本文選題：紅色毛癬菌 + 維生素D��； 參考：《河北醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:有研究顯示,人類對(duì)紅色毛癬菌普遍易感,全球90%的皮膚癬菌病是由紅色毛癬菌引起的,發(fā)病高峰集中在夏季,大多不引起明顯的炎癥反應(yīng),而呈現(xiàn)為慢性遷延病程,嚴(yán)重影響著患者生活質(zhì)量[1]。維生素D是一類固醇類衍生物,可對(duì)人體免疫系統(tǒng)產(chǎn)生重要的影響,其作用越來(lái)越受到人們的關(guān)注[2],維生素D通過(guò)與維生素D受體(VDR)結(jié)合發(fā)揮生理作用。VDR屬于類固醇激素/甲狀腺激素受體超家族成員,是一種配體依賴的核轉(zhuǎn)錄因子。皮膚是機(jī)體內(nèi)所需維生素D的重要源泉,可以在太陽(yáng)光的照射下由皮膚內(nèi)儲(chǔ)備的前體物質(zhì)維生素D3原轉(zhuǎn)化而來(lái)。維生素D的活性形式1,25(OH)2D3,在夏季血清水平較冬季為高,夏季維生素D的存貯有一個(gè)上升趨勢(shì),這可導(dǎo)致前炎癥因子的下降,從而導(dǎo)致機(jī)體對(duì)病原微生物產(chǎn)生免疫耐受。IL-12是一種異二聚體促炎因子,可通過(guò)誘導(dǎo)γ干擾素的產(chǎn)生、促進(jìn)Th1細(xì)胞的分化,起到強(qiáng)大的促炎癥作用[3]。IL-10可對(duì)多種免疫細(xì)胞的生物活性及這些細(xì)胞釋放的細(xì)胞因子的生物效應(yīng)產(chǎn)生抑制作用,其生理功能就是限制和終止炎癥反應(yīng)。因此,IL-10在限制宿主對(duì)病原菌的免疫過(guò)程中起重要作用[4,5]。本實(shí)驗(yàn)通過(guò)紅色毛癬菌體外刺激角質(zhì)形成細(xì)胞,檢測(cè)VDR與IL-10、IL-12的表達(dá)并對(duì)其表達(dá)水平進(jìn)行相關(guān)分析,進(jìn)而探討紅色毛癬菌感染過(guò)程中是否通過(guò)VDR信號(hào)途徑介導(dǎo)機(jī)體對(duì)紅色毛癬菌的免疫耐受導(dǎo)致疾病慢性遷延。方法:1實(shí)驗(yàn)用細(xì)胞的培養(yǎng)HaCaT細(xì)胞購(gòu)自上海賽笠生物科技有限公司編號(hào):MXC138。HaCaT細(xì)胞的培養(yǎng)使用90%DMEM+10%FBS+1%青鏈霉素混合劑配制而成的完全培養(yǎng)基。將HaCaT細(xì)胞制成單細(xì)胞懸液后,進(jìn)行細(xì)胞爬片,培養(yǎng)5天后,取出爬片,丙酮固定,進(jìn)行HE染色及免疫組織化學(xué)SP法檢測(cè)HaCaT細(xì)胞角蛋白10的表達(dá)。2實(shí)驗(yàn)用菌株實(shí)驗(yàn)菌株采用河北醫(yī)科大學(xué)第二醫(yī)院真菌室保存菌種。3菌株的培養(yǎng)及菌懸液的配制將紅色毛癬菌在PDA上活化三次,28℃培養(yǎng)7—14天。加入含0.1%吐溫80的無(wú)菌水,吹打混合均勻,然后用組織研磨器將菌懸液研磨,血球計(jì)數(shù)板計(jì)數(shù),調(diào)整濃度為4-6×105CFU/mL。4實(shí)驗(yàn)分組與處理實(shí)驗(yàn)分為對(duì)照組和實(shí)驗(yàn)組。將HaCaT細(xì)胞培養(yǎng)1周后取出,棄原培養(yǎng)基,PBS洗滌3遍后,用0.25%胰蛋白酶將細(xì)胞從培養(yǎng)瓶底部消化下來(lái),加入完全培養(yǎng)基終止消化,離心所收集到的細(xì)胞,用完全培養(yǎng)基調(diào)終濃度至1-2×106/mL,轉(zhuǎn)種到24孔培養(yǎng)板中。繼續(xù)培養(yǎng)24小時(shí)后行分組處理。實(shí)驗(yàn)組每孔中加入濃度為4-6×105CFU/mL的菌懸液1mL,對(duì)照組每孔加入1mL含0.1%吐溫80無(wú)菌水1mL。依次在2h、4h、8h、16h采集實(shí)驗(yàn)組和對(duì)照組的上清液。-20℃凍存待測(cè)。每個(gè)時(shí)間點(diǎn)均設(shè)3個(gè)平行孔。5ELISA法測(cè)各組IL-10、IL-12的分泌水平復(fù)溫實(shí)驗(yàn)組和對(duì)照組所收集到的上清液,依照所購(gòu)買試劑盒使用指南,分別測(cè)定細(xì)胞培養(yǎng)上清液中的IL-10、IL-12表達(dá)水平。6Westernblot法測(cè)各組VDR的表達(dá)水平PBS溶液清洗長(zhǎng)有HaCaT細(xì)胞的24孔板,刮下細(xì)胞轉(zhuǎn)移至EP管內(nèi),離心后添加裂解液RIPA裂解細(xì)胞,提取總蛋白,SDS-PAGE凝膠電泳,轉(zhuǎn)至PVDF膜,脫脂牛奶封閉完成之后,分別孵育VDR(1:1000)與β-actin(1:1000)一抗及山羊抗兔/小鼠IgG二抗(1:3000),最后用雙色紅外激光掃描儀進(jìn)行掃膜,ImageJ軟件分析結(jié)果。7統(tǒng)計(jì)學(xué)分析SPSS13.0統(tǒng)計(jì)學(xué)分析軟件,所得結(jié)果以均數(shù)±標(biāo)準(zhǔn)差表示,組間比較所使用的統(tǒng)計(jì)學(xué)方法是單因素方差分析,組內(nèi)不同時(shí)間點(diǎn)的兩兩比較所使用的統(tǒng)計(jì)學(xué)方法為SNK-q檢驗(yàn),VDR與IL-10、IL-12的相關(guān)性分析所使用的統(tǒng)計(jì)學(xué)方法為Pearson相關(guān)分析法,P0.05為有統(tǒng)計(jì)學(xué)意義。結(jié)果:1HaCaT細(xì)胞培養(yǎng)的形態(tài)學(xué)觀察倒置顯微鏡下觀察,細(xì)胞生長(zhǎng)狀態(tài)良好,多數(shù)細(xì)胞貼壁,折光性強(qiáng),圓形,細(xì)胞膜完整并且向外伸出2至3個(gè)偽足。5天后細(xì)胞呈現(xiàn)多角形,融合成片,外觀呈鋪路石樣排列。HaCaT細(xì)胞HE染色結(jié)果可見其胞質(zhì)豐富,呈粉紅色,核大呈藍(lán)紫色。免疫組化結(jié)果顯示HaCaT細(xì)胞的角蛋白10在胞膜及胞質(zhì)中呈陽(yáng)性表達(dá)。2上清液IL-10表達(dá)水平的ELISA法測(cè)定結(jié)果在實(shí)驗(yàn)的2h、4h、8h、16h,實(shí)驗(yàn)組IL-10表達(dá)水平分別為1427.71±15.952、1562.09±12.24、1826.57±16.12、2363.807±53.465pg/mL,IL-10的表達(dá)水平隨著時(shí)間的延長(zhǎng)而增加。對(duì)照組在2h、4h、8h、16h的IL-10表達(dá)水平分別為1100.58±2.46、1160.8±7.044、1285.07±5.185、1400.96±11.657pg/mL,IL-10的表達(dá)水平隨著時(shí)間的延長(zhǎng)而增加。IL-10在實(shí)驗(yàn)組的表達(dá)水平比對(duì)照組高,F=6.591,P=0.042(P0.05),差異有統(tǒng)計(jì)學(xué)意義。3上清液IL-12表達(dá)水平的ELISA法測(cè)定結(jié)果在實(shí)驗(yàn)的2h、4h、8h、16h,實(shí)驗(yàn)組IL-12表達(dá)水平分別為296.85±0.6、285.43±2.03、275.19±6.03、249.18±3.72pg/mL,IL-12的表達(dá)水平隨著時(shí)間的延長(zhǎng)而減少。