红色毛癣菌对角质形成细胞VDR与IL-10、IL-12表达的影响及相关关系的实验研究

发布时间：2018-04-22 17:49

本文选题：红色毛癣菌 + 维生素D　；参考：《河北医科大学》2017年硕士论文

【摘要】：目的:有研究显示,人类对红色毛癣菌普遍易感,全球90%的皮肤癣菌病是由红色毛癣菌引起的,发病高峰集中在夏季,大多不引起明显的炎症反应,而呈现为慢性迁延病程,严重影响着患者生活质量[1]。维生素D是一类固醇类衍生物,可对人体免疫系统产生重要的影响,其作用越来越受到人们的关注[2],维生素D通过与维生素D受体(VDR)结合发挥生理作用。VDR属于类固醇激素/甲状腺激素受体超家族成员,是一种配体依赖的核转录因子。皮肤是机体内所需维生素D的重要源泉,可以在太阳光的照射下由皮肤内储备的前体物质维生素D3原转化而来。维生素D的活性形式1,25(OH)2D3,在夏季血清水平较冬季为高,夏季维生素D的存贮有一个上升趋势,这可导致前炎症因子的下降,从而导致机体对病原微生物产生免疫耐受。IL-12是一种异二聚体促炎因子,可通过诱导γ干扰素的产生、促进Th1细胞的分化,起到强大的促炎症作用[3]。IL-10可对多种免疫细胞的生物活性及这些细胞释放的细胞因子的生物效应产生抑制作用,其生理功能就是限制和终止炎症反应。因此,IL-10在限制宿主对病原菌的免疫过程中起重要作用[4,5]。本实验通过红色毛癣菌体外刺激角质形成细胞,检测VDR与IL-10、IL-12的表达并对其表达水平进行相关分析,进而探讨红色毛癣菌感染过程中是否通过VDR信号途径介导机体对红色毛癣菌的免疫耐受导致疾病慢性迁延。方法:1实验用细胞的培养HaCaT细胞购自上海赛笠生物科技有限公司编号:MXC138。HaCaT细胞的培养使用90%DMEM+10%FBS+1%青链霉素混合剂配制而成的完全培养基。将HaCaT细胞制成单细胞悬液后,进行细胞爬片,培养5天后,取出爬片,丙酮固定,进行HE染色及免疫组织化学SP法检测HaCaT细胞角蛋白10的表达。2实验用菌株实验菌株采用河北医科大学第二医院真菌室保存菌种。3菌株的培养及菌悬液的配制将红色毛癣菌在PDA上活化三次,28℃培养7—14天。加入含0.1%吐温80的无菌水,吹打混合均匀,然后用组织研磨器将菌悬液研磨,血球计数板计数,调整浓度为4-6×105CFU/mL。4实验分组与处理实验分为对照组和实验组。将HaCaT细胞培养1周后取出,弃原培养基,PBS洗涤3遍后,用0.25%胰蛋白酶将细胞从培养瓶底部消化下来,加入完全培养基终止消化,离心所收集到的细胞,用完全培养基调终浓度至1-2×106/mL,转种到24孔培养板中。继续培养24小时后行分组处理。实验组每孔中加入浓度为4-6×105CFU/mL的菌悬液1mL,对照组每孔加入1mL含0.1%吐温80无菌水1mL。依次在2h、4h、8h、16h采集实验组和对照组的上清液。-20℃冻存待测。每个时间点均设3个平行孔。5ELISA法测各组IL-10、IL-12的分泌水平复温实验组和对照组所收集到的上清液,依照所购买试剂盒使用指南,分别测定细胞培养上清液中的IL-10、IL-12表达水平。6Westernblot法测各组VDR的表达水平PBS溶液清洗长有HaCaT细胞的24孔板,刮下细胞转移至EP管内,离心后添加裂解液RIPA裂解细胞,提取总蛋白,SDS-PAGE凝胶电泳,转至PVDF膜,脱脂牛奶封闭完成之后,分别孵育VDR(1:1000)与β-actin(1:1000)一抗及山羊抗兔/小鼠IgG二抗(1:3000),最后用双色红外激光扫描仪进行扫膜,ImageJ软件分析结果。7统计学分析SPSS13.0统计学分析软件,所得结果以均数±标准差表示,组间比较所使用的统计学方法是单因素方差分析,组内不同时间点的两两比较所使用的统计学方法为SNK-q检验,VDR与IL-10、IL-12的相关性分析所使用的统计学方法为Pearson相关分析法,P0.05为有统计学意义。