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前B细胞克隆增强因子在大鼠急性呼吸窘迫综合征中的作用研究

发布时间:2018-06-24 17:48

  本文选题:前B细胞克隆增强因子 + 急性呼吸窘迫综合征 ; 参考:《重庆医科大学》2013年硕士论文


【摘要】:背景:ARDS是ICU中的常见病和多发病,各种因素如肺部感染、脓毒血症、重症急性胰腺炎、创伤、输血等均可导致ARDS的发生,是机体过度炎症反应在肺部的表现。随着医学的不断发展,ARDS的发生率和死亡率较以前明显降低,,但形式仍不容乐观,因此,ARDS发病机制和治疗手段研究成为众多学者们关注的热点,现有研究也显示ARDS的发生发展机制十分复杂,涉及众多的信号通路及细胞因子。 前B细胞克隆增强因子(Pre-B-Cell Colony Enhancing Factor),又称为内脂素(visfatin),或烟酰胺磷酸核糖转移酶(Nampt),是近年来发现的主要由成熟脂肪细胞和激活的炎症细胞分泌,全身多个器官均可表达的炎症因子。目前有体外细胞实验研究发现炎症刺激可以使肺泡上皮细胞、肺微动脉内皮细胞等大量表达PBEF,而减少PBEF的表达则可减轻上述细胞由炎症刺激引起的相关损害,这表明PBEF可能涉及包括肺微血管损伤、肺泡上皮细胞损伤、通透性增加等在内有关ARDS的多个病理生理过程,同时也有学者通过对犬类、鼠类的不同ARDS模型进行研究后认为PBEF是ARDS的潜在生物标志,因此PBEF可能成为研究ARDS的发病机制和治疗ARDS的新靶点。 目的:以油酸尾静脉注射法建立大鼠ARDS模型,并行药物干预,观察前B细胞克隆增强因子对ARDS大鼠肺组织病理学评分、湿干重比及动脉血氧分压的影响;对ARDS大鼠肺泡灌洗液中TNF-α及IL-1β含量的影响;对ARDS大鼠肺组织TNF-α、IL-1β mRNA和ICAM-1、VCAM-1mRNA及蛋白表达以及NF-κB p65蛋白表达的影响;通过肺组织病理学评分、湿干重比、炎症因子及粘附因子表达及肺泡灌洗液中炎症因子含量的变化,结合已有的体外实验所观察到的现象,试图证实前B细胞克隆增强因子在ARDS中的作用,为进一步研究ARDS的发病机制和病理生理过程以及治疗措施提供新的思路和依据。 方法:40只成年健康雄性SD大鼠,体质量(214±26)g,按随机数字法分为空白对照组(C组)、模型组(OA组)、药物干预组(D组)、溶媒对照组(S组),OA组、D组及S组大鼠以油酸尾静脉注射法复制ARDS模型,剂量为0.15ml/kg,D组及S组大鼠于造模前24小时、12小时及半小时腹腔内注射药物,D组以10mg/kg腹腔内注射PBEF抑制剂FK866,S组注射FK866等体积溶媒二甲亚砜。通过观察大鼠呼吸频率、皮肤及口唇颜色和活动能力等初步判定造模是否成功。造模成功6小时后以3.5%水合氯醛以10ml/kg腹腔内注射麻醉,麻醉成功后先分离出气管,剖腹从腹主动脉处抽动脉血行血气分析检测氧分压(mmHg),开胸观察肺组织颜色及形态,结扎右肺门,行气管切开置入导管后行左肺肺泡灌洗术,并收集肺泡灌洗液,离心取上清液备用,测定炎症因子TNF-α、IL-1β含量(ng/L);收集完毕后取材,右肺上叶立即固定于福尔马林中,行病理学、免疫组化检测,右肺中叶行湿干重比检测,右肺下叶立即冻存于-80℃冰箱或者液氮中备用,一部分以RT-PCR及RT-qPCR方法检测炎症因子TNF-α、IL-1β及粘附因子ICAM-1、VCAM-1mRNA的表达,另一部分以Western Blot方法检测NF-κB p65蛋白的表达。 结果: 1.行为学及外表观察 C组大鼠一般情况好,较为活泼,无毛发竖立等现象,口唇及皮肤呈现粉红色,无呼吸困难;OA组、D组及S组大鼠造模后短时间内即出现口唇及皮肤紫绀,全身毛发竖立,活动减少,呼吸频率显著增加,与C组比较差异均有统计学意义(p0.001):OA组与S组呼吸频率最高,但两者比较差异没有统计学意义(p0.05),D组呼吸频率较OA组与S组低,差异有统计学意义(均p0.05),各组均无死亡。 2.动脉血氧分压 C组大鼠动脉血氧分压为(103.2±11.2)mmHg,与其他各组比较差异有统计学意义(均p0.001),D组动脉血氧分压为(73.8±6.8)mmHg,与OA组及S组比较差异有统计学意义(p0.05),OA组动脉血氧分压为(59.6±6.7)mmHg,S组动脉血氧分压为(59.8±11.0)mmHg,OA组及S组比较差异无统计学意义(p0.05)。 