Effect Of Suckling Pig Kupffer Cells On Hepatocyte Function,Proliferation And Apoptosis In Co-culture System

Objective:To establish a stable,efficient,and rapid experimental procedure for separating suckling pig hepatocytes and kupffer cells,explore the effect of kupffer cells on hepatocyte function,proliferation and apoptosis and lay the foundation for the application and related research of co-culture of primary hepatocytes and kupffer cells in vitro.Methods:(1)Using modified Seglen two-step perfusion method to separate suckling pig hepatocytes,and combined with Percoll density gradient centrifugation to kupffer cells,and then the Transwall indirect co-culture system was constructed.The primary hepatocytes of suckling pigs are randomly divided into two groups:hepatocyte alone culture group(single group)and hepatocyte-kupffer cell co-culture group at a ratio of 4:1(co-culture group);(2)Immunofluorescence was used to identify hepatocytes and kupffer cells,under an inverted phase microscope,cells were examined for morphology and growth status in each groups;(3)The cell supernatant of each group was harvested every 24h,then detecting the changes of aspartate aminotransferase and glucose by using the automatic biochemical instrument and albumin in supernatant by ELISA;(4)Harvested number of hepatocytes of each group in the first,third,fifth and seventh day,respectively.Then the flow cytometry was used to detect the rate of cell apoptosis in each groups,and detecting the cell proliferation and vitality by Enzyme-linked immunoassay.Results:(1)Using this method,each suckling pig can obtain the number of hepatocytes(1.24 ±1.65)× 1010/whole liver;according to the trypan blue exclusion method,the hepatocyte survival rate is(92.4±2.55)%.The yield of kupffer cells was(1.65±0.64)×109/whole liver,and the purity was up to(93 ± 2.26)%.In 10 days of morphological observation,the hepatocytes grew well in the single group,but began to apoptosis 7 days later and all of them died within 2 weeks;and the hepatocytes in the co-culture group grew better comparing with the single group,they began to apoptosis 10 days later and all of them died within 3 weeks.(2)Except that the albumin level in the supernatant of the co-culture group was significantly lower than that of the single group on the 1st,the difference was statistically significant(P<0.05),and it was higher than the single group on the 3rd and 7th days,and the difference was statistically significant Significance(P<0.05).(3)The results showed that in CCK-8 method,the proliferative activity of co-culture group on the 1st,3rd,5th and 7th day was higher than that of single culture group,the difference is statistically significant(P<0.05).(4)Compared the apoptosis rate of the two groups,the method described that the apoptosis rate in the single culture group was higher than that in the co-culture group,and the difference was statistically significant(P<0.05).Conclusions:This experiment successfully established the experimental process of modified Seglen perfusion method to separate suckling pig hepatocytes,and combined with Percoll density gradient centrifugation to kupffer cell.This method is stable,efficient,and fast.These results indicate that kupffer cells and hepatocytes can be co-cultured under appropriate culture conditions,and co-culture of hepatocytes and kupffer cells in a 4:1 ratio can increase the synthesis function of hepatocytes,promote hepatocyte proliferation,and inhibit their apoptosis.