纳米多孔PLLA膜对晚期内皮祖细胞行为的影响  

EFFECTS OF NANOPOROUS PLLA SCAFFOLD ON LATE ENDOTHELIAL PROGENITOR CELLS BEHAVIOR

作　　者：卢辉俊[1] 冯章启[2] 乔彤[1] 刘晨[1] 顾忠泽[2] 刘长建[1] 

机构地区：[1]南京大学医学院附属鼓楼医院血管外科,南京210008 [2]东南大学生物电子学国家重点实验室

出　　处：《中国修复重建外科杂志》2009年第2期166-172,共7页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：江苏省卫生厅重大科研资助项目(K200609)~~

摘　　要：目的观察晚期内皮祖细胞(endothelial progenitor cells,EPCs)在纳米多孔PLLA膜表面黏附、增殖的情况,为优化组织工程血管支架材料提供一种新途径。方法新西兰大白兔,体重2.5～3.0 kg,雌雄不限。取兔外周血分离和培养晚期EPCs。采用静电纺丝技术制备纳米无序纤维、有序纤维及超级有序纤维,行低温等离子体技术改性及Ⅰ型胶原表面涂覆,制备无序膜、有序膜及超级有序膜。实验分为A(单纯细胞)、B(无序膜)、C(普通膜)、D(有序膜)、E(超级有序膜)组,将原代晚期EPCs(1×105个/mL)复合至支架材料培养,3、5、7、9、11、13、15和17 d后MTT法检测细胞增殖能力;实验分A(无序膜)、B(普通膜)、C(有序膜)、D(超级有序膜)4组,取浓度为1×105个/mL的第3代晚期EPCs悬液3 mL滴加至膜上,复合培养4、12和24 h后沉淀法检测细胞黏附率,1、3、7 d测定细胞增殖率,并于复合培养后4、24、72 h观察细胞形态变化。结果纳米PLLA纤维直径300～400 nm,孔隙率>90%。培养后3、5、7、9、11、13、15和17 d,各组A值均随培养时间延长而增高,细胞生长良好;A、B组间各时间点比较差异无统计学意义(P>0.05),培养后5～17 d,C、D、E组与A、B组间比较差异均有统计学意义(P<0.05)。复合培养4 h,A组黏附率高于B、C、D组,差异有统计学意义(P<0.05);12 h及24 h,B、C、D组黏附率明显高于A组,差异有统计学意义(P<0.05);各组4 h与12、24 h比较差异均有统计学意义(P<0.05),12 h与24 h比较差异无统计学意义(P>0.05)。复合培养1、3和7 d,B、C、D组增殖率明显高于A组,差异有统计学意义(P<0.05);B、C、D组间除培养后1 d,其余各时间点比较差异均有统计学意义(P<0.05)。形态学观察示细胞在各支架膜上生长良好,A组及B组细胞生长较散在、杂乱;C、D组细胞沿纤维定向附着、伸展、增殖并分泌ECM,D组细胞结构保持更好。结论纳米多孔PLLA有序膜及超级有序膜支架能促进�Objective To observe the adhesion and proliferation of late endothelial progenitor cells （EPCs） planted on nanoporous PLLA scaffold in vitro and to provide a new approach that optimizes tissue engineered material. Methods Male and female New Zealand rabbits （weight 2.5-3.0 kg） were used. Isolated late EPCs from rabbit peripheral blood were cultured. Electrostatic spinning technique was adopted to prepare misaligned nanofibers, aligned nanofibers and super-aligned nanofibers, and low temperature plasma technique was applied to prepare misaligned membrane, aligned membrane and super-aligned membrane. After being divided into group A （cells only）, B （misaligned membrane）, C （normal membrane）, D （aligned membrane） and E （super-aligned membrane）, the primary late EPCs （1 × 10^5/mL） were cultured on scaffolds and MTT method was used to detect cell proliferation ability at 3, 5, 7, 9, 11, 13, 15 and 17 days after culture. After being divided into group A （misaligned membrane）, B （normal membrane）, C （aligned membrane） and D （superaligned membrane）, precipitation method was applied to detect cell adhesion rate at 4, 12 and 24 hours after compound culture, and the morphologic changes of cells were observed at 4, 24 and 72 hours after compound culture. Results Fiber diameters in nanofibrous PLLA scaffolds were 300-400 nm, with a porosity rate of above 90%. At 3, 5, 7, 9, 11, 13, 15 and 17 days after culture, A value of each group was increased with time and the cells in each group grew well, showing there was no significant difference between group A and group B at each time point （P 〉 0.05 ）; during the period of 7-15 days after culture, the difference between groups C, D and E and groups A and B was significant （P 〈 0.05）. At 4 hours after compound culture, the adhesion rate of group A was superior to that of groups B, C and D （P 〈 0.05）; at 12 and 24 hours after compound culture, the adhesion rate of groups B, C and D was remarkably higher than that of g

关 键 词：组织工程血管 晚期内皮祖细胞 纳米多孔PLLA 支架材料 

分 类 号：R318.08[医药卫生—生物医学工程] R322[医药卫生—基础医学]