3.肺组织病理学及湿干重比 C组大鼠肺组织在肉眼下观察呈粉红色,未见明显充血、出血及肿胀,光镜下观察为正常肺组织结构;OA组大鼠肺组织在肉眼观察下呈暗红色、肿胀,可见明显出血、充血,光镜下可见肺组织为典型的ARDS病理学表现;D组大鼠肺组织肉眼观察仍可见肿胀及充血、出血,但程度较OA组轻,光镜下可见肺组织仍有与OA组相同的病理改变,但受损面积明显减小;S组大鼠肺组织肉眼观与显微镜观察结果与OA组类似。而C组湿干重比为4.174±0.311,与其他各组比较差异有统计学意义(均p0.001),D组湿干重比为5.855±0.200,与OA组及S组比较差异有统计学意义(p0.05),OA组湿干重比为6.527±0.239,S组湿干重比为6.664±0.324,OA组及S组比较差异无统计学意义(p0.05)。 4.免疫组化法检测肺组织PBEF、ICAM-1、VCAM-1蛋白相对表达量 免疫组化检测可在所有ARDS大鼠肺组织的支气管粘膜上皮、血管内皮、肺泡壁及肺泡水肿液中发现PBEF蛋白表达。与C组比较,OA组、S组PBEF、ICAM-1、VCAM-1蛋白相对表达量明显升高,差异有统计学意义(均p0.001),而D组各指标蛋白相对表达量较OA组及S组有所减少,但较C组高,差异有统计学意义(均p0.05)。 5.肺泡灌洗液中炎症因子TNF-α及IL-1β含量 C组大鼠炎症因子含量均较其他组低,两者分别与其他各组比较差异有统计学意义(p0.05或p0.001),OA组与S组BALF中炎症因子含量最多,但OA组及S组比较两者差异均无统计学意义(p0.05)。D组各炎症因子含量较OA组及S组低,差异均有统计学意义(p0.05) 6.RT-PCR法半定量及RT-qPCR法定量检测肺组织PBEF、TNF-α、IL-1β、ICAM-1及VCAM-1mRNA表达水平 RT-PCR法半定量检测各指标结果显示,C组各指标mRNA相对表达量最低,较其他各组差异有统计学意义(p0.001或p0.05),D组各指标mRNA相对表达量较OA组及S组低,差异具有统计学意义(p0.05或p0.001),OA组及S组各指标mRNA相对表达量最高,但两者相比差异没有统计学意义(p0.05),RT-qPCR法检测结果与RT-PCR法检测结果所得出结果类似。 7.Western Blot法半定量检测肺组织中NF-κB p65蛋白表达水平 Western Blot法半定量检测各指标结果显示,C组NF-κB p65相对表达量最低,较其他各组差异有统计学意义(均p0.001),D组相对表达量较OA组及S组低,差异具有统计学意义(p0.05或p0.001),OA组及S组相对表达量最高,但两者相比差异没有统计学意义(p0.05)。 结论: 1、PBEF能促进ARDS大鼠肺组织炎症因子TNF-α及IL-1β的表达和生成; 2、PBEF能促进ARDS大鼠肺组织粘附分子ICAM-1及VCAM-1的表达; 3、PBEF能促进ARDS大鼠肺组织NF-κB的激活和表达。 因此, PBEF可能通过激活炎症反应,促进炎症因子和粘附分子表达等途径造成肺组织的炎性损伤以及炎性细胞的浸润和迁移等,在ARDS发生发展中起重要作用。
[Abstract]:Background : ARDS is common disease and multiple diseases in ICU . Various factors such as pulmonary infection , sepsis , severe acute pancreatitis , trauma , transfusion , etc . can lead to ARDS . With the development of medicine , the incidence and mortality rate of ARDS is much lower than before , but the form is still not optimistic . Therefore , the pathogenesis and treatment of ARDS have become the focus of many scholars , and the existing research also shows that the development mechanism of ARDS is very complex , involving many signal pathways and cytokines .

