大鼠颈部及口底淋巴管畸形动物模型的实验研究

发布时间：2018-06-06 04:54

本文选题：淋巴管畸形 + 动物模型　；参考：《武汉大学》2011年博士论文

【摘要】：淋巴管畸形(LM)是发生于儿童及成人的一种良性病变,其确切的发病机制目前还处于争论之中,多数学者认为与淋巴管生成紊乱相关。手术治疗有较高复发率,广泛的微囊型病变尚无有效的治疗手段。淋巴管畸形发生的病理机制一直是人们研究的难点,且研究进展相对比较缓慢,一个重要原因就是对于淋巴管畸形缺少有效的体内模型。为了研究淋巴管畸形的发病机制,对比分析评价不同治疗方法的效果,建立淋巴管畸形的动物模型已成为必需。理想的淋巴管畸形的动物模型应能够很好的模拟发生于人的淋巴管畸形的临床及病理学特点。已有研究显示利用弗氏不完全佐剂(FIA)能够成功建立淋巴管畸形的动物模型。但是,他们所建立的动物模型病变较小,且局限在小鼠腹膜区,不能表现人类头颈部淋巴管畸形的特点。为了研究头颈部淋巴管畸形的发病机制等,我们利用弗氏不完全佐剂建立大鼠口底和颈部淋巴管畸形的动物模型,通过组织学、免疫组织化学、扫描电镜及拉曼光谱分析等评价手段分析所形成病变的特点；同时,我们也比较了不同免疫佐剂诱导形成的淋巴管畸形的类型及病变大小；并在免疫佐剂中添加一定量的VEGF-C,以初步探讨血管内皮生长因子-C (VEGF-C)在淋巴管畸形生成与发展中的可能作用。第一部分应用弗氏不完全佐剂诱导大鼠颈部及口底淋巴管畸形的形成目的：利用弗氏不完全佐剂(FIA)诱导大鼠颈部及口底淋巴管畸形的形成,由此建立一种简单且具有高度可重复性的淋巴管畸形的动物模型。方法：将16只雌性Wistar大鼠随机分成四组(FIA-N组、FIA-F组、PBS组、BLANK组)。其中,FIA-N组在大鼠颈部皮下注射0.2 ml FIA乳剂,FIA-F组在大鼠口底黏膜下注射0.2 ml FIA乳剂,PBS组在大鼠颈部皮下及口底黏膜下均注射0.2 ml磷酸盐缓冲液(PBS),BLANK组不进行任何处理。两周后各组大鼠受试部位重复注射一次。两月后处死动物,记录大体标本所见,并取注射部位组织,行组织学、免疫组织化学染色观察以及透射电镜观察。结果：在两次注射并观察两月后,FIA-N组大鼠颈部皮下形成透明或半透明囊泡状病变,其内充满透明或半透明液体内容物；FIA-F组大鼠口底黏膜区形成白色斑块样病变；PBS与BLANK组未有肉眼可见病变形成。颈部皮下及口底黏膜下所形成的病变,在光镜下均呈现为大小不等的囊泡状病变,经免疫组化染色,囊泡状病变囊壁内衬细胞均阳性表达淋巴管内皮细胞标志物LYVE-1及VEGFR-3。囊壁纵行切片透射电镜图片显示囊壁内衬为不连续排列的单层内皮细胞,呈椭圆形或梭形,无细胞间连接,也未见完整的基底膜与周细胞。结论：FIA可在大鼠颈部皮下及口底黏膜下形成淋巴管畸形病变,此模型可在临床及病理表现方面成功的模拟发生于人头颈部的淋巴管畸形,且此方法简单,可重复性较高。第二部分不同免疫佐剂诱导大鼠颈部皮下形成淋巴管畸形的比较目的：比较不同免疫佐剂形成的淋巴管畸形的特点,探讨免疫佐剂在诱导淋巴管畸形中所起到的可能作用。方法：将28只雌性Wistar大鼠随机分成七组(FIA组、FCA组、MF59组、WO组、AH组、PBS组、BLANK组),按分组将5种不同免疫佐剂(弗氏不完全佐剂FIA、弗氏完全佐剂FCA、MF59佐剂、白油佐剂、氢氧化铝佐剂)分别注射于大鼠颈部皮下各0.2 ml,PBS组在大鼠颈部皮下注射0.2 ml PBS,BLANK组不作任何处理。