In this study the mouse tumor necrosis factor-alpha (TNF-α) was prepared by stimulating macrophage cell line RAW 264.7 with lipopoly-saccharide (LPS) obtained from Escheria coli strain 055:B5.

No description available.

No description available.

No description available.

Liver X receptor (LXR) is a member of the nuclear hormone receptor superfamily that is activated by hydroxylated cholesterol derivatives referred to as oxysterols. It has also been shown to play a crucial role in regulating cholesterol trafficking and lipid metabolism in liver and macrophages. Furthermore, LXR. has also been directly implicated in the reduction of atherosclerosis in several murine models of the disease by virtue of its ability to promote reverse cholesterol efllux from intima-resident lipid-loaded macrophages. While roles for LXR in monocyte biology have focused primarily on cholesterol trafficking, evidence for other functions for the receptor outside of its traditional role as a mediator of cholesterol homeostasis is lacking. Presented herein is evidence that LXR also serves as a mediator of cytokine expression. This work has shown that treatment of human peripheral blood monocytes or monocytic THP-1 cells with the LXR ligand 22(R)-hydroxycholesterol (22R-HC), in combination with 9-cis-retinoic acid (9cRA), a ligand for the LXR. heterodimerization partner retinoid X receptor (RXR), results in the specific induction of the potent pro-apoptotic and pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-α.). Promoter analysis, inhibitor studies, and order-of-addition experiments demonstrated that TNF-α. induction by 22R-HC and 9cRA occurs by a novel two-step process. The initial step involves 22R-HC-dependent induction of TNF-α. mRNA, and intracellular accumulation of TNF-alpha protein, mediated by binding of LXRα/RXRα to an LXR response element at position -879 of the TNF-α promoter. Subsequent cell release of TNF-alpha protein occurs via a separable RXR-dependent step that requires de novo transcription and protein synthesis. Furthermore, the RXR-dependent secretory event can be mimicked by agents that induce monocytic differentiation like phorbol esters that culminate in RXR activation by a pathway that does not require exogenous ligand. In this context, RXR was also shown to be a down stream target of the protein kinase C (PKC) signal transduction cascade, that results in the activation of RXR and the induction of secretory factor(s) which facilitate secretion of LXR-derived TNF-α. These studies have provided evidence that should help to expand the currently known role for LXR in monocyte biology and have furthermore identified a new role for RXRs in promoting the secretion of soluble factors like cytokines. Furthermore, in light of reports that show LXR activity promotes a reduction in atherosclerosis, it stands to reason that this regulatory circuit of LXR-dependent production of TNF -α from monocytes would similarly contribute to the attenuation of atherosclerosis 𝘪𝘯 𝘷𝘪𝘷𝘰. / Thesis / Master of Science (MSc)

Tumor necrosis factor-a (TNFa) is primarily a macrophagederived cytokine. TNFa, in vitro, kills or inhibits growth of approximately one third of surveyed transformed cell lines dincluding the L929 and WEHI 164 murine fibrosarcoma cell lines. Very little is known about the mechanisms of TNFa action. However, recently, it has been theorized that TNFa has no activity of its own and that the receptor for TNFa on the cell surface, when properly triggered, activates the cellular mechanisms which may result in the cell's death. The objective of this study was to produce an antiidiotypic antibody to TNFa to be used as a tool to study the mechanisms of TNFa action. A hybridoma that secretes an antiidiotypic antibody to TNFa (Ab2J1) has been produced and isolated following standard procedures. This antibody was found to be of isotype IgG2a as determined by an indirect ELISA test. The Ab2J1 exhibited TNFa target cell-killing capabilities in vitro. The TNFa-resistant cell lines, SP2jO and NS-1 were resistant to Ab2J1 and TNFa sensitive cells, L929 and WEHI 164, were sensitive to Ab2J1. The cell killing activity of both TNFa and Ab2f3 could be neutralized by a monoclonal anti-TNFa antibody. Both TN Fa and Ab2f3 acted in parallel having an effect on the killing of Brucella abortus strain RB51 by peritoneal macrophages, whereas neither TNFa nor Ab2f3 had an effect on the killing of strain 2308 by macrophages. These results, again indicate that TNFa and Ab2f3 have parallel dbactericidal effects and that Ab2f3 is capable of mimicking TNFa activity. The Ab2J1 was further characterized by gel electrophoresis and Western blot and was found to have two subunits of 25 and 50 kDa molecular weights similar to IgG. This anti-idiotypic antibody to TNFa may help in understanding the mechanisms of the cytotoxic activity of TNFa. / Master of Science

