發(fā)布時間：2018-04-25 18:39

  本文選題：盆底功能障礙性疾病 + 膠原代謝�。� 參考：《武漢大學(xué)》2016年博士論文

【摘要】：盆底功能障礙性疾病(pelvic floor disorder, PFD),主要包括盆底器官脫垂(pelvic organ prolapse, POP)和壓力性尿失禁(stress urinary incontinence, SUI),是全世界范圍內(nèi)中老年女性常見的疾病,是由于盆底支持組織薄弱造成的盆腔器官如子宮、陰道等下降移位引發(fā)的器官位置及功能異常。雖然PFD不會危及患者生命,但嚴(yán)重影響患者日常生活、活動以及生活質(zhì)量大幅度下降,對于患者心理健康也造成及其重要的危害。雖然PFD發(fā)病的具體作用機(jī)制至今不明,但以膠原代謝紊亂為主的細(xì)胞外基質(zhì)(extracellular matrix, ECM)成分異常構(gòu)成PFD的分子及生物學(xué)基礎(chǔ)。眾多研究證明在盆底支持組織如子宮主、骶韌帶以及陰道壁組織中膠原含量失衡。盆底支持組織主要是由富含膠原的致密結(jié)締組織組成。以膠原為主的盆底結(jié)締組織具有穩(wěn)定性和順應(yīng)性,能保持陰道的穩(wěn)定性和可塑性。Ⅰ型膠原與組織牢固程度密切相關(guān),而Ⅲ型膠原與組織的彈性相關(guān)。PFD與其他結(jié)締組織病相似,都起源于分子水平的膠原改變。成纖維細(xì)胞是盆底結(jié)締組織的主要成分和細(xì)胞類型,能夠直接對周圍的機(jī)械刺激環(huán)境做出反應(yīng),具有機(jī)械信號反應(yīng)性,能分泌膠原等ECM,對保持盆底組織自穩(wěn)、修復(fù)和重建有重要作用。在正常機(jī)體中,盆底結(jié)締組織中成纖維細(xì)胞調(diào)節(jié)膠原的合成與分解代謝,二者保持動態(tài)平衡。當(dāng)膠原的合成與分解代謝平衡被破壞,膠原含量以及結(jié)構(gòu)改變,導(dǎo)致膠原功能異常,從而出現(xiàn)一系列病理生理變化,可能導(dǎo)致PFD。因此,盆底成纖維細(xì)胞發(fā)生膠原代謝紊亂與POP發(fā)病密切相關(guān),是在分子生物學(xué)水平研究PFD發(fā)病機(jī)制的基礎(chǔ)。由于經(jīng)歷妊娠、分娩等自然的生理過程,女性盆底支持組織處在不斷變化的復(fù)雜的力學(xué)環(huán)境中。研究顯示孕產(chǎn)次數(shù)以及陰道分娩次數(shù)是PFD發(fā)病的重要危險因素。此外,長期便秘以及重物提舉也與PFD發(fā)病密切相關(guān)。研究顯示PFD患者的盆底支持組織和細(xì)胞的生物力學(xué)特性發(fā)生異常改變。PFD的發(fā)病與其盆底支持組織受到腹腔內(nèi)壓力增高或直接作用于盆底支持組織的機(jī)械擠壓相關(guān)。盡管證據(jù)支持機(jī)械力的作用是PFD發(fā)病的重要原因,但是聯(lián)系物理力學(xué)與PFD的具體分子機(jī)制仍然不清楚。POP患者盆底支持組織呈現(xiàn)高水平的氧化應(yīng)激(oxidative stress, OS)狀態(tài)。氧化損傷標(biāo)志物8-OHdG和4-HNE在POP患者盆底支持組織子宮骶韌帶(uterosacral ligament, USL)中含量升高,抗氧化酶GPx1的表達(dá)顯著降低。上述證據(jù)提示OS與POP的發(fā)病密切相關(guān)。