促进香猪子宫内膜损伤后功能性修复的实验研究

发布时间：2018-03-12 14:24

本文选题：胶原支架　切入点：碱性成纤维生长因子　出处：《南京大学》2014年硕士论文　论文类型：学位论文

【摘要】：研究背景：在临床上,感染或宫腔操作均可引起子宫内膜损伤,造成内膜再生障碍,引起宫腔粘连、闭经等,并成为继发性不孕重要原因之一。然而现阶段的治疗方法只能部分改善粘连,对内膜的重建再生并没有有效的手段。随着组织工程的研究深入,最新的组织工程材料为修复受损内膜提供了新的治疗前景。本课题组利用胶原支架复合bFGF在大鼠子宫全层损伤修复中得到令人兴奋的结果。本研究将着重于建立小型香猪子宫内膜损伤模型以及利用组织工程方法对损伤后内膜进行修复。第一章胶原支架复合骨髓间充质干细胞和胶原复合碱性成纤维生长因子对小型香猪子宫壁全层损伤后新鲜创面修复作用目的：研究空白胶原支架,复合bFGF胶原支架,复合香猪BMSC胶原支架对小型香猪子宫全层2cm×1cm机械性损伤创面的修复作用。方法：本研究包括小型香猪39只,共分为四组：自然修复组(子宫损伤后不进行干预修复),胶原支架/PBS组(子宫损伤后用空白胶原膜修复),胶原支架/CBD-bFGF组(子宫损伤后用复合CBD-bFGF的胶原支架修复),胶原支架/BMSC组,(子宫损伤后用复合小型香猪骨髓间充质干细胞的胶原支架修复),每组再分为切片组和生育组(单侧损伤和双侧损伤)。术后90天开腹观察子宫修复状况,有无外翻,腹腔粘连程度,宫腔是否通畅等。将无外翻、创面修复完全的子宫取出,进行组织学检测,包括HE染色,免疫组化染色等。结果：(1)各组子宫均出现不同程度内膜外翻,以自然修复组(90.3%)尤为严重,其余各组分别为空白胶原膜组(55%),胶原支架/CBD-bFGF组(63%),胶原支架／BMSC组(61%),各胶原支架修复组创面愈合率间无显著性差异；(2)对比单侧损伤和双侧损伤,除自然修复组外(创面愈合率分别为9.7%和0),其余各组均为单侧修复率高于双侧,空白胶原膜(66.7%和25%),胶原支架/CBD-bFGF组(36.7%和16.7%),胶原支架/BMSC组(44.4%和37.5%)；(3)HE和免疫组化显示：胶原支架/PBS、胶原支架/CBD-bFGF组和胶原支架/BMSC组的肌层厚度优于自然修复组,但三组两者之间没有明显差异。子宫内膜厚度四组之间没有显著性差异。结论：当前采用的香猪子宫全层损伤大小及手术方式不适合进一步的干预研究,需进一步探究减轻手术部位炎症反应的方法；也需要探索香猪子宫全层损伤合适的大小。以确定能够反应子宫损伤干预效果的香猪模型。第二章建立稳定有效的小型香猪子宫内膜损伤模型目的：建立合适的大动物子宫内膜损伤模型为进一步探究临床组织工程修复子宫内膜奠定基础。方法：性成熟小型香猪4只麻醉后,暴露双侧子宫,剪开三面创口,创面大小分组设置为：长为2cm、3cm、4cm,宽为约管径1/2-2/3。根据子宫长度,每侧设置2-3个创面。分离浆膜与内膜,剪去内膜并将浆膜缝合于创缘。术后8周和12周分别开腹观察子宫与周围组织粘连情况,创面修复效果,管腔是否塌陷(通畅度)。将创面愈合的子宫取出并进行组织学检测,包括HE染色和免疫组化染色。结果：(1)改进后的模型相对于子宫全层损伤模型,腹腔黏连严重程度明显减轻,即使有少量创面粘连,也多为易于分离的疏松膜性粘连；克服了创面内膜外翻,不愈合等情况。所有术后的子宫管腔均通畅。(2)术后8周,2cm、3cm的创面修复已有新生内膜组织包括：有一定内膜厚度,有血管和腺体形成,还有少量环形肌纤维长入。(3)术后12周,2cm创面修复已近于正常组织；3cm创面内膜厚度、血管数量低于2cm创面,腺体结构不成熟；4cm创面挛缩,管腔塌陷,仅有单层上皮细胞形成,肌层结构紊乱,纤维增生显著,瘢痕化明显。结论：通过改进后的手术方法能够建立较为稳定成熟的小型香猪子宫内膜损伤模型。
[Abstract]:Background: in clinical infection, or intrauterine operation can cause endometrial damage, causing intimal aplasia caused by intrauterine adhesions, amenorrhea, and become one of the important causes of secondary infertility. However only treatment at this stage of the improved adhesion, reconstruction and regeneration of endometrium. With no effective means of tissue engineering provides in-depth, promising new organization of the new engineering materials for repair of damaged endometrium. The research group using collagen scaffolds bFGF get exciting results in rat uterus full-thickness injury repair. This study will focus on the establishment of miniature pig endometrial damage model and using tissue engineering methods of damage after the endometrial repair. The first chapter collagen scaffold combined with bone marrow mesenchymal stem cells and collagen combined with basic fibroblast growth factor on uterine wall injury in minipigs After the repair effect of fresh tissue Objective: To study the blank composite collagen scaffold, bFGF scaffold, BMSC scaffold composite repair effect of pig of miniature pig uterus total layer 2cm * 1cm mechanical injury wound. Methods: This study included 39 miniature pigs were divided into four groups: natural repair group (uterine injury is not intervention), repair of collagen scaffold /PBS group (uterine injury with blank collagen membrane repair, collagen scaffold (/CBD-bFGF group) after uterine injury with collagen scaffold to repair composite CBD-bFGF), group /BMSC (composite collagen scaffold, miniswines bone marrow mesenchymal stem cells with collagen scaffold to repair the uterus after injury, n = 16) as the slice group and the control group (unilateral and bilateral injury injury). After 90 days of open observation of uterine repair status, there is no valgus, the degree of adhesion, intrauterine patency. No