by Shan Sze Wan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 145-166). / Abstracts in English and Chinese. / Title --- p.i / Abstract --- p.ii / 摘要 --- p.v / Acknowledgements --- p.vii / Table of Contents --- p.viii / List of Abbreviations --- p.xiv / List of Figures --- p.xvii / List of Tables --- p.xx / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- What are the general functions of cytokines? --- p.2 / Chapter 1.2 --- What is TNP-α? --- p.4 / Chapter 1.3 --- Actions of TNF-α --- p.5 / Chapter 1.4 --- General functions of TNF-α in astrocytes --- p.6 / Chapter 1.5 --- TNF-α receptors (TNF-Rs) --- p.8 / Chapter 1.6 --- Second messengers induced by TNP-α --- p.10 / Chapter 1.7 --- Glial Cells --- p.11 / Chapter 1.7.1 --- Oligodendroglia --- p.12 / Chapter 1.7.2 --- Brain Macrophages (Microglia) --- p.12 / Chapter 1.7.3 --- Astrocytes --- p.14 / Chapter 1.7.3.1 --- Functions of astrocytes --- p.15 / Chapter 1.8 --- "Brain injury, astrogliosis and scar formation" --- p.20 / Chapter 1.9 --- β-Adrenergic receptors (β-ARs) --- p.21 / Chapter 1.9.1 --- The active functional unit: the receptor complex --- p.22 / Chapter 1.9.2 --- General functions and distribution of β-ARs --- p.22 / Chapter 1.10 --- Functions of β-ARs in astrocytes --- p.24 / Chapter 1.10.1 --- Regulations of astrogliosis by β-ARs --- p.24 / Chapter 1.10.1.1 --- β-ARs are expressed in normal optic nerves and up-regulated after nerve crush --- p.24 / Chapter 1.10.1.2 --- Injury-induced alterations in endogenous catecholamine leads to enhanced β-AR activation --- p.25 / Chapter 1.10.1.3 --- β-AR blockade suppresses glial scar formation --- p.25 / Chapter 1.10.1.4 --- β-AR agonists affect the proliferation of astrocytes in normal brain --- p.26 / Chapter 1.11 --- Manganese Superoxide Dismutase (MnSOD) --- p.27 / Chapter 1.11.1 --- MnSOD is the target gene of NF-kB --- p.29 / Chapter 1.11.2 --- Induction of MnSOD by proinflammatory cytokines in rat primary astrocytes --- p.29 / Chapter 1.11.3 --- SMase and ceramides induce MnSOD in various cell types --- p.30 / Chapter 1.12 --- Why do we use C6 glioma cells? --- p.31 / Chapter 1.13 --- Aims and Scopes of this project --- p.32 / Chapter Chapter 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.36 / Chapter 2.1.1 --- Cell Line --- p.36 / Chapter 2.1.2 --- Cell Culture Reagents --- p.36 / Chapter 2.1.2.1 --- Complete Dulbecco´ةs modified Eagle medium (CDMEM) --- p.36 / Chapter 2.1.2.2 --- Rosewell Park Memorial Institute (RPMI) medium --- p.37 / Chapter 2.1.2.3 --- Phosphate buffered saline (PBS) --- p.37 / Chapter 2.1.3 --- Recombinant cytokines --- p.38 / Chapter 2.1.4 --- Chemicals for signal transduction study --- p.38 / Chapter 2.1.4.1 --- Modulators of protein kinase C (PKC) --- p.38 / Chapter 2.1.4.2 --- Modulator of protein kinase A (PKA) --- p.39 / Chapter 2.1.4.3 --- β-Adrenergic agonist and antagonist --- p.39 / Chapter 2.1.5 --- Antibodies --- p.40 / Chapter 2.1.5.1 --- Anti-TNF-receptor type 1 (TNF-R1) antibody --- p.40 / Chapter 2.1.5.2 --- Anti-TNF-receptor type 2 (TNF-R2) antibody --- p.41 / Chapter 2.1.5.3 --- Anti-βi-adrenergic receptor (βl-AR) antibody --- p.42 / Chapter 2.1.5.4 --- Anti-β2-adrenergic receptor (β2-AR) antibody --- p.42 / Chapter 2.1.5.5 --- Antibody conjugates --- p.43 / Chapter 2.1.6 --- Reagents for RNA isolation --- p.43 / Chapter 2.1.7 --- Reagents for reverse transcription-polymerase chain reaction (RT-PCR) --- p.43 / Chapter 2.1.8 --- Reagents for electrophoresis --- p.45 / Chapter 2.1.9 --- Reagents and buffers for Western blot --- p.45 / Chapter 2.1.10 --- Other chemicals and reagents --- p.47 / Chapter 2.2 --- Maintenance of rat C6 glioma cell line --- p.47 / Chapter 2.3 --- RNA isolation --- p.48 / Chapter 2.3.1 --- Measurement of RNA yield --- p.49 / Chapter 2.4 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.50 / Chapter 2.5 --- Western blot analysis --- p.52 / Chapter Chapter 3 --- RESULTS / Chapter 3.1 --- Effect of TNF-α on the expression of TNF-receptors (TNFRs) in C6 glioma cells --- p.55 / Chapter 3.1.1 --- Effect of TNF-α on TNF-R1 and -R2 mRNA expression