臺灣博碩士論文加值系統

眼角膜（cornea）是眼睛對抗外來物的第一道防線，覆蓋角膜的上皮細胞（corneal epithelial cells）負責保護角膜間質並維持角膜的透亮。角膜上皮細胞的凋亡（apoptosis）是包括乾眼症、物理或化學性的傷害、低劑量的紫外線傷害、隱形眼鏡造成的角膜水腫及角膜感染等多種眼角膜疾病最終的病理變化。乙四型胸腺素（thymosin beta-4; Tb4）由四十三個氨基酸所組成，廣泛存在於細胞內，除了調節細胞的骨架與移行外，其在不同的細胞及不同的環境下，具有不同的生理功能，近年來的研究顯示外加乙四型胸腺素可以保護眼角膜上皮細胞、結膜上皮細胞及心肌細胞免於凋亡，但其作用機轉仍不清楚，因此本研究乃深入探討外加乙四型胸腺素之抑制眼角膜細胞凋亡的機轉。
本研究使用經SV40病毒不朽化之人類眼角膜上皮細胞為模型，外加自大腸桿菌純化之N-端帶有六個組氨酸（Histidine-tag）的乙四型胸腺素，經免疫螢光染色分析後，發現該胜肽於二小時內即進入細胞（內在化, internalization）。分別以Fas Ligand（FasL）及過氧化氫（H2O2）引發眼角膜上皮細胞因活化死亡受體（death receptor）的外在路徑(extrinsic pathway）或粒線體的內在路徑（intrinsic pathway）造成凋亡時，發現外加乙四型胸腺素藉由抑制蛋白質分解酶（caspase）-8、9、3的活化以降低該細胞之凋亡，有趣的是，當該細胞經胞飲作用抑制劑cytochalasin D處理後，不但乙四型胸腺素的內在化受到抑制，且其抗凋亡的作用也喪失。顯示乙四型胸腺素的內在化是其協助眼角膜上皮細胞對抗凋亡所必需。
再者，外加乙四型胸腺素可經由降低細胞內氧化壓力（oxidative stress）而抑制因過氧化氫造成粒線體路徑的活化。在正常狀態下，該胜肽不但可以增加錳超氧化物歧化酶(Mn-SOD)及銅/鋅超氧化物歧化酶(Cu/Zn-SOD)的蛋白質表現量；也能在有過氧化氫的環境下，進一步增強錳超氧化物歧化酶和過氧化氫酶（catalase）的基因表現及蛋白質表現量；反之，以過氧化氫酶抑制劑3-amino-1,2,4-triazole同時處理細胞，則會阻斷乙四型胸腺素對過氧化氫造成細胞傷害的保護。
綜言之，本研究証明外加的乙四型胸腺素的內在化是抗眼角膜細胞凋亡的必要條件，其抑制氧化壓力/內在路徑所引發的細胞凋亡機制很可能是藉由活化重要的抗氧化酶的表現來降低細胞內氧化壓力，以增加其存活。

Cornea surface, the first-line defense of the eye, is covered with corneal epithelial cells which functionally protect corneal stroma from injury and maintain the transparency of stroma. Apoptosis is the ultimate pathomechanism of many corneal diseases including dry eye, mechanical or chemical injury, ultraviolet ray injury, contact lens-induced corneal edema and infection. Thymosin beta-4 (Tb4) is a ubiquitous peptide consisting of 43 amino acids that has pleiotropic functions including an anti-apoptotic effect on corneal epithelial cells when exogenously administered. However, the protective mechanism of Tb4 on cornea remains largely unknown. The purpose of this study is to elucidate the anti-apoptotic mechanism(s) of exogenous Tb4 on human corneal epithelial cells.
In this study, SV-40 immortalized human corneal epithelial (HCE-T) cells were incubated with recombinant Tb4 produced by E. coli. Immunofluorescence staining demonstrated that exogenous Tb4 entered HCE-T cells within 2 hours (i.e. internalization). Fas Ligand (FasL) and hygrogen peroxide (H2O2) were used to trigger extrinsic and intrinsic pathway-mediated apoptosis on HCE-T cells, respectively. Exogenous Tb4 inhibited the activation of caspase-8 and -3 induced by FasL as well as caspase-9 and -3 triggered by H2O2. Internalization of this peptide into HCE-T cells was found to be essential for its apoptosis prevention since disruption of the cellular entry of Tb4 by cytochalasin D abrogated its cytoprotective effects.
Moreover, Tb4 could reduce intracellular ROS levels induced by H2O2. Tb4 not only stimulated the expression of manganese superoxide dismutase (Mn-SOD) and copper/zinc SOD (Cu/Zn-SOD) in normal physiological conditions, but also enhanced the transcription and translation of both MnSOD and catalase induced by H2O2. Finally, the addition of 3-amino-1,2,4-triazole (AT), a catalase inhibitor, abrogated the protective effect of Tb4 against H2O2-induced oxidative damage.
In conclusion, this study demonstrated that internalization is essential for the anti-apoptotic effects of exogenous Tb4 on HCE-T cells; and its protection against oxidative damage/intrinsic pathway-mediated apoptosis can be attributed to the up-regulation of crucial anti-oxidative enzymes.

