本文選題：口腔扁平苔蘚 + 動物模型��； 參考：《河北醫(yī)科大學(xué)》2017年碩士論文

【摘要】：第一部分口腔扁平苔蘚動物模型的初步建立目的:采用口腔扁平苔蘚患者病變組織勻漿輔以弗氏佐劑針刺劃痕法,在NOD/SCID小鼠頰黏膜建立口腔扁平苔蘚的動物模型。并嘗試在應(yīng)用人PBL建立具有人免疫學(xué)特性的NOD/SCID小鼠模型的基礎(chǔ)之上建立口腔扁平苔蘚的動物模型。方法:1 OLP-NOD/SCID小鼠模型的建立用少許OLP患者病變組織來制備組織勻漿。選取12只NOD/SCID小鼠,鼠齡4周,雌性,體重20~25 g,動物體形豐滿,發(fā)育正常,行動迅速,反應(yīng)靈敏,被毛濃密有光澤,食欲良好。飼養(yǎng)和各項實驗操作均在動物實驗中心SPF級的層流實驗室進行。將其隨機分為2組,每組6只。第1組為對照組,采用生理鹽水針刺NOD/SCID小鼠頰黏膜4周,隔兩天針刺一次。第2組為實驗組,采用OLP患者病變組織勻漿輔以弗氏佐劑針刺頰黏膜4周,隔兩天針刺一次。處理4周處死,于作用處頰黏膜取材。制成HE染色的病理切片,在光學(xué)顯微鏡下觀察病理變化。2 Hu-PBL-NOD/SCID小鼠模型建立及鑒定2.1將18只NOD/SCID小鼠隨機分為3組,每組6只;第1組,每只腹腔注射人外周血淋巴細(xì)胞2×107/0.5ml,作為人免疫重建低劑量組;第2組,每只腹腔注射人外周血淋巴細(xì)胞4×107/0.5ml,作為人免疫重建高劑量組;第3組,每只腹腔注射0.5ml生理鹽水,作為空白對照組。2.2從生物學(xué)特性和免疫學(xué)特性兩個方面對小鼠模型進行鑒定:每天觀察小鼠的生物學(xué)體征;第8周摘眼球取血,利用流式細(xì)胞儀檢測小鼠外周血中人CD3+、CD4+T淋巴細(xì)胞。結(jié)果:1 OLP-NOD/SCID小鼠模型的建立肉眼觀察實驗組針刺劃痕組織勻漿后,針刺部位黏膜充血,質(zhì)地脆弱。鏡下觀察實驗組病理結(jié)果:黏膜上皮不全角化,棘細(xì)胞層增生,基底細(xì)胞可見輕度空泡變性,上皮釘突不規(guī)則延長,固有層散在淋巴細(xì)胞浸潤,但固有層還未出現(xiàn)大量淋巴細(xì)胞浸潤帶的OLP典型病理表現(xiàn)。肉眼觀察對照組黏膜無明顯變化,鏡下觀察病理結(jié)果:黏膜上皮過度角化,固有層未見炎細(xì)胞浸潤。2 Hu-PBL-NOD/SCID小鼠模型建立及鑒定第1組小鼠到第8周營養(yǎng)狀態(tài)、精神狀態(tài)良好;通過流式細(xì)胞儀檢測小鼠外周血中人CD3+、CD4+T淋巴細(xì)胞雙陽性表達率為3.03±0.47%,表達量不足,未能建立起Hu-PBL-NOD/SCID小鼠模型。第2組到第8周后發(fā)生GVHR反應(yīng),并且死亡1只;通過流式細(xì)胞儀檢測小鼠外周血中人CD3+、CD4+T淋巴細(xì)胞雙陽性表達率為36.13±4.79%。雖然淋巴細(xì)胞已達到一定數(shù)量,但小鼠出現(xiàn)了GVHR。第3組小鼠體健,營養(yǎng)狀態(tài)、精神狀態(tài)良好,通過流式細(xì)胞儀未檢測到小鼠外周血中有人CD3+、CD4+T淋巴細(xì)胞表達。第二部分化濕行瘀清熱方治療口腔扁平苔蘚患者前后血清中差異蛋白的定量研究目的:本課題小組前期應(yīng)用雙向熒光差異凝膠電泳及質(zhì)譜技術(shù)成功鑒定出化濕行瘀清熱方治療口腔扁平苔蘚患者前、后的血清差異蛋白,發(fā)現(xiàn)人抗凝血酶Ⅲ、維生素D結(jié)合蛋白等在治療OLP前后的血清中存在差異表達,并通過Western-blot給予了初步的半定量驗證。為進一步探討化濕行瘀清熱方對口腔扁平苔蘚的影響作用,本研究擬采用ELISA技術(shù)在蛋白水平上行進一步準(zhǔn)確的定量研究,為OLP可能的發(fā)病機制及化濕行瘀清熱方的臨床應(yīng)用提供理論依據(jù)。方法:選取20例OLP患者,采集治療前和經(jīng)化濕行瘀清熱方治療8周后的靜脈血,靜置30min后離心,吸取上層血清。采用ELISA對OLP患者治療前后血清中差異蛋白的表達情況進行定量研究。結(jié)果:ELISA結(jié)果顯示人抗凝血酶Ⅲ,維生素D結(jié)合蛋白在口腔扁平苔蘚患者治療后的血清中濃度較治療前均下調(diào)(P0.05)。結(jié)論:1本實驗采用組織勻漿+弗氏佐劑針刺劃痕后的病變部位已出現(xiàn)黏膜上皮不全角化,棘細(xì)胞層輕度增生,基底細(xì)胞輕度液化變性,上皮釘突不規(guī)則延長,固有層散在淋巴細(xì)胞浸潤的病理表現(xiàn),較前期實驗的黏膜上皮出現(xiàn)棘層增生,固有層散在炎細(xì)胞浸潤的病理改變,更加接近OLP的病理表現(xiàn)。2本實驗注射人PBL 2×107/0.5ml后表達量不足,未能成功建立人PBL-NOD/SCID鼠模型,而注入人PBL 4×107/0.5ml后則發(fā)生GVHR反應(yīng)。3維生素D結(jié)合蛋白,人抗凝血酶Ⅲ在口腔扁平苔蘚患者治療后血清中的表達均降低,這兩種蛋白表達量的差異與化濕行瘀清熱方對口腔扁平苔蘚的作用機制有一定關(guān)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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R781.5;R-332

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