口腔扁平苔藓动物模型的初步建立及化湿行瘀清热方治疗口腔扁平苔藓患者前后血清中差异蛋白的定量研究

发布时间：2018-06-01 20:10

本文选题：口腔扁平苔藓 + 动物模型　；参考：《河北医科大学》2017年硕士论文

【摘要】：第一部分口腔扁平苔藓动物模型的初步建立目的:采用口腔扁平苔藓患者病变组织匀浆辅以弗氏佐剂针刺划痕法,在NOD/SCID小鼠颊黏膜建立口腔扁平苔藓的动物模型。并尝试在应用人PBL建立具有人免疫学特性的NOD/SCID小鼠模型的基础之上建立口腔扁平苔藓的动物模型。方法:1 OLP-NOD/SCID小鼠模型的建立用少许OLP患者病变组织来制备组织匀浆。选取12只NOD/SCID小鼠,鼠龄4周,雌性,体重20~25 g,动物体形丰满,发育正常,行动迅速,反应灵敏,被毛浓密有光泽,食欲良好。饲养和各项实验操作均在动物实验中心SPF级的层流实验室进行。将其随机分为2组,每组6只。第1组为对照组,采用生理盐水针刺NOD/SCID小鼠颊黏膜4周,隔两天针刺一次。第2组为实验组,采用OLP患者病变组织匀浆辅以弗氏佐剂针刺颊黏膜4周,隔两天针刺一次。处理4周处死,于作用处颊黏膜取材。制成HE染色的病理切片,在光学显微镜下观察病理变化。2 Hu-PBL-NOD/SCID小鼠模型建立及鉴定2.1将18只NOD/SCID小鼠随机分为3组,每组6只;第1组,每只腹腔注射人外周血淋巴细胞2×107/0.5ml,作为人免疫重建低剂量组;第2组,每只腹腔注射人外周血淋巴细胞4×107/0.5ml,作为人免疫重建高剂量组;第3组,每只腹腔注射0.5ml生理盐水,作为空白对照组。2.2从生物学特性和免疫学特性两个方面对小鼠模型进行鉴定:每天观察小鼠的生物学体征;第8周摘眼球取血,利用流式细胞仪检测小鼠外周血中人CD3+、CD4+T淋巴细胞。结果:1 OLP-NOD/SCID小鼠模型的建立肉眼观察实验组针刺划痕组织匀浆后,针刺部位黏膜充血,质地脆弱。镜下观察实验组病理结果:黏膜上皮不全角化,棘细胞层增生,基底细胞可见轻度空泡变性,上皮钉突不规则延长,固有层散在淋巴细胞浸润,但固有层还未出现大量淋巴细胞浸润带的OLP典型病理表现。肉眼观察对照组黏膜无明显变化,镜下观察病理结果:黏膜上皮过度角化,固有层未见炎细胞浸润。2 Hu-PBL-NOD/SCID小鼠模型建立及鉴定第1组小鼠到第8周营养状态、精神状态良好;通过流式细胞仪检测小鼠外周血中人CD3+、CD4+T淋巴细胞双阳性表达率为3.03±0.47%,表达量不足,未能建立起Hu-PBL-NOD/SCID小鼠模型。第2组到第8周后发生GVHR反应,并且死亡1只;通过流式细胞仪检测小鼠外周血中人CD3+、CD4+T淋巴细胞双阳性表达率为36.13±4.79%。虽然淋巴细胞已达到一定数量,但小鼠出现了GVHR。第3组小鼠体健,营养状态、精神状态良好,通过流式细胞仪未检测到小鼠外周血中有人CD3+、CD4+T淋巴细胞表达。第二部分化湿行瘀清热方治疗口腔扁平苔藓患者前后血清中差异蛋白的定量研究目的:本课题小组前期应用双向荧光差异凝胶电泳及质谱技术成功鉴定出化湿行瘀清热方治疗口腔扁平苔藓患者前、后的血清差异蛋白,发现人抗凝血酶Ⅲ、维生素D结合蛋白等在治疗OLP前后的血清中存在差异表达,并通过Western-blot给予了初步的半定量验证。为进一步探讨化湿行瘀清热方对口腔扁平苔藓的影响作用,本研究拟采用ELISA技术在蛋白水平上行进一步准确的定量研究,为OLP可能的发病机制及化湿行瘀清热方的临床应用提供理论依据。方法:选取20例OLP患者,采集治疗前和经化湿行瘀清热方治疗8周后的静脉血,静置30min后离心,吸取上层血清。采用ELISA对OLP患者治疗前后血清中差异蛋白的表达情况进行定量研究。结果:ELISA结果显示人抗凝血酶Ⅲ,维生素D结合蛋白在口腔扁平苔藓患者治疗后的血清中浓度较治疗前均下调(P0.05)。结论:1本实验采用组织匀浆+弗氏佐剂针刺划痕后的病变部位已出现黏膜上皮不全角化,棘细胞层轻度增生,基底细胞轻度液化变性,上皮钉突不规则延长,固有层散在淋巴细胞浸润的病理表现,较前期实验的黏膜上皮出现棘层增生,固有层散在炎细胞浸润的病理改变,更加接近OLP的病理表现。2本实验注射人PBL 2×107/0.5ml后表达量不足,未能成功建立人PBL-NOD/SCID鼠模型,而注入人PBL 4×107/0.5ml后则发生GVHR反应。3维生素D结合蛋白,人抗凝血酶Ⅲ在口腔扁平苔藓患者治疗后血清中的表达均降低,这两种蛋白表达量的差异与化湿行瘀清热方对口腔扁平苔藓的作用机制有一定关系。
[Abstract]:The first part of the animal model of oral lichen planus: to establish the animal model of oral lichen planus in the buccal mucosa of NOD/SCID mice by using the pathological tissue homogenate of oral lichen planus with the needling method of Freund's adjuvant acupuncture, and try to establish the basis for the establishment of the NOD/SCID mouse model with human immunological characteristics in the application of human PBL. Methods: the animal model of oral lichen planus was established. Methods: 1 OLP-NOD/SCID mice model was established with a few OLP patients' pathological tissue to prepare tissue homogenate. 12 NOD/SCID mice were selected for 4 weeks, female and body weight 20~25 G. The animals have full body shape, normal development, quick action, quick reaction, luster and good appetite. And all the experimental operations were carried out in the laminar flow laboratory at the SPF level in the animal experimental center. They were randomly divided into 2 groups, 6 in each group. First groups were used as the control group. The cheek mucosa of the NOD/SCID mice was needled by physiological saline for 4 weeks and the needle was needled for two days. The second groups were used as the experimental group, and the pathological tissue homogenate of the patients with OLP was used for 4 weeks of buccal mucosa with Freund's adjuvant and septum septum. Two days of acupuncture at one time. Treatment for 4 weeks and sacrificed on the buccal mucosa of action. The pathological section of HE staining was made. Under the optical microscope, the pathological changes of.2 Hu-PBL-NOD/SCID mice were established and identified. 18 mice were randomly divided into 3 groups, 6 of each group, and first groups, each intraperitoneal injection of peripheral blood lymphocytes 2 x 107/0.5ml. The low dose group was rebuilt for human immunization; the second group, each intraperitoneal injection of peripheral blood lymphocytes of 4 x 107/0.5ml, was used as the high dose group for human immune reconstruction; the third group, each intraperitoneal injection of 0.5ml saline, was used as the blank control group to identify the mice models from two aspects of biological and immunological characteristics: daily observation of the mice's birth. Physical signs; eighth weeks of picking eyeballs and taking blood, using flow cytometry to detect the human CD3+ and CD4+T lymphocytes in the peripheral blood of mice. Results: after the