【作者】 刘源；

【导师】 唐其柱；

【作者基本信息】 武汉大学 ， 内科学， 2015， 博士

【摘要】 背景心力衰竭发生发展的基本机制是心室重构,病理解剖上主要表现为心肌细胞肥大、凋亡以及心肌间质纤维化,而间质纤维化的主要特点为细胞外基质(Extracellularmatrix, ECM)过度沉积和间质细胞如肌成纤维细胞的增殖,进而导致心肌组织中毛细血管密度降低,心室顺应性也降低,伴随着压力负荷的持续存在,心室逐渐由舒张功能障碍转变为收缩功能障碍。目前研究认为,在心脏病理过程中,成纤维细胞的来源具有异质性,除心脏本身固有的成纤维细胞会发生增殖活化外,其他成分如内皮细胞、上皮细胞、循环纤维细胞、单核细胞、周细胞均可转化为成纤维细胞,目前调控成纤维细胞转化的具体机制尚未阐明,因此找到调控转变的关键因子可以为以后的临床治疗心脏纤维化,改善心室重构提供可靠的靶点。Toll样受体5(Toll like receptor 5, TLR5)是Toll样受体家族成员之一,其不仅在免疫细胞上有表达(单核／巨噬细胞、未成熟的树突状细胞),在其他非免疫细胞上如心肌细胞、血管内皮细胞上也均有表达。近年研究认为,Toll样受体除与其相应的配体结合发生反应外,其也具有危险信号监测的作用。有研究显示TLR5与其配体鞭毛蛋白结合后会引起心肌功能障碍,此外,TLR5在肺纤维化肺泡上皮细胞发生上皮向间质转化(Epithelial-mesenchymal transition, EMT)发挥促进作用。然而,TLR5在压力负荷诱导的心室重构中的作用尚不明确。目的探讨TLR5对压力负荷诱导的小鼠心室重构的影响,并阐明其可能机制。方法第一部分：通过胸主动脉缩窄(Aortic banding, AB)术构建压力负荷诱导的小鼠心室重构模型。通过Western Blot (WB)及实时荧光定量(Real-time) PCR检测并对比假手术(sham)组及AB术后1W、4W、8W小鼠心脏中TLR5表达的差异；第二部分：采用1μM血管紧张素Ⅱ (Angiotensin II, Ang Ⅱ)分别刺激新生SD大鼠心肌细胞和H9c2细胞24h建立心肌细胞肥大模型,检测TLR5在刺激前后蛋白或基因表达变化有无差异；采用10ng/ml转化生长因子β1 (transforming growth factor-β1, TGF-β1)刺激人脐静脉内皮细胞(Human Umbilical Vein Endothelial Cells, HUVEC-12) 72h建立上皮-间质转化(Endothelial-to-mesenchymal transition, EndMT)模型,检测TLR5在刺激前后蛋白及基因表达有无差异；第三部分：采用TLR5基因敲除(Knock out, KO)小鼠和C57BL/6 (Wild type, WT)小鼠为实验对象,运用AB术构建压力负荷诱导的心室重构模型。通过对比心重/体重比(Heartweight/Body weight, HW/BW)、肺重/体重比(Lung weight/Body weight, LW/BW)和心重/胫骨长度比(Heart weight/ Tibial length, HW/TL)来评估小鼠心脏及肺脏的重量改变；通过心脏的大体拍照和心脏组织切片的苏木精-伊红染色法(HE)及麦胚凝集素(WGA)染色评估小鼠心脏体积及心肌细胞横截面积(Cardiomyocyte cross sectional area, CSA)的改变；运用超声心动图评估小鼠的心功能的变化；通过心脏组织切片的天狼星红(Picrosirius red, PSR)染色评估小鼠心肌间质胶原含量的变化；运用Real time-PCR检测心肌肥厚和心肌纤维化标志物mRNA表达量的改变；第四部分：运用AB术构建压力负荷诱导的心室重构模型,检测sham组C57小鼠及AB术后1W、2W、4W、8W炎性因子nRNA (IL-1、IL-6、TNFα)的动态表达水平；采用TLR5KO小鼠和WT小鼠为实验对象,运用AB术构建压力负荷诱导的心室重构模型,运用Realtime-PCR检测炎性因子及代表炎症反应增强扩大标志物的mRNA表达量；采用免疫荧光染色评估巨噬细胞浸润情况；第五部分：运用AB术构建压力负荷诱导的心室重构模型,检测sham组小鼠及AB术后1W、2W、4W、8W血小板内皮细胞黏附分子(CD31)及血管内皮生长因子(Vascular Endothelial Growth Factor, VEGF) mRNA的动态变化及两者之间的相关性；采用TLR5 KO小鼠和WT小鼠为实验对象,运用AB术构建压力负荷诱导的心室重构模型,WB检测CD31 α平滑肌动蛋白(α-SMA)及波形蛋白(vimentin)表达量的差异；免疫荧光双染检测两组中CD31/α-SMA及CD31/Collagen Ⅲ的改变；WB检测不同组中Smad2/Smad3的磷酸化情况；Real time-PCR检测在EndMT过程中发挥关键作用转录因子的表达情况；采用质粒Ad-TLR5转染HUVEC-12细胞建立过表达模型,通过TGF-β1刺激72h建立EndMT模型,光镜下观察细胞的大体形态改变；WB检测CD31、α-SMA 及 vimentin表达量改变；免疫荧光双染检测CD31/vimentin的改变情况；结晶紫实验检测细胞的迁移速度。结果第一部分：C57小鼠AB术后,TLR5基因及蛋白的表达量在1W、4W及8W时较sham组均明显升高,术后1周升高最为明显,之后升高幅度逐渐下降；第二部分：Ang Ⅱ刺激新生SD大鼠心肌细胞和H9c2细胞24h后,TLR5基因(前者)及蛋白(后者)在刺激前后表达无差异；TGF-β1刺激HUVEC-12 72h后,TLR5蛋白及基因表达水平较刺激前均明显上调；第三部分：取材结果显示：AB术后,WT组HW/BW、HW/TL均明显升高,而KO组两项指标则显著降低；大体拍照显示：AB术后,WT组心脏体积明显增大,但KO组显著降低；HE及WGA染色显示：AB术后,WT组CSA明显增大,但KO组显著降低；超声心动图结果显示：AB术后,WT组左心室后壁厚度、室间隔厚度、左心室舒张末内径、左心室收缩末内径均明显升高,而KO组则明显降低,WT组左心室射血分数和短轴缩短率在AB后显著下降,而KO组则明显升高；心肌肥厚标志物检测结果显示：WT组AB术后心钠素(ANP)、B型利钠肽(BNP)和肌球蛋白重链β (β-MHC)的mRNA水平显著增高,而KO组则明显降低,肌球蛋白重链α (α-MHC)在WT组AB后表达水平降低,而KO组则明显升高；PSR染色显示：WT组AB术后心肌细胞间质胶原含量增多,而KO组则明显降低；心肌纤维化标记物检测结果显示：WT组AB术后Ⅰ型胶原蛋白α (Collagen 1α)、 Ⅲ型胶原蛋白α (CollagenⅢα)、纤维连接蛋白(Fibronectin)、结缔组织生长因子(connective tissue growth factor, CTGF)、 vimentin、α-SMA、TGF-β1、成纤维细胞特异蛋白1(FSP1)的mRNA表达水平增高,而KO组则明显降低；第四部分：与sham组相比,AB后不同时间点(1W、2W、4W、8W)心肌组织中IL-1、IL-6、TNFa的mRNA含量均明显增高,以AB 2W时最高。