ISOLATION OF STEM CELLS FOR THE KIDNEY AND EXPERIMENTAL TRIAL FOR THE REGENERATIVE MEDICINE

肾脏干细胞的分离及再生医学的实验研究

• 批准号: 13470211
• 负责人: IMAI Enyu
• 金额: $ 9.47万
• 依托单位: OSAKA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13470211/
• 关键词: REGENERATION; STEM CELL; GFP; RENAL FAILURE; TUBULAR EPITHELIAL CELL; SP CELL; RETINOIC ACID; NEPHROTIC SYNDROME; 骨髄幹細胞; 糸球体上皮細胞; GFPラット; PDGF

Using transgenic rats which express GFP (Green Fluorescent Protein) throughout the body, we established chimeric rats which carry GFP(+) cells in all progeny of bone marrow. This system allowed us to find that bone marrow-derived cells contributed to the regeneration of mesangial cell in a self limiting acute glomerulonephritis model. We also showed that bone mar row cells were able to differentiate into mesangial cells in vitro. Both in vivo and in vitro, Platelet-derived growth factor-B was critical for the generation of bone marrow-derived mesangial cells. We also established a method to purify precursor cells/stem cells for mesangial cells from crude bone marrow preparation by using an unwoven mesh filter system. We purified side population (SP) cells from adult rat kidneys, and characterized them. In short, we proved that SP cells were not the stem cells for mesangial cells or tubular epithelial cells even though they clearly existed in the kidney. In order to find genes which are critical for the nephrogenesis, we compared a set of RNA obtained from neonatal rat kidneys to another set of RNA obtained from adult rat kidneys. By the suppression subtractive hybridization method, we found that retinaldehyde dehydrogenase type 2 (RALDH2) was highly enriched in the comma-shaped body and S-shaped body, and that RALDH2 was upregulated during the differentiation of glomerular epithelial cells. We also developed a new method which allows us to purify RNA from the nephrogenic zone of neonatal rat kidneys. Our extensive analysis revealed that the local concentration of retinoic acid that is produced by RALDH2 plays a critical role to repair injured podocytes, and that retinoic acid directly regulates the transcription of nephrin. Our further analysis about RALDH2 and other newly identified genes are under way.

使用全身表达GFP（绿色荧光蛋白）的转基因大鼠，我们建立了在所有骨髓后代中携带GFP（+）细胞的嵌合大鼠。这一系统使我们能够发现骨髓来源的细胞在自限性急性肾小球肾炎模型中有助于系膜细胞的再生。我们还发现骨髓细胞在体外能够分化为系膜细胞。无论在体内还是体外，血小板源性生长因子-B对骨髓源性系膜细胞的生成都是至关重要的。我们还建立了一种方法，纯化前体细胞/干细胞的系膜细胞从粗骨髓制备，通过使用无纺网过滤系统。我们纯化侧群（SP）细胞从成年大鼠肾脏，并进行了表征。总之，我们证明了SP细胞不是系膜细胞或肾小管上皮细胞的干细胞，尽管它们明确存在于肾脏中。为了找到对肾发生至关重要的基因，我们比较了从新生大鼠肾脏获得的一组RNA和从成年大鼠肾脏获得的另一组RNA。通过抑制性消减杂交技术，我们发现2型视黄醇脱氢酶（RALDH 2）在肾小球S形小体和逗号小体中高度富集，并且在肾小球上皮细胞分化过程中RALDH 2表达上调。我们还开发了一种新的方法，使我们能够从新生大鼠肾脏的肾区纯化RNA。我们广泛的分析表明，由RALDH 2产生的视黄酸的局部浓度对修复受损的足细胞起着关键作用，并且视黄酸直接调节nephrin的转录。我们对RALDH 2和其他新发现的基因的进一步分析正在进行中。

项目成果

Ito T, Suzuki A, Okabe M, Imai E, et al.: "Application of bone marrow derived stem cells in experimental nephrology"Exp Nephrol. 9. 444-450 (2001)

Ito T、Suzuki A、Okabe M、Imai E 等人：“骨髓干细胞在实验肾病学中的应用”Exp Nephrol。

Imai E.: "Gene therapy approach in renal disease in the 21st century"Nephrol Dial Transplant. 16 Suppl 5. 26-34 (2001)

Imai E.：“21 世纪肾脏疾病的基因治疗方法”肾拨号移植。

Isaka Y, Imai E.: "Future strategy: gene therapy for diabetic nephropathy"Contrib Nephrol. 127-132 (2001)

Isaka Y、Imai E.：“未来策略：糖尿病肾病的基因治疗”Contrib Nephrol。

Ito T, Suzuki A, Imai E, et al.: "Tornado extraction : a method to enrich and purify RNA from the nephrogenic zone of the neonatal rat kidney"Kidney Int. 62. 763-769 (2002)

Ito T、Suzuki A、Imai E 等人：“龙卷风提取：一种从新生大鼠肾脏的肾源区富集和纯化 RNA 的方法”Kidney Int。

Horio M, Ito A, Matsuoka Y, Moriyama T, et al.: "Apoptosis induced by hypertonicity in Madin Darley canine kidney cells : protective effect of betaine"Nephrol Dial Transplant. 16. 483-490 (2001)

Horio M、Ito A、Matsuoka Y、Moriyama T 等人：“Madin Darley 犬肾细胞高渗诱导的细胞凋亡：甜菜碱的保护作用”肾移植。