Expression of Trp gene family in vascular system.

Yip Ham.Thesis (M.Phil.)--Chinese University of Hong Kong, 2001.Includes bibliographical references (leaves 132-141).Abstracts in English and Chinese.Acknowledgement --- p.iAbbreviations --- p.iiAbstract --- p.iii摘要 --- p.vChapter Chapter 1: --- Introduction --- p.5Chapter 1.1 --- Calcium Signaling --- p.5Chapter 1.1.1 --- Importance of Calcium to Life Forms --- p.5Chapter 1.1.2 --- Calcium Channels in Excitable and Non-excitable Cells --- p.6Chapter 1.2 --- Vascular Endothelial Cells --- p.8Chapter 1.2.1 --- General Functions --- p.8Chapter 1.2.2 --- Calcium signaling in Endothelial Cells --- p.9Chapter 1.3 --- Capacitative Calcium Entry (CCE) or Store-operated Calcium Entry (SOC) --- p.10Chapter 1.3.1 --- Definition --- p.10Chapter 1.3.2 --- Endoplasmic Reticulum (ER) as the Main Intracellular Calcium Stores --- p.10Chapter 1.3.3 --- Types of Experiments leading to the Identification of SOCs --- p.11Chapter 1.3.4 --- Emptying the Internal Calcium Store --- p.11Chapter 1.3.4.1 --- Inhibition of Calcium ATPase --- p.11Chapter 1.3.4.2 --- IP3 Triggered Release of Calcium --- p.12Chapter 1.3.5 --- "Store-operated Calcium Current, Icrac" --- p.15Chapter 1.3.6 --- Different Types of SOCs in Animal Cells --- p.16Chapter 1.4 --- Transient Receptor Potential (Trp) Gene & Transient Receptor Potential Like (Trpl) Gene in Drosophila --- p.17Chapter 1.4.1 --- Discoverery of Trp and Trpl --- p.17Chapter 1.4.2 --- Expression Studies of Drosophila Trp and Trpl --- p.19Chapter 1.4.2.1 --- Trp and Trpl form Channels but only Trp is Store Operated --- p.19Chapter 1.4.2.2 --- Co-expression Studies of Trp and Trpl --- p.20Chapter 1.5 --- Molecular Cloning and Expression of Mammalian Trp Homologues --- p.21Chapter 1.5.1 --- Seven Human Homologus of Trp were found --- p.21Chapter 1.5.2 --- Expression Pattern of mammalian Trp Homologues in Different Tissues --- p.23Chapter 1.5.3 --- Expression Studies of Mammalian Trp Homologues Yields Contradictory Results --- p.27Chapter 1.5.3.1 --- Trpl --- p.27Chapter 1.5.3.2 --- Trp2 --- p.28Chapter 1.5.3.3 --- Trp3 --- p.29Chapter 1.5.3.4 --- Trp4 --- p.30Chapter 1.5.3.5 --- Trp5 --- p.31Chapter 1.5.3.6 --- Trp6 --- p.31Chapter 1.5.3.7 --- Trp7 --- p.31Chapter 1.5.3.8 --- "Activation of Trp3, Trp6 and Trp7 by Diacylglycerol (DAG)" --- p.32Chapter 1.5.3.9 --- Functional Consequence after Co-expression of Trp Homologues --- p.34Chapter 1.5.3.10 --- Antisense Strategy to Determine the Functional Subunits of Store-operated Channels --- p.35Chapter 1.5.3.11 --- Possible Reasons for the Contradictory Results of Trp Homologues When Expressed in a Heterologous System --- p.36Chapter 1.6 --- Aims Of Study --- p.37Chapter Chapter 2. --- Materials and Methods --- p.38Chapter 2.1 --- Cell Culture --- p.38Chapter 2.2 --- Total RNA extraction from HCAEC 5286 --- p.39Chapter 2.3 --- Reverse Transcription from Cultured Human Coronary Artery Endothelial Cell Line HCAEC 5286 --- p.40Chapter 2.4 --- Polymerase Chain Reaction (PCR) of Partial Trp Gene Fragments --- p.41Chapter 2.5 --- Separation and