【作者】 肖椰蕾；

【导师】 赵渝；

【作者基本信息】 重庆医科大学 ， 外科学（专业学位）， 2023， 博士

【摘要】 背景使用补片进行腹外疝修补术已经成为国际公认的手术标准。虽然补片已经成为目前应用最为广泛的补片材料之一,但它仍存在不可避免的弊端,如引起脏器粘连、肠梗阻,甚至肠外瘘等。为了优化聚丙烯补片聚丙烯的不足,提升其生物相容性,“改良的”聚丙烯补片被不断的开发出来,如在聚丙烯表面添加钛涂层。这种钛化聚丙烯补片不仅保持了聚丙烯补片原有的轻质、柔软,还改善了它的亲水性和亲细胞性等特性。然而,目前针对钛化聚丙烯补片的实验研究还甚少且富有争议。为了更全面的认识钛化聚丙烯补片,我们开展了临床实验、动物实验以及细胞实验三个层面的研究。方法第一部分的回顾性临床实验,收集重庆医科大学附属第一医院疝外科,使用补片进行腹股沟疝修补术的患者病例资料并进行随访,分析比较了倾向性得分匹配后的患者的围术期、并发症、疼痛以及生活质量等资料。受临床实际限制,这一部分中所纳入比较的补片是轻型钛化聚丙烯补片与两款临床上常用的两种聚丙烯补片(Lichtenstein术中的普通聚丙烯补片与TAPP术中的3D聚丙烯补片),而在后续的动物与细胞实验中,选择了临床研究中与轻型钛化聚丙烯补片物理参数十分相近的普通聚丙烯补片作为横向比较。不仅如此,同时纳入了同一制造商生产的超轻型和中重型钛化聚丙烯补片作为纵向比较,以研究不同载钛量对实验结果的影响。第二部分的动物实验,以新西兰白兔为动物模型,通过在其腹内斜肌与腹外斜肌间、腹膜壁层(前腹壁)、降结肠浆膜层和股神经下方安置补片,以观察补片的抗收缩性能、抗粘连性能、炎症水平以及对神经的影响,并判断钛涂层是否改善了聚丙烯材料的生物组织相容性及其作用规律。第三部分实验中,我们对第二部分中所观察到的补片收缩现象进行机制性的探索。通过动物腹膜标本与细胞实验,观察了补片对腹膜组织和腹膜间皮细胞(Human Peritoneal Mesothelial cells,HMr SV5)转化生长因子β1(Transform growth factorβ,TGF-β1)表达的影响,同时模拟了炎症诱导的HMr SV5上皮间质化(Epithelial-mesenchymal transition,EMT)的发生,并尝试通过TAK1(TGF-beta-activated kinase-1)信号通路来解释补片影响HMr SV5 EMT的机制。最后,第四部分的细胞实验模拟了疝修补术的细胞环境,通过对细胞增殖与凋亡进行检测,判断补片对细胞生长的影响,并在共聚焦显微镜和电镜下直接确认了细胞在补片表面的粘附与生长情况。结果第一部分的临床数据提示,不论是在Lichtenstein术还是经腹腹膜前疝修补术(Transabdominal Preperitoneal Prosthesis,TAPP)的围手术期,轻型钛化聚丙烯补片并没有明显的优势。但在TAPP术后3个月至1年内,使用轻型钛化聚丙烯补片的患者在活动状态下出现异物感和慢性疼痛的概率更少,尽管1年后这种优势已不再明显。此外,在TAPP术后,使用轻型钛化聚丙烯补片的患者在生活质量评分中的Vitality、Social Functioning和Reported Health Transition方面具有一定的优势性。在第二部分的动物体内实验中,观察到轻型钛化聚丙烯补片较普通聚丙烯补片在抗收缩率性能和抗粘连性能方面均具有不同程度的优势,并且不同载钛量的钛化聚丙烯补片的表现呈现规律性的变化。此外,我们还发现钛涂层的添加能减轻聚丙烯材料对神经的损伤。第三部分的实验结果表明,相较于普通聚丙烯补片,轻型钛化聚丙烯补片可以降低TGF-β1在组织和细胞中的表达。同时,我们成功地以激活的人单核/巨噬细胞诱导了HMr SV5发生EMT,并发现补片会诱使HMr SV5 EMT程度的加重,但轻型钛化聚丙烯补片的程度略轻于普通聚丙烯补片。结合所观察到的现象,我们选择了与TGF-β1和EMT两者均有关联性的TAK1信号通路展开了研究,并观察到钛化聚丙烯补片在p-TAK1、p-JNK和p-p38蛋白表达中的优势,我们认为钛化聚丙烯补片可以通过影响TAK1信号通路进而调控HMr SV5的EMT。第四部分细胞实验模拟了疝修补术的细胞环境,并提示了钛涂层的添加虽然抑制了细胞的增殖,但也减轻了细胞的凋亡程度,即降低了聚丙烯材料对细胞生长的刺激。加上钛元素本身的“生物惰性”与“亲水亲细胞性”,使得细胞能更多的粘附于钛化聚丙烯补片表面,进而更快的形成单层间皮细胞层,并且随载钛量的增加细胞粘附数量越多。结论临床研究提示,轻型钛化聚丙烯补片在TAPP术后3个月至1年内,能够减少患者活动状态下的异物感和慢性疼痛,并且提高了患者某些方面的生活质量。动物及细胞实验提示了,钛化聚丙烯补片的钛涂层能在一定程度上改善聚丙烯材料某些方面的生物相容性,并且不同载钛量的钛化聚丙烯补片的作用结果不同。其在炎症水平、抗收缩率性能和抗粘连性能方面较聚丙烯补片均具有不同程度的优势,并且能减轻聚丙烯材料对神经的损伤。同时,钛涂层能可以降低聚丙烯引起的组织与细胞中TGF-β1的表达,虽然钛化聚丙烯补片同样会诱使腹膜间皮细胞EMT程度的加重,但其程度略轻于聚丙烯补片,并且这可能是通过影响TAK1信号通路而实现的。我们推断这也可能是钛化聚丙烯补片抗收缩机理的一部分。不仅如此,钛涂层的添加还会改变聚丙烯材料对细胞增殖和凋亡造成影响,加上钛的“生物惰性”与“亲细胞亲水性”,使得细胞在其表面的粘附更佳,能更快的形成单层间皮细胞层,并且我们推测这可能与钛化聚丙烯补片造成的粘连更少相关。更多还原

【Abstract】 Background The use of mesh in hernia repair surgery has become an internationally recognized surgical standard.While polypropylene mesh is among the most widely used materials,it still has drawbacks such as organ adhesions,intestinal obstruction,and extraintestinal fistulas.To improve the biocompatibility of polypropylene mesh,titanized polypropylene mesh has been developed.This mesh not only maintains the lightweight and softness of polypropylene,but also improves its hydrophilicity and cell affinity.However,there is limited and controversial experimental research on titanized polypropylene mesh.In order to gain a more comprehensive understanding of its properties,we conducted clinical trials,animal experiments,and cell experiments.MethodIn the first part,we collected data on patients who underwent inguinal