【摘要】：第一章電熱損傷法建立新西蘭大白兔宮腔粘連模型目的:宮腔粘連(Intrauterine adhesions,IUAs)指因為各種因素造成子宮內(nèi)膜基底層損傷,在創(chuàng)傷愈合過程中,子宮內(nèi)膜表面出現(xiàn)局部瘢痕或形成粘連帶粘連在一起,造成宮腔部分或完全閉塞,臨床表現(xiàn)為月經(jīng)失調(diào)、盆腔痛等,嚴(yán)重者可能導(dǎo)致閉經(jīng)、不孕、自發(fā)性流產(chǎn)等結(jié)果,對女性生育功能有著重大的影響,是導(dǎo)致女性不孕的重要因素。然而目前其發(fā)病機(jī)制及治療方法仍未明確和統(tǒng)一,因此建立一個穩(wěn)定有效的宮腔粘連動物模型,則成為開展相關(guān)研究的前提和基礎(chǔ)。本文旨在利用機(jī)械損傷和電熱損傷法構(gòu)建兔IUA模型,觀察比較兩種方法的建模效果。方法:42只成年雌性新西蘭大白兔隨機(jī)分為機(jī)械損傷組和電熱損傷組,每組21只。兩組分別以機(jī)械刮宮和醫(yī)用多功能高頻電刀電灼損傷兔一側(cè)子宮內(nèi)膜模擬宮腔粘連形成,以另一側(cè)子宮作為自身對照,分別比較損傷側(cè)和對照側(cè)子宮病理變化、腺體數(shù)量、纖維化面積及胚胎個數(shù),評估兩種方法建模效果及造成的子宮內(nèi)膜損傷對兔生育力的影響。其中機(jī)械損傷組兔的損傷側(cè)子宮為M-A組,對照側(cè)子宮為M-B組;電熱損傷組兔的損傷側(cè)子宮為E-A組,對照側(cè)子宮為E-B組。分別在損傷后7d、14d和28d收集兔雙側(cè)子宮組織,行HE和Massoon染色觀察兩側(cè)子宮內(nèi)膜病理改變,并對兩側(cè)子宮內(nèi)膜的腺體個數(shù)和內(nèi)膜纖維化面積比進(jìn)行統(tǒng)計學(xué)分析和比較。另外將損傷后7d的雌兔與成年雄兔合籠,14天后觀察比較兩側(cè)子宮胚胎個數(shù)。結(jié)果:兩種建模方法均未見明顯纖維粘連帶形成。病理組織學(xué)觀察顯示機(jī)械損傷組術(shù)后7d,M-A組損傷內(nèi)膜腺體數(shù)量較B組減少,間質(zhì)水腫、白細(xì)胞浸潤,毛細(xì)血管充血、阻塞,可見少量新生毛細(xì)血管;術(shù)后14d和30d,M-A組和M-B組子宮內(nèi)膜切片HE染色鏡下顯示并沒有明顯的差異,損傷后的子宮內(nèi)膜已完全修復(fù)。電熱損傷組建模術(shù)后7d,與E-B組相比,E-A組宮腔皺襞減少,表面稍平坦,基本被柱狀上皮細(xì)胞覆蓋,腺體數(shù)量明顯減少,間質(zhì)水腫、白細(xì)胞浸潤,毛細(xì)血管充血、阻塞,可見少量新生毛細(xì)血管;術(shù)后14d,E-A組宮腔表面皺襞仍少,表面較平坦,上皮基本全部被柱狀上皮細(xì)胞覆蓋,腺體個數(shù)仍較少,間質(zhì)水腫不再明顯,可見少量白細(xì)胞浸潤,新生毛細(xì)血管增多;術(shù)后28d,E-A組子宮內(nèi)膜形態(tài)學(xué)基本恢復(fù)正常,可見較多新生腺體,未見間質(zhì)充血和淋巴細(xì)胞浸潤。機(jī)械組損傷后7d,M-A組子宮內(nèi)膜腺體數(shù)量較M-B組減少,差異有統(tǒng)計學(xué)意義(P0.05);機(jī)械損傷后14d、30d,M-A組子宮內(nèi)膜腺體數(shù)量與M-B組相比差異無統(tǒng)計學(xué)意義。電熱損傷后7d和14d,E-A組子宮內(nèi)膜腺體數(shù)量較E-B組減少,差異均有統(tǒng)計學(xué)意義(P0.05);而電熱損傷后30d E-A組子宮內(nèi)膜腺體數(shù)量與E-B組相比差異無統(tǒng)計學(xué)意義。