Preliminary Study Of Osmotic Water Permeability Diversification In Primary Trophoblast Cultures From AQP1-knockout Mice

Maternal-fetal fluid balance is critically important during pregnancy and amniotic fluid is essential for fetal growth and development. As a pregnant temporary organ, the placenta plays a key role in regulation of fluid transfer and nutrition metabolism during pregnancy.Aquaporins (AQPs) are specific water channels that allow the rapid transcellular movement of water in response to osmotic/hydrostatic pressure gradients. Up to now, thirteen members have been defined in this family from mammals and some physiological functions have been shown. Several AQPs have been identified expression in the placenta and fetal membranes in human, murine and ovine, suggesting that AQPs may play important roles in pregnant physiology, especially in maternal-fetal fluid balance.Our previous researches have identified some changed phenotypes in AQP1-knockout pregnant mice, providing direct evidences that AQP1 plays important roles in pregnancy and amniotic fluid circulation. This study established the primary culture system of trophoblast cells in AQP1-knockout (AQP1-/- )and wild type (AQP1+/+) group; the measurement of water permeability in two groups provided direct functional evidence that AQP1-mediated plasma membrane water permeability played an important role in Maternal-fetal fluid balance. In addition, the preliminary study showed the different migration ability between the two group trophoblast cells, which provided a theoretical basis for the AQP1 gene in the role of obstetric disease and gestational trophoblastic disease. Part OneStudy of osmotic water permeability diversification in primary trophoblast cultures from AQP1-knockout pregnant mice Chapter IPrimary cell culture and identification of AQP1+/+ and AQP1-/- trophoblast Objective:To obtain AQP1+/+ and AQP1-/- trophoblast cells suitable for future experiment by primary cell culture in vitro.Method:Homozygous AQP1 knockout mice and wild type mice were mated respectively. The day a copulation plug was found was designated as gestational day 1 (1GD). Placental tissues were separately chosen from wild-type CD1 and AQP1-knockout pregnant mice of 12 GD. Primary culture of mouse trophoblast cells was carried out by tissue-culture method, and the subsequent serial passage of cells performed by trypsin digestion. Trophoblast cells were identified by cytokeratin 7 immunohistochemical staining, which is as a specific cytoskeletal protein.Result:Cells grew from the minced tissue the next day, and cells grew spreading after days. Cell passage could be performed at about 15 days later. The trophoblast cells of two groups did not show an obvious difference in gross size, appearance or proliferation rate. Cytokeratin 7 staining showed that the purity of the cultured cells exceeded 95%.Conclusion:The primary culture system of trophoblast cells in two groups was successfully established. The trophoblast cells obtained thereby may well satisfy subsequent experiment. Chapter IIMeasurement of water permeability in AQP1+/+ and AQP1-/- trophoblast Objective:Our previous study has demonstrated that the maternal-fetal fluid balance of AQP1-knockout (AQP1-/-) pregnant mice was impaired. The goal of this study was to measure osmotic water permeability in primary cultures of mouse trophoblast cells to define quantitatively the functional role of AQP1 in water transport across the trophoblast plasma membrane.Method:1. The expression of AQP1 gene in trophoblast cells of wild type was confirmed by immunofluorescence.2. The osmotic water permeability of trophoblast cells plasma membranes was measured by a calcein fluorescence quenching method in response to osmotic gradients.Result:1. Immunofluorescence showed that AQP1 expressed in trophoblast cells membrane of wild type.2. Results of the osmotic water permeability in two groups are followed. The osmotic water permeability of AQP1-/- trophoblast was significantly lower than AQP1+/+ group (P=0.009), only 57% of wild type group in response to hypotonic challenge; and its osmotic water permeability was also lower than AQP1+/+ group (P=0.036), approximate 64% of wild type group in response to hypertonic challenge.Conclusion:The results indicated the osmotic water permeability of AQP1-KO trophoblast cells was significantly lower than that of wild type group. It provides direct functional evidence that AQP1-mediated plasma membrane water permeability plays an important role in Maternal-fetal fluid balance. Part TwoPreliminary study of migration in AQP1-knockout trophoblast Objective:To observe the migration ability in vitro cultured AQP1+/+ and AQP1-/- mouse trophoblast cells after scratch wound.Method:Secondary cultured AQP1+/+ and AQP1-/- trophoblast cells were used to prepare scratch wound model, the diversity of migration were observed after scratch wounding.Result:Scratch wound assays showed a significant reduction in the number of migration cells in AQP1-knockout trophoblast compared to wild type group.Conclusion:AQP1 plays an important role in trophoblast cell migration, especially in the migration of post-traumatic repair.