Swelam Wael, Ida-Yonemochi Hiroko, Maruyama Satoshi, Ohshiro Kazufumi, Cheng Jun, Saku Takashi
Department of Tissue Regeneration and Reconstruction, Division of Oral Pathology, Niigata University Graduate School of Medical and Dental Sciences, 2-5274 Gakkocho-dori, Niigata 951-8514, Japan.
Virchows Arch. 2005 Jun;446(6):653-62. doi: 10.1007/s00428-005-1219-1. Epub 2005 Apr 23.
To better understand the poorly vascularized background of the stroma of pleomorphic adenomas, we attempted to determine the expression of molecules related to blood vessels and hypoxic conditions in pleomorphic adenoma. Surgical specimens and tumor cells in primary culture of salivary pleomorphic adenomas were used for immunohistochemistry for CD31, vascular endothelial growth factor (VEGF) and its receptors Flk-1 and Flt-1, as well as for hypoxia markers, such as hypoxia-inducible factor-1alpha (HIF-1alpha) and lactate dehydrogenase-1 (LDH). At the same time, alternative splicing modes of the VEGF gene and expression levels of the HIF-1alpha gene were analyzed in surgical specimens by means of reverse-transcription polymerase chain reaction (RT-PCR) and direct sequencing of the PCR products. In addition to co-immunolocalization with CD31+ vascular endothelial cells, VEGF and its receptors were demonstrated in normal duct epithelial and myoepithelial cells as well as in tumor cells in ductal structures and in myxochondroid stromata. Immunolocalizations for HIF-1alpha and LDH were confirmed in the VEGF-positive area. Immunofluorescence signals for VEGF and others were confirmed in pleomorphic adenoma cells in culture. RT-PCR results showed that there were at least four splicing modes of the VEGF gene, among which VEGF(121) was most enhanced, and higher HIF-1alpha levels in pleomorphic adenomas. The results suggest that pleomorphic adenoma cells produce VEGF in several functional forms for their own proliferation or differentiation, and that the VEGF expression is controlled by hypoxic circumstances of poorly vascularized pleomorphic adenomas.
为了更好地理解多形性腺瘤基质血管化不良的背景，我们试图确定多形性腺瘤中与血管和缺氧状态相关分子的表达情况。唾液多形性腺瘤手术标本及原代培养的肿瘤细胞用于CD31、血管内皮生长因子（VEGF）及其受体Flk-1和Flt-1的免疫组织化学检测，以及缺氧标志物如缺氧诱导因子-1α（HIF-1α）和乳酸脱氢酶-1（LDH）的检测。同时，通过逆转录聚合酶链反应（RT-PCR）和PCR产物直接测序分析手术标本中VEGF基因的可变剪接模式和HIF-1α基因的表达水平。除了与CD31+血管内皮细胞共免疫定位外，VEGF及其受体在正常导管上皮和肌上皮细胞以及导管结构和黏液软骨样基质中的肿瘤细胞中也有表达。在VEGF阳性区域证实了HIF-1α和LDH的免疫定位。在培养的多形性腺瘤细胞中证实了VEGF等的免疫荧光信号。RT-PCR结果显示VEGF基因至少有四种剪接模式，其中VEGF(121)增强最为明显，且多形性腺瘤中HIF-1α水平较高。结果表明，多形性腺瘤细胞产生多种功能形式的VEGF以促进自身增殖或分化，且VEGF的表达受血管化不良的多形性腺瘤缺氧环境的调控。