對(duì)照組在2h、4h、8h、16h的IL-12表達(dá)水平分別為294.85±4.601、317.36±7.026、340.123±18.905、385.050±6.593pg/mL,IL-12的表達(dá)水平隨著時(shí)間的延長(zhǎng)而增加。實(shí)驗(yàn)組的IL-12表達(dá)水平明顯低于對(duì)照的IL-12表達(dá)水平,F=7.012,P=0.038(P0.05),差異有統(tǒng)計(jì)學(xué)意義。4WesternBlot法測(cè)人永生化角質(zhì)形成細(xì)胞(HaCaT)中VDR蛋白的表達(dá)水平在實(shí)驗(yàn)的2h、4h、8h、16h,實(shí)驗(yàn)組VDR蛋白表達(dá)水平灰度比值分別為0.394±0.011、0.468±0.019、0.458±0.021、0.616±0.156。對(duì)照組在2h、4h、8h、16h的VDR蛋白表達(dá)水平分別為0.27±0.014、0.294±0.015、0.301±0.002、0.413±0.01。實(shí)驗(yàn)組和對(duì)照組的VDR蛋白表達(dá)水平均隨時(shí)間的延長(zhǎng)而表達(dá)增加,但實(shí)驗(yàn)組VDR蛋白上升的水平高于對(duì)照組,F=8.404,P=0.027(P0.05),差異有統(tǒng)計(jì)學(xué)意義。5紅色毛癬菌作用于HaCaT細(xì)胞后VDR的表達(dá)水平分別與IL-10、IL-12的表達(dá)水平的相關(guān)分析結(jié)果Pearson相關(guān)分析結(jié)果顯示VDR蛋白表達(dá)水平與IL-10表達(dá)水平呈正相關(guān),r=0.955,P=0.045(P0.05),差異有統(tǒng)計(jì)學(xué)意義。VDR蛋白表達(dá)水平與IL-12表達(dá)水平呈負(fù)相關(guān),r=-0.968,P=0.032(P0.05),差異有統(tǒng)計(jì)學(xué)意義。結(jié)論:1紅色毛癬菌刺激角質(zhì)形成細(xì)胞后,IL-10的表達(dá)水平呈時(shí)間依賴性上升趨勢(shì),IL-12的表達(dá)水平呈時(shí)間依賴性下降趨勢(shì)。2紅色毛癬菌刺激角質(zhì)形成細(xì)胞后,VDR蛋白表達(dá)呈時(shí)間依賴性上升趨勢(shì)。3紅色毛癬菌刺激角質(zhì)形成細(xì)胞后,VDR表達(dá)與IL-10的表達(dá)呈正相關(guān),而與IL-12的表達(dá)呈負(fù)相關(guān)。綜上所述,紅色毛癬菌刺激角質(zhì)形成細(xì)胞后,隨作用時(shí)間的延長(zhǎng),VDR及IL-10表達(dá)上升,而IL-12表達(dá)下降。推測(cè)紅色毛癬菌可能通過(guò)VDR途徑促進(jìn)角質(zhì)形成細(xì)胞分泌IL-10以及抑制IL-12表達(dá),進(jìn)而導(dǎo)致機(jī)體對(duì)紅色毛癬菌產(chǎn)生免疫耐受,這可能是疾病遷延不愈原因之一。
[Abstract]:Objective: studies have shown that human beings are generally susceptible to Trichophyton rubre. 90% of the global dermworm disease is caused by Trichophyton rubre. The peak of the disease is concentrated in the summer, most of which do not cause obvious inflammatory response, but it is a chronic course of disease, which seriously affects the quality of life of the patients [1]. vitamin D, a steroid derivative, and can be used for people. The body immune system has an important effect, its role is becoming more and more concerned about [2], vitamin D is combined with vitamin D receptor (VDR) to play a physiological role,.VDR is a member of the steroid hormone / thyroid hormone receptor superfamily, a ligand dependent nuclear transcription factor. The skin is an important source of vitamin D in the body, It can be converted from the precursor substance vitamin D3 stored in the skin under the light of the sun. The active form of vitamin D, 1,25 (OH) 2D3, is higher in summer than in winter. In summer, the storage of vitamin D has an upward trend, which can lead to the descending of the pro-inflammatory factors, thus causing the organism to be immune to the pathogenic microorganism. .IL-12 is a different two polymer pro-inflammatory factor, which can induce the production of interferon gamma, promote the differentiation of Th1 cells, and play a strong pro-inflammatory effect, [3].IL-10 can inhibit the biological activity of many immune cells and the biological effects of these cells. The physiological function is to restrict and terminate the anti inflammation. Therefore, IL-10 plays an important role in limiting the host's immune process to pathogenic bacteria. [4,5]. stimulation of keratinocytes by Trichophyton rubre in vitro, the expression of VDR and IL-10, IL-12, and the correlation analysis of its expression level, and then to explore whether the organism is mediated by VDR signal pathway in the process of Trichophyton rubrum infection. The immune tolerance of Trichophyton rubrum causes chronic deferred disease. Methods: 1 HaCaT cells cultured in vitro were purchased from Shanghai saichi Biological Technology Co., Ltd. number: MXC138.HaCaT cells were cultured with 90%DMEM+10%FBS+1% green streptomycin mixture as a complete medium. HaCaT cells were made into single cell suspension and fined. 