Koh Jae Woong, Shin Young Joo, Oh Joo Youn, Kim Mee Kum, Ko Jung Hwa, Hwang Jeong Min, Wee Won Ryang, Lee Jin Hak
Department of Ophthalmology, Chosun University College of Medicine, Gwangju, and Seoul National University Hospital Clinical Research Institute, Korea.
Curr Eye Res. 2007 Jul-Aug;32(7-8):611-6. doi: 10.1080/02713680701459441.
To investigate the change of tissue inhibitor of metalloproteinase (TIMP) in cryopreserved amniotic membranes (AM) according to preservation time, and to evaluate the expression of TIMP in freeze-dried AM.
Cryopreserved or fresh AMs were incubated in dispase II for two hours at 37 degrees C and their epithelial cells were scraped with a cell scraper. Remaining stromal AM was minced and frozen in liquid nitrogen, and then treated with 0.1% diethyl pyrocarbonate. The mRNA levels of TIMP-1 and -2 were determined by reverse transcription-polymerase chain reaction (RT-PCR) in epithelial and stromal cells of fresh AM, AMs cryopreserved for 6 and 12 months, and freeze dried AM, respectively. Western blot analysis and immunohistochemical staining were performed to assess the expression of TIMP-1 in fresh, cryopreserved, and freeze dried AMs.
RT-PCR revealed that mRNAs of TIMP-1 and -2 were expressed in the amniotic epithelial cells of both fresh and cryopreserved AMs, while the stromal cells of fresh or cryopreseved AMs and freeze-dried AM showed higher expression of TIMP-1 than TIMP-2 mRNA. On Western blot analysis, the level of TIMP-1 was more in fresh AMs than in cryopreserved or freeze-dried AM, but it was not statistically significant.
TIMP-1 was expressed in cryopreserved AMs until 12 months, and the amount of expression was comparable to that in fresh AMs.
研究金属蛋白酶组织抑制剂（TIMP）在冷冻保存羊膜（AM）中随保存时间的变化，并评估TIMP在冻干羊膜中的表达。
将冷冻保存或新鲜的羊膜在37℃下用dispase II孵育两小时，并用细胞刮匙刮去其上皮细胞。将剩余的羊膜基质切碎并在液氮中冷冻，然后用0.1%焦碳酸二乙酯处理。分别通过逆转录-聚合酶链反应（RT-PCR）测定新鲜羊膜、冷冻保存6个月和12个月的羊膜以及冻干羊膜的上皮细胞和基质细胞中TIMP-1和-2的mRNA水平。进行蛋白质免疫印迹分析和免疫组织化学染色以评估TIMP-1在新鲜、冷冻保存和冻干羊膜中的表达。
RT-PCR显示，新鲜和冷冻保存羊膜的羊膜上皮细胞中均表达TIMP-1和-2的mRNA，而新鲜或冷冻保存羊膜以及冻干羊膜的基质细胞中TIMP-1的表达高于TIMP-2 mRNA。蛋白质免疫印迹分析显示，新鲜羊膜中TIMP-1的水平高于冷冻保存或冻干羊膜，但差异无统计学意义。
TIMP-1在冷冻保存12个月的羊膜中均有表达，且表达量与新鲜羊膜相当。
