愈合期与正常内侧副韧带细胞生物学特性差别比较的实验研究

Biological differences between healing and normal medial collateral ligament fibroblasts

[目的]探讨愈合期膝内侧副韧带与正常内侧副韧带成纤维细胞增殖及α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)合成能力的差别。[方法]选取成年雄性SD大鼠12只,体重350～375g,无菌切取正常内侧副韧带与愈合期韧带,以眼科剪剪成1mm×1mm×lmm大小,并将其置于培养瓶中,0.25%胰蛋白酶消化传代制备成纤维细胞。取第3代MCL细胞,培养24、48h后,采用二甲基噻唑二苯基四唑溴盐(MTT)法测定细胞增殖。另外,通过Western-Blot法测定各组细胞α-SMA的表达。[结果]培养24、48h后,MTT法测得愈合期韧带细胞增殖的吸光度值分别为0.49±0.08和0.53±0.07,与正常韧带细胞比较差异均有统计学意义(P<0.05)。Western Blot结果显示,愈合期韧带细胞α-SMA表达量明显高于正常韧带细胞,是正常韧带细胞的1.7倍,差异有统计学意义(P<0.05)。[结论]愈合期韧带细胞增殖及α-SMA表达能力均明显较正常韧带细胞强,这为了解韧带愈合过程以及如何能提高韧带愈合质量在细胞及分子水平提供了充分的理论基础。

[ Objective] To examine the difference of proliferation and a-smooth muscle actin （a-SMA） expression between healing and normal medial collateral ligament （MCL） fibmblasts. [Method]Ten adult,male,Sprague-Dawley rats weighing between 350 and 375 g were used in this study. Normal and healing MCL were cut into small pieces in aseptic conditions, and then placed and cultured in culture chamber. Fibroblasts were passaged with 0.25% trypsin. After 24 and 48 hours incubation, MTT was used to measure the cell proliferation. The production of a-SMA was measured by Western Blot. [ Result] After 24 and 48 hours incubation, healing fib roblasts showed absorbency values of 0.49 ±0. 080 and 0.53±0.07 respectively. II was found that healing fibroblasts proliferated faster than normal fibroblasts （ P 〈 0.05 ）. Healing fibroblasts had 1.7 fold greater protein expression of a-SMA than normal fibroblasts （P 〈0.05 ）. [ Conclusion] Healing fibroblasts possess a faster proliferation and higher a-SMA protein expression than normal fibroblasts. The findings provide a cellular and molecular basis for realizing the mechanism and improving the quality of healing process.