CONTENTS

page
I. ABSTRACT 6
II. INTRODUCTION 11
III. MATERIALS AND METHODS 17
Cell Culture 17
Preparation of the Recombinant Tb4 17
Preparation of the Anti-Tb4 Polyclonal Antibody 19
Reagent 20
a. Apoptosis Inducers
b. Endocytosis Inhibitor
c. ROS Scavenger
d. Catalase Inhibitor
Western Blot Analysis 22
a. Recombinant Tb4
b. Anti-ROS Enzymes and apoptosis-related proteins
MTT and MTS Assays 24
Immunofluorescence Staining 24
a. Internalization of Exogenous Tb4
b. Caspase Activation
Flowcytometry 26
a. Surface Expression of Fas
b. Fluorogenic Substrate Cleavage Assay
c. Intracellular ROS Level
Colorimetric Substrate Cleavage Assay 27
RNA Isolation 27
Conventional and Quantitative RT-PCR Analysis 28
Mitochondrial Activity Analysis 29
a. Mitochondrial-Specific Gene Expression
b. Mitochondrial DNA Copy Number
c. Oxidative Phosphorylation-Related Protein
d. ATP Production
e. Oxygen Consumption
f. Mitochondrial Mass
Statistics 33
IV. RESULTS 34
Recombinant Tb4 is recognized by antibodies against both histidine tag and Tb4. 35
FasL and H2O2 trigger death of HCE-T cells. 35
Exogenous Tb4 prevents HCE-T cells from FasL- and H2O2-induced apoptosis. 37
Exogenous Tb4 enters HCE-T cells. 37
Cytochalasin D blocks the internalization of Tb4 in HCE-T cells. 38
Internalization is essential for the anti-apoptotic effect of exogenous Tb4. 38
Tb4 does not alter surface expression of Fas on HCE-T cells. 40
Tb4 does not alter the expression of Bax and Bcl-2 in HCE-T cells. 40
Tb4 does not change mitochondrial functions in HCE-T cells. 40
Apoptosis triggered by H2O2 is mediated through the intrinsic pathway as a consequence of increased intracellular oxidative stress. 41
Tb4 reduces intracellular oxidative stress in HCE-T cells elicited by H2O2. 42
Tb4 may not be a ROS scavenger. 42
Tb4 increases the expression of several anti-oxidative enzymes in HCE-T cells. 43
Catalase inhibitor counteracts the ROS-reducing effects of exogenous Tb4. 44
V. DISCUSSION 45
VI. REFERENCES 54
VII. FIGURES 71
Figure 1. Preparation of the recombinant histidine-tagged Tb4 and a polyclonal anti-Tb4 antibody. 72
Figure 2. Exogenous Tb4 protects HCE-T cells against FasL-induced apoptosis. 73
Figure 3. Exogenous Tb4 protects HCE-T cells against H2O2-induced apoptosis. 74
Figure 4. Exogenous Tb4 enters HCE-T cells. 75
Figure 5. Cytochalasin D blocks the internalization of Tb4 in HCE-T cells. 76
Figure 6. Cytochalasin D blocks the antiapoptotic effect of exogenous Tb4. 77
Figure 7. Cytochalasin D reduces the suppressive effect of exogenous Tb4 on caspase activation. 78
Figure 8. Tb4 does not alter surface expression of Fas on HCE-T cells. 79
Figure 9. Tb4 does not affect Bax and Bcl-2 expression on HCE-T cells. 80
Figure 10. Apoptosis by H2O2 is mediated through an intrinsic pathway. 81
Figure 11. H2O2-elicited elevation of intracellular ROS is suppressed by Tb4. 82
Figure 12. Elevation of intracellular ROS induced by H2O2 is suppressed by exogenous Tb4 but not immediately. 83
Figure 13. Tb4 increases protein expression of several anti-oxidative enzymes. 84
Figure 14. Tb4 up-regulates gene expression of several anti-oxidative enzymes. 85
Figure 15. Catalase inhibitor abrogates the ROS-reducing effects of Tb4. 86
VIII. SUPPLEMENTARY FIGURES 87
Supplementary figure 1. Tb4 is not toxic to HCE-T cells at the concentration of 20 �慊/ml. 88
Supplementary figure 2. Dose-dependent protective effect of exogenous Tb4 on HCE-T cells. 89
Supplementary figure 3. Optimal concentration of cytochalasin D is determined by cytotoxicity test. 90
Supplementary figure 4. Apoptosis of HCE-T cells triggered by high concentration of H2O2 is mediated through both extrinsic and intrinsic pathway. 91
Supplementary figure 5. Neither Tb4 nor H2O2 alters the expression of some mitochondrial-specific genes. 92
Supplementary figure 6. Tb4 does not affect the levels of several oxidative phosphorylation-related proteins. 93
Supplementary figure 7. Neither Tb4 nor H2O2 alters ATP production in HCE-T cells. 94
Supplementary figure 8. Neither Tb4 nor H2O2 alters oxygen consumption of HCE-T cells. 95
Supplementary figure 9. Neither Tb4 nor H2O2 changes mitochondrial mass in HCE-T cells. 96
Supplementary figure 10. Neither Tb4 nor H2O2 alters mitochondrial DNA copy number in HCE-T cells. 97
IX. CONCLUSION 98
X. PROSPECT 101
XI. APPENDIX 105
Ho JH, Chuang CH, Ho CY, Shih YR, Lee OK, Su Y. Internalization is essential for the anti-apoptotic effects of exogenous thymosin beta-4 on human corneal epithelial cells. Invest Ophth Vis Sci. 2007;48: 27-33. 106
Ho JH, Tseng KC, Ma WH, Chen KH, Lee OK, Su Y. Thymosin beta-4 up-regulates anti-oxidative enzymes and protects human cornea epithelial cells against oxidative damage. Brit J Ophthalmol 2008 (accepted) 113

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