本文選題：盆底器官脫垂 + Ⅰ型膠原纖維�。� 參考：《復旦大學》2014年博士論文

【摘要】：盆底器官脫垂性疾病(POP)是中老年婦女常見的疾病,嚴重影響了其生活質量,然而,其發(fā)病機制尚不明確。因此有關其發(fā)病機制的研究一直是婦產科研究學者關注的焦點和亟待解決的難題。作為盆底支持結構的主要成分,盆底結締組織的病理生理變化與POP的發(fā)生發(fā)展密切相關。盆底結締組織主要由成纖維細胞為主的細胞成分和膠原纖維為主的細胞基質成分組成。在正常人體結締組織中,由成纖維細胞調節(jié)膠原纖維的合成、降解,二者達到動態(tài)平衡。然而當此平衡被打破,膠原纖維在重塑過程中發(fā)生結構和功能的異常,而發(fā)生了相應的病理生理變化,如盆底器官的脫垂。近年來研究發(fā)現,糖化終末產物(Advanced Glycated End-products, AGEs)和其受體(Receptor of AGEs, RAGE)結合后,啟動細胞內多種信號傳導途徑,參與纖維細胞的增殖、凋亡和膠原的合成、分解代謝,進而調節(jié)及原纖維的重塑。它廣泛地涉及到了多種系統(tǒng)和組織的病理生理變化,比如皮膚老化、心血管損傷和重構、糖尿病腎臟疾病、骨骼重構、牙齦增生、疤痕增生等。在皮膚中,AGEs與RAGE結合后,可通過ROS、NO、神經酰胺、P38、JNK-MAP激酶等信號機制的活化促進成纖維細胞的凋亡。重要的是AGEs與RAGE結合后可通過上述信號傳導機制激活NF-K B (nuclear factor-k-gene binding)影響成纖維細胞合成和分泌膠原纖維。同時AGEs結合RAGE后還能影響細胞基質的降解,如促進MMP-1和TIMP-1的合成和分泌。1996年Jackson設想：由于盆底結締組織AGEs的增加,一方面使得盆底膠原纖維容易斷裂,盆底結締缺乏應有的生物力學性能;另一方面,AGEs的增加妨礙了盆底膠原纖維的代謝平衡,新生膠原無法成功塑形并發(fā)揮應有的力學性能,從而導致了盆底功能障礙的發(fā)生。上述理論似乎合理地解釋了盆底功能障礙性疾病的發(fā)病機制,但尚缺乏足夠的證據。因此,為了進一步AGEs-RAGE與盆底器官脫垂之間的關系,本研究項目設計并完成了以下的實驗。第一部分AGEs、RAGE和Collagen Ⅰ在盆底器官脫垂疾病陰道壁的表達[研究目的]研究AGEs、RAGE和collagen Ⅰ在盆底器官脫垂性疾病陰道壁的表達及其與POP的關系。[研究對象和方法]我院2012年因盆底器官脫垂POP-Q評分Ⅲ度及以上需行全子宮切除術者為研究組,共44例；因其他疾病需行全子宮切除術者為對照組,共46例。采用免疫組化和Western Blooting的方法,檢測研究組和對照組陰道壁穹窿部位AGEs、RAGE 和 Collagen Ⅰ的表達,采用RAGE全基因測序的方法檢測24例研究組和25例對照組RAGE全基因序列,尋找存在臨床意義SNP位點。[研究結果]盆底器官脫垂組陰道壁內collagen Ⅰ含量低于對照組,且隨著年齡的增加其含量也下降(P0.05);盆底器官脫垂性疾病陰道壁內AGEs含量高于對照組(p0.05)；盆底器官脫垂性疾病陰道壁組織內RAGE的含量與對照組無統(tǒng)計學差異(P0.05)；RAGE全基因測序后發(fā)現兩個潛在的SNP(Single Nucleotide Polymorphism)位點,但均位于內含子,且與外顯子相距較遠,在剪接的過程中被丟棄的可能很大,因此存在有臨床意義的SNP位點的可能性較低。[結論]研究顯示AGEs的提高和collagen Ⅰ的下降與POP的發(fā)生存在相互對應關系,提示AGEs量的改變與盆底器官的脫垂存在相關性。第二部分AGEs-RAGE通路在盆底成纖維細胞Ⅰ型膠原代謝中的作用[研究目的]研究AGEs-RAGE通路在盆底成纖維細胞Ⅰ型膠原代謝中的作用[研究對象和方法]取研究組和對照組陰道壁組織各3例,進行成纖維細胞的原代培養(yǎng)、鑒定,采用SRB的方法檢測AGEs對成纖維細胞的促進凋亡的作用,再用WB 和 real time PCR的方法檢測AGEs對成纖維細胞分泌collagen Ⅰ、MMP-1、 TIMP-1、RAG E的蛋白和合成]nRNA的影響。[研究結果]盆底器官脫垂組成纖維細胞在AGEs的作用下更容易出現調亡(25mmg/L),對照組則需要AGEs增加到一定的濃度才出現凋亡(75mg/L)。盆底器官脫垂組成纖維細胞合成分泌較原纖維下降(p0.05),合成和分泌MMP-1上升(p0.05)；在mRNA水平,TIMP-mRNA隨著AGEs的增加出現下降,RAGE出現緩慢上升而后下降,但其蛋白的分泌和和合成無顯著變化；而對照組在接受不同濃度的AGEs作用后,其collagenⅠ下降和MMP-1上升幅度不如盆底脫垂組細胞明顯,或沒有變化,TIMP-1和RAGE也無變化。[研究結論]結果提示AGEs容易使盆底器官脫垂患者的成纖維細胞出現凋亡,更容易使collagen Ⅰ表達下降,MMP-1表達上升。第三部分AGEs-RAGE通路在POP成纖維細胞膠原代謝中的信號機制的研究[研究目的]AGEs-RAGE通路在POP成纖維細胞膠原代謝中的信號機制的研究。[研究方法和步驟]采用WB的方法檢測AGEs作用盆底脫垂組成纖維細胞的RAGE、MAPK-p38、NF-kB-p65信號通路的變化。分別采用SiRN、SB203580、 PDTC阻斷RAGE、MAPK-p38、NF-kB-p65信號分子。最后用real time PCR的方法驗證。[研究結果]首先經過篩選發(fā)現,P-p38在加藥后16分鐘出現高峰,P-p65在加藥后1小時出現高峰。然后選用自行設計的SiR NA將RAGE阻斷,Sb203580阻斷MAPK, PDTC阻斷NF-kB。研究發(fā)現,阻斷RAGE后,其下游的P38,NF-kB被阻斷,下游的作用產物如collagen Ⅰ 和 Mmp-1也被阻斷；僅僅阻斷P38 不能阻斷NF-kB及其下游的產物,阻斷NF-kB分子能影響其下游的產物的合成。最后利用real time PCR從mRNA水平間各個阻斷節(jié)點,結果與免疫印跡結果相對應。[結論]AGEs與RAGE結合后,通過激活P-p38、P-p65,引起膠原纖維合成和分泌的下降、MMp-1合成和分泌的上升,可造成結締組織膠原纖維含量的下降,但MAPK-P38并非是此水平的唯一信號分子。第四部分AGEs-RAGE通路在SD大鼠腹壁缺損修復過程中的變化[研究目的]研究AGEs-RAGE通路在SD大鼠腹壁缺損修復過程中的變化和作用,研究AGEs-RAGE通路與collagen Ⅰ重塑以及缺損修復效果的關系。[研究方法和步驟]剛成年的SD大鼠人造腹壁缺損,然后利用三種不同的方法進行缺損修補,即聚丙烯網片、SIS生物合成網片、雙側腹直肌直接對縫。