TLR4在移植静脉内膜增生机制中作用的研究

发布时间：2018-04-18 01:37

本文选题：TLR4 + 静脉移植　；参考：《吉林大学》2015年博士论文

【摘要】：目的：构建TLR4siRNA重组质粒并转染大鼠旁路移植静脉，研究TLR4在移植静脉内膜增生过程中的调节作用。探讨TLR4对炎性细胞因子IL-1β、IL-6、TNF-a、抑制粘附分子ICAM-1、VCAM-1表达的调节作用，并研究沉默TLR4基因对Prx1过表达诱导的细胞侵袭、迁移和增殖能力的影响。方法：建立SD大鼠颈动脉旁路移植模型，450只大鼠随机分为五组：空白对照组；阴性对照组；TLR4siRNA溶液灌注组；TLR4siRNA蛋白胶组；TLR4siRNA溶液灌注+TLR4siRNA蛋白胶组。分别于术后1、3、7、14及28天获取移植静脉，每个时间点18只。为了揭示TLR4在静脉移植内膜增生过程中是否参与调节作用，我们用Real-time PCR检测术后1、3、7天获取的移植静脉中TLR4及炎症细胞因子IL-1β、IL-6、TNF-α的mRNA表达水平。术后1、3、7、14及28天获取移植静脉行组织切片，HE染色测量内膜厚度。 Western blot检测大鼠移植静脉组血管内膜增生组Prx1的表达；逆转录病毒转染VSMC细胞，构建Prx1稳定高表达的VSMC细胞系。MTT实验检测Prx1过表达对VSMC细胞增殖能力的影响；Transwell实验检测Prx1过表达对VSMC细胞侵袭能力的影响；Wound-healing实验检测Prx1过表达对VSMC细胞组迁移能力的影响。在Prx1过表达细胞组中同时转染TLR4干扰质粒，MTT实验检测TLR4基因沉默对VSMC细胞增殖的影响；Wound-healing实验检测TLR4基因沉默对VSMC细胞组迁移能力的影响。结果：（1）随着移植静脉血管内膜的增生、水肿，TLR4基因表达量显著升高，并在移植的第3天达到高峰（p0.05,n=6），并与第7天，第14天相比，无明显差异（p0.05,n=6）；（2）与正常静脉相比，移植静脉中的IL-1β、IL-6、TNF-a、ICAM-1、VCAM-1及TLR4mRNA基因表达水平在术后1周内明显升高。与对照组相比，TLR4siRNA溶液灌注组，TLR4siRNA蛋白胶组，TLR4siRNA溶液灌注+TLR4siRNA蛋白胶组炎症细胞因子IL-1β、IL-6、TNFα、ICAM-1、VCAM-1及TLR4基因表达水平在术后1、3、7天均显著下降；（3）从TLR4siRNA溶液灌注组，TLR4siRNA蛋白胶组，TLR4siRNA溶液灌注+TLR4siRNA蛋白胶组的结果上看，TLR4siRNA的处理方法没有影响TLR4siRNA对IL-1β、IL-6、TNF-a、ICAM-1、VCAM-1的基因表达的抑制作用。 Prx1在血管内膜增生组中的表达明显高于对照组。VSMC细胞Prx1过表达组细胞的穿膜数量为（150±17）/视野，明显高于对照组的细胞穿膜数量（40±5）/视野。VSMC细胞Prx1过表达组细胞的迁移距离明显高于对照组。Prx1过表达组的细胞存活数在第1、2、3天分别为（5.625±0.1）×105、（8.9±0.737）×105、（10.635±0.065）×105，显著高于对照组细胞在第1、2、3天的细胞存活数（3.0±0.025）×105、（4.1±0.035）×105、（5.06±0.023）×105。说明Prx1过表达促进了VSMC细胞的增殖能力。TLR4SiRNA干扰+Prx1过表达组的VSMC细胞迁移距离明显低于Prx1过表达组细胞的迁移距离。TLR4SiRNA干扰+Prx1过表达组的细胞存活数在第1、2、3天分别为（3.824±0.43）×105、（5.395±0.6）×105、（6.456±0.28）×105，显著低于Prx1过表达组在第1、2、3天的细胞存活数（5.825±0.12）×105、（9.1±0.5375）×105、（10.735±0.075）×105。结论：大鼠旁路静脉移植术后，，出现明显的急性炎症反应，内膜增生明显，TLR4及炎症细胞因子表达水平显著升高，参与负向调节作用。降低TLR4的表达能够抑制移植静脉内膜增生，抑制炎性细胞因子IL-1β、IL-6、TNF-a的表达，抑制粘附分子ICAM-1、VCAM-1的表达。 Prx1过表达促进了VSMC的侵袭、迁移及增殖能力；沉默TLR4基因后能够减弱Prx1过表达对VSMC的诱导作用，沉默TLR4基因显著抑制了VSMC的侵袭、迁移和增殖能力。说明Prx1依赖于TLR4促进VSMC的侵袭、迁移和增殖能力，TLR4有望成为血管内膜增生诊断、治疗及预后评估的新靶点。
[Abstract]:Objective:
To construct the recombinant plasmid of TLR4siRNA was transfected into rat vein graft bypass, the role of regulating TLR4 in the process of intimal hyperplasia of vein transplantation. To discuss the effects of TLR4 on inflammatory cytokines IL-1 beta, IL-6, TNF-a, inhibition of adhesion molecule ICAM-1, regulate the expression of VCAM-1, and to study the gene silence of TLR4 overexpression induced cell invasion of Prx1. Migration and proliferation.
Method:
The establishment of SD rat carotid artery bypass graft model, 450 rats were randomly divided into five groups: control group; negative control group; TLR4siRNA group TLR4siRNA solution perfusion; fibrin glue group; TLR4siRNA solution perfusion +TLR4siRNA fibrin glue group. Respectively after 1,3,7,14 and 28 days for vein graft, 18 rats at each time point in order to reveal whether TLR4 is involved in the regulation of action in the process of vein graft intimal hyperplasia, we use TLR4 and inflammatory cytokines IL-1 get 1,3,7 days Real-time PCR detection after transplantation in intravenous beta, IL-6, the expression level of TNF- alpha mRNA. Vein graft tissue sections for 1,3,7,14 and for 28 days after operation, HE staining to measure the intima the thickness.
The expression of Western blot was detected in the rat vein graft intimal hyperplasia group group Prx1; retrovirus transfected VSMC cell, VSMC cell line was established by.MTT assay, Prx1 high expression of stable Prx1 overexpression on the proliferation ability of VSMC cells; Transwell assay was used to detect the effect of overexpression of Prx1 on invasion of VSMC cells; Wound-healing assay Prx1 effect on the expression of VSMC cell migration ability. In the Prx1 overexpressing cell group and transfected with TLR4 plasmid, MTT assay to detect the effect of TLR4 gene silencing on the proliferation of VSMC cells; Wound-healing assay of TLR4 gene silencing on VSMC cells migration.
