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，这些组织是如何在没有血液流动的情况下维持的，人们知之甚少。在无血管组织中，我们重点研究了髓核(NP)，即椎间盘的无血管成分。为了探索在NP中表达哪些分子，我们在NP和其他11种不同的组织中进行了微阵列分析。有趣的是，我们发现血管内皮生长因子(VEGF)在NP组织中的表达高于其他组织。为了分析血管内皮生长因子在NP中的作用，我们通过Western blotting和RT-PCR分析证实了VEGF在NP组织中的表达。鼻咽癌组织中均检测到血管内皮生长因子的剪接变异体。低氧反应基因GLUT-1和PGK-1以及血管内皮生长因子在NP细胞中的表达高于其他组织，NP为低氧标志物，匹莫硝唑为阳性组织。此外，体外…提示，在低氧条件下，np细胞中血管内皮生长因子的表达上调，而在常氧条件下则不表达。提示缺氧可促进神经节细胞血管内皮生长因子的表达。在VEGF受体中，RT-PCR检测到Flt-1而不是Flk-1在NP中表达，这一结果在Flt-1-LacZ和Flk-1-LacZ敲入小鼠NP的LacZ染色中得到了证实。这些结果提示，在NP细胞中，血管内皮生长因子可能通过Flt-1作为自分泌或旁分泌环发挥抗凋亡作用。在体外，加入Flt-1-Fc可增加凋亡细胞的数量，但不能增加CD4-Fc的作用。因此，抗凋亡可能是血管内皮生长因子在NP细胞中的作用之一。在MRI分析中，与人类一样，大鼠的腰椎间盘T2高信号强度，即健康的NP，随着年龄的增长而降低。由于T2高信号随增龄而减弱，NP中VEGFmRNA的表达下调，这与软骨形成标志物聚集素和II型胶原的表达下调有关。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的存活中发挥作用。目前，我们已经开始利用Flt-1-TK/TK小鼠对其在NP细胞中的血管内皮生长因子的功能进行分析，Flt-1信号转导缺陷是由于Flt-1的胞浆结构域被破坏。较少

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