目的探究溶质载体家族7成员5(solute carrier family 7 member 5,SLC7A5)促进转化生长因子-β1(transforming growth factor-β1,TGF-β1)诱导的人肺成纤维细胞活化增殖的作用。方法人肺成纤维细胞用TGF-β1孵育24h,使用PCR-Array方法...目的探究溶质载体家族7成员5(solute carrier family 7 member 5,SLC7A5)促进转化生长因子-β1(transforming growth factor-β1,TGF-β1)诱导的人肺成纤维细胞活化增殖的作用。方法人肺成纤维细胞用TGF-β1孵育24h,使用PCR-Array方法筛选靶基因。使用沉默慢病毒sh-SLC和重表达慢病毒r-SLC转染成纤维细胞,利用Western Blot实验和qRT-PCR实验观察目标基因SLC7A5和成纤维细胞活化指标α平滑肌肌动蛋白(alpha smooth muscle actin,α-SMA)、成纤维细胞特异性蛋白(fibroblast specific protein,FSP)、Ⅰ型胶原(typeⅠcollagen,COL-1)合成。qRT-PCR实验检测mTOR信号通路分子。CCK-8方法检测各组成纤维细胞增殖速度。流式细胞术检测各组细胞周期变化情况。结果与对照组PBS的细胞相比,TGF-β1实验组的HLFs中SLC7A5高表达,沉默SLC7A5基因降低SLC7A5、α-SMA、FSP和COL-1表达量以及mTOR和4EBP1的转录水平,抑制成纤维细胞增殖速度,G1期比例增加,S期降低,而回复实验表明重新表达SLC7A5基因的r-SLC能够逆转以上现象。结论SLC7A5可能通过mTOR信号通路促进TGF-β1诱导的人肺成纤维细胞活化增殖,SLC7A5有望成为肺纤维化疾病中抑制成纤维细胞活化增殖的靶点。展开更多
Tooth morphogenesis is orchestrated by a complex interplay of signaling pathways and transcription factors that control cell proliferation,apoptosis,and differentiation,with the Wnt/β-catenin signaling pathway playin...Tooth morphogenesis is orchestrated by a complex interplay of signaling pathways and transcription factors that control cell proliferation,apoptosis,and differentiation,with the Wnt/β-catenin signaling pathway playing a pivotal role.However,the comprehensive regulatory mechanisms of Wnt/β-catenin signaling remain largely unclear.Smad7,a key antagonist of the TGF-βsuperfamily,is essential for maintaining tissue homeostasis and ensuring proper cellular function.Our previous study has demonstrated that Smad7 knockout in mice leads to impaired proliferative property of tooth germ cells,resulting in small molars.Here,we identified SMAD7 expression in human dental papilla and dental pulp,colocalized with β-CATENIN and cell proliferationrelated proteins.RNA sequencing analysis revealed a significant reduction in Wnt signaling activity in Smad7-deficient mouse tooth germs.Using lentivirus transfection,we established SMAD7-knockdown human dental papilla stem cells,which manifested remarkably blunt proliferation rate,along with diminished Wnt signaling activity.In vivo transplantation investigations further revealed the indispensable role of SMAD7 in dentin formation.Mechanistically,we revealed that β-CATENIN interacts with P-SMAD2/3 and SMAD7 through co-immunoprecipitation and yeast two-hybrid assays.Inhibition of TGF-β pathway or disruption of SMAD7/β-CATENIN transcription factor complex formation potently impacted Wnt/β-catenin activities,indicating both direct and indirect regulatory mechanisms.These findings highlight the critical role of SMAD7 in the proliferation and diffe rentiation of human dental stem cells,which could contribute to dental tissue regeneration and engineering.展开更多
文摘目的探究溶质载体家族7成员5(solute carrier family 7 member 5,SLC7A5)促进转化生长因子-β1(transforming growth factor-β1,TGF-β1)诱导的人肺成纤维细胞活化增殖的作用。方法人肺成纤维细胞用TGF-β1孵育24h,使用PCR-Array方法筛选靶基因。使用沉默慢病毒sh-SLC和重表达慢病毒r-SLC转染成纤维细胞,利用Western Blot实验和qRT-PCR实验观察目标基因SLC7A5和成纤维细胞活化指标α平滑肌肌动蛋白(alpha smooth muscle actin,α-SMA)、成纤维细胞特异性蛋白(fibroblast specific protein,FSP)、Ⅰ型胶原(typeⅠcollagen,COL-1)合成。qRT-PCR实验检测mTOR信号通路分子。CCK-8方法检测各组成纤维细胞增殖速度。流式细胞术检测各组细胞周期变化情况。结果与对照组PBS的细胞相比,TGF-β1实验组的HLFs中SLC7A5高表达,沉默SLC7A5基因降低SLC7A5、α-SMA、FSP和COL-1表达量以及mTOR和4EBP1的转录水平,抑制成纤维细胞增殖速度,G1期比例增加,S期降低,而回复实验表明重新表达SLC7A5基因的r-SLC能够逆转以上现象。结论SLC7A5可能通过mTOR信号通路促进TGF-β1诱导的人肺成纤维细胞活化增殖,SLC7A5有望成为肺纤维化疾病中抑制成纤维细胞活化增殖的靶点。
基金supported by the National Key Research and Development Program of China to W.Tian (2022YFA1104400)the National Natural Science Foundation of China to T.Chen (82100959)a grant from the Sichuan Science and Technology Program to Z.Liu (2024YFFK0068)。
文摘Tooth morphogenesis is orchestrated by a complex interplay of signaling pathways and transcription factors that control cell proliferation,apoptosis,and differentiation,with the Wnt/β-catenin signaling pathway playing a pivotal role.However,the comprehensive regulatory mechanisms of Wnt/β-catenin signaling remain largely unclear.Smad7,a key antagonist of the TGF-βsuperfamily,is essential for maintaining tissue homeostasis and ensuring proper cellular function.Our previous study has demonstrated that Smad7 knockout in mice leads to impaired proliferative property of tooth germ cells,resulting in small molars.Here,we identified SMAD7 expression in human dental papilla and dental pulp,colocalized with β-CATENIN and cell proliferationrelated proteins.RNA sequencing analysis revealed a significant reduction in Wnt signaling activity in Smad7-deficient mouse tooth germs.Using lentivirus transfection,we established SMAD7-knockdown human dental papilla stem cells,which manifested remarkably blunt proliferation rate,along with diminished Wnt signaling activity.In vivo transplantation investigations further revealed the indispensable role of SMAD7 in dentin formation.Mechanistically,we revealed that β-CATENIN interacts with P-SMAD2/3 and SMAD7 through co-immunoprecipitation and yeast two-hybrid assays.Inhibition of TGF-β pathway or disruption of SMAD7/β-CATENIN transcription factor complex formation potently impacted Wnt/β-catenin activities,indicating both direct and indirect regulatory mechanisms.These findings highlight the critical role of SMAD7 in the proliferation and diffe rentiation of human dental stem cells,which could contribute to dental tissue regeneration and engineering.
