【作者】 赵颖；

【导师】 夏志军；

【作者基本信息】 中国医科大学 ， 妇产科学， 2021， 博士

【摘要】 目的:盆底器官脱垂(pelvic organ prolapse,POP)是影响许多女性生活质量的主要健康问题,是由于多种原因引起的盆底支持结构的损伤,老化,松弛,继而导致的子宫及阴道前后壁的膨出。随着盆腔器官脱垂症状的出现,对女性的心理健康也会产生负面影响。POP是老年女性的多发病,就发病诱因来说,其与妊娠分娩相关,但具体发病机制仍然不清楚。众所周知,盆底支持组织包括盆底周围的筋膜韧带和结缔组织,在骨盆支持系统中起着重要作用,细胞外基质(extracellular matrix,ECM)的数量、结构是组织稳定性的关键因素。盆底的解剖和功能完整性与盆底支持结构密切相关,保证了机械支撑和器官的稳定性。韧带和肌肉组织在骨盆底上起到悬挂阴道和子宫的作用,这种结构类似于“吊床”结构:当肌肉收缩时,韧带会变的伸展,使骨盆器官更加稳固。如果组织没有足够的胶原骨架支撑,韧带就会锚定在一个不稳定的锚定点,整个结构将向下塌陷。越来越多的研究表明POP与细胞外基质的改变有关。有人认为POP中胶原蛋白和弹性蛋白的产生都发生了改变。也有研究认为,组织中胶原纤维的数量减少能够降低盆底韧带和筋膜等组织的支撑力量。但是目前尚不清楚这些改变是否是盆底功能障碍的结果,或者更确切地说是盆底功能障碍的原因。胶原蛋白通常以不溶性纤维的形式存在,参与维持组织完整性。此外,它能抵抗高强度的张力,是决定结缔组织韧性的重要因素。盆底结缔组织主要包括Ⅰ型胶原(Collagen I,ColⅠ)和Ⅲ型胶原(Collagen III,ColⅢ)。在POP的病理生理过程中,胶原和基质金属蛋白酶(matrix metalloproteinases,MMP)起着关键的作用,因为在阴道壁可以观察到I型和III型胶原的转换以及MMPs的同时激活。据报道,POP中ColⅠ和ColⅢ的含量降低,研究表明,胶原降解增加是POP中胶原含量降低的主要原因。P44/42信号通路即ERK(extracellular signal-regulated kinase)信号通路,是MAPK信号通路的成员。POP阴道组织中ColⅠ和ColⅢ的表达减少可能是由于MAPK和NF-κB信号通路参与胶原合成和降解。也有研究指出,固有肌层内稳态的改变和脱垂的发展被证明是由于ERK1/2活化增强导致平滑肌细胞增殖增加所致。转化生长因子β1(transforming growth factor-β1,TGF-β1)是已知的成纤维细胞产生ECM蛋白的刺激因子,并介导成纤维细胞对机械应力的反应,诱导的信号通路主要由Smads蛋白介导,但目前的研究表明TGF-β1也可不依赖于Smads信号。除了经典的TGF-β1/Smad信号通路,TGF-β1还能以非Smad信号途径激活,如MAPK信号通路,PI3K/Akt信号通路等。我们推测P44/42 MAPK通路可能参与盆腔器官脱垂患者盆底支持组织改变的机制,为了进一步研究P44/42 MAPK通路是否参与TGF-β1对盆底支持结构中CTGF及胶原代谢,我们设计并完成了以下实验。研究方法:选取25例POP组以及25例对照组患者子宫主韧带组织,利用Western blot、免疫组织化学以及RT-PCR的方法,检测并对比两组患者子宫主韧带组织中TGF-β1、P44/42、P-P44/42、Collagen I、Collagen III、MMP9、TIMP1、CTGF表达的差异。应用贴壁法和酶消化法原代培养POP患者子宫主韧带组织成纤维细胞,免疫细胞化学法对成纤维细胞进行鉴定。应用外源性TGF-β1及其抑制剂SB431542处理成纤维细胞,MTT法及Annexin V,FITC细胞凋亡检测细胞增殖以及凋亡的变化,WB法检测P44/42、P-P44/42、CTGF、Collagen I、Collagen III及相关蛋白的表达,RT-PCR法检测CTGF及上述胶原相关蛋白m RNA表达,Elisa法检测上清中CTGF、Collagen I、Collagen III的分泌;应用P44/42抑制剂PD98059处理成纤维细胞后,再外源性加入TGF-β1,MTT法及Annexin V,FITC细胞凋亡检测细胞增殖以及凋亡的变化,WB法检测CTGF及上述胶原及相关蛋白的表达,RT-PCR法检测细胞中各种相关m RNA表达。通过flexcell-4000柔性基底加载系统对原代培养的3-4代子宫主韧带成纤维细胞进行机械拉伸,鬼笔环肽染色观察细胞拉伸后细胞骨架的改变。以10%的机械力作用成纤维细胞12小时、24小时,WB检测TGF-β1、P44/42、P-P44/42、CTGF、Collagen I、Collagen III蛋白的表达,RT-PCR方法检测上述蛋白m RNA的表达。应用P44/42抑制剂PD98059处理成纤维细胞,同时应用10%的机械力拉伸成纤维细胞12小时,WB检测P44/42、P-P44/42、CTGF、Collagen I、MMP9、TIMP1的表达变化,RT-PCR方法检测上述蛋白m RNA的表达。结果:POP患者的子宫主韧带组织中检测结果发现TGF-β1表达低于照组,磷酸化P44/42的表达,POP组低于对照组。POP组患者子宫主韧带组织MMP9表达高于对照组,而CTGF、Collagen I、Collagen III及TIMP1的表达低于对照组。原代培养子宫主韧带成纤维细胞,外源性使用5ng/ml的TGF-β1进行处理成纤维细胞,处理后与对照组比较,细胞增殖和凋亡检测结果可见成纤维细胞的增殖增加,凋亡减少,P-P44/42的相对表达量增加,CTGF、Collagen I、Collagen III、TIMP1的表达增加,MMP9的表达量下降,而应用TGF-β1抑制剂SB431542处理成纤维细胞,可见成纤维细胞增殖减低,凋亡增加,P-P44/42的相对表达降低,CTGF、Collagen I、Collagen III、TIMP1的表达降低,MMP9的表达量增加。应用P44/42的抑制剂PD98059及TGF-β1共同处理成纤维细胞,PD98059能够降低TGF-β1促进成纤维细胞增殖的作用,并使细胞凋亡增加,与TGF-β1单独处理组相比,CTGF、Collagen I、Collagen III、TIMP1的表达降低,MMP9的表达量增加。对成纤维细胞进行0.5Hz,10%的机械力拉伸12h及24h后,TGF-β1、P-P44/42、CTGF、Collagen I的表达均较未拉伸组增加,但两组间没有明显差异。Collagen III的表达在机械拉伸12小时后未出现明显改变,而拉伸24小时后可见明显升高。以P44/42抑制剂PD98059作用成纤维细胞,同时给予10%的机械力拉伸细胞12小时,可以发现PD98059能够降低机械力所引起的CTGF、Collagen I的增多,并增加MMP9的表达。结论:1.POP患者子宫主韧带中P-P44/42、TGF-β1、CTGF、Collagen I、Collagen III、TIMP1呈低表达,而MMP9呈高表达。2.TGF-β1可以通过P44/42 MAPK信号通路调节子宫主韧带成纤维细胞的增殖、凋亡以及CTGF、胶原及其相关蛋白的代谢。3.一定强度的机械力对成纤维细胞CTGF和胶原的代谢起到正向调节作用,并可通过P44/42 MAPK信号通路来调控表达,一定强度的机械拉伸对盆底支持组织起到促进胶原代谢的保护性作用。更多还原

