| Objective: To research the mechanism of double function of tTG in hepatocellular injury and proliferation through observing the expression and distribution of tTG in vitro, enriching the information of signal transduction, offering some new theory on the prevention and cure of liver disease to refer.Methods: Rat primary hepatocytes were stimulated with ethanol at a low concentration to make hepatocellular injury, and PNLP, Putr, CsA were added separately to determine their effects on ethanol -induced injury. The morphological changes of the hepatocytes were observed by invert microscope; The cellular proliferation was measured by MTT; The cellular apoptosis was observed by Hoechst33342 fluorescent-cytochemistry; TTG protein expression was determinated by immunocytochemistry; TTG distribution in cellular different position was tested by Western blotting.Results: (1) MTT assay showed that hepatocyte proliferation activity was inhibited by ethanol when the cells were incubated with ethanol for 24h at a final concentration of 200mM (There is a significant difference between group control and group ethanol, P<0.01) and was promoted by PNLP (200uM) or Putr (lOOuM) or CsA (lOuM) , especially in the group PNLP and CsA the cell proliferation was significantly enhanced (compared with group ethanol, P<0.01) . (2) Observing and counting the apoptic cells by Hoechst fluorescent-cytochemistry showed that the apoptotic cells were observed at a final concentration of 50mM of ethanol treatmentfor 18h to rat hepatocyte. The cell apoptotic rate was in group ethanol 15.4%, in group PNLP (200uM) 9.3%, in group Putr (lOOuM) 5.4%, in group CsA (lOuM) 4.6%, in group control 2.9% respectively. The group ethanol was more higher than the other groups (There are different significantly, P<0.01 orP<0.05).(3)The image analysis of tTG immunocytochemistry showed that the expression of tTG in rat hepatocyte was significantly enhanced at the final concentration of SOmM of ethanol treatment for 18h. The integral optical density in each group displayed by image analysis of tTG immunocytochemistry was different, group ethanol 97.632, group PNLP(200uM) 103.87, both was more higher than the group control(64.557)(There are different significantly, PO.01) , and group Putr (lOOuM) 54.595, group CsA (lOuM) 40.464, compared with group ethanol separately was lower significantly (There are different significantly, P<0.01) . (4) TTG content in cellular plasma protein which was extracted after 18h with different treatment to rat hepatocyte tested by Western blotting showed that the average optical density of PAGE bands by tTG electrophoresis was 0.81 in group control, 0.90 in group ethanol (SOmM) , 0.84 in group PNLP (200uM) , 0.66 in group Putr (ImM) , 0.77 in group CsA (3uM) , 0.75 in group CsA (5uM) respectively. Group ethanol was more higher than the other groups (There are different significantly, P<0.01 or P<0.05; but not group PNLP) , and group CsA indicated a negative dependence relationship with CsA's concentration in definite range. (5) TTG content of cell nuclear protein which was extracted after 18h with different treatment to rat hepatocyte tested by Western blotting showed that the average optical density of PAGE bands by tTG electrophoresis were different, group ethanol (SOmM) 1.02, was more higher than group control 0.9 (There is a significant difference between group control and group ethanol, PO.05), and group Putr (ImM) 0.74 was more lower than group ethanol (There is a significant difference between group Putr and group ethanol, P<0.01 )o (6) TTG content of cell membrane protein which was extracted after 18h with different treatment to rat hepatocyte tested by Western blotting showed that the average optical density of PAGE bands by tTG electrophoresis was 0.25 in group control, 0.19 in group ethanol (lOOmM) , 0.32 in group ethanol plus PNLP(100mM+200uM), 0.33 in group PNLP (200uM) respectively. Group ethanol plus PNLP was more higher than group ethanol or group control (There is different significantly, P<0.01 or PO.05) , and group e... |
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