Gene expression profiling and corresponding sub-classification of fibroblastic cells within neoplastic and non-neoplastic diseased conditions

本研究的目的是基因表达谱分析和相应的肿瘤和非肿瘤疾病条件下的成纤维细胞的亚分类。(1)使用石蜡包埋切片和CD 34、波形蛋白（Vim）、podoplanin/D2-40（pod）、细胞角蛋白8（CK 8）、α-平滑肌肌动蛋白（ASMA）和高分子量钙调蛋白（HCD）的单克隆抗体对胃和结直肠肿瘤性和非肿瘤性疾病进行免疫组织化学分析。提出了7种成纤维细胞; i）正常组织中的CD 34 +Vim+ pod + CK 8-ASMA-HCD-细胞，ii）在癌侵袭粘膜下层、急性炎症和近期局部循环障碍以及照射后组织的早期中的CD 34-Vim+pod+ CK 8-ASMA-HCD-细胞，iii）在侵袭性腺癌的促纤维增生基质和胃肠腺瘤的腺周区域中的CD 34-Vim+pod+ CK 8-ASMA+ HCD-细胞，iv）瘢痕组织中的CD 34-Vim+ pod-CK 8-ASMA + HCD-细胞， 关于我们 vi）正常结肠直肠隐窝中的CD 34-Vim+ pod-CK 8-ASMA+HCD+细胞（隐窝周成纤维细胞），vii）胃肠腺瘤的腺周区域中的CD 34-Vim+ pod-CK 8-ASMA+HCD-细胞。(2)为了确认和/或修改成纤维细胞的拟定亚型，应使用细胞类型特异性细胞表面标记物和磁珠（磁珠法）从新鲜的正常和患病组织中快速分离细胞（成纤维细胞、淋巴细胞等）。在此基础上，进行基因表达系列分析和基因芯片分析。(3)在进行磁珠法时，选择良好的成纤维细胞表面标记物是最重要的。正常成纤维细胞和内皮细胞在细胞表面均表达CD 34，但上皮细胞、成熟淋巴细胞和其他间充质细胞不表达;正常成纤维细胞在内皮细胞被CD 31抗体去除的部分中通过CD 34检测。(4)对于癌症相关成纤维细胞（CA-Fib），不存在特异性细胞表面标志物。大多数癌相关成纤维细胞在其细胞膜上表达膜截短的基质金属蛋白酶（MT 1-MMP）。MT 1-MMP的特异性抗体适用于CA-Fib。少

The purpose of this study is gene expression profiling analysis and corresponding sub-classification of fibroblastic cells within neoplastic and non-neoplastic diseased conditions.(1) Immunohistochemical analysis using paraffin-embedded sections and monoclonal antibodies to CD34, vimentin(vim), podoplanin/D2-40(pod), cytokeratin 8(CK8), alpha-smooth muscle actin (ASMA), and high molecular weight caldesmon (HCD) was performed in gastric and colorectal neoplastic and non-neoplastic diseases. Seven kinds of fibroblastic cells were proposed; i) CD34+vim+pod-CKS-ASMA-HCD-cells in normal tissue, ii) CD34-vim+pod+CK8-ASMA-HCD-cells in early phase of cancer invasion to submucosa, acute inflammation and recent localized circulatory disturbance, and post-irradiated tissue, iii) CD34-vim+pod+CK8-ASMA+HCD-cells in desmoplastic stroma of invasive adenocarcinomas and periglandular areas of gastrointestinal adenomas, iv) CD34-vim+pod-CK8-ASMA+HCD-cells in scar tissue, v) CD34-vim+pod+CK8+ASMA+HCD-cel … More ls seen in subserosal tissue involved by neoplastic and non-neoplastic diseases, vi) CD34-vim+pod-CK8-ASMA+HCD+ cells in normal colorectal crypts (pericryptal fibroblast), vii) CD34-vim+pod-CK8-ASMA+HCD- cells in peri-glandular areas of gastrointestinal adenomas.(2) To confirm and/or revise the proposed subtypes of fibroblastic cells, cell isolation (fibroblastic cells, lymphocytes, etc) should be hurried from the fresh normal and diseased tissues, using cell type specific cell surface markers and magnetic beads (magnetic bead method). And then, both serial analysis of gene expression and DNA microarray analysis should be done.(3) In performing the magnetic beads method, to choose good cell surface markers for fibroblasts is most important. Both normal fibroblasts and endothelial cells express CD34 in the cell surface, but epithelial cells, mature lymphocytes and other mesenchymal cells does not; normal fibroblast are detected by CD34 in a fraction which endothelial cells are removed by CD31 antibody.(4) No specific cell surface markers exist for cancer-associated fibroblasts (CA-Fib). Most of the cancer-associated fibroblasts express membrane-truncated matrix metalloproteinase (MT1-MMP) in their cell membrane. Specific antibodies to MT1-MMP are suitable for CA-Fib. Less