Hayashi Miyuki, Hayashi Yasuhito, Liu Chia-Yang, Tichelaar Jay W, Kao Winston W Y
Department of Ophthalmology, University of Cincinnati, Cincinnati, OH 45267-0527, USA.
Mol Vis. 2005 Mar 16;11:201-7.
Available evidence suggests that fibroblast growth factor 7 (FGF7, also known as keratinocyte growth factor, KGF) serves as a paracrine growth factor modulating corneal epithelial cell proliferation. In the present study, we used a binary inducible transgenic mouse model to examine the role of FGF7 on corneal epithelium proliferation.
A keratocyte specific 3.2 kb murine keratocan promoter (Kerapr) was used to prepare Kerapr-rtTA transgenic (Kr) mice that constitutively overexpress reverse tetracycline transcription activator (rtTA) by cornea stromal keratocytes. The Kr mice were crossed with tet-O-FGF7 mice to produce Kr/tet-O-FGF7 bitransgenic mice. Expression of human FGF7 (hFGF7) was induced by the administration of doxycycline via intraperitoneal injection and/or feeding mice doxycycline in drinking water and chow. Overexpression of hFGF7 was confirmed by RT-PCR and western blot. BrdU incorporation was used to determine cell proliferation.
The rtTA mRNA and protein were constitutively expressed by the cornea with or without doxycycline induction, whereas hFGF7 was detected only in Kr/tet-O-FGF7 bitransgenic mice upon induction by doxycycline. Examination of induction kinetics in adult Kr/tet-O-FGF7 bitransgenic mice after a single intraperitoneal injection of doxycycline revealed that hFGF7 mRNA expression was detected 12 h after doxycycline administration, peaked at 36 h, was sustained up to 48 h, and declined thereafter. The elevated level of hFGF7 expression coincided with hyperproliferation of corneal epithelial cells. In bitransgenic mice, the number of BrdU labeled cells increased after 36 and 48 h of transgene induction compared to controls of noninduced bitransgenic or doxycycline treated single transgenic mice. The BrdU labeling index was 33+/-9.2 positive cells per corneal section for Kr/tet-O-FGF7 bitransgenic mice and 25+/-9.3 for tet-O-FGF7 single transgenic mice at 36 h post-doxycycline treatment. However, the excess FGF7 driven by doxycycline induction did not produce severe perturbation of corneal epithelium homeostasis.
Our results demonstrate that the doxycycline inducible system is effective in regulating transgene expression in corneal stroma of Kr/tet-O-FGF7 bitransgenic mice. However, the development of pathology resulting from the overexpression of transgenes may depend on whether the amount of transgene product present is sufficient to alter the homeostasis of the targeted tissues.
现有证据表明，成纤维细胞生长因子7（FGF7，也称为角质形成细胞生长因子，KGF）作为旁分泌生长因子调节角膜上皮细胞增殖。在本研究中，我们使用二元诱导转基因小鼠模型来研究FGF7对角膜上皮增殖的作用。
使用角膜细胞特异性的3.2 kb小鼠角膜蛋白聚糖启动子（Kerapr）制备Kerapr-rtTA转基因（Kr）小鼠，该小鼠由角膜基质角膜细胞组成性过表达反向四环素转录激活因子（rtTA）。将Kr小鼠与tet-O-FGF7小鼠杂交，产生Kr/tet-O-FGF7双转基因小鼠。通过腹腔注射给予强力霉素和/或在饮用水和食物中喂给小鼠强力霉素来诱导人FGF7（hFGF7）的表达。通过RT-PCR和蛋白质印迹法确认hFGF7的过表达。使用BrdU掺入法测定细胞增殖。
无论是否有强力霉素诱导，rtTA mRNA和蛋白均由角膜组成性表达，而hFGF7仅在强力霉素诱导的Kr/tet-O-FGF7双转基因小鼠中检测到。对成年Kr/tet-O-FGF7双转基因小鼠单次腹腔注射强力霉素后的诱导动力学检查显示，强力霉素给药后12小时检测到hFGF7 mRNA表达，在36小时达到峰值，持续至48小时，此后下降。hFGF7表达水平的升高与角膜上皮细胞的过度增殖一致。在双转基因小鼠中，与未诱导的双转基因或强力霉素处理的单转基因小鼠的对照相比，转基因诱导36小时和48小时后，BrdU标记的细胞数量增加。强力霉素处理后36小时，Kr/tet-O-FGF7双转基因小鼠的BrdU标记指数为每角膜切片33±9.2个阳性细胞，tet-O-FGF7单转基因小鼠为25±9.3个。然而，强力霉素诱导驱动的过量FGF7并未对角膜上皮稳态产生严重干扰。
我们的结果表明，强力霉素诱导系统可有效调节Kr/tet-O-FGF7双转基因小鼠角膜基质中的转基因表达。然而，转基因过表达导致的病理发展可能取决于存在的转基因产物量是否足以改变靶组织的稳态。
