子宫性不孕相关兔宫腔粘连模型及人离体子宫灌注研究
发布时间：2018-08-20 14:41
【摘要】：第一章电热损伤法建立新西兰大白兔宫腔粘连模型目的:宫腔粘连(Intrauterine adhesions,IUAs)指因为各种因素造成子宫内膜基底层损伤,在创伤愈合过程中,子宫内膜表面出现局部瘢痕或形成粘连带粘连在一起,造成宫腔部分或完全闭塞,临床表现为月经失调、盆腔痛等,严重者可能导致闭经、不孕、自发性流产等结果,对女性生育功能有着重大的影响,是导致女性不孕的重要因素。然而目前其发病机制及治疗方法仍未明确和统一,因此建立一个稳定有效的宫腔粘连动物模型,则成为开展相关研究的前提和基础。本文旨在利用机械损伤和电热损伤法构建兔IUA模型,观察比较两种方法的建模效果。方法:42只成年雌性新西兰大白兔随机分为机械损伤组和电热损伤组,每组21只。两组分别以机械刮宫和医用多功能高频电刀电灼损伤兔一侧子宫内膜模拟宫腔粘连形成,以另一侧子宫作为自身对照,分别比较损伤侧和对照侧子宫病理变化、腺体数量、纤维化面积及胚胎个数,评估两种方法建模效果及造成的子宫内膜损伤对兔生育力的影响。其中机械损伤组兔的损伤侧子宫为M-A组,对照侧子宫为M-B组;电热损伤组兔的损伤侧子宫为E-A组,对照侧子宫为E-B组。分别在损伤后7d、14d和28d收集兔双侧子宫组织,行HE和Massoon染色观察两侧子宫内膜病理改变,并对两侧子宫内膜的腺体个数和内膜纤维化面积比进行统计学分析和比较。另外将损伤后7d的雌兔与成年雄兔合笼,14天后观察比较两侧子宫胚胎个数。结果:两种建模方法均未见明显纤维粘连带形成。病理组织学观察显示机械损伤组术后7d,M-A组损伤内膜腺体数量较B组减少,间质水肿、白细胞浸润,毛细血管充血、阻塞,可见少量新生毛细血管;术后14d和30d,M-A组和M-B组子宫内膜切片HE染色镜下显示并没有明显的差异,损伤后的子宫内膜已完全修复。电热损伤组建模术后7d,与E-B组相比,E-A组宫腔皱襞减少,表面稍平坦,基本被柱状上皮细胞覆盖,腺体数量明显减少,间质水肿、白细胞浸润,毛细血管充血、阻塞,可见少量新生毛细血管;术后14d,E-A组宫腔表面皱襞仍少,表面较平坦,上皮基本全部被柱状上皮细胞覆盖,腺体个数仍较少,间质水肿不再明显,可见少量白细胞浸润,新生毛细血管增多;术后28d,E-A组子宫内膜形态学基本恢复正常,可见较多新生腺体,未见间质充血和淋巴细胞浸润。机械组损伤后7d,M-A组子宫内膜腺体数量较M-B组减少,差异有统计学意义(P0.05);机械损伤后14d、30d,M-A组子宫内膜腺体数量与M-B组相比差异无统计学意义。电热损伤后7d和14d,E-A组子宫内膜腺体数量较E-B组减少,差异均有统计学意义(P0.05);而电热损伤后30d E-A组子宫内膜腺体数量与E-B组相比差异无统计学意义。机械损伤后7d,M-A组内膜纤维化面积比与M-B组相比差异无统计学意义;电热损伤后7d,E-A组内膜纤维化面积比较E-B组增大,差异有统计学意义(P0.05)。M-A组子宫胚胎个数与M-B组相比差异无统计学意义;E-A组子宫胚胎个数较E-B组减少,差异有统计学意义(P0.05)。结论:利用机械损伤法很难建立稳定有效的兔宫腔粘连模型。尽管未见明显纤维粘连带形成,电热损伤造成的胚胎个数的减少说明采用电热损伤法建立的兔宫腔粘连模型在损伤后7-14天是稳定有效的。第二章人离体子宫冷灌注及冷缺血保存的安全区间目的:离体子宫保存是子宫移植过程中需要解决的一项关键问题,良好科学的子宫灌注和保存方法是保证子宫移植成功的重要前提。本文旨在探讨人离体子宫利用组氨酸-色氨酸-酮戊二酸盐(Histidine-tryptophane-ketoglutarate,HTK)液冷灌注及冷缺血保存的安全区间,为子宫移植的相关研究及临床应用提供理论依据和参考。方法:样本为人离体子宫7例,来源于行子宫切除术的宫颈癌患者。其中3例用HTK液在低温条件下经子宫动脉灌注,测量并记录一定灌注压力所对应的灌注高度,建立灌注高度与灌注压之间的数学关系模型;4例在适当灌注高度下进行短时间冷灌注,然后立刻取子宫内膜及肌组织分别于HTK保存液和生理盐水(normal saline,NS)中保存,两组又根据保存时间(0、3、6、24h)的不同分为 Oh 组、HTK 3h 组、HTK 6h 组、HTK 24h 组、NS 3h 组、NS 6h 组和 NS 24h组,利用光学和电子显微镜观察各组子宫组织细胞形态学改变,测定并比较各组子宫平滑肌收缩能力,分析离体子宫短时灌注后冷缺血保存的最佳时限。结果:灌注压90～140mmHg对应的灌注高度为62～122cm,理论灌注高度H与灌注压P关系公式为H(m)=(?)(?)。与Oh组比较,HTK3h组和HTK6h组的子宫组织在光学和电子显微镜下形态学结构均未见明显改变,但HTK24h组及所有的NS组的子宫组织均出现不同程度的细胞水肿、细胞间失去联系、线粒体肿胀及染色质粗染等不可逆的退行性改变;HTK3h、HTK6h和HTK24h三组子宫肌组织的肌收缩力差异无统计学意义(P=0.772);而与相同时段HTK组标本相比,NS3h、6h和24h组子宫肌组织的肌收缩力均明显降低(P0.05)。结论:人类离体子宫组织在低温环境下利用HTK液灌注的安全高度是62～122cm,理论灌注高度H与灌注压P关系模型公式为H(m)=(?)人离体子宫短时灌注后于4℃ HTK液中保存可耐受冷缺血时间至少6h。
[Abstract]:Chapter 1 Establishment of intrauterine adhesions (IUAs) model in New Zealand rabbits by electrothermal injury Objective: Intrauterine adhesions (IUAs) refers to the injury of endometrial basal layer caused by various factors. In the process of wound healing, local scars or adhesion bands appear on the surface of endometrium, resulting in partial or complete occlusion of the uterine cavity. Clinical manifestations are menstrual disorders, pelvic pain, etc. Severe cases may lead to amenorrhea, infertility, spontaneous abortion and other results, which have a significant impact on female reproductive function, is an important factor leading to female infertility. Methods: 42 adult female New Zealand white rabbits were randomly divided into mechanical injury group and electrothermal injury group with 21 rabbits in each group. The pathological changes, the number of glands, the area of fibrosis and the number of embryos on the injured side and the control side of the uterus were compared, and the effects of the two methods on the fertility of rabbits were evaluated. The injured uterus of mechanically injured rabbits was M-A group, the control uterus was M-B group, the injured uterus of electrothermal injured rabbits was E-A group and the control uterus was E-B group. The bilateral uterus tissues of rabbits were collected 7 days, 14 days and 28 days after injury, and the