Wu Xia, Zhang Yu-Rong, Zhu Xiao-Ning, Wang Jing
Department of Integrated Traditional Chinese and Western Medicine, Southwest Medical University, Luzhou 646000.
Hepatobiliary Department, The Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University, Luzhou 646000, China.
Zhongguo Ying Yong Sheng Li Xue Za Zhi. 2022 Mar;38(2):187-192. doi: 10.12047/j.cjap.6261.2022.033.
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. 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. 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 (＜ 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 (＜0.05). 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.
通过从非酒精性脂肪性肝炎（NASH）大鼠中分离纯化原代肝细胞和原代库普弗细胞，建立体外NASH原代细胞模型，为NASH研究中的细胞实验提供可靠的技术支持。选取40只SD大鼠，随机分为对照组和NASH组。对照组大鼠喂普通饲料，NASH组大鼠喂高脂饲料（88%基础饲料+10%猪油+2%胆固醇）。6-8周后，使用NASH评分表，病理观察肝组织切片脂肪变性+小叶内炎症+气球样变性评分≥4分，表明大鼠NASH模型成功建立。然后用胶原酶原位灌注法分离纯化NASH大鼠的原代肝细胞。通过CK-18和CD68免疫荧光及墨汁吞噬试验对细胞进行鉴定。用油红O染色检测脂质蓄积情况，测定丙氨酸氨基转移酶（ALT）和天冬氨酸氨基转移酶（AST）含量以评估NASH大鼠原代肝细胞的肝功能。通过蛋白质免疫印迹法检测原代库普弗细胞炎症因子的表达。最后，将原代肝细胞和原代库普弗细胞按6:1的比例共培养并在显微镜下观察。成功分离纯化了NASH原代肝细胞和原代库普弗细胞。油红O染色显示，与对照组相比，NASH组原代肝细胞有明显的脂肪沉积，且NASH组原代肝细胞中AST和ALT水平显著高于对照组，表明有明显的肝损伤（＜0.05）。蛋白质免疫印迹结果显示，原代库普弗细胞中TNF-α、IL-1β和MCP-1水平显著高于对照组（＜0.05）。通过胶原酶原位灌注成功分离出NASH大鼠的原代肝细胞和原代库普弗细胞。同时，成功建立了比例共培养的大鼠体外原代细胞NASH模型。