【Abstract】 Objective: Pelvic organ prolapse(POP)is a major health problem that affects the quality of life of many women.It is caused by a variety of reasons,such as damage,aging,relaxation of pelvic floor support structure,and then the bulge of uterus and vaginal wall.With the decrease of pelvic organs,the symptoms of bladder,intestine and prolapse appeared.In addition to physical symptoms,it also has a negative impact on women’s mental health.It is a common disease in elderly women.As far as the predisposing factors are concerned,it is related to pregnancy and childbirth,but the specific pathogenesis is still unclear.As we all know,pelvic floor supporting tissue includes fascia,ligament and connective tissue around the pelvic floor,which plays an important role in pelvic support system.The quantity,structure and tissue of extracellular matrix(ECM)are the key factors of tissue stability.The anatomical and functional integrity of the pelvic floor is closely related to the pelvic floor connecting stent,which ensures the mechanical support and organ stability.Ligaments and muscles hang the vagina and uterus from the pelvis.This structure can be effectively compared with the structure of suspension bridge: when the muscles contract,the ligaments will stretch,making the pelvic organs form and firm.If the tissue does not have enough collagen skeleton to support it,the ligament will anchor on an unformed mass and the whole structure will collapse downward.More and more studies show that POP is related to the change of extracellular matrix.Some people think that the production of collagen and elastin in POP has changed.Some studies suggest that if the number of collagen fibers in the tissue is reduced,it can reduce the supporting force of the basin ligament,fascia and other tissues.However,it is not clear whether these changes are the result of pelvic floor dysfunction,or more precisely the cause of pelvic floor dysfunction.Collagen usually exists in the form of insoluble fiber,which is involved in maintaining tissue integrity.In addition,it can resist high tensile strength,which is an important factor to determine the toughness of connective tissue.The connective tissue of pelvic floor mainly includes collagen I(Col I)and collagen III(Col III).Collagen and matrix metalloproteinases(MMP)play a key role in the pathophysiological process of POP,because the conversion of Col I and Col III and the simultaneous activation of MMPs can be observed in the