本文選題：膿毒癥急性腎損傷 + 糖皮質激素　； 參考：《昆明醫(yī)科大學》2017年碩士論文

【摘要】：[目的]膿毒癥急性腎損傷(septic acute kidney injury,SAKI)是重癥監(jiān)護室中常見的一種危重疾病,其發(fā)病率及死亡率居高不下,但發(fā)病機制尚未明確。其中,細胞凋亡學說在膿毒癥急性腎損傷中的作用越來越受到重視,多項臨床及動物實驗顯示,膿毒癥發(fā)生時,機體處于相對腎上腺皮質功能不全(relative adrenal insufficiency,RAI)的狀態(tài),小劑量糖皮質激素可以保護SAKI中的腎功能。本實驗擬構建SAKI小鼠模型,采用Ac-DEVD-CHO(Caspase-3特異性抑制劑)聯合小劑量甲強龍早期干預,通過ELISA檢測小鼠血清中炎癥因子TNF-α、急性腎損傷標志物NGAL(中性粒細胞明膠酶相關載脂蛋白,載脂蛋白-2)以及Western Blot檢測腎臟凋亡相關蛋白Bax、Bcl-2表達,比較二者單獨干預與聯合干預措施對SAKI小鼠模型的腎保護作用及機制,為臨床治療SAKI提供新的治療靶點和理論依據。[方法]清潔級雄性C57BL/6小鼠72只,采用盲腸結扎穿孔(Cecal Ligation and Puncture,CLP)法,構建SAKI模型,隨機分為6組(n=12),①假手術對照組(sham group);②盲腸結扎穿孔組(CLP group);③DMSO對照組(DMSO group);④小劑量甲潑尼龍早期干預組(GC group);⑤Caspase-3抑制劑Ac-DEVD-CHO早期干預組(CHO group);⑥Caspase-3抑制劑Ac-DEVD-CHO聯合小劑量甲強龍組(CHO+GC group)。各組術后都給予37℃生理鹽水0.3ml/10g股部皮下注射行液體復蘇。六組小鼠分別于術后6h、12h、24h通過摘眼球法取血,使用ELISA方法測定血清中的TNF-α、NGAL濃度;同時各組使用磷酸鹽緩沖液灌注后取出小鼠腎臟,使用Western-Blot方法測定腎臟中凋亡蛋白Bax、Bcl-2的表達。ELISA結果分析使用酶標儀在450nm波長下測量各孔的光密度(optical density,OD)值,Western-Blot條帶圖像灰度值測量分析采用Gel-pro軟件包分析。數據統計分析采用SPSS20.0版軟件包(SPSS Company,Chicago,IllinoisUSA)進行;數據均以均數土標準差(x±s)表示,統計方法采用重復測量資料的方差分析,P≤0.05為差異有統計學意義。[結果]1.各組小鼠血清TNF-α及血清NGAL結果:6h、12h、24h小鼠CLP組和DMSO對照組血清TNF-α及血清NGAL表達無明顯差異(P0.05)。6hCLP組、CHO組TNF-α較Sham組有明顯升高(P0.01);GC組較CLP組TNF-α有明顯降低(P0.01),GC+CHO組較GC組有降低趨勢,較Sham組有升高趨勢,但無統計學差異(PO.05);12h、24hSham組血清TNF-α較6h組有升高趨勢,但無統計學差異(P0.05);12hCLP組較6hCLP組血清TNF-α有明顯升高,24hCLP組較12h CLP組血清TNF-α有明顯升高(P0.01);12hGC組較12hCLP組有明顯降低(P0.01);12hGC+CHO組較12hGC組有明顯降低(P0.01);12hGC組和GC+CHO組較6h組有升高趨勢,但無統計學差異(P0.05);24hGC組較24hCLP組有明顯降低(P0.01);24hGC+CHO組較24h GC組有明顯降低(P0.01);24hGC組和GC+CHO組較12h組有升高趨勢,但無統計學差異(P0.05)。12hCLP組較6hCLP組血清NGAL有明顯升高(P0.01),24hCLP組較12hCLP組血清NGAL有明顯降低(P0.01),與6hCLP手術組無統計學差異(P0.05),各個時間組CLP組較Sham組NGAL都有明顯升高(P0.01),GC組較CLP組血清NGAL有降低趨勢,但無統計學差異(P0.05);CHO組較CLP組血清NGAL有明顯降低(P0.01),GC+CHO組較CHO組有降低趨勢,但無統計學差異(P0.05)。2.各組小鼠腎組織凋亡相關蛋白Bax及Bcl-2表達結果:6h和12h的Bax/actin比值比較中,CLP組最高,GC+CHO組最低,其中CLP組與Sham組比較Bax表達升高有統計學意義(P0.05)。CHO組及GC+CHO組與CLP組比較Bax表達降低差異有統計學意義(P0.05)。GC+CHO組與DMSO組比較Bax表達降低有統計學意義(P0.05)。GC+CHO組較CHO組有降低趨勢,但差異無統計學意義(P0.05)。24h的Bax/β-actin比值比較中,Sham組最高,CLP組其次,GC+CHO組較CLP組升高有統計學意義(P0.05),CHO組較CLP組降低趨勢,其余各組間均無統計學差異(P0.05)。6h和12h的Bcl-2/β-actin比值比較中,GC+CHO組最高,CHO組其次,DMSO組最低。CHO組及GC+CHO組與DMSO組比Bcl-2表達增高均有統計學意義(P0.05);GC+CHO組較CHO組有升高趨勢,但差異無統計學意義(P0.05)。24h的Bcl-2/β-actin比值比較中,GC+CHO組最高,CHO組其次,CLP組最低。各組間均無統計學差異(P0.05),GC+CHO組較CHO組有升高趨勢,但差異無統計學意義(P0.05)。[結論]1.單獨使用小劑量甲強龍早期干預膿毒癥相關腎損傷小鼠模型,可以減少炎性因子TNF-α的釋放,減輕小鼠SAKI模型的腎損傷;2.單獨使用Caspase-3抑制劑Ac-DEVD-CHO早期干預小鼠膿毒癥急性腎損傷模型,可以減輕小鼠腎臟細胞凋亡,進而減輕小鼠SAKI模型的腎損傷;3.Caspase-3抑制劑Ac-DEVD-CHO和小劑量甲強龍聯合應用時,比單獨使用其中一種藥物能更有效的減少小鼠膿毒癥急性腎損傷模型腎臟細胞凋亡、炎癥因子TNF-α釋放,降低血清NGAL,更好的減輕小鼠SAKI模型的腎損傷。
