新型丝素蛋白多孔支架复合兔髓核细胞体外构建组织工程化髓核的实验研究  

A study of porous silk fibroin scaffolds with rabbit nucleus pulposus cells for the in vitro construction of tissue- engineered nucleus pulposus

作　　者：曾超[1,2] 朱美峰[3] 徐宝山[1] 杨强[1] 王连永[3] 李秀兰[4] 张杨[4] 马信龙[1] 夏群[1] 杜立龙 丁晓明[1,2] 刘英[5] 

机构地区：[1]天津市天津医院脊柱外科,天津市300211 [2]天津医科大学研究生院,天津市300070 [3]南开大学生命科学院,天津市300071 [4]天津市天津医院骨研所细胞工程室,天津市300211 [5]天津市南开医院急诊科,300100

出　　处：《中国矫形外科杂志》2014年第11期1018-1024,共7页Orthopedic Journal of China

基　　金：国家自然科学基金课题资助(编号:81272046;31000432);中国博士后科学基金资助项目(编号:2011M500530;2012T50235);天津市卫生局科技基金重点资助项目(编号:2013KR16);天津市卫生局攻关课题(编号:10KG113)

摘　　要：[目的]观察以新型丝素蛋白多孔支架复合兔髓核细胞体外构建组织工程化髓核的可行性。[方法]分离培养兔髓核细胞,与丝素蛋白多孔支架在体外复合培养,建立组织工程化髓核模型,通过扫描电镜、HE染色、甲苯胺蓝染色、Ⅱ型胶原免疫组化以及酶联免疫吸附测定观察细胞在支架上1周和3周的生长及增殖情况。[结果]培养1周后,扫描电镜显示细胞呈球状均匀地贴附在支架内部。细胞-支架复合体HE染色可见支架内部有大量髓核细胞,甲苯胺兰染色阳性,Ⅱ型胶原免疫组化染色阳性。培养3周后,扫描电镜显示细胞成层黏附于支架表面,细胞重叠生长,分泌大量细胞外基质,HE染色可见支架内部有大量髓核细胞填满支架孔隙并分泌大量细胞外基质,甲苯胺兰染色阳性,Ⅱ型胶原免疫组化染色阳性。酶联免疫吸附测定:3周组蛋白多糖含量(52.4±4.5)ng/ml明显高于1周组(29.3±3.6)ng/ml,P<0.05,3周组的II型胶原含量(24.3±1.8)ng/ml明显高于1周组(15.16±1.5)ng/ml,P<0.05。[结论]新型丝素蛋白多孔支架复合兔髓核细胞体外培养生长良好,分泌大量类似髓核样细胞外基质,可以用于体外构建组织工程化髓核。[ Objectivel To observe the feasibility of porous silk fibroin scaffolds with rabbit nucleus pulposus cells for the in vitro construction of tissue - engineered nucleus pulposus. [ Methods ] Rabbit nucleus pulposus cells were isolated in vitro and compositely cultured with porous silk fibroin scaffolds to obtain tissue - engineered nucleus pulposus. The growth and prolifer- ation of the cells in the scaffold at 1 week and 3 weeks were observed using scanning electron microscopy, hematoxylin and eosin （H ＆ E） staining, toluidine blue staining, type Ⅱ collagen immunohistochemistry, and enzyme linked immunosorbent assay （ELISA） . [ Results] After 1 week of culture, scanning electron microscopy revealed that the cells evenly attached to the scaf- fold. H ＆ E staining of the cell - scaffold complex revealed a large number of nucleus pulposus cells growing in the scaffold. The results of toluidine blue staining and type Ⅱ collagen immunohistochemical staining were positive. After 3 weeks of culture, scan- ning electron microscopy revealed that the cells showed overlapping growth, formed layers that adhered to the surface of the scaf-fold, and secreted large amounts of extracellular matrix. H ＆ E staining revealed a large number of nucleus pulposus cells filled within the pores of the scaffold, which secreted large amounts of extracellular matrix. The results of toluidine blue staining and type II collagen immunohistochemical staining were positive. ELISA in- dicated a significantly higher proteoglycan content and type II col- lagen content after 3 weeks than after 1 week （52.4 ± 4. 5 ng/ mLvs. 29.3 ± 3.6ng/mLand24.3 ± 1.8ng/mLvs. 15.16 ±1.5 ng/mL, respectively ; P 〈 0. 05 ） . [ Conclusion ] The rabbit nucleus pulposus cells grew well in vitro with the new por-ous silk fibroin scaffold and secreted large amounts of nucleus pulposus - like extracellular matrix; therefore, this scaffold can be used for the in vitro construction of tissue - engineered nucleus pulposus.

关 键 词：丝素蛋白 髓核细胞 组织工程 增殖 

分 类 号：R318.08[医药卫生—生物医学工程]