5 days after culture, it was taken out for 5 days to take out creeping pieces, acetone was fixed, HE staining and immunohistochemical SP method were used to detect the expression of keratin 10 in HaCaT cells. The strain of strain.2 in the fungal chamber of the second hospital of Hebei Medical University and the preparation of the bacterial suspension were activated for three times on PDA and 28 C at 28. After 7 to 14 days, the aseptic water containing 0.1% Twain 80 was added, and the mixing was evenly mixed. Then the bacterial suspension was grinded with a tissue grinder, the blood cell count board was counted, the concentration was 4-6 * 105CFU/mL.4 and the experiment group was divided into the control group and the experimental group. The HaCaT cells were cultured for 1 weeks, abandoned the original medium, and after the PBS washing 3 times, 0.25% pancreas eggs were used after the 3 times of washing. The white enzyme digested the cells from the bottom of the culture bottle and added the complete medium to terminate the digestion. The cells collected by the centrifuge were collected in the complete culture medium to 1-2 x 106/mL and transferred to the 24 hole culture plate. The cells were continued to be cultured for 24 hours. The experimental group had a concentration of 4-6 x 105CFU/mL in each hole of the experimental group, and the control group per hole. 1mL containing 0.1% Twain 80 aseptic water 1mL. was stored in 2H, 4h, 8h, 16h and the supernatant of the control group at.-20 degrees. Each time point had 3 parallel holes.5ELISA method to measure the IL-10, the secretion level of IL-12 and the control group, the supernatant collected by the control group, respectively, according to the purchase Kit Guide, respectively. Cell culture supernatant IL-10, IL-12 expression level.6Westernblot method to measure the expression level of VDR in each group PBS solution cleaning the 24 pore plate of HaCaT cells and transferred to EP tube. After centrifugation, the lysate RIPA lysate cells were added, the total protein was extracted, SDS-PAGE gel electrophoresis, to PVDF membrane, after the skimmed milk was closed, incubated respectively. VDR (1:1000) and beta -actin (1:1000) resistance and Goat anti rabbit / mouse IgG two resistance (1:3000), and finally using a dual color infrared laser scanner to sweep the film, ImageJ software analysis results of.7 statistics analysis SPSS13.0 statistical analysis software, the results are expressed in mean number of standard deviation, the statistical method used among groups is a single factor variance. Analysis, the statistical method used in the 22 comparison of different time points in the group was SNK-q test. The statistical method used in the correlation analysis of VDR and IL-10 and IL-12 was Pearson correlation analysis, and P0.05 was statistically significant. Results: the morphological observation of 1HaCaT cell culture was observed under inverted microscope, the cell growth was good and most of the cells were fine. Cell wall, strong refraction, round, cell membrane integrity and outstretched 