然后在術后3、9、15、21個月觀察缺損修復的情況,包括修復部位宏觀觀察、電鏡超微結構的觀察、修復部位力學性能的檢測,最后檢測修復部位collagen Ⅰ、AGEs、 RAGE表達的變化和區(qū)別。分析AGEs-RAGE通路與collagen Ⅰ和修復效果之間的相互關系。[研究結果] SIS生物合成網片組仿真度好、柔軟,但出現膨出較其他組增多(p0.05)；力學性能檢測其修復部位最大抗張力最低(p0.05)；電鏡下發(fā)現膠原纖維增生較慢、稀疏,成纖維細胞、局部血管增生管徑較�。欢覚z測到修復部位collagen Ⅰ含量最低、AGEs含量最高(p0.05),而RAGE的表達無明顯差異(p0.05)。AGEs在術后先出現上升,而后出現下降;collagen Ⅰ含量在術后早期各組基本相似,但在術后中遠期SIS網片植入組collagen Ⅰ含量最低。[結論]術后早期SIS網片的降解是缺損部位膨出的直接原因,中晚期膠原代謝異常促進了局部膨出；AGEs抑制了局部膠原纖維代謝的合成,加速了膠原纖維的降解促進了局部膨出；而且在術后15月以后AGEs已基本降低到正常范圍,但其妨礙膠原代謝的作用后果已造成。全文結果根據前述各部分結果可知：1, AGEs-RAGE通路參與并抑制了盆底器官脫垂患者陰道壁膠原纖維合成,加速其降解過程；2, AGE-RAGE通路通過激活MAPK. NF-kB信號分子發(fā)揮作用,影響了盆底結締組織較遠代謝,但這并不是唯一的信號途徑；3, AGEs發(fā)揮作用多在損傷修復早期,并妨礙修復的順利進行,數月后可能開始降至正常范圍。本研究為盆底器官的脫垂提供了理論依據,即AGE-RAGE通路通過抑制膠原纖維合成、促進膠原纖維的降解,進一步促進了POP的發(fā)生；也為盆底器官脫垂的治療奠定了理論基礎。
[Abstract]:Pelvic floor organ prolapse (POP) is a common disease in middle-aged and elderly women, which seriously affects the quality of life. However, its pathogenesis is not clear. Therefore, the research on its pathogenesis has been the focus of attention and urgent problems to be solved by the researchers in gynecology and obstetrics. As the main component of the pelvic floor support structure, the pelvic floor connective tissue is the main component of the pelvic floor support structure. The pathophysiological changes are closely related to the occurrence and development of POP. The pelvic floor connective tissue is mainly composed of fibroblast - based cell components and collagen fiber based matrix components. In normal human connective tissue, fibroblasts regulate the synthesis and degradation of collagen fibers, and the two can achieve dynamic balance. However, this balance is beaten. In the process of remodeling, the structure and function of the collagen fibers occur during the remodeling process, and the corresponding pathophysiological changes, such as the prolapse of the pelvic floor organs, have occurred. In recent years, it was found that after the combination of Advanced Glycated End-products (AGEs) and its receptor (Receptor of AGEs, RAGE), a variety of signal transduction pathways in the cells were started. The proliferation of fibroblasts, the synthesis of apoptosis and collagen, catabolism, and regulation and the remodeling of the fibrous fibers. It is widely involved in the pathophysiological changes of various systems and tissues, such as skin aging, cardiovascular damage and remodeling, diabetic kidney disease, skeletal restructure, gingival hyperplasia, scar hyperplasia, etc. in the skin, AGEs and RAGE junctions After combination, the activation of ROS, NO, ceramide, P38, JNK-MAP kinase can promote the apoptosis of fibroblasts. It is important that AGEs and RAGE can activate NF-K B (nuclear factor-k-gene binding) through the above-mentioned signaling mechanism and affect the formation and secretion of collagen fibers in fibroblasts. Degradation of rounded cell matrix, such as promoting the synthesis and secretion of MMP-1 