因此,本研究使用人盆底成纖維細(xì)胞——人子宮骶韌帶成纖維細(xì)胞(human uterosacral ligament fibroblast, hUSLF)建立機(jī)械力加載模型和氧化應(yīng)激模型,檢測兩種模型下膠原代謝的改變,并使用抗氧化劑N-乙酰半胱氨酸(N-acetyl-L-cysteine, NAC)處理成纖維細(xì)胞,觀察在抗氧化作用下對膠原代謝的影響。本研究旨在深入研究機(jī)械力以及氧化應(yīng)激在PFD發(fā)病中的作用以及二者的相互關(guān)系,探索PFD發(fā)病的病理生理機(jī)制,為臨床防治PFD提供理論依據(jù)。第一部分機(jī)械力在人盆底成纖維細(xì)胞膠原代謝中的作用目的：觀察機(jī)械力作用于hUSLF后,膠原及其代謝相關(guān)酶和調(diào)節(jié)因子的含量變化；探究機(jī)械力在PFD發(fā)病相關(guān)的膠原代謝紊亂中的作用。材料和方法：對因非POP以及SUI良性婦科疾病行子宮切除術(shù)的16位患者在術(shù)中取USL組織塊,對新鮮獲取的組織立即行原代培養(yǎng)成纖維細(xì)胞,取3～8代細(xì)胞進(jìn)行機(jī)械力加載,使用頻率0.1 Hz,大小為5333肛(培養(yǎng)板形變量為4 mm)的力作用于細(xì)胞4 h。采用實時定量逆轉(zhuǎn)錄PCR (qRT-PCR)和Western Blot分別檢測I型、III型膠原的含量以及代謝相關(guān)的酶MMP-2、MMP-9以及促ECM合成的轉(zhuǎn)化生長因子TGF-β1蛋白的表達(dá)情況,使用細(xì)胞流式技術(shù)檢測hUSLF后細(xì)胞凋亡情況的變化,用熒光顯微鏡觀察熒光染色后的細(xì)胞骨架蛋白F-actin的表達(dá)和分布情況,觀察hUSLF受到較大的機(jī)械負(fù)荷后對膠原代謝、細(xì)胞凋亡以及細(xì)胞骨架的影響,證明機(jī)械力負(fù)荷與PFD發(fā)病的關(guān)系。結(jié)果：1.機(jī)械力作用于hUSLF后,I型、III型膠原含量在mRNA水平(p0.05)和蛋白水平(p0.05)均有顯著下降。2.機(jī)械力作用后,成纖維細(xì)胞分泌的降解膠原的酶MMP-2和MMP-9均顯著升高(p0.05),促進(jìn)膠原合成和穩(wěn)定的TGF-β1表達(dá)量下降(p0.05)。3. 成纖維細(xì)胞凋亡率在機(jī)械力負(fù)荷后升高(p0.05)。4.細(xì)胞骨架在機(jī)械力的作用下,排列規(guī)則,但表達(dá)量變少,并且骨架蛋白松弛。結(jié)論：1. 機(jī)械力作用于人盆底成纖維細(xì)胞后干擾I型、III型膠原的合成,膠原合成與分解水平失衡,導(dǎo)致膠原含量減少。2.機(jī)械力作用下成纖維細(xì)胞凋亡水平增加,成纖維細(xì)胞數(shù)量減少；較大的機(jī)械力負(fù)荷導(dǎo)致傳遞和轉(zhuǎn)化機(jī)械信號的細(xì)胞骨架松弛,骨架蛋白水平降低。3.人盆底成纖維細(xì)胞膠原代謝異常與機(jī)械力負(fù)荷相關(guān)。第二部分氧化應(yīng)激在人盆底成纖維細(xì)胞膠原代謝中的作用目的：觀察hUSLF處于高水平氧化應(yīng)激狀態(tài)下,膠原及其代謝相關(guān)酶和調(diào)節(jié)因子的含量變化；給予氧化應(yīng)激模型細(xì)胞抗氧化劑NAC預(yù)處理,進(jìn)一步探究機(jī)體細(xì)胞內(nèi)高氧化應(yīng)激水平與PFD發(fā)病相關(guān)的膠原代謝紊亂的關(guān)系。材料和方法：同第一部分實驗取材以及檢測指標(biāo),對因非POP以及SUI的良性婦科疾病行子宮切除術(shù)的患者在術(shù)中取USL組織塊,對新鮮獲取的組織立即行原代培養(yǎng)成纖維細(xì)胞,取3～8代細(xì)胞建立高水平氧化應(yīng)激模型,使用0.4 mM H2O2處理細(xì)胞4小時后,檢測細(xì)胞內(nèi)氧化應(yīng)激損傷標(biāo)志物ROS水平以及8-OHdG含量,鑒定模型是否成功。