静脉注入胆红素对新生大鼠脾磷酸化TAK1和磷酸化IKK蛋白表达的影响

发布时间：2018-08-04 19:06

【摘要】：目的：建立新生SD大鼠高胆红素血症模型，探讨静脉注入胆红素对脾Toll样受体/核因子κB（TLR/NF-κB）信号通路中磷酸化转化生长因子-β激活激酶1（TAK1，Transforming growth factor-β activatedkinase-1）和磷酸化核因子κB抑制因子激酶（IKK，Inhibitor of nuclearfactor kB kinase）蛋白表达的影响。方法：⒈实验分组：选用144只清洁级7～8d新生SD大鼠，雌雄不限，体重12.0～15.0g，随机分为空白对照组（Ⅰ）、脂多糖对照组（LPS，Ⅱ）、15mg/kg胆红素对照组（Ⅲ）、15mg/kg胆红素+LPS组（Ⅳ a）、30mg/kg胆红素+LPS组（Ⅳ b）和50mg/kg胆红素+LPS组（Ⅳ c），共6组，每组24只，每组又分为2h、5h和24h3个时间点，每个时间点8只。⒉建立新生SD大鼠高胆红素血症模型：⑴乙醚麻醉新生SD大鼠，酒精消毒颈部，暴露一侧颈静脉，给予不同剂量胆红素（15mg/kg、30mg/kg和50mg/kg）颈静脉注入；空白对照组和LPS对照组给予生理盐水0.1ml颈静脉注入；缝合颈部切口；⑵在颈静脉注入1h后，空白对照组和15mg/kg胆红素对照组给予生理盐水0.05ml腹腔注入，不注射LPS；其余各组1h后给予LPS1mg/kg腹腔注入；⑶分别于颈静脉注入后2h、5h、24h采血，测血浆胆红素浓度；⑷处死动物，，4％缓冲甲醛心脏灌注做内固定后快速取脾脏，固定、脱水、透明、包埋制成石蜡标本，采用免疫组织化学法检测脾磷酸化TAK1和磷酸化IKK蛋白的表达情况。结果：⒈新生SD大鼠在注入胆红素5～10min后皮肤出现不同程度的黄染，呈金黄色，1h后皮肤黄染减退；在注入1h时，15mg/kg、30mg/kg和50mg/kg胆红素组血浆总胆红素浓度95％的可信区间分别为：（106.31，123.49）μmol/L、（196.58，238.90）μmol/L和（325.15，349.07）μmol/L，且胆红素注入剂量与血浆总胆红素浓度呈正相关（rs=0.9452，P＜0.01）。2.胆红素对脾磷酸化TAK1和磷酸化IKK蛋白表达的影响：⑴低浓度范围（106.31，123.49μmol/L）的胆红素单独作用可抑制脾磷酸化TAK1和磷酸化IKK蛋白的表达（P＜0.05）；2h即可观察到这种抑制作用且作用最强，5h作用减弱，24h作用消失。⑵胆红素对LPS刺激磷酸化TAK1和磷酸化IKK蛋白表达有抑制作用（P＜0.05），且随着胆红素浓度的升高，抑制作用增强；2h时这种抑制作用最强，5h作用减弱，24h时低浓度范围胆红素的制作用消失，中、高浓度[（196.58,238.90）μmol/L和（325.15,349.07）μmol/L]范围胆红素的抑制作用仍然存在。3.磷酸化TAK1和磷酸化IKK蛋白表达水平与血浆总胆红素浓度的相关分析结果:⑴在注入胆红素2h和5h时，磷酸化TAK1表达水平与血浆总胆红素浓度呈负相关（r分别为-0.9067和-0.9198，P＜0.01）；在24h时，磷酸化TAK1表达水平与血浆中胆红素浓度无相关关系（r=0.1373，P＞0.05）。⑵在注入胆红素2h和5h时，磷酸化IKK蛋白表达水平与血浆总胆红素浓度呈负相关（r分别为-0.8247和-0.8479，P＜0.01）；在24h时，磷酸化IKK蛋白表达水平与血浆总胆红素浓度无相关关系（r=0.1077，P＞0.05）4.磷酸化IKK与磷酸化TAK1IOD（SUM）值的相关分析结果：在2h、5h和24h时，磷酸化IKK与磷酸化TAK1IOD（SUM）值呈正相关（r分别为0.8739、0.9014和0.8487，P＜0.01）。结论：1.胆红素静脉注入新生SD大鼠体内可以成功制作高胆红素血症动物模型。2.低浓度范围胆红素即可抑制LPS刺激脾磷酸化TAK1和磷酸化IKK蛋白的表达，且随着胆红素浓度的升高，抑制作用越明显，持续时间越长。结果提示，高胆红素血症病例免疫功能低下，可能与胆红素影响免疫细胞TLRs信号通路中磷酸化TAK1和磷酸化IKK蛋白的表达有关。
[Abstract]:Objective: to establish a neonatal SD rat model of hyperbilirubinemia, and to explore the effect of bilirubin on spleen Toll like receptor / nuclear factor kappa B (TLR/NF- kappa B) signaling pathway of phosphorylated TGF - beta activated kinase 1 (TAK1, Transforming growth factor- beta activatedkinase-1) and phosphorylated nuclear factor kappa B inhibitory factor kinase The effect of clearfactor kB kinase protein expression. Methods: a group of 144 clean 7 to 8D neonatal SD rats, male and female, and body weight 12 to 15.0g, were randomly divided into blank control group (I), lipopolysaccharide control group (LPS, II), 15mg/kg bilirubin control group (III), 15mg/kg bilirubin +LPS group (IV A), 30mg/kg bilirubin group (IV) And 50mg/kg bilirubin +LPS group (IV C), a total of 6 groups, each group of 24, each group was divided into 2h, 5h and 24h3 time points, each time point 8. Establish a newborn SD rat model of hyperbilirubinemia: 1 ether anesthetized newborn SD rats, alcohol disinfection neck, exposure to one side jugular vein, giving different doses of bilirubin (15mg/kg, 30mg/kg and 50mg/kg) jugular vein Injection; blank control group and LPS control group were injected with 0.1ml jugular vein in normal saline and suture neck incision. (2) after injection of 1H into the jugular vein, the blank control group and the 15mg/kg bilirubin control group were given 0.05ml intraperitoneal injection without LPS; the other groups were injected with LPS1mg/kg intraperitoneally after 1h; (3) injection of the jugular vein in the jugular vein, respectively. After 2h, 5h, 24h, plasma bilirubin concentration was measured. 