2 to 3 pseudo feet.5 days after the appearance of polygons, fused into slices, the appearance of paving stone like.HaCaT cells HE staining results show that the cytoplasm is rich, pink, blue purple. Immunohistochemical results show that HaCaT cell keratin 10 in the cytoplasm and cytoplasm of cytoplasm. The results of positive expression of IL-10 expression in.2 supernatant were determined by ELISA method in the experimental 2h, 4h, 8h, 16h, and the expression level of IL-10 in the experimental group was 1427.71 + 15.9521562.09 + 12.241826.57 + 16.122363.807 + 53.465pg/mL, and the expression level increased with the time. 100.58 + 2.461160.8 + 7.0441285.07 + 5.1851400.96 + 11.657pg/mL, the expression level of IL-10 increased with time, and the expression level of.IL-10 in the experimental group was higher than that of the control group, F=6.591, P=0.042 (P0.05). The difference was statistically significant in IL-12 expression of.3 supernatant The expression level was 296.85 + 0.6285.43 + 2.03275.19 + 6.03249.18 + 3.72pg/mL respectively. The expression level of IL-12 decreased with the prolongation of time. The expression level of IL-12 in 2H, 4h, 8h and 16h was 294.85 + 4.601317.36 + 7.026340.123 + + +. The expression level of IL-12 in the test group was significantly lower than that of the control IL-12 expression level, F=7.012, P=0.038 (P0.05), the difference was statistically significant, the level of the expression of VDR protein in the human immortalized keratinocyte (HaCaT) by.4WesternBlot method was in the experimental 2h, 4h, 8h, 16h, the ratio of the level of the level of the protein expression was 0.394 +. The expression level of VDR protein in 2H, 4h, 8h, 16h in 458 + 0.021,0.616 + 0.156. control group was 0.27 + 0.014,0.294 + 0.015,0.301 + 0.002,0.413 + 0.01. experiment group and control group, the expression of VDR protein expression increased with time. Statistical significance.5 expression level of Trichophyton rhytinea in HaCaT cells was related to the expression level of IL-10 and IL-12 respectively. The results of Pearson correlation analysis showed that the expression level of VDR protein was positively correlated with the level of IL-10 expression, r=0.955, P=0.045 (P0.05), and the difference was statistically significant in the expression of the.VDR protein and the expression of IL-10. The level showed negative correlation, r=-0.968, P=0.032 (P0.05), and the difference was statistically significant. Conclusion: after the 1 Trichophyton rubrum stimulated keratinocytes, the expression level of IL-10 showed a time dependent trend, the expression level of IL-12 was time dependent and.2 red Trichophyton rubrum stimulated keratinocyte, and the expression of VDR protein was time dependent. On the rise of.3, the expression of VDR was positively correlated with the expression of IL-10, but negatively correlated with the expression of IL-12. To sum up, the expression of VDR and IL-10 was increased with the time of stimulation of Trichophyton rubrum, and the expression of IL-12 decreased with the prolongation of the action time. It is suggested that Trichophyton rubrum may be through VDR pathway. Promoting the secretion of IL-10 from keratinocytes and inhibiting the expression of IL-12, which may lead to immune tolerance to Trichophyton rubrum, may be one of the causes of the disease.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R756

