發(fā)布時間：2018-06-06 04:54

  本文選題：淋巴管畸形 + 動物模型　； 參考：《武漢大學(xué)》2011年博士論文

【摘要】：淋巴管畸形(LM)是發(fā)生于兒童及成人的一種良性病變,其確切的發(fā)病機制目前還處于爭論之中,多數(shù)學(xué)者認為與淋巴管生成紊亂相關(guān)。手術(shù)治療有較高復(fù)發(fā)率,廣泛的微囊型病變尚無有效的治療手段。 淋巴管畸形發(fā)生的病理機制一直是人們研究的難點,且研究進展相對比較緩慢,一個重要原因就是對于淋巴管畸形缺少有效的體內(nèi)模型。為了研究淋巴管畸形的發(fā)病機制,對比分析評價不同治療方法的效果,建立淋巴管畸形的動物模型已成為必需。 理想的淋巴管畸形的動物模型應(yīng)能夠很好的模擬發(fā)生于人的淋巴管畸形的臨床及病理學(xué)特點。已有研究顯示利用弗氏不完全佐劑(FIA)能夠成功建立淋巴管畸形的動物模型。但是,他們所建立的動物模型病變較小,且局限在小鼠腹膜區(qū),不能表現(xiàn)人類頭頸部淋巴管畸形的特點。 為了研究頭頸部淋巴管畸形的發(fā)病機制等,我們利用弗氏不完全佐劑建立大鼠口底和頸部淋巴管畸形的動物模型,通過組織學(xué)、免疫組織化學(xué)、掃描電鏡及拉曼光譜分析等評價手段分析所形成病變的特點；同時,我們也比較了不同免疫佐劑誘導(dǎo)形成的淋巴管畸形的類型及病變大小；并在免疫佐劑中添加一定量的VEGF-C,以初步探討血管內(nèi)皮生長因子-C (VEGF-C)在淋巴管畸形生成與發(fā)展中的可能作用。 第一部分應(yīng)用弗氏不完全佐劑誘導(dǎo)大鼠頸部及口底淋巴管畸形的形成 目的：利用弗氏不完全佐劑(FIA)誘導(dǎo)大鼠頸部及口底淋巴管畸形的形成,由此建立一種簡單且具有高度可重復(fù)性的淋巴管畸形的動物模型。方法：將16只雌性Wistar大鼠隨機分成四組(FIA-N組、FIA-F組、PBS組、BLANK組)。其中,FIA-N組在大鼠頸部皮下注射0.2 ml FIA乳劑,FIA-F組在大鼠口底黏膜下注射0.2 ml FIA乳劑,PBS組在大鼠頸部皮下及口底黏膜下均注射0.2 ml磷酸鹽緩沖液(PBS),BLANK組不進行任何處理。兩周后各組大鼠受試部位重復(fù)注射一次。兩月后處死動物,記錄大體標本所見,并取注射部位組織,行組織學(xué)、免疫組織化學(xué)染色觀察以及透射電鏡觀察。結(jié)果：在兩次注射并觀察兩月后,FIA-N組大鼠頸部皮下形成透明或半透明囊泡狀病變,其內(nèi)充滿透明或半透明液體內(nèi)容物；FIA-F組大鼠口底黏膜區(qū)形成白色斑塊樣病變；PBS與BLANK組未有肉眼可見病變形成。頸部皮下及口底黏膜下所形成的病變,在光鏡下均呈現(xiàn)為大小不等的囊泡狀病變,經(jīng)免疫組化染色,囊泡狀病變囊壁內(nèi)襯細胞均陽性表達淋巴管內(nèi)皮細胞標志物L(fēng)YVE-1及VEGFR-3。囊壁縱行切片透射電鏡圖片顯示囊壁內(nèi)襯為不連續(xù)排列的單層內(nèi)皮細胞,呈橢圓形或梭形,無細胞間連接,也未見完整的基底膜與周細胞。結(jié)論：FIA可在大鼠頸部皮下及口底黏膜下形成淋巴管畸形病變,此模型可在臨床及病理表現(xiàn)方面成功的模擬發(fā)生于人頭頸部的淋巴管畸形,且此方法簡單,可重復(fù)性較高。 第二部分不同免疫佐劑誘導(dǎo)大鼠頸部皮下形成淋巴管畸形的比較 目的：比較不同免疫佐劑形成的淋巴管畸形的特點,探討免疫佐劑在誘導(dǎo)淋巴管畸形中所起到的可能作用。方法：將28只雌性Wistar大鼠隨機分成七組(FIA組、FCA組、MF59組、WO組、AH組、PBS組、BLANK組),按分組將5種不同免疫佐劑(弗氏不完全佐劑FIA、弗氏完全佐劑FCA、MF59佐劑、白油佐劑、氫氧化鋁佐劑)分別注射于大鼠頸部皮下各0.2 ml,PBS組在大鼠頸部皮下注射0.2 ml PBS,BLANK組不作任何處理。兩周后各組大鼠受試部位重復(fù)注射一次。兩月后處死動物,記錄大體標本所見,并切取注射部位所形成的病變組織,測量計算各組病變的體積后,行組織學(xué)、免疫組織化學(xué)染色觀察。