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Molecular analysis of cell growth regulating factor in retinal, capillary endothelial cells

视网膜、毛细血管内皮细胞细胞生长调节因子的分子分析

基本信息

批准号：
12672206
负责人：
HOSOYA Ken-ichi
金额：
$ 2.05万
依托单位：
TOYAMA MEDICAL AND PHARMACEUTICAL UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12672206/
关键词：
inner blood-retinal barrier retinal endothelial cell line retinal pericyte cell line cell growth regulating factor conditionally immortalized cell line co-culture system xc oxidative stress condition TGFβ1 条件的不死化細胞株液性因子血管新生 syste

项目摘要

Conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB) and retinal pericyte cell line (TR-rPCT) were established from transgenic rats harboring the temperature-sensitive simian virus 40 large T-antigen gene. The paracrine interaction between TR-iBRB and TR-rPCT cells has been investigated to elucidate the cell growth regulating factor in pericyte. TR-iBRB cells were seeded on the insert of Transwell chamber and TR-rPCT cell were seeded under the insert (contact co-culture model) and on the well chamber (non-contact co-culture model). The conditioned medium for TR-rPCT cells was also used to observe the cell growth of TR-iBRB cells. The growth of TR-iBRB cells was significantly slowed down with increasing concentration of TR-rPCT conditioned medium. In contact co-culture model, the growth of TR-iBRB cells was arrested 4 days after the beginning of the co-culture. These results suggest that some factor released from pericyte cells is involved in regulating the cell growth in retinal capillary endothelial cells.The [^<14>C] L-cystine uptake by TR-iBRB cells appeared to be mediated through a saturable Na^+-independent process with a Michaelis-Menten constant of 9.2 μM, suggesting system xc, which consists of XCT and 4F2hc mRNA, mediated L-cystine uptake. After 100 μM diethyl maleate treatment, which is a model agent for oxidative stress, the XCT mRNA level and L-cystine uptake activity in TR-iBRB cells were enhanced in a time-dependent manner. Concomitantly, the glutathione concentration in TR-iBRB cells was increased. In contrast, the 4F2hc mRNA level was unchanged up to 24 hours and was induced for more than 24 hours by DEM pretreatment. These findings suggest L-cystine transport is activated by induction of system xc under the oxidative stress conditions at the inner blood-retinal barrier.

以携带猴病毒40大t抗原基因的转基因大鼠为材料，建立了条件永生化大鼠视网膜毛细血管内皮细胞系（TR-iBRB）和视网膜周细胞细胞系（TR-rPCT）。研究了TR-iBRB和TR-rPCT细胞的旁分泌相互作用，以阐明细胞生长调节因子在周细胞中的作用。将TR-iBRB细胞接种于Transwell室的插入物上，将TR-rPCT细胞接种于插入物（接触共培养模型）和孔室（非接触共培养模型）下。同时采用TR-rPCT细胞条件培养基观察TR-iBRB细胞的生长情况。随着TR-rPCT条件培养基浓度的增加，TR-iBRB细胞的生长明显减慢。在接触共培养模型中，共培养开始4天后，TR-iBRB细胞的生长被阻止。这些结果提示，视网膜毛细血管内皮细胞的生长可能与周细胞释放的某种因子有关。r - ibrb细胞摄取[^<14 bb0 C] l -胱氨酸可能是通过一个不依赖于Na^+的饱和过程介导的，Michaelis-Menten常数为9.2 μM，表明由XCT和4F2hc mRNA组成的系统xc介导了l -胱氨酸的摄取。100 μM马来酸二乙酯作为氧化应激模型剂处理后，TR-iBRB细胞XCT mRNA水平和l -胱氨酸摄取活性呈时间依赖性增强。同时，TR-iBRB细胞中的谷胱甘肽浓度升高。相比之下，4F2hc mRNA水平在24小时内保持不变，并通过DEM预处理诱导超过24小时。这些发现表明，在氧化应激条件下，l -胱氨酸运输是通过诱导血视网膜屏障内的xc系统激活的。