機(jī)械損傷后7d,M-A組內(nèi)膜纖維化面積比與M-B組相比差異無統(tǒng)計學(xué)意義;電熱損傷后7d,E-A組內(nèi)膜纖維化面積比較E-B組增大,差異有統(tǒng)計學(xué)意義(P0.05)。M-A組子宮胚胎個數(shù)與M-B組相比差異無統(tǒng)計學(xué)意義;E-A組子宮胚胎個數(shù)較E-B組減少,差異有統(tǒng)計學(xué)意義(P0.05)。結(jié)論:利用機(jī)械損傷法很難建立穩(wěn)定有效的兔宮腔粘連模型。盡管未見明顯纖維粘連帶形成,電熱損傷造成的胚胎個數(shù)的減少說明采用電熱損傷法建立的兔宮腔粘連模型在損傷后7-14天是穩(wěn)定有效的。第二章人離體子宮冷灌注及冷缺血保存的安全區(qū)間目的:離體子宮保存是子宮移植過程中需要解決的一項關(guān)鍵問題,良好科學(xué)的子宮灌注和保存方法是保證子宮移植成功的重要前提。本文旨在探討人離體子宮利用組氨酸-色氨酸-酮戊二酸鹽(Histidine-tryptophane-ketoglutarate,HTK)液冷灌注及冷缺血保存的安全區(qū)間,為子宮移植的相關(guān)研究及臨床應(yīng)用提供理論依據(jù)和參考。方法:樣本為人離體子宮7例,來源于行子宮切除術(shù)的宮頸癌患者。其中3例用HTK液在低溫條件下經(jīng)子宮動脈灌注,測量并記錄一定灌注壓力所對應(yīng)的灌注高度,建立灌注高度與灌注壓之間的數(shù)學(xué)關(guān)系模型;4例在適當(dāng)灌注高度下進(jìn)行短時間冷灌注,然后立刻取子宮內(nèi)膜及肌組織分別于HTK保存液和生理鹽水(normal saline,NS)中保存,兩組又根據(jù)保存時間(0、3、6、24h)的不同分為 Oh 組、HTK 3h 組、HTK 6h 組、HTK 24h 組、NS 3h 組、NS 6h 組和 NS 24h組,利用光學(xué)和電子顯微鏡觀察各組子宮組織細(xì)胞形態(tài)學(xué)改變,測定并比較各組子宮平滑肌收縮能力,分析離體子宮短時灌注后冷缺血保存的最佳時限。結(jié)果:灌注壓90～140mmHg對應(yīng)的灌注高度為62～122cm,理論灌注高度H與灌注壓P關(guān)系公式為H(m)=(?)(?)。與Oh組比較,HTK3h組和HTK6h組的子宮組織在光學(xué)和電子顯微鏡下形態(tài)學(xué)結(jié)構(gòu)均未見明顯改變,但HTK24h組及所有的NS組的子宮組織均出現(xiàn)不同程度的細(xì)胞水腫、細(xì)胞間失去聯(lián)系、線粒體腫脹及染色質(zhì)粗染等不可逆的退行性改變;HTK3h、HTK6h和HTK24h三組子宮肌組織的肌收縮力差異無統(tǒng)計學(xué)意義(P=0.772);而與相同時段HTK組標(biāo)本相比,NS3h、6h和24h組子宮肌組織的肌收縮力均明顯降低(P0.05)。結(jié)論:人類離體子宮組織在低溫環(huán)境下利用HTK液灌注的安全高度是62～122cm,理論灌注高度H與灌注壓P關(guān)系模型公式為H(m)=(?)人離體子宮短時灌注后于4℃ HTK液中保存可耐受冷缺血時間至少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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R711.6

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