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L. Chua, H. He, S. C. G. Tseng; Purification of HC·HA Complex and Presence of HA, II and TSG-6 in Human Chorionic Membrane. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4861.
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We have previously demonstrated the presence of hyaluronan (HA), inter-α-trypsin inhibitor (IαI) and tumor necrosis factor-stimulated gene-6 protein (TSG-6) in human amniotic membrane (AM), and successfully purified from AM extract HC•HA complex formed by a covalent linkage between heavy chains (HC) and hyaluronan (HA). HC•HA exerts anti-inflammatory and anti-scarring action. Herein, we investigate whether HA, IαI, TSG-6, and particularly HC•HA complex are also present in human chorionic membrane (CH).
The presence of HA, IαI and TSG-6 in cryopreserved human CH were detected by immunostaining with specific probe or antibodies. CH from three donors were extracted with PBS, and subjected to two rounds of ultra-centrifugation in CsCl/4M guanidine HCl to purify HC•HA complex as reported for AM extract. The amount and sizes of HA were determined by HA ELISA and agarose gel electrophoresis analyses, respectively. The covalent linkage between HA and HC in the CH extract was determined by 0.05M NaOH treatment followed by Western blotting with anti-IαI antibody.
HA ELISA and immunostaining showed that HA, IαI and TSG-6 were present in the CH. The concentration of HA in the CH extract after purification was 0.72 ± 0.02 µg/ml, which was significantly lower than 41.2 ± 5.3 µg/ml in AME. In contrast, CH extract contained significantly more proteins (1826 ± 60 µg/ml) compared to AME (214 ± 36 µg/ml). These proteins could be separated by the first ultracentrifugation (400 ± 92 µg/ml) and further reduced by the second ultracentrifugation (123 ± 56 µg/ml) during purification of HC•HA complex. The covalent linkage between HC of IαI and HA in the purified HC•HA complex was confirmed to be a NaOH-sensitive bond by Western Blot analysis. These results were consistently noted in CH extracts obtained from all three donors.
HA, IαI and TSG-6 are present in human CH. HC•HA complex present in human CH can be purified in a significantly lesser amount as compared to that purified from human AM. Further studies are needed to determine the source of HC•HA complex and their therapeutic potential in exerting anti-inflammatory, anti-scarring and anti-angiogenic actions.
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