對氧化應(yīng)激模型組給予10 mM抗氧化劑NAC預(yù)處理,觀察在NAC存在或不存在時,對氧化應(yīng)激模型組細(xì)胞進(jìn)行檢測。采用實時定量逆轉(zhuǎn)錄PCR (qRT-PCR)和Western Blot分別檢測I型、III型膠原的含量以及代謝相關(guān)的酶MMP-2、MMP-9以及促ECM合成的轉(zhuǎn)化生長因子TGF-β1蛋白的表達(dá)情況,使用細(xì)胞流式技術(shù)檢測hUSLF處于高水平氧化應(yīng)激水平時細(xì)胞凋亡情況的變化,用熒光顯微鏡觀察熒光染色后的細(xì)胞骨架蛋白F-actin的表達(dá)和分布情況,觀察處于高水平氧化應(yīng)激狀態(tài)和在氧化應(yīng)激受到抑制的狀態(tài)時,hUSLF在膠原代謝、細(xì)胞凋亡以及細(xì)胞骨架等方面的變化,證明機(jī)體的組織和細(xì)胞高氧化應(yīng)激狀態(tài)與PFD發(fā)病的關(guān)系。結(jié)果：1.鑒定hUSLF的高水平氧化應(yīng)激模型,檢測細(xì)胞內(nèi)ROS水平和8-OHdG含量,與對照組細(xì)胞相比,二者水平大幅度升高。2.處于高氧化應(yīng)激狀態(tài)的hUSLF.I型、Ⅲ型膠原含量在mRNA水平(p0.001)和蛋白水平(p0.01)均有顯著下降。3.高水平氧化應(yīng)激狀態(tài)的成纖維細(xì)胞分泌的降解膠原的酶MMP-2和MMP-9均顯著升高(p0.01),促進(jìn)膠原合成和穩(wěn)定的TGF-β1表達(dá)量下降(p0.001)。4.成纖維細(xì)胞凋亡率在高氧化應(yīng)激水平時升高(p0.001)。5.細(xì)胞骨架在細(xì)胞處于高氧化應(yīng)激狀態(tài)時,排列不規(guī)律,表達(dá)量變少,并且骨架蛋白松弛。6. 給予抗氧化劑NAC預(yù)處理氧化應(yīng)激模型組細(xì)胞后,成纖維細(xì)胞內(nèi)ROS水平和8-OHdG含量大幅度降低,氧化應(yīng)激狀態(tài)被抑制。7.給予抗氧化劑NAC預(yù)處理氧化應(yīng)激模型組細(xì)胞后,成纖維細(xì)胞膠原含量增加(p0.01),細(xì)胞凋亡減少(p0.01),細(xì)胞骨架蛋白表達(dá)量升高。結(jié)論：1.人盆底成纖維細(xì)胞處于高水平氧化應(yīng)激水平時,抑制Ⅰ型、Ⅲ型膠原的合成,膠原合成與分解水平失衡,導(dǎo)致膠原含量減少。2.高氧化應(yīng)激狀態(tài)的成纖維細(xì)胞,凋亡水平增加,成纖維細(xì)胞數(shù)量減少；氧化應(yīng)激影響傳遞和轉(zhuǎn)化機(jī)械信號的細(xì)胞骨架,細(xì)胞骨架松弛,骨架蛋白含量降低。3.人盆底成纖維細(xì)胞的膠原代謝異常與盆底細(xì)胞內(nèi)高水平氧化應(yīng)激狀態(tài)相關(guān)。4.當(dāng)盆底成纖維細(xì)胞內(nèi)氧化應(yīng)激狀態(tài)受到抑制后,由高氧化應(yīng)激水平所引起的細(xì)胞內(nèi)代謝變化得到逆轉(zhuǎn)。第三部分機(jī)械力與氧化應(yīng)激在人盆底成纖維細(xì)胞膠原代謝異常中的作用關(guān)系目的：對hUSLF的加載模型給予抗氧化劑NAC預(yù)處理,與未經(jīng)NAC預(yù)處理的模型組對比,觀察NAC預(yù)處理后兩種模型下,Ⅰ型、Ⅲ型膠原及其代謝相關(guān)酶和調(diào)節(jié)因子的含量變化；研究在與PFD發(fā)病密切相關(guān)的膠原代謝異常中,機(jī)械力負(fù)荷與高水平氧化應(yīng)激狀態(tài)的作用以及相互關(guān)系,探究PFD發(fā)病的可能病理生理機(jī)制。