透明质酸对大鼠肺泡巨噬细胞和肺成纤维细胞分泌蛋白酶的影响

目的:研究不同大小透明质酸对大鼠肺泡巨噬细胞和肺成纤维细胞分泌基质金属蛋白酶（MMPs）的诱导作用及对细胞周围胶原收缩和降解的影响。方法:Wistar 大鼠肺泡灌洗获取肺泡巨噬细胞，Wistar 乳鼠肺组织块培养获取肺成纤维细胞；透明质酸（简称HA）分为4个分子量：100 kD、200 kD、500 kD、1 000 kD；将两种细胞混悬液分别与胶原溶液、不同分子量的透明质酸溶液混匀形成胶原凝胶后将凝胶漂浮于培养液中连续培养72 h；每24 h 记录凝胶面积；留取第1个24 h的培养上清液应用酶联免疫吸附法检测MMPs；72 h后收集凝胶，应用分光光度测定法进行羟脯氨酸检测。结果:（1）不同分子量HA都可刺激肺泡巨噬细胞分泌MMP-9、MMP-12和MMP-13，与对照组比差异显著(P<0.05)；其中以200 kD HA作用最强。（2）不同分子量HA都可刺激肺成纤维细胞分泌MMP-1、MMP-9和MMP-13，与对照组比较，P<0.05；其中以100、200 kD HA作用最为显著。（3）凝胶形成后24 h，100、200、500 kD HA使肺泡巨噬细胞凝胶面积明显缩小，与对照组比较有显著差异（P<0.05)，并且这种作用一直持续到72 h。（4）凝胶形成后24 h， 100、200、500 kD的HA使肺成纤维细胞凝胶面积明显缩小，与1 000 kD HA组及对照组比较有显著差异(P<0.05))；72 h后，100、200 kD HA使凝胶面积进一步缩小，与500、1 000 kD HA组及对照组比较，P<0.05。（5）4种分子量的HA与肺泡巨噬细胞共同孵育72h后，凝胶中羟脯氨酸含量与对照组比较均有显著减少，其中100、200 kD HA组羟脯氨酸含量降低最显著(P<0.05)。（6）4种分子量的HA与成纤维细胞共同孵育72h后，凝胶中羟脯氨酸含量与对照组比较均显著减少，且不同分子量之间差异显著(P<0.05)。结论：（1）HA可以刺激大鼠肺泡巨噬细胞和肺成纤维细胞分泌多种与胶原降解相关的MMPs；其中200 kD HA作用最强。（2）HA可以促进两种细胞凝胶收缩，以100、200、500 kD的HA作用最显著。（3）HA可以降解细胞凝胶中的胶原，使其羟脯氨酸含量减低，以100、200 kD HA作用最强。

Effects of hyaluronan on secretion of MMPs from murine alveolar macrophage and lung fibroblast

AIM:To investigate the effects of different molecular weights of hyaluronan (HA) on secretion of matrix metalloproteinases (MMPs) from macrophages and fibroblasts as well as contraction and degradation of collagen gels.METHODS:(1) Alveolar macrophages were obtained from lungs of normal Wistar rats by bronchoalveolar lavage.Lung fibroblasts were obtained by performing primary culture of lung tissue which was acquired from rat born first day.(2) HA was divided into 4 different molecular weights (MW):100 kD, 200 kD, 500 kD and 1 000 kD.(3) Mixing collagen, HA and cell suspension so that the final mixture resulted in 0.75 g/L collagen, 5×108 cells/L macrophages or fibroblasts.The mixture was cast into 24-well plates.Gelation occurred within 30 min, after which gels were released from the wells and floated in culture medium.(4) The areas of collagen gels were measured every 24 h, and the media were changed everyday.(5) The supernatants of culture media were collected after first 24 h and the concentration of MMPs was measured using an enzymed-linked immunosorbent assay (ELISA).(6) Collection of collagen gel after 72 h, spectrophotometry was used to measure hydroxyproline.RESULTS:(1) HA with different MW induced MMP-9, 12, 13 releases from alveolar macrophages, among which 200 kD had the most significant effect.(2) HA with different MW induced MMP-1, 9, 13 releases from lung fibroblasts, among which HA of 100 kD and 200 kD had the most significant effect.(3) HA with MW 100 kD, 200 kD and 500 kD augmented macrophage-mediated collagen gel contraction at 24 h, and the effect continued for 72 h.(4) HA with MW 100 kD, 200 kD and 500 kD augmented fibroblast-mediated collagen gel contraction at all time points, but only HA of 100 kD and 200 kD had significant effect compared with that of 500 kD and 1 000 kD after 72 h.(5) Over three-day culture period, addition of HA resulted in decrease of hydroxyproline in macrophage collagen gel, HA with 100 kD and 200 kD MW had significant effect than that of 500 kD and 1 000 kD.(6) Over three-day culture period, HA added resulted in decrease of hydroxyproline in fibroblast collagen gel, 200 kD HA had significant effect than that of others.CONCLUSION:(1) HA induces MMPs secretion from macrophages and fibroblasts when casted into collagen gel, among which HA with MW 200 kD has the strongest effect.(2) HA augmentes the contraction of collagen gels in both cells, and HA of 100 kD, 200 kD and 500 kD has the most significant effect.(3) Hyaluronan results in the degradation of the collagen within the gel; HA of 100 kD and 200 kD decreases the hydroxyproline to the lowest level.