Purification of PCR Products --- p.43Chapter 2.5.1 --- Separation --- p.43Chapter 2.5.2 --- Purification --- p.43Chapter 2.6 --- Confirmation of PCR Products --- p.44Chapter 2.7 --- Molecular Cloning of Trp Gene Family --- p.45Chapter 2.7.1 --- "Cloning of HTrpl, HTrp3, HTrp4,HTrp5,HTrp6, HTrp7" --- p.45Chapter 2.7.1.1 --- Polishing the Purified PCR Products --- p.47Chapter 2.7.1.2 --- Determination of the Amount of Polished PCR Products --- p.47Chapter 2.7.1.3 --- Inserting the PCR Products into the pPCR-Script Amp SK(+)Cloning Vector (Ligation) --- p.48Chapter 2.7.1.4 --- Transformation --- p.48Chapter 2.7.1.5 --- Preparing Glycerol Stocks Containing the Bacterial Clones --- p.49Chapter 2.7.1.6 --- Plasmid DNA Preparation --- p.49Chapter 2.8.1.7 --- Clones Confirmation --- p.50Chapter 2.8 --- In situ Hybridization --- p.54Chapter 2.8.1 --- Probe Preparation --- p.54Chapter 2.8.1.1 --- Trp1 Probe --- p.54Chapter 2.8.1.2 --- Trp3 Probe --- p.58Chapter 2.8.1.3 --- Trp4 Probe --- p.61Chapter 2.8.1.4 --- Trp5 Probe --- p.62Chapter 2.8.1.5 --- Trp6 Probe --- p.63Chapter 2.8.1.6 --- Trp7 Probe --- p.65Chapter 2.8.1.7 --- Control Probe --- p.66Chapter 2.8.2 --- Testing of DIG-Labeled RNA Probes --- p.66Chapter 2.8.3 --- Paraffin Sections Preparation --- p.67Chapter 2.8.4 --- In Situ Hybridization: Pretreatment --- p.67Chapter 2.8.5 --- "Pre-hybridization, Hybridization and Post-hybridization" --- p.68Chapter 2.8.5.1 --- Pre-Hybridization --- p.68Chapter 2.8.5.2 --- Hybridization --- p.68Chapter 2.8.5.3 --- Post-Hybridization --- p.69Chapter 2.8.6 --- Colorimetric Detection of Human Trps mRNA --- p.69Chapter 2.9 --- Northern Hybridization --- p.70Chapter 2.9.2 --- Labelling of Riboprobe with 32P --- p.70Chapter 2.9.3 --- Prehybridization and Hybridization with Radiolabeled RNA Probes --- p.73Chapter Chapter 3. --- Results --- p.74Chapter 3.1 --- Polymerase Chain Reaction (PCR) of Partial Trp Gene Fragments --- p.74Chapter 3.2.1 --- Expression of TRPs RNA in Human Coronary Artery --- p.78Chapter 3.2.1.1 --- Expression of Trp Transcripts in Tunica Intima and Media --- p.79Chapter 3.2.1.2 --- Expression of Trp Transcripts in the Tunica Adventitia --- p.88Chapter 3.2.2 --- Expression of TRPs RNA in Human Cerebral Artery --- p.97Chapter 3.2.2.1 --- Expression of Trp Transcripts in Tunica Intima and Media --- p.97Chapter 3.3 --- Northern Blot Analysis of Human Trp5 RNA in Human Multiple Tissue Blot --- p.115Chapter Chapter 4: --- Discussion --- p.117Chapter 4.1 --- Co-expression of Trps in Vascular Tissues --- p.117Chapter 4.1.1 --- Expression of Trps in Endothelia --- p.117Chapter 4.1.2 --- In Smooth Muscle Cells --- p.118Chapter 4.2 --- Trp Channel and Store-operated Channel in Endothelial Cells --- p.119Chapter 4.3 --- Heteromultimerization of Trps Subtypes --- p.120Chapter 4.4 --- Northern Blot Analysis --- p.124Chapter 4.5 --- Potential Physiological Functions of Trps --- p.125Chapter 4.6 --- Trp Channels as a Therapeutic Target? --- p.128Chapter 4.7 --- Technical Aspects in the Present Studies --- p.129Chapter 4.8 --- Conclusion --- p.131Reference --- p.13