材料和方法：同第一部分實驗取材以及檢測指標(biāo),對因非POP以及SUI良性婦科疾病行子宮切除術(shù)的患者在術(shù)中取USL組織塊,對新鮮獲取的組織立即行原代培養(yǎng)成纖維細(xì)胞,取3～8代細(xì)胞建立機(jī)械力加載模型,在建模前對細(xì)胞加10 mM抗氧化劑NAC預(yù)處理。應(yīng)用熒光染色觀察細(xì)胞內(nèi)氧化應(yīng)激損傷標(biāo)志物ROS以及8-OHdG的含量,采用實時定量逆轉(zhuǎn)錄PCR (qRT-PCR)和Western Blot分別檢測Ⅰ型、Ⅲ型膠原的含量以及代謝相關(guān)的酶MMP-2、MMP-9以及促ECM合成的轉(zhuǎn)化生長因子TGF-β1蛋白的表達(dá)情況,使用細(xì)胞流式技術(shù)檢測hUSLF細(xì)胞凋亡情況的變化,用熒光顯微鏡觀察熒光染色后的細(xì)胞骨架蛋白F-actin的表達(dá)和分布情況,觀察hUSLF在膠原代謝、細(xì)胞凋亡以及細(xì)胞骨架等方面的變化,證明機(jī)械力與氧化應(yīng)激在PFD發(fā)病相關(guān)的膠原代謝異常中的作用和關(guān)系。結(jié)果：1.機(jī)械力負(fù)荷hUSLF后,成纖維細(xì)胞處于高氧化應(yīng)激狀態(tài),細(xì)胞內(nèi)氧化應(yīng)激損傷標(biāo)志物ROS和8-OHdG水平顯著升高。2.抗氧化劑NAC阻止機(jī)械力負(fù)荷hUSLF后導(dǎo)致的ROS和8-OHdG水平升高。3.抗氧化劑NAC抑制機(jī)械力負(fù)荷hUSLF造成的I型和III型膠原含量降低(p 0.05)、MMP-2和MMP-9升高(p0.05)以及TGF-β1表達(dá)量下降(p0.05)。4. 抗氧化劑NAC作用后,機(jī)械力加載模型的成纖維細(xì)胞凋亡率顯著降低(p0.05)。5.抗氧化劑NAC作用后,機(jī)械力加載模型中細(xì)胞骨架在表達(dá)量升高。結(jié)論：1.機(jī)械力作用于人盆底成纖維細(xì)胞后細(xì)胞內(nèi)氧化應(yīng)激水平顯著升高,呈現(xiàn)高水平氧化應(yīng)激狀態(tài)。2.抗氧化劑NAC能有效抑制人盆底成纖維細(xì)胞的高氧化應(yīng)激狀態(tài),并且抗氧化劑NAC能大幅度降低承受機(jī)械力負(fù)荷的人盆底成纖維細(xì)胞內(nèi)的高氧化應(yīng)激水平。3.抗氧化劑NAC逆轉(zhuǎn)由機(jī)械力負(fù)荷以及高氧化應(yīng)激水平造成的I型和III型膠原含量降低、MMP-2和MMP-9升高、TGF-β1表達(dá)量下降、細(xì)胞凋亡增加以及細(xì)胞骨架蛋白表達(dá)量減少。4. 外界機(jī)械力負(fù)荷作用于人盆底支持組織成纖維細(xì)胞,誘導(dǎo)細(xì)胞內(nèi)高水平氧化應(yīng)激狀態(tài),造成膠原代謝發(fā)生異常,膠原含量降低,進(jìn)而可能導(dǎo)致PFD的發(fā)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.

【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R711.5

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