银屑病中钙浓度对HaCaT细胞增殖及白细胞介素6表达、分泌的影响研究

发布时间：2018-06-26 00:55

  本文选题：银屑病 + 钙离子　； 参考：《河北大学》2017年硕士论文

【摘要】：银屑病为皮肤科常见病,其发病机制复杂,多认为与遗传、免疫、感染、外界环境等因素有关。临床上银屑病的治疗方法虽多,但也只能达到暂时缓解的目的,且银屑病病程较长,反复发作不易根治,严重危害人们的身心健康。白细胞介素(interleukin,IL)6是一种重要的细胞因子,在银屑病患者皮损处表达增高并可被角质形成细胞合成分泌。钙离子作为细胞内重要的第二信使参与机体凝血、肌肉收缩、心脏节律正常等多种生理功能外,对角质形成细胞的增殖、分化也有一定影响。为了探讨钙离子与IL-6之间是否存在相关性,本研究通过观察不同浓度钙离子对体外培养人角质形成细胞株HaCaT增殖的影响及对IL-6 mRNA表达、蛋白分泌的影响初步分析两者间的相关性。目的:探究钙离子对人角质形成细胞株HaCaT增殖的影响及对IL-6mRNA表达、蛋白分泌的影响。方法:用加入不同浓度钙离子的洛斯维RPMI1640培养液[钙离子终浓度分别为0.3毫摩尔/升(mmol/L)、0.5mmol/L、1mmol/L]与RPMI1640培养液(对照组)分别培养HaCaT细胞。培养HaCaT细胞24h、48h时,二甲基噻唑二苯基四盐(MTT)法检测细胞增殖情况;荧光定量聚合酶链反应(qPCR)法检测钙离子对HaCaT细胞IL-6信使核糖核酸(messenger ribonucleic acid,mRNA)表达的影响;酶联免疫吸附法(ELISA)检测不同钙离子浓度培养条件下HaCaT细胞上清液中IL-6的分泌量;免疫细胞化学法观察细胞中IL-6的表达,其结果用专业图像分析软件(Image Pro Plus)6.0进行分析。采用独立样本t检验、单因素方差分析和皮尔逊(Pearson)相关分析进行统计学处理。结果:MTT结果显示,在设定浓度下HaCaT细胞的增殖率随着钙离子浓度的增加而增高;与对照组相比,各浓度组培养24h时HaCaT细胞增殖率分别为9.65%?0.57%、29.47%?0.73%、52.18%?1.13%(t=29.08,69.12,79.52,P均0.01);培养48h时各浓度组HaCaT细胞增殖率分别为10.23%?0.51%、34.17%?1.14%、57.96%?0.21%(t=35.36,51.67,87.37,P均0.01)。各浓度组培养HaCaT细胞24h时,IL-6 mRNA表达量及其蛋白分泌量随钙离子浓度增加而升高(F=133.07,121.68,P0.01);培养HaCaT细胞48h时,IL-6 mRNA表达量及其蛋白分泌量随钙离子浓度增加而升高(F=151.17,78.64,P0.001)。免疫细胞化学结果显示,培养HaCaT细胞24h、48h时,IL-6平均光密度随钙离子浓度增加而升高(F=113.22,202.05,P0.01)。相关性分析结果显示,培养HaCaT细胞24h时,钙离子与IL-6 mRNA表达和蛋白分泌均呈正性相关(r=0.97、r=0.83,P0.01),培养HaCaT细胞,48h时,钙离子与IL-6 mRNA表达和蛋白分泌均呈正性相关(r=0.98、0.91,P0.01)。结论:在设定浓度下,钙离子可促进HaCaT细胞的体外增殖,并可在mRNA和蛋白水平促进IL-6的表达和分泌。这可能是银屑病皮损处钙浓度增高导致角质形成细胞增殖和炎症反应加重的原因。
[Abstract]:Psoriasis is a common disease in the Department of dermatology. Its pathogenesis is complicated, and most of them are related to the factors of heredity, immunity, infection and external environment. Although the treatment of psoriasis is more and more, it can only achieve the purpose of temporary relief, and the course of psoriasis is long, repeated attacks are not easy to cure, and it seriously endangers people's physical and mental health. Interleukin (inter) Leukin, IL) 6 is an important cytokine, which is expressed in the skin lesions of patients with psoriasis and can be synthesized and secreted by keratinocytes. Calcium ions, as an important second messenger in the cells, participate in the body's coagulation, muscle contraction, and normal cardiac rhythm, and have a certain effect on the proliferation and differentiation of keratinocytes. To investigate whether there is a correlation between calcium ion and IL-6, the effect of calcium ions on the proliferation of human keratinocyte cell line HaCaT in vitro and the effect of IL-6 mRNA expression and protein secretion on the proliferation of human keratinocyte strain were investigated. Influence and effect on the expression of IL-6mRNA and protein secretion. Methods: the culture solution of RPMI1640 (0.3 milmole / liter (mmol/L), 0.5mmol/L, 1mmol/L] and RPMI1640 culture solution) was cultured with different concentration of calcium ions, and HaCaT fine cells were cultured respectively. HaCaT cells 24h, 48h, two methyl thiazole two phenyl four salt (MTT) assay of cell proliferation; fluorescence quantitative polymerase chain reaction (qPCR) method to detect the effect of calcium ion on the expression of IL-6 messenger RNA (messenger ribonucleic acid, mRNA) in HaCaT cells; enzyme linked immunosorbent assay (ELISA) detection of IL-6 secretion in the supernatant of HaCaT cells under different calcium concentration cultures; immunization The expression of IL-6 in the cells was observed by the method of study. The results were analyzed with professional image analysis software (Image Pro Plus) 6. Independent sample t test, single factor ANOVA analysis and Pearson (Pearson) correlation analysis were used for statistical processing. Results: MTT results showed that the proliferation rate of HaCaT cells increased with the increase of calcium ion concentration under the set concentration. The proliferation rate of HaCaT cells in each concentration group was 9.65%? 0.57%, 29.47%? 0.73%, 52.18%? 1.13% (t=29.08,69.12,79.52, P 0.01), compared with the control group, and the proliferation rates of HaCaT cells in each concentration group were 10.23%? 0.51%, 34.17%? 1.14%, 57.96%? 0.21% (t=35.36,51.67,87.37, P are all 0.01) at the time of 48h culture. Each concentration group was cultured for HaCaT fine. When the cell 24h, the expression of IL-6 mRNA and its protein secretion increased with the increase of calcium ion concentration (F=133.07121.68, P0.01). When the HaCaT cell 48h was cultured, the mRNA expression of IL-6 and its protein secretion increased with the increase of calcium ion concentration (F=151.17,78.64, P0.001). The density increased with the increase of calcium ion concentration (F=113.22202.05, P0.01). The results of correlation analysis showed that the calcium ions were positively correlated with the expression of IL-6 mRNA and protein secretion (r=0.97, r=0.83, P0.01) when cultured HaCaT cells 24h (r=0.97, r=0.83, P0.01), and 48h, calcium ions were positively correlated with the expression of IL-6 and protein secretion. 1). Conclusion: at the set concentration, calcium ions can promote the proliferation of HaCaT cells in vitro, and promote the expression and secretion of IL-6 at the level of mRNA and protein. This may be the cause of the increase of calcium concentration in the skin lesions of psoriasis and the aggravation of the proliferation of keratinocytes and the aggravation of the inflammatory reaction.
【学位授予单位】：河北大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R758.63

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