Centre for Tissue Engineering and Regenerative Medicine, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia.
Department of Physiology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia.
Adv Exp Med Biol. 2024;1450:59-76. doi: 10.1007/5584_2023_777.
Conditioned medium from cultured fibroblast cells is recognized to promote wound healing and growth through the secretion of enzymes, extracellular matrix proteins, and various growth factors and cytokines. The objective of this study was to profile the secreted proteins present in nasal fibroblast conditioned medium (NFCM). Nasal fibroblasts isolated from human nasal turbinates were cultured for 72 h in Defined Keratinocytes Serum Free Medium (DKSFM) or serum-free F12: Dulbecco's Modified Eagle's Medium (DMEM) to collect conditioned medium, denoted as NFCM_DKSFM and NFCM_FD, respectively. SDS-PAGE was performed to detect the presence of protein bands, followed by MALDI-TOF and mass spectrometry analysis. SignalP, SecretomeP, and TMHMM were used to identify the secreted proteins in conditioned media. PANTHER Classification System was performed to categorize the protein according to protein class, whereas STRING 10 was carried out to evaluate the predicted proteins interactions. SDS-PAGE results showed the presence of various protein with molecular weight ranging from ~10 kDa to ~260 kDa. Four protein bands were identified using MALDI-TOF. The analyses identified 104, 83, and 7 secreted proteins in NFCM_FD, NFCM_DKSFM, and DKSFM, respectively. Four protein classes involved in wound healing were identified, namely calcium-binding proteins, cell adhesion molecules, extracellular matrix proteins, and signaling molecules. STRING10 protein prediction successfully identified various pathways regulated by secretory proteins in NFCM. In conclusion, this study successfully profiled the secreted proteins of nasal fibroblasts and these proteins are predicted to play important roles in RECs wound healing through various pathways.
条件培养基来源于培养的成纤维细胞，被认为通过分泌酶、细胞外基质蛋白和各种生长因子和细胞因子来促进伤口愈合和生长。本研究的目的是分析鼻成纤维细胞条件培养基（NFCM）中存在的分泌蛋白。从人鼻鼻甲中分离的鼻成纤维细胞在无血清角质形成细胞培养基（DKSFM）或无血清 F12：杜尔贝科改良伊格尔培养基（DMEM）中培养 72 小时，以收集条件培养基，分别表示为 NFCM_DKSFM 和 NFCM_FD。进行 SDS-PAGE 以检测蛋白质条带的存在，然后进行 MALDI-TOF 和质谱分析。使用 SignalP、SecretomeP 和 TMHMM 识别条件培养基中的分泌蛋白。使用 PANTHER 分类系统根据蛋白质类别对蛋白质进行分类，而 STRING 10 则用于评估预测蛋白的相互作用。SDS-PAGE 结果显示存在各种分子量范围从10 kDa 到260 kDa 的蛋白质。使用 MALDI-TOF 鉴定了 4 个蛋白质条带。分析鉴定出 NFCM_FD、NFCM_DKSFM 和 DKSFM 中分别有 104、83 和 7 种分泌蛋白。鉴定出 4 种参与伤口愈合的蛋白质类，即钙结合蛋白、细胞粘附分子、细胞外基质蛋白和信号分子。STRING10 蛋白预测成功识别了 NFCM 中分泌蛋白调节的各种途径。总之，本研究成功地对鼻成纤维细胞的分泌蛋白进行了分析，这些蛋白预测通过各种途径在 RECs 伤口愈合中发挥重要作用。