in C6 cells --- p.56 / Chapter 3.1.2 --- The signaling systems mediating TNP-α-induced TNF-R2 expression in C6 cells --- p.57 / Chapter 3.1.2.1 --- The involvement of PKC in TNF-α-induced TNF-R2 expression in C6 cells --- p.57 / Chapter 3.1.2.2 --- Effect of PMA on the TNF-R protein levels in C6 cells --- p.63 / Chapter 3.1.2.3 --- Effect of Ro31 on the TNF-α-induced TNF-R protein level in C6 cells --- p.65 / Chapter 3.1.2.4 --- Effect of PKA activator on the level of TNF-R2 mRNA in C6 cells --- p.67 / Chapter 3.2 --- Effect of TNP-α on the expression of β1- and β2-adrenergic receptors (β1- and β2-ARs) in C6 glioma cells --- p.69 / Chapter 3.2.1 --- Effect of TNF-α on β1- and β2-ARs mRNA expression in C6 cells --- p.70 / Chapter 3.2.2 --- The signaling systems mediating TNF-α-induced β1- and β2-AR expression in C6 cells --- p.70 / Chapter 3.2.2.1 --- The involvement of PKC mechanism between TNF-α and β-ARs in C6 cells --- p.71 / Chapter 3.2.2.2 --- Effect of PMA on the β1- and β2-ARs protein level in C6 cells --- p.76 / Chapter 3.2.2.3 --- Effect of Ro31 on the TNF-α-induced β1- and β2-AR protein levels in C6 cells --- p.78 / Chapter 3.2.2.4 --- Effect of dbcAMP on the levels of βl- and β2-ARs mRNA in C6 cells --- p.80 / Chapter 3.3 --- Relationship between TN1F-R2 and β-adrenergic mechanism in C6 cells --- p.82 / Chapter 3.3.1 --- Effects of isproterenol and propranolol on endogenous TNF-α mRNA levels in C6 cells --- p.82 / Chapter 3.3.2 --- Effects of isoproterenol and propranolol on TNF-R2 mRNA levels in C6 cells --- p.83 / Chapter 3.3.3 --- Effects of β1-agonist and antagonist on endogenous TNF-α mRNA expression in C6 cells --- p.87 / Chapter 3.3.4 --- Effects of β1-agonist and antagonist on TNF-R2 mRNA expression in C6 cells --- p.91 / Chapter 3.3.5 --- Effects of β2-agonist and antagonist on endogenous TNF-α mRNA in C6 cells --- p.93 / Chapter 3.3.6 --- Effects of β2-agonist and antagonist on TNF-R2 mRNA in C6 cells --- p.100 / Chapter 3.4 --- Effect ofTNF-α on the expression of a transcriptional factor nuclear factor kappa B (NF-kB) in C6 glioma cells --- p.102 / Chapter 3.4.1 --- Effect ofTNF-α on NF-kB (p50) mRNA expression in C6 cells --- p.106 / Chapter 3.4.2 --- Effect of β-agonist and antagonist on NF-kB (p50) mRNA expression in C6 cells --- p.108 / Chapter 3.4.3 --- Effect of PMA and Ro31 on the levels of NF-kB mRNA in C6 cells --- p.109 / Chapter 3.5 --- Effects of TNF-α on the expression of manganese superoxide dismutase (MnSOD) in C6 glioma cells --- p.111 / Chapter 3.5.1 --- Effects of TNF-α on MnSOD and Cu-ZnSOD mRNAs expression in C6 cells --- p.114 / Chapter 3.5.2 --- Effects of β-agonist and β-antagonist on MnSOD mRNA expression in C6 cells --- p.115 / Chapter 3.5.3 --- Effects of PKC activator and inhibitor on the levels of MnSOD mRNA in C6 cells --- p.117 / Chapter Chapter 4 --- DISCUSSION AND CONCLUSION / Chapter 4.1 --- Effects of TNF-α on the expression of TNF-receptors (TNFRs) in C6 glioma cells --- p.122 / Chapter 4.2 --- Effects of TNF-a on the expression of β1- and β2-adrenergic receptors (β1 and β2-ARs) in C6 glioma cells --- p.126 / Chapter 4.3 --- Relationship between TNF-α and β-adrenergic mechanism in C6 cells --- p.128 / Chapter 4.4 --- Effects of TNF-α on the expression of a transcriptional factor nuclear factor kappa B (NF-kB) in C6 glioma cells --- p.131 / Chapter 4.5 --- Effects of TNF-α on the expression of manganese superoxide dismutase (MnSOD) in C6 glioma cells --- p.133 / Chapter 4.6 --- Possible sources of β-agonists --- p.136 / Chapter 4.7 --- Conclusions --- p.137 / Appendix A --- p.143 / References --- p.145

by Wong Kin Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 348-373). / Abstracts in English and Chinese. / Abstract --- p.ii / 撮要 --- p.iv / Acknowledgements --- p.vi / Table of Contents --- p.vii / List of Abbreviations --- p.xviii / List of Tables --- p.xxi / List of Figures --- p.xxii / Chapter CHAPTER 1. --- INTRODUCTION / Chapter 1.1. --- Events happened in brain injury --- p.1 / Chapter 1.2. --- An alternate approach based on neuronal regeneration --- p.3 / Chapter 1.3. --- Fate of astrocytes after brain injury --- p.4 / Chapter 1.3.1. --- General information of astrocytes --- p.4 / Chapter 1.3.2. --- Functions of astrocytes --- p.5 / Chapter 1.4. --- Factors relate to astrocytes proliferation --- p.7 / Chapter 1.4.1. --- TNF-α --- p.8 / Chapter 1.4.2. --- β adrenergic mechanism and astrocyte proliferation --- p.11 / Chapter 1.5. --- Cell cycle-related proteins --- p.13 / Chapter 1.5.1. --- Maturation promoting factor (MPF) --- p.15 / Chapter 1.5.2. --- Early G1 phase --- p.16 / Chapter 1.5.3. --- Retinoblastoma protein (pRb) --- p.18 / Chapter 1.5.4. --- Cyclin-dependent kinase (cdk) activating kinase (Cak) --- p.19 / Chapter 1.5.5. --- "Cyclin, cdks, cki" --- p.20 / Chapter 1.5.5.1. --- Cyclins --- p.20 / Chapter 1.5.5.1.1. --- Cyclin D --- p.21 / Chapter 1.5.5.1.2. --- Cyclin E --- p.22 / Chapter 1.5.5.1.3. --- Cyclin A --- p.23 / Chapter 1.5.5.1.4. --- Cyclin B --- p.23 / Chapter 1.5.5.2. --- Cyclin-dependent kinases (cdks) --- p.24 / Chapter 1.5.5.3. --- Cyclin-dependent kinase inhibitor (cki) --- p.24 / Chapter 1.5.5.3.1. --- INK4 proteins (inhibitors of cdk-4 and cdk-6) --- p.25 / Chapter 1.5.5.3.2. --- p21 family proteins --- p.25 / Chapter 1.5.5.3.2.1. --- p21 --- p.25 / Chapter 1.5.5.3.2.2. --- p27 --- p.25 / Chapter 1.6. --- Apoptosis related proteins --- p.26 / Chapter 1.6.1. --- bcl-2 family --- p.26 / Chapter 1.6.1.1. --- bcl-2 --- p.26 / Chapter 1.6.1.2. --- bcl-x --- p.27 / Chapter 1.6.1.3. --- bcl-xα --- p.27 / Chapter 1.6.1.4. --- bcl-w --- p.28 / Chapter 1.6.1.5. --- Myeloid cell leukemia factor 1 (Mcl-1) --- p.28 / Chapter 1.7. --- C6 glioma cell line --- p.28 / Chapter 1.8. --- Aim of this project --- p.30 / Chapter CHAPTER 2. --- MATERIALS & METHODS / Chapter 2.1. --- Materials / Chapter 2.1.1. --- Rat C6 glioma cell line --- p.32 / Chapter 2.1.2. --- Cell culture materials preparation / Chapter 2.1.2.1. --- Complete Dulbecco's Modified Medium (cDMEM) --- p.32 / Chapter 2.1.2.2. --- Serum-free Dulbecco's Modified Medium (sDMEM) --- p.33 / Chapter 2.1.2.3. --- Phosphate buffered saline (PBS) --- p.33 / Chapter 2.1.3. --- Drug preparation / Chapter 2.1.3.1. --- Recombinant cytokines --- p.34 / Chapter 2.1.3.2. --- Antibodies / Chapter 2.1.3.2.1. --- Antibodies used in expression analysis --- p.34 / Chapter 2.1.4. --- Antibodies used in Western blotting --- p.34 / Chapter 2.1.5. --- Reagents for RNA isolation --- p.36 / Chapter 2.1.6. --- Reagents for reverse transcription-polymerase chain reaction (RT-PCR) --- p.36 / Chapter 2.1.7. --- Reagents for Electrophoresis --- p.38 / Chapter 2.1.8. --- Reagents and buffers for Western blotting --- p.38 / Chapter 2.1.9. --- Other chemicals and reagents --- p.39 / Chapter 2.2. --- Methods / Chapter 2.2.1. --- Maintenance of C6 cells --- p.39 / Chapter 2.2.2. --- Preparation of cells for assays --- p.40 / Chapter 2.2.3. --- Drugs preparation --- p.40 / Chapter 2.2.4. --- Determination of RNA expression by RT-PCR analysis / Chapter 2.2.4.1. --- RNA extraction --- p.41 / Chapter 2.2.4.2. --- Spectrophotometric Quantitation of DNA and RNA --- p.43 / Chapter 2.2.4.3. --- RNA gel electrophoresis --- p.43 / Chapter 2.2.4.4. --- Reverse transcription-polymerase chain reaction (RT- PCR) --- p.43 / Chapter 2.2.4.5. --- Separation of PCR products by agarose gel electrophoresis --- p.43 / Chapter 2.2.4.6. --- Quantification of band density --- p.45 / Chapter 2.2.4.7. --- Restriction enzyme (RE) digestion --- p.45 / Chapter 2.2.5. --- Determination of protein expression by Western blotting / Chapter 2.2.5.1. --- Total protein extraction --- p.46 / Chapter 2.2.5.2. --- Western blotting analysis --- p.46 / Chapter CHAPTER 3. --- RESULTS / Chapter 3.1. --- Effects of TNF-α on cell cycle related genes and proteins expression --- p.49 / Chapter 3.1.1. --- Effects of TNF-α on the time courses of cyclin D1 gene and protein expression --- p.49 / Chapter 3.1.2. --- Effect of TNF-α on the time course of cyclin D2 gene expression --- p.50 / Chapter 3.1.3. --- Effects of TNF-α on the time courses of cyclin D3 gene and protein expression --- p.53 / Chapter 3.1.4. --- Effects of TNF-α on the time courses of cdk-4 gene and protein expression --- p.55 / Chapter 3.1.5. --- Effects of TNF-α on the time courses of cyclin E gene and protein expression --- p.55 / Chapter 3.1.6. --- Effects of TNF-α on the time courses of cdk-2 gene and protein expression --- p.58 / Chapter 3.1.7. --- Effects of TNF-α on the time courses of p15 gene and protein expression --- p.61 / Chapter 3.1.8. --- Effects of TNF-α on the time courses of p27 gene and protein expression --- p.61 / Chapter 3.1.9. --- Effects of TNF-α on the time courses of p21 gene and protein expression --- p.64 / Chapter 3.1.10. --- Effects of TNF-α on the time courses of p130 gene and protein expression --- p.66 / Chapter 3.1.11. --- Effects of TNF-α on the time courses of Cak gene and protein expression --- p.66 / Chapter 3.1.12. --- Effects of TNF-α on the time courses of cyclin H gene and protein expression --- p.68 / Chapter 3.1.13. --- Effects of TNF-α on the time courses of cyclin B gene and protein expression- --- p.71 / Chapter 3.1.14. --- Effect of TNF-α on the time course of bcl-2 protein expression --- p.71 / Chapter 3.1.15. --- Effects of TNF-α on the time courses of bcl-XL gene and protein expression --- p.73 / Chapter 3.1.16. --- Effect of TNF-α on the time course of bcl-xα gene expression --- p.73 / Chapter 3.1.17. --- Effects of TNF-α on the time courses of bcl-w gene and protein expression --- p.76 / Chapter 3.1.18. --- Effects of TNF-α on the time courses of Mcl-1 gene expression --- p.76 / Chapter 3.2. --- Effects of TNF-R1 and -R2 on cell cycle related genes and proteins expression --- p.81 / Chapter 3.2.1. --- Effects of blocking TNF-R1/ -R2 on the time courses of cyclin D1 gene and protein expression --- p.81 / Chapter 3.2.2. --- Effect of blocking TNF-R1/ -R2 on the time course of cyclin D2 gene expression --- p.82 / Chapter 3.2.3. --- Effects of blocking TNF-R1/ -R2 on the time courses of cyclin D3 gene and protein expression --- p.85 / Chapter 3.2.4. --- Effects of blocking TNF-R1/ -R2 on the time courses of cdk-4 gene and protein expression --- p.90 / Chapter 3.2.5. --- Effects of blocking TNF-R1/ -R2 on the time courses of cyclin E gene and protein expression --- p.93 / Chapter 3.2.6. --- Effects of blocking TNF-R1/ -R2 on the time courses of cdk-2 gene and protein expression --- p.93 / Chapter 3.2.7. --- Effects of blocking TNF-R1/ -R2 on the time courses of p15 gene and protein expression --- p.96 / Chapter 3.2.8. --- Effects of blocking TNF-R1/ -R2 on the time courses of p27 gene and protein expression --- p.99 / Chapter 3.2.9. --- Effects of blocking TNF-R1/ -R2 on the time courses of p21 gene and protein expression --- p.103 / Chapter 3.2.10. --- Effects of blocking TNF-R1/ -R2 on the time courses of pl30 gene and protein expression --- p.106 / Chapter 3.2.11. --- Effect of blocking TNF-R1/ -R2 on the time course of Cak gene expression --- p.110 / Chapter 3.2.12. --- Effects of blocking TNP-R1/ -R2 on the time courses of cyclin H gene and protein expression --- p.110 / Chapter 3.2.13. --- Effects of blocking TNF-R1/ -R2 on the time courses of cyclin B gene and protein expression --- p.112 / Chapter 3.2.14. --- Effect of blocking TNF-R1/ -R2 on the time course of bcl-2 protein expression --- p.116 / Chapter 3.2.15. --- Effects of blocking TNF-R1/ -R2 on the time courses of bcl-xL gene and protein expression --- p.119 / Chapter 3.2.16. --- Effect of blocking TNF-R1/ -R2 on the time course of bcl-xα gene expression --- p.122 / Chapter 3.2.17. --- Effects of blocking TNF-R1/ -R2 on the time courses of bcl-w gene and protein expression --- p.124 / Chapter 3.2.18. --- Effect of blocking TNF-R1/ -R2 on the time course of Mcl-1 gene expression --- p.124 / Chapter 3.3. --- "Effects of other cytokines (IL-6, IL-lα, IL-lβ, IFγ) on cell cycle related genes and proteins expression" --- p.129 / Chapter 3.3.1. --- "Effects of TNF-α, IL-6, IL-lα, IL-lβ, IFγ on cyclin D1 gene and protein expression" --- p.129 / Chapter 3.3.2. --- "Effects of TNF-a, IL-6, IL-lα, IL-lβ, IFγ on cyclin D2 gene and protein expression" --- p.132 / Chapter 3.3.3. --- "Effects of TNF-α, IL-6, IL-lα, IL-1β, IFγ on cyclin D3 gene and protein expression" --- p.136 / Chapter 3.3.4. --- "Effects of TNF-α, IL-6, IL-lα, IL-1β, IFγ on cdk-4 gene and protein expression" --- p.140 / Chapter 3.3.5. --- "Effects of TNF-α, IL-6, IL-lα, IL-1β, IFγ on cyclin E gene and protein expression" --- p.144 / Chapter 3.3.6. --- "Effects of TNF-α, IL-6, IL-lα, IL-1β, IFγ on cdk-2 gene and protein expression" --- p.148 / Chapter 3.3.7. --- "Effects of TNF-α, IL-6, IL-lα, IL-1β, IFγ on pl5 gene and protein expression" --- p.152 / Chapter 3.3.8. --- "Effects of TNF-α, IL-6, IL-lα, IL-1β, IFγ on p27 gene and protein expression" --- p.152 / Chapter 3.3.9. --- "Effects of TNF-α, IL-6, IL-lα, IL-ip, IFγ on p21 gene and protein expression" --- p.159 / Chapter 3.3.10. --- "Effects of TNF-α, IL-6, IL-lα, IL-lβ, IFγ on pl30 gene and protein expression" --- p.162 / Chapter 3.3.11. --- "Effects of TNF-α, IL-6, IL-lα, IL-lp, IFγ on Cak gene expression" --- p.166 / Chapter 3.3.12. --- "Effects of TNF-α, IL-6, IL-lα, IL-1β, IFy on cyclin H gene and protein expression -" --- p.170 / Chapter 3.3.13. --- "Effects of TNF-α, IL-6, IL-lα, IL-1β, IFγ on cyclin B gene and protein