and TIMP-1, and secreting.1996 Jackson envisaged that the increase of AGEs in the pelvic floor connective tissue makes the pelvic floor collagen fibers break easily and the pelvic floor connective lacks due biomechanical properties; on the other hand, AGEs increases the metabolic balance of the pelvic floor collagen fibers, and the new gum These theories seem to reasonably explain the pathogenesis of pelvic floor dysfunction, but lack sufficient evidence. Therefore, this research project has been designed and completed to further the relationship between AGEs-RAGE and pelvic organ prolapse. The following experiments were made. Part 1: expression of AGEs, RAGE and Collagen I in the vaginal wall of the pelvic organ prolapse disease [Objective] to study the expression of AGEs, RAGE and collagen I in the vaginal wall of the pelvic organ prolapse and its relationship with POP. [object and methods] in 2012, the POP-Q score of pelvic floor organ prolapse in our hospital and the degree of POP-Q Total hysterectomy was required for the study group, a total of 44 cases, and 46 cases were treated with total hysterectomy for other diseases. The immunohistochemical and Western Blooting methods were used to detect the expression of AGEs, RAGE and Collagen I in the vaginal wall of the study group and the control group, and 24 cases were examined by the method of full gene sequencing of RAGE. Study group and 25 cases of control group RAGE whole gene sequence, find the existence of clinical significance SNP loci. [results] the content of collagen I in the vaginal wall of pelvic organ prolapse group was lower than that of the control group, and the content of the vaginal wall decreased with age (P0.05), and the content of AGEs in the vaginal wall of pelvic floor organ prolapse was higher than that of the control group (P0.05). There was no significant difference in the content of RAGE in the vaginal wall of the vertical disease with the control group (P0.05); two potential SNP (Single Nucleotide Polymorphism) loci were found after the whole gene sequencing of RAGE, but they were all located in the intron, and were far away from the exons, and could be discarded during the splicing process, so there was a clinical SNP. The possibility of the loci is low. [Conclusion] the study shows that the improvement of AGEs and the decrease of collagen I and the occurrence of POP suggest the correlation between the changes of AGEs and the prolapse of the pelvic organs. The role of the second part of the AGEs-RAGE pathway in the type I collagen metabolism of the pelvic floor fibroblasts is to study the AGEs-RAGE passage. The role of the road in the type I collagen metabolism of the pelvic floor fibroblasts [object and method] was taken from 3 cases of the study group and the control group, 3 cases of the vaginal wall tissue, and the primary culture of fibroblasts was carried out. The effect of AGEs on the apoptosis