透明质酸对人胚胎肺成纤维细胞胶原凝胶的作用及其途径

目的了解透明质酸(HA)片段是否促进人胚胎肺成纤维细胞分泌基质金属蛋白酶(MMPs),探讨HA与细胞的相互作用对胶原收缩和降解的影响,初步研究HA与肺成纤维细胞作用的受体途径。方法1)取人胚胎肺组织块进行肺成纤维细胞培养;2)实验分为5组:CD44抗体组(C组)、整合素β1(Integralβ1)抗体组(B组)、IgG1对照抗体组(I组)、HA组(H组)和蒸馏水对照组(M组);3)肺成纤维细胞分别与CD44抗体、Integralβ1抗体或IgG1抗体孵育30min;4)将细胞悬液与胶原溶液、透明质酸溶液(200000、0.1g/L)混匀,共同孵育,待形成凝胶后将凝胶漂浮于培养液中,连续培养72h;5)每24h记录凝胶面积,留取第1个24h的培养上清液应用酶联免疫吸附法检测MMPs,72h后收集凝胶,应用分光光度测定法进行羟脯氨酸检测;6)应用免疫细胞化学法检测肺成纤维细胞透明质酸CD44受体。结果1)5个组的凝胶面积均随时间的推移而逐渐变小,C组和B组的凝胶面积在各个时间点与I组和H组比较都有显著增加,差异有统计学意义(P=0.000),与对照组比较差异无统计学意义。2)肺成纤维细胞胶原凝胶中羟脯氨酸的含量以C组和M组最高,2组与B组、I组及H组比较差异有统计学意义(P<0.05)。3)肺成纤维细胞C组和B组MMP-9的水平明显低于H组和I组(P<0.05),与M组比较差异无统计学意义;C组MMP-1的水平明显低于B组、H组和I组(P<0.05),与M组比较差异无统计学意义。4)HA可促进肺成纤维细胞CD44受体的表达,以相对分子质量为200000的片段作用最强。结论HA对人肺成纤维细胞MMPs的分泌有促进作用,加剧了胶原凝胶的收缩和降解,这种作用可被CD44抗体所抑制,也可被Integrinβ抗体部分抑制。

Effects and Pathways of Effects of Hyaluronan on Collagen Gel from Human Fetal Lung Fibroblasts

Objective Low molecular weight HA was added into fibroblasts collagen gels to investigate the effect of HA on induction of matrix metalloproteinases （MMPs） and contraction and degradation of collagen gel, to explore the role of CD44 and integrin β1 in the above effects. Methods 1 ） Lung fibroblasts were obtained by performing the primary human fetal lung tissue culture. 2 ） Experiments were divided into five groups： anti-CD44-Ab group（ C）, anti-integrin β1-Ab group （B）, IgG1 control group （I）, HA group （H） and double distilled H20 control group （M）. 3 ） Fibroblasts were pretreated for 30 minutes with concentration of 25 mg/L of antibodies specific for CD44 receptor, integrin β1 or nonimmune IgG1. 4） Mixing collagen, 2.5 times DMEM, HA and cell suspension, the mixture was cast into 24-well plates. Gelation occurred within 30 minutes, after which gels were released from the well and floated in culture medium. 5） The areas of collagen gels were measured every 24 hours, the supernatants of culture media were collected after first 24 hours and the concentration of MMPs is measured using an enzyme-linked immunosorbent assay （ ELISA）, collection of collagen gel after 72 hours, spectrophotometry was used to measure hydroxyproline. 6） Immunocytochemistry was used to analyze the expression of CD44. Results 1 ） HA increased the contraction of collagen gel of fibroblasts, while C group and B group attenuated the gel contraction significantly（ P = 0. 000）. 2） The content of hydroxyproline in fibroblasts collagen gel reduced significantly in B group, I group and H group（P 〈0.05）. 3） C group, B group and M group secreted less MMP-9 than other two groups from fibroblasts（P 〈0.05） ; while only C group and M group secreted less MMP-1 than other three groups（ P 〈 0.05 ）. 4 ） Immunocytochemistry showed HA with different MW increasing the expression of CD44 receptor of fibroblasts, among which HA of 200 000 had the highest effect. Conclusion Hyaluronan induced