【作者】 戴文娟；

【导师】 刘卓拉；

【作者基本信息】 山西医科大学 ， 呼吸内科， 2011， 博士

【摘要】 研究目的:1建立机械性创伤大鼠模型;TNF-a、肾上腺素损伤大鼠模型;拉贝洛尔干预大鼠模型。2观察机械性创伤时肺组织中FoxA1 mRNA和蛋白质的表达改变及肺组织中肺泡Ⅱ型上皮细胞的凋亡。3观察TNF-a、肾上腺素对大鼠肺泡Ⅱ型上皮细胞FoxA1 mRNA、蛋白质表达的影响及大鼠肺泡Ⅱ型上皮细胞的凋亡。4研究TNF-a、肾上腺素对大鼠肺组织中FoxA1 mRNA、蛋白质表达的影响及大鼠肺泡Ⅱ型上皮细胞的凋亡。研究方法:1机械性创伤对大鼠肺组织FoxA1mRNA和蛋白及大鼠肺泡Ⅱ型上皮细胞凋亡的影响建立非致死性机械性创伤模型:麻醉大鼠后,将其置于Noble-Collip创伤仪(直径12英寸),以40 rpm的转速,旋转200圈,造成大鼠非致死性创伤。以ELISA法检测大鼠血清中TNF-a、肾上腺素含量;使用实时荧光定量PCR和免疫蛋白印迹检测创伤组和伪创伤组大鼠肺组织FoxA1 mRNA的表达的变化;对大鼠肺组织切片进行TUNEL和肺泡Ⅱ型上皮细胞特异性标志物SP-C染色检测大鼠肺组织中凋亡的肺泡Ⅱ型上皮细胞。2 TNF-a、肾上腺素体内实验对肺泡Ⅱ型上皮细胞的影响建立TNF-a、肾上腺素损伤离体细胞模型:100ng/mlTNF-a和2000ng/ml盐酸肾上腺素刺激大鼠肺泡Ⅱ型上皮细胞。运用荧光定量PCR、免疫蛋白印迹检测TNF-a、肾上腺素对大鼠肺泡Ⅱ型上皮细胞FoxA1 mRNA、蛋白质表达,同时运用流式细胞术检测大鼠肺泡Ⅱ型上皮细胞的凋亡。3 TNF-a、肾上腺素体内实验对肺泡Ⅱ型上皮细胞的影响建立TNF-a、肾上腺素损伤大鼠模型:向大鼠腹腔分别注射TNF-α(2.5ug/kg)、盐酸肾上腺素(1ml/kg);拉贝洛尔干预模型:在给大鼠灌胃盐酸拉贝洛尔后3h、6h、24h后再放入Noble-Collip创伤仪以40 r/min转动200圈造成机械性创伤。运用免疫蛋白印迹检测这三种模型中大鼠肺泡Ⅱ型上皮细胞FoxA1蛋白质表达,通过TUNEL和SP-C免疫组化染色对比,检测大鼠肺组织中肺泡Ⅱ型上皮细胞的凋亡,同时运用ELISA法检测三种大鼠模型中血清中TNF-a、肾上腺素含量。研究结果:1机械性创伤对大鼠肺组织FoxA1mRNA和蛋白及大鼠肺泡Ⅱ型上皮细胞凋亡的影响1.1机械性创伤对大鼠分泌肾上腺素和TNF-α的影响以ELISA法检测大鼠血清中TNF-a、肾上腺素含量,创伤后各时间点血清中肾上腺素和TNF-α含量逐渐上升,分别于创伤后6h和3h达高峰,峰值为(1.7560±0.1311)ng/ml和135.0898±1.5349(ng/l),差异有统计学意义(P<0.01)1.2机械性创伤致继发性肺损伤对FoxA1 mRNA和蛋白质表达的影响使用实时荧光定量PCR和免疫蛋白印迹检测创伤组和伪创伤组大鼠肺组织FoxA1 mRNA的表达的变化。结果显示创伤组FoxA1 mRNA表达较伪创伤组增高,FoxA1蛋白表达在创伤组均高于伪创伤组,于创伤后24h达到高峰(P<0.01)。1.3机械性创伤对大鼠肺泡Ⅱ型上皮细胞凋亡的影响TUNEL和肺泡Ⅱ型上皮细胞特异性标志物SP-C染色结果表明大鼠创伤后出现肺泡Ⅱ型上皮细胞凋亡。2 TNF-a、肾上腺素对离体大鼠肺泡Ⅱ型上皮细胞的影响2.1 TNF-a、肾上腺素对大鼠肺泡Ⅱ型上皮细胞FoxA1mRNA的表达影响使用实时荧光定量PCR和免疫蛋白印迹检测各组大鼠肺泡Ⅱ型上皮细胞FoxA1 mRNA和蛋白质的表达的变化。结果显示盐酸肾上腺素、TNF-a刺激后FoxA1mRNA和蛋白表达明显上升,24小时达到高峰。差异有统计学意义(P<0.01)。2.2 TNF-a、肾上腺素对肺泡Ⅱ型上皮细胞凋亡的影响采用流式细胞术检测了100ng/mlTNF-a和2000ng/ml盐酸肾上腺素刺激大鼠肺泡Ⅱ型上皮细胞后的凋亡率,结果表明,两组刺激12小时后大鼠肺泡Ⅱ型上皮细胞凋亡率显著上升,与对照组比较差异有统计学意义。3 TNF-a、肾上腺素体内实验对肺泡Ⅱ型上皮细胞的影响3.1 TNF-a、肾上腺素对肺泡Ⅱ型上皮细胞的影响使用实时荧光定量PCR和western blot检测各组FoxA1蛋白的表达情况,结果显示,FoxA1蛋白表达在TNF-a注射组、盐酸肾上腺素注射组均高于生理盐水对照组,于创伤后6h达到高峰,差异有统计学意义(P<0.01)以ELISA法检测大鼠血清中TNF-a、肾上腺素含量,结果显示,TNF-a注射组、盐酸肾上腺素注射组与生理盐水对照组相比含量明显增高,差异有统计学意义(p<0.01) TUNEL和肺泡Ⅱ型上皮细胞特异性标志物SP-C染色结果表明腹腔注射TNF-a、盐酸肾上腺素出现肺泡Ⅱ型上皮细胞凋亡,并且于12小时到达高峰。3.2拉贝洛尔对大鼠肺泡Ⅱ型上皮细胞的影响以western blot检测(β-actin为内参对照)各组中FoxA1蛋白的表达情况,FoxA1蛋白表达在盐酸拉贝洛尔灌胃组均低于创伤组,差异有统计学意义(P<0.01)。血清中肾上腺素含量的检测结果:拉贝洛尔干预组与创伤组相比含量明显降低,差异有统计学意义(p<0.01);血清中TNF-α含量的检测结果:拉贝洛尔干预组与创伤组相比含量显著明显降低,差异有统计学意义(p<0.01)。TUNEL和肺泡Ⅱ型上皮细胞特异性标志物SP-C染色结果表明拉贝洛尔干预组与创伤组相比肺泡Ⅱ型上皮细胞凋亡明显减少。研究结论:1机械性创伤使大鼠血清中TNF-a、肾上腺素水平显著升高并且大鼠肺组织中FoxA1表达上调,导致肺泡Ⅱ型上皮细胞凋亡。2 TNF-a、肾上腺素刺激的肺泡Ⅱ型上皮细胞中FoxA1表达上调。FoxA1在TNF-a、肾上腺素诱导的肺泡Ⅱ型上皮细胞凋亡中具有重要作用。3肾上腺素、TNF-a水平增高引起的肺损伤类似于机械性创伤所导致的继发性肺损伤,并且出现肺泡Ⅱ型上皮细胞凋亡。4拉贝洛尔可以通过抑制交感神经活性减少肾上腺素的释放,降低TNF-a的水平,减少肺泡Ⅱ型上皮细胞凋亡从而减轻机械性创伤所导致的继发性肺损伤。5机械性创伤可以引起机体TNF-a、肾上腺素水平升高,其中TNF-a、肾上腺素又有相互促进作用,加重肺损伤程度,使肺泡Ⅱ型上皮细胞凋亡。6拉贝洛尔对机械性创伤导致的继发性肺损伤有保护作用。更多还原

