Schepens Eye Research Institute, Harvard Medical School, Boston, MA 02114, USA.
Invest Ophthalmol Vis Sci. 2012 May 14;53(6):2897-903. doi: 10.1167/iovs.11-8269.
To evaluate the effect of location and size of biopsy on phenotype and proliferative capacity of cultured rat conjunctival epithelial cells.
Pieces of conjunctiva were used from six areas: superior and inferior areas of bulbus, fornix, and tarsus of male Sprague-Dawley rats (n = 6). Explants were grown in RPMI 1640 with 10% fetal bovine serum on coverslips for 8 days or assayed for colony-forming efficiency (n = 9). Analysis included immunofluorescence microscopy and outgrowth measurements with ImageJ software. The Mann-Whitney test and Spearman's rank-order correlation test were used.
Superior (23.9 ± 2.9-fold growth) and inferior (22.4 ± 1.2-fold growth) forniceal tissues yielded significantly more outgrowth with respect to explant size than superior bulbar (13.4 ± 1.9-fold growth; P < 0.05 and P < 0.01, respectively), inferior bulbar (13.6 ± 1.6-fold growth; P = 0.01 and P < 0.01, respectively), and inferior tarsal tissues (14.0 ± 1.3-fold growth; P = 0.01). Outgrowth size correlated positively with explant size (r(s) = 0.54; P < 0.001), whereas explant size correlated negatively with fold growth (r(s) = 0.36; P < 0.001). Superior forniceal cells displayed higher colony-forming efficiency (3.6% ± 0.9%) than superior bulbar (1.1% ± 0.3%; P < 0.05) and inferior bulbar cells (1.6% ± 0.8%; P < 0.05). Percentage of p63+ and PCNA+ cells correlated positively with explant and outgrowth size.
Small forniceal conjunctival explants grow the most effectively; however, for transplantation purposes, the loss of p63+ and PCNA+ cells with small explants must be considered.
评估活检部位和大小对培养的大鼠结膜上皮细胞表型和增殖能力的影响。
使用雄性 Sprague-Dawley 大鼠的 6 个部位（球结膜的上下部、穹窿部、睑板和跗骨）的结膜组织（n = 6）。将培养物在 RPMI 1640 中生长，其中含有 10%的胎牛血清，在盖玻片上培养 8 天或进行集落形成效率分析（n = 9）。分析包括免疫荧光显微镜和使用 ImageJ 软件进行的生长测量。采用曼-惠特尼检验和斯皮尔曼等级相关检验。
与球结膜相比，穹窿部（分别为 23.9 ± 2.9 倍和 22.4 ± 1.2 倍）的组织产生的生长量明显更大（P < 0.05 和 P < 0.01），而球结膜（13.4 ± 1.9 倍）和睑板下部（13.6 ± 1.6 倍）则较小（P = 0.01 和 P < 0.01），睑板下部（14.0 ± 1.3 倍）则较小（P = 0.01）。生长量与培养物大小呈正相关（r(s) = 0.54；P < 0.001），而培养物大小与倍数生长呈负相关（r(s) = 0.36；P < 0.001）。上穹窿部细胞的集落形成效率较高（3.6% ± 0.9%），而上穹窿部细胞（1.1% ± 0.3%；P < 0.05）和下穹窿部细胞（1.6% ± 0.8%）则较低。p63+和 PCNA+细胞的百分比与培养物和生长量呈正相关。
小的穹窿结膜培养物生长最有效；但是，对于移植目的，必须考虑到小培养物中 p63+和 PCNA+细胞的丢失。
