Ba Pengfei, Zhang Xiaojuan, Yu Miao, Li Linxia, Duan Xiaoyu, Wang Mingying, Lv Shuyan, Fu Guo, Yang Pishan, Yang Chengzhe, Sun Qinfeng
Department of Periodontology, School of Stomatology, Shandong University, Jinan, Shandong 250012, P.R. China.
Shandong Provincial Key Laboratory of Oral Tissue Regeneration, Shandong University, Jinan, Shandong 250012, P.R. China.
Oncol Lett. 2019 Sep;18(3):2484-2490. doi: 10.3892/ol.2019.10556. Epub 2019 Jul 4.
The aim of the present study was to investigate the differential biological characteristics between cancer-associated fibroblasts (CAFs) and peri-tumor fibroblasts (PTFs) in tongue squamous cell carcinoma (TSCC). The primary CAFs and PTFs from TSCC were obtained and purified. Cell morphology was observed, and the expression of α-smooth muscle actin (α-SMA), vimentin and cytokeratin 19 (CK19) was detected by immunohistochemistry (IHC). The percentage of α-SMA positive cells in CAFs and PTFs was calculated separately, and α-SMA expression was further confirmed by western blot analysis. Cell viability and the expression of matrix metalloproteinase 2 (MMP2), stromal cell derived factor1 (SDF-1) and transforming growth factor β1 (TGFβ1) in the purified fibroblasts was detected separately. CAFs and PTFs were attained and purified. Compared with PTFs, CAFs were long-fusiform shaped cells with reduced cytoplasm and variable size. CAFs crowded together in a disorderly manner when the cell density was increased, but this phenomenon did not occur with PTFs. IHC results verified that there was no significant difference between CAFs and PTFs in the percentage of cells staining positive for CK19 and vimentin (P>0.05); the percentage of positive staining cells for α-SMA in CAFs was significantly higher compared with that in PTFs (P<0.001) Western blot analysis showed that α-SMA expression in CAFs was 4.3-fold higher compared with that in PTFs (P<0.001). A Cell Counting Kit-8 assay indicated that the viability of CAFs increased significantly compared with that in the PTFs (P<0.05). Reverse transcription-quantitative polymerase chain reaction and ELISA analysis showed that the expression of MMP2, SDF-1 and TGF β1 in CAFs was higher compared with that in PTFs (P<0.05). CAFs are distinguishable from PTFs with respect to their morphology, cellular phenotype, cell viability and pro-carcinogenic cytokine expression.
本研究的目的是调查舌鳞状细胞癌（TSCC）中癌相关成纤维细胞（CAF）和肿瘤周围成纤维细胞（PTF）之间的生物学差异特征。获取并纯化来自TSCC的原代CAF和PTF。观察细胞形态，并通过免疫组织化学（IHC）检测α平滑肌肌动蛋白（α-SMA）、波形蛋白和细胞角蛋白19（CK19）的表达。分别计算CAF和PTF中α-SMA阳性细胞的百分比，并通过蛋白质印迹分析进一步确认α-SMA的表达。分别检测纯化的成纤维细胞中的细胞活力以及基质金属蛋白酶2（MMP2）、基质细胞衍生因子1（SDF-1）和转化生长因子β1（TGFβ1）的表达。获取并纯化CAF和PTF。与PTF相比，CAF为长梭形细胞，细胞质减少且大小不一。当细胞密度增加时，CAF以无序方式聚集在一起，但PTF未出现这种现象。IHC结果证实，CAF和PTF中CK19和波形蛋白染色阳性细胞的百分比无显著差异（P>0.05）；CAF中α-SMA阳性染色细胞的百分比显著高于PTF（P<0.001）。蛋白质印迹分析表明，CAF中α-SMA的表达比PTF高4.3倍（P<0.001）。细胞计数试剂盒-8检测表明，与PTF相比，CAF的活力显著增加（P<0.05）。逆转录定量聚合酶链反应和酶联免疫吸附测定分析表明，CAF中MMP2、SDF-1和TGFβ1的表达高于PTF（P<0.05）。CAF在形态、细胞表型、细胞活力和促癌细胞因子表达方面与PTF不同。
