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Molecular and cellular approaches to the treatment of degenerative lumbar disc disease

治疗退行性腰椎间盘疾病的分子和细胞方法

基本信息

批准号：
17591595
负责人：
CHIBA Kazuhiro
金额：
$ 2.24万
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：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

项目摘要

There are several avascular tissues such as intervertebral disc, cartilage and lens. However, how these tissues are maintained without blood flow is poorly understood. Among the avascular tissues, we focused on nucleus pulposus (NP), avascular component of intervertebral disc. To explore what molecules are expressed in NP, microarray analysis was performed in NP and other eleven different tissues. Interestingly, we found that Vascular Endothelial Growth Factor (VEGF) was highly expressed in NP when compared with other tissuesTo analyse role of VEGF in NP, VEGF expression in NP was confirmed by western blotting and RT-PCR analysis. All splice variants of VEGF were detected in NP. -Expressions of hypoxic response genes such as GLUT-1 and PGK-1 as well as VEGF were higher in NP cells than other tissues, and NP was hypoxic marker, pimonidazole, positive tissues. Moreover, VEGF expression in NP cells was up-regulated under hypoxic conditions and not in normoxic conditions in vitro suggestin … More g that VEGF expression in NP is promoted by hypoxia. Among VEGF receptors, the expression of Flt-1 but not Flk-1 was detected in NP by RT-PCR analysis, and this finding was confirmed by LacZ staining of NP in Flt-1-lacZ and Flk-1-lacZ knock-in mice. These results suggest that VEGF may function as an anti-apoptotic factor in NP cells through Flt-1 as autocrine or paracrine loop. In in vitro, numbers of apoptotic cells was increased by addition of Flt-1-Fc but not by CD4-Fc. Thus, anti-apoptosis may be one of functions of VEGF in NP cells. In MRI analysis, T2 high signal intensity of intervertebral disc, which indicate a healthy NP, decreased with aging in rat as seen in human. As T2 high signal decreased with aging, the VEGF mRNA expression in NP was down-regulated, which also correlated with down-regulation of expression of chondrogenic markers such as aggrecan and type II collagen.Taken together, our results demonstrate that VEGF-Flt-1 signal may play role in NP survival in autocrine/paracrine manner. Now, we have started analysis on function of VEGF in NP cells using Flt-1-TK/TK mice, which has Flt-1 signaling deficiency because of disruption of cytoplasmic domain of Flt-1. Less

有几种无血管组织，如椎间盘、软骨和晶状体。然而，人们对这些组织如何在没有血流的情况下维持状态知之甚少。在无血管组织中，我们重点关注髓核（NP），即椎间盘的无血管成分。为了探索 NP 中表达的分子，在 NP 和其他 11 种不同组织中进行了微阵列分析。有趣的是，我们发现与其他组织相比，血管内皮生长因子（VEGF）在NP中高表达。为了分析VEGF在NP中的作用，通过蛋白质印迹和RT-PCR分析证实了NP中VEGF的表达。在 NP 中检测到 VEGF 的所有剪接变体。 -NP细胞中GLUT-1、PGK-1等缺氧反应基因以及VEGF的表达高于其他组织，NP为缺氧标志物，哌莫硝唑，阳性组织。此外，在体外缺氧条件下，NP细胞中的VEGF表达上调，而在常氧条件下则不然，这表明缺氧会促进NP中的VEGF表达。在 VEGF 受体中，通过 RT-PCR 分析在 NP 中检测到 Flt-1 而不是 Flk-1 的表达，并且通过 Flt-1-lacZ 和 Flk-1-lacZ 敲入小鼠中 NP 的 LacZ 染色证实了这一发现。这些结果表明VEGF可能通过Flt-1作为自分泌或旁分泌环在NP细胞中发挥抗凋亡因子的作用。在体外，添加 Flt-1-Fc 会增加凋亡细胞的数量，但添加 CD4-Fc 则不会增加凋亡细胞的数量。因此，抗凋亡可能是VEGF在NP细胞中的功能之一。在 MRI 分析中，大鼠椎间盘的 T2 高信号强度（表明 NP 健康）随着年龄的增长而降低，就像在人类中看到的那样。由于T2高信号随着年龄的增长而减弱，NP中的VEGF mRNA表达下调，这也与软骨形成标志物如聚集蛋白聚糖和II型胶原蛋白的表达下调相关。综上所述，我们的结果表明VEGF-Flt-1信号可能以自分泌/旁分泌的方式在NP存活中发挥作用。现在，我们开始使用 Flt-1-TK/TK 小鼠分析 VEGF 在 NP 细胞中的功能，该小鼠由于 Flt-1 胞质结构域的破坏而具有 Flt-1 信号传导缺陷。较少的