Kallistatin对脓毒症大鼠血清炎症介质及肺组织细胞凋亡的作用

发布时间：2018-03-26 00:43

本文选题：人激肽释放酶结合蛋白　切入点：细胞凋亡　出处：《南华大学》2015年硕士论文

【摘要】：目的:探讨人激肽释放酶结合蛋白(Kallistatin,KS)重组体腺病毒(Ad-KS)对脓毒症大鼠血清炎症介质及肺组织细胞凋亡的作用。方法:1、清洁级健康雄性SD大鼠32只,体重280±20g,随机分成4组,分别为假手术组(SC组)、脓毒症组(SEP组)、Ad-KS处理组(KS组)、空腺病毒(Ad-Null)对照组(AC组)。盲肠穿孔结扎术(cecal ligation and puncture,CLP)制备大鼠脓毒症模型。2、KS组大鼠CLP术前48h经大鼠尾静脉注射Ad-KS进行基因转染,使大鼠体内表达人KS蛋白。AC组注射Ad-Null腺病毒作为对照。3、所有存活大鼠术后24h经腹主动脉采血行血气分析,经心脏采血行血常规、血生化,留取血清备用,放血法处死大鼠,快速取肺组织。4、苏木精-伊红染色(HE)观察肺组织病理形态改变。ELISA法检测血清炎症介质TNF-α、IL-6及IL-10水平,Western Blot法检测肺组织Caspase-3、Bax及Bcl-2的表达。TUNEL检测观察肺组织细胞凋亡情况。结果:1、肺组织常规病理切片及HE染色发现,SC组肺泡结构完整,间质未见增厚水肿,无明显中性粒细胞及红细胞浸润。SEP组、AC组肺泡萎陷,肺泡腔缩小,肺间质增厚、充血水肿,间质内大量中性粒细胞、红细胞浸润,部分可见肺实变。KS组与SEP组、AC组比较,肺泡萎陷程度减轻,间质充血水肿及浸润减轻。2、ELISA检测血清炎症介质水平,SEP组、AC组血清促炎因子TNF-α、IL-6较SC组升高(P0.05),但抗炎因子IL-10无明显升高。KS组血清TNF-α、IL-6较SEP组、AC组下降,抗炎因子IL-10升高(P0.05)。3、Western blot检测肺组织凋亡相关蛋白表达,SEP组、AC组肺组织Caspase-3表达较SC组明显增加(P0.05)。KS组肺组织Caspase-3表达较SEP组、AC组减少。促凋亡蛋白Bax及抗凋亡蛋白Bcl-2表达在SC组、SEP组、AC组间无明显差异。KS组其Bax表达较其他三组减少,Bcl-2表达增加(P0.05)。4、TUNEL观察肺组织细胞凋亡情况,SEP组、AC组较SC组肺组织细胞凋亡增加,KS组与SEP组、AC组相比凋亡减少。SEP组与AC组间无明显差异。结论:1、Kallistatin重组体腺病毒降低脓毒症大鼠血清TNF-α、IL-6水平、升高IL-10水平,发挥抗炎作用。2、Kallistatin重组体腺病毒抑制脓毒症大鼠肺组织细胞凋亡及Caspase-3的表达,减轻肺损伤。
[Abstract]:Objective: to investigate the effects of human kallikrein binding protein (Kallistatin- KS) recombinant adenovirus (Ad-KS) on inflammatory mediators in serum and apoptosis of lung tissue in septic rats. Methods: thirty two healthy male SD rats of clean grade, weighing 280 卤20 g, were randomly divided into 4 groups. Sepsis model was established in sepsis rats 48 h before CLP in the sham operation group, the sepsis group and Ad-KS treatment group, Ad-Null group and AC group, respectively. Cecal ligation and puncture group was ligated by cecal perforation and ligation of caecum. The rat model of sepsis was induced by injection of CLP 48 hours before operation. Ad-KS gene transfection, The Ad-Null adenovirus was injected into the control group of rats expressing human KS protein in vivo. All the surviving rats were collected blood from abdominal aorta for blood gas analysis 24 hours after operation, and blood routine examination, blood biochemistry, blood biochemistry and serum collection were performed through the heart, and the rats were killed by bloodletting method. Lung tissue. 4 and hematoxylin-eosin staining.) to observe the pathological changes of lung tissue. Elisa to detect the level of serum inflammatory mediators TNF- 伪, IL-6 and IL-10. Western Blot method to detect the expression of Caspase-3, Bax and Bcl-2 in lung tissue. Tunel to detect the apoptosis of lung tissue cells. Results 1. Routine pathological sections of lung tissue and HE staining showed that the alveolar structure of SC group was intact. There was no thickening edema, no obvious neutrophil and erythrocyte infiltration. In the SEP group, alveolar collapse, alveolar lumen shrinkage, interstitial thickening, hyperemia edema, neutrophil infiltration and erythrocyte infiltration were observed in the AC group. Compared with the AC group of SEP group, the degree of alveolar collapse was reduced in KS group and SEP group. The level of serum inflammatory mediators in SEP group was higher than that in SC group (P 0.05), but the serum TNF- 伪 IL-6 level in KS group was lower than that in SEP group. Expression of apoptosis-related protein in lung tissue Caspase-3 expression in the AC group was significantly higher than that in the SC group. The expression of Caspase-3 in the lung tissue of the P0.05 + KS group was significantly lower than that in the SEP group, and the expression of Bax and Bcl-2 in the lung tissue was significantly lower than that in the control group (P < 0.05). The expression of apoptosis-related protein (Bax) and anti-apoptotic protein (Bcl-2) in the lung tissue were detected by Western blot. There was no significant difference between the two groups. The expression of Bax in KS group was lower than that in the other three groups. The increase of Bcl-2 expression was observed by Tunel. The apoptosis of lung tissue was observed by Tunel. The apoptosis of lung tissue in AC group was higher than that in SC group. The apoptosis of KS group was higher than that of SEP group. There was no significant difference between SEP group and AC group. Conclusion the recombinant adenovirus of Kallistatin can reduce the level of TNF- 伪 and IL-6 in serum of septic rats. To increase the level of IL-10 and to play an anti-inflammatory effect. 2. The recombinant adenovirus of Kallistatin inhibits the apoptosis of lung tissue and the expression of Caspase-3 in septic rats, and alleviates lung injury.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R459.7

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