Expression of cell proliferation and apoptosis biomarkers in pterygia and normal conjunctiva

Abstract

Purpose: To analyze the expression of apoptosis and cell proliferation molecules in pterygium tissues of Chinese patients.

Methods: Thirty-three pterygia were surgically removed using the bare sclera procedure, and 23 normal bulbar conjunctivas were also obtained. Formalin-fixed, paraffin-wax-embedded tissues were analyzed by immunohistochemistry with anti- proliferating cell nuclear antigen (PCNA), K(i)-67 (a proliferating cell marker), mutant p53 (mP53), Bcl-2 associated X-protein (BAX), B-cell lymphoma gene 2 (Bcl-2), and caspase-3 antibodies. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) analysis was used to analyze the apoptotic cells.

Results: Our study revealed that the positive rate of PCNA and K(i)-67 significantly increased in the pterygium samples compared to the normal conjunctiva samples. In the molecules involved in apoptosis, the results showed that the positive rate of Bcl-2 and mP53 significantly increased in the pterygium samples. However, no difference was found between the pterygium and normal conjunctiva samples in the expression of Bax and caspase-3. Through TUNEL analysis, apoptotic cells were seen in the entire width of the epithelial layer in normal conjunctivas but were found mainly confined to the outer layer of the epithelial cells in pterygia.

Conclusions: The finding of high levels of cellular proliferation and low levels of cellular apoptosis in pterygia confirmed that both cell apoptosis and proliferation are known to play an important role in human pterygium pathogenesis.

Figure 1

Immunohistochemical staining for K_i-67 and PCNA positive cells in pterygium and normal conjunctiva samples. Positive K_i-67 immunostaining showed higher nuclear staining in pterygia (A) than in normal conjunctivas (B). PCNA immunostaining showed a higher nuclear staining in pterygia (C) than in normal conjunctivas (D). All slides were counterstained with Mayer’s hematoxylin. Original magnifications: A-D, 400×.

Figure 2

Immunohistochemical staining for Bcl-2, Bax, mutant p53, and TUNEL analysis positive cells in pterygium and normal conjunctiva samples. Human pterygium tissues (A, C, E, G); Human normal conjunctiva tissues (B, D, F, H). Bcl-2 showed strong expression in the cytoplasm throughout the entire width of the epithelial layer in pterygium (A). No expression was shown in the epithelial layer of Bcl-2 in normal conjunctiva (B). Bax showed strong expression in the cytoplasm throughout the entire width of the epithelial layer in both pterygium (C) and normal conjunctiva (D). mP53 immunostaining showed nuclear staining in the pterygium epithelial layer (E), but no expression in normal conjunctiva (F). For TUNEL analysis, the positive cells in pterygium showed nuclear staining in the basal layer of the epithelium (G). In normal conjunctiva, the positive cells showed nuclear staining in the whole width of the epithelial layer (H). All slides were counterstained with Mayer’s hematoxylin. Original magnifications: A-H, 400×.