of fibroblasts was detected by SRB method, and the WB and real time PCR were used to detect the formation of AGEs on the fibroblasts. The effects of vitamin C on collagen I, MMP-1, TIMP-1, RAG E protein and synthesis of]nRNA. [results] the fibrous cells of pelvic floor organ prolapse are more susceptible to apoptosis (25mmg/L) under the action of AGEs, and the control group needs AGEs to increase to a certain concentration to appear apoptosis (75mg/L). The synthesis and secretion of fibrous cells in pelvic floor organ prolapse are produced. Compared with the decrease of primary fiber (P0.05), the synthesis and secretion of MMP-1 increased (P0.05); at the level of mRNA, TIMP-mRNA decreased with the increase of AGEs, and the RAGE appeared slowly and then declined, but the secretion and synthesis of the protein had no significant change, while the control group was less than the collagen I drop and MMP-1 increase after the action of AGEs in different concentrations. The cells of the pelvic prolapse group were obvious or unchanged, and there was no change in TIMP-1 and RAGE. [Conclusion] the results suggest that AGEs is easy to induce apoptosis in the fibroblasts of patients with pelvic organ prolapse, which makes the expression of collagen I decline and the expression of MMP-1 rise. The signal mechanism of the third part AGEs-RAGE pathway in the collagen metabolism of POP fibroblasts The study of the signal mechanism of]AGEs-RAGE pathway in the collagen metabolism of POP fibroblasts. [methods and steps] the WB method was used to detect the changes in the RAGE, MAPK-p38, NF-kB-p65 signaling pathways of AGEs in the pelvic floor prolapse, using SiRN, SB203580, PDTC to block RAGE, MAPK-p38, and signals. Finally, the real time PCR method was used to verify. [results] first after screening, it was found that P-p38 appeared at the peak after 16 minutes after the addition of the drug. P-p65 appeared at the peak after 1 hours after the addition. Then, the RAGE was blocked by a self-designed SiR NA, Sb203580 blocked MAPK, and PDTC blocked the NF-kB. research. The downstream products such as collagen I and Mmp-1 are also blocked; only blocking P38 can not block the products of NF-kB and its downstream, blocking NF-kB molecules can affect the synthesis of downstream products. Finally, real time PCR is used to block each node from mRNA level. The result is corresponding to the result of immunoblotting. [conclusion]AGEs and RAGE are combined, The decrease in the synthesis and secretion of collagen fibers by activating P-p38, P-p65, and the rise of MMp-1 synthesis and secretion can cause a decline in the content of collagen fibers in connective tissue, but MAPK-P38 is not the only signal molecule at this level. The changes of the fourth part of the AGEs-RAGE pathway in the repair of abdominal wall defect in SD rats [research purposes] study AGEs-RAG The changes and effects of E pathway in the