Establishment of gene therapy targeted for renal mesangial and proximal tubular cells

针对肾系膜和近端肾小管细胞的基因治疗的建立

• 批准号: 12671049
• 负责人: HAYASHI Matsuhiko
• 金额: $ 1.98万
• 依托单位: Keio 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-12671049/
• 关键词: NFκB; Experimental glomerulonephritis; Adenovirus; Proximal tubules; IκB; Interstitial damage

(1) Development of single-chane monoclonoal antibody against mesangial cellsTo develop single-chain monoclonal antibody against mesangial cells, Balb/c mouse were immunized with rat and human mesangial cells. From these immunized mouse spleen, CDNA library of phage-display type monoclonal antibody was developed. Currently, second-screening of this library is being performed, although specific antibody has not been obtained, yet.(2) Gene transfer of truncated IκBα to renal proximal cells in renal impairment ratsAs renal interstitial injury model, albumin-loaded rats were used. Gene transfer of truncated IκBα, which inhibits nuclear factor κB (NFκB), key molecule for inflammation, was performed with recombinant adenovirus via renal artery. Adenovirus transferred reporter gene into proximal cells, efficiently, and transferred truncated IκBα abolished tubulo-interstitial changes by albumin loading. From these results, it was suggested that inhibition of NFκB prevented renal damage in this model, we also examined a possible drug for the treatment of renal diseases. We found that, tranilast, which is known to inhibit NFκB in several cell lines, could inhibit NFκB activation in mesangial cells, suggesting that this drug may be useful for clinical settings.To express transferred gene more efficiently, we analyzed promoter activity of vitamin D 1α-hydroxylase, which is predominantly expressed in proximal tubules. This study showed that region of 105 bp upstream of the initiation codon has promoter activity for expression in proximal tubular cell line, LLC-PK1. Furthermore, TCF-1 binding motif in this region is shown to play important roles in promoter activity.

（1）开发针对弥赛亚细胞的单髓单克隆抗体开发针对弥赛亚细胞的单链单克隆抗体，用大鼠和人类弥赛亚细胞免疫BALB/C小鼠。从这些免疫刺激的小鼠脾脏中，开发了噬菌体播种型单克隆抗体的cDNA库。目前，尽管尚未获得特异性抗体，但仍在执行该文库的第二筛选。（2）（2）在肾脏损伤的拉塔斯肾脏间质损伤模型中，截短的IκBα向肾脏代理细胞的基因转移，但使用了白蛋白负载的大鼠。通过肾动脉进行重组腺病毒，抑制注射核因子κB（NFκB）的截短IκBα的基因转移。腺病毒将记者基因有效地转移到代理细胞中，并通过白蛋白载荷转移了截短的IκBα消除的tubulo-Interstitial变化。从这些结果中，提出对NFκB的抑制阻止了该模型的肾脏损害，我们还检查了一种可能治疗肾脏疾病的药物。我们发现，已知可以抑制几个细胞系中NFκB的透明剂可以抑制内键细胞中的NFκB激活，这表明该药物可能对临床环境有用。为了更有效地表达基因，我们分析了维生素D1α-羟基酶的启动子活性，这是占主导地位的pepantepearnal in prominiminiminalsiminalsiminalsiminarsiminalsiminarsiminalsiminalsiminals in proximinal in proximinal in proximinals in proximinalsiminaine proximinimininail proximinimininail的启动。这项研究表明，倡议密码子上游的105 bp区域具有启动子活性，可在近端结核细胞系有限责任公司LLC-PK1中表达。此外，该区域中的TCF-1结合基序被证明在启动子活性中起着重要作用。

项目成果

Hayashi M, et al.: "The effects of calcium channel blockers on nuclear factor kappa B activation in the mesangium cells"Hypertens Res. 23(5). 521-525 (2000)

Hayashi M 等人：“钙通道阻滞剂对系膜细胞核因子 kappa B 激活的影响”Hypertens Res。

Yoshida T, et al.: "Dietary phosphorus deprivation induces 25-hydroxyvitamin D_3 1 alpha-hydroxylase gene expression"Endocrinology. 142(5). 1720-1726 (2001)

Yoshida T 等人：“膳食磷缺乏诱导 25-羟基维生素 D_3 1 α-羟化酶基因表达”内分泌学。

Tsuganezawa H, et al.: "A new member of the HCO_3 transporter superfamily is an apical anion exchanger of beta-intercalated cells in the kidney"J Biol Chem. 276(11). 8180-8189 (2001)

Tsuganezawa H 等人：“HCO_3 转运蛋白超家族的新成员是肾脏中 β 嵌入细胞的顶端阴离子交换剂”J Biol Chem。

Yoshida T, et al.: "Identification of a renal proximal tubular cell-specific enhancer in the mouse 25-hydroxyvitamin D 1α-hydroxylase"J Am Soc Nephrol. (In press).

Yoshida T 等人：“小鼠 25-羟基维生素 D 1α-羟化酶中肾近端肾小管细胞特异性增强剂的鉴定”J Am Soc Nephrol。

Shimizu-Hirota R, et al.: "Regulation of vascular proteoglycan synthesis by angiotensin II type 1 and type 2 receptors"J Am Soc Nephrol. 12 (12). 2609-2615 (2001)

Shimizu-Hirota R 等人：“血管紧张素 II 1 型和 2 型受体对血管蛋白聚糖合成的调节”J Am Soc Nephrol。