【作者】 曹成波；

【导师】 周岩冰；

【作者基本信息】 青岛大学 ， 外科学， 2009， 硕士

【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;p<0.05);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;p<0.05;AST t=2.632,2.446,3.090,2.895;p<0.05),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。更多还原

【Abstract】 Objective Through co-culture Kupffer cells with hepatocyte in vitro,to obeserve the growth、morphology and metabolism of hepatocytes and establish experimental cell model.To investigat the effect of glucose on albumin mRNA expression in rat hepatocytes in vitro.Methods(1) In-situⅣcollagenase two-step perfusion method was used to digest rat liver,low speed centrifugalization was applied to isolate parenchymal hepatic cells and densitygradient centrifugation by percoll fluid to isolate Kupffer cells respectively.Culture hepatocyte cells alone and /or co-culture with Kupffer cells in the proportion six to one,the growth,morphous of hepatocytes were observed under the lightmicroscope and the level of albumin and ALT、AST in the culture supernatant were detected at 12 hours intervals,the concentrations of IL-1、IL-6 and TNF-a in the supernatant were also assayed at 36h.(2) Hepatocyte cells were inoculated into culture plate to co-culture with Kupffer cells in the proportion six to one;The cells were incubated in mediums containing 3.9 mmol/L, 7.0 mmol/L,11.1 mmol/L and 22.2 mmol/L glucose,among which 3.9 mmol/L glucose group was taken as control.The albumin mRNA in hepatocytes was assessed by reverse transcription-polymerase chain reaction(RT-PCR) and the albumin IL-1、IL-6 and TNF-αin the supernatant were measured at 4h,24h after intervention.Results The growth and developed to normal hepatocytes morphous of hepatocytes culture alone were quick,and hepatocytes could survive for 15 days,when hepatocytes co-culture with kupffer cells that proliferate slower than that culture alone. The hepatocytes could survive for 10 days.In culture alone group,the albumin level is significant higher than that in co-culture group at 24 h、36 h、48 h、60 h(t = 2.980, 3.139,2.551,2.605;p<0.05);The level of ALT、AST in hepatocytes culture alone supernatant was significant lower than those in co-culture group at 24h、36h、48h、60h(ALT t = 3.055,2.666,2.824,3.108;p<0.05;AST t = 2.632,2.446,3.090,2.895; p<0.05),IL-1、IL-6 and TNF-αcould be detected in co-culture groups,while were absent in culture alone groups at 36h.(2) There were no significant diffence of IL-1、IL-6、TNF-αin the culture supernatant among different groups,compared with control group,the level of albumin and the expression of albumin mRNA in high concentration glucose groups had no significant decreace at 4h;The level of IL-1、IL-6、TNF-αin high concentration glucose groups supernatant was significantly higher than those in control groups at 24h,the level of albumin and the expression of albumin mRNA in high concentration glucose groups were decreased significantly, and that was in a dose-related manner.Conclusion(l) Hepatocytes co-culture with Kupffer cells can be carried out under eligible culture condition,the cytokines secretion function of Kupffer cell and albumin synthesis function of hepatocytes are maintained.(2) High concentration glucose could indirectly inhibit the transcription of albuminogene through cytokines generation such as IL-1、IL-6 and TNF-α.更多还原