valgus, wound repair complete uterus take Out for histological examination, including HE staining, immunohistochemical staining. Results: (1) there are different degrees of uterus intimal eversion, the natural repair group (90.3%) is particularly serious, the remaining groups were divided into blank collagen membrane group (55%), /CBD-bFGF group (63%) collagen scaffolds, collagen scaffold / group BMSC (61%), the collagen scaffold to repair the wound healing group had no significant difference between the rate; (2) comparison of unilateral injury and bilateral injury, in addition to the natural repair group (wound healing rates were 9.7% and 0), the other groups were unilateral repair rate is higher than the double side blank, collagen membrane (66.7% and 25% /CBD-bFGF group), collagen scaffold (36.7% and 16.7%), group /BMSC collagen scaffold (44.4% and 37.5%); (3) HE and immunohistochemistry showed that: /PBS collagen scaffold, muscle layer thickness is better than that of /CBD-bFGF group and collagen scaffold collagen scaffold /BMSC group natural recovery group, but no significant differences between the three groups. Both the uterus The endometrial thickness had no significant difference between the four groups. Conclusion: the pig uterus full-thickness injury size and operation mode is not suitable for further intervention studies, further explore methods to reduce surgical site inflammation; also need to explore the size of full-thickness damage suitable pig uterus. To determine the effect of pig model can uterine injury intervention the reaction. The second chapter is to establish a stable and effective miniswines endometrial injury model objective: to establish an animal model of endometrial lesion suitable for further clinical study of tissue engineering to repair endometrium foundation. Methods: 4 anesthetized mature miniature pigs after exposure of bilateral uterine, cut three wound, wound size packet set as long as 2cm: 3cm, 4cm, width of about 1/2-2/3. diameter according to the length of uterus, each side set up 2-3 wounds. Separating the serosa and intima, cut in film and The suture to the wound edge. Serous after 8 weeks and 12 weeks were performed to observe the uterine adhesion with surrounding tissue, the effect of wound repair, lumen collapse (patency). The wound healing of uterus and was removed for histological examination, including HE staining and immunohistochemical staining. Results: (1) improved the model is compared with the whole layer of the uterus damage model, adhesion severity significantly reduced, even if there is a small wound adhesion, mostly for loose membrane adhesion easy separation; overcome the wound healing without intimal eversion. All postoperative uterine lumen was unobstructed. (2) 8 weeks after surgery, 2cm 3cm has wound repair of neointimal tissue including: certain endometrial thickness with blood vessels and glands, as well as a small amount of circular muscle fiber ingrowth. (3) after 12 weeks, 2cm has been close to the normal tissue wound repair; wound 3cm endometrial thickness, the number of blood vessels below 2cm wound, Glandular structure is not mature; 4cm wound contracture, lumen collapse, only a monolayer of epithelial cells, muscle layer structure disorder, fiber hyperplasia, scarring significantly. Conclusion: the modified surgical approach to establish minipig endometrial damage model is more stable and mature.

【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R711.74

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