机械力诱导高水平氧化应激在盆底功能障碍性疾病中作用机制的研究

发布时间：2018-04-25 18:39

本文选题：盆底功能障碍性疾病 + 胶原代谢　；参考：《武汉大学》2016年博士论文

【摘要】：盆底功能障碍性疾病(pelvic floor disorder, PFD),主要包括盆底器官脱垂(pelvic organ prolapse, POP)和压力性尿失禁(stress urinary incontinence, SUI),是全世界范围内中老年女性常见的疾病,是由于盆底支持组织薄弱造成的盆腔器官如子宫、阴道等下降移位引发的器官位置及功能异常。虽然PFD不会危及患者生命,但严重影响患者日常生活、活动以及生活质量大幅度下降,对于患者心理健康也造成及其重要的危害。虽然PFD发病的具体作用机制至今不明,但以胶原代谢紊乱为主的细胞外基质(extracellular matrix, ECM)成分异常构成PFD的分子及生物学基础。众多研究证明在盆底支持组织如子宫主、骶韧带以及阴道壁组织中胶原含量失衡。盆底支持组织主要是由富含胶原的致密结缔组织组成。以胶原为主的盆底结缔组织具有稳定性和顺应性,能保持阴道的稳定性和可塑性。Ⅰ型胶原与组织牢固程度密切相关,而Ⅲ型胶原与组织的弹性相关。PFD与其他结缔组织病相似,都起源于分子水平的胶原改变。成纤维细胞是盆底结缔组织的主要成分和细胞类型,能够直接对周围的机械刺激环境做出反应,具有机械信号反应性,能分泌胶原等ECM,对保持盆底组织自稳、修复和重建有重要作用。在正常机体中,盆底结缔组织中成纤维细胞调节胶原的合成与分解代谢,二者保持动态平衡。当胶原的合成与分解代谢平衡被破坏,胶原含量以及结构改变,导致胶原功能异常,从而出现一系列病理生理变化,可能导致PFD。因此,盆底成纤维细胞发生胶原代谢紊乱与POP发病密切相关,是在分子生物学水平研究PFD发病机制的基础。由于经历妊娠、分娩等自然的生理过程,女性盆底支持组织处在不断变化的复杂的力学环境中。研究显示孕产次数以及阴道分娩次数是PFD发病的重要危险因素。此外,长期便秘以及重物提举也与PFD发病密切相关。研究显示PFD患者的盆底支持组织和细胞的生物力学特性发生异常改变。PFD的发病与其盆底支持组织受到腹腔内压力增高或直接作用于盆底支持组织的机械挤压相关。尽管证据支持机械力的作用是PFD发病的重要原因,但是联系物理力学与PFD的具体分子机制仍然不清楚。POP患者盆底支持组织呈现高水平的氧化应激(oxidative stress, OS)状态。氧化损伤标志物8-OHdG和4-HNE在POP患者盆底支持组织子宫骶韧带(uterosacral ligament, USL)中含量升高,抗氧化酶GPx1的表达显著降低。上述证据提示OS与POP的发病密切相关。因此,本研究使用人盆底成纤维细胞——人子宫骶韧带成纤维细胞(human uterosacral ligament fibroblast, hUSLF)建立机械力加载模型和氧化应激模型,检测两种模型下胶原代谢的改变,并使用抗氧化剂N-乙酰半胱氨酸(N-acetyl-L-cysteine, NAC)处理成纤维细胞,观察在抗氧化作用下对胶原代谢的影响。本研究旨在深入研究机械力以及氧化应激在PFD发病中的作用以及二者的相互关系,探索PFD发病的病理生理机制,为临床防治PFD提供理论依据。第一部分机械力在人盆底成纤维细胞胶原代谢中的作用目的：观察机械力作用于hUSLF后,胶原及其代谢相关酶和调节因子的含量变化；探究机械力在PFD发病相关的胶原代谢紊乱中的作用。材料和方法：对因非POP以及SUI良性妇科疾病行子宫切除术的16位患者在术中取USL组织块,对新鲜获取的组织立即行原代培养成纤维细胞,取3～8代细胞进行机械力加载,使用频率0.1 Hz,大小为5333肛(培养板形变量为4 mm)的力作用于细胞4 h。采用实时定量逆转录PCR (qRT-PCR)和Western Blot分别检测I型、III型胶原的含量以及代谢相关的酶MMP-2、MMP-9以及促ECM合成的转化生长因子TGF-β1蛋白的表达情况,使用细胞流式技术检测hUSLF后细胞凋亡情况的变化,用荧光显微镜观察荧光染色后的细胞骨架蛋白F-actin的表达和分布情况,观察hUSLF受到较大的机械负荷后对胶原代谢、细胞凋亡以及细胞骨架的影响,证明机械力负荷与PFD发病的关系。结果：1.机械力作用于hUSLF后,I型、III型胶原含量在mRNA水平(p0.05)和蛋白水平(p0.05)均有显著下降。2.机械力作用后,成纤维细胞分泌的降解胶原的酶MMP-2和MMP-9均显著升高(p0.05),促进胶原合成和稳定的TGF-β1表达量下降(p0.05)。3. 成纤维细胞凋亡率在机械力负荷后升高(p0.05)。4.细胞骨架在机械力的作用下,排列规则,但表达量变少,并且骨架蛋白松弛。结论：1. 机械力作用于人盆底成纤维细胞后干扰I型、III型胶原的合成,胶原合成与分解水平失衡,导致胶原含量减少。2.机械力作用下成纤维细胞凋亡水平增加,成纤维细胞数量减少；较大的机械力负荷导致传递和转化机械信号的细胞骨架松弛,骨架蛋白水平降低。3.人盆底成纤维细胞胶原代谢异常与机械力负荷相关。第二部分氧化应激在人盆底成纤维细胞胶原代谢中的作用目的：观察hUSLF处于高水平氧化应激状态下,胶原及其代谢相关酶和调节因子的含量变化；给予氧化应激模型细胞抗氧化剂NAC预处理,进一步探究机体细胞内高氧化应激水平与PFD发病相关的胶原代谢紊乱的关系。材料和方法：同第一部分实验取材以及检测指标,对因非POP以及SUI的良性妇科疾病行子宫切除术的患者在术中取USL组织块,对新鲜获取的组织立即行原代培养成纤维细胞,取3～8代细胞建立高水平氧化应激模型,使用0.4 mM H2O2处理细胞4小时后,检测细胞内氧化应激损伤标志物ROS水平以及8-OHdG含量,鉴定模型是否成功。对氧化应激模型组给予10 mM抗氧化剂NAC预处理,观察在NAC存在或不存在时,对氧化应激模型组细胞进行检测。采用实时定量逆转录PCR (qRT-PCR)和Western Blot分别检测I型、III型胶原的含量以及代谢相关的酶MMP-2、MMP-9以及促ECM合成的转化生长因子TGF-β1蛋白的表达情况,使用细胞流式技术检测hUSLF处于高水平氧化应激水平时细胞凋亡情况的变化,用荧光显微镜观察荧光染色后的细胞骨架蛋白F-actin的表达和分布情况,观察处于高水平氧化应激状态和在氧化应激受到抑制的状态时,hUSLF在胶原代谢、细胞凋亡以及细胞骨架等方面的变化,证明机体的组织和细胞高氧化应激状态与PFD发病的关系。结果：1.鉴定hUSLF的高水平氧化应激模型,检测细胞内ROS水平和8-OHdG含量,与对照组细胞相比,二者水平大幅度升高。2.处于高氧化应激状态的hUSLF.I型、Ⅲ型胶原含量在mRNA水平(p0.001)和蛋白水平(p0.01)均有显著下降。3.高水平氧化应激状态的成纤维细胞分泌的降解胶原的酶MMP-2和MMP-9均显著升高(p0.01),促进胶原合成和稳定的TGF-β1表达量下降(p0.001)。4.成纤维细胞凋亡率在高氧化应激水平时升高(p0.001)。5.