青—老年大鼠肾脏交互移植模型的构建及机体内环境衰老关键因子的研究

发布时间：2018-07-17 03:50

【摘要】：目的：通过建立青年-老年大鼠肾脏交互移植模型,观察内环境对肾脏衰老的影响.应用非标记定量蛋白质组学方法筛选与衰老显著相关蛋白,验证并探讨其对衰老影响的可能作用机制。方法：①通过肾脏原位切除和供肾原位移植术方法,把年轻大鼠的肾脏移植到年老大鼠,同时把年老大鼠的肾脏移植到年轻大鼠,术后体内生长6个月后,建立青年-老年大鼠肾脏交互移植模型；②观察交互移植后肾脏病理(HE.PAS染色)、组织衰老(β-gal染色)、炎症因子(TNF-α和IL-1p)的变化情况；③用差异蛋白质组分析筛选参与肾脏衰老的蛋白,并在Wi38细胞进行验证其参与细胞衰老的作用通路。结果：①成功建立青年-老年大鼠肾脏交互移植模型,移植后肾脏血运正常,肾脏移植后存活；②与青年大鼠肾脏比较,移植到老年大鼠的青年肾脏HE染色、PAS染色肾脏病理无明显改变,β-gal染色增加,炎症因子TNF-α和IL-1p表达增加；与老年大鼠肾脏比较,移植到青年大鼠的老年肾脏HE染色、PAS染色肾脏病理无明显改变,β-gal染色降低,炎症因子TNF-α和IL-1p表达降低；③蛋白质组学技术比较肾脏移植各组的差异表达蛋白,共鉴定到了279个差异倍数2以上的差异蛋白。最终筛选出16个有与ATP能量代谢、凋亡、细胞死亡有关的蛋白质,蛋白-蛋白相互作用分析提示Sod1与多个蛋白有相互作用。与20代Wi38细胞比较：40代Wi38细胞Sod1表达降低、p16表达升高。Sod1过表达时,Ac-NF-κB表达降低,细胞衰老减轻；Sod1敲除后Ac-NF-κB表达升高,细胞衰老加重。结论：1.本研究成功建立青年-老年大鼠肾脏交互移植模型,术后移植肾脏血运好,存活率高。2.内环境可以影响肾脏衰老状态,青年-老年大鼠肾脏交互移植模型中,内环境可以使老年肾脏年轻化、年轻肾脏衰老化。3.通过蛋白质组学分析发现Sod1参与(抑制)肾脏衰老过程,通过抑制Ac-NF-κB拮抗参与衰老重要通路NF-κB通路。
[Abstract]:Aim: to observe the effect of internal environment on renal aging by establishing a model of kidney transplantation in young and aged rats. The unlabeled quantitative proteomics method was used to screen the proteins associated with senescence, and the possible mechanism of its effect on senescence was verified and discussed. Methods the kidney of the young rat was transplanted to the old rat by orthotopic nephrectomy and orthotopic transplantation of the donor kidney. At the same time, the kidney of the aged rat was transplanted to the young rat. After 6 months of operation, the kidney was transplanted to the young rat. The renal pathological changes (HE.PAS staining), tissue senescence (尾 -gal staining) and inflammatory factors (TNF- 伪 and IL-1p) were observed in the kidney of young and aged rats. The protein involved in renal senescence was screened by differential proteome analysis. The pathway involved in cell senescence was verified in Wi38 cells. Results the kidney cross-transplantation model of young and old rats was successfully established by 1: 1. The renal blood flow was normal after transplantation, and the kidney survival after transplantation was compared with that of young rats. There were no significant changes in renal pathology, 尾 -gal staining and the expression of inflammatory cytokines TNF- 伪 and IL-1p in young kidney grafted into aged rats by HE staining and pas staining. The expression of TNF- 伪 and IL-1p was decreased in the aged kidney of young rats by HE staining, pas staining, 尾 -gal staining, and the protein expression of TNF- 伪 and IL-1p. A total of 279 differentially expressed proteins were identified. Finally, 16 proteins related to ATP energy metabolism, apoptosis and cell death were screened. The protein-protein interaction analysis indicated that Sod1 interacted with many proteins. Compared with 20 generation Wi38 cells, the expression of Sod1 in Wi38 cells was decreased. The expression of p16 was increased and the expression of Ac-NF- 魏 B was decreased when Sod1 was overexpressed. The senescence of cells decreased after Sod1 knockout, and the expression of Ac-NF- 魏 B increased, and the senescence of cells increased. Conclusion 1. In this study, the model of kidney transplantation was established successfully in young and old rats. The graft blood was good after operation, and the survival rate was high. 2. The internal environment can affect the aging state of the kidney. In the model of kidney transplantation between young and aged rats, the internal environment can make the old kidney younger and the young kidney aging. 3. Through proteomics analysis, it was found that Sod1 was involved in (inhibit) renal senescence, and through inhibition of Ac-NF- 魏 B antagonism, Sod1 was involved in NF- 魏 B pathway.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R692;R-332

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