【摘要】：目的：建立新生大鼠高膽紅素血癥模型，探討靜脈注入膽紅素對脾髓樣分化因子88（Myeloid differentiation factor88，MyD88）和磷酸化p38MAPK（phospho-p38MAP Kinase，p-p38MAPK）蛋白表達及凋亡的影響。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（Ⅳa）、30mg/kg膽紅素+LPS組（Ⅳb）和50mg/kg膽紅素+LPS組（Ⅳc），共6組，每組24只，每組又分為2h、5h、24h三個時間點，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固定后快速取脾臟，固定、脫水、透明、包埋制成石蠟標本，采用免疫組織化學法檢測脾MyD88和磷酸化p38MAPK蛋白的表達，TUNEL染色原位法檢測脾細胞凋亡情況。結果：⒈新生SD大鼠在注入膽紅素5～10min后皮膚出現不同程度的黃染，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％的置信區(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（rs=0.9452，P＜0.01）。⒉膽紅素對脾MyD88蛋白表達的影響：⑴低濃度范圍（106.31，123.49μmol/L）的膽紅素和LPS單獨作用可以促進脾細胞MyD88的表達（P＜0.01），但膽紅素弱于LPS的促進作用（P＜0.01）；5h即可觀察到膽紅素的這種促進作用，24h作用消失；⑵低濃度范圍的膽紅素對LPS刺激MyD88表達的影響不明顯（P＞0.05）；⑶中、高濃度范圍[（196.58，238.90）和（325.15，349.07）μmol/L]的膽紅素對LPS刺激MyD88表達有抑制作用（P＜0.01），，且隨著濃度的升高，抑制作用增強；2h即可觀察到這種抑制作用，5h作用明顯，24h時中等濃度范圍的膽紅素的抑制作用消失，高濃度范圍的抑制作用仍然存在（P＜0.01）。⒊膽紅素對脾p-p38MAPK蛋白表達的影響：⑴低濃度范圍的膽紅素和LPS單獨作用可促進脾細胞p38MAPK的磷酸化（P＜0.01），但弱于LPS的促進作用（P＜0.01）；2h即可觀察到膽紅素的這種促進作用，5h作用增強，24h作用仍存在；⑵中、低濃度范圍的膽紅素對LPS刺激p-p38MAPK表達有抑制作用（P＜0.01），且濃度越高，抑制作用越明顯；2h即可觀察到這種抑制作用，5h作用增強，24h作用消失；⑶高濃度范圍的膽紅素對LPS刺激p-p38MAPK表達有促進作用（P＜0.01）；5h時即可觀察到這種促進作用，24h作用減弱。⒋MyD88和p-p38MAPK與膽紅素濃度的相關分析結果：⑴2h、5h和24h的MyD88IOD（SUM）值與膽紅素濃度呈負相關（r分別為：－0.856、－0.791和－0.875，P＜0.01），即隨著膽紅素濃度的升高，抑制脾細胞表達MyD88的作用越明顯；⑵2h的p-p38MAPK IOD（SUM）值與膽紅素濃度無相關關系（r=－0.123，P＞0.05）；5h的p-p38MAPK IOD（SUM）值與膽紅素濃度呈正相關（r=0.897，P＜0.01）；24h的p-p38MAPK IOD（SUM）值與膽紅素濃度呈正相關（r=0.827，P＜0.01）。表明在5h和24h時，隨著膽紅素濃度的升高，p-p38MAPK的表達有增加的趨勢。⒌膽紅素對脾細胞凋亡的影響：⑴低濃度范圍的膽紅素組凋亡率增加（P＜0.01）；⑵中、低濃度范圍的膽紅素與LPS共同作用可減少脾細胞凋亡（P＜0.01）；高濃度范圍的膽紅素與LPS共同作用可使細胞凋亡增多（P＜0.01）。結論：⒈低濃度膽紅素對LPS刺激MyD88表達的影響不明顯；中、高濃度的膽紅素對MyD88表達有抑制作用，這種抑制作用呈濃度依賴性，隨著濃度的升高，抑制作用增強，持續(xù)時間延長。⒉中、低濃度膽紅素可抑制p38MAPK的磷酸化，高濃度則促進p38MAPK的磷酸化，且隨著濃度的升高，作用時間延長。⒊高濃度膽紅素對脾細胞有毒性作用，細胞凋亡增多。提示膽紅素對免疫細胞的影響可能與調節(jié)TLRs信號通路中MyD88的表達和p38MAPK的磷酸化及誘導細胞的凋亡有關。
[Abstract]:AIM: To establish a hyperbilirubinemia model of neonatal rats and investigate the effects of intravenous bilirubin on the expression of myeloid differentiation factor 88 (MyD88) and phospho-p38MAP Kinase (p-p38MAPK) protein and apoptosis of spleen in neonatal rats. They were randomly divided into blank control group (I), lipopolysaccharide control group (LPS, II), 15 mg / kg bilirubin control group (III), 15 mg / kg bilirubin + LPS group (IVa), 30 mg / kg bilirubin + LPS group (IVb) and 50 mg / kg bilirubin + LPS group (IVc), totally 6 groups, 24 rats in each group. Each group was divided into 2 hours, 5 hours, 24 hours three time points, 8 rats at each time point. Establishment of hyperbilirubinemia model in neonatal SD rats: _Ether anesthesia neonatal SD rats, alcohol disinfection of the neck, exposure of one side of the jugular vein, given different doses of bilirubin (15mg/kg, 30mg/kg and 50mg/kg) jugular vein injection; blank control group and LPS control group given 0.1ml normal saline jugular vein injection; suture the neck incision; _in jugular vein One hour after injection, the blank control group and 15 mg/kg bilirubin control group were injected with 0.05 ml normal saline intraperitoneally, without LPS injection; the other groups were injected with LPS 1mg/kg intraperitoneally after 1 hour; _Blood was collected at 2 hours, 5 hours and 24 hours after jugular vein injection, and plasma bilirubin concentration was measured; _Animals were sacrificed and spleen was quickly taken after 4% buffered formaldehyde cardiac perfusion