本文關(guān)鍵詞： 腎小管上皮細(xì)胞 組蛋白 烏司他丁 線粒體 凋亡 自噬　出處：《南方醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景膿毒癥急性腎損傷是危重病人常見(jiàn)的臨床并發(fā)癥之一,可作為一項(xiàng)獨(dú)立危險(xiǎn)因素影響患者預(yù)后,給患者及社會(huì)帶來(lái)巨大負(fù)擔(dān)。膿毒癥AKI病理生理學(xué)發(fā)病機(jī)制復(fù)雜,其中越來(lái)越多的研究顯示:腎小管上皮細(xì)胞凋亡及細(xì)胞自噬功能異常在膿毒癥AKI發(fā)病過(guò)程中起重要作用。近來(lái),新的研究發(fā)現(xiàn):細(xì)胞外組蛋白作為損傷相關(guān)模式分子(damage-associated molecular patterns,DAMPs),是介導(dǎo)膿毒癥器官功能障礙的重要介質(zhì),包括膿毒癥AKI。我們前期工作發(fā)現(xiàn):組蛋白可以線粒體凋亡途徑誘導(dǎo)腎小管上皮細(xì)胞凋亡。那么改善組蛋白誘導(dǎo)的腎小管上皮細(xì)胞損傷也許是防治膿毒癥AKI的新思路。烏司他丁(Ulinastatin,UTI)是從尿液中分離純化而來(lái)的一種廣譜蛋白酶抑制劑,大量臨床研究證實(shí)烏司他丁對(duì)膿毒癥、急性肺損傷、重癥急性胰腺炎、部分肝切除術(shù)后肝功能、心肺旁路術(shù)后急性腎損傷、術(shù)后認(rèn)知功能障礙、多器官能障礙綜合征等具有良好療效,而具體機(jī)制有待進(jìn)一步研究。眾多研究顯示烏司他丁具有廣泛的生物學(xué)效應(yīng),如穩(wěn)定細(xì)胞溶酶體膜、抑制溶酶體酶的釋放、抑制炎癥瀑布樣反應(yīng)、清除氧自由基、保護(hù)線粒體等機(jī)制發(fā)揮器官保護(hù)作用。那么,烏司他丁對(duì)組蛋白誘導(dǎo)的腎小管上皮細(xì)胞損傷起保護(hù)作用,值得探究。研究目的1.明確烏司他丁(UTI)是否對(duì)組蛋白(CTH)誘導(dǎo)的腎小管上皮細(xì)胞損傷起保護(hù)作用;2.初步探討烏司他丁(UTI)減少組蛋白(CTH)誘導(dǎo)腎小管上皮細(xì)胞損傷的可能作用機(jī)制。研究方法1.用CCK-8法檢測(cè)不同濃度組蛋白及一定組蛋白濃度處理不同時(shí)間,UTI及CTH對(duì)HK-2細(xì)胞活性的影響。2.Annexin V FITC/PI雙染流式細(xì)胞儀檢測(cè)UTI對(duì)組蛋白誘導(dǎo)HK-2細(xì)胞凋亡的影響。3.Hoechst染色倒置熒光顯微鏡觀察細(xì)胞核形態(tài)學(xué)變化。5.Rhodamine123染色流式細(xì)胞儀檢測(cè)線粒體膜電位(MMP)變化。6.DCFH-DA染色流式細(xì)胞儀檢測(cè)活性氧(ROS)的產(chǎn)生。7.Western blot法測(cè)定Bcl-2、Cleaved Casepase-3、LC3Ⅱ、Beclin1 蛋白表達(dá)水平.研究結(jié)果1.CTH能明顯降低HK-2細(xì)胞活性,具有濃度、時(shí)間依賴性,CTH IC50為64.25ug/ml;不同濃度UTI組(500U/ml、1000U/ml、2000U/ml)對(duì)組蛋白誘導(dǎo)的HK-2細(xì)胞凋亡率均有保護(hù)作用,但組間比較差異無(wú)統(tǒng)計(jì)學(xué)意義;高濃度UTI(2000U)對(duì)HK-2細(xì)胞活性無(wú)明顯抑制作用;2.Annexin V FITC/PI雙染證實(shí)CTH能引起HK-2細(xì)胞發(fā)生凋亡,而UTI能減少CTH引起的HK-2細(xì)胞凋亡(p0.01)。3.Hoechst染色結(jié)果顯示:CTH能引起HK-2細(xì)胞發(fā)生凋亡,細(xì)胞核固縮濃染,致密濃染點(diǎn)明顯增多;而UTI能減少CTH引起的HK-2細(xì)胞凋亡,致密濃染點(diǎn)減少。4.Rhodamine123染色結(jié)果顯示:CTH能引起HK-2細(xì)胞線粒體膜電位下降,而UTI能減弱CTH引起的線粒體膜電位下降。5.DCFH-DA染色發(fā)現(xiàn):CTH能引起HK-2細(xì)胞活性氧(ROS)產(chǎn)生增多,而UTI能減弱CTH引起的活性氧生成。6.Western blot法測(cè)得:CTH能引起HK-2細(xì)胞Cleaved Caspase-3、LC3Ⅱ、Beclin1蛋白表達(dá)上升,Bcl-2蛋白表達(dá)下降(p0.05);而UTI能下調(diào)CTH引起的LC3Ⅱ、Beclin1、Cleaved Caspase-3蛋白表達(dá)上升、上調(diào)Bcl-2蛋白表達(dá)(p0.05);研究結(jié)論UTI對(duì)組蛋白誘導(dǎo)的HK-2細(xì)胞損傷起保護(hù)作用,其機(jī)制可能與維持線粒體膜電位、清除ROS、上調(diào)抗凋亡蛋白Bcl-2蛋白表達(dá)、抑制Caspase-3活化從而抑制細(xì)胞凋亡及調(diào)節(jié)自噬功能相關(guān)。
[Abstract]:The research background of sepsis induced acute kidney injury is one of the common clinical complications in critically ill patients, can be used as an independent risk factors influencing the prognosis of the patients, which brings great burden to patients and society. Sepsis AKI pathophysiological mechanism is complex, which more and more research has shown that apoptosis and cell function of autophagy in renal tubular epithelial cells may play an important role in sepsis pathogenesis of AKI. Recently, a new study found that extracellular histones as damage associated molecular patterns (damage-associated, molecular, patterns, DAMPs) is an important mediator of sepsis and organ dysfunction, including sepsis and AKI. our previous work found that histones can mitochondrial apoptosis pathways to induce apoptosis of renal tubular epithelial cells. Then improve the renal tubular epithelial cell injury induced by histone maybe is a new way of prevention and treatment of sepsis and AKI. Ulinastatin (Uli Nastatin, UTI) is a broad-spectrum protease inhibitor purified from the urine and a large number of clinical studies confirmed the effects of ulinastatin on sepsis, acute lung injury, severe acute pancreatitis, liver function after partial hepatectomy, acute kidney injury after