AB 8W后,WT组IL-1、IL-6、TNFa的mRNA表达水平明显增高,而KO组则明显降低；免疫荧光结果显示,WT组心肌中有大量巨噬细胞浸润,而KO组心肌中未见到巨噬细胞浸润,此外,KO小鼠中巨噬细胞炎性蛋白(MIP-2)的mRNA水平明显低于WT组；触发和放大炎症反应的标记物结果显示,WT组髓过氧化物酶(MPO)、髓系细胞触发受体-1(TREM-1)及其配体细胞外受体蛋白DAP-12的mRNA水平均明显升高,而KO组则明显降低；第五部分：与sham相比,AB后不同时间点(1W、2W、4W)心肌组织中CD31和VEGF的mRNA表达量逐渐增高,而8W时两项指标较4W时均明显下降,两项指标的变化具有正相关。AB 8W后,WB结果显示：与WT组相比,KO组CD31显著增高,a-SMA、vimentin两项指标则显著降低；免疫荧光双染的结果显示：WT组小鼠心脏中部分血管内皮细胞的标记物CD31消失,仅间质的标记物a-SMA或Collagen Ⅲ为阳性表达,KO小鼠心脏中CD31表达正常,CD31与a-SMA/Collagen III有部分重合；影响EndMT的可能机制结果显示,WT组Smad2/3的磷酸化水平明显增高,信号通路下游的转录调控因子snail 1、snail2、twist 1、twist 2及神经性钙粘附蛋白(N-cadherin) 的 mRNA水平也明显增高,而KO组中以上指标均明显下降。4组细胞经过不同干预后,光镜显微镜下可见：Control组细胞为鹅卵石样形状,细胞之间连接比较紧密,间隙较小,TGF-β1组细胞则呈狭长型,细胞之间较松散,间隙较大,Ad-TLR5+TGF-β1组细胞也呈现狭长型,排列较为紊乱,细胞间的间隙较TGF-β1组更大,Ad-TLR5组细胞形态则与Control组相似；Western blot结果显示：与Control组相比,TGF-β1组CD31显著下调、α-SMA和vimentin则显著上调；与TGF-β1组相比,Ad-TLR5+ TGF-β1组CD31则下调更为显著,a-SMA和vimentin上调更为显著；与Control组相比,单加TLR5组三项指标相比无差异；免疫荧光结果显示：Control组CD31表达较强,仅有少量的vimentin表达,TGF-β1组中CD31表达较弱,vimentin表达有上调,Ad-TLR5+TGF-β1组中CD31表达进一步减弱,vimentin表达则更为明显,而Ad-TLR5组CD31和vimentin的表达水平与Control组相似；结晶紫实验结果显示,在6h,12h,24h三个时间点均发现Ad-TLR5+TGF-β1组增殖较为活跃,划痕间隙较TGF-β1组明显缩窄。结论1.压力负荷诱导的心室重构可增加心肌组织中TLR5的表达；2.TLR5基因敲除可改善压力负荷诱导的心肌肥厚和心肌纤维化；3.TLR5基因敲除可改善压力负荷诱导的炎症反应及巨噬细胞浸润；4.TLR5基因通过促进EndMT促进心肌纤维化。更多还原

【Abstract】 BackgroundPathological cardiac remodeling which is mainly characterized by enlargement and apoptosis of myocyte and cardiac fibrosis, eventually lead to heart failure. Cardiac fibrosis is an accumulation of excessive extracellular matrix proteins (ECM) in the myocardium and is associated with a decreased extent of microvasculature and increased stiffness, initially associated with diastolic dysfunction that frequently progresses to systolic dysfunction.While many previous studies have found that except the resident fibroblasts, cardiac fibroblasts are a heterogeneous population in the development of cardiac disease, and are likely derive from various distinct tissue niches such as endothelial, epcardium, fibrocytes from the bone marrow, monocytes and pericytes. Since the molecular mechanisms have not been clearly elucidated, a better understanding of the factors that regulate cardiac fibrosis could reveal potential therapeutic targets for treating cardiac remodeling.TLR5 is a member of Toll like receptors family, it expressed not only in immune cells (monocytes/macrophages, immature dendritic cells) but also in