pathological changes of endometrium on both sides were observed by HE and Mason staining. The number of embryos on both sides of the uterus was observed and compared 14 days later. Results: No obvious fibrous adhesions were found in the two modeling methods. Histopathological observation showed that the number of injured endometrial glands in the M-A group was 7 days after operation. Compared with group B, there were less interstitial edema, leukocyte infiltration, capillary congestion, obstruction and a small number of new capillaries. At 14 and 30 days after operation, there was no significant difference between M-A group and M-B group. The injured endometrium was completely repaired by HE staining. Less, slightly flat surface, basically covered by columnar epithelial cells, glands significantly reduced in number, interstitial edema, leukocyte infiltration, capillary congestion, obstruction, visible a small number of new capillaries; 14 days after surgery, the E-A group of uterine cavity surface folds are still less, the surface is flat, epithelial cells are basically covered by columnar epithelial cells, the number of glands is still less, stroma. Edema was no longer evident, a small amount of leukocyte infiltration and neocapillary increased; 28 days after surgery, endometrial morphology of E-A group returned to normal, more neonatal glands were seen, no interstitial congestion and lymphocyte infiltration. 7 days after injury, the number of endometrial glands in M-A group was significantly lower than that in M-B group (P 0.05). The number of endometrial glands in E-A group was significantly lower than that in E-B group at day 7 and 14 after electrothermal injury (P 0.05), but there was no significant difference between E-A group and E-B group at day 30 after electrothermal injury. On the 7th day, there was no significant difference between M-A group and M-B group in the area ratio of endometrial fibrosis; on the 7th day after electrothermal injury, the area of endometrial fibrosis in E-A group was larger than that in E-B group (P 0.05). CONCLUSION: It is difficult to establish a stable and effective rabbit model of intrauterine adhesions by mechanical injury. Although no obvious fibrous adhesions were observed, the reduction of embryo number caused by electrothermal injury indicates that the rabbit model of intrauterine adhesions established by electrothermal injury is stable and effective 7-14 days after injury. Chapter 2 Cold perfusion and cold perfusion of human uterus in vitro Objective: Ischemic preservation of uterus in vitro is a key problem to be solved in the process of uterine transplantation. A good and scientific method of uterine perfusion and preservation is an important prerequisite for the success of uterine transplantation. Methods: Seven human uterus specimens were collected from 7 patients with cervical cancer who underwent hysterectomy. Three of them were perfused with HTK solution through uterine artery under hypothermia condition, and the perfusion was measured and recorded. The mathematical model of the relationship between the perfusion height and the perfusion pressure was established according to the perfusion height corresponding to the pressure; 4 cases were perfused with cold perfusion for a short time at a suitable perfusion height, and then the endometrium and muscle tissue were immediately stored in HTK solution and normal saline (NS), respectively. The two groups were divided into two groups according to the storage time (0, 3, 6, 24 h). Oh group, HTK 3H group, HTK 6h group, HTK 24h group, NS 3H group, NS 6h group and NS 24h group were used to observe the morphological changes of uterine cells in each