狼疮肾炎中活性维生素D对单核细胞趋化蛋白-1表达的影响

发布时间：2018-04-21 17:48

本文选题：系统性红斑狼疮 + 狼疮肾炎　；参考：《川北医学院》2017年硕士论文

【摘要】：目的:1.通过检测系统性红斑狼疮(systemic lupus erythematosus,SLE)患者血清25-(OH)D3水平、外周血单个核细胞(PBMC)单核细胞趋化因子-1(monocyte chemotactic protein-1,MCP-1)mRNA表达及24小时尿MCP-1水平,并分析血清25-(OH)D3水平与其他两者之间的相关性,探讨活性维生素D对狼疮肾炎(lupus nephritis,LN)患者MCP-1表达水平的影响。2.以人肾系膜细胞(human renal mesangial cells,HRMCs)为研究对象,以脂多糖(lipopolysaccharide,LPS)及抗dsDNA抗体为刺激因素,以1α,25-二羟维生素D3(1α,25-dihydroxyvitamin D3,1α,25-(OH)2D3)为干预处理因素,观察抗dsDNA抗体对HRMCs及1α,25-(OH)2D3对MCP-1表达水平的影响,探讨LN疾病发生发展的可能机制,同时为从免疫调节方面提供临床改善LN提供理论依据。方法:1.收集30例健康体检者、80例nLN患者和73例LN患者血清、全血及24小时尿,通过电化学发光免疫分析法(electrochemiluminescence immunoassay,ECLIA)法测定各组血清25-(OH)D3水平,利用实时定量反转录-聚合酶链反应(RT-qPCR)方法检测各组外周血PBMC中MCP-1mRNA的表达水平,通过酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测各组尿液中MCP-1水平,并分析MCP-1mRNA的表达水平、尿液中MCP-1水平与血清25-(OH)D3水平的关系。2.体外培养人肾系膜细胞,接种于6孔板中待细胞生长至80%左右融合后,用无血清肾系膜细胞培养基37℃、5%co2预培养6h,使其处于同步化状态后进行以下各组实验。(1)加入lps至终浓度0.1μg/ml,同时加或不加不同浓度1α,25-(oh)2d3(10-7mol/l、10-8mol/l、10-9mol/l、10-10mol/l)干预24小时和1α,25-(oh)2d310-7mol/l干预不同时间(3h、6h、12h、24h及48h)。(2)加入三种抗dsdna单克隆抗体(163p.64、163p.77及452s.160,各10μg/ml)组,分别刺激24小时、48小时及72小时。(3)加入163p.77不同浓度(1μg/ml、5μg/ml、10μg/ml、15μg/ml)组,刺激72小时。(4)加入163p.7710μg/ml,同时加或不加不同浓度1α,25-(oh)2d3不