| [Objective]To establish a stable and effective standard procedure for isolate hepatocytes and Kupffer cells of SD rat. Through co-culture Kupffer cells with hepatocyte, to understand the influence of Kupffer cells on the growth, morphology and metabolism of hepatocyte. In order to provide the data for optimal deploy primary culture hepatocyte in vitro.[Methods] In-situ IV collagenase two-step perfusion method to digest SD rat liver, apply low speed centrifugalization to isolate parenchymal hepatic cells and densitygradient centrifugation by percoll fluid to isolate Kupffer cells respectively . Inoculate hepatocyte cells into 6 holes culture plate to culture alone and/or co-culture with Kupffer cells in the proportion six to one , the growth, morphous of hepatocyte are observed under the light microscope and the level of albumin and glucose in the culture supernatant are detected by automatic biochemistry meter , and compare synthesis function and metabolism of hepatocytes culture alone with co-culture with Kupffer cells.[Results]The mean amount of isolated hepatocytes from each rat is (2.03±0.26) x10~8, and the alive rate of the cells is (91.7±2.11) %.The mean amount of isolated Kupffer cells from each rat is (3.19±0.20) xlO7, the purity of kupffer cells is 85-92% in average. The primary adherent culture hepatocytes are observed over 192 hours, the hepatocytes of culture alone their growth and developed to nomal hepatocytes morphous quickly, 2 weeks later the cells death occurs, after 21 days the hepatocytes culture alone are totaly death. The hepatocytes of co-culture with kupffer cells proliferate slower than that culture alone. They beginning to death 48 hours later, and totally death after 10 days.Supernatant was collected and tested the level of albumin and glucose at 24 hours intervals. In culture alone group, the albumin level is significant higher than inco-culture group at 481k 961k 1201k 1441k 168h (1.722±0.123vsl.640±0.127, 1.808±0.097vsl.580±0.084,1.680±0.070vsl.540±0.085,1.850±0.082vsl.720±0.042, 1.644±0.067vsl.525±0.035 respectively, independent-sample t test, p<0.05), however, has no significant deviation at 721k 192h (1. 648±0. 049vsl. 650±0.141, 1.650 ±0. 045vsl. 650+0. 028 respectively, independent-sample t test, p>0.05 ). Although the level of glucose in hepatocyte culture alone supernatant is significant higher than in co-culture group at 961k 1441k 168h (10. 33+0. 350vs9.60±0. 300, 9.51 ± 0.221vs8. 85 ± 0. 025 , - 9. 38 + 0.198vs8. 72 + 0. 339 respectively, independent-sample t tes, p<0.05) , has no significant deviation at other time point (8.81 ±0.154vs8. 73 + 0.224, 9.92 ± 0. 220vs9. 48 + 0. 398 , 9.62 + 0. Illvs9.32 ± 0.254 , 9.04 ± 0.011vs8.71 ± 0.117 respectively , independent-sample t tes, p>0.05) .[Conclusion] (l)Established a stable and effective standard procedure based on the method of IV collagenase two-step perfusion by meas of infer-liver inferior caval vein as inflow tract for isolate SD rat hepatocytes and combined with percoll fluid densitygradient centrifugation for isolate Kupffer cells successfully;(2)Primary adherent co-culture hepatocytes with Kupffer cells in the proportion six to one successfully performed , suggested that the hepatocytes co-culture with Kupffer cells can be carried out under eligible culture condition, but the growth> albumin synthesis function^ glucose metabolism of hepatocytes will be influenced in simple synchronous adherent coculture. These data offered that when hepatocyte coculture with Kupffer cell in vitro, further to optimize the opportunity ,way, and to build more suitable coculture system is needed.
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