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 萬(wàn)春輝;杜先智;;維生素D誘導(dǎo)自噬對(duì)巨噬細(xì)胞清除結(jié)核分枝桿菌的作用[J];中國(guó)免疫學(xué)雜志;2015年04期

2 曹雨娜;張虹;;維生素D抗腫瘤作用的研究進(jìn)展[J];中國(guó)臨床藥學(xué)雜志;2014年02期

3 畢愛芬;裴德翠;胡漢斌;湛昌文;丘雪峰;;25-羥維生素D水平與妊娠期細(xì)菌性陰道病發(fā)生率的關(guān)系[J];中外醫(yī)學(xué)研究;2013年15期

4 彭靜;曹祥山;邱國(guó)強(qiáng);孫冠星;;1,25(OH)_2維生素D_3對(duì)人樹突狀細(xì)胞成熟及其介導(dǎo)免疫耐受的影響[J];中國(guó)實(shí)驗(yàn)血液學(xué)雜志;2012年03期

5 牟金金;楊敏;柳汝明;周俊翔;唐堯;;維生素D輔助治療肺結(jié)核的系統(tǒng)評(píng)價(jià)[J];中國(guó)藥房;2011年28期

6 嚴(yán)媚;溫丙昭;曹琰;江明;;1,25-(OH)_2D_3及復(fù)方丹參對(duì)大鼠異基因骨髓移植后造血重建及T細(xì)胞亞群的影響[J];西安交通大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2010年03期

7 丁曉飛;張青麗;郝莉;李冰燕;張?jiān)隼?;1,25(OH)_2D_3對(duì)巨噬細(xì)胞吞噬金黃色葡萄球菌過(guò)程的影響[J];營(yíng)養(yǎng)學(xué)報(bào);2010年02期

8 陳江華,姜睿,何強(qiáng),祝恒成,王惠萍,金娟,陳瑩;維生素D衍生物聯(lián)合供者骨髓細(xì)胞輸注誘導(dǎo)異基因大鼠心臟移植免疫耐受[J];中華器官移植雜志;2004年01期

9 章愛斌;1,25-(OH)_2D_3的免疫抑制作用及其在器官移植中的應(yīng)用(文獻(xiàn)綜述)[J];國(guó)外醫(yī)學(xué).外科學(xué)分冊(cè);2003年05期

10 李德發(fā),劉煥龍,席鵬彬,陳勇,李宇紅;維生素D_3對(duì)斷奶仔豬生長(zhǎng)性能和免疫機(jī)能的影響[J];中國(guó)農(nóng)業(yè)大學(xué)學(xué)報(bào);2001年05期

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