致SLE患者贫血的红细胞凋亡损伤研究

发布时间：2018-05-25 18:14

本文选题：系统性红斑狼疮 + 红细胞凋亡　；参考：《安徽医科大学》2017年硕士论文

【摘要】：研究背景系统性红斑狼疮(SLE)患者随病情进展可出现不同程度的贫血症状,贫血率可高达70%以上,慢性病性贫血最常见。SLE患者因伴有自由基代谢障碍,活性氧类聚集从而形成机体过氧化状态。研究证实红细胞在氧化压力、能量供给不足、感染等因素作用下凋亡率增加。探讨红细胞凋亡损伤可能是对研究SLE患者贫血病因的科学补充。目的研究系统性红斑狼疮(SLE)贫血患者红细胞凋亡损伤,并探讨其可能的发生机制。方法本研究分别以SLE患者和健康献血员作为实验组和对照组,分为红细胞实验、血浆孵育红细胞实验和血浆抗氧化能力实验三部分。红细胞实验为研究两组对象红细胞凋亡损伤。分别收集20例两组对象的新鲜全血,测量血细胞参数。常规分离出红细胞,制成一定浓度的红细胞悬液。经Annexin V-FITC荧光染色,利用流式细胞仪检测细胞凋亡率及细胞体积大小,同时用激光共聚焦显微镜观察凋亡细胞的形态;经荧光探针Fluo-3/AM和DCFH-DA标记,利用流式细胞仪检测细胞内钙离子浓度和胞内活性氧类含量;荧光标记二抗-流式法检测胞内神经酰胺含量。分别比较两组各项指标差异。血浆孵育红细胞实验为研究SLE患者血浆对红细胞调亡损伤的影响,分别收集20例两组对象新鲜全血,分离出的血浆用以孵育健康人O型红细胞。24小时后用相同方法检测两组红细胞凋亡率及体积、胞内钙离子浓度、神经酰胺和活性氧类含量,比较两组差异。另外利用酶标仪测量比较两组血浆总抗氧化能力差异。结果实验组血红蛋白含量及血细胞比容均明显低于正常对照组,差异有统计学意义(P0.001);红细胞实验中,实验组细胞凋亡率、胞内钙离子浓度显著高于对照组,差异有统计学意义(P0.001);实验组胞内活性氧类含量明显高于对照组(P0.01);相比健康献血员,患者红细胞体积缩小,差异有统计学意义(P0.01);两组细胞胞内神经酰胺含量无明显差异;激光共聚焦荧光显微镜下可以观察到凋亡红细胞膜上的FITC荧光信号。血浆孵育红细胞实验中,实验组细胞凋亡率、胞内钙离子浓度显著高于对照组,差异有统计学意义(P0.001,P0.05);细胞大小、胞内神经酰胺含量以及活性氧类均无明显差异。两组血浆总抗氧化能力也无显著差异。结论红细胞凋亡增加是引起SLE患者贫血的原因之一。胞内钙离子浓度增加以及细胞自身活性氧类代谢障碍可能是红细胞凋亡的发生机制。
[Abstract]:Background: patients with systemic lupus erythematosus (SLEs) may develop anemia symptoms of varying degrees with the progression of the disease, the anemia rate can be as high as more than 70%. The most common chronic anemia. SLE patients with free radical metabolic disorders. Reactive oxygen species accumulate to form a state of peroxidation. The results showed that the apoptosis rate of erythrocytes increased under the action of oxidative pressure, insufficient energy supply and infection. To explore the damage of erythrocyte apoptosis may be a scientific supplement to study the etiology of anemia in patients with SLE. Objective to study the damage of erythrocyte apoptosis in anemia patients with systemic lupus erythematosus (SLEA) and its possible mechanism. Methods in this study, SLE patients and healthy blood donors were divided into three parts: erythrocyte experiment, plasma incubating erythrocyte experiment and plasma antioxidant capacity experiment. Erythrocyte apoptosis injury was studied in two groups. Fresh whole blood was collected from 20 cases of two groups and blood cell parameters were measured. Red blood cells were routinely separated and made into a certain concentration of red blood cell suspension. The apoptosis rate and cell volume were detected by flow cytometry by Annexin V-FITC fluorescence staining, and the morphology of apoptotic cells was observed by confocal laser microscope. The apoptotic cells were labeled with fluorescence probe Fluo-3/AM and DCFH-DA. Intracellular calcium concentration and intracellular reactive oxygen species were detected by flow cytometry and ceramide content was detected by fluorescence labeled second antibody flow cytometry. The difference of each index between the two groups was compared. In order to study the effect of plasma on erythrocyte apoptosis injury in patients with SLE, fresh whole blood samples were collected from 20 patients in two groups. The isolated plasma was used to incubate healthy human type O red blood cells for 24 hours. The apoptosis rate and volume of erythrocytes, intracellular calcium concentration, ceramide and reactive oxygen species contents in the two groups were measured by the same method, and the differences were compared between the two groups. In addition, the difference of plasma total antioxidant capacity between the two groups was measured by enzyme labeling instrument. Results the hemoglobin content and blood cell volume in the experimental group were significantly lower than those in the normal control group (P 0.001), the apoptosis rate and intracellular calcium concentration in the experimental group were significantly higher than those in the control group. The content of reactive oxygen species in the experimental group was significantly higher than that in the control group (P 0.01), the volume of erythrocytes in the patients was smaller than that in the healthy blood donors, and the difference was statistically significant (P 0.01), and there was no significant difference in the content of ceramide between the two groups. The FITC fluorescence signals on the membrane of apoptotic erythrocytes can be observed under laser confocal fluorescence microscope. The apoptosis rate and intracellular calcium concentration in the experimental group were significantly higher than those in the control group (P 0.001 P 0.05), but there was no significant difference in cell size, ceramide content and reactive oxygen species. There was no significant difference in total antioxidant capacity between the two groups. Conclusion the increase of erythrocyte apoptosis is one of the causes of anemia in SLE patients. The increase of intracellular Ca ~ (2 +) concentration and the metabolic disturbance of reactive oxygen species may be the mechanism of erythrocyte apoptosis.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R593.241;R446.11

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