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

有几种无血管组织，如椎间盘、软骨和晶状体。然而，在没有血液流动的情况下，这些组织是如何维持的，人们知之甚少。在无血管组织中，我们着重于髓核（NP），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种不同的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中的表达较高。为了分析VEGF在NP中的作用，我们通过western blotting和RT-PCR分析证实了VEGF在NP中的表达。在NP中检测到所有的VEGF剪接变体。- NP细胞中GLUT-1、PGK-1等缺氧反应基因及VEGF表达高于其他组织，NP为缺氧标志物、吡硝唑阳性组织。此外，在缺氧条件下，NP细胞中VEGF的表达上调，而在体外常氧条件下，VEGF的表达上调，这表明缺氧可以促进NP细胞中VEGF的表达。在VEGF受体中，RT-PCR检测到NP中Flt-1的表达，而Flk-1的表达未被检测到，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。这些结果提示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可增加凋亡细胞的数量，而CD4-Fc则不能。因此，抗凋亡可能是VEGF在NP细胞中的功能之一。MRI分析显示，大鼠椎间盘T2高信号强度与人类一样，随着年龄的增长而下降，表明NP健康。随着年龄的增长，T2高信号降低，NP中VEGF mRNA表达下调，这也与聚集蛋白、II型胶原等软骨标志物表达下调相关。综上所述，我们的研究结果表明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，我们使用Flt-1-TK/TK小鼠开始分析VEGF在NP细胞中的功能，因为Flt-1的胞质结构域被破坏而导致Flt-1信号缺失。少

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