自组装多肽纳米凝胶支架三维培养前软骨干细胞的实验研究  

PROLIFERATION AND CHONDROGENIC DIFFERENTIATION OF PRECARTILAGINOUS STEM CELLS IN SELFASSEMBLING PEPTIDE NANOFIBER SCAFFOLDS

作　　者：罗文[1] 范建楠[2] 叶川[2] 

机构地区：[1]贵阳医学院研究生院贵阳,550004 [2]贵阳医学院附属医院骨科

出　　处：《中国修复重建外科杂志》2012年第12期1505-1511,共7页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：贵州省卫生厅基金资助项目(gzwkj2012-1-055);贵州省科学技术厅基金资助项目(LG[2012]051)~~

摘　　要：目的构建新型自组装多肽纳米凝胶支架RGDmx,探讨支架的细胞相容性及其对前软骨干细胞(precartilaginous stem cells,PSCs)增殖和向软骨细胞方向分化的影响。方法取新生SD大鼠四肢长骨干骺端组织分离培养PSCs,采用免疫磁珠分选系统分选纯化原代细胞,并进行鉴定。取KLD-12、KLD-12-PRG多肽冻干粉,以体积比1∶1复合构建RGDmx。将纯化后的第3代PSCs接种至KLD-12(对照组)和RGDmx(实验组)培养,1、3、7、14 d用细胞计数(cell counting kit,CCK)-8法检测支架细胞增殖-毒性;制备不同混合比(0、20%、40%、60%、80%、100%)RGDmx,观察其对PSCs增殖的影响;采用无血清软骨形成培养液(complete chondrogenic medium,CCM)诱导两组支架内PSCs向软骨细胞方向分化,培养14 d时行甲苯胺蓝染色法鉴定,并用RT-PCR法检测两组软骨分化特异性基因Ⅱ型胶原和蛋白聚糖的表达情况。结果成功分离纯化获得成纤维细胞生长因子受体3表达阳性细胞,免疫组织化学染色及免疫荧光染色鉴定为PSCs。CCK-8检测结果显示,复合培养后实验组细胞吸光度(A)值随培养时间延长逐步提高,7 d达峰值;其中7、14 d时细胞A值高于对照组(P<0.05);复合培养7 d时,混合比为40%组细胞A值较其他混合比组高,差异有统计学意义(P<0.05)。CCM诱导培养14 d时,两组支架内细胞甲苯胺蓝染色均呈阳性;RT-PCR检测示实验组细胞Ⅱ型胶原和蛋白聚糖mRNA表达水平均高于对照组,差异有统计学意义(P<0.05)。结论自组装多肽纳米凝胶支架RGDmx具有良好的细胞相容性,可有效促进PSCs增殖及向软骨细胞方向分化,是组织工程较理想的细胞载体系统。Objective To construct a new type of self-assembling peptide nanofiber scaffolds--RGDmx, and to study the cell compatibility of the new scaffolds and the proliferation and chondrogenic differentiation of precartilaginous stem cells （PSCs） in scaffolds. Methods PSCs were separated and purified from newborn Sprague Dawley rats by magnetic activated cell sorting and indentified by immunohistochemistry and immunofluorescent staining. The RGDmx were constructed by mixing KLD-12 and KLD-12-PRG at volume ratio of 1 ： 1. PSCs at passage 3 were seeded into the KLD-12 scaffold （control group） and RGDmx scaffold （experimental group）. The proliferation of PSCs in 2 groups were observed with the method of cell counting kit （CCK） -8 after 1, 3, 7, and 14 days after culture. The RGDmx were constructed by mixing KLD-12-PRG and KLD-12 at different volume ratios of 0, 20%, 40%, 60%, 80%, and 100% and the proliferation of PSCs was also observed. The complete chondrogenic medium （CCM） was used to induce chondrogenic differentiation of PSCs in different scaffolds. The differentiation of PSCs was observed by toluidine blue staining and RT-PCR assay. Results PSCs were separated and purified successfully, which were identified by immunohistochemistry and immunofluorescent staining methods. The results of CCK-8 showed that the absorbance （A） value in the experimental group increased gradually and reached the highest at 7 days; the A value in the experimental group was significantly higher than that in the control group at 7 days and 14 days （P 〈 0.05）. Meanwhile, the A value in the RGDmx scaffold with a volume ratio of 40% was significantly higher than those in others （P 〈 0.05）. After 14 days of induction culture with CCM, the toluidine blue staining results were positive in 2 groups; the results of RT-PCR showed that the expression levels of collagen type II and the aggrecan in the experimental group were significantly higher than those in the control group （P 〈 0.05）. Conclusion The self-ass

关 键 词：组织工程支架 自组装多肽纳米凝胶支架 前软骨干细胞 关节软骨缺损修复 

分 类 号：R318.08[医药卫生—生物医学工程]