hernia repair using meshes at the First Affiliated Hospital of Chongqing Medical University and followed up to analyze and compare perioperative,complications,pain,and postoperative quality of life data after propensity score matching.Due to clinical limitations,this section includes a comparison between lightweight titanized polypropylene mesh and two commonly used polypropylene meshes in clinical practice(ordinary polypropylene mesh in Lichtenstein surgery and 3D polypropylene mesh in TAPP surgery).In subsequent animal and cell experiments,we have chosen the ordinary polypropylene mesh in first part with physical parameters similar to that of the lightweight titanized polypropylene mesh as a horizontal comparison.Additionally,we included titanized polypropylene meshes with different titanium loadings by the same manufacturer as a vertical comparison.In the second part of the animal experiment,we used New Zealand white rabbits as animal models and placed different meshes between the external oblique abdominal muscle and the internal oblique abdominal muscle,in the parietal peritoneum(anterior abdominal wall),on the serous layer of the descending colon,and on the underside of the femoral nerve to observe their anti-contractility,anti-adhesiveness,inflammation levels,and effect on nerves.We assessed whether titanium coating improved the biocompatibility of polypropylene mesh and its regularity.In the following third part of the experiment,we attempted to explore the mechanisms underlying the observed contraction phenomenon in the second part.We observed the effect of the meshes on the peritoneal tissue and peritoneal mesothelial cell(Human Peritoneal Mesothelial cells,HMr SV5)expression of TGF-β1(Transform growth factorβ,TGF-β1)through animal specimens and cell experiments.We also simulated inflammation-induced epithelial-mesenchymal transition(Epithelialmesenchymal transition,EMT)of HMr SV5 and attempted to explain the mechanism of the mesh’s effect on EMT through the TAK1 signaling pathway.In the final part,we simulated the cell environment of hernia repair surgery and assessed the effect of the mesh on cell through the detection of proliferation and apoptosis.We directly confirmed the cell adhesion and growth status under a confocal microscope and electron microscope.ResultsIn the first retrospective clinical trial,the data showed that lightweight titanized polypropylene mesh has no significant advantage in the perioperative period,either Lichtenstein surgery or TAPP surgery.However,within 3 months to 1 year after TAPP surgery,patients who used lightweight titanized polypropylene mesh have a lower probability of foreign body sensation and chronic pain during activity,although this advantage was no longer significant after 1 year.In addition,after TAPP surgery,patients who used lightweight titanized polypropylene mesh may have certain advantages in Vitality,Social Functioning,and Reported Health Transition in terms of their quality of life.In the second part animal experiments,we observed that the lightweight titanized polypropylene mesh had varying degrees of advantages in terms of anti-shrinkage and anti-adhesion,and the