Gene Function Of Aquaporin Water Channels

Aquaporins(AQPs) were found as a family of intrinsic membrane protein in the earlyof 1990' that function as special water-transporting channels at the membrane ofeukaryocyte and prokaryocyte. Since AQP1 was first cloned from the membrane oferythrocyte, rapid and serial progress has been made in the study on AQPs' structure andfunction. Till now, thirteen members have been defined from mammals and the study on theirgene structure, expression regulation, chromosome location, tissue distribution, andphysiological function has been furthered. It has been confirmed that AQPs play an importantphysiological part in body fluid transport. The study in relation to AQP1 gene function isfurther. In our study, the role of AQP1 in tumor angiogenesis has been studied and the establishmentof EYFP-V163S transgenic mice for in vivo water transport studies explored. AQP1,which is the only intrinsic membrane protein expressed at endothelium ofnormal tissues functioning as water-transporter , has been reported to express highly inmicrovessels of many different type tumors indicating the possible involvement of AQP1 intumor angiogenesis. In our study ,AQP1-knockout mice on the genetic background ofC57BL/6J were injected subcutaneously with about 2×105cell of B16F10 melanomarespectively. After 12 days, tumors were harvested and weighed, fixed with 10% formalin.The results demonstrated that nearly 30% retarded growth of inoculated melanoma in AQP1knockout mice compared to tumors in wild-type mice. Immunohistochemistry of melanomasections revealed strong AQP1 protein labeling in endothelium of tumor blood vessels inwild-type mice and negative labeling of AQP1 in the counterpart structures inAQP1-knockout mice. On the same tumor sections stained by hematoxylin, melanoma cellsfrom islands with microvessels in the center. Based on CD31 protein labeling, it is easily seenthat high-density and thin microvessels(142/mm2) locate in the center of tumor islands inwild-type mice, whereas sparse and enlarged vessels(47/mm2) are evident in tumor islands inAQP1 knockout mice coincident with larger necrotic area in outer layer of the islands. Theseresults provide definitive evidence that lacking of water channel AQP1 results in defectivetumor angiogenesis and retarded tumor growth that may be involved in insufficient bloodsupply and/or abnormal transendothelial fluid transport. The underlying molecular andcellular mechanisms are worth further investigation.Another task for our study is to establish the transgenic mice model expressing thefluorescent protein EYFP-V163S sensitive to Cl- for study trans-membrane transporting ofwater and Cl - in vivo, Which appears theoreticically important because Until now, theresearch on water and Cl - trans-membrane transporting is only carried out by studying oncultured cells for the lack of endo-investigation means of water and Cl - transporting. theknowledge acquired about water and Cl – channel was mainly based on study of cultured cells. The directly research means is starved for for furthering investingation ontrans-membrane transporting of water and Cl - and perfecting theory about the function ofwater and Cl – channels,.Developing vivi-means appears more important .and indispensably,especially with the transition from gene-era to physiological function-era. The establishmentof EYFP-V163S transgenic mice model will bring it into realization that measure directly waterand Cl - channel function in different tissues by perfusing in vivo tissues with halides anddetecting dynamically the change of fluorescent. At the same time,.cells from tissues ofEYFP-V163S transgenic mice could be cultured as cell models for screening inhibitors andactivator of water aquaporins and Cl – channels. In this study, the fluorescent protein EYFP-V163S has been targeted to ROSA26 locusat the genome of E14 ES cell and established several ES lines expressing EYFP-V163Ssteadily, the targeting vector based on positive and negative selection was used which led thepositive rate to 60%.Two chimeras were obtained but the transgene trans...