The Effects of Hypoxia on Development of Ovine Fetal Liver Cells and Its RAS Mechanisms
|Course||The embryonic physiological and perinatal Basic Medical|
|Keywords||hypoxia ovine fetal liver cell cell culture hepatocyte function cell cycle apoptosis AngⅡ AT1R AT2R protein expression|
Part I Effects of hypoxia on development of ovine fetal liver cells Objectives: To investigate the effects of hypoxia on ovine fetal liver cell development.Methods: Hepatocytes were isolated and in primary culture, with the method of liver collagenase perfusion through fetal umbilical vein followed by density gradient centrifugation. Hypoxia-injured hepatocyte model was established to be the hypoxia group, and the non-injured primary cultured hepatocyte was the control group. Ultrastructural changes of the hepatocytes were observed with the transmission electron microscopy. Concentrations of total protein （TP）, Albumin （ALB）, Creatinine （CREA）, Urea nitrogen （BUN）, Aspartate transferase （AST）, Alanine transaminase （ALT）, and Lactic acid dehydrogenase （LDH） in culture medium were determined by the automatic biochemical analyzer. Cell cycle and apoptosis of the hepatocytes were detected using the flow cytometry.Results: In hepatocytes of the hypoxia group, some ultrastructural changes such as: cytoplasmic vacuolization, endoplasmic reticulum dilation, mitochondria swelling, and mitochondrial crista swelling and rupturing, were observed with the transmission electron microscopy. In culture medium of the hypoxia group, concentrations of TP and ALB significantly decreased （p<0.05）, whereas concentrations of AST and LDH significantly increased （p<0.01）, compared to that of control. However, there was no difference of concentrations of CREA, BUN, and ALT in culture medium between hypoxia and control group. Moreover, compared with the control group, proportion of cells in S phase significantly decreased （p<0.05）, and proportion of apoptotic cells significantly increased （p<0.05） in the hypoxia group. Conclusions: Hypoxia could induce some ultrastructural changes, affect certain cellular functions, inhibit proliferation, and enhance apoptosis in the cultured ovine fetal liver cells.PartⅡEffects of hypoxia on RAS of ovine fetal liver cellsObjectives: To investigate changes of RAS in ovine fetal liver cells affected by hypoxiaMethods: Ovine fetal liver cells cultured for three days were randomly divided into six groups according to the respective treatments: 1) Control; 2) AngⅡ; 3) Hypoxia; 4) Hypoxia+AngⅡ; 5) Losartan+AngⅡ; 6) PD123319+AngⅡ(drug concentrations were all 10-6mol/L). Cell cycle of liver cells was detected by flow cytometry. Ovine fetal liver cells cultured for three days were randomly divided into four groups according to the respective treatments: 1) Control; 2) AngⅡ; 3) Hypoxia; 4) Hypoxia+AngⅡ(concentration of AngⅡwas 10-6mol/L). Apioptosis of liver cells was determined by flow cytometry. Protein expressions of AT1R and AT2R in liver cells were detected by Western Blotting.Results: In hypoxia, AngⅡ, and Hypoxia+AngⅡgroups, proportions of cells in S phase were significantly decreased （p<0.05,p<0.01,p<0.01）, while proportions of apoptosis were significantly increased （p<0.05）, compared to that of control group. In Hypoxia+AngⅡgroup, proportion of cells in S phase was significantly decreased compared to that of control group （p<0.05）, but was not significantly different to that of AngⅡgroup （p>0.05）; however, proportion of apoptosis was not significantly different to that of control group （p>0.05）, but was significantly increased compared to that of AngⅡgroup （p<0.05）. Compared to that of control group, proportion of cells in S phase was significantly decreased in AngⅡand Losartan+AngⅡgroups （p<0.01）, but not significantly changed in PD123319+AngⅡgroup （p>0.05）. Compared to that of AngⅡgroup, proportion of cells in S phase was not significantly changed in Losartan+AngⅡgroup （p>0.05）, but significantly increased in PD123319+AngⅡ group （p<0.05）. AT1R protein expressions in Hypoxia and AngⅡgroups were not significantly different to that of control group （p>0.05）. However, compared to that of control group, AT2R protein expression was significantly increased in Hypoxia group （p<0.05）, but not significantly changed in AngⅡgroup （p>0.05）.Conclusions: Hypoxia and AngⅡboth could inhibit proliferation and enhance apoptosis in ovine fetal liver cells which might be associated with AT2R but not AT1R.