AGEs-RAGE通路在盆底器官脱垂疾病中的研究

发布时间：2018-06-09 02:09

本文选题：盆底器官脱垂 + Ⅰ型胶原纤维　；参考：《复旦大学》2014年博士论文

【摘要】：盆底器官脱垂性疾病(POP)是中老年妇女常见的疾病,严重影响了其生活质量,然而,其发病机制尚不明确。因此有关其发病机制的研究一直是妇产科研究学者关注的焦点和亟待解决的难题。作为盆底支持结构的主要成分,盆底结缔组织的病理生理变化与POP的发生发展密切相关。盆底结缔组织主要由成纤维细胞为主的细胞成分和胶原纤维为主的细胞基质成分组成。在正常人体结缔组织中,由成纤维细胞调节胶原纤维的合成、降解,二者达到动态平衡。然而当此平衡被打破,胶原纤维在重塑过程中发生结构和功能的异常,而发生了相应的病理生理变化,如盆底器官的脱垂。近年来研究发现,糖化终末产物(Advanced Glycated End-products, AGEs)和其受体(Receptor of AGEs, RAGE)结合后,启动细胞内多种信号传导途径,参与纤维细胞的增殖、凋亡和胶原的合成、分解代谢,进而调节及原纤维的重塑。它广泛地涉及到了多种系统和组织的病理生理变化,比如皮肤老化、心血管损伤和重构、糖尿病肾脏疾病、骨骼重构、牙龈增生、疤痕增生等。在皮肤中,AGEs与RAGE结合后,可通过ROS、NO、神经酰胺、P38、JNK-MAP激酶等信号机制的活化促进成纤维细胞的凋亡。重要的是AGEs与RAGE结合后可通过上述信号传导机制激活NF-K B (nuclear factor-k-gene binding)影响成纤维细胞合成和分泌胶原纤维。同时AGEs结合RAGE后还能影响细胞基质的降解,如促进MMP-1和TIMP-1的合成和分泌。1996年Jackson设想：由于盆底结缔组织AGEs的增加,一方面使得盆底胶原纤维容易断裂,盆底结缔缺乏应有的生物力学性能;另一方面,AGEs的增加妨碍了盆底胶原纤维的代谢平衡,新生胶原无法成功塑形并发挥应有的力学性能,从而导致了盆底功能障碍的发生。上述理论似乎合理地解释了盆底功能障碍性疾病的发病机制,但尚缺乏足够的证据。因此,为了进一步AGEs-RAGE与盆底器官脱垂之间的关系,本研究项目设计并完成了以下的实验。第一部分AGEs、RAGE和Collagen Ⅰ在盆底器官脱垂疾病阴道壁的表达[研究目的]研究AGEs、RAGE和collagen Ⅰ在盆底器官脱垂性疾病阴道壁的表达及其与POP的关系。[研究对象和方法]我院2012年因盆底器官脱垂POP-Q评分Ⅲ度及以上需行全子宫切除术者为研究组,共44例；因其他疾病需行全子宫切除术者为对照组,共46例。采用免疫组化和Western Blooting的方法,检测研究组和对照组阴道壁穹窿部位AGEs、RAGE 和 Collagen Ⅰ的表达,采用RAGE全基因测序的方法检测24例研究组和25例对照组RAGE全基因序列,寻找存在临床意义SNP位点。[研究结果]盆底器官脱垂组阴道壁内collagen Ⅰ含量低于对照组,且随着年龄的增加其含量也下降(P0.05);盆底器官脱垂性疾病阴道壁内AGEs含量高于对照组(p0.05)；盆底器官脱垂性疾病阴道壁组织内RAGE的含量与对照组无统计学差异(P0.05)；RAGE全基因测序后发现两个潜在的SNP(Single Nucleotide Polymorphism)位点,但均位于内含子,且与外显子相距较远,在剪接的过程中被丢弃的可能很大,因此存在有临床意义的SNP位点的可能性较低。[结论]研究显示AGEs的提高和collagen Ⅰ的下降与POP的发生存在相互对应关系,提示AGEs量的改变与盆底器官的脱垂存在相关性。第二部分AGEs-RAGE通路在盆底成纤维细胞Ⅰ型胶原代谢中的作用[研究目的]研究AGEs-RAGE通路在盆底成纤维细胞Ⅰ型胶原代谢中的作用[研究对象和方法]取研究组和对照组阴道壁组织各3例,进行成纤维细胞的原代培养、鉴定,采用SRB的方法检测AGEs对成纤维细胞的促进凋亡的作用,再用WB 和 real time PCR的方法检测AGEs对成纤维细胞分泌collagen Ⅰ、MMP-1、 TIMP-1、RAG E的蛋白和合成]nRNA的影响。[研究结果]盆底器官脱垂组成纤维细胞在AGEs的作用下更容易出现调亡(25mmg/L),对照组则需要AGEs增加到一定的浓度才出现凋亡(75mg/L)。