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Objective: To study the effect of cyclic adenosine monophosphate on aquaporin 8 (AQP8) expression and distribution in human amnion-derived WISH cells., Methods: Human amnion-derived WISH cells were cultured. The cells were divided into control group and study group at random. The study group was established by exposure to various concentrations of 8-Br-cAMP. Western analysis was used to quantify AQP8 expression levels. RT-PCR was used to quantify AQP8 mRNA expression levels. Immunofluorescence was used to determine the localization of AQP8 in WISH cells., Results: With increase of cAMP, AQP8 mRNA and protein expressions significantly increased in WISH cells in vitro. When the concentration of cAMP reached 200 micromol/L, AQP8 mRNA and protein expressions were highest. Incubation with cAMP (200 micromol/L) for 2 hours resulted in a 2-fold increase in AQP8 mRNA level, and incubation for 8 hours resulted in a peak. After incubation for 16 hours AQP8 mRNA level began to descend, and after 24 hours it decreased to baseline. Incubation with cAMP for 8 hours AQP8 protein level began to increase. Incubation for 24 hours resulted in a peak in AQP8 protein level, and after incubation for 48 hours it began to decline. By immunofluorescence microscopy after incubation with cAMP (200 micromol/L) cells, AQP8 labeling in plasma membrane was enhanced and intracellular AQP8 labeling was decreased., Conclusion: The cAMP triggers translocation of AQP8 from cytosol to the plasma membrane via vesicle-transporting related protein instead of AQP8 itself. cAMP may upregulate the transcription of target gene protein kinase A. The cAMP may be the critical regulatory medium of AQP8 in WISH cells.

Objective: To evaluate the influence of up-regulation of glucose regulated protein 78 (GRP 78) induced by 2-deoxyglucose (2DG) on fetal rat cerebral neuron apoptosis following intrauterine distress and the unification of endoplasmic reticulum and mitochondrium., Methods: (1) Fetal rat intrauterine distress model was established and rats were divided into normal group (N = 10), ischemia- reperfusion(IR) group (n = 40) and treatment group (n = 40, injection of 2DG into pregnant rats' abdomen after operation ). (2) Neuron apoptosis and the influence of 2DG on apoptosis was detected by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. The expression of GRP78, caspase-9, -12, and cytoron C protein were detected by western blot technique., Results: (1) The number of TUNEL positive neuron in normal group was 4.3 +/- 1.8/mm2. The expression of GRP78, caspase-9, -12, cytoron C in cytoplasm were 0.012 +/- 0.003, 0.004 +/- 0.003, 0.006 +/- 0.002, 0.012 +/- 0.005, respectively. (2) The number of TUNEL positive neuron in the IR group were 43.6 +/- 11.4/mm2 (reperfusion 3 h), 64.4 +/- 9.3/mm2 (reperfusion 6 h), 74.2 +/- 12.1/mm2 (reperfusion 12 h), 97.3 +/- 8.9/ mm2 (reperfusion 24 h), respectively. They were significantly more than that in normal group (P < 0.05). The expression of GRP78 at corresponding times in IR group were 0.092 +/- 0.008 (reperfusion 3 h), 0.078 +/- 0.006 (reperfusion 6 h), 0.054 +/- 0.009 (reperfusion 12 h), 0.038 +/- 0.007 (reperfusion 24 h), respectively. The expression of cytoron C in cytoplasm at corresponding times in IR group were 0.040 +/- 0.006 (reperfusion 3 h), 0.076 +/- 0.009 (reperfusion 6 h), 0.108 +/- 0.005 (reperfusion 12 h), 0.089 +/- 0.008 (reperfusion 24 h), respectively. The expression of caspase-9 at corresponding times in IR group were 0.042 +/- 0.003 ( reperfusion 3 h), 0.086 +/- 0.007 (reperfusion 6 h), 0.142 +/- 0.006 (reperfusion 12 h), 0.112 +/- 0.009 (reperfusion 24 h), respectively. The expression of caspase-12 at corresponding times in IR group were 0.076 +/- 0.006 (reperfusion 3 h), 0.113 +/- 0.010 (reperfusion 6 h), 0.125 +/- 0.005 (reperfusion 12 h), 0.057 +/- 0.008 (reperfusion 24 h), respectively. They were significantly higher than that in normal group (P < 0.05). (3) The number of TUNEL positive neuron in the treatment group were 19.4 +/- 10.6/mm2 (reperfusion 3 h), 26.4 +/- 12.3 /mm2 (reperfusion 6 h), 39.3 +/- 13.3/mm2 (reperfusion 12 h), 49.3 +/- 13.6/mm2 (reperfusion 24 h), respectively. They were significantly lower than that in IR group, but more than that in normal group (P < 0.05). The expression of GRP78 at corresponding times in the treatment group were 0.158 +/- 0.012 (reperfusion 3 h), 0.175 +/- 0.005 (reperfusion 6 h), 0.125 +/- 0.013 (reperfusion 12 h), 0.079 +/- 0.004 (reperfusion 24 h), respectively. They were significantly higher than that in IR group and normal group (P < 0.05) . The expression of cytoron C in cytoplasm at corresponding times in IR group were 0.026 +/- 0.002 (reperfusion 3 h), 0.042 +/- 0.008 (reperfusion 6 h), 0.062 +/- 0.007 (reperfusion 12 h), 0.045 +/- 0.004 (reperfusion 24 h), respectively. The expression of caspase-9 at corresponding times in IR group were 0.033 +/- 0.002 (reperfusion 3 h), 0.063 +/- 0.005 (reperfusion 6 h), 0.092 +/- 0.005 (reperfusion 12 h), 0.068 +/- 0.008 (reperfusion 24 h), respectively. The expression of caspase-12 at corresponding times in IR group were 0.061 +/- 0.004 (reperfusion 3 h), 0.068 +/- 0.009 (reperfusion 6 h), 0.072 +/- 0.007 (reperfusion 12 h), 0.054 +/- 0.005 (reperfusion 24 h), respectively. They were significantly lower than that in IR group, but higher than that in normal group (P < 0.05)., Conclusions: Fetal rat cerebral neuron apoptosis following intrauterine distress is associated with the action of endoplasmic reticulum and mitochondrium. Up-regulation of GRP78 induced by 2DG counteracts primary cellular damage caused by endoplasmic reticulum stress. 2DG plays a protective role for fetal rat cerebral neuron following intrauterine distress.

Objective: To investigate the alteration on the expression levels of protein and messenger RNA (mRNA) of aquaporin-8 (AQP8) in the hepatocytes of pregnant rats with intrahepatic cholestasis induced by ethinylestradiol., Methods: A total number of 20 15-day pregnant rats were randomly divided into two groups: control group, intrahepatic cholestasis of pregnancy (ICP) group. In ICP group, rats were subcutaneously injected with ethinylestradiol (2.5 mg x kg(-1) x d(-1)) for 5 days to make the model of ICP. In control group, rats received subcutaneous injection of appropriate volume of propylene glycol for 5 days. The protein expression and subcellular localization of AQP8 in the hepatocytes of pregnant rats were detected using immunohistochemical methods. The mRNA expression of AQP8 in the hepatocytes was assayed by RT-PCR method., Results: The model of ICP of pregnant rats was made successfully. Expressions of AQP8 protein and mRNA were detected in two groups. AQP8 protein level in ICP group was 10.8 +/- 2.4, significantly decreased than in control group 17.1 +/- 2.2 (P < 0.05). AQP8 mRNA level in ICP group was 1.07 +/- 0.11, significantly higher than in control group 0.80 +/- 0.11 (P < 0.05)., Conclusion: Down-regulated AQP8 protein expression and up-regulated AQP8 mRNA expression in the hepatocytes of pregnant rats with intrahepatic cholestasis may contribute to the pathogenesis of ICP.