4. Killed animals. After 4% buffers of formaldehyde heart perfusion, the spleen was quickly removed, fixed, dehydrated, transparent, and embedded into paraffin specimens. The expression of phosphorylated TAK1 and phosphorylated IKK protein was detected by immunohistochemistry. Results: the newborn SD rats were injected with bilirubin 5. After 10min, the skin was yellow dyed with golden yellow, and the skin yellow dyed after 1h. When 1H was injected, the confidence interval of the plasma total bilirubin concentration of 15mg/kg, 30mg/kg and 50mg/kg bilirubin group was (106.31123.49) mu mol/L, (196.58238.90) mu mol/L and (325.15349.07) micronux, and the dose of bilirubin injection and the total plasma were total. The effect of bilirubin concentration (rs=0.9452, P < 0.01).2. bilirubin on the expression of phosphorylated TAK1 and phosphorylated IKK protein of spleen: (1) the single effect of the bilirubin in the low concentration range (106.31123.49 mu mol/L) can inhibit the expression of spleen phosphorylated TAK1 and phosphorylated IKK protein (P < 0.05); 2H can observe the inhibitory effect and the strongest effect. The effect of 5h was weakened, and the effect of 24h disappeared. (2) bilirubin inhibited the expression of phosphorylated TAK1 and phosphorylated IKK protein by LPS (P < 0.05), and with the increase of bilirubin concentration, the inhibitory effect was enhanced. The inhibitory effect of the bilirubin was strongest, the action of 5h weakened, and the production of bilirubin in the low concentration range at 24h was vanished, and high concentration [(196.58238.90] [(196.58238.90]) The inhibitory effects of mol/L and (325.15349.07) mol/L] range bilirubin still exist in the correlation analysis of.3. phosphorylation TAK1 and the expression level of phosphorylated IKK protein and plasma total bilirubin concentration. (1) the level of phosphorylation TAK1 expression is negatively correlated with the concentration of plasma total bilirubin when injected with bilirubin 2H and 5h (R is -0.9067 and -0.9198, respectively). P < 0.01); at 24h, there is no correlation between the expression level of phosphorylated TAK1 and the concentration of bilirubin in plasma (r=0.1373, P > 0.05). (2) the expression level of phosphorylated IKK protein is negatively correlated with the concentration of plasma total bilirubin when injected with bilirubin 2H and 5h (R is -0.8247 and -0.8479, P < 0.01), and the level of phosphorylated protein expression and blood Correlation analysis of the concentration of total bilirubin (r=0.1077, P > 0.05) 4. phosphorylated IKK and phosphorylated TAK1IOD (SUM) values: at 2h, 5h and 24h, there is a positive correlation between phosphorylation IKK and phosphorylation TAK1IOD (SUM) value (R is 0.8487, 0.01). Conclusion: 1. bilirubin intravenous injection can be successful in neonatal rats. The low concentration of bilirubin in the animal model of hyperbilirubinemia can inhibit the expression of LPS stimulation of the spleen phosphorylated TAK1 and phosphorylated IKK protein, and with the increase of bilirubin concentration, the more obvious the inhibition effect, the longer the duration. The results suggest that the immune function of the cases of hyperbilirubinemia is low, and it may affect the immune cell T with bilirubin. The expression of phosphorylated TAK1 and phosphorylated IKK protein is related to LRs signaling pathway.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R722.17

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