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

本研究的目的是分析肿瘤和非肿瘤病变条件下纤维母细胞的基因表达谱和相应的亚分类。(1)采用石蜡包埋切片和CD34、vimentin（vim）、podoplanin/D2-40（pod）、细胞角蛋白8（CK8）、α -平滑肌肌动蛋白（ASMA）、高分子量caldesmon （HCD）单克隆抗体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CD34+vim+pod- CK8-ASMA- hcd细胞，ii)癌症早期侵袭粘膜下层、急性炎症和近期局限性循环障碍以及辐照后组织中的CD34-vim+pod+CK8-ASMA- hcd细胞，iii)侵袭性腺癌和胃肠道腺瘤腺周区间质中的CD34-vim+pod+CK8-ASMA+ hcd细胞，iv)疤痕组织中的CD34-vim+pod+CK8-ASMA+ hcd细胞，v) CD34-vim+pod+CK8+ASMA+HCD-细胞…在肿瘤和非肿瘤性疾病累及的浆膜下组织中更多见，vi) CD34-vim+pod-CK8-ASMA+HCD+细胞在正常的结肠隐窝（隐窝周围成纤维细胞）中，vii) CD34-vim+pod-CK8-ASMA+HCD-细胞在胃肠道腺瘤的腺周区域。(2)为了确认和/或修改所提出的成纤维细胞亚型，应尽快从新鲜的正常和病变组织中分离细胞（成纤维细胞、淋巴细胞等），使用细胞类型特异性细胞表面标记物和磁珠（磁珠法）。然后进行基因表达序列分析和DNA微阵列分析。(3)在磁珠法中，为成纤维细胞选择良好的细胞表面标记物是最重要的。正常成纤维细胞和内皮细胞表面均表达CD34，而上皮细胞、成熟淋巴细胞等间充质细胞表面不表达CD34；内皮细胞被CD31抗体去除后，用CD34检测正常成纤维细胞。(4)癌症相关成纤维细胞（CA-Fib）不存在特异性的细胞表面标记物。大多数癌症相关成纤维细胞在其细胞膜上表达膜截断基质金属蛋白酶（MT1-MMP）。针对MT1-MMP的特异性抗体适合于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