【Abstract】 Objects:1. Establishment of Non-lethal Mechanical trauma (MT) model and pulmonary injury model in rats. To establish labetalol intervention rats model using labetalol.2. Non-lethal MT model was used to detect mRNA and protein expression of FoxA1 in pulmonary tissues and typeⅡpenumonocyte apoptosis.3. Cell model in vitro was used to detect the effects of TNF-αand epinephrine on mRNA and protein expression of FoxA1 in pulmonary tissues and typeⅡpenumonocyte apoptosis.4. pulmonary injury model was used to detect detect the effects of TNF-αand epinephrine on mRNA and protein expression of FoxA1 in pulmonary tissues and typeⅡpenumonocyte apoptosis.Methods:1.The effects of MT on mRNA and protein expression of FoxA1 in pulmonary tissues and typeⅡpenumonocyte apoptosis.Establishment of Non-lethal Mechanical trauma (MT) model in rats. After anesthetized, rats were placed in a Noble-Collip drum (12-in.-diameter) and subjected to 200 revolutions at a rate of 40 rpm to induce a non-lethal MT. TNFαand epinephrine levels were observed by ELISA method. Expression of FoxA1 was detected by RT-PCR and western-blotting in MT group and control group. TUNEL and anti-SP-C staining were used to measure typeⅡpenumonocyte apoptpsis in rat lung tissue following mechanical trauma.2. The effects of TNFαand epinephrine on mRNA and protein expression of FoxA1 in typeⅡpenumonocyte in vitroEstablishment of pulmonary injury model in typeⅡpenumonocyte ,Expression of FoxA1 was detected by RT-PCR and western-blotting in groups. Apoptosis rate of typeⅡpenumonocyte after 100ng/mlTNF-αand 2000ng/ml epinephrine induction was detected by flow cytometry.3. The effects of TNFαand epinephrine on typeⅡpenumonocyte in vivoEstablishment of TNF-α, epinephrine injury rat model : Rats were injected intraperitoneally to TNF-α(2.5ug/kg), epinephrine hydrochloride (1ml/kg). To establish labetalol intervention rats model using labetalol. In the labetalol hydrochloride to rats after gavage 3h, 6h, 24h after the instrument into the Noble-Collip trauma to 40 r / min rotation caused by mechanical trauma of 200 laps.Expression of FoxA1 was detected by western-blotting in three groups. TNFαand epinephrine levels were observed by ELISA method. TUNEL and anti-SP-C staining were used to measure typeⅡpenumonocyte apoptpsis in rat lung tissue Results:1. The effects of MT on mRNA and protein expression of FoxA1 in pulmonary tissues and typeⅡpenumonocyte apoptosis.1.1 The effects of MT on secretion of TNF-αand epinephrine in rat’s serum.TNFαand epinephrine levels were observed by ELISA method. Compared with normal of that, TNFαand epinephrine levels were increased at each time point after MT(P<0.01), the highest level was happened at 6h and 8h after MT, (1.7560±0.1311)ng/ml and 135.0898±1.5349(ng/l), separately.1.2 The effects of MT Secondary pulmonary injury on mRNA and protein expression of FoxA1Expression of FoxA1 was detected by RT-PCR and western-blotting in MT group and control group. Compared with control group, mRNA expression of FoxA1 was increased; protein expression of FoxA1 was increased with highest level at 24h after MT(P<0.01).1.3 The effects of MT on typeⅡpenumonocyte