performance of titanized polypropylene mesh with different titanium loadings showed regular changes.Additionally,we found that the addition of titanium can reduce the nerve damage caused by polypropylene.The third part of the experiment results showed that,compared with the polypropylene mesh,the lightweight titanized polypropylene mesh reduced the expression of TGF-β1 in tissues and cells.At the same time,we successfully induced HMr SV5 to undergo EMT by co-culture with activating monocyte/macrophages and found that the mesh aggravated the degree of EMT,but the degree of lightweight titanized polypropylene mesh was slightly lighter than that of polypropylene mesh.Based on the observed phenomena,we chose to explore the TAK1 signaling pathway,which is associated with both TGF-β1 and EMT,and observed the advantages of titanized polypropylene mesh in the expression of p-TAK1,p-JNK,and p-p38 proteins,which suggests that titanized polypropylene mesh may affect the EMT of HMr SV5 by influencing the TAK1 signaling pathway.The final part of the cell experiment simulated the cellular environment of hernia repair surgery and showed that although the addition of titanium coating inhibited cell proliferation,it also reduced the degree of cell apoptosis,that is,reduced the stimulation of polypropylene material on cell growth.In addition to the “biological inertia”and “hydrophilic cellphilicity” of titanium element,cells showed more likely to adhere to the surface of the titanized polypropylene mesh,and more quickly form a single layer of mesothelial cell layer,and the number of adherent cells increased with the increase of titanium loadings.ConclusionOur clinical studies suggest that lightweight polypropylene mesh reduced foreign body sensation and chronic pain in patients during 3 months to 1year after TAPP surgery,and improve certain aspects of their quality of life.Then,through animal and cell experiments,we think that the titanium coating can improve the biocompatibility of certain aspects of the polypropylene mesh to a certain extent,and the effect of titanized polypropylene mesh with different titanium loadings is different.It has varying degrees of advantages over polypropylene mesh in inflammation level,anti-shrinkage performance,and anti-adhesion performance,and is more likely to reduce the nerve damage by polypropylene mesh.In addition,titanized polypropylene mesh can reduce the expression of TGF-β1 in tissue,although it also induces an increase in the degree of HMr SV5 EMT,the degree of which is slightly lower than that of polypropylene mesh,and this may be achieved by affecting the TAK1 signaling pathway.We conclude that this may also be a part of the anti-shrinkage mechanism of titanated polypropylene mesh.Moreover,the addition of titanium coating also affects cell proliferation and apoptosis,with the addition of “biological inertia” and “hydrophilic cellphilicity” of titanium element,cells showed more likely to adhere to the surface of the titanized polypropylene mesh,and more quickly form a single layer of mesothelial cell layer.And we speculate that this may be related to less adhesion caused by titanated polypropylene meshes.更多还原