| Trans-membrane transportation of water is closely related with the biological response and activity. AQP (aquaporin) is an important molecule in water transportation. The first AQP was reported in 1988 and until 1997 the first molecule in water transportation was named as aquaporin. It has been proved that polar water transports through the bi-layer lipid cellular membrane by water channel transportation protein. The change of the function of AQPs is critical in water metabolism. It means further studies of these proteins might provide us new therapy in the diseases related to water metabolism disorder.To date, 11 members in water membrane channel protein (transporter) family, designated as AQPO-10, have been cloned and characterized in human, with their distinct tissue specificity. Both AQP1 and AQP4 are mostly expressed in brain, and AQP4 especially expressed on both sides of BBB (brain blood barrier). Different modification on AQPs can regulate the permeability of cellular membranes. It has been reported that there is no clear abnormality in AQP4 knock-out mice. When they are challenged with ischemia, the cerebral edema is even lighter than wild type mice. Since AQP4 is a water selective channel, the modification on cerebral AQP4 may provide a new direction on the treatment of cerebral edema.Few studies have been done on cerebral AQP4 in the world, and there is none in China. All of them are published after year 2000, and most are in high quality journals. The following is what we have learned in this field.1. AQP4 gene cloning and construction in expression vectorPurpose: AQP4 gene cloning and construction in the expression vector can provide us a research tool. Methods: total RNA was isolated from rat cerebellar cortex, random primer was used for cDNA synthesis, and specific primers was used for RT-PCR to amplify AQP4. Sense primer 1 is: 5'- GTT ATC ATG GGA AAC TGG GAA A;Anti-sense primer 1: 5'-TAC CTC TCC AGA CGA GTC CTT C; Sense primer 2: 5'- TAT CGA ATT CAT GAG TGS CGG AGC TGC AGC GAG GCG; Anti-sense primer 2: 5'-TAT GCG GCC GCT CAT ACA GAA GAT AAT ACC TCT. PCR product amplified by the first pair of primers was inserted into pCRI, while the product amplified by second one was digested with EcoR I and Not I. Then after complete digestion the DNA was inserted into pcDNA3.l/Zeo(+). Results: The PCR products amplified by the first and second pair of primers were 294bp and 993bp, respectively. The first one was from 664 to 957 of AQP4 complete sequence, the second was whole sequence (Genebank: U14007). These two fragments were cloned into the pCRI and pcDNA3.1 Zeo (+), respectively. Both of them were sequenced at Sanggong, shanghai. The sequencing results were the same as reported. Conclusion: We successfully cloned AQP4 gene and also constructed into the vector, which can be used for further study.2. The expression of AQP4 in normal rat brain.Purpose: To examine the expression level of cerebral AQP4 in rat brain. Methods: In situ hybridization with AQP4 cRNA probe (made in our lab) and immunohistochemistry staining with anti-AQp4 monoclonal antibody provided by ADI (USA) were performed on cryostal brain tissue slide from healthy adult SD rats. Results: Immunohistochemistry staining showed that positive staining cells mostly were glia cells, capillary endothelial cells, ependymal cells of cerebral ventricle, and choroid plexus. All the positive cells were stained at the cell membrane. Most importantly, on glia cells the expression of AQP4 was mainly on the side of cell membrane contacting with capillary vessel endothelial cells. In situ hybridization showed a wide distribution on glia cells, capillary endothelial cells, cerebellar Purkinje's cells, brain stem nucleus, ependymal cells of cerebral ventricle, choroid plexus, also hippocampal neurons, some hypothalamic neurons and some cortex neurons. Conclusion: AQP4 was widely expressed in rat brain, highly related with endothelial cells and glia cells, which were closely connected with blood brain barrier, related with ependymal...
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