[Abstract]:[Objective] sepsis acute kidney injury (septic acute kidney injury, SAKI) is a common disease in critically ill ICU, high morbidity and mortality, but the pathogenesis is still unclear. Among them, the theory of apoptosis in sepsis and acute kidney injury in the clinical display more attention. And a number of animal experiments, sepsis occurs when the body is in relative adrenal insufficiency (relative adrenal, insufficiency, RAI) of the state, a small dose of glucocorticoid can protect renal function in SAKI. This study intends to construct a mouse model of SAKI, with Ac-DEVD-CHO (Caspase-3 inhibitor) combined with low-dose methylprednisolone early intervention by inflammatory cytokines TNF- ELISA detection in serum alpha, NGAL markers of acute kidney injury (neutrophil gelatinase associated lipocalin, apolipoprotein -2) and Western Blot detection of renal The dirty of apoptosis related protein Bax, Bcl-2 expression, compared with the two individual intervention and combined interventions on a mouse model of SAKI renal protective effect and mechanism for the treatment of SAKI to provide therapeutic targets and theoretical basis. Methods: male C57BL/6 mice were new 72, cecal ligation perforation (Cecal Ligation and Puncture. CLP) method to construct the model of SAKI, were randomly divided into 6 groups (n=12), sham operation control group (sham group); the cecal ligation and puncture group (CLP group); the DMSO control group (DMSO group); the early small dose methylprednisolone intervention group (GC group) and Caspase-3 inhibitor Ac-DEVD-CHO; early intervention group (CHO group); the Caspase-3 inhibitor Ac-DEVD-CHO combined with low dose methylprednisolone group (CHO+GC group). Each group were given normal saline 0.3ml/10g 37 C femoral subcutaneous injection for fluid resuscitation. The small group of six rats respectively after 6h, 12h, 24h by eyeball method Blood serum levels of TNF- were determined using the method of ELISA alpha, NGAL concentration; at the same time the groups using phosphate buffer after reperfusion kidney was removed, the determination of apoptosis protein Bax in kidney using the Western-Blot method, the optical density using eliasa measurement of each hole in the 450nm wavelength analysis results the expression of.ELISA Bcl-2 (optical density OD) the value of Western-Blot band image gray value measurement and analysis using Gel-pro software package. The analysis using SPSS20.0 software package for statistical analysis of data (SPSS Company, Chicago, IllinoisUSA); data are presented as mean + standard deviation (x + s), variance analysis using statistical methods for repeated measurement data, P is less than or equal to 0.05 for the difference there was statistical significance. Results the serum]1. TNF- mice and serum NGAL results: 6h, 12h, 24h in CLP group and DMSO control group of serum TNF- and serum NGAL expression had no significant difference (P0.05) in.6hCLP group, CHO group, TNF- alpha 杈僑ham緇勬湁鏄庢樉鍗囬珮(P0.01);GC緇勮緝CLP緇凾NF-偽鏈夋槑鏄鵑檷浣，

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