vaginal wall.It has been reported that the content of Col I and Col III in POP is decreased.Studies show that the increase of collagen degradation is the main reason for the decrease of collagen content in pop.P44 / 42 signaling pathway,namely ERK(extracellular signal regulated kinase)signaling pathway,is a member of MAPK signaling pathway.It has been proved that the increase of ERK1/2 activation leads to the increase of smooth muscle cell proliferation.It has also been proved that the decreased expression of Col I and Col III in POP vaginal tissue may be due to MAPK and NF-κ B signaling pathways involved in collagen synthesis and degradation.Transforming growth factor-β1(TGF-β1)is a known stimulator for fibroblasts to produce ECM protein,and mediates the response of fibroblasts to mechanical stress.The induced signal pathway is mainly mediated by Smads protein,but the current research shows that TGF-β1 can also be independent of Smads signal.In addition to the classical TGF-β1/Smad signaling pathway,TGF-β1 can also be activated by non Smad signaling pathways,such as MAPK signaling pathway,PI3K/Akt signaling pathway,etc.We speculate that P44/42 MAPK pathway may be involved in the mechanism of pelvic floor support tissue changes in patients with pelvic organ prolapse.In order to further study whether P4442 MAPK pathway is involved in TGF-β1 on the metabolism of CTGF and collagen in pelvic floor support structure,we designed and completed the following experiments.Methods: Using Western blot,immunohistochemistry and RT-PCR methods,25 cases of POP group and 25 cases of control group were selected,TGF-β1,P44/42,P-P44/42,Collagen I,Collagen III,MMP9,TIMP1 and CTGF were detected and compared between the two groups.The primary culture of fibroblasts from the cardinal ligament of uterus was carried out by adherence method and enzyme digestion method,and the fibroblasts were identified by immunocytochemistry.The fibroblasts were treated with TGF-β1 and its inhibitor SB431542.The proliferation and apoptosis of fibroblasts were detected by MTT Assay and flow cytometry.The expressions of P442,P-P442,CTGF,Collagen I,Collagen III and related proteins were detected by WB assay,the m RNA expressions of CTGF and Collagen-related protein were detected by RT-PCR,the secretion of CTGF,Collagen I and Collagen III were detected by Elisa assay.The fibroblasts were treated with P44/42 inhibitor PD98059,then exogenous TGF-β1 was added,cell proliferation and apoptosis were detected by MTT assay and flow cytometry assay,CTGF and collagen-related proteins were detected by WB assay,and m RNA expression was detected by RT-PCR.Flexcell-4000 flexible substrate loading system was used to mechanically stretch the primary 3-4 generation uterine cardinal ligament fibroblasts,and the cytoskeleton was observed by