盆底器官脱垂组成纤维细胞合成分泌较原纤维下降(p0.05),合成和分泌MMP-1上升(p0.05)；在mRNA水平,TIMP-mRNA随着AGEs的增加出现下降,RAGE出现缓慢上升而后下降,但其蛋白的分泌和和合成无显著变化；而对照组在接受不同浓度的AGEs作用后,其collagenⅠ下降和MMP-1上升幅度不如盆底脱垂组细胞明显,或没有变化,TIMP-1和RAGE也无变化。[研究结论]结果提示AGEs容易使盆底器官脱垂患者的成纤维细胞出现凋亡,更容易使collagen Ⅰ表达下降,MMP-1表达上升。第三部分AGEs-RAGE通路在POP成纤维细胞胶原代谢中的信号机制的研究[研究目的]AGEs-RAGE通路在POP成纤维细胞胶原代谢中的信号机制的研究。[研究方法和步骤]采用WB的方法检测AGEs作用盆底脱垂组成纤维细胞的RAGE、MAPK-p38、NF-kB-p65信号通路的变化。分别采用SiRN、SB203580、 PDTC阻断RAGE、MAPK-p38、NF-kB-p65信号分子。最后用real time PCR的方法验证。[研究结果]首先经过筛选发现,P-p38在加药后16分钟出现高峰,P-p65在加药后1小时出现高峰。然后选用自行设计的SiR NA将RAGE阻断,Sb203580阻断MAPK, PDTC阻断NF-kB。研究发现,阻断RAGE后,其下游的P38,NF-kB被阻断,下游的作用产物如collagen Ⅰ 和 Mmp-1也被阻断；仅仅阻断P38 不能阻断NF-kB及其下游的产物,阻断NF-kB分子能影响其下游的产物的合成。最后利用real time PCR从mRNA水平间各个阻断节点,结果与免疫印迹结果相对应。[结论]AGEs与RAGE结合后,通过激活P-p38、P-p65,引起胶原纤维合成和分泌的下降、MMp-1合成和分泌的上升,可造成结缔组织胶原纤维含量的下降,但MAPK-P38并非是此水平的唯一信号分子。第四部分AGEs-RAGE通路在SD大鼠腹壁缺损修复过程中的变化[研究目的]研究AGEs-RAGE通路在SD大鼠腹壁缺损修复过程中的变化和作用,研究AGEs-RAGE通路与collagen Ⅰ重塑以及缺损修复效果的关系。[研究方法和步骤]刚成年的SD大鼠人造腹壁缺损,然后利用三种不同的方法进行缺损修补,即聚丙烯网片、SIS生物合成网片、双侧腹直肌直接对缝。然后在术后3、9、15、21个月观察缺损修复的情况,包括修复部位宏观观察、电镜超微结构的观察、修复部位力学性能的检测,最后检测修复部位collagen Ⅰ、AGEs、 RAGE表达的变化和区别。分析AGEs-RAGE通路与collagen Ⅰ和修复效果之间的相互关系。[研究结果] SIS生物合成网片组仿真度好、柔软,但出现膨出较其他组增多(p0.05)；力学性能检测其修复部位最大抗张力最低(p0.05)；电镜下发现胶原纤维增生较慢、稀疏,成纤维细胞、局部血管增生管径较小；而且检测到修复部位collagen Ⅰ含量最低、AGEs含量最高(p0.05),而RAGE的表达无明显差异(p0.05)。AGEs在术后先出现上升,而后出现下降;collagen Ⅰ含量在术后早期各组基本相似,但在术后中远期SIS网片植入组collagen Ⅰ含量最低。[结论]术后早期SIS网片的降解是缺损部位膨出的直接原因,中晚期胶原代谢异常促进了局部膨出；AGEs抑制了局部胶原纤维代谢的合成,加速了胶原纤维的降解促进了局部膨出；而且在术后15月以后AGEs已基本降低到正常范围,但其妨碍胶原代谢的作用后果已造成。全文结果根据前述各部分结果可知：1, AGEs-RAGE通路参与并抑制了盆底器官脱垂患者阴道壁胶原纤维合成,加速其降解过程；2, AGE-RAGE通路通过激活MAPK. NF-kB信号分子发挥作用,影响了盆底结缔组织较远代谢,但这并不是唯一的信号途径；3, AGEs发挥作用多在损伤修复早期,并妨碍修复的顺利进行,数月后可能开始降至正常范围。本研究为盆底器官的脱垂提供了理论依据,即AGE-RAGE通路通过抑制胶原纤维合成、促进胶原纤维的降解,进一步促进了POP的发生；也为盆底器官脱垂的治疗奠定了理论基础。
[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.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R711.2

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