expression" --- p.174 / Chapter 3.3.14. --- "Effects of TNF-α, IL-6, IL-lα, IL-1β, IFγ on bcl-2 gene and protein expression" --- p.178 / Chapter 3.3.15. --- "Effects of TNF-a, IL-6, IL-lα, IL-1β, IFγ on bcl-xL gene and protein expression" --- p.178 / Chapter 3.3.16. --- "Effects of TNF-α, IL-6, IL-lα, IL-1β, IFγ on bcl-xα gene expression" --- p.184 / Chapter 3.3.17. --- "Effects of TNF-α, IL-6, IL-lα, IL-lβ, IFγ on bcl-w gene and protein expression" --- p.187 / Chapter 3.3.18. --- "Effects of TNF-α, IL-6, IL-lα, IL-1β, IFγ on Mcl-1 gene expression" --- p.191 / Chapter 3.4. --- Effects of P-ARs on cell cycle related genes expression --- p.194 / Chapter 3.4.1. --- Effects of β-AR agonists and antagonists on cyclin D1 gene expression --- p.195 / Chapter 3.4.2. --- Effects of β-AR agonists and antagonists on cyclin D2 gene expression --- p.198 / Chapter 3.4.3. --- Effects of β-AR agonists and antagonists on cyclin D3 gene expression --- p.201 / Chapter 3.4.4. --- Effects of β-AR agonists and antagonists on cdk-4 gene expression --- p.204 / Chapter 3.4.5. --- Effects of β-AR agonists and antagonists on cyclin E gene expression --- p.207 / Chapter 3.4.6. --- Effects of β-AR agonists and antagonists on cdk-2 gene expression - --- p.210 / Chapter 3.4.7. --- Effects of β-AR agonists and antagonists on p15 gene expression --- p.213 / Chapter 3.4.8. --- Effects of β-AR agonists and antagonists on p27 gene expression --- p.216 / Chapter 3.4.9. --- Effects of β-AR agonists and antagonists on p21 gene expression --- p.219 / Chapter 3.4.10. --- Effects of β-AR agonists and antagonists on p130 gene expression --- p.222 / Chapter 3.4.11. --- Effects of β-AR agonists and antagonists on Cak gene expression --- p.225 / Chapter 3.4.12. --- Effects of β-AR agonists and antagonists on cyclin H gene expression --- p.228 / Chapter 3.4.13. --- Effects of β-AR agonists and antagonists on cyclin B gene expression --- p.231 / Chapter 3.4.14. --- Effects of β-AR agonists and antagonists on bcl-XL gene expression --- p.233 / Chapter 3.4.15. --- Effects of β-AR agonists and antagonists on bcl-xα gene expression --- p.236 / Chapter 3.4.16. --- Effects of β-AR agonists and antagonists on bcl-w gene expression --- p.239 / Chapter 3.4.17. --- Effects of β-AR agonists and antagonists on Mcl-1 gene expression --- p.243 / Chapter CHAPTER 4. --- DISCUSSION & CONCLUSION --- p.247 / Chapter 4.1. --- Effects of TNF-α on the induction of cell cycle regulatory genes/proteins expression --- p.248 / Chapter 4.2. --- Effects of TNF-α on bcl-2 family apoptotic inhibitor genes expression --- p.250 / Chapter 4.3. --- The TNF-R subtype(s) responsible for the TNF-a-induced cell cycle regulatory genes and proteins expression --- p.251 / Chapter 4.4. --- Is the TNF-α-induced cell cycle regulatory genes and proteins expression cytokine specific? --- p.253 / Chapter 4.5. --- The relationship between TNF-α and β-adrenergic mechanism in C6 cell proliferation --- p.254 / Chapter 4.6. --- General Discussion --- p.256 / Chapter 4.7. --- Possible treatments for brain injury --- p.258 / APPENDIX --- p.259 / REFERENCES --- p.348

有證據顯示基質細胞在腫瘤的發生發展中可以發揮重要的作用，基質細胞可以提供適宜腫瘤細胞增殖的腫瘤微環境。腫瘤相關成纖維細胞是一種特殊的與腫瘤生成高度相關的基質細胞。而通过我们的论证，小鼠胚胎成纖維細胞可以作為一種腫瘤相關成纖維細胞的細胞模型。 / 腫瘤誘導因子（TIF）是本實驗室在成瘤實驗中發現的一種新的倉鼠CXC 趨化因子。基于蛋白質序列的分析，TIF 属于Gro CXC 趨化因子家族。這個家族主要通過激活其受體CXCR2 來發揮作用。為了研究TIF 在腫瘤發生中的作用，我們在CHO-K1 細胞中建立了過表達TIF 的穩定細胞株。 / 我們發現共同注射的永生化MEF 與過表達TIF 的D12 細胞導致了腫瘤生長的抑制。為了研究這種現象，重組TIF 蛋白在大腸桿菌中表達，并且用鎳柱進行了提純。純化的蛋白被用于處理CHO-K1 細胞與永生化MEF。我們發現高水平的TIF 可以導致CXCR2 下游的Erk 磷酸化水平下降。其可能的機制為CXCR2 在高水平的TIF 作用下的脫敏作用。同時高水平TIF 可以導致永生化MEF 中CD133 水平的下降。因此，CXCR2 脫敏為TIF 導致腫瘤抑制的可能機制。 / Lines of evidence indicate that stromal cell is one of the determinants in tumor formation by providing a favorable microenvironment for the growth of cancer cells. Cancer associated fibroblast (CAF) is a special form of stromal cells which are shown to be derived from bone marrow. Upon reaching the tumor, the bone marrow-derived mesenchymal stem cells differentiate into CAF, which secrets various growth factors and cytokines to promote cancer growth. Furthermore, genetic study shows that CAF displays p53 mutations and other genetic changes. / Tumor induced factor (TIF) is a CXC chemokine that is originally identified from a xenograft tumor. Sequence analysis suggests TIF is a family member of the Gro CXC chemokines, and exerts its cellular function via activating CXCR2 receptors. In order to investigate the functional roles of TIF, a stable cell line over-expressing TIF