同時抽取FIA組、FCA組所形成大囊泡液體內(nèi)容物,與FIA、FCA乳劑進行比較,行拉曼光譜分析。結(jié)果：FIA、FCA與WO組大鼠于頸部皮下形成透明或半透明的大囊型病變,周圍伴有數(shù)量不等透明微囊泡散在分布,MF59組僅形成透明的微囊型病變,AH組在頸部注射部位皮膚及皮下組織發(fā)生破潰結(jié)痂,PBS組與BLANK組無肉眼可見病變形成。將各組病變體積進行比較,FIA與FCA組所形成病變明顯較WO組及MF59組大。組織學(xué)觀察顯示各組頸部皮下所形成病變在光鏡下均表現(xiàn)為大小不等的囊泡,各組間無明顯差別。免疫組化染色結(jié)果顯示,各囊泡囊壁內(nèi)襯細胞均陽性表達淋巴管內(nèi)皮細胞標志物L(fēng)YVE-1及VEGFR-3。抽取囊泡內(nèi)容物后發(fā)現(xiàn),FIA組與FCA組所形成的大囊泡內(nèi)容物呈現(xiàn)為半透明乳狀液體,經(jīng)靜置約12小時后,表現(xiàn)為與FIA、FCA乳劑同樣的靜置分層現(xiàn)象。拉曼光譜分析圖譜顯示,其液體內(nèi)容物的成分基本為蛋白質(zhì)與脂類,其圖譜在波形與峰位置兩方面均與FIA、FCA乳劑的拉曼光譜圖類似。結(jié)論：不同的免疫佐劑(氫氧化鋁佐劑除外)注射后均可誘導(dǎo)大鼠頸部皮下形成淋巴管畸形病變,但所形成的病變在囊泡數(shù)量上以及病變體積上有所不同。FIA與FCA乳劑可作為誘導(dǎo)大鼠淋巴管畸形形成的首選藥物。大囊型病變可能是由于佐劑的儲庫作用形成,微囊型病變可能是由于佐劑的免疫刺激作用形成。 第三部分血管內(nèi)皮細胞生長因子-C在淋巴管畸形生成與發(fā)展中的可能作用 目的：探討血管內(nèi)皮細胞生長因子C (VEGF-C)在淋巴管畸形發(fā)生和發(fā)展中的可能作用。方法：將28只雌性Wistar大鼠隨機分成七組(FIA組、FIA-V組、FCA組、FCA-V組、PBS-V組、PBS組、BLANK組),按照分組分別給予不同的注射劑0.2 ml(FIA,添加VEGF-C的FIA,FCA,添加VEGF-C的FCA,用PBS稀釋的VEGF-C), PBS組在頸部皮下注射0.2 ml PBS,BLANK組不作任何處理。兩周后各組大鼠受試部位重復(fù)注射一次。兩月后處死動物,記錄大體標本所見,并切取注射部位病變組織,測量計算各組病變組織體積,并行組織學(xué)、免疫組織化學(xué)染色觀察。結(jié)果：FIA組、FIA-V組、FCA組、FCA-V組大鼠頸部皮下均形成了肉眼可見的大囊泡病變,且添加了VEGF-C的FIA-V組及FCA-V組所形成的病變在囊泡數(shù)量上和病變體積上均較FIA組及FCA組要多且大。然而,僅注射VEGF-C的PBS-V組的大鼠頸部皮下則未形成肉眼可見病變。組織學(xué)觀察顯示各組頸部皮下所形成病變在光鏡下均表現(xiàn)為大小不等的囊泡,各組間無明顯組織學(xué)差別；僅注射VEGF-C的PBS-V組在光鏡下仍未見明顯病變產(chǎn)生。免疫組化染色結(jié)果顯示,各囊泡內(nèi)襯細胞均陽性表達淋巴管內(nèi)皮細胞標志物L(fēng)YVE-1及VEGFR-3,各組間無明顯差別。結(jié)論：VEGF-C的作用在于促進淋巴管畸形的發(fā)展而不是誘導(dǎo)其發(fā)生。
[Abstract]:Lymphatic malformation (LM) is a benign disease occurring in children and adults. The exact mechanism of the disease is still in dispute. Most scholars believe that it is associated with lymphatic disorders. Surgical treatment has a high recurrence rate, and there is no effective treatment for a wide range of Microcystics.
