细胞焦亡在SLE炎症反应中作用及滋阴清热药影响研究

发布时间：2018-09-02 09:15

【摘要】：目的:1、通过观察狼疮鼠、脂多糖刺激狼疮鼠及健康鼠肾组织细胞焦亡因子和调控基因NLRP3表达差异,探讨细胞焦亡信号通路及调控因子与SLE炎症反应的关系;2、通过观察狼疮鼠肾组织细胞焦亡因子、调控因子NLRP3在滋阴清热药作用下的表达变化,探讨滋阴清热药对SLE炎症相关细胞焦亡因子和调控因子NLRP3的作用,并以电镜观察肾小球损害情况,研究滋阴清热药对SLE肾组织的治疗作用,为滋阴清热药控制SLE炎症反应研究提供思路及方法。方法:1、SPF级B6.MRL-Faslpr/Jnju雌性狼疮鼠20只,SPF级C57BL/6JN雌性健康鼠10只,周龄均为8-10周;按体重及生长状况把模型鼠随机分为:(1)脂多糖组(L组)(n=10):脂多糖按8mg·kg-1剂量腹腔注射,两天一次,干预6周。(2)模型组(M组)(n=10):与脂多糖等容积生理盐水腹腔注射,两天一次,干预6周。健康鼠为(3)空白组(C组)(n=10):与脂多糖等容积生理盐水腹腔注射,两天一次,干预6周。用药时观察记录各小鼠体重、活动、体貌等变化情况;6周后取肾组织应用免疫组化、及荧光定量PCR检测各组肾组织细胞焦亡因子Caspase-1、IL-1β和调控因子NLRP3表达差异。2、SPF级B6.MRL-Faslpr/Jnju雌性狼疮鼠20只,SPF级C57BL/6JN雌性健康鼠10只,周龄均为8-10周;按体重及生长状况把模型鼠随机分为:(1)中药组(T组)(n=10):滋阴清热药按20 g·kg-1·d-1剂量灌胃(人等效剂量的2倍),干预6周。(2)模型组(M组)(n=10):每天予滋阴清热药等容积灌胃无菌蒸馏水,干预6周。健康鼠为(3)空白组(C组)(n=10):每天予滋阴清热药等容积灌胃无菌蒸馏水,干预6周。用药时观察记录各组小鼠体重、活动、体貌等变化情况。6周后取肾组织电镜观察肾小球病理情况,免疫组化及荧光定量PCR检测各组肾组织细胞焦亡因子Caspase-1、IL-1β和调控因子NLRP3表达差异,及足细胞靶抗原Nephrin表达变化。结果:1、免疫组化结果显示LPS组和模型组Caspase-1和IL-1β表达阳性位点比空白组多,染色更深;LPS组后阳性位点比模型组多,黄染更明显;平均光密度值比较,模型组Caspase-1、l L-1β表达比空白组高,差异有统计学意义(p0.05);LPS组比模型组高(p0.05),而对比于空白组更明显(p0.01);荧光定量PCR结果显示模型组Caspase-1、lL-1β和调控因子NLRP3肾皮质mRNA表达量比空白组高,差异有统计学意义(p0.05),而LPS组表达比模型组高,差异有统计意义(p0.05)。2、滋阴清热药治疗后,中药组IL-1β免疫组化染色明显比模型组浅,阳性位点有所减少;平均光密度值和荧光定量PCRmRNA表达结果均显示中药组IL-1β比模型组低,差异有统计学意义(p0.05),中药组Caspase-1染色和模型组差异不大,平均光密度值和mRNA表达和模型组数据差别无统计学意义(p0.05),但中药组数值比模型组低,有降低趋势;荧光定量PCR结果显示中药组NLRP3mRNA表达量数值比模型组低,但差异无统计意义(p0.05);免疫组化结果显示中药组Nephrin表达区域分布较模型组增强,但仍比空白组少,平均光密度值中药组比模型组高(P0.05)。电镜显示中药组可改善肾皮质区内肾小球和肾小管变性,足细胞密度结果显示中药组比模型组升高(P0.05),但中药组和模型组与空白组对比均减少(P0.05)。与空白相比,模型组和中药组足突宽度明显增加(P0.01),而中药组较模型组缩小(P0.05)。结论:1、细胞焦亡因子及调控因子NLRP3可能是SLE炎症反应产生的重要途径。2、滋阴清热药可降低细胞焦亡因子及调控因子NLRP3表达,同时能减轻肾脏足细胞损伤及足细胞裂孔膜相关分子Nephrin蛋白表达降低,这可能是滋阴清热药控制SLE炎症反应的重要途径。
[Abstract]:Objective: 1. To investigate the relationship between the expression of NLRP3 and the inflammatory response of SLE in lupus mice, lupus mice and healthy mice by observing the difference of expression of NLRP3 and cytokines in lupus mice and healthy mice kidney tissues stimulated by lipopolysaccharides. To explore the effect of Ziyin Qingre on inflammatory related cytokines and regulatory factor NLRP3 in SLE, and to observe the damage of glomerulus by electron microscope, and to study the therapeutic effect of Ziyin Qingre on renal tissue of SLE. 20 lupus mice and 10 SPF grade C57BL/6JN female healthy mice aged 8-10 weeks were randomly divided into two groups according to body weight and growth status: (1) lipopolysaccharide group (L group) (n=10): lipopolysaccharide was injected intraperitoneally at a dose of 8 mg 65 Pre-6 weeks. Healthy mice were divided into (3) blank group (group C) (n=10): intraperitoneal injection