细胞骨架在细胞处于高氧化应激状态时,排列不规律,表达量变少,并且骨架蛋白松弛。6. 给予抗氧化剂NAC预处理氧化应激模型组细胞后,成纤维细胞内ROS水平和8-OHdG含量大幅度降低,氧化应激状态被抑制。7.给予抗氧化剂NAC预处理氧化应激模型组细胞后,成纤维细胞胶原含量增加(p0.01),细胞凋亡减少(p0.01),细胞骨架蛋白表达量升高。结论：1.人盆底成纤维细胞处于高水平氧化应激水平时,抑制Ⅰ型、Ⅲ型胶原的合成,胶原合成与分解水平失衡,导致胶原含量减少。2.高氧化应激状态的成纤维细胞,凋亡水平增加,成纤维细胞数量减少；氧化应激影响传递和转化机械信号的细胞骨架,细胞骨架松弛,骨架蛋白含量降低。3.人盆底成纤维细胞的胶原代谢异常与盆底细胞内高水平氧化应激状态相关。4.当盆底成纤维细胞内氧化应激状态受到抑制后,由高氧化应激水平所引起的细胞内代谢变化得到逆转。第三部分机械力与氧化应激在人盆底成纤维细胞胶原代谢异常中的作用关系目的：对hUSLF的加载模型给予抗氧化剂NAC预处理,与未经NAC预处理的模型组对比,观察NAC预处理后两种模型下,Ⅰ型、Ⅲ型胶原及其代谢相关酶和调节因子的含量变化；研究在与PFD发病密切相关的胶原代谢异常中,机械力负荷与高水平氧化应激状态的作用以及相互关系,探究PFD发病的可能病理生理机制。材料和方法：同第一部分实验取材以及检测指标,对因非POP以及SUI良性妇科疾病行子宫切除术的患者在术中取USL组织块,对新鲜获取的组织立即行原代培养成纤维细胞,取3～8代细胞建立机械力加载模型,在建模前对细胞加10 mM抗氧化剂NAC预处理。应用荧光染色观察细胞内氧化应激损伤标志物ROS以及8-OHdG的含量,采用实时定量逆转录PCR (qRT-PCR)和Western Blot分别检测Ⅰ型、Ⅲ型胶原的含量以及代谢相关的酶MMP-2、MMP-9以及促ECM合成的转化生长因子TGF-β1蛋白的表达情况,使用细胞流式技术检测hUSLF细胞凋亡情况的变化,用荧光显微镜观察荧光染色后的细胞骨架蛋白F-actin的表达和分布情况,观察hUSLF在胶原代谢、细胞凋亡以及细胞骨架等方面的变化,证明机械力与氧化应激在PFD发病相关的胶原代谢异常中的作用和关系。结果：1.机械力负荷hUSLF后,成纤维细胞处于高氧化应激状态,细胞内氧化应激损伤标志物ROS和8-OHdG水平显著升高。2.抗氧化剂NAC阻止机械力负荷hUSLF后导致的ROS和8-OHdG水平升高。3.抗氧化剂NAC抑制机械力负荷hUSLF造成的I型和III型胶原含量降低(p 0.05)、MMP-2和MMP-9升高(p0.05)以及TGF-β1表达量下降(p0.05)。4. 抗氧化剂NAC作用后,机械力加载模型的成纤维细胞凋亡率显著降低(p0.05)。5.抗氧化剂NAC作用后,机械力加载模型中细胞骨架在表达量升高。结论：1.机械力作用于人盆底成纤维细胞后细胞内氧化应激水平显著升高,呈现高水平氧化应激状态。2.抗氧化剂NAC能有效抑制人盆底成纤维细胞的高氧化应激状态,并且抗氧化剂NAC能大幅度降低承受机械力负荷的人盆底成纤维细胞内的高氧化应激水平。3.抗氧化剂NAC逆转由机械力负荷以及高氧化应激水平造成的I型和III型胶原含量降低、MMP-2和MMP-9升高、TGF-β1表达量下降、细胞凋亡增加以及细胞骨架蛋白表达量减少。4. 外界机械力负荷作用于人盆底支持组织成纤维细胞,诱导细胞内高水平氧化应激状态,造成胶原代谢发生异常,胶原含量降低,进而可能导致PFD的发生。
[Abstract]:Pelvic floor disorder (PFD), which mainly includes pelvic floor organ prolapse (pelvic organ prolapse, POP) and stress urinary incontinence (stress urinary incontinence, SUI). It is a common disease in the middle aged women worldwide and the pelvic organs such as the uterus and vagina caused by the weak pelvic floor support tissue. The position and function of the organ caused by the reduction of displacement. Although PFD does not endanger the patient's life, it seriously affects the daily life, activity and the quality of life greatly decreased, and it also causes the patient's mental health and its important harm. Although the specific mechanism of PFD is unknown, the cells are mainly collagen metabolic disorders. The abnormalities of the extracellular matrix (ECM) composition constitute the molecular and biological basis of PFD. Numerous studies have shown that the collagen content of the pelvic floor supporting tissues such as the uterus, the sacral ligament and the vaginal wall is unbalanced. The pelvic floor support is mainly composed of collagen rich connective tissue. It has stability and compliance with the stability and plasticity of the vagina. Type I collagen is closely related to the firmness of tissue, and type III collagen is similar to other connective tissue diseases, similar to other connective tissue diseases, all of which originate at the molecular level of collagen change. Fibroblasts are the main components and cell types of the pelvic floor connective tissue. Direct response to the surrounding