两周后各组大鼠受试部位重复注射一次。两月后处死动物,记录大体标本所见,并切取注射部位所形成的病变组织,测量计算各组病变的体积后,行组织学、免疫组织化学染色观察。同时抽取FIA组、FCA组所形成大囊泡液体内容物,与FIA、FCA乳剂进行比较,行拉曼光谱分析。结果：FIA、FCA与WO组大鼠于颈部皮下形成透明或半透明的大囊型病变,周围伴有数量不等透明微囊泡散在分布,MF59组仅形成透明的微囊型病变,AH组在颈部注射部位皮肤及皮下组织发生破溃结痂,PBS组与BLANK组无肉眼可见病变形成。将各组病变体积进行比较,FIA与FCA组所形成病变明显较WO组及MF59组大。组织学观察显示各组颈部皮下所形成病变在光镜下均表现为大小不等的囊泡,各组间无明显差别。免疫组化染色结果显示,各囊泡囊壁内衬细胞均阳性表达淋巴管内皮细胞标志物LYVE-1及VEGFR-3。抽取囊泡内容物后发现,FIA组与FCA组所形成的大囊泡内容物呈现为半透明乳状液体,经静置约12小时后,表现为与FIA、FCA乳剂同样的静置分层现象。拉曼光谱分析图谱显示,其液体内容物的成分基本为蛋白质与脂类,其图谱在波形与峰位置两方面均与FIA、FCA乳剂的拉曼光谱图类似。结论：不同的免疫佐剂(氢氧化铝佐剂除外)注射后均可诱导大鼠颈部皮下形成淋巴管畸形病变,但所形成的病变在囊泡数量上以及病变体积上有所不同。FIA与FCA乳剂可作为诱导大鼠淋巴管畸形形成的首选药物。大囊型病变可能是由于佐剂的储库作用形成,微囊型病变可能是由于佐剂的免疫刺激作用形成。第三部分血管内皮细胞生长因子-C在淋巴管畸形生成与发展中的可能作用目的：探讨血管内皮细胞生长因子C (VEGF-C)在淋巴管畸形发生和发展中的可能作用。方法：将28只雌性Wistar大鼠随机分成七组(FIA组、FIA-V组、FCA组、FCA-V组、PBS-V组、PBS组、BLANK组),按照分组分别给予不同的注射剂0.2 ml(FIA,添加VEGF-C的FIA,FCA,添加VEGF-C的FCA,用PBS稀释的VEGF-C), PBS组在颈部皮下注射0.2 ml PBS,BLANK组不作任何处理。两周后各组大鼠受试部位重复注射一次。两月后处死动物,记录大体标本所见,并切取注射部位病变组织,测量计算各组病变组织体积,并行组织学、免疫组织化学染色观察。结果：FIA组、FIA-V组、FCA组、FCA-V组大鼠颈部皮下均形成了肉眼可见的大囊泡病变,且添加了VEGF-C的FIA-V组及FCA-V组所形成的病变在囊泡数量上和病变体积上均较FIA组及FCA组要多且大。然而,仅注射VEGF-C的PBS-V组的大鼠颈部皮下则未形成肉眼可见病变。组织学观察显示各组颈部皮下所形成病变在光镜下均表现为大小不等的囊泡,各组间无明显组织学差别；仅注射VEGF-C的PBS-V组在光镜下仍未见明显病变产生。免疫组化染色结果显示,各囊泡内衬细胞均阳性表达淋巴管内皮细胞标志物LYVE-1及VEGFR-3,各组间无明显差别。结论：VEGF-C的作用在于促进淋巴管畸形的发展而不是诱导其发生。
[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.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392

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