ABSTRACT
<p><b>OBJECTIVE</b>To investigate the measurements of gene expressing at a single hepatocyte level.</p><p><b>METHODS</b>Individual hepatocyte was isolated from cryostat tissue section using laser microdissection technique. To detect the mRNA expressed by single hepatocyte, RNA was extracted, reversely transcribed to cDNA and amplified by nested polymerase chain reaction (PCR).</p><p><b>RESULTS</b>Single cell was microdissected from cryostat tissue using an ultraviolet laser micromanipulator. The RNA could be extracted from the isolated cell(s), and the RT-PCR production could be observed after electrophoresis, whose quantitation was compatible with the number of cells.</p><p><b>CONCLUSION</b>Combining laser microdissection and nested RT-PCR can monitor gene expression at a single cell level in vivo.</p>
Subject(s)
Humans , Dissection , Gene Expression Profiling , Hepatocytes , Metabolism , Lasers , RNA, Messenger , Reverse Transcriptase Polymerase Chain Reaction , MethodsABSTRACT
<p><b>OBJECTIVES</b>To study cartilage oligomeric matrix protein (COMP) mRNA and protein expression in normal pancreas, chronic pancreatitis (CP), and pancreatic cancer tissues.</p><p><b>METHODS</b>Tissues from 15 cases of normal pancreas, 14 cases of chronic pancreatitis and 14 cases of pancreatic cancer were analyzed by Northern blot, Western blot, in situ hybridization and immunohistochemistry.</p><p><b>RESULTS</b>COMP mRNA signals and immunoreactivity were strongly present in the cytoplasm of degenerating acinar cells in CP tissues as well as in CP-like lesions in pancreatic cancer tissues. In contrast, COMP expression was weak to absent in the cytoplasm of cancer cells in pancreatic cancer tissues, and in ductal cells and islet cells in normal pancreatic tissues.</p><p><b>CONCLUSION</b>COMP is preferentially expressed in degenerating acinar cells in CP and in CP-like areas in pancreatic cancer, suggesting a potential role of this molecular in acinar cell dysfunction in CP.</p>
Subject(s)
Humans , Blotting, Northern , Blotting, Western , Cartilage Oligomeric Matrix Protein , Dimerization , Extracellular Matrix Proteins , Chemistry , Genetics , Metabolism , Glycoproteins , Chemistry , Genetics , Metabolism , Immunohistochemistry , In Situ Hybridization , Matrilin Proteins , Pancreas , Metabolism , Pathology , Pancreatic Neoplasms , Metabolism , Pathology , Pancreatitis, Chronic , Metabolism , Pathology , RNA, Messenger , Genetics , MetabolismABSTRACT
Objective To investigate the role of substance P (SP) and its neurokinin-1 receptor (NK-1R) in the pathophysiologic process of Crohn′s disease. Methods In 23 surgical patients of Crohn′s disease and 24 healthy controls, reverse transcription polymerase chain reaction (RT-PCR) was used to determine the mRNA expression of NK-1R, Western blot analysis was used to determine NK-1R protein expression levels, and immunohistochemistry was used to localize expression of NK-1R. Results Compared with normal gut NK-1R mRNA and NK-1R protein in Crohn′s disease were overexpressed. In Crohn′s disease moderate to strong intestinal NK-1R immunoreactivity was found in the lamina propria mononuclear cells, lymphoid follicles, and the surface and crypt epithelium, lymphoid follicles, submucosal vessels, smooth muscle and myoenteric plexus neurones. Conclusions In cases of Crohn′s disease, overexpression of NK-1R may disturb neuropeptides loop balance, and may be involved in the pathophysiological change in this disease.