mechanical stimulus environment, with mechanical signal responsiveness, can secrete collagen and other ECM, to maintain the pelvic floor tissue self-stability, repair and reconstruction is important. In the normal body, the pelvic floor connective tissue fibroblasts regulate the synthesis and catabolism of collagen, the two maintain dynamic balance. When collagen synthesis and differentiation The metabolic balance is destroyed, the collagen content and structure change, which leads to the abnormal function of collagen, which leads to a series of pathophysiological changes, which may lead to PFD., so the disorder of collagen metabolism in the pelvic floor fibroblasts is closely related to the pathogenesis of POP. It is the basis of the study of the pathogenesis of PFD at the molecular biology level. In natural physiological processes, female pelvic floor support is in a constantly changing and complex mechanical environment. Studies have shown that the number of pregnant women and the number of vaginal delivery are important risk factors for the onset of PFD. In addition, long-term constipation and heavy lifting are also closely related to the pathogenesis of PFD. The study shows the birth of pelvic floor support tissue and cells in PFD patients. Abnormal changes in physical properties of.PFD are associated with increased abdominal pressure in the pelvic floor or mechanical extrusion directly acting on pelvic floor support. Although evidence supports the role of mechanical force as an important cause of the pathogenesis of PFD, the specific molecular mechanism associated with physical mechanics and PFD remains unclear on the.POP patient's basin. A high level of oxidative stress (oxidative stress, OS) state. Oxidative damage markers, 8-OHdG and 4-HNE, were elevated in the uterine sacral ligament (uterosacral ligament, USL) in the pelvic floor support of POP patients, and the expression of antioxidant enzyme GPx1 was significantly reduced. The above evidence suggests that OS is closely related to the pathogenesis of POP. Using human pelvic fibroblasts - human uterosacral ligament fibroblast (hUSLF), a mechanical loading model and an oxidative stress model were established to detect the changes in the collagen metabolism under two models and use the antioxidant N- acetyl cysteine (N-acetyl-L-cysteine, NAC) to treat fibroblasts. This study aims to investigate the effects of oxidative stress on the metabolism of collagen. The purpose of this study is to investigate the role of mechanical force and oxidative stress in the pathogenesis of PFD and the relationship between the two, explore the pathophysiological mechanism of PFD and provide theoretical basis for the clinical prevention and control of PFD. Objective: To observe the changes of collagen and its metabolic related enzymes and regulatory factors after the action of mechanical force on hUSLF; explore the role of mechanical force in the disorder of collagen metabolism related to the pathogenesis of PFD. Materials and methods: 16 