Table 1.
Primer Sequence Pairs Used for Quantitative Real-Time PCR.
Figure 1.
Conjunctival epithelial changes in explant cultures.
Hematoxylin & Eosin staining showed increases in conjunctival epithelial cell layers in airlift culture. Their number particularly increased from day 8 to day 14, resulting in an undulated basal epithelial plane and digital invasion. In contrast, the cell layers of submerged culture did not show significant increase. Bar represents 200 µm.
Figure 2.
Conjunctival epithelial squamous metaplasia in airlift cultures.
(A) P63 staining showed positive nuclei in all basal cells and some suprabasal cells in normal human conjunctiva (D0). In the airlift culture, p63 expression increased throughout the conjunctival epithelial layers at D2, while the superficial cell layers lost p63 expression from D12 to D14. (B) K16 positive cells were not present in normal conjunctival epithelium (D0). In the airlift culture, K16 positive cells emerged in the suprabasal layer from D2 and gradually increased and spread to superficial layers from day 4 to day 14. (C) K19 expression in the full thickness freshly isolated ex vivo conjunctival epithelial cells (D0). K19 expression gradually decreased in the airlift culture after 4 days of cultivation. There were K19 positive cell clusters in the conjunctival stroma. (D) K10 staining was negative in normal conjunctiva before culturing (D0), but became positive in superficial cell layers at day 2 and day 4, and gradually increased to lower layers from day 8 to day 14. (E) Pax6 expressed in full thickness of normal conjunctival epithelial cells (D0), and it decreased in the basal and supra-basal layers in airlift culture from D2 to D14. Bars represent 100 µm.
Figure 3.
Cytokeratin gene expression level changes in airlift cultures.
Realtime PCR showed that K16 (A) and K10 (C) gene expression progressively increased from day 8 to day 14, whereas K19 (B) gene expression significantly decreased on day 8 and day 14 in airlift cultures. *P<0.05.
Figure 4.
Wnt/β-catenin signaling pathway activation in conjunctival epithelium of airlift explant cultures.
β-catenin (column A) and phosphor-β-catenin (Ser-552, column B) staining were performed at different time points after airlifting culture. β-catenin mostly anchored to the cell membrane and showed very low expression levels in cytoplasm of normal conjunctival epithelium (D0). Phosphor-β-catenin was only found in the cell membrane of basal conjunctival epithelium. Cytoplasmic accumulation of β-catenin and phosphor β-catenin in conjunctival epithelium in airlift explants started at day 2, and then gradually decreased after day 6. Nuclear translocation of β-catenin and phosphor β-catenin was seen in the basal and suprabasal layers of conjunctival epithlial cells from day 2 to day 6 (see inserts). At days 12 and 14, β-catenin and the majority of phosphor-β-catenin has translocated to the cell membrane. Inserts are high power magnification of specific locations. Bar represents 100 µm.
Figure 5.
Mucin expression in airlift conjunctival explant cultures.
MUC5AC (A) showed scattered expression in normal human conjunctiva, which decreased after 2 days airlift culture, and became negative from day 8 to 14. MUC19 (B) expressed in the full thickness freshly isolated healthy ex vivo conjunctival epithelium. It dramatically decreased after 4 days airlift culture and was undetectable from day 8. MUC4 (C) expressed in the full thickness of epithelial cells in normal conjunctiva. It declined from day 4 in airlift culture. MUC16 (D) expressed in the full thickness of freshly isolated healthy ex vivo conjunctival epithelium. In the airlift group, MUC16 expressed in superficial and some suprabasal epithelial cells from D2 to D14. Its expression was stronger in the superficial squamous shaped cells at day 14. Bars represent 100 µm.
Figure 6.
Mucin gene expression level changes in airlift cultures.
Realtime PCR showed that MUC5AC (A), MUC19 (B) and MUC4 (C) gene expression significantly decreased on day 8 and day 14, whereas MUC16 (D) mRNA level significantly increased on day 14 in airlift cultures. *P<0.05.
Figure 7.
Apoptosis of conjunctival epithelial Cells and stromal cells in airlift cultures.
Lanes A and C were counterstained with the nuclear of DAPI dye. Lanes B and D show apoptotic cells based on green fluorescence using the TUNEL assay in the same visual field. Apoptotic cells appeared in both the epithelium and stroma from day 2 to day 14 in airlift cultures, while there were no apoptotic cells in freshly isolated healthy ex vivo conjunctival tissue before culture (D0), and only sporadic apoptotic cells in submerged explants throughout the culture duration (SUB D2 and SUB D14). Bar represents 200 µm.
Figure 8.
Increased proinflammatory mediators in conditioned media of airlift cultures.
IL-1β concentration was maintained at a low level from day 0 (0.97±0.08 pg/mL) to day 6 (1.03±0.16 pg/mL) in airlift culture, while it increased at day 8 (3.89±1.41 pg/mL, P<0.001), and remained high level at day 12 (3.50±1.33 pg/mL, P<0.001). The TNF-α concentration increased after 6 days of airlift culture (8.81±0.26 pg/mL at day 0, 38.71±7.88 pg/mL at day 6, P<0.001) and declined at day 10. MMP-9 remained at a low level from day 0 to day 4, and gradually increased from day 6 to day 12 in airlift culture. (0.27±0.08 ng/mL, 230.61±138.47 ng/mL, 571.78±196.06 ng/mL, 796.49±216.05 ng/mL, and 749.28±307.97 ng/mL in airlift culture for 0, 6, 8, 10 and 12 days respectively). In submerged cultures, no dramatic increases of IL-1β and TNF-α were detected, while MMP-9 only increased significantly after 10 days of culture (195.95±66.07 ng/mL at D10 and 230.99±107.46 ng/mL at D12, P<0.001). However, MMP-9 concentrations remained lower than those in airlift culture from day 6 to day 12 (P<0.001).