nonimmune cells, including cardiomyocytes and vascular endothelial cells. Recent researches demonstrated that, in addition to sensing of pathogens, TLRs are now demonstrated to sense host ligands as part of a wider role in the monitoring of danger signals. Researches have indicated that TLR5 can trigger cardiac innate immune responses and caused acute contractile dysfunction when combined with its ligand flagellin, and TLR5 can promote Epithelial-mesenchymal transition (EMT) in alveolar epithelial cells during pulmonary fibrosis. However, the role of TLR5 in the pressure overload-induced cardiac remodeling is still not elaborated. MethodsPart one:We used a model of aortic banding (AB)-induced cardiac remodeling, and detect the protein and mRNAexpression of TLR5. After AB for indicated time points (0W、1W、4W、8W), compared the expression of TLR5 with the sham operation group;Part two:Myocardial cells of newborn SD rats and H9c2 cardiomyocytes were both stimulated by 1 μM Angiotensin Ⅱ (Ang Ⅱ) for 24h, detect the TLR5 expression before and after the stimulus; Human Umbilical Vein Endothelial Cells (HUVEC-12) were stimulated by lOng/ml TGF-β1 for 72h to induce endothelial-to-mesenchymal transition (EndMT), also detect the TLR5 expression before and after the stimulus; Part three:TLR5 knockout (KO) mice and wild type (WT) mice were used in this part. The approach for mice modeling is the same as part one. Heart weight/body weight (HW/BW), lung weight/body weight (LW/BW) and heart weight/tibial length (HW/TL) were used to assess weight change of heart and lung of mice; the picture of whole heart was used to assess the volume of heart, HE and WGA staining was used to evaluate the cross-sectional area (CSA) of cardiomyocytes; echocardiography was used in the assessment of cardiac function; PSR staining was used to evaluate the collagen volume of LV. Real time-PCR was used to detect the mRNA expression of cardiac hypertrophy and fibrosis;Part four:TLR5 knockout (KO) mice and wild type (WT) mice were used in this part. The approach for mice modeling is the same as part one. After C57 mice received AB for the indicated time points (0W、1W、2W、4W、8W), compared the expression of inflammatory factors (IL-1、 IL-6、TNFα) with the sham operation group; 8W later, Real time-PCR was used to detect the mRNA expression of inflammatory factors and markers which represented the cardiac inflammatory phenotype triggered and expanded; immunofluorescence staining was used to detect the macrophages infiltration;Part five:(1) TLR5 knockout (KO) mice and wild type (WT) mice were used in this part. The approach for mice modeling is the same as part one. After WT mice received AB for the indicated time points (0W、1W、2W、4W、8W), compared the mRNA