Objective: To investigate whether damaged human amniotic epithelial cells (HAEC) could be repaired on the matrix of formulated fibrin clot in vitro and the effects of epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and transforming growth factor beta(1) (TGF-beta(1)) on the proliferation of HAEC., Methods: Ring drill was used to drill the HAEC layer on culture sheets to make quantified models of damaged HAEC, on which the lacks were then covered with fibrin clot. Subsequently, EGF (EGF group), bFGF (bFGF group) and TGF-beta(1) (TGF-beta(1) group) of different concentration were added into the sheets respectively. After the predesigned culturing time, the growing and transiting conditions of HAEC were observed under inverted microscope after Giemsa stain. Also, the proliferating conditions of HAEC were detected by using 5-bromodeoxyuridine (BrdU)., Results: In all groups, HAEC could transit toward damaged area on fibrin clot and grow there. Higher transiting speed and larger cell numbers were observed in the EGF and bFGF groups followed by the control group, while the TGF-beta(1) group showed the relatively poorer results. Proliferating rates of HAEC were 17.8%, 28.0%, 35.3%, 51.6%, 34.1%, 34.2% and 26.0% respectively by EGF of different cultured concentration (1.0 ng/ml, 5.0 ng/ml, 10.0 ng/ml, 20.0 ng/ml, 40.0 ng/ml, 80.0 ng/ml and 160.0 ng/ml). Proliferating rates of HAEC were 18.0%, 35.7%, 43.0%, 52.7%, 67.4%, 43.6% and 30.5% respectively by bFGF of different cultured concentration (1.0 ng/ml, 5.0 ng/ml, 10.0 ng/ml, 20.0 ng/ml, 40.0 ng/ml, 80.0 ng/ml and 160.0 ng/ml). Compared with the control group, EGF groups (EGF concentration ranging from 10 ng/ml to 80 ng/ml) and bFGF groups (bFGF concentration ranging from 5 ng/ml to 80 ng/ml) showed better proliferating effects of HAEC (P < 0.05), especially the 20 ng/ml EGF group and 40 ng/ml bFGF group had the best proliferating results among their own respective groups (P < 0.05). Proliferating rates of HAEC were 17.1%, 15.1%, 9.3%, 6.2%, 4.8%, 3.6%, 2.0% and 1.2% respectively by TGF-beta(1) of different cultured concentration (0.1 ng/ml, 0.2 ng/ml, 0.4 ng/ml, 0.8 ng/ml, 1.6 ng/ml, 3.2 ng/ml, 6.4 ng/ml and 12.8 ng/ml). Proliferating rates of HAEC in TGF-beta(1) groups (TGF-beta(1) concentration ranging from 0.8 ng/ml to 12.8 ng/ml) were significantly lower than that in the control group (P < 0.05)., Conclusions: HAEC could transit and grow on the matrix of fibrin clot and repair the damaged area. EGF and bFGF could obviously stimulate HAEC proliferation, while TGF-beta(1) might have the inhibitive effects.

Objective: This study was to evaluate the grading of intrahepatic cholestasis of pregnancy (ICP)., Methods: All 278 ICP cases were collected in the First Affiliated Hospital of Chongqing Medical University between Jan 1999 and May 2003. They were graded into mild ICP and severe ICP based on liver function test. Outcome of pregnancy and perinatal mortality were assessed., Results: One hundred and ninety two cases were diagnosed as mild ICP while 86 cases were as severe ICP. There was significant difference in the average time of expectant therapy (10.3 +/- 4.8 vs 19.1 +/- 6.4 days) and gestational weeks (35.8 +/- 1.6 vs 37.7 +/- 1.9) between severe ICP and mild ICP groups (P < 0.01 and P < 0.001). The incidences of meconium passage, newborn asphyxia and admissions to neonatal intensive care unit in severe ICP were significantly higher than that in mild ICP. These were 38.4% vs 9.4%, 12.8% vs 4.2%, and 30.2% vs 9.4%, respectively. The difference of perinatal mortality between the patients with ICP diagnosed by using and not using the grading system (0.7% - 1.2% vs 3.9% - 6.0%) was statistically significant (P < 0.01)., Conclusion: Grading of ICP based on liver function test may improve perinatal prognosis.