*Dacryology Service, Ophthalmic Plastics Surgery; †Sudhakar and Sreekant Stem Cell Biology Laboratory; and ‡Center for Epidemiology and Biostatistics, LV Prasad Eye Institute, Banjara Hills, Hyderabad, Andhra Pradesh, India.
Ophthalmic Plast Reconstr Surg. 2013 Nov-Dec;29(6):469-74. doi: 10.1097/IOP.0b013e3182a23086.
To establish primary cultures of human nasal mucosal fibroblasts (HNMFs) and to test the effect of varying concentrations of mitomycin C (MMC) and treatment durations on cellular proliferation and viability of the fibroblasts.
Laboratory investigation.
Nasal mucosa harvested from patients undergoing a dacryocystorhinostomy was used to establish primary cultures by explant culture method. Cells were expanded and frozen at every passage, and passage 3 cells were used for further experiments. The cells were then treated with different concentrations of mitomycin C (0.1-0.5 mg/ml) for different time periods (3, 5, and 10 minutes). Cell viability was checked by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Cellular proliferation index was determined with bromodeoxyuridine immunostaining. Apoptotic index was measured using annexin A5 affinity assay, propidium iodide staining, and 4',6-diamidino-2-phenylindole counterstaining. The actin cytoskeletons of fibroblasts were studied using phalloidin staining.
The doubling time of cultured HNMFs is approximately 24 hours. Similarly, 0.4 mg/ml beyond 5 minutes and 0.5 mg/ml concentration at all time points were lethal and caused extensive cell death when compared with controls. A concentration of 0.2 mg/ml for 3 minutes of exposure prevented cell proliferation of HNMF cells by inducing cell cycle arrest, without causing extensive apoptosis.
The minimum effective concentration appears to be 0.2 mg/ml for 3 minutes. This in vitro study could be the starting point for further clinical and histopathologic studies to validate its clinical usefulness.
建立人鼻腔黏膜成纤维细胞（HNMF）的原代培养，并检测不同浓度丝裂霉素 C（MMC）和作用时间对成纤维细胞增殖和活力的影响。
实验室研究。
通过组织块培养法从接受泪囊鼻腔吻合术的患者中获取鼻黏膜，建立原代培养。细胞在每一代都进行扩增和冷冻，并在第 3 代时用于进一步实验。然后，用不同浓度的丝裂霉素 C（0.1-0.5mg/ml）作用不同时间（3、5 和 10 分钟）处理细胞。用 MTT（3-[4,5-二甲基噻唑-2-基]-2,5-二苯基四唑溴盐）法检测细胞活力。用溴脱氧尿嘧啶核苷免疫染色检测细胞增殖指数。用 annexin A5 亲和测定法、碘化丙啶染色和 4',6-二脒基-2-苯吲哚复染法测量细胞凋亡指数。用鬼笔环肽染色研究成纤维细胞的肌动蛋白细胞骨架。
培养的 HNMF 的倍增时间约为 24 小时。同样，与对照组相比，0.4mg/ml 超过 5 分钟和 0.5mg/ml 浓度的药物在所有时间点均具有致命性，并导致广泛的细胞死亡。暴露于 0.2mg/ml 浓度 3 分钟可通过诱导细胞周期停滞来阻止 HNMF 细胞的增殖，而不会引起广泛的细胞凋亡。
最小有效浓度似乎为 0.2mg/ml，作用时间为 3 分钟。这项体外研究可能是进一步的临床和组织病理学研究的起点，以验证其临床应用价值。
