PD-1在HBV转基因小鼠急性肝损伤中的表达及意义

Expression and significance of programmed death 1(PD-1) in the acute liver injury of HBV transgenic mic

目的观察HBV转基因小鼠急性肝损伤过程中肝脏程序性死亡受体-1(PD-1)的动态变化情况,初步探讨HBV转基因小鼠在急性肝损伤的免疫变化机制。方法 40只健康C57BL/6小鼠随机分为正常对照组(n=20)及四氯化碳(CCL4)干预组(n=20);另选取20只SPF级转基因HBsAg阳性鼠为转基因HBsAg阳性CCL4组。正常对照组随机分为4组,按0.5mL/kg腹腔注射橄榄油溶液,0、24、48、72h4个时间点取标本。CCL4干预组、转基因HBsAg阳性CCL4组一次性给予0.5mL/kg腹腔注射10%CCL4橄榄油溶液,分别于0、24、48、72h每组随机挑选5只小鼠,采集肝脏标本,用免疫组织化学方法检测每组小鼠肝脏组织中PD-1的表达,采用实时荧光定量PCR法检测PD-1mRNA的表达。结果正常对照组PD-1阴性表达,其余各组肝脏组织中PD-1表达水平有随时间变化的趋势并且时间因素的作用随着分组的不同而不同。48、72h时,转基因HBsAg阳性CCL4组PD-1表达的累积光密度(IOD)值明显高于CCL4干预组及正常对照组,差异有统计学意义(P<0.05)。24、48、72h时,CCL4干预组、转基因HBsAg阳性CCL4组PD-1表达的IOD值均高于0h,CCL4干预组24h时PD-1表达最高,转基因HBsAg阳性CCL4组48h时PD-1表达最高(P<0.05)。PD-1mRNA表达水平有随时间变化的趋势并且时间因素的作用随着分组的不同而不同。除正常对照组PD-1mRNA表达随时间没有改变,从24~72h,CCL4干预组和转基因HBsAg阳性CCL4组PD-1mRNA的表达量明显增高,均高于0h时。48、72h时,转基因HBsAg阳性CCL4组表达最高,与CCL4干预组比较有统计学意义(P<0.05)。CCL4干预组PD-1mRNA表达明显高于正常对照组(P<0.05)。结论 HBV转基因小鼠24h即可出现急性肝损伤,尤其是HBsAg表达阳性的转基因小鼠损伤较CCL4干预的小鼠严重。转基因HBsAg阳性小鼠PD-1在48、72h明显升高,发生强烈的免疫反应。

Objective To observe the dynamic expression of programmed death 1(PD-1)in the acute liver injury of HBV transgenic mice,and to pilot investigate the possible immune mechanism of acute liver injury in HBV transgenic mice.Methods Healthy male C57BL/6 mice were randomly divided into normal control group(n =20)and carbon tetrachloride(CCL4)group(n=20).Twenty HBsAg positive mice were selected as transgenic HBsAg Positive CCL4 group.The normal control group were randomly divided into four groups:the control group was intraperitoneally injected with 0.5 mL/kg olive oil solution,and the specimens were taken at 0,24,48 and 72 h.CCL4 intervention group(n =20)and transgenic HBsAg positive CCL4 group :intraperitoneal injection of 10% CCL4 olive oil was administered with 0.5 mL/kg,and the livers were sampled at 0,24,48 and 72,The protein and mRNA of PD-1 in the liver of each group was detected by immunohistochemistry and real-time quantitative PCR,respectively.Results The expression of PD-1 was negative in the normal control group.The positive immunostaining of PD-1 in the liver tissues were mainly present in the cytoplasm of liver.There was a trend that the expression of PD-1 varies with the time.At 48,72 h,the IOD of PD-1 expression in transgenic HBsAg positive CCL4 group was shown to be significantly higher than that in CCL4 intervention group(P <0.05).The expression of PD-1 in CCL4 intervention group and transgenic HBsAg positive CCL4 group at 24,48,72 hwas higher than control group.PD-1 expression was the highest in CCL4 intervention group at 24 hand in HBsAg-positive CCL4 intervention group at 48 h(P <0.05).PD-1 mRNA expression levels had a tendency to change with time and the effect of time factors differed among groups.From 24 hto 72 h,the expression level of PD-1 mRNA in CCL4 intervention group and transgenic HBsAg-positive CCL4 intervention group was significantly higher than that at 0 h.Compared with CCL4 intervention group,the expression of HBsAg positive CCL4 group was the highest at 48,72 h(P <0.05).The expression of PD-1 mRNA in CCL4-treated group was significantly higher than normal control group(P <0.05).Conclusion HBV transgenic mice showed acute liver injury 24 hlater.In particular,HBsAg-positive transgenic mice has more severe injury than CCL4 intervention group.The expression of PD-1 was observed to be high in transgenic HBsAg-positive mice at 48 and 72 h,with the occurrence of a strong immune response.