结果:1HaCaT细胞培养的形态学观察倒置显微镜下观察,细胞生长状态良好,多数细胞贴壁,折光性强,圆形,细胞膜完整并且向外伸出2至3个伪足。5天后细胞呈现多角形,融合成片,外观呈铺路石样排列。HaCaT细胞HE染色结果可见其胞质丰富,呈粉红色,核大呈蓝紫色。免疫组化结果显示HaCaT细胞的角蛋白10在胞膜及胞质中呈阳性表达。2上清液IL-10表达水平的ELISA法测定结果在实验的2h、4h、8h、16h,实验组IL-10表达水平分别为1427.71±15.952、1562.09±12.24、1826.57±16.12、2363.807±53.465pg/mL,IL-10的表达水平随着时间的延长而增加。对照组在2h、4h、8h、16h的IL-10表达水平分别为1100.58±2.46、1160.8±7.044、1285.07±5.185、1400.96±11.657pg/mL,IL-10的表达水平随着时间的延长而增加。IL-10在实验组的表达水平比对照组高,F=6.591,P=0.042(P0.05),差异有统计学意义。3上清液IL-12表达水平的ELISA法测定结果在实验的2h、4h、8h、16h,实验组IL-12表达水平分别为296.85±0.6、285.43±2.03、275.19±6.03、249.18±3.72pg/mL,IL-12的表达水平随着时间的延长而减少。对照组在2h、4h、8h、16h的IL-12表达水平分别为294.85±4.601、317.36±7.026、340.123±18.905、385.050±6.593pg/mL,IL-12的表达水平随着时间的延长而增加。实验组的IL-12表达水平明显低于对照的IL-12表达水平,F=7.012,P=0.038(P0.05),差异有统计学意义。4WesternBlot法测人永生化角质形成细胞(HaCaT)中VDR蛋白的表达水平在实验的2h、4h、8h、16h,实验组VDR蛋白表达水平灰度比值分别为0.394±0.011、0.468±0.019、0.458±0.021、0.616±0.156。对照组在2h、4h、8h、16h的VDR蛋白表达水平分别为0.27±0.014、0.294±0.015、0.301±0.002、0.413±0.01。实验组和对照组的VDR蛋白表达水平均随时间的延长而表达增加,但实验组VDR蛋白上升的水平高于对照组,F=8.404,P=0.027(P0.05),差异有统计学意义。5红色毛癣菌作用于HaCaT细胞后VDR的表达水平分别与IL-10、IL-12的表达水平的相关分析结果Pearson相关分析结果显示VDR蛋白表达水平与IL-10表达水平呈正相关,r=0.955,P=0.045(P0.05),差异有统计学意义。VDR蛋白表达水平与IL-12表达水平呈负相关,r=-0.968,P=0.032(P0.05),差异有统计学意义。结论:1红色毛癣菌刺激角质形成细胞后,IL-10的表达水平呈时间依赖性上升趋势,IL-12的表达水平呈时间依赖性下降趋势。2红色毛癣菌刺激角质形成细胞后,VDR蛋白表达呈时间依赖性上升趋势。3红色毛癣菌刺激角质形成细胞后,VDR表达与IL-10的表达呈正相关,而与IL-12的表达呈负相关。综上所述,红色毛癣菌刺激角质形成细胞后,随作用时间的延长,VDR及IL-10表达上升,而IL-12表达下降。推测红色毛癣菌可能通过VDR途径促进角质形成细胞分泌IL-10以及抑制IL-12表达,进而导致机体对红色毛癣菌产生免疫耐受,这可能是疾病迁延不愈原因之一。
[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.

【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】：R756

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