expression CD31 and VEGF; Western Blot was used to detect CD31, a-SMA, vimentin and the phosphorylation of Smad2/Smad3; immunofluorescence staining was used to detect the change of CD31/a-SMA and CD31/Collagen Ⅲ; Real time-PCR was used to detect the exression of transcription factors which play the key role during EndMT; (2) HUVEC-12 with and without Ad-TLR5 transfection was used in this part, different groups were stimulated by TGF-β1 for 72h, Cell morphological changes were observed under light microscope; Western Blot was used to detect CD31, a-SMA and vimentin; immunofluorescence staining was used to detect the change of CD31/vimentin; the migration ability of different groups was obtained by an in vitro scratch wound assay.ResultsPart one:The level of TLR5 protein and mRNA were both highly increased 1 week after AB compared with the sham-operated group, increased expression was also found at 4 and 8 weeks after AB with particularly high expression at 1 week;Part two:TLR5 was expressed in all of the three cell lines, but there was no evident change of the TLR5 mRNA in the neonatal rat cardiac myocytes and TLR5 protein in the H9c2 cardiomyoblasts induced by Ang Ⅱ despite hypertrophic response was evident; TLR5 mRNA and protein expression were both upregulated when the HUVEC-12 were treated with lOng/ml TGF-β1 for 3 consecutive days;Part three:Results of the HW/BW, LW/BW and HW/TL ratios and the cardiomyocyte cross sectional area (CSA) were all strikingly decreased in the AB induced TLR5 KO mice compared to the WT mice, the gross hearts, H&E and WGA staining results also confirmed the role of TLR5 deficiency on cardiac remodeling; after 8 weeks of AB, TLR5 KO mice demonstrated significant attenuation of wall thickness, chamber dilation, myocardial compliance and hemodynamics compared with the WT group; the mRNA expression of cardiac remolding markers demonstrated lower levels of ANP, BNP and (3-MHC while higher levels of a-MHC in AB-induced TLR5 deficiency mice compared with the wild-type mice; AB-induced cardiac fibrosis (perivascular and interstitial) was significantly lower in global TLR5 KO mice versus WT, Our data demonstrated that these indicators of tissue mRNA expression [collagen la, collagen IIIα, fibronectin, connective tissue growth factor (CTGF), vimentin, a-SMA, TGF-β1, fibroblast specific protein (FSP1)] were all reduced in TLR5 KO mice;Part four:Sham-operated mice exhibited low level mRNA expression of IL-1, IL-6 and TNF-a, After 1 week of AB, mRNA expression of IL-1, IL-6 and TNF-a in WT myocardial were all upregulated, peaking at 2 weeks, and gradually reduced.8 weeks after AB, it was still statistically higher in the AB group compared with the Sham-operated group, while, in the TLR5 