3种不同组织来源的组织工程韧带种子细胞的体外评价

Comparison of 3 kinds of cells as cell sources for tissue engineering ligament

目的体外比较和评价由韧带、滑膜和骨髓组织3种不同来源细胞作为组织工程韧带的种子细胞的功能特征。方法分别从大鼠的膝关节前交叉韧带(Anterior cruciate ligament,ACL)、膝关节滑膜(synovium)和股骨骨髓中分离出前交叉韧带成纤维细胞(ACL fibroblasts,AF)、滑膜成纤维细胞(synovial fibroblasts,SF)和骨髓间质细胞(bone marrowstromal cells,BMSC),在体外培养3、5和7d,然后收集细胞,分离细胞总DNA和总RNA,通过细胞总DNA含量分析,评价细胞的增殖性能,利用总RNA,通过RT-PCR扩增Ⅰ、Ⅲ胶原目标基因,然后通过电泳比较细胞分泌表达Ⅰ、Ⅲ型胶原蛋白的mRNA的水平。结果在取材方面BMSC显示出明显的分离操作简便性;在增殖性能方面,体外培养后3、5和7d,细胞总DNA定量分析BMSCS均明显优于AF和SF(P<0.01);第5和第7d,SF明显优于AF(P<0.05);在韧带功能评价方面,RT-PCR和电泳结果显示培养后的3种细胞在第7d均表达Ⅰ、Ⅲ型胶原蛋白,BMSCs表达水平明显高于其它2种来源的细胞。结论BMSCs与AF和SF相比来源丰富且操作简便,具有良好的增殖活性和分泌表达Ⅰ、Ⅲ型胶原蛋白细胞外基质等特性,符合韧带细胞的生物学活性特征,适用于组织工程韧带的种子细胞。

Objective To compare and evaluate 3 kinds of cells from hgament, synovium and bone marrow tissues as cell sources for tissue engineering hgament.Methods and methods Firstly,Isolate the cells Anterior crueiate ligament （ACL） cells （AF） ,synovial fibroblasts （SF） and bone marrow stromal cells（BMSC） from ACL, synovium of knee joint and bone marrow of femur respectively of rat,and then the cell culture was carried out for 3,5 and 7 days.The total DNA and RNA was isolated,and the quantitative analysis of total DNA for evaluation of cell proliferation, and the total RNA was used to amplify the target genes of collagen Ⅰ andⅢ with RTPCR, then the electrophoresis was performed for characterizing collagen Ⅰ and Ⅲ gene expressions. Results BMSC showed the apparente convenient on operation of isolation, and showed the best cell proliferation compare with AF and SF by the quantitative analysis of total DNA（ P 〈 0.01 ） at the time point of 3,5 and 7 days after culture, and SF showed significance batter than AF at the 5 and 7 days after culture（P 〈 0.05） .On the side of the function of ligament, all the 3 kinds of cells showed collagen ⅠandlⅢ expression at the time point of 7 days after culture, and the BMSC was the highest compare with AF and SF by the analysis of collagen Ⅰ and Ⅲ gene expressions based on RT-PCR. Conclusion Comparing with the AF and SF, BMSC has the characters of convenient operation, rich source, has the properties on cell proliferation and expression of collagen Ⅰ and Ⅲ. In this study, BMSC showed biological properties of ligament cells and refer to be the cell source＇for tissue engineering ligament.