新型丝素蛋白支架复合兔髓核细胞体内初步构建组织工程髓核的研究  

Preliminary Construction of Tissue Engineering Nucleus Pulposus Combining Silk Fibroin Porous Scaffold with Rabbit Nucleus Pulposus Cells

作　　者：赵家宁[1] 徐宝山[2] 曾超[1] 杨强[2] 马信龙[2] 张春秋[3] 李秀兰[2] 张扬[2] 

机构地区：[1]天津医科大学研究生院,300070 [2]天津市天津医院 [3]天津理工大学

出　　处：《天津医药》2014年第11期1076-1079,I0002,共5页Tianjin Medical Journal

基　　金：国家自然科学基金资助项目(81272046;31300798;31000432);中国博士后科学基金项目(2011M500530;2012T50221);天津市卫生局科技基金(2013KR16)

摘　　要：目的探讨新型丝素蛋白多孔支架复合经PKH26标记的兔髓核细胞初步构建组织工程髓核的可行性。方法消化分离兔髓核细胞,培养获取P1代细胞,对P1代髓核细胞行番红O以及Ⅱ型胶原免疫组织化学染色;PKH26标记兔髓核细胞,MTT法检测标记前后髓核细胞增殖情况,将标记后的细胞接种支架,体外培养4 d后,将细胞-支架复合物植入裸鼠皮下,体内培养12周后,进行活体荧光成像技术检测、HE染色、甲苯胺蓝染色、番红O染色和Ⅱ型胶原免疫组化染色。结果 P1代髓核细胞番红O染色阳性,Ⅱ型胶原免疫组织化学染色阳性;标记后细胞荧光强度分布均匀,标记前后髓核细胞光密度(OD)值比较差异无统计学意义(P>0.05);活体成像技术显示裸鼠皮下植入的细胞-支架复合体呈现强烈荧光;HE染色见多孔支架内壁有大量髓核细胞填满并分泌大量细胞外基质;甲苯胺蓝染色、番红O染色及Ⅱ型胶原免疫组化染色均呈阳性,细胞周围大量细胞外基质分泌。结论以新型丝素蛋白多孔支架复合兔髓核细胞经体内培养形成的类髓核样组织,可用于组织工程化髓核的构建。Objective To investigate the feasibility of construction of tissue engineering nucleus pulposus by combining the novel silk fibroin porous scaffold with PKH26 labeled rabbit nucleus pulposus cells. Methods Rabbit nucleus pulposus cells were isolated and cultured, then the passage 1 nucleus pulposus cells were stained with safranin O and typeⅡcollagen immunohistochemical staining. The isolated rabbit nucleus pulposus cells were labeled with PKH26. MTT assay was used for examining the proliferation of the nucleus pulposus cells before and after labeling. Labeled cells were inoculated in the scaffold, cultured for 4 days and then the cell-scaffold complexes were implanted subcutaneously into nude mice. After 12 weeks of in vivo culture, the cell-scaffold complexes were detected by in vivo imaging technology, H＆E staining, toluidine blue staining, safranin O staining and collagen typeⅡimmunohistochemical staining. Results Safranin O staining and typeⅡcollagen immunohistochemical staining of the passage 1 nucleus pulposus cells were positive. The fluores？cence intensity of labeled cell was distributed, and the difference of OD value of nucleus pulposus cells was not statistically significant before and after labeling （P〉0.05）. The in vivo imaging technique showed a strong fluorescencea in porous scaf？fold. H＆amp;E staining of cell-scaffold complexes showed that the scaffolds were filled with a large number of nucleus pulposus cells and large amount of extracellular matrix. Toluidine blue staining, safranin O staining and typeⅡcollagen immunohistochemical staining were positive, and large amount of extracellular matrix was secreted around the cells. Conclusion The new silk fibroin porous scaffold with rabbit nucleus pulposus cells in vivo culture formed nucleus pulposus like tissue, which can be used for construction of tissue engineering nucleus.

关 键 词：丝素蛋白 组织工程 免疫组织化学 髓核 PKH26 

分 类 号：R329-33[医药卫生—人体解剖和组织胚胎学] R349.89[医药卫生—基础医学]