Toll样受体信号通路对异种心脏移植中的心肌细胞凋亡影响的初步研究

发布时间：2018-03-09 00:29

本文选题：异种移植　切入点：心肌细胞凋亡　出处：《苏州大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的:在心脏移植后,急性血管性排斥反应导致心肌细胞间质出血、水肿,周围中性粒细胞浸润,血管内皮细胞死亡,最终细胞凋亡。本实验拟研究Toll样受体通路及其下游通路核因子-κB(NF-κB)在心肌细胞凋亡中的作用。方法:在体外培养C57BL6小鼠的心肌细胞及SD大鼠和BALB/C小鼠的脾细胞。在体外建立C57BL6小鼠到BALB/C小鼠和SD大鼠的心脏移植细胞模型。将实验分为六组,A组:C57BL6小鼠心肌细胞+PBS液(PBS组);B组:C57BL6小鼠心肌细胞+TNF-α(TNF-α组);C组:C57BL6小鼠心肌细胞+BALB/C小鼠脾细胞(Mouse SC组);D组:C57BL6小鼠心肌细胞+BALB/C小鼠脾细胞+BALB/C小鼠血清(Mouse SC+S组);E组:C57BL6小鼠心肌细胞+SD大鼠脾细胞(Rat SC组);F组:C57BL6小鼠心肌细胞+SD大鼠脾细胞+SD大鼠血清(Rat SC+S组),共培养时间为108h。运用Annexin-V/PI法检测各组C57BL6小鼠心肌细胞凋亡,比较同种和异种免疫原对心肌细胞刺激效应的差别。RT-PCR法检测A组、C组、D组、E组、F组心肌细胞TLR-1、TLR-2、TLR-3、TLR-4、TLR-5、TLR-7、TLR-9的表达,筛选出被激活的Toll样受体。利用相应的Toll样受体的阻断抗体抑制激活的Toll样受体,Annexin-V/PI法检测心肌细胞凋亡,Western-blot法检测其下游通道NF-κB的表达变化。结果:运用酶消化法培养C57BL6小鼠的心肌细胞,细胞活性好,纯度达80-90%。供心心肌细胞凋亡检测实验中,TNF-α组、Rat SC组、Rat SC+S组的心肌细胞凋亡比例达71.31±7.71%、70.05±5.73%、68.61±4.71%,与PBS组、Mouse SC组、Mouse SC+S组相比,心肌细胞凋亡程度有明显差异(P0.001)。检测未经处理的C57BL6小鼠心肌细胞的各Toll样受体信号的表达,均在相对较低水平,呈静息状态。各实验组中,在Rat SC组和Rat SC+S组心肌细胞的TLR-2和TLR-3的表达明显增高(P0.05),其中TLR-3的增高更加明显(P0.05)。运用TLR-3抗体来抑制心肌细胞TLR-3信号通路后,供心心肌细胞的凋亡比例减少(P0.05),NF-κB p65蛋白的表达量降低(P0.05)。结论:1.在小鼠-大鼠心脏移植细胞模型中,TLR-2和TLR-3是异种免疫原诱导的心肌细胞凋亡过程中所激活的主要Toll受体种类,而TLR-3是受激活最明显的受体。2.通过抑制TLR-3受体信号通路的激活,可抑制下游的NF-κB表达,减轻心肌细胞凋亡。
[Abstract]:Objective: after cardiac transplantation, acute vascular rejection resulted in interstitial hemorrhage, edema, neutrophil infiltration and vascular endothelial cell death. The purpose of this study was to investigate the role of nuclear factor-魏 B-魏-魏 B in cardiomyocyte apoptosis. Methods: cardiomyocytes of C57BL6 mice and spleen cells of SD rats and BALB/C mice were cultured in vitro. The model of heart transplantation cells from C57BL6 mice to BALB/C mice and SD rats was established in vitro. The experiment was divided into six groups: group A, group 1: C57BL6 mice cardiomyocytes, PBS solution group, group B, group B: TNF- 伪 + TNF- 伪 group, group C, group C, group C, group C, group BALB/C, cardiomyocytes of C57BL6 mouse cardiomyocytes: TNF- 伪, TNF- 伪, TNF- 伪, TNF- 伪, TNF- 伪 and TNF- 伪. Murine splenocytes / mice / mice / mice / mice / / D / / BALB/C / C57BL6 / BALB/C mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / / / mice / mice / mice / mice / mice /. The co-culture time of SD rat SC S group was 108 h. Myocardial cell apoptosis was detected by Annexin-V/PI assay in C57BL6 mice. RT-PCR was used to detect the expression of TLR-1, TLR-2, TLR-3, TLR-4, TLR-5, TLR-7 and TLR-9 in cardiomyocytes of group A, C, C, D, E and F, respectively, by comparing the effects of allogeneic and xenogeneic immunogen on cardiomyocytes. The activated Toll like receptors were screened. The expression of NF- 魏 B in the downstream channel of cardiomyocytes was detected by using the Annexin-V / Pi method to detect the expression of NF- 魏 B in the downstream channels of the activated Toll like receptors by blocking antibodies against the corresponding Toll like receptors. Results: the enzyme digestion method was used to detect the expression of NF- 魏 B. Cultured cardiomyocytes of C57BL6 mice, The percentage of cardiomyocyte apoptosis in TNF- 伪 group was 71.31 卤7.71 and 70.05 卤5.731.61 卤4.71 in rat SC group, which was higher than that in PBS group, compared with mouse SC group in TNF- 伪 group, and the percentage of apoptosis in rat SC group was 71.31 卤7.71 in TNF- 伪 group, and 68.61 卤4.71 in mouse SC group in TNF- 伪 group. There was a significant difference in the degree of cardiomyocyte apoptosis. The expression of Toll like receptors in untreated C57BL6 mouse cardiomyocytes was detected at a relatively low level and in a resting state. In Rat SC group and Rat SC S group, the expression of TLR-2 and TLR-3 in cardiomyocytes was significantly higher than that in Rat SC group, especially in TLR-3 group. TLR-3 antibody was used to inhibit the TLR-3 signal pathway in cardiomyocytes. The ratio of apoptosis in donor cardiomyocytes decreased the expression of P0.05- 魏 B p65 protein. Conclusion: 1. TLR-2 and TLR-3 are the main types of Toll receptors activated by xenogen-induced cardiomyocyte apoptosis in murine rat heart transplantation cell model. By inhibiting the activation of TLR-3 receptor signaling pathway, TLR-3 can inhibit the expression of NF- 魏 B downstream and attenuate cardiomyocyte apoptosis.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R654.2

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