group by optical and electron microscopy. The contractile ability of uterine smooth muscle in each group was measured and compared, and the optimal time limit of cold ischemia preservation after short-term perfusion was analyzed. Compared with Oh group, the uterine tissue of HTK3h group and HTK6h group had no obvious morphological changes under optical and electron microscopy, but the uterine tissue of HTK24h group and all NS groups had different degrees of cell edema and intercellular loss. There was no significant difference in the contractility of uterine myometrium between HTK3h, HTK6h and HTK24h groups (P = 0.772), but the contractility of uterine myometrium in NS3h, 6h and 24h groups was significantly lower than that in HTK group (P 0.05). The safe height of tissue perfused with HTK solution at low temperature was 62-122 cm. The relationship between theoretical perfusion height H and perfusion pressure P was modeled as H(m)=(?) The tissue could be preserved in HTK solution at 4 (?) for at least 6 hours after short-term perfusion of human uterus.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R711.6
[Abstract]:Chapter 1 Establishment of intrauterine adhesions (IUAs) model in New Zealand rabbits by electrothermal injury Objective: Intrauterine adhesions (IUAs) refers to the injury of endometrial basal layer caused by various factors. In the process of wound healing, local scars or adhesion bands appear on the surface of endometrium, resulting in partial or complete occlusion of the uterine cavity. Clinical manifestations are menstrual disorders, pelvic pain, etc. Severe cases may lead to amenorrhea, infertility, spontaneous abortion and other results, which have a significant impact on female reproductive function, is an important factor leading to female infertility. Methods: 42 adult female New Zealand white rabbits were randomly divided into mechanical injury group and electrothermal injury group with 21 rabbits in each group. The pathological changes, the number of glands, the area of fibrosis and the number of embryos on the injured side and the control side of the uterus were compared, and the effects of the two methods on the fertility of rabbits were evaluated. The injured uterus of mechanically injured rabbits was M-A group, the control uterus was M-B group, the injured uterus of electrothermal injured rabbits was E-A group and the control uterus was E-B group. The bilateral uterus tissues of rabbits were collected 7 days, 14 days and 28 days after injury, and the pathological changes of endometrium on both sides were observed by HE and Mason staining. The number of embryos on both sides of the uterus was observed and compared 14 days later. Results: No obvious fibrous adhesions were found in the two modeling methods. Histopathological observation showed that the number of injured endometrial glands in the M-A group was 7 days after operation. Compared with group B, there were less interstitial edema, leukocyte infiltration, capillary congestion, obstruction and a small number of new capillaries. At 14 and 30 days after operation, there was no significant difference between M-A group and M-B group. The injured endometrium was completely repaired by HE staining. Less, slightly flat surface, basically covered by columnar epithelial cells, glands significantly reduced in number, interstitial edema, leukocyte infiltration, capillary congestion, obstruction, visible a small number of new capillaries; 14 days after surgery, the E-A group of uterine cavity surface folds are still less, the surface is flat, epithelial cells are basically covered by columnar epithelial cells, the number of glands is still less, stroma. Edema was no longer evident, a small amount of leukocyte infiltration and neocapillary increased; 28 days after surgery, endometrial morphology of E-A group returned to normal, more neonatal glands were seen, no interstitial congestion and lymphocyte infiltration. 