cardiopulmonary bypass surgery, postoperative cognitive dysfunction and multiple organ disorder syndrome has good curative effect, but the specific mechanism needs further study. Many studies show that Ulinastatin has extensive biological effects, such as stable cell lysosomal membrane, inhibiting the release of lysosomal enzymes, inhibit the inflammatory cascade reaction, scavenging oxygen free radicals and protect the mitochondria function of organ protection. Then, ulinastatin the renal tubular epithelial cell injury induced by histone plays a protective role, it is worth exploring. The purpose of the study is to clear 1. ulinastatin (UTI) on protein group (CTH) of renal tubular epithelial cell injury induced by Protection; 2. to investigate the effects of ulinastatin (UTI) reduced histone (CTH) induced by the possible mechanism of renal tubular epithelial cell injury. Methods 1. by CCK-8 method under different concentrations of protein and histone concentrations detected in different time, an inverted fluorescent microscope. The morphological changes of the cell nucleus.5.Rhodamine123 staining flow cytometry. The mitochondrial membrane the potential effect of.3.Hoechst UTI cells and CTH on HK-2 cell activity on.2.Annexin V FITC/PI double staining flow cytometry on histone UTI induced apoptosis in HK-2 cells staining (MMP) changes of.6.DCFH-DA staining flow cytometry was used to detect the reactive oxygen species (ROS) generation.7.Western blot Cleaved method of determination of Bcl-2, Casepase-3, LC3 II, the expression of Beclin1 protein. Results 1.CTH significantly decreased the activity of HK-2 cells, with the concentration, the time dependence of CTH IC50 64.25ug/ ml; different concentration of UTI groups (50 0U/ml, 1000U/ml, 2000U/ml) rate has a significant protective effect on the apoptosis of HK-2 cells induced by the protein group, but no statistically significant difference between groups; high concentration of UTI (2000U) had no obvious inhibitory effect on the activity of HK-2 cells; 2.Annexin V FITC/PI staining confirmed that CTH can cause the occurrence of apoptosis of HK-2 cells, HK-2 cell apoptosis and UTI reduction caused by CTH (P0.01).3.Hoechst staining results showed that CTH can induce HK-2 cell apoptosis, karyopyknosis hyperchromatic, dense staining points increased significantly; HK-2 and UTI can reduce the apoptosis induced by CTH, dense stain decreased.4.Rhodamine123 staining showed that CTH can cause mitochondrial membrane potential of HK-2 cells decreased. The mitochondrial membrane potential and UTI can reduce the CTH induced decrease of.5.DCFH-DA staining showed that CTH could induce HK-2 cells reactive oxygen species (ROS) increased production of reactive oxygen species, and UTI may attenuate CTH induced a.6.Western blot method: CTH can induce the HK-2 cell Cleaved Caspase-3, LC3 II, Beclin1 protein expression increased, Bcl-2 expression decreased (P0.05); and UTI can down regulate the CTH induced by LC3 II, Beclin1, Cleaved increased the expression of Caspase-3 protein, up-regulated the expression of Bcl-2 protein (P0.05); to study the protective effects of UTI on HK-2 cells conclusion protein induced the injury group, the mechanism may be related to maintaining mitochondrial membrane potential and clearance of ROS, increase the expression of anti apoptosis protein Bcl-2 protein, inhibit the activation of Caspase-3 and inhibition of apoptosis and regulation of autophagy related functions.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R459.7

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