荨麻提取物诱导骨膜细胞增殖分化:构建组织工程骨及超微结构的变化  

Urtica extracts induce periosteal cell proliferation and differentiation: tissue-engineered bone construction and ultrastructural changes

作　　者：徐兵 柳园 Xu Bing;Liu Yuan(the People’s Hospital of Longhua,Shenzhen 518109,Guangdong Province,China;Shenzhen Longhua District Central Hospital,Shenzhen 518110,Guangdong Province,China)

机构地区：[1]深圳市龙华区人民医院,广东省深圳市518109 [2]深圳市龙华区中心医院,广东省深圳市518110

出　　处：《中国组织工程研究》2018年第12期1805-1810,共6页Chinese Journal of Tissue Engineering Research

基　　金：江西省科技计划项目(20134BBG00009)~~

摘　　要：背景:荨麻作为一种传统的中草药,目前认为其主要的药理作用是抗炎、抗风湿等作用,其对骨膜细胞的效果的相关研究较少。目的:探讨荨麻诱导人骨膜细胞增殖及分化特性,并诱导其体外构骨,为荨麻运用于骨组织工程提供理论依据。方法:实验取材于成人锁骨。体外组织块培养法获得骨膜原代细胞,恒温培养扩增后,取第3代骨膜细胞种植于细胞培养板中。实验分为3组,实验组分别加入10-6,10-5及10-4 g/L荨麻提取物,阳性对照组加入50μg/L骨形态发生蛋白7,对照组仅加入纯净水。分别在第1,4,7和10天MTT法检测骨膜细胞增殖情况,碱性磷酸酶染色和Von Kossa染色检测碱性磷酸酶和钙结节的表达。分别将10-4 g/L荨麻提取物及骨形态发生蛋白7加入到含有聚己内酯三维支架中和骨膜细胞复合培养构建组织工程骨,培养24 d,电镜下观察不同组细胞形态学情况。结果与结论:(1)MTT结果显示各时间点荨麻提取物不同浓度组和阳性对照组的吸光度值均显著高于对照组(P<0.05),荨麻提取物不同浓度组的各时间点吸光度值相比,10-4 g/L组>10-5 g/L组>10-6 g/L组(P<0.05),表明10-4 g/L为最佳浓度;(2)实验组各时间点碱性磷酸酶和钙结节的染色阳性率与阳性对照组相比,差异无显著性意义(P>0.05),但2组各时间点表达量均显著高于对照组(P<0.05);(3)电镜下观察实验组细胞膜表面有大量微绒毛状突起,线粒体基质致密有少量嵴,细胞增殖和分化活跃。且阳性对照组和荨麻组细胞内线粒体数量显著多于对照组(P<0.05);(4)结果提示,荨麻能促进骨膜细胞增殖,同时能诱导骨膜细胞分化为成骨细胞,可作为组织工程中的诱导剂。另外,荨麻可诱导骨膜细胞在聚己内酯三维支架中扩增并构建组织工程骨。BACKGROUND:Urtica,a traditional Chinese herbal,holds anti-inflammatory and anti-rheumatic effects,but its influence on periosteal cells is little reported.OBJECTIVE:To investigate the proliferation and differentiation of human periosteal cells induced by urtica and to construct tissue-engineered bone by periosteal cells in vitro,thereby providing theoretical basis for the application of urtica in bone tissue engineering.METHODS:Human clavicle periosteum was obtained.Primary periosteal cells were cultured by tissue culture method in vitro,passaged,and the 3 generations of periosteal cells were seeded into cell culture plate,and treated with urtica extracts(10-6,10-5 and 10-4 g/L,experimental group),50μg/L bone morphogenetic protein-7(positive control group)or purified water(control group).The absorbance values of periosteal cells were measured by MTT assay,and the expression levels of alkaline phosphatase and calcium nodules were detected by alkaline phosphatase staining and Von Kossa staining at 1,4,7 and 9 days after culture.10-4 g/L urtica extracts and bone morphogenetic protein-7 were added into the three-dimensional polyclonal lactone scaffold and periosteal cells to construct tissue-engineered bone.After 24 days of culture,the morphological changes of periosteal cells were observed under electron microscope.RESULTS AND CONCLUSION:MTT results revealed that the absorbance values of periosteal cells in the experimental and positive control groups were significantly higher than those in the control group at different time points(P<0.05);the order of absorbance values was as follows:10-4 group>10-5 group>10-6 g/L group(P<0.05),suggesting 10-4 g/L was the optimal concentration.The positive rates of alkaline phosphatase and calcium nodules in the experimental and positive control groups were significantly higher than those in the control group(P<0.05),but the experimental and positive control groups did not differ significantly(P>0.05).In the experimental group,there were abundant microvilli on the cell membrane,t

关 键 词：荨麻 骨膜细胞 细胞增殖 碱性磷酸酶 骨组织构建 钙结节 聚己内酯支架 骨形态发生蛋白7 成骨细胞 荨麻科 细胞增殖 组织工程 

分 类 号：R318[医药卫生—生物医学工程]