靜脈注入膽紅素對新生大鼠脾磷酸化TAK1和磷酸化IKK蛋白表達的影響
發(fā)布時間：2018-08-04 19:06
【摘要】：目的：建立新生SD大鼠高膽紅素血癥模型，探討靜脈注入膽紅素對脾Toll樣受體/核因子κB（TLR/NF-κB）信號通路中磷酸化轉(zhuǎn)化生長因子-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）蛋白表達的影響。方法：⒈實驗分組：選用144只清潔級7～8d新生SD大鼠，雌雄不限，體重12.0～15.0g，隨機分為空白對照組（Ⅰ）、脂多糖對照組（LPS，Ⅱ）、15mg/kg膽紅素對照組（Ⅲ）、15mg/kg膽紅素+LPS組（Ⅳ a）、30mg/kg膽紅素+LPS組（Ⅳ b）和50mg/kg膽紅素+LPS組（Ⅳ c），共6組，每組24只，每組又分為2h、5h和24h3個時間點，每個時間點8只。⒉建立新生SD大鼠高膽紅素血癥模型：⑴乙醚麻醉新生SD大鼠，酒精消毒頸部，暴露一側(cè)頸靜脈，給予不同劑量膽紅素（15mg/kg、30mg/kg和50mg/kg）頸靜脈注入；空白對照組和LPS對照組給予生理鹽水0.1ml頸靜脈注入；縫合頸部切口；⑵在頸靜脈注入1h后，空白對照組和15mg/kg膽紅素對照組給予生理鹽水0.05ml腹腔注入，不注射LPS；其余各組1h后給予LPS1mg/kg腹腔注入；⑶分別于頸靜脈注入后2h、5h、24h采血，測血漿膽紅素濃度；⑷處死動物，，4％緩沖甲醛心臟灌注做內(nèi)固定后快速取脾臟，固定、脫水、透明、包埋制成石蠟標(biāo)本，采用免疫組織化學(xué)法檢測脾磷酸化TAK1和磷酸化IKK蛋白的表達情況。結(jié)果：⒈新生SD大鼠在注入膽紅素5～10min后皮膚出現(xiàn)不同程度的黃染，呈金黃色，1h后皮膚黃染減退；在注入1h時，15mg/kg、30mg/kg和50mg/kg膽紅素組血漿總膽紅素濃度95％的可信區(qū)間分別為：（106.31，123.49）μmol/L、（196.58，238.90）μmol/L和（325.15，349.07）μmol/L，且膽紅素注入劑量與血漿總膽紅素濃度呈正相關(guān)（rs=0.9452，P＜0.01）。2.膽紅素對脾磷酸化TAK1和磷酸化IKK蛋白表達的影響：⑴低濃度范圍（106.31，123.49μmol/L）的膽紅素單獨作用可抑制脾磷酸化TAK1和磷酸化IKK蛋白的表達（P＜0.05）；2h即可觀察到這種抑制作用且作用最強，5h作用減弱，24h作用消失。⑵膽紅素對LPS刺激磷酸化TAK1和磷酸化IKK蛋白表達有抑制作用（P＜0.05），且隨著膽紅素濃度的升高，抑制作用增強；2h時這種抑制作用最強，5h作用減弱，24h時低濃度范圍膽紅素的制作用消失，中、高濃度[（196.58,238.90）μmol/L和（325.15,349.07）μmol/L]范圍膽紅素的抑制作用仍然存在。3.磷酸化TAK1和磷酸化IKK蛋白表達水平與血漿總膽紅素濃度的相關(guān)分析結(jié)果:⑴在注入膽紅素2h和5h時，磷酸化TAK1表達水平與血漿總膽紅素濃度呈負(fù)相關(guān)（r分別為-0.9067和-0.9198，P＜0.01）；在24h時，磷酸化TAK1表達水平與血漿中膽紅素濃度無相關(guān)關(guān)系（r=0.1373，P＞0.05）。⑵在注入膽紅素2h和5h時，磷酸化IKK蛋白表達水平與血漿總膽紅素濃度呈負(fù)相關(guān)（r分別為-0.8247和-0.8479，P＜0.01）；在24h時，磷酸化IKK蛋白表達水平與血漿總膽紅素濃度無相關(guān)關(guān)系（r=0.1077，P＞0.05）4.磷酸化IKK與磷酸化TAK1IOD（SUM）值的相關(guān)分析結(jié)果：在2h、5h和24h時，磷酸化IKK與磷酸化TAK1IOD（SUM）值呈正相關(guān)（r分別為0.8739、0.9014和0.8487，P＜0.01）。結(jié)論：1.膽紅素靜脈注入新生SD大鼠體內(nèi)可以成功制作高膽紅素血癥動物模型。2.低濃度范圍膽紅素即可抑制LPS刺激脾磷酸化TAK1和磷酸化IKK蛋白的表達，且隨著膽紅素濃度的升高，抑制作用越明顯，持續(xù)時間越長。結(jié)果提示，高膽紅素血癥病例免疫功能低下，可能與膽紅素影響免疫細(xì)胞TLRs信號通路中磷酸化TAK1和磷酸化IKK蛋白的表達有關(guān)。
[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.
【學(xué)位授予單位】：瀘州醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R722.17
本文編號：2164845
[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.
【學(xué)位授予單位】：瀘州醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R722.17
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