establishment of the 1 OLP-NOD/SCID mice model, the experimental group was established by the naked eye observation group, and the acupuncture site mucosa was congested and the texture was fragile. The pathological results of the experimental group were observed under the microscope: the mucous epithelium was not all keratinized and spinous. In the cell layer, the basal cells showed mild vacuolar degeneration, the epithelial peg irregular extension, the lamina propria scattered in the lymphocyte infiltration, but the lamina propria did not have a large number of OLP typical pathological manifestations of lymphocyte infiltration zone. No obvious changes were observed in the mucosa of the control group. Inflammatory cell infiltration.2 Hu-PBL-NOD/SCID mice model established and identified first groups of mice to eighth weeks of nutrition state, good mental state; through the flow cytometry test the mouse peripheral blood CD3+, the CD4+T lymphocyte double positive expression rate is 3.03 + 0.47%, the expression is insufficient, the Hu-PBL-NOD/SCID mouse model is not built. Second groups to eighth weeks later. GVHR reaction, and 1 deaths, were detected by flow cytometry. The double positive expression rate of CD4+T lymphocytes in the peripheral blood of mice was 36.13 + 4.79%., although the number of lymphocytes had reached a certain number, but the mice appeared in the GVHR. third groups of mice, with good nutritional status and good mental state. The mice were not detected by the flow cytometry. The expression of CD3+ and CD4+T lymphocyte in the blood. The purpose of quantitative study on the difference protein in the serum of patients with oral lichen planus treated by second divisions of the recipe for removing stasis and clearing heat: the group has successfully identified the treatment of the patients with oral lichen planus before and after the application of bi-directional fluorescence differential gel electrophoresis and mass spectrometry. The differential expression of human antithrombin III and vitamin D binding protein in the serum before and after the treatment of OLP was found, and the preliminary semi quantitative verification was given through Western-blot. The purpose of this study was to explore the effect of the recipe for removing blood stasis and clearing heat on the oral lichen planus. This study was to use ELISA technique at the protein level. A precise quantitative study was carried out to provide a theoretical basis for the possible pathogenesis of OLP and the clinical application of the recipe for removing blood stasis and clearing heat. Methods: 20 patients with OLP were selected to collect the venous blood before and after the treatment for 8 weeks after the treatment, and then the 30min was centrifuged and the upper layer serum was absorbed. The serum of the patients with OLP before and after the treatment of OLP was used. Results: ELISA results showed that the serum concentrations of human antithrombin III and vitamin D binding protein in oral lichen planus were lower than those before treatment (P0.05). Conclusion: the 1 experiments with tissue homogenate + Freund adjuvant needles have been found to have mucosal epithelial insufficiency Keratinization, acanthocyst hyperplasia, mild liquefaction and degeneration of basal cells, irregular extension of epithelial piling process, pathological manifestation of diffusion of lamina propria in lymphocyte infiltration, hyperplasia of spinous layer in mucous epithelium in earlier experiment, pathological changes of propria in inflammatory cell infiltration, closer to OLP's pathological manifestation,.2 experiment injection of PBL 2 x 107/0.5 The expression of ml was insufficient and the human PBL-NOD/SCID rat model was not established successfully. The GVHR reaction,.3 vitamin D binding protein, and the expression of human antithrombin III in the patients with oral lichen planus were reduced after the injection of PBL 4 x 107/0.5ml. The difference of the expression of the two proteins in the oral lichen planus was the difference between the expression of the protein and the prescription of the decoction of removing blood stasis and clearing heat on the oral lichen planus There is a certain relationship between the mechanism of action.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R781.5;R-332

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