apoptosisThe results of TUNEL and SP-C staining showed that apopotosis of typeⅡpenumonocyte increased.2. The effects of TNFαand epinephrine on mRNA and protein expression of FoxA1 in typeⅡpenumonocyte in vitro2.1 The effects of TNFαand epinephrine on mRNA expression of FoxA1 in typeⅡpenumonocyteExpression of FoxA1 was detected by RT-PCR and western-blotting in groups. mRNA and protein expression of FoxA1 was increased with highest level at 24h after MT after TNFαand epinephrine induction(P<0.01).2.1 The effects of TNFαand epinephrine on typeⅡpenumonocyte apoptosisApoptosis rate of typeⅡpenumonocyte after 100ng/mlTNF-αand 2000ng/ml epinephrine induction was detected by flow cytometry. Compared with control goup, apoptosis rate of typeⅡpenumonocyte increased 12h after induction.3. The effects of TNFαand epinephrine on typeⅡpenumonocyte in vivo 3.1 The effects of TNFαand epinephrine on typeⅡpenumonocyteExpression of FoxA1 was detected by western-blotting in groups. Compared with control goup, expression of FoxA1 in TNF-αgroup and epinephrine group increased with highest level at 24h after MT(P<0.01); TNF-αand epinephrine levels were observed by ELISA method. Compared with control group, TNFαand epinephrine levels were increased (P<0.01); The results of TUNEL and SP-C staining showed that apopotosis of typeⅡpenumonocyte increased after injection of TNFαand epinephrine, the highest apoptosis rate was happened 12h after injection.3.2 The effects of labetalol on typeⅡpenumonocyte Protein expression of FoxA1 was detected by western-blotting in groups. Compared with control goup, expression of FoxA1 decreased in labetalol groups(P<0.01); Compared with control goup, TNF-αand epinephrine levels were decreased in labetalol groups(P<0.01); The results of TUNEL and SP-C staining showed that apopotosis of typeⅡpenumonocyte decreased in labetalol groups compared with pulmonary injury groups.Conclusions:1. Mechanical trauma led to increase of TNF-α、epinephrine levels in rat’s serum and FoxA1 expression. Mechanical trauma led to apopotosis of typeⅡpenumonocyte.2. FoxA1 expression was increased in typeⅡpenumonocyte after induction of TNF-αand epinephrine. FoxA1 was play a critical role in apopotosis of typeⅡpenumonocyte induced by TNFαand epinephrine.3. Secondary pulmonary injury induced by MT was similar to pulmonary injury caused by excessive TNF-αand epinephrine level, and led to apopotosis of typeⅡpenumonocyte.4. Labetalol inhibited gangliated nerve activity, decreasing epinephrine and TNF-αsecretion, reducing apopotosis of typeⅡpenumonocyte, and lessening pulmonary injury.5. Secondary pulmonary injury induced by MT was related with excessive TNF-αand epinephrine level, Which TNF-a, have mutually reinforcing effects of epinephrine and increased lung injury, and led to apopotosis of typeⅡpenumonocyte.6. Labetalol has a protective effect on Secondary pulmonary injury induced by MT.更多还原