in hamster CHO-K1 was established. / To explore the cancer-stromal interactions in xenograft, mouse embryonic fibroblast (MEF) were used as a study model for CAF. MEF was sub-cultured by a conventional protocol that was used for developing the NIH3T3 cells. Based on the growth patterns and expressions of cell markers, growth of MEF can be divided into three stages: the early stage, the senescent stage and the immortalized stage. Our results suggested that MEF might mirror the various developmental stages of CAF. / To examine the contributions of MEF in tumorigenesis, CHO-K1 cells and MEF were co-injected into nude mice. Intriguingly, MEF that in senescent and immortalized stages, rather than in early stage, promoted tumor formation. A possibility arose that the contribution of senescent and immortalized MEF in promoted tumorigenesis may due to CD133 and CXCL1, as the expression of CD133 and CXCL1 in senescent and immortalized MEF were higher than that of MEF in early stage. Moreover, as MEF could gradually develop into a fibroblast promoted tumor formation, MEF could be used as a crucial model to illustrate the origination and development of CAF. / Surprisingly, in nude mice co-injected with immortalized MEF with TIF-overexpressing D12 cells, suppression instead of promotion of tumor growth was found. In order to explore the underlined mechanism of tumor suppression, recombinant TIF protein was purified based on a bacterial expression system. Using purified TIF protein to treat CHO-K1 cells and MEF, it was found that low concentration of TIF promoted Erk phosphorylation but high concentration of TIF suppressed it, which might resulted from desensitization of CXCR2 receptors. Reduction of Erk phosphorylation resulted in decreased proliferation in CHO-K1 cells and alleviated expression of CD133 in MEF, which could be the mechanisms for TIF-induced tumor suppression in nude mice. / Taken together, a CAF model was established to examine the function of TIF in tumor-fibroblast interactions. Mechanistic studies indicated that TIF-induced tumor suppression in nude mice was mediated via desensitization of CXCR2 receptors by high concentration of TIF in the tumor microenvironment. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Qi, Wei. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 189-206). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Tumorigenesis --- p.4 / Chapter 1.1.1 --- Virus transformation --- p.4 / Chapter 1.1.2 --- Proto-oncogene and oncogene --- p.5 / Chapter 1.1.3 --- Tumor suppressor gene --- p.7 / Chapter 1.1.4 --- Epigenetic alteration --- p.9 / Chapter 1.1.5 --- Cancer stem cell --- p.11 / Chapter 1.1.6 --- Tumor microenvironment --- p.14 / Chapter 1.2 --- Cancer associated fibroblast (CAF) --- p.17 / Chapter 1.2.1 --- Markers for CAF --- p.17 / Chapter 1.2.2 --- CAF and normal fibroblast --- p.20 / Chapter 1.2.3 --- CAF, a important player in tumor growth --- p.22 / Chapter 1.2.4 --- CAF and angiogenesis --- p.23 / Chapter 1.2.5 --- CAF and tumor invasion --- p.25 / Chapter 1.3 --- Chemokine --- p.27 / Chapter 1.3.1 --- Structure of chemokine --- p.27 / Chapter 1.3.2 --- Chemokine and cell Recruitment --- p.30 / Chapter 1.3.3 --- Chemokine and tumor microenvironment --- p.30 / Chapter 1.4 --- Tumor Induced Factor and its induced tumor suppression --- p.38 / Chapter 1.5 --- The aims of the project --- p.47 / Chapter Chapter Two --- Purification of Tumor Induced Factor / Chapter 2.1 --- Introduction --- p.49 / Chapter 2.2 --- Materials --- p.52 / Chapter 2.2.1 --- Chemical --- p.52 / Chapter 2.2.2 --- Enzyme --- p.52 / Chapter 2.2.3 --- Antibody --- p.52 / Chapter 2.3 --- Method --- p.53 / Chapter 2.3.1 --- Overview of protein expression system --- p.53 / Chapter 2.3.2 --- Purification of Trx-His₆-S-TIF protein --- p.54 / Chapter 2.3.3 --- BCA assay --- p.60 / Chapter 2.3.4 --- SDS-PAGE --- p.60 / Chapter 2.3.5 --- Western blotting --- p.61 / Chapter 2.3.6 --- Preparation of pET28/His₆-Sumo-TIF bacterial expression vector --- p.62 / Chapter 2.3.7 --- Optimization of culture condition for BL21 expressed His₆-Sumo-TIF protein --- p.67 / Chapter 2.3.8 --- Purification of His₆-Sumo-TIF protein --- p.68 / Chapter 2.3.9 --- Homology model of TIF --- p.68 / Chapter 2.4 --- Results --- p.69 / Chapter 2.4.1 --- Purification of Trx-His₆-S-TIF --- p.70 / Chapter 2.4.2 --- Optimization of purification protocol of His₆-Sumo-TIF --- p.71 / Chapter 2.4.3 --- Large scale purification of mature TIF --- p.75 / Chapter 