[Isolation and co-culture of primary hepatocytes and primary Kupffer cells from rats with nonalcoholic steatohepatitis]

Objective: By isolating and purifying primary hepatocytes and primary Kupffer cells from rats with nonalcoholic steatohepatitis (NASH), and establishing the primary cell model of NASH in vitro, to provide reliable technical support for cell experiment in the study of NASH. Methods: Forty SD rats were selected and randomly divided into the control group and the NASH group. The rats in the control group were fed with common feed, and the rats in the NASH group were fed with a high-fat diet (88% basal feed + 10% lard + 2% cholesterol). After 6-8 weeks, using the NASH score table, the liver tissue section steatosis + intralobular inflammation + ballooning degeneration score ≥ 4 points under pathological observation, indicating that the rat NASH model was successfully established. And the primary hepatocytes of NASH rats were isolated and purified by collagenase in situ perfusion. Cells were identified by CK-18 and CD68 immunofluorescence and ink swallowing test. The lipid accumulation was tested by Oil red O staining, and the contents of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined to evaluate the liver function in primary hepatocytes of NASH rats. The expressions of inflammatory factors of primary Kupffer cells were detected by Western blot. Finally, primary hepatocytes and primary Kupffer cells were co cultured at the ratio of 6:1 and observed under microscope. Results: NASH primary hepatocytes and primary Kupffer cells were successfully isolated and purified. Compared with the control group, Oil red O staining showed that the primary hepatocytes of the NASH group had obvious fat deposition, and the AST and ALT levels in the primary hepatocytes of the NASH group were significantly higher than those of the control group, indicating obvious liver damage (P＜ 0.05). The Western blot result showed that the levels of TNF-α, IL-1β and MCP-1 in primary Kupffer cells was significantly higher than the control group (P＜0.05). Conclusion: The primary hepatocytes and primary Kupffer cells of NASH rats were isolated successfully by collagenase in situ perfusion. At the same time, a proportional co-culture rat in vitro primary cell NASH model was successfully established.