deficiency group subject to AB, pro-inflammatory cytokines and MIP-2 mRNA expression were significantly lower than WT, immunofluorescence assays also demonstrated that macrophages infiltration was lower in TLR5 deficiency group; there was an increasing mRNA expression of MPO, TREM-1and its ITAM-containing adaptor DAP12 in myocardium in the WT AB group, while in the AB-induced TLR5 KO group, this phenomenon was significantly reversal.Part five:After 1 week of AB, significant CD31 and vascular endothelial growth factor (VEGF) mRNA upregulation was noted in the pressure-overload hearts and gradually increased, peaking at 4 weeks, and reduced at 8 weeks, we further found that CD31 mRNA expression levels were positively correlated with VEGF mRNA expression levels in the pressure-overload hearts; 8 weeks after AB, WB demonstrated that myocardium of TLR5 deficiency had significantly higher levels of CD31 than WT while levels of a-SMA and vimentin and were significantly lower; It was also confirmed by co-localization of CD31/a-SMA and CD31/CollagenⅢ; p-Smad2 and p-Smad3 were both decreased in the AB-induced TLR5 deficiency mice compared with the WT group; Our results also showed that some transcription factors which play an important role during EndMT like snail, snail2, TWIST1, TWIST2, N-cadherin were all reduced in TLR5 deficiency mice; In basal-medium cultures, the HUVEC-12 arrayed into a cobblestone-like structure, and gradually transformed into fusiform structure when stimulated by TGF-β1 for 3 days, the situation will get worse when treated the HUVEC-12 transfected with Ad-TLR5 by TGF-β1, while, there was no effect on cell structure when added Ad-TLR5 only; The results of WB demonstrated that, HUVEC-12 stimulated by TGF-β1 had significantly lower levels of CD31 than the control group while levels of a-SMA and vimentin were significantly higher. The situation will get worse when treated the HUVEC-12 transfected with Ad-TLR5 by TGF-β1, CD31 level was further reduced while a-SMA and vimentin were further higher compared with the HUVEC-12 treated by TGF-β1 group. There was no effect on the change of cell marker when added Ad-TLR5 only, It was also confirmed by co-localization of CD31/vimentin; when treated the HUVEC-12 transfected with Ad-TLR5 by TGF-β1, cells migrated even faster than the only TGF-β1 induced group. Conclusion 1. Cardiac remodeling increase the expression of TLR5;2. TLR5 knockout attenuates cardiac hypertrophy, interstitial fibrosis and dysfunction induced by pressure overload;3. TLR5 knockout attenuates inflammation and macrophage infiltration induced by pressure overload;4. TLR5 promotes cardiac fibrosis induced by pressure overload through promoting endothelial-mesenchymal transition更多还原