2.4.4 --- Homology modeling of TIF --- p.80 / Chapter 2.5 --- Discussion --- p.83 / Chapter Chapter 3 --- Three Stages Hypothesis / Chapter 3.1 --- Introduction --- p.86 / Chapter 3.2 --- Material --- p.93 / Chapter 3.2.1 --- Chemical --- p.93 / Chapter 3.2.2 --- Enzyme --- p.93 / Chapter 3.2.3 --- Animal --- p.93 / Chapter 3.2.4 --- Antibody --- p.94 / Chapter 3.3 --- Methods --- p.95 / Chapter 3.3.1 --- Isolate MEF from 13.5 days mouse embryo --- p.95 / Chapter 3.3.2 --- Culture of MEF following 3T3 protocol --- p.96 / Chapter 3.3.3 --- X gal staining --- p.96 / Chapter 3.3.4 --- Analysis of MEF cell size and complexity by flow cytometry --- p.98 / Chapter 3.3.5 --- MTT assay --- p.98 / Chapter 3.3.6 --- Analysis of CD133 by flow cytometry --- p.99 / Chapter 3.3.7 --- ROS detected by DCFH-DA fluorescent probe --- p.99 / Chapter 3.3.8 --- Double staining of cancer stem cell marker and ROS fluorescent probe --- p.100 / Chapter 3.3.9 --- Reverse transcription --- p.101 / Chapter 3.3.10 --- Analysis CXCL1 mRNA expression level by PCR --- p.102 / Chapter 3.3.11 --- Gelatin zymography --- p.103 / Chapter 3.3.12 --- In-vivo tumorigenicity assay --- p.104 / Chapter 3.4 --- Results --- p.106 / Chapter 3.4.1 --- Three Stages of MEF --- p.106 / Chapter 3.4.2 --- X gal staining --- p.106 / Chapter 3.4.3 --- Flow cytometric analysis of cell diameter and cellular complexity of MEF --- p.109 / Chapter 3.4.4 --- MTT assay --- p.109 / Chapter 3.4.5 --- CD 133 expression of MEF detected by flow cytometry --- p.110 / Chapter 3.4.6 --- Reactive oxygen species of MEF detected by flow cytometry --- p.118 / Chapter 3.4.7 --- The level of ROS and CD133 of MEF detected by flow cytometry stimultaneously --- p.121 / Chapter 3.4.8 --- TIF treatment reduces the small CSC subpopulation in senescent stage MEF --- p.124 / Chapter 3.4.9 --- Increased CXCL1 expression in senescent stage and immortalized stage MEF --- p.125 / Chapter 3.4.10 --- Matrix metalloproteinase 2 activities in different stages of MEF . --- p.129 / Chapter 3.4.11 --- In vivo tumorigenicity assay --- p.130 / Chapter 3.5 --- Discussion --- p.133 / Chapter Chapter Four --- Biphasic Effect of TIF in Cancer-Fibroblasts Interaction / Chapter 4.1 --- Introduction --- p.140 / Chapter 4.2 --- Material --- p.143 / Chapter 4.2.1 --- Chemical --- p.144 / Chapter 4.2.2 --- Kit and Instrument --- p.144 / Chapter 4.2.3 --- Antibody --- p.144 / Chapter 4.3 --- Method --- p.145 / Chapter 4.3.1 --- Purification of TIF-His₆-Flag --- p.145 / Chapter 4.3.2 --- Western blotting to detect purified TIF-His₆-Flag --- p.145 / Chapter 4.3.3. --- Measurement of cell proliferation by cell counting --- p.145 / Chapter 4.3.4 --- MTT assay --- p.146 / Chapter 4.3.5 --- Western blotting to detect pErk and total Erk --- p.146 / Chapter 4.3.6 --- Soft agar assay --- p.148 / Chapter 4.3.7 --- Gelatinase detection --- p.148 / Chapter 4.3.8 --- Wound healing assay --- p.149 / Chapter 4.3.9 --- Colony formation assay --- p.149 / Chapter 4.3.10 --- Detection of CD133 by flow cytometry --- p.150 / Chapter 4.4 --- Results --- p.151 / Chapter 4.4.1 --- Purification of TIF-His₆-Flag --- p.151 / Chapter 4.4.2 --- Reduced cell proliferation of D12 in long time culture --- p.153 / Chapter 4.4.3 --- Reduced metabolic activities of D12 cells in time culture --- p.155 / Chapter 4.4.4. --- TIF-CXCR2-pErk signal axis in CHO cells --- p.155 / Chapter 4.4.5 --- Bigger colonies formed by D12 cells in soft agar assay --- p.161 / Chapter 4.4.6 --- TIF-CXCR2-pErk-MMP9 signal pathway in D12 cells --- p.162 / Chapter 4.4.7 --- Reduced migration of D12 cells --- p.164 / Chapter 4.4.8 --- Reduced cell invasion of D12 cells --- p.165 / Chapter 4.4.9 --- Reduced colony number of D12 cells in colony formation assay --- p.168 / Chapter 4.4.10 --- Bi-phasic “bell shape“ bi-phasic response on Erk activation of TIF in CHO-K1 cells --- p.169 / Chapter 4.4.11 --- Bi-phasic “bell shape“ effect of TIF to pErk in immortalized MEFs --- p.172 / Chapter 4.4.12 --- Reduced CD133 in immortalized MEF by high concentration of TIF --- p.173 / Chapter 4.5 --- Discussion --- p.177 / Chapter Chapter Five --- General Discussion / Chapter 5.1 --- Project Summary --- p.183 / Chapter 5.2 --- Significances of the project --- p.185 / Chapter 5.3 --- Future work --- p.188

Thesis (Ph. D.)--West Virginia University, 2006. / Title from document title page. Document formatted into pages; contains x, 189 p. : ill. (some col.). Includes abstract. Includes bibliographical references.