【摘要】：目的:1、通過觀察狼瘡鼠、脂多糖刺激狼瘡鼠及健康鼠腎組織細胞焦亡因子和調控基因NLRP3表達差異,探討細胞焦亡信號通路及調控因子與SLE炎癥反應的關系;2、通過觀察狼瘡鼠腎組織細胞焦亡因子、調控因子NLRP3在滋陰清熱藥作用下的表達變化,探討滋陰清熱藥對SLE炎癥相關細胞焦亡因子和調控因子NLRP3的作用,并以電鏡觀察腎小球損害情況,研究滋陰清熱藥對SLE腎組織的治療作用,為滋陰清熱藥控制SLE炎癥反應研究提供思路及方法。方法:1、SPF級B6.MRL-Faslpr/Jnju雌性狼瘡鼠20只,SPF級C57BL/6JN雌性健康鼠10只,周齡均為8-10周;按體重及生長狀況把模型鼠隨機分為:(1)脂多糖組(L組)(n=10):脂多糖按8mg·kg-1劑量腹腔注射,兩天一次,干預6周。(2)模型組(M組)(n=10):與脂多糖等容積生理鹽水腹腔注射,兩天一次,干預6周。健康鼠為(3)空白組(C組)(n=10):與脂多糖等容積生理鹽水腹腔注射,兩天一次,干預6周。用藥時觀察記錄各小鼠體重、活動、體貌等變化情況;6周后取腎組織應用免疫組化、及熒光定量PCR檢測各組腎組織細胞焦亡因子Caspase-1、IL-1β和調控因子NLRP3表達差異。2、SPF級B6.MRL-Faslpr/Jnju雌性狼瘡鼠20只,SPF級C57BL/6JN雌性健康鼠10只,周齡均為8-10周;按體重及生長狀況把模型鼠隨機分為:(1)中藥組(T組)(n=10):滋陰清熱藥按20 g·kg-1·d-1劑量灌胃(人等效劑量的2倍),干預6周。(2)模型組(M組)(n=10):每天予滋陰清熱藥等容積灌胃無菌蒸餾水,干預6周。健康鼠為(3)空白組(C組)(n=10):每天予滋陰清熱藥等容積灌胃無菌蒸餾水,干預6周。用藥時觀察記錄各組小鼠體重、活動、體貌等變化情況。6周后取腎組織電鏡觀察腎小球病理情況,免疫組化及熒光定量PCR檢測各組腎組織細胞焦亡因子Caspase-1、IL-1β和調控因子NLRP3表達差異,及足細胞靶抗原Nephrin表達變化。結果:1、免疫組化結果顯示LPS組和模型組Caspase-1和IL-1β表達陽性位點比空白組多,染色更深;LPS組后陽性位點比模型組多,黃染更明顯;平均光密度值比較,模型組Caspase-1、l L-1β表達比空白組高,差異有統(tǒng)計學意義(p0.05);LPS組比模型組高(p0.05),而對比于空白組更明顯(p0.01);熒光定量PCR結果顯示模型組Caspase-1、lL-1β和調控因子NLRP3腎皮質mRNA表達量比空白組高,差異有統(tǒng)計學意義(p0.05),而LPS組表達比模型組高,差異有統(tǒng)計意義(p0.05)。2、滋陰清熱藥治療后,中藥組IL-1β免疫組化染色明顯比模型組淺,陽性位點有所減少;平均光密度值和熒光定量PCRmRNA表達結果均顯示中藥組IL-1β比模型組低,差異有統(tǒng)計學意義(p0.05),中藥組Caspase-1染色和模型組差異不大,平均光密度值和mRNA表達和模型組數(shù)據(jù)差別無統(tǒng)計學意義(p0.05),但中藥組數(shù)值比模型組低,有降低趨勢;熒光定量PCR結果顯示中藥組NLRP3mRNA表達量數(shù)值比模型組低,但差異無統(tǒng)計意義(p0.05);免疫組化結果顯示中藥組Nephrin表達區(qū)域分布較模型組增強,但仍比空白組少,平均光密度值中藥組比模型組高(P0.05)。電鏡顯示中藥組可改善腎皮質區(qū)內腎小球和腎小管變性,足細胞密度結果顯示中藥組比模型組升高(P0.05),但中藥組和模型組與空白組對比均減少(P0.05)。與空白相比,模型組和中藥組足突寬度明顯增加(P0.01),而中藥組較模型組縮小(P0.05)。結論:1、細胞焦亡因子及調控因子NLRP3可能是SLE炎癥反應產(chǎn)生的重要途徑。2、滋陰清熱藥可降低細胞焦亡因子及調控因子NLRP3表達,同時能減輕腎臟足細胞損傷及足細胞裂孔膜相關分子Nephrin蛋白表達降低,這可能是滋陰清熱藥控制SLE炎癥反應的重要途徑。
[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.
【學位授予單位】：廣州醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R259

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