It is suggested that PBEF may be a potential biomarker for ARDS , and PBEF may be a potential biomarker for ARDS , and PBEF may be a new target for the study of ARDS and ARDS .

Objective : To establish a rat ARDS model by intravenous injection of oleic acid tail vein , and observe the effect of pre - B cell clone enhancement factor on pathological score , wet dry weight ratio and arterial oxygen partial pressure in ARDS rats .
Effect of TNF - 伪 and IL - 1尾 in alveolar lavage fluid of ARDS rats
The effects of TNF - 伪 , IL - 1尾 mRNA and ICAM - 1 , VCAM - 1 mRNA and protein expression and the expression of NF - 魏B in the lung tissues of ARDS rats were studied .
In order to further study the role of preB cell clone enhancement factor in ARDS and to provide a new idea and basis for further research on the pathogenesis and pathological process of ARDS and the treatment measures , the role of the former B cell clone enhancement factor in ARDS was tried to confirm the role of the former B cell clone enhancement factor in ARDS .

Methods : Forty adult healthy male SD rats were randomly divided into two groups : blank control group ( group C ) , model group ( OA group ) , drug intervention group ( group D ) , vehicle control group ( S group ) , OA group , group D and S group .
The expression of TNF - 伪 , IL - 1尾 and ICAM - 1 and VCAM - 1 mRNA were detected by RT - PCR and RT - qPCR .

Results :

1 . Behavior and appearance observation

In group C , the general condition of the rats was good , more active , no hair was erected , the lip and skin were pink , and there was no difficulty in breathing ;
There was significant difference between OA group and group S group ( p < 0.05 ) . The difference between OA group and group S was significant ( p < 0.05 ) .

2 . Arterial blood oxygen partial pressure

The arterial oxygen partial pressure in group C was ( 103.2 卤 11.2 ) mmHg , the difference was statistically significant compared with other groups ( p < 0.05 ) . The arterial oxygen partial pressure in OA group was ( 59.6 卤 6.7 ) mmHg , and the arterial oxygen partial pressure in group S was ( 59.8 卤 11.0 ) mmHg , and there was no significant difference between OA group and S group ( p < 0.05 ) .

3 . Lung tissue pathology and wet dry weight ratio

In group C , the lung tissues were pink without obvious congestion , hemorrhage and swelling , and the normal lung tissue structure was observed under the light microscope .
The lung tissues of OA group showed dark red , swelling , obvious hemorrhage , congestion , and lung tissue was typical ARDS pathological manifestation under light microscope .
In group D , swelling and congestion and bleeding were observed in the lung tissues of the rats , but the degree of bleeding was lower than that in OA group , but the lung tissue still had the same pathological changes as OA group , but the damaged area was obviously reduced ;
Compared with OA group , the wet dry weight ratio of group C was 5.855 卤 0.200 , and the wet dry weight ratio in group D was 6.527 卤 0.239 , and the wet dry weight ratio in group S was 6.664 卤 0.324 , and there was no significant difference between the OA group and S group ( p < 0.05 ) .

4 . The expression of PBEF , ICAM - 1 and VCAM - 1 in lung tissues was detected by immunohistochemistry .

Compared with group C , the relative expression of PBEF , ICAM - 1 and VCAM - 1 in OA group and S group was significantly higher than that of OA group and S group ( P < 0.01 ) .

5 . TNF - 伪 and IL - 1尾 in alveolar lavage fluid

In group C , the inflammatory factors were lower in group C than in other groups ( p < 0.05 or p < 0.001 ) . There was no statistical difference between OA group and S group ( p < 0.05 ) . The inflammatory factors in group D were lower than those in group OA and S group ( p < 0.05 ) .

6.RT - PCR and RT - qPCR were used to detect the levels of PBEF , TNF - 伪 , IL - 1尾 , ICAM - 1 and VCAM - 1 mRNA in lung tissues .

RT-PCR娉曞崐瀹氶噺妫

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