臺灣博碩士論文加值系統

青光眼 (Glaucoma) 病徵是視網膜神經節細胞 (retinal ganglion cells, RGC)受到損傷後的漸進性喪失，阻礙視神經傳導，造成視力喪失與失明的疾病。青光眼的成因有許多來源，與基因、眼房液堆積或者外力擠壓拉扯等因素均有相關性，確切發病機制還不完全清楚，多數證據顯示以上因素導致眼內壓力升高是引發視神經損傷的主要危險因素。過去的研究多以眼內高滲透壓之動物模式探討青光眼的發病過程，缺乏高滲透壓對於視網膜神經節細胞生理功能與分機轉的研究及探討。因此，本研究擬以高滲透壓處理分化自人類胚胎幹細胞的視網膜神經節細胞，探討並完成以下三大目標 : (1) 高滲透壓對於細胞的生理功能之影響，包含電生理表現與細胞分泌之生長相關因子之改變; (2) 壓力相關之分子機轉，尤其著重於神經系統於滲透壓改變時相對應的離子通道TRPV1，探討TRPV1活化後造成的離子通道的改變、支持細胞生長之重要因子生成與分泌的改變; (3) 由機轉中發展出胞內滲透壓相關孔道或受體的阻斷劑、或者其下游之抑制劑、活化劑，釐清眼內壓升高導致的高滲透壓模式下RGC存活率、生理功能是否均回復之現象。本研究發現高滲透壓導致TRPV1的過度表達可能會損害RGC細胞，並利用高劑量的NaCl來模擬高滲透壓建立hiPSC-RGC細胞的損傷模式，並發現TRPV1通道的活化。活化後的TRPV1下游通過立即促進BDNF的產生來保護RGC細胞免受受傷。 同時，TRPV1還增強了鈣離子的流入，隨後激活了一系列自噬分子。 一旦平衡被破壞，TRPV1過度表達會導致細胞死亡。此外實驗結果顯示通過I-RTX和H89，證明TRPV1從ER轉移到細胞膜是細胞存活所必需的，但過表達的TRPV1最終會導致細胞死亡。本研究結果期望可以提供未來開發青光眼治療藥物新選擇。

Glaucoma is one kind of retinal diseases. The main symptoms are severe atrophic optic nerves and irreversible progressive loss of retinal ganglion cells (RGCs). Many factors may induce the occurrence of Glaucoma, including genes, ocular fluid accumulation or external squeeze pull and other factors, while elevated intraocular pressure (IOP) is the major risk factor for glaucoma. So far, most of studies were focused on the influence of IOP on trabecular meshwork but not RGCs, whereas accompanied dystrophy of RGCs would lead to progressive visual loss very quickly. Hence, it is needed to develop combinative therapeutics for retarding the deleterious of RGCs. In the past, many studied showed that pathogenesis of glaucoma was accompanied with high osmotic pressure, which disturbed the cellular adhesion and calcium balance by increasing the expression of glaucoma relative genes, such as CYP1B1. However, fewer studies mentioned the effects of the alteration of osmolarity on RGCs. Therefore, this study intends to through administrating the hiPSCs derived retinal ganglion cells with high osmotic pressure to explore and accomplish the following three goals: (1) To validate the effect of osmotic pressure influences on the genetic expression, i.e. glaucoma related genes and the physiological function of RGCs, including electrophysiological manifestations and neural growth factors secretion. (2) To detect the responses of pressure-related molecules, i.e. ion channel TRPV1 or TRPV4 and their downstream protein expression which is corresponding to the function and morphological regulation of RGCs. (3) To reduce the activation of pressure-stimulated molecules by applying RGCs with the channel or receptor blockers, or its downstream inhibitors, activators for further validating the essential role of our targeted pressure-relative molecules in mediating cellular fluid/transmission and the secretion of neural growth factors, i.e. BDNF. Meanwhile, via the inhibition of the targeted pressure-related molecules, it will be benefit to recover the functions and morphology of RGCs under osmolarity stimulation. Furthermore, our study reveals that TRPV1 expression is the key player in maintaining the early survival of high osmolality-induced damaged hiPSC-derived RGCs and a downstream member of activated TRPV1 functions to protect RGC cells from injuries by promoting the immediate generation of BDNF. Meanwhile, TRPV1 also enhanced the influx of calcium followed by the activation of a cascade of autophagy molecules. I-RTX and H89 demonstrated that the translocation of TRPV1 from ER to cell membrane is essential for cell survival, and the overexpression of TRPV1 would lead to ultimate cell death. This study is expected to provide new options for future development of glaucoma treatment.

摘要 i
ABSTRACT iii
CONTENT V
LIST OF FIGURES Viii
LIST OF ABBREVIATIONS iX
Chapter 1. Introduction 1
1.1 Introduction of RGC 2
1.2 Introduction of Glaucoma 3
1.3 Eye hyperosmolarity 4
1.4 Transient receptor potential cation channel subfamily V member 1 (TRPV1) 5
1.5 iPSC derived RGCs as a platform 7
1.6. The rationale of this study 8
Chapter 2. Materials and Methods 9
2.1 Cell culture 10
2.1.1 Culture of RGC-5 10
2.1.2 Culture of hiPSCs 10
2.1.3 Differentiation of hiPS cells to RGCs 10
2.1.4 High osmolality treatment 11
2.2 Western blot 12
2.3 Immunofluorescence 12
2.4 Cell viability (MTT Assay) 13
2.5 Cell culture medium osmolality test 13
2.6 Electrophysiological Analysis 13
2.7 ELISA 14
2.8 In vivo eye pressure measurement 14
2.9 Statistical analysis 15
Chapter 3. Results 16
3.1 Induction of hiPSC-derived RGCs 17
3.2 Hyperosmotic stress response of RGC cells 18
3.3 Autophagy reaction in response to hyperosmotic stress in hiPSC-derived RGCs 19
3.5 BDNF secretion from H89 treated RGCs 23
3.6 H89 attenuated NaCl induced high osmolarity in vivo. 23
3.7 The summary of high osmolarity caused damages through activating TRPV1 on RGC 24
Chapter 4. Discussion 25
Chapter 6. Conclusion 30
Reference 33
Table and figures 38


LIST OF FIGURES
Figure 1. Induction of iPSC-derived RGCs 39
Figure 2. Hyperosmotic stress response of RGC cells 42
Figure 3. Hyperosmotic stress response of RGC cells fuction 45
Figure 4. Autophagy reaction in response to hyperosmotic stress in hiPSC-derived RGCs 49
Figure 5. BDNF secretion from H89 treated RGCs 52
Figure 6. H89 attenuated NaCl induced high osmolarity in vivo 53
Figure 7. The summary of high osmolarity caused damages through activating TRPV1 on RGC 54

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