| Objective:To culture human orbital fibroblast in vitro, and to observe the effects of hyaluronidase on proliferation of orbital fibroblast and secrection of hyaluronic acid in orbital fibroblast. Methods:1. orbital fibroblasts were cultured by means of monolayer culture, then observe the proliferative status and morphological characteristic by HE pigmentation, identify the cells by immuneohistochemical measurement.2. FBs were plated in 96-well plates and cultured in medium with 10% fetal bovine serum for 48h and without fetal bovine serum for 24h. Then FBs were divided into control group and test groups of hyaluronidase at different density(0, 5, 10, 25, 50, 100U/ml) at random. After the cells were cultured for 24h, 48h, 72h respectively, the morphological change was observed, cell proliferation and secrection of HA were determined by MTT and radioimmunoassay.Results:1 . Orbital fibroblast developed well and passed stably, the result of immuneohistochemical appraisal was positive.2. After cultured in the medium supplemented with different density of hyaluronidase for 24h, 48, 72h, the proliferation of the cells was not inhibited atdifferent degree. (P > 0.05).3. The result of radioimmunoassay of HA indicated that hyaluronidase at every concentration after treating for 24h> 48h and 72h,decreased the secretion of HA significantly(P < 0.05). And with the density and time increasing, the quantity of HA in supematants was more decreased. Conclusion:1. The orbital fibroblasts were cultured successfully passed stably, it can be the model for researching thyroid associated ophthalmopathy.2. hyaluronidase can not inhibit the prolixferation of FBs significantly,but it can decreased the quantity of HA in dose-dependmenK time-dependment manner, it provide the theoretic evidence for the clinical application of hyaluronidase. |
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