Result:
(1) with intimal hyperplasia of the transplanted vein, edema, TLR4 gene expression was significantly increased, and reached a peak in third days of transplantation (P0.05, n=6), and seventh days, fourteenth days compared with no significant difference (P0.05, n=6); (2) compared with the normal veins, veins of Yi Zhijing IL-6, TNF-a, IL-1 beta, ICAM-1, VCAM-1 and TLR4mRNA gene expression level in 1 weeks after surgery was significantly increased. Compared with the control group, TLR4siRNA solution perfusion group, TLR4siRNA protein gel group, TLR4siRNA solution perfusion +TLR4siRNA fibrin glue group of inflammatory cytokines IL-6, IL-1 beta, TNF alpha, ICAM-1, VCAM-1 and TLR4 gene expression the level at 1,3,7 days after operation were significantly decreased; (3) from TLR4siRNA solution perfusion group, TLR4siRNA glue group, TLR4siRNA solution perfusion +TLR4siRNA fibrin glue group results, the processing method of TLR4siRNA has no effect on IL-1 TLR4siRNA IL-6, TNF-a, beta, ICAM-1, inhibition of VCAM-1 gene expression The effect of the system.
The expression of Prx1 in intimal hyperplasia was significantly higher than the control group of.VSMC cells Prx1 expression of the number of transmembrane cells to the group (150 + 17) / vision, significantly higher than the control group in the cell membrane numbers (40 + 5) /.VSMC cell migration distance of vision over expression of Prx1 cells was significantly higher than the control group.Prx1 over expression group the number of survival cells on day 1,2,3 respectively (5.625 + 0.1) * 105 (8.9 + 0.737) * 105 (10.635 + 0.065) * 105, significantly higher than the control group cells on day 1,2,3 cell survival (3 + 0.025) * 105 (4.1 + 0.035) * 105, (5.06 + 0.023) * 105. indicated that overexpression of Prx1 promoted VSMC cell migration from the proliferation of.TLR4SiRNA +Prx1 cells overexpressing VSMC interference group was significantly lower than that in Prx1 cells the migration distance of.TLR4SiRNA interference in +Prx1 overexpression group the number of survival cells on day 1,2,3 respectively (3.824 + 0.43) * 105 (5.395 + 0.6). X 105, (6.456 + 0.28) * 105, significantly lower than the cell survival of Prx1 over expression group (5.825 + 0.12) x 105, (9.1 + 0.5375) x 105, (10.735 + 0.075) * 105.
Conclusion:
Rat vein bypass transplantation, acute inflammatory reaction obviously, endometrial hyperplasia significantly, the expression of TLR4 and inflammatory cytokines increased significantly in negative regulation. Reducing the expression of TLR4 can inhibit intimal hyperplasia of vein grafts, inhibition of inflammatory cytokine IL-1 beta, IL-6, TNF-a expression, inhibition of adhesion molecule ICAM-1, the expression of VCAM-1.
Overexpression of Prx1 promoted VSMC invasion, migration and proliferation; after silencing TLR4 gene can decrease Prx1 overexpression induced effect on VSMC, TLR4 gene silencing significantly inhibited VSMC invasion, migration and proliferation. Prx1 relies on TLR4 to promote VSMC invasion, migration and proliferation ability, TLR4 is expected to become a diagnosis of vascular intimal hyperplasia, a new target evaluation of treatment and prognosis.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R654.2

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