The pathological mechanism of lymphatic malformation has been a difficult problem in people's research, and the research progress is relatively slow. One of the important reasons is the lack of effective body model for lymphatic malformation. In order to study the pathogenesis of lymphatic malformation, the effect of different treatment methods is compared and analyzed, and the animal model of lymphatic malformation is established. It has become a necessity.
The ideal animal model of lymphatic malformation should be able to simulate the clinical and pathological characteristics of the human lymphatic malformation. Studies have shown that the animal model of lymphatic malformation can be successfully established by FIA. However, the animal model of the animal model is smaller and limited to the peritoneal region of mice. It can not show the characteristics of human head and neck lymphatic malformations.
In order to study the pathogenesis of the head and neck lymphatic malformation, we used Freund's incomplete adjuvant to establish animal models of the deformity of the mouth and neck of the rat, and analyzed the characteristics of the disease by histology, immunohistochemistry, scanning electron microscopy and Raman spectroscopy. At the same time, we also compared different immunity. The types of lymphatic malformation induced by the adjuvant and the size of the lesions, and a certain amount of VEGF-C were added to the immune adjuvant to explore the possible role of vascular endothelial growth factor -C (VEGF-C) in the formation and development of lymphatic malformation.
The first part is the formation of lymphatic malformations in the neck and mouth floor in rats by incomplete Freund's adjuvant.
Objective: to induce the formation of lymphatic malformation of the neck and the bottom of the mouth of the rat by FIA, a simple and highly repeatable animal model of lymphatic malformation was established. Methods: 16 female Wistar rats were randomly divided into four groups (group FIA-N, group FIA-F, PBS and BLANK). Among them, the FIA-N group was in the neck of the rat. 0.2 ml FIA emulsion was injected subcutaneously in group FIA-F and 0.2 ml FIA emulsion was injected under the oral mucosa of rats. Group PBS was injected with 0.2 ml phosphate buffer solution (PBS) under the subcutaneous and suboral mucosa of the rat, and no treatment was performed in group BLANK. After two weeks, the rats were injected again and again. The animals were killed after two months, and the specimens were recorded in general. The tissue, histological, immunohistochemical staining and transmission electron microscopy were observed. Results: after two injections and two months of observation, a transparent or semitransparent vesicular lesion was formed in the neck of the FIA-N group, with transparent or translucent liquid content, and a white plaque in the suboral mucosa of group FIA-F rats. There were no visible lesions in the PBS and BLANK groups. The lesions formed under the subcutaneous and suboral submucosa of the neck were all vesicular lesions of different sizes under the light microscope. By immunohistochemical staining, the cell lining cells in the cystic wall of the vesicular lesion were all positive expression of the lymphatic endothelial cell marker of the lymphatic endothelium and the wall of the VEGFR-3. capsule. The images of electron microscope showed that the inner lining of the cyst wall lined with discontinuous lining was oval or spindle shaped, without intercellular connection and no complete basement membrane and pericytes. Conclusion: FIA can form lymphatic malformation under the subcutaneous and suboral mucosa of the rat's neck, and this model can be successfully simulated in clinical and pathological manifestations. It is simple and reproducible in human head and neck lymphatic malformations.