and internal fixation. The expression of MyD88 and phosphorylated p38 MAPK protein in spleen was detected by immunohistochemistry, and the apoptosis of spleen cells was detected by TUNEL staining in situ. The confidence intervals of 95% of plasma total bilirubin concentration in 15 mg/kg, 30 mg/kg and 50 mg/kg bilirubin groups were (106.31, 123.49) micromol/L, (196.58, 238.90) micromol/L and (325.15, 349.07) micromol/L, respectively, and the dose of bilirubin was positively correlated with plasma total bilirubin concentration (rs = 0.9452, P < 0.01). The effect of low concentration of bilirubin and LPS on myD88 expression in spleen cells (P < 0.01), but the effect of bilirubin was weaker than that of LPS (P < 0.01); the effect of bilirubin on myD88 expression was observed in 5 hours, and disappeared in 24 hours; and the effect of low concentration of bilirubin on LPS-stimulated myD88 expression in spleen cells (P < 0.01). _Bilirubin in high concentration range [(196.58,238.90) and (325.15,349.07) micromol/L] inhibited the expression of MyD88 stimulated by LPS (P _The effect of bilirubin on the expression of p-p38 MAPK protein in spleen: _Bilirubin and LPS at low concentration alone could promote the phosphorylation of p38 MAPK in spleen cells (P < 0.01), but was weaker than that of LPS (P < 0.01); _Bilirubin at low concentration inhibited the expression of p-p38 MAPK stimulated by LPS (P < 0.01), and the higher the concentration was, the more obvious the inhibition was; the inhibition was observed at 2 h, the effect was enhanced at 5 h, and disappeared at 24 h; _Bilirubin at high concentration range inhibited the expression of p-p38 MAPK stimulated by LPS. _MyD88 and p-p38 MAPK were negatively correlated with bilirubin concentration (r: - 0.856, - 0.791, and - 0.875, respectively) at 2h, 5h and 24h (P < 0.01). The expression of MyD88 in spleen cells was more obvious; _2 h p-p38 MAPK IOD (SUM) value had no correlation with bilirubin concentration (r = - 0.123, P > 0.05); 5 h p-p38 MAPK IOD (SUM) value was positively correlated with bilirubin concentration (r = 0.897, P < 0.01); 24 h p-p38 MAPK IOD (SUM) value was positively correlated with bilirubin concentration (r = 0.827, P < 0.01). With the increase of bilirubin concentration, the expression of p-p38 MAPK increased. _The effect of bilirubin on apoptosis of spleen cells: _Apoptosis rate of low concentration of bilirubin group increased (P < 0.01); _In low concentration of bilirubin combined with LPS can reduce the apoptosis of spleen cells (P < 0.01); in high concentration range of bilirubin and LPS can reduce the apoptosis of spleen cells (P < 0.01); CONCLUSION: Low concentration of bilirubin has no obvious effect on LPS-induced MyD88 expression, and high concentration of bilirubin inhibits MyD88 expression in a concentration-dependent manner. With the increase of concentration, the inhibitory effect increases and the duration prolongs. The phosphorylation of p38 MAPK was inhibited, and the phosphorylation of p38 MAPK was promoted at high concentration, and the action time was prolonged with the increase of concentration. _The toxic effect of high concentration bilirubin on spleen cells and the increase of apoptosis were observed. Apoptosis is related to cell apoptosis.
【學位授予單位】：瀘州醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R722.17

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