Joko Takeshi, Nanba Daisuke, Shiba Fumio, Miyata Kazunori, Shiraishi Atsushi, Ohashi Yuichi, Higashiyama Shigeki
Department of Ophthalmology, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime, Japan.
Mol Vis. 2007 Apr 27;13:649-58.
To determine whether the promyelocytic leukemia zinc finger (PLZF) protein, a transcriptional repressor and negative regulator during cell cycling, plays a role in the proliferation of cultured human corneal endothelial cells (HCECs).
The expressions of the mRNA and the protein of PLZF were determined by real-time PCR and western blot analysis, respectively. The changes in the expression of the PLZF gene of cultured HCECs were investigated at different times after cell-cell contacts were disrupted by incubation with EDTA. The cell proliferation rate was assessed with a real-time cell electronic sensing (RT-CES) system after cultured HCECs were infected with either PLZF or LacZ encoding adenovirus vector (Ad-PLZF or Ad-LacZ). The PLZF-regulating genes were analyzed by DNA microarray analysis in cultured HCECs infected with Ad-PLZF.
The expression of the mRNA of PLZF was first detected when the cultured HCECs became confluent, and the relative amount of PLZF mRNA continued to increase for up to 5 days as the cell-cell contacts were formed more firmly. On the other hand, the expression of the mRNA of PLZF decreased about 20 fold 3 h after EDTA exposure, and gradually returned to the original level as the cell-cell contacts were reformed at 72 h after the exposure. The assessment using the RT-CES system showed that the proliferation of cultured HCECs was inhibited for up to 72 h when infected by Ad-PLZF. DNA microarray analysis revealed that the transforming growth factor-beta stimulated clone 22 (TSC-22) gene was up-regulated by 2.32 fold when infected by Ad-PLZF.
These findings indicate that the expression of PLZF in HCECs is closely related to the formation of cell-cell contacts, and that PLZF may play a role in suppressing their proliferation.
确定早幼粒细胞白血病锌指（PLZF）蛋白，一种细胞周期中的转录抑制因子和负调节因子，是否在培养的人角膜内皮细胞（HCECs）增殖中发挥作用。
分别通过实时PCR和蛋白质印迹分析确定PLZF的mRNA和蛋白质表达。在用EDTA孵育破坏细胞间接触后，在不同时间研究培养的HCECs中PLZF基因表达的变化。在用编码PLZF或LacZ的腺病毒载体（Ad-PLZF或Ad-LacZ）感染培养的HCECs后，使用实时细胞电子传感（RT-CES）系统评估细胞增殖率。通过DNA微阵列分析在感染Ad-PLZF的培养HCECs中分析PLZF调节基因。
当培养的HCECs汇合时首次检测到PLZF的mRNA表达，并且随着细胞间接触更牢固地形成，PLZF mRNA的相对量持续增加长达5天。另一方面，EDTA暴露后3小时，PLZF的mRNA表达下降约20倍，并在暴露后72小时随着细胞间接触重新形成而逐渐恢复到原始水平。使用RT-CES系统的评估表明，当被Ad-PLZF感染时，培养的HCECs的增殖被抑制长达72小时。DNA微阵列分析显示，当被Ad-PLZF感染时，转化生长因子-β刺激克隆22（TSC-22）基因上调2.32倍。
这些发现表明，HCECs中PLZF的表达与细胞间接触的形成密切相关，并且PLZF可能在抑制其增殖中发挥作用。
