Abstract：
OBJECTIVE: In the current investigation, the effects of the mTOR inhibitors, Rapa and Torin1 on the TGF-β2-induced conjunctival fibrogenesis were studied.
METHODS: Experimental research.
METHODS: 2D and 3D cultures of HconF were subjected to the following analyses; (1) planar proliferation evaluated by TEER (2D), (2) Seahorse metabolic analyses (2D), (3) subepithelial proliferation evaluated by the 3D spheroids\' size and hardness, and (4) the mRNA expression of ECM proteins and their regulators (2D and 3D).
RESULTS: Rapa or Torin1 both significantly increased planar proliferation in the non-TGF-β2-treated 2D HconF cells, but in the TGF-β2-treated cells, this proliferation was inhibited by Rapa and enhanced by Torin1. Although Rapa or Torin1 did not affect cellular metabolism in the non-TGF-β2-treated HconF cells, mTOR inhibitors significantly decreased and increased the mitochondrial respiration and the glycolytic capacity, respectively, under conditions of TGF-β2-induced fibrogenesis. Subepithelial proliferation, as evidenced by the hardness of the 3D spheroids, was markedly down-regulated by both Rapa and Torin1 independent of TGF-β2. The mRNA expressions of several ECM molecules and their regulators fluctuated in the cases of 2D vs 3D and TGF-β2 untreated vs treated cultures.
CONCLUSIONS: The present findings indicate that mTOR inhibitors have the ability to increase and to reduce planar and subepithelial proliferation in HconF cells, depending on the inhibitor being used.
METHODS: Experimental research.
METHODS: 2D and 3D cultures of HconF were subjected to the following analyses; (1) planar proliferation evaluated by TEER (2D), (2) Seahorse metabolic analyses (2D), (3) subepithelial proliferation evaluated by the 3D spheroids\' size and hardness, and (4) the mRNA expression of ECM proteins and their regulators (2D and 3D).
RESULTS: Rapa or Torin1 both significantly increased planar proliferation in the non-TGF-β2-treated 2D HconF cells, but in the TGF-β2-treated cells, this proliferation was inhibited by Rapa and enhanced by Torin1. Although Rapa or Torin1 did not affect cellular metabolism in the non-TGF-β2-treated HconF cells, mTOR inhibitors significantly decreased and increased the mitochondrial respiration and the glycolytic capacity, respectively, under conditions of TGF-β2-induced fibrogenesis. Subepithelial proliferation, as evidenced by the hardness of the 3D spheroids, was markedly down-regulated by both Rapa and Torin1 independent of TGF-β2. The mRNA expressions of several ECM molecules and their regulators fluctuated in the cases of 2D vs 3D and TGF-β2 untreated vs treated cultures.
CONCLUSIONS: The present findings indicate that mTOR inhibitors have the ability to increase and to reduce planar and subepithelial proliferation in HconF cells, depending on the inhibitor being used.
摘要：
目的：在目前的调查中，mTOR抑制剂的作用，研究了Rapa和Torin1对TGF-β2诱导的结膜纤维化的影响。
方法：实验研究。
方法：对HconF的2D和3D培养物进行以下分析：（1）通过TEER（2D）评估平面增殖，（2）海马代谢分析（2D），(3)通过3D球状体的大小和硬度评估上皮下增殖，和（4）ECM蛋白及其调节因子的mRNA表达（2D和3D）。
结果：Rapa或Torin1均显着增加非TGF-β2处理的2DHconF细胞的平面增殖，但是在TGF-β2处理的细胞中，Rapa抑制了这种增殖，Torin1增强了这种增殖。尽管Rapa或Torin1不影响非TGF-β2处理的HconF细胞的细胞代谢，mTOR抑制剂显著降低和增加线粒体呼吸和糖酵解能力，分别,在TGF-β2诱导的纤维发生的条件下。上皮下增生,正如3D球体的硬度所证明的那样，Rapa和Torin1明显下调，与TGF-β2无关。在2Dvs3D和TGF-β2未处理vs处理培养物中，几种ECM分子及其调节剂的mRNA表达波动。
结论：目前的研究结果表明，mTOR抑制剂能够增加和减少HconF细胞的平面和上皮下增殖，取决于所使用的抑制剂。
方法：实验研究。
方法：对HconF的2D和3D培养物进行以下分析：（1）通过TEER（2D）评估平面增殖，（2）海马代谢分析（2D），(3)通过3D球状体的大小和硬度评估上皮下增殖，和（4）ECM蛋白及其调节因子的mRNA表达（2D和3D）。
结果：Rapa或Torin1均显着增加非TGF-β2处理的2DHconF细胞的平面增殖，但是在TGF-β2处理的细胞中，Rapa抑制了这种增殖，Torin1增强了这种增殖。尽管Rapa或Torin1不影响非TGF-β2处理的HconF细胞的细胞代谢，mTOR抑制剂显著降低和增加线粒体呼吸和糖酵解能力，分别,在TGF-β2诱导的纤维发生的条件下。上皮下增生,正如3D球体的硬度所证明的那样，Rapa和Torin1明显下调，与TGF-β2无关。在2Dvs3D和TGF-β2未处理vs处理培养物中，几种ECM分子及其调节剂的mRNA表达波动。
结论：目前的研究结果表明，mTOR抑制剂能够增加和减少HconF细胞的平面和上皮下增殖，取决于所使用的抑制剂。
