The Effect Of Glucose On Albumin MRNA Expression In Rats Hepatocytes In Vitro

Objective Through co-culture Kupffer cells with hepatocyte in vitro,to obeserve the growth,morphology and metabolism of hepatocytes and establish experimental cell model.To investigat the effect of glucose on albumin mRNA expression in rat hepatocytes in vitro.Methods(1) In-situⅣcollagenase two-step perfusion method was used to digest rat liver,low speed centrifugalization was applied to isolate parenchymal hepatic cells and densitygradient centrifugation by percoll fluid to isolate Kupffer cells respectively.Culture hepatocyte cells alone and /or co-culture with Kupffer cells in the proportion six to one,the growth,morphous of hepatocytes were observed under the lightmicroscope and the level of albumin and ALT,AST in the culture supernatant were detected at 12 hours intervals,the concentrations of IL-1,IL-6 and TNF-a in the supernatant were also assayed at 36h.(2) Hepatocyte cells were inoculated into culture plate to co-culture with Kupffer cells in the proportion six to one;The cells were incubated in mediums containing 3.9 mmol/L, 7.0 mmol/L,11.1 mmol/L and 22.2 mmol/L glucose,among which 3.9 mmol/L glucose group was taken as control.The albumin mRNA in hepatocytes was assessed by reverse transcription-polymerase chain reaction(RT-PCR) and the albumin IL-1,IL-6 and TNF-αin the supernatant were measured at 4h,24h after intervention.Results The growth and developed to normal hepatocytes morphous of hepatocytes culture alone were quick,and hepatocytes could survive for 15 days,when hepatocytes co-culture with kupffer cells that proliferate slower than that culture alone. The hepatocytes could survive for 10 days.In culture alone group,the albumin level is significant higher than that in co-culture group at 24 h,36 h,48 h,60 h(t = 2.980, 3.139,2.551,2.605;p<0.05);The level of ALT,AST in hepatocytes culture alone supernatant was significant lower than those in co-culture group at 24h,36h,48h,60h(ALT t = 3.055,2.666,2.824,3.108;p<0.05;AST t = 2.632,2.446,3.090,2.895; p<0.05),IL-1,IL-6 and TNF-αcould be detected in co-culture groups,while were absent in culture alone groups at 36h.(2) There were no significant diffence of IL-1,IL-6,TNF-αin the culture supernatant among different groups,compared with control group,the level of albumin and the expression of albumin mRNA in high concentration glucose groups had no significant decreace at 4h;The level of IL-1,IL-6,TNF-αin high concentration glucose groups supernatant was significantly higher than those in control groups at 24h,the level of albumin and the expression of albumin mRNA in high concentration glucose groups were decreased significantly, and that was in a dose-related manner.Conclusion(l) Hepatocytes co-culture with Kupffer cells can be carried out under eligible culture condition,the cytokines secretion function of Kupffer cell and albumin synthesis function of hepatocytes are maintained.(2) High concentration glucose could indirectly inhibit the transcription of albuminogene through cytokines generation such as IL-1,IL-6 and TNF-α.