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

利用在全身表达绿色荧光蛋白的转基因大鼠，我们建立了在所有子代骨髓中携带绿色荧光蛋白（+）细胞的嵌合大鼠。该系统使我们发现骨髓来源的细胞有助于自限性急性肾小球肾炎模型中系膜细胞的再生。我们还发现，骨髓细胞能够在体外分化为系膜细胞。在体内和体外实验中，血小板源性生长因子- b对骨髓源性系膜细胞的生成至关重要。我们还建立了一种利用非织造网过滤系统从粗骨髓制备物中纯化系膜细胞前体细胞/干细胞的方法。我们从成年大鼠肾脏中纯化了侧群（SP）细胞，并对其进行了表征。总之，我们证明SP细胞不是系膜细胞或小管上皮细胞的干细胞，尽管它们明显存在于肾脏中。为了找到对肾形成至关重要的基因，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。通过抑制减法杂交方法，我们发现视黄醛脱氢酶2型（RALDH2）在逗号形体和s形体中高度富集，并且在肾小球上皮细胞分化过程中RALDH2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肾源区纯化RNA。我们的广泛分析表明，RALDH2产生的局部维甲酸浓度对损伤足细胞的修复起关键作用，维甲酸直接调节肾素的转录。我们对RALDH2和其他新发现基因的进一步分析正在进行中。

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.