repair of abdominal wall defect in SD rats, study the relationship between AGEs-RAGE pathway and collagen I remodeling and defect repair. [methods and steps] the artificial abdominal wall defect of adult SD rats, and then use three different methods to repair the defect, namely, polypropylene mesh, SIS biosynthesis net, double The lateral rectus muscles were directly seams. Then the defects were observed in 3,9,15,21 months after the operation, including the macroscopic observation of the repair site, the observation of the ultrastructure of the electron microscope, the detection of the mechanical properties of the repair parts, and the changes of the expression of the repair site collagen I, AGEs, RAGE and the region. The AGEs-RAGE pathway and collagen I and the repair effect were analyzed. The relationship between the SIS biosynthesis mesh group was good and soft, but the swelling was more than the other groups (P0.05); the maximum tensile strength of the repair site was lowest (P0.05) by mechanical properties (P0.05). The content of collagen I was the lowest, and the content of AGEs was the highest (P0.05), but the expression of RAGE had no significant difference (P0.05).AGEs first appeared and then decreased, and the content of collagen I was basically similar in the early post operation groups, but the minimum content of collagen I was in the middle and long term after operation. [Conclusion] the decrease of SIS net in the early postoperative period was reduced. The solution is the direct cause of the swelling of the defect site. The abnormal collagen metabolism in the middle and late stages promotes local swelling; AGEs inhibits the synthesis of the metabolism of local collagen fibers, accelerates the degradation of collagen fibers and promotes local expansion, and AGEs has been reduced to normal confines after 15 months of operation, but the consequences of its hindering collagen metabolism have been created. According to the results mentioned above, 1, the AGEs-RAGE pathway participates in and inhibits the synthesis of collagen fibers in the vaginal wall of patients with pelvic organ prolapse and accelerates the degradation process; 2, the AGE-RAGE pathway plays a role by activating the MAPK. NF-kB signal molecules, affecting the distal metabolism of the pelvic floor connective tissue, but this is not the only signal. This study provides a theoretical basis for the prolapse of pelvic floor organs, which means that the AGE-RAGE pathway promotes the degradation of collagen fibers by inhibiting the synthesis of collagen fibers and further promoting the occurrence of POP, and also the basin of the AGEs. The basis of the treatment of the prolapse of the bottom organs has been laid.
【學位授予單位】：復旦大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R711.2

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相關期刊論文 前1條

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本文編號：1998320