葡萄糖对大鼠肝细胞白蛋白基因表达的影响

发布时间：2018-10-23 16:48

【摘要】： 目的首先观察大鼠Kupffer细胞与肝实质细胞体外共同培养时肝实质细胞生长、形态及功能状况,建立实验细胞模型。进一步观察不同浓度葡萄糖对大鼠肝细胞白蛋白基因表达的影响。方法(1)原位二步Ⅳ型胶原酶灌注法联合Percoll液密度梯度离心法分离肝细胞与Kupffer细胞;体外进行肝细胞单独培养、肝细胞与Kupffer细胞按6:1比例共同培养,观察不同情况下肝细胞生存时间和形态,每隔24h检测培养上清中白蛋白和ALT、AST的水平,并在36h放免法检测上清液TNF-α、IL-1、IL-6含量。(2)用终浓度分别为3.9mmol/L,7.0mmol/L,11.1mmol/L,22.2mmol/L的葡萄糖处理联合培养体系,以3.9mmol/L组作为对照组,分别于4h、24h留取细胞上清液,全自动生化仪检测白蛋白浓度,放免法检测上清液TNF-α、IL-1、IL-6含量,提取肝细胞RNA及逆转录聚合酶链法(RT-PCR)检测白蛋白mRNA含量。结果(1)单独培养组肝细胞的生长、增殖迅速,并向正常肝细胞的形态演变,肝细胞可培养存活至15d;共同培养组肝细胞细胞生长增殖缓慢,细胞可培养存活至10d。混合培养组上清白蛋白水平在24、36、48、60h比单独培养组低(t=2.980,3.139,2.551,2.605;p0.05);混合培养组上清ALT、AST水平在24、36、48、60h高于于单独培养组(ALT t=3.055,2.666,2.824,3.108;p0.05;AST t=2.632,2.446,3.090,2.895;p0.05),联合培养组Kupffer细胞保持IL-1、IL-6和TNF-α分泌功能,而单独培养组未检测到IL-1、IL-6和TNF-α。(2)不同浓度葡萄糖处理肝细胞4h后,各组肝细胞上清中IL-1,IL-6,TNF水平与对照组相比差异无统计学意义,与此同时,各组肝细胞上清中白蛋白水平与肝细胞白蛋白mRNA表达与对照组相比下降不明显;而培养24h以后,各组肝细胞上清中IL-1,IL-6,TNF水平与对照组相比上升明显,并且,随着葡萄糖浓度的增加肝细胞上清中IL-1,IL-6,TNF浓度不断上升,并且呈现剂量依赖关系,各组肝细胞上清中白蛋白水平与肝细胞白蛋白mRNA表达与对照组相比下降明显,呈现剂量依赖关系,与肝细胞上清中IL-1,IL-6,TNF水平变化一致。结论(1)肝实质细胞和Kupffer细胞在适宜的培养条件下可进行共同培养,kupffer细胞分泌细胞因子的功能和肝细胞白蛋白合成分泌功能保持良好,可用于实验研究。(2)高浓度葡萄糖糖能促进Kupffer细胞释放IL-1、IL-6和TNF-α,间接抑制大鼠肝细胞白蛋白mRNA的表达。
[Abstract]:Objective to observe the growth, morphology and function of rat Kupffer cells and hepatic parenchymal cells in vitro. To investigate the effect of glucose concentration on the expression of albumin gene in rat hepatocytes. Methods (1) Hepatocytes and Kupffer cells were isolated by in situ two-step collagenase perfusion combined with Percoll liquid density gradient centrifugation. Hepatocytes were cultured separately in vitro and co-cultured with Kupffer cells at 6:1. The survival time and morphology of hepatocytes were observed under different conditions. The levels of albumin and ALT,AST in supernatants were detected every 24 hours, and the contents of TNF- 伪 and IL-1,IL-6 in supernatants were detected by radioimmunoassay at 36h. (2) the co-culture system was treated with glucose with final concentration of 3.9mmol / L 7.0mmol / L 11.1mmol 路L ~ (-1) 路L ~ (-2) mmol 路L ~ (-1) 路L ~ (-1) glucose. 3.9mmol/L group was used as control group. The supernatant was collected at 4 h or 24 h. The concentration of albumin was detected by automatic biochemical instrument, the content of TNF- 伪 and IL-1,IL-6 in supernatant was detected by radioimmunoassay, the content of albumin mRNA was detected by RNA extraction from hepatocytes and by reverse transcriptase polymerase chain reaction (RT-PCR). Results (1) the growth and proliferation of hepatocytes in the single culture group were rapid, and the morphology of the hepatocytes evolved to normal. The hepatocytes could survive to 15 days, while the co-cultured group could grow slowly, and the cells could survive to 10 days. The supernatant albumin level in the mixed culture group was lower than that in the single culture group for 60 h (t2.980,3.139U 2.139U 2.551U 2.605p0.05), and the ALT,AST level in the supernatant of the mixed culture group was higher than that in the single culture group (ALT t3.0552.6662.8243.108p0.05), and the Kupffer cells in the co-culture group maintained the function of IL-1,IL-6 and TNF- 伪 secretion. However, there was no IL-1,IL-6 and TNF- 伪. (2) after 4 hours of glucose treatment, the level of IL-1,IL-6,TNF in the supernatant of hepatocytes in each group was not significantly different from that in the control group, and at the same time, there was no significant difference between the two groups. The levels of albumin in the supernatant of hepatocytes and the expression of albumin mRNA in the hepatocytes of each group were not significantly decreased compared with those of the control group, but after 24 hours of culture, the level of IL-1,IL-6,TNF in the supernatant of hepatocytes in each group was significantly higher than that in the control group. The concentration of IL-1,IL-6,TNF in the supernatant of hepatocytes increased with the increase of glucose concentration, and showed a dose-dependent relationship. The levels of albumin in the supernatants and the expression of albumin mRNA in hepatocytes decreased significantly in each group compared with those in the control group. There was a dose-dependent relationship, which was consistent with the change of IL-1,IL-6,TNF level in the supernatant of hepatocytes. Conclusion (1) Hepatic parenchymal cells and Kupffer cells can be co-cultured under suitable culture conditions. The function of cytokines secreted by kupffer cells and the synthesis and secretion of albumin in hepatocytes remain good. (2) High concentration glucose could promote the release of IL-1,IL-6 and TNF- 伪 from Kupffer cells and indirectly inhibit the expression of albumin mRNA in rat hepatocytes.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

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