The second part is a comparison of different immune adjuvants inducing lymphatic malformations in the neck of rats.
Objective: To compare the characteristics of lymphatic malformation formed by different immune adjuvant and explore the possible role of immune adjuvant in inducing lymphatic malformation. Methods: 28 female Wistar rats were randomly divided into seven groups (group FIA, group FCA, group MF59, WO, AH, PBS, BLANK), and 5 different immune adjuvant (F uncomplete adjuvant F) were divided into groups. IA, Freund's complete adjuvant FCA, MF59 adjuvant, white oil adjuvant, aluminum hydroxide adjuvant were injected 0.2 ml subcutaneously in the neck of rats. Group PBS was injected subcutaneously in the neck of rats 0.2 ml PBS, and no treatment was done in group BLANK. After two weeks, the rats were repeatedly injected one time. The animals were executed two months later, the gross specimens were recorded and the injection department was cut. At the same time, the volume of the lesion was measured and measured by immunohistochemical staining. FIA group, group FCA and group of FIA, FCA emulsion were compared with FIA and FCA emulsion. The result: FIA, FCA and WO group rats formed transparent or translucent large capsule subcutaneously in the neck of the group of WO. In the group MF59, group AH and group BLANK had no visible lesions in the skin and subcutaneous tissue of the neck, and there were no visible lesions in the group PBS and the BLANK group. The pathological changes of each group were compared, and the pathological changes in the group of FIA and FCA were obviously more than that of the WO group and the MF59 group. The histological observation showed that the lesions formed under the neck of the neck of each group were all the vesicles of different sizes, and there was no significant difference between each group. The results of immunohistochemical staining showed that the positive expression of lymphatic endothelial cell markers LYVE-1 and VEGFR-3. were found in the vesicle wall lining cells, and the FIA group and the FCA group were found. The content of the large vesicle is presented as a semitransparent emulsion. After 12 hours of static, the content of the content is the same as the FIA, FCA emulsion. The Raman spectrum analysis atlas shows that the composition of the liquid content is basically protein and lipid, and its atlas is the Raman spectrum of the FIA and FCA emulsion in the waveform and peak position. Similar. Conclusion: different immune adjuvant (except for aluminum hydroxide adjuvant) can induce subcutaneous lymphatic malformation in the neck of rats, but the difference in the number of vesicles and the volume of.FIA and FCA can be the first choice to induce the formation of lymphatic malformation in rats. The microencapsulated lesion may be due to the immune stimulation of adjuvants due to the formation of adjuvant storage.
The third part is the possible role of vascular endothelial growth factor -C in the formation and development of lymphatic malformations.
Objective: To investigate the possible role of vascular endothelial growth factor C (VEGF-C) in the occurrence and development of lymphatic malformation. Methods: 28 female Wistar rats were randomly divided into seven groups (group FIA, FIA-V, FCA, FCA-V, PBS-V, PBS, BLANK). With VEGF-C FCA and PBS diluted VEGF-C), group PBS was injected subcutaneously 0.2 ml PBS in the neck, and no treatment in group BLANK. After two weeks, the rats were repeatedly injected one time. The animals were killed after two months. The animals were killed and the pathological tissue of the injection site was cut and the volume of the lesion tissue was measured, the histology and immunity were calculated. Results: in group FIA, group FIA-V, group FCA, and group FCA-V, the large cysts of large vesicles were found in the neck of the rats, and the number and volume of the FIA-V and FCA-V groups added to VEGF-C were more than that of the FIA group and the FCA group. However, only the PBS-V group of the VEGF-C was injected into the rat neck. Histological observation showed that the lesions formed under the neck of the neck of each group were all the vesicles of different sizes under the light microscope, and there was no obvious histological difference between each group. No obvious pathological changes were found in the group of VEGF-C only PBS-V under the light microscope. Positive expression of lymphatic endothelial cell markers LYVE-1 and VEGFR-3, no significant difference between each group. Conclusion: the role of VEGF-C is to promote the development of lymphatic malformation, not to induce its occurrence.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R392

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本文編號：1985204