白藜芦醇恢复去分化正常髓核细胞表型的实验研究  

RESTORING PHENOTYPE OF DEDIFFERENTIATED NORMAL NUCLEUS PULPOSUS CELLS BY RESVERATROL

作　　者：沈皆亮[1] 胡侦明[1] 钟小明[1] 王大武[1] 徐林飞[1] 

机构地区：[1]重庆医科大学附属第一医院骨科,重庆400016

出　　处：《中国修复重建外科杂志》2013年第5期547-553,共7页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金面上项目(81171751)~~

摘　　要：目的研究体外单层培养与藻酸盐微球立体培养对人正常髓核细胞分化的影响,并探讨白藜芦醇(resveratrol,RES)恢复藻酸盐微球立体培养下去分化髓核细胞表型的调节机制。方法取6例腰椎爆裂骨折患者自愿捐赠的正常髓核组织分离培养髓核细胞,并行细胞鉴定;取单层培养的第1、3、5、7代髓核细胞分别行形态学观察、β-半乳糖苷酶(senescence-associatedβ-galactosidase,SA-β-gal)染色观察细胞衰老情况及甲苯胺蓝染色观察蛋白多糖表达。分别取单层培养和藻酸盐微球立体培养的第1代髓核细胞检测细胞增殖情况。取立体培养1周的第7代髓核细胞随机分为立体培养空白组(A组)、RES组(B组)、沉默交配型信息调节因子2同源蛋白1(silent mating type information regulation 2homolog 1,SIRT1)-小干扰RNA(small interfering RNA,siRNA)+RES组(C组)、阴性对照-siRNA+RES组(D组),另设置髓核细胞单层培养空白组(E组),行相应培养后采用Western blot检测SIRT1、聚蛋白多糖(Aggrecan)和Ⅱ型胶原蛋白表达,实时荧光定量PCR检测SIRT1 mRNA表达水平。结果细胞鉴定提示培养细胞为髓核细胞。形态学观察及SA-β-gal、甲苯胺蓝染色示,在体外连续单层培养条件下,正常髓核细胞易发生去分化,且随着传代次数增加趋势愈明显。藻酸盐微球立体培养48 h内髓核细胞增殖显著低于单层培养(P<0.05),但能显著提高去分化髓核细胞SIRT1、Ⅱ型胶原与Aggrecan蛋白的表达,E组与A组比较差异有统计学意义(P<0.05);与A组相比,B组3种蛋白表达显著提高(P<0.05)。C组SIRT1 mRNA和蛋白表达均受明显抑制,显著低于B、D组(P<0.05),Ⅱ型胶原与Aggrecan蛋白表达也显著低于B、D组(P<0.05)。结论连续体外单层培养条件下,髓核细胞增殖速度较快,但在培养过程中易发生去分化现象;而在藻酸盐微球立体培养条件下,RES可以恢复去分化髓核细胞表型,合成更多细胞外基质,这一机制与Objective To investigate the effects of in-vitro monolayer culture and three-dimensional （3-D） alginate microsphere culture on the differentiation of normal human nucleus pulposus cells （NPCs）, and to discuss the regulatory mechanism of restoring the phenotype of dedifferentiated NPCs by culturing resveratrol （RES） in 3-D alginate microsphere. Methods Normal human nucleus pulposus tissues were harvested for culture and identification of NPCs from 6 patients with burst lumbar vertebra fracture. NPCs at passages 1, 3, 5, and 7 in the in-vitro monolayer culture were harvested to observe the morphology, cell aging, and proteoglycan expression. The cell proliferation rates of NPCs at passage 1 in-vitro in monolayer culture and in 3-D alginate microsphere culture were detected. NPCs at passage 7 were randomly divided into 3-D alginate microsphere control group （group A）, RES group （group B）, silent mating type information regulation 2 homolog 1 （SIRT1）- small interfering RNA （siRNA） ＋ RES group （group C）, and negative control-siRNA ＋ RES group （group D）; and NPCs in the in-vitro monolayer culture was monolayer control group （group E）. After corresponding treatment, Western blot was used for determining the protein expressions of SIRT1, Aggrecan, and collagen type II; real-time fluorescence quantitative PCR was used for detecting SIRT1 mRNA expression. Results The cultured cells were identified to be NPCs. Morphological observation, senescence-associated β-galactosidase （SA-β-gal） staining, and toluidine blue staining showed that dedifferentiation of normal NPCs tended to occur under continuous in-vitro monolayer culture, which was more obvious with increase of passage number. NPCs in 3-D alginate microsphere culture showed significantly lower proliferation rate than NPCs in the in-vitro monolayer culture （P 〈 0.05）, but it could significantly improve the protein expressions of collagen type II and Aggrecan in dedifferentiated NPCs, showing significantly difference

关 键 词：髓核细胞 细胞表型 单层培养 藻酸盐微球立体培养 白藜芦醇 

分 类 号：R681.5[医药卫生—骨科学]