with normal saline of the same volume as lipopolysaccharide, once every two days, intervention for 6 weeks. There were 20 SPF grade B6. MRL - Faslpr / Jnju female lupus mice and 10 SPF grade C57BL / 6JN female healthy mice aged from 8 to 10 weeks. Group (n = 10): the rats were given the same volume of sterile distilled water every day for 6 weeks. the healthy rats were (3) blank group (group C) (n = 10). the rats were given the same volume of sterile distilled water every day for 6 weeks. the changes of weight, activity and body appearance of each group were observed and recorded. Results: 1. Immunohistochemistry showed that there were more positive sites of Caspase-1 and IL-1 beta in LPS group and model group than in blank group, and the positive sites were deeper in LPS group. Compared with the blank group, the expression of Caspase-1, L-1 beta in the model group was higher than that in the model group (p0.05); the expression of Caspase-1, L-1 beta in the LPS group was higher than that in the model group (p0.05), but more obvious than that in the blank group (p0.01); the expression of Caspase-1, L-1 beta and regulatory factor NLRP3 mRNA in the renal cortex of the model group was detected by fluorescence quantitative PCR. Compared with the blank group, the expression of IL-1beta in LPS group was higher than that in model group, and the difference was statistically significant (p0.05). There was no significant difference in Caspase-1 staining and model group. There was no significant difference in average optical density and mRNA expression between model group and traditional Chinese medicine group (p0.05), but the value of traditional Chinese medicine group was lower than that of model group, and the expression of NLRP3 mRNA in traditional Chinese medicine group was lower than that of model group. The results of immunohistochemistry showed that the expression of Nephrin in TCM group was stronger than that in model group, but still less than that in blank group. The average optical density of TCM group was higher than that in model group (P 0.05). Compared with the blank group, the width of the foot process in the model group and the traditional Chinese medicine group increased significantly (P 0.01), while the width of the foot process in the traditional Chinese medicine group decreased (P 0.05). It can reduce the expression of NLRP3 and apoptosis factor, and reduce the injury of renal podocytes and the expression of Nephrin, a podocyte hiatus membrane-related molecule. This may be an important way for Ziyin Qingre Drug to control SLE inflammation.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R259

【参考文献】