Expression of MRP14 gene is frequently down-regulated in Chinese human esophageal cancer

Migration inhibitory factor-related protein 14 (MRP 14) is one of calcium-binding proteins,referred as S 100A9. The heterodimeric molecule formed by MRP 14 with its partner MRP8 (S 100A8) is the major fatty acid carrier in neutrophils.The MRP8/14 complex has been also implicated in the intracellular transport of arachidonic acid and its precursors in keratinocytes. We show here the involvement of MRP14 in human esophageal cancer. In an initial study,mRNA differential display - reverse transcription polymerase chain reaction (DD-PCR) was performed with two esophageal carcinomas,one esophageal adenocarcinoma and matched normal adjacent mucosa. DD-PCR with the arbitrary primer OPA3 showed that one cDNA band was highly expressed in normal tissues,but disappeared or substantially decreased in tumor counterparts. It was later identified to be the 3'-end of migration inhibitory factor-related protein 14 (MRP14).Northern blotting,RT-PCR and Western blotting corroborated the down-regulation of MRP14 in 58/64 squamous cell carcinomas and 2/2 adenocarcinomas as compared with adjacent normal epithelia of the esophagus. MRP14 was undetectable in 3/3 esophageal-carcinoma cell lines. Immunochemistry demonstrated that expression of MRP14 was restricted to normal esophageal epithelia. No mutation was found in the genomic DNA of the MRP14 gene by PCR and directed DNA sequencing. Our finding suggested that the reduction of MRP14 expression is a frequent event in Chinese human esophageal cancer.

Glial cell-derived neurotrophic factor mRNA expression in a rat model of spinal cord injury following bone marrow stromal cell transplantation

BACKGROUND： Several animal experiments utilizing bone marrow stromal cell （BMSC） transplantation for the treatment of spinal cord injury have proposed a hypothesis that BMSC transplantation effects are associated with increased glial cell-derived neurotrophic factor （GDNF） expression. OBJECTIVE： To confirm the effects of BMSC transplantation on GDNF mRNA expression in rats with spinal cord injury by reverse transcription-polymerase chain reaction （RT-PCR）. DESIGN, TIME AND SETTING： The present molecular, cell biology experiment was performed at the Key Laboratory of Children＇s Congenital Malformation, Ministry of Health of China ＆ Department of Developmental Biology, Basic Medical College, China Medical University between March 2006 and May 2007. MATERIALS： Sixty healthy Wistar rats aged 2-4-months and of either gender were included in this study. Spinal cord injury was induced in all rats by hemisection of T9 on the left side. RT-PCR kits were purchased from TaKaRa Company, China. Type 9600 RCR amplifier was provided by Perkin Elmer Company, USA. METHODS： Three rats were selected for BMSC culture and subsequent transplantation （after three passages）. Of the remaining 57 rats, nine were selected for sham-operation （sham-operated group）, where only the T9 spinal cord was exposed without hemisection. A total of 48 rats were randomly and evenly divided into BMSC transplantation and model groups. In the BMSC transplantation group, following spinal cord injury induction, each rat was administered a BMSC suspension tbrougb two injection sites selected on the gray and white matter boundary caudally and cephalically, seperately and near to injury site in the spinal cord. The model group received an equal volume of PBS through the identical injection sites. MAIN OUTCOME MEASURES： At 24 and 72 hours, as well as at 7 days, following spinal cord injury, the spinal cord at the T9 segment was removed. Eight rats were allocated to each time point in the BMSC transplantation and model groups, with three rats allocated to the sham-operated group. GDNF mRNA expression was semiquantitatively analyzed by RT-PCR. RESULTS： The sham-operated group exhibited extremely low GDNF mRNA expression. GDNF mRNA expression significantly increased at 24 hours after spinal cord injury, reached a peak level at 72 hours, and slowly decreased thereafter. However, it remained higher than normal levels at 7 days （P 〈 0.05）. At all time points following spinal cord injury, GDNF mRNA expression was significantly greater in the BMSC transplantation group than in the model group （P 〈 0.05）. CONCLUSION： Transplantation of BMSCs into the injured spinal cord up-regulated GDNF mRNA expression, thereby promoting repair of the injured spinal cord.

Changes of ECM and CAM gene expression profile in the cirrhotic liver after HCV infection: Analysis by cDNA expression array

AIM: We aimed to observe the expression of extracellular matrix (ECM) and cellular adhesion molecules (CAM) in cirrhotic liver tissues after hepatitis C virus (HCV) infection. METHODS: Twelve patients with post HCV inflammatory liver cirrhosis were selected to evaluate their liver function and other virological, pathological parameters. Then three specimens of cirrhotic patients whose health assessment results and laboratory data were similar and three normal liver specimens explanted from liver grafts prepared for liver transplantation were chosen for investigating gene expression of ECM and CAM using cDNA expression array. RESULTS: The cDNA array assay revealed 36.7% (36/96)of genes with changes, in which 26.3% (26/96) was up regulated and 10.1% (10/96) was down-regulated. Integrin (ITGA), collagen (COL), ADAMTS were identified as the characteristic changes of ECM and CAM gene expression levels. ITGA were demonstrated β1 and β2 sub-section changed in liver cirrhosis.CONCLUSION: ECM and CAM play an important role inthe progression of liver cirrhosis after HCV infection. The capital mechanism is related to the inflammatory cellsinfiltration, the activation and transformation of ECM producing cells and the imbalance between production and elimination of ECM.

Application of atomic force microscopy in blood research

AIM: To find suitable solutions having lesser granules and keeping erythrocytes in normal shapes under atomic force microscopy (AFM).METHODS: Eight kinds of solutions, 1% formaldehyde,PBS buffer (pH7.2), citrate buffer (pH6,0), 0.9% NaCl,5% dextrose, TAE, 1640 medium and 5% EDTA-K2, were selected from commonly used laboratory solutions, and venous blood from a healthy human volunteer was drawn and anticoagulated with EDTA-K2. Before scanned by AFM (NanoScopeⅢa SPM, Digital Instruments, Santa Barbara,CA), a kind of intermixture was deposited on freshly cleaved mica and then dried in the constant temperature cabinet (37 ℃).RESULTS: One percent formaldehyde, citrate buffer, 5%dextrose, TAE, were found to keep human erythrocytes in normal shape with few particles. Processed by these solutions, fine structures of human erythrocyte membrane were obtained.CONCLUSION: One percent formaldehyde, citrate buffer,5% dextrose and TAE may be applied to disposeerythrocytes in AFM. The results may offer meaningful data for clinical diagnosis of blood by AFM.