEGF between group A,B, C and D(0.059+/-0.012,0.057+/-0.016,0.059+/-0.018,0.057+/-0.014, F=0.060,P =0.980).There was statistical difference in content of FGF-2 between group A,B,C and D(215.83+/-19.56,565.83+/-47.14,228.33+/-17.67,445.00+/- 17.69 pg/mL, F=317.377,P =0.000).The content of FGF-2 was higher in group B compared to the other 3 groups and was higher in group D compared to group A and group C( P=0.009,P=0.010,P=0.025;P=0.013,P =0.017).There was no statistical difference in content of FGF-2 between group A and group C( P =0.050). Conclusion: Both human OBs and HUVEC,co-cultured in nHAC composite,can adhere,grow and proliferate well.OTF can not promote the proliferation of HUVEC of OBs-HUVECs co-culture system in nHAC composite.The proliferation of HUVEC may be related to FGF-2 secreted by OBs.
作者
李晋玉
俞兴
姜俊杰
赵学千
孙旗
郑晨颖
白春晓
刘楚吟
贾育松
LI Jinyu;YU Xing;JIANG Junjie;ZHAO Xueqian;SUN Qi;ZHENG Chenying;BAI Chunxiao;LIU Chuyin;JIA Yusong(Dongzhimen Hospital of Beijing University of Traditional Chinese Medicine,Beijing 100700,China;Institute of Basic Research in Clinical Medicine of China Academy of Chinese Medical Sciences,Beijing 100700,China)
出处
《中医正骨》
2019年第7期1-8,共8页
The Journal of Traditional Chinese Orthopedics and Traumatology
基金
国家自然科学基金项目(81503601
81603517)
北京中医药大学东直门医院“青苗人才培养计划”项目(DZMYS-201802)
北京中医药大学2018年度基本科研业务项目(2018-JYBZZ-JS096)
中华中药学会青年人才托举工程项目(CACM-2018-QNRC-C08)
关键词
骨碎补
黄酮
细胞增殖
血管内皮细胞
成骨细胞
共同培养技术
羟基磷灰石类
胶原
内皮生长因子
成纤维细胞生长因子2
组织工程
drynaria fortune
flavone
cell proliferation
vascular endothelial cell
osteoblasts
coculture techniques
hydroxyapatites
collagen
endothelial growth factors
fibroblast growth factor 2
tissue engineering