phalloidin staining.The fibroblasts were stretched by 10% mechanical stretch for 12 hours and 24 hours,TGF-β1,P44/42,P-P44/42,CTGF,Collagen I,Collagen III protein were detected by WB,and m RNA expression was detected by RT-PCR.The fibroblasts were treated with P44/42 inhibitor PD98059 and the fibroblasts were stretched by 10% mechanical stretch for 12 hours.The expressions of P44/42,P-P44/42,CTGF,Collagen I,MMP9 and TIMP1 were detected by WB and RT-PCR.Results: The expression of TGF-β1 and P-P44/42 in cardinal ligament of uterus in POP group was lower than that in control group.In POP group,the expression of MMP9 was higher than that in control group,but the expression of CTGF,Collagen I,Collagen III and TIMP1 was lower than that in control group.Compared with the control group,fibroblast proliferation and apoptosis were increased,and the relative expression of P-P44/42 was increased in primary culture and TGF-β1with 5 ng/ml exogenous addition,the expression of CTGF,Collagen I,Collagen III and Timp1 increased,while the expression of MMP9 decreased.Sb431542 could decrease the proliferation and apoptosis of fibroblasts,and the expression of P-P44/42 decreased,the expression of CTGF,Collagen I,Collagen III and TIMP1 decreased,while the expression of MMP9 increased.PD98059,an inhibitor of P44/42,was used to treat fibroblasts before TGF-β1 treatment.PD98059 could decrease the effect of TGF-1 on fibroblast proliferation and increase apoptosis,the expression of CTGF,Collagen I,Collagen III and TIMP1 decreased,while the expression of MMP9 increased.The TGF-β1,P-P44/42,CTGF and Collagen I expression were increased in fibroblasts treated with 0.5Hz,10% mechanical stretch for 12 and 24 hours,but there was no significant difference between the two groups.The expression of Collagen III did not change after 12 hours of mechanical stretch,but increased after 24 hours of mechanical stretch.P44/42 inhibitor PD98059 could inhibit the proliferation of CTGF and Collagen I and increase the expression of MMP9 in fibroblasts treated with 10% mechanical stretch for 12 hours.Conclusion: 1.The expression of P-P44/42,TGF-β1,CTGF,Collagen I,Collagen III and Timp1 in the cardinal uterine ligament of patients with POP was lower,while the expression of MMP9 was higher than control group.2.TGF-β1 can regulate the proliferation,apoptosis and the metabolism of connective tissue growth factor,Collagen and their related proteins through P44/42 MAPK signal pathway.3.The the mechanical stretch of certain intensity plays a positive regulation on the metabolism of CTGF and Collagen in fibroblasts,and the expression of CTGF and Collagen can be regulated by P44/42 MAPK signal pathway,a certain intensity of mechanical stretch on the pelvic floor support tissue to promote Collagen metabolism of the protective role.更多还原