7 days after injury, the number of endometrial glands in M-A group was significantly lower than that in M-B group (P 0.05). The number of endometrial glands in E-A group was significantly lower than that in E-B group at day 7 and 14 after electrothermal injury (P 0.05), but there was no significant difference between E-A group and E-B group at day 30 after electrothermal injury. On the 7th day, there was no significant difference between M-A group and M-B group in the area ratio of endometrial fibrosis; on the 7th day after electrothermal injury, the area of endometrial fibrosis in E-A group was larger than that in E-B group (P 0.05). CONCLUSION: It is difficult to establish a stable and effective rabbit model of intrauterine adhesions by mechanical injury. Although no obvious fibrous adhesions were observed, the reduction of embryo number caused by electrothermal injury indicates that the rabbit model of intrauterine adhesions established by electrothermal injury is stable and effective 7-14 days after injury. Chapter 2 Cold perfusion and cold perfusion of human uterus in vitro Objective: Ischemic preservation of uterus in vitro is a key problem to be solved in the process of uterine transplantation. A good and scientific method of uterine perfusion and preservation is an important prerequisite for the success of uterine transplantation. Methods: Seven human uterus specimens were collected from 7 patients with cervical cancer who underwent hysterectomy. Three of them were perfused with HTK solution through uterine artery under hypothermia condition, and the perfusion was measured and recorded. The mathematical model of the relationship between the perfusion height and the perfusion pressure was established according to the perfusion height corresponding to the pressure; 4 cases were perfused with cold perfusion for a short time at a suitable perfusion height, and then the endometrium and muscle tissue were immediately stored in HTK solution and normal saline (NS), respectively. The two groups were divided into two groups according to the storage time (0, 3, 6, 24 h). Oh group, HTK 3H group, HTK 6h group, HTK 24h group, NS 3H group, NS 6h group and NS 24h group were used to observe the morphological changes of uterine cells in each group by optical and electron microscopy. The contractile ability of uterine smooth muscle in each group was measured and compared, and the optimal time limit of cold ischemia preservation after short-term perfusion was analyzed. Compared with Oh group, the uterine tissue of HTK3h group and HTK6h group had no obvious morphological changes under optical and electron microscopy, but the uterine tissue of HTK24h group and all NS groups had different degrees of cell edema and intercellular loss. There was no significant difference in the contractility of uterine myometrium between HTK3h, HTK6h and HTK24h groups (P = 0.772), but the contractility of uterine myometrium in NS3h, 6h and 24h groups was significantly lower than that in HTK group (P 0.05). The safe height of tissue perfused with HTK solution at low temperature was 62-122 cm. The relationship between theoretical perfusion height H and perfusion pressure P was modeled as H(m)=(?) The tissue could be preserved in HTK solution at 4 (?) for at least 6 hours after short-term perfusion of human uterus.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R711.6
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