Higa Kazunari, Shimmura Shigeto, Shimazaki Jun, Tsubota Kazuo
Cornea Center, Ichikawa General Hospital, Tokyo Dental College, Chiba, Japan.
Cornea. 2005 Mar;24(2):206-12. doi: 10.1097/01.ico.0000133999.45262.83.
To demonstrate the role of intrinsic hyaluronic acid (HA) in the entrapment of inflammatory cells by amniotic membrane (AM) in vitro.
HA concentration in AM was analyzed by the sandwich protein binding assay, and the approximate molecular weight was measured by gel filtration chromatography. To localize HA in AM tissue, toluidine blue staining with and without hyaluronidase treatment was performed. Adhesion of the lymphocyte cell lines, Jurkat and Molt 4, and peripheral blood mononuclear cells (PBMC) to AM and HA-coated glass slides was analyzed in an in vitro binding assay. Flow cytometry was performed to quantify the expression of the HA receptor, CD44, in Jurkat, Molt 4, and PBMC.
HA was present in high levels in the stroma of AM, also demonstrated by intense staining with toluidine blue. Staining was inhibited by both hyaluronidase treatment and acidic pH. Molt 4, which constitutively expressed CD44, bound to AM and HA-coated slides significantly more than Jurkat cells (CD44-). Adhesion of Molt 4 was inhibited by pretreatment with both soluble HA and anti-CD44 antibody. LPS- or TNF-alpha-treated PBMC also bound to AM and HA-coated slides and was inhibited by pretreatment with an anti-CD44 antibody.
HA in AM stroma may play an important role in the entrapment of inflammatory cells including lymphocytes when used as a patch in ocular surface disease.
在体外证明内源性透明质酸（HA）在羊膜（AM）捕获炎性细胞中的作用。
采用夹心蛋白结合分析法分析AM中的HA浓度，通过凝胶过滤色谱法测量其近似分子量。为了在AM组织中定位HA，进行了有无透明质酸酶处理的甲苯胺蓝染色。在体外结合试验中分析淋巴细胞系Jurkat和Molt 4以及外周血单核细胞（PBMC）与AM和HA包被载玻片的黏附情况。采用流式细胞术定量分析Jurkat、Molt 4和PBMC中HA受体CD44的表达。
HA在AM基质中含量很高，甲苯胺蓝强烈染色也证明了这一点。透明质酸酶处理和酸性pH均抑制染色。组成性表达CD44的Molt 4与AM和HA包被载玻片的结合明显多于Jurkat细胞（CD44阴性）。可溶性HA和抗CD44抗体预处理均抑制Molt 4的黏附。脂多糖或肿瘤坏死因子-α处理的PBMC也与AM和HA包被载玻片结合，抗CD44抗体预处理可抑制这种结合。
当用作眼表疾病的贴片时，AM基质中的HA可能在捕获包括淋巴细胞在内的炎性细胞中起重要作用。
