乌司他丁对组蛋白诱导肾小管上皮细胞（HK-2）损伤的保护作用及机制

发布时间：2018-02-01 07:30

本文关键词： 肾小管上皮细胞组蛋白乌司他丁线粒体凋亡自噬　出处：《南方医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景脓毒症急性肾损伤是危重病人常见的临床并发症之一,可作为一项独立危险因素影响患者预后,给患者及社会带来巨大负担。脓毒症AKI病理生理学发病机制复杂,其中越来越多的研究显示:肾小管上皮细胞凋亡及细胞自噬功能异常在脓毒症AKI发病过程中起重要作用。近来,新的研究发现:细胞外组蛋白作为损伤相关模式分子(damage-associated molecular patterns,DAMPs),是介导脓毒症器官功能障碍的重要介质,包括脓毒症AKI。我们前期工作发现:组蛋白可以线粒体凋亡途径诱导肾小管上皮细胞凋亡。那么改善组蛋白诱导的肾小管上皮细胞损伤也许是防治脓毒症AKI的新思路。乌司他丁(Ulinastatin,UTI)是从尿液中分离纯化而来的一种广谱蛋白酶抑制剂,大量临床研究证实乌司他丁对脓毒症、急性肺损伤、重症急性胰腺炎、部分肝切除术后肝功能、心肺旁路术后急性肾损伤、术后认知功能障碍、多器官能障碍综合征等具有良好疗效,而具体机制有待进一步研究。众多研究显示乌司他丁具有广泛的生物学效应,如稳定细胞溶酶体膜、抑制溶酶体酶的释放、抑制炎症瀑布样反应、清除氧自由基、保护线粒体等机制发挥器官保护作用。那么,乌司他丁对组蛋白诱导的肾小管上皮细胞损伤起保护作用,值得探究。研究目的1.明确乌司他丁(UTI)是否对组蛋白(CTH)诱导的肾小管上皮细胞损伤起保护作用;2.初步探讨乌司他丁(UTI)减少组蛋白(CTH)诱导肾小管上皮细胞损伤的可能作用机制。研究方法1.用CCK-8法检测不同浓度组蛋白及一定组蛋白浓度处理不同时间,UTI及CTH对HK-2细胞活性的影响。2.Annexin V FITC/PI双染流式细胞仪检测UTI对组蛋白诱导HK-2细胞凋亡的影响。3.Hoechst染色倒置荧光显微镜观察细胞核形态学变化。5.Rhodamine123染色流式细胞仪检测线粒体膜电位(MMP)变化。6.DCFH-DA染色流式细胞仪检测活性氧(ROS)的产生。7.Western blot法测定Bcl-2、Cleaved Casepase-3、LC3Ⅱ、Beclin1 蛋白表达水平.研究结果1.CTH能明显降低HK-2细胞活性,具有浓度、时间依赖性,CTH IC50为64.25ug/ml;不同浓度UTI组(500U/ml、1000U/ml、2000U/ml)对组蛋白诱导的HK-2细胞凋亡率均有保护作用,但组间比较差异无统计学意义;高浓度UTI(2000U)对HK-2细胞活性无明显抑制作用;2.Annexin V FITC/PI双染证实CTH能引起HK-2细胞发生凋亡,而UTI能减少CTH引起的HK-2细胞凋亡(p0.01)。3.Hoechst染色结果显示:CTH能引起HK-2细胞发生凋亡,细胞核固缩浓染,致密浓染点明显增多;而UTI能减少CTH引起的HK-2细胞凋亡,致密浓染点减少。4.Rhodamine123染色结果显示:CTH能引起HK-2细胞线粒体膜电位下降,而UTI能减弱CTH引起的线粒体膜电位下降。5.DCFH-DA染色发现:CTH能引起HK-2细胞活性氧(ROS)产生增多,而UTI能减弱CTH引起的活性氧生成。6.Western blot法测得:CTH能引起HK-2细胞Cleaved Caspase-3、LC3Ⅱ、Beclin1蛋白表达上升,Bcl-2蛋白表达下降(p0.05);而UTI能下调CTH引起的LC3Ⅱ、Beclin1、Cleaved Caspase-3蛋白表达上升、上调Bcl-2蛋白表达(p0.05);研究结论UTI对组蛋白诱导的HK-2细胞损伤起保护作用,其机制可能与维持线粒体膜电位、清除ROS、上调抗凋亡蛋白Bcl-2蛋白表达、抑制Caspase-3活化从而抑制细胞凋亡及调节自噬功能相关。
[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.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R459.7

【参考文献】