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Development of gene theropy for glaucoma by neaws of modulation of proteoglycan praduction in aqueous outflow rocite

通过调节房水流出液中蛋白多糖的产生来开发青光眼基因疗法

基本信息

批准号：
08457464
负责人：
TANIHARA Hidenobu
金额：
$ 4.74万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

We have investigated our hypothesis on mechanism of primary angly open glaucoma, that an interactive relationship between TGF-beta2 in aqueous humor and proteoglycan (PG) expression in trabecular meshwork (TM) cells, may play crucial roles in regulation of aqueous outflow resistance.Gene expression of the PG core protein was screened in human and bovine cultured TM cells by reverse transcription -initiated polymerase chain reaction (RT-PCR) and the RT-PCR experiments showed that cDNA fragments of the three PGs (decorin, lumican and fibromodulin). Subsequently, the TM cells were cultured with TGF-beta2, and the changes of gene expressions of the PGs were investigated. As a result, the expression of three PGs (decorin, lumican and fibromodulin) were up-regulated by TGF-beta2 stimulation.In addition, we assessed TGF-beta2 in aqueous humor which were collected from variable glaucomatous patients and non-glaucomatous (cataractous) patients with a specific-capture ELISA.Our study revealed that increased levels of TGF-beta2 in aqueous humor was found only in eyes with POAG,but not with EXS and PACG.These findings suggested that increased levels of TGF-beta2 may be different from other types of glaucoma.Furthrmore, we attempted to create a POAG model by means of TGF-beta2 gene transfer and resultant increase of proteolglycan production in TM cells, and in autocrine and/or paracrine manners of production. We have succeeded to efficiently introduce genes to primate and human TM cells by the HVJ liposome method. This results showed the potential of this method for a somatic gene therapy of the trabecular meshwork to treat glaucoma.These result suggested the potential role of increased levels of TGF-beta2 in aqueous humor and resultant increase of proteolglycan production in the pathogenesis of primary open angle glaucoma, and the gene transfer methods may be useful to treat glaucoma.

我们已经研究了我们关于原发性开角型青光眼机制的假设，即房水中TGF-β 2与小梁网（TM）细胞中蛋白聚糖（PG）表达之间的相互作用关系，采用逆转录-聚合酶链反应（RT-PCR）技术，在人和牛培养的TM细胞中筛选PG核心蛋白的基因表达。RT-PCR结果显示，获得了核心蛋白聚糖（decorin）、光蛋白聚糖（lumican）和纤调蛋白（fibromodulin）的cDNA片段。随后，将TM细胞与TGF-β 2共同培养，并观察PG基因表达的变化。结果，三种PG的表达此外，我们评估了从不同类型的青光眼患者和非青光眼患者收集的房水中的TGF-β 2，我们的研究显示，只有在患有POAG的眼睛中才发现房水中TGF-β 2水平的增加，本研究通过将TGF-β_2基因导入TM细胞，使TM细胞蛋白多糖的分泌增加，并以自分泌和/或旁分泌的方式表达，建立了POAG模型。我们已经成功地通过HVJ脂质体方法将基因有效地引入灵长类动物和人TM细胞。本研究结果表明，小梁网体细胞基因治疗青光眼的方法是可行的，提示青光眼患者房水中TGF-β_2水平的升高及由此引起的蛋白多糖的增加在原发性开角型青光眼的发病机制中可能起着重要的作用，基因转移方法可能对青光眼的治疗有一定的意义。