patients who underwent hysterectomy for non POP and SUI benign gynecologic diseases were taken USL tissue during the operation. The tissue took the primary culture into fibroblasts and took 3~8 generations of cells for mechanical loading, using the frequency of 0.1 Hz and the force of 5333 anus (culture plate variable 4 mm) on cell 4 h., using real-time quantitative reverse transcription PCR (qRT-PCR) and Western Blot to detect I type, the content of III type collagen and the metabolism related enzyme MMP-2, M. MP-9 and the expression of TGF TGF- beta 1 protein synthesized by ECM, the changes in apoptosis of hUSLF cells were detected by flow cytometry. The expression and distribution of cytoskeleton protein F-actin after fluorescence staining were observed by fluorescence microscopy. The effect of hUSLF on collagen metabolism and apoptosis after large mechanical load were observed. As well as the influence of cytoskeleton, the relationship between mechanical load and PFD was proved. Results: after 1. mechanical force acting on hUSLF, I, III type collagen content was significantly reduced by.2. mechanical force at mRNA level (P0.05) and protein level (P0.05), and the enzyme MMP-2 and MMP-9 increased significantly (P0.05) and promoted by fibroblasts. Collagen synthesis and stable TGF- beta 1 expression decreased (P0.05) the apoptosis rate of.3. fibroblasts increased after mechanical load (P0.05).4. cytoskeleton was arranged under the action of mechanical force, but the expression was less, and the skeleton protein was relaxed. Conclusion: 1. mechanical force acts on the human pelvic fibroblasts and interferes with I type, and the combination of III collagen As a result, the level of collagen synthesis and decomposition is unbalanced, which leads to the increase of the apoptosis level of fibroblasts and the decrease in the number of fibroblasts under the action of.2. mechanical force. The large mechanical load leads to the relaxation of the cytoskeleton in the transmission and transformation of mechanical signals, and the decrease of the skeletal protein level in.3. human pelvic floor fibroblasts and the abnormal collagen metabolism. Mechanical load related. Second the role of oxidative stress in the collagen metabolism of human pelvic floor fibroblasts in order to observe the changes in the content of collagen and its metabolic related enzymes and regulating factors under the high level of oxidative stress in hUSLF, and to further explore the cell in the body by the oxidation stress model cell antioxidant NAC preconditioning. The relationship between the level of high oxidative stress and the disorder of collagen metabolism associated with PFD. Materials and methods: with the first part of the experimental materials and detection indexes, the USL tissue blocks were taken during the operation of the patients undergoing hysterectomy for non POP and SUI benign gynecologic diseases, and 3~8 of the freshly acquired groups were cultured for primary fibroblasts. A high level oxidative stress model was established on behalf of the cells. After 4 hours using 0.4 mM H2O2 cells, the level of ROS and 8-OHdG content of oxidative stress damage markers were detected. The model group was pretreated with 10 mM antioxidant NAC, and the oxidative stress model group was observed in the presence or absence of NAC. Cells were detected. Real-time quantitative reverse transcription PCR (qRT-PCR) and Western Blot were used to detect the content of I type, III type collagen, and metabolic related enzymes MMP-2, MMP-9, and the expression of the TGF TGF- beta 1 protein, which was synthesized by ECM, and cell apoptosis was detected by flow cytometry at high level of oxidative stress. The expression and distribution of cytoskeleton protein F-actin after fluorescence staining were observed by fluorescence microscopy, and the changes in collagen metabolism, apoptosis and cytoskeleton were observed at high levels of oxidative stress and inhibition of oxidative stress, and the high oxidation of tissues and cells in the body should be demonstrated by the changes in the high level of oxidative stress and the inhibition of oxidative stress. Results: 1. the high level oxidative stress model of hUSLF was identified and the level of ROS and 8-OHdG in the cells were detected. Compared with the control group, the level of.2. was significantly higher in the hUSLF.I type of high oxidative stress, and the content of type III collagen decreased significantly in mRNA level (p0.001) and protein level (P0.01). .3. the enzyme MMP-2 and MMP-9 secreted by fibroblasts in high level oxidative stress state increased significantly (P0.01), promoting collagen synthesis and stable TGF- beta 1 expression decreased (p0.001) the apoptosis rate of.4. fibroblasts increased at high oxidative stress water (p0.001).5. cell skeleton in high oxidative stress state. The level of ROS and 8-OHdG in the fibroblasts was greatly reduced and the oxidative stress was inhibited by.7. to the oxidative stress model group cells, and the collagen content of fibroblasts increased after the oxidative stress was inhibited by.7., and the oxidative stress was suppressed by the antioxidant NAC pretreated with antioxidant NAC. Add (P0.01), decrease in apoptosis (P0.01) and increase the expression of cytoskeleton protein. Conclusion: when the 1. human pelvic fibroblasts are at high level of oxidative stress, the synthesis of type I, type III collagen, collagen synthesis and decomposition level are unbalance, resulting in the decrease of collagen content in.2. high oxidative stress state, and the increase of apoptosis level. The number of fibroblasts decreased; oxidative stress affects the cytoskeleton, cytoskeleton, cytoskeleton, cytoskeleton, and decreased collagen metabolism in.3. human pelvic floor fibroblasts related to the high level of oxidative stress in the pelvic floor cells.4. when the oxidative stress in the pelvic floor fibroblasts is inhibited, The changes in intracellular metabolism caused by high oxidative stress level were reversed. Third the relationship between mechanical force and oxidative stress in the abnormal collagen metabolism of human pelvic floor fibroblasts: the hUSLF loading model was pretreated with antioxidant NAC, compared with the model group that had not been pretreated with NAC, and two kinds of pretreatment were observed. Under the model, type I, type I, type III collagen and its metabolic related enzymes and regulatory factors change; study the role and interaction of mechanical load and high level oxidative stress in the abnormal collagen metabolism related to the pathogenesis of PFD, and explore the possible pathologic mechanism of PFD. Materials and methods: the first part of the experiment Materials and detection indexes were taken for USL tissue blocks in patients undergoing hysterectomy for non POP and SUI benign gynecologic diseases. Primary cultured fibroblasts were performed for fresh tissues, and 3~8 generation of cells were used to establish a mechanical load model. Before modeling, the cells were pretreated with 10 mM antioxidant NAC. The fluorescence staining was used to observe the application of fluorescence staining. The content of ROS and 8-OHdG in intracellular oxidative stress damage markers were detected by real-time quantitative reverse transcription PCR (qRT-PCR) and Western Blot, respectively, the content of type III collagen, metabolism related enzymes MMP-2, MMP-9, and the expression of the TGF TGF- beta 1 protein, which were synthesized by ECM, and the cell flow technique was used to detect hUSLF. The changes in apoptosis, the expression and distribution of cytoskeleton F-actin after fluorescence staining were observed by fluorescence microscopy, and the changes of hUSLF in collagen metabolism, cell apoptosis and cytoskeleton were observed. The effect and relationship of mechanical force and oxidative stress on the abnormal metabolism of PFD in the pathogenesis of PFD were proved. After the mechanical load hUSLF, the fibroblasts were in high oxidative stress state, and the levels of ROS and 8-OHdG in the intracellular oxidative stress damage markers increased significantly. The level of ROS and 8-OHdG increased by the.2. antioxidant NAC, which prevented the mechanical load hUSLF, and the.3. antioxidant NAC inhibited the I type and the collagen content caused by the hUSLF loading of the mechanical load (0.). 05), when MMP-2 and MMP-9 increased (P0.05) and TGF- beta 1 expression decreased (P0.05).4. antioxidant NAC, the apoptosis rate of fibroblasts in mechanical loading model decreased significantly (P0.05).5. antioxidant NAC action, and the expression of cytoskeleton in mechanical loading model increased. Conclusion: 1. mechanical force acts on human pelvic fibroblasts. The level of oxidative stress in the cells increased significantly, showing a high level of oxidative stress..2. antioxidant NAC can effectively inhibit human pelvic floor fibroblasts.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R711.5

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