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本研究通过建立小鼠急性移植物抗宿主病(aGVHD)模型,研究aGVHD发病过程中补体系统的活化对病理过程的影响。选用近交系C57BL/6(H-2Kb)小鼠和BALB/c(H-2Kd)小鼠作为实验动物。模型组以前者作为供鼠,后者作为受鼠,给予受鼠8Gy60Coγ线全身照射后4-6小时进行骨髓细胞+脾细胞移植;对照组为同基因移植组。通过观察小鼠的体表特征、生存期以及体重变化跟踪模型组动物aGVHD的发生发展。采用流式细胞术鉴定移植后模型小鼠的基因型;运用酶联免疫吸附试验(ELISA)从蛋白水平检测整个发病过程中器官组织C3a、C5a等补体相关成分的分泌表达,同时运用荧光定量PCR技术从基因水平检测补体相关基因的表达变化;通过苏木精伊红(HE)染色对aGVHD所导致的组织损伤进行病理学分析,同时利用免疫荧光(IF)等实验技术检测补体相关成分在发病小鼠相应器官组织中的沉积,初步探讨其与组织病理损伤的内在联系。结果表明:模型组小鼠表现出典型的aGVHD特征:食欲下降、体重减轻、皱毛、弓背、活动减少、脱毛等;与对照组相比,模型组小鼠靶器官(肝脏)组织中补体相关成分的表达无论从蛋白水平还是基因水平上均有明显升高(p<0.05);组织病理学分析结果显示,模型组小鼠的靶器官组织中(肝脏的中央静脉和门管区)有显著的炎性细胞浸润,并且在浸润部位有明显的补体成分C3的沉积。结论:在小鼠aGVHD发病过程中,肝组织中的补体系统持续性地过度活化,产生的补体相关成分沉积于受损的肝组织内,沉积部位有明显的炎性细胞浸润,这说明补体系统的过度活化可能与aGVHD所导致的病理损伤有密切联系。
In this study, aGVHD model was established to study the effect of complement system activation on the pathological process of aGVHD. Inbred strain C57BL / 6 (H-2Kb) mice and BALB / c (H-2Kd) mice were selected as experimental animals. In the model group, the former was used as the donor and the latter as the recipient, and the bone marrow cells + spleen cells were transplanted 4-6 hours after the whole body irradiation of the 8Gy60Coγ line of the rats; the control group was the same gene transplantation group. The appearance and development of aGVHD in model group were observed by observing the body surface characteristics, survival time and body weight of mice. The genotypes of the model mice after transplantation were identified by flow cytometry. The secretory expression of C3a, C5a and other complement-related components during the whole course of the disease was detected by enzyme-linked immunosorbent assay (ELISA) Technique was used to detect the expression of complement-related genes at the genetic level. Pathological analysis of aGVHD-induced tissue damage was performed by hematoxylin-eosin (HE) staining. At the same time, the immunofluorescence (IF) Mouse corresponding organ tissue deposition, preliminary study of its intrinsic relationship with histopathological damage. The results showed that: model mice showed typical features of aGVHD: loss of appetite, weight loss, wrinkles, bow, activity, hair removal, etc .; compared with the control group, the model group of target organ (liver) The expression of related components was significantly increased at the protein level and the gene level (p <0.05). Histopathological analysis showed that there were significant differences in the target organ tissues (central vein and portal area of the liver) Of inflammatory cell infiltration, and in the infiltration of significant deposition of complement component C3. CONCLUSION: During the development of mouse aGVHD, the complement system in liver tissue is continuously over-activated, the complement-related components produced deposit in the damaged liver tissue with obvious infiltration of inflammatory cells in the deposition site, indicating that the complement system The overactivation of aGVHD may be closely related to the pathological damage.