Effects of amniotic membrane suspension in human corneal wound healing in vitro
Effects of amniotic membrane suspension in human corneal wound healing in vitro
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发表时间：
2009-11
期刊：
影响因子：
2.2
通讯作者：
Jin A Choi;Hyun-Jin Jin-Hyun-Jin-Jin-2256478452;Samhyun Jung;Eunkyung Yang;Jun-Sub Choi;So-Hyang Chung;Choun-Ki Joo
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作者：
Jin A Choi;Hyun-Jin Jin-Hyun-Jin-Jin-2256478452;Samhyun Jung;Eunkyung Yang;Jun-Sub Choi;So-Hyang Chung;Choun-Ki Joo
目的探讨羊膜悬液对角膜创伤愈合，尤其是对上皮细胞增殖和迁移的影响。方法将人角膜上皮细胞（HCECs）分别培养于含不同浓度AM（5%和30%）的培养液、含Dulbecco改良Eagle培养基：营养混合物F-12（阴性对照）和含Dulbecco改良Eagle培养基：营养混合物F-12（阳性对照）的血清中。为了评估AM的迁移潜力，通过手动刮取HCEC和细胞粘附分子（E-钙粘蛋白）的免疫细胞化学染色进行迁移测定。采用逆转录-聚合酶链反应（RT-PCR）和蛋白质印迹法（Western blot）检测基质金属肽酶9（MMP 9）和粘附分子（E-钙粘蛋白、纤连蛋白）的相对表达。5-溴脱氧尿苷（5-Bromo-2-deoxyuridine，BrdU）掺入法检测AM增殖活性，Western blot检测增殖细胞核抗原（proliferating cell nuclear antigen，PCNA）。此外，酶联免疫吸附试验（ELISA）用于测量AM悬浮液中促有丝分裂生长因子（表皮生长因子[EGF]、角质形成细胞生长因子[KGF]、肝细胞生长因子[HGF]和碱性成纤维细胞生长因子[bFGF]）的蛋白浓度。结果随着AM浓度的增加，细胞迁移率增加，30% AM处理组和阳性对照组与阴性对照组相比，差异有统计学意义（p<0.05）。RT-PCR结果显示AM可上调MMP 9基因的表达，下调E-cadherin和FN基因的表达。Western blot分析表明，MMP 9的表达显着较高的AM治疗组，而在AM治疗组的E-钙粘蛋白和纤连蛋白的表达水平显着降低。免疫细胞化学显示大量的E-钙粘蛋白附近的伤口边缘损伤后24小时的AM治疗组。增殖试验显示，AM使BrdU阳性细胞计数/总细胞计数（标记指数）增加至统计学显著程度（在30% AM和阳性对照组中p<0.05）。Western blot分析显示，细胞周期相关蛋白PCNA的表达随着AM处理的结果逐渐增加。ELISA结果显示，我们的AM悬浮液含有4种生长因子（HGF，EGF，KGF和FGF）。HGF的量特别大，其次是EGF。结论AM混悬液对角膜上皮损伤的愈合有促进作用，且随着AM浓度的增加，角膜上皮的迁移和增殖明显增加。
Purpose To investigate the biochemical mechanism of amniotic membrane (AM) suspension on corneal wound healing, particularly on epithelial proliferation and migration. Methods Human corneal epithelial cells (HCECs) were cultured in media with different concentrations of AM suspension (5% and 30%), Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (negative control), and serum containing Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (positive control). In an effort to evaluate the migratory potential of AM, migration assays were conducted via the manual scraping of HCECs and immunocytochemical staining of cell adhesion molecules (E-cadherin). The relative expression of matrix metallopeptidase 9 (MMP9) and adhesion molecules (E-cadherin, fibronectin) was determined via reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. The proliferative potential of AM was evaluated via a proliferation assay using 5-Bromo-2-deoxyuridine (BrdU) incorporation and western blot analysis for proliferating cell nuclear antigen (PCNA). In addition, enzyme-linked immunosorbent assay (ELISA) was used to measure the protein concentrations of mitogenic growth factors (epidermal growth factor [EGF],keratinocyte growth factor [KGF], hepatocyte growth factor [HGF], and basic fibroblast growth factor [bFGF]) in AM suspensions. Results Migration assay rates were enhanced as AM concentrations increased, with statistically significant changes seen in 30% AM-treated and positive control cells, compared to negative control cells (p<0.05). RT-PCRs revealed that the expression of the MMP9 gene was upregulated by AM, and the expressions of E-cadherin and fibronectin genes were downregulated by AM. Western blot analysis demonstrated significantly higher MMP9 expression in AM-treated groups, versus significantly lower levels of E-cadherin and fibronectin expression in AM-treated groups. Immunocytochemistry showed large quantities of E-cadherin near the wound edges after 24 h of injury in the AM-treated groups. The proliferation assay showed that the BrdU positive cell counts/total cell counts (labeling index) were augmented by AM to a statistically significant degree (p<0.05 in the 30% AM and positive control groups). Western blot analysis showed that the expression cell cycle-associated protein, PCNA, increased gradually as a result of AM treatment. ELISA showed that our AM suspension contained 4 growth factors (HGF, EGF, KGF, and FGF). The amount of HGF was especially large, followed by that of EGF. Conclusions These results demonstrate that the suspension form of AM maintains its beneficial effect on corneal epithelial wound healing in vitro, and that AM suspension leads to significant increases in corneal epithelial migration and proliferation with increasing AM concentrations.