PKCβ抑制剂对大鼠肾脏缺血再灌注损伤后M1及M2型巨噬细胞浸润的影响

发布时间：2018-02-20 18:29

本文关键词： 缺血再灌注损伤炎症因子 PKCβ抑制剂巨噬细胞　出处：《遵义医学院》2017年硕士论文　论文类型：学位论文

【摘要】：目的:通过建立不同大鼠肾缺血再灌注损伤模型,分析PKCβ抑制剂对大鼠肾缺血再灌注损伤后M1及M2型巨噬细胞表面标记物及介质在肾组织的表达情况,为进一步研究PKCβ抑制剂减轻肾缺血再灌注损伤的机制提供理论基础。方法:构建不同的大鼠肾缺血再灌注损伤模型,按处理不同分为肾缺血再灌注损伤非灌洗组(A组)、肾缺血再灌注损伤预灌洗组(B组)、PKCβ抑制剂治疗组(C组)、假手术组(D组)。A、B、D组术前一天予以生理盐水1ml/只灌胃,C组术前一天予以PKCβ抑制剂1ml/只灌胃。第二日将A、B、C组打开腹腔充分暴露双侧肾脏后去掉右侧肾脏,A、C组左侧肾脏予以血管夹钳夹左侧肾动脉阻断肾脏血供60min后再恢复血流灌注24h;B组于缺血前予生理盐水对左侧肾脏进行灌洗冲尽残留血细胞后以同样的方法缺血60min后再恢复血液灌注24h;D组仅行开腹、关腹手术。24h后处死大鼠分别取左侧肾脏标本进行以下检测:1.免疫组织化学法分别检测各组诱导型一氧化氮合酶(iNOS)和白介素-12(IL-12)表达以分析M1型巨噬细胞在肾脏的浸润情况;2.免疫荧光染色法分别检测各组巨噬细胞细胞标记物(CD197/CD163)表达以分析M1及M2型巨噬细胞在肾脏的浸润情况;3.荧光定量PCR法检测各组树突状细胞相关性C型植物血凝素-1(Dectin-1)、1-型精氨酸酶(Arg-1)mRNA表达分析M2型巨噬细胞在肾脏的浸润情况。结果:1.免疫组织化学法显示iNOS主要表达于髓质外带近端肾小管上皮细胞胞浆及包膜上,而IL-12主要表达于肾髓质的肾小管间质中,部分表达于肾小球系膜细胞及肾小管上皮细胞胞浆中。iNOS、IL-12在D组表达很少,在A、B组显著增加,并且A、B组表达较C组明显增高(P0.01);A组表达高于B组(P0.01);iNOS在C组表达较D组明显增高(P0.01),但是IL-12在C组与D组表达无差异(P0.05)。2.免疫荧光染色显示CD197、CD163主要表达在肾小管肾间质中,CD197在D组表达较少,A组表达显著增高,并且A组表达较B、C组增高(P0.01);B组与C组比较二者无明显差异(P0.05);D组表达较A、B、C组降低(P0.01)。CD163在C组表达较A、B、D组显著增高(P0.01);A、B、D组相互比较均无明显差异(P0.05)。3.荧光定量PCR显示Dectin-1及Arg-1 mRNA在D组表达很少,C组表达明显增高,C组表达较A、B、D组明显增高(P0.05),A、B、D组相互比较均无明显差异(P0.05)。结论:1.缺血前预灌洗可减少肾缺血再灌注损伤后M1型巨噬细胞的浸润;2.PKCβ抑制剂可减少肾缺血再灌注损伤后M1型巨噬细胞的浸润、增加抗炎型M2型巨噬细胞的表达来促进组织修复,减轻炎症反应,但具体机制暂不清楚,需后续研究进一步证实。
[Abstract]:Objective: to study the expression of PKC 尾 inhibitor on the surface markers and mediators of M1 and M2 macrophages after renal ischemia-reperfusion injury in different rats. To provide a theoretical basis for further study on the mechanism of PKC 尾 inhibitor in alleviating renal ischemia-reperfusion injury. Methods: different renal ischemia-reperfusion injury models were established in rats. According to the treatment, the rats were divided into two groups: group A (non-lavage group), group B (prelavage group), group C (PKC 尾 inhibitor treatment group), group D (sham operation group), group D (group D): 1 ml of normal saline before operation / group C (intragastric instillation of normal saline only). One day before operation, PKC 尾 inhibitor 1ml / g was perfused into the stomach. On 2nd, the patients in group A were given intraperitoneal full exposure of the bilateral kidneys, and the left kidneys were removed from the right kidney. The left kidneys in the group A were removed by clamp clamp and the left renal artery was clamped to block the renal blood supply for 60 minutes before resuming the blood supply. In group B, the left kidney was washed with saline for 24 h before ischemia, and the residual blood cells were washed out in the same way. After 60 minutes of ischemia, the rats in group D were restored to hemoperfusion for 24 hours. Group D only underwent laparotomy. 24 hours after abdominal closure operation, the rats were sacrificed for the following detection: 1. Immunohistochemical method was used to detect the expression of inducible nitric oxide synthase (iNOS) and interleukin-12 IL-12 (IL-12) in order to analyze the infiltration of M1 macrophages in the kidney. Immunofluorescence staining method was used to detect the expression of macrophage marker CD197 / CD163 in order to analyze the infiltration of M1 and M2 macrophages in the kidney. Fluorescence quantitative PCR method was used to detect the dendritic cell associated C type plant in each group. Expression of procoagulin-1 Dectin-1- argininase (Arg-1N) mRNA in the kidney the infiltration of M2 macrophage in kidney was analyzed. Results 1. Immunohistochemistry showed that iNOS was mainly expressed in the cytoplasm and capsule of proximal tubular epithelial cells of medullary outer band. However, IL-12 was mainly expressed in the tubulointerstitial of renal medulla, partly in glomerular Mesangial cells and tubule epithelial cells. The expression of iNOSIL-12 in the cytoplasm of glomerular Mesangial cells and renal tubular epithelial cells was very small in group D, but increased significantly in group A and B. Moreover, the expression of IL-12 in group A was significantly higher than that in group C (P 0.01). The expression of IL-12 in group C was significantly higher than that in group D, but there was no difference in the expression of IL-12 between group C and group D. Immunofluorescence staining showed that CD197 CD163 was mainly expressed in renal tubulointerstitium. The expression of CD197 in group D was significantly higher than that in group A. Moreover, there was no significant difference between group A and group C in the expression of P0.01C and group C. There was no significant difference between group A and group C in the expression of P0.01C. CD163 in group A was significantly higher than that in group A (P 0.01). There was no significant difference between group A and group C in the expression of P0.05U. 3. Fluorescence quantitative PCR showed Dectin-1 and Arg-1. The expression of mRNA in group D was less than that in group C. The expression of mRNA in group C was significantly higher than that in group A (P 0.05). Conclusion: 1. Pre-lavage before ischemia can reduce the infiltration of M1 macrophages after renal ischemia-reperfusion injury. PKC 尾 inhibitor can reduce the infiltration of M1 macrophages after renal ischemia reperfusion injury. The expression of anti-inflammatory M2 macrophages was increased to promote tissue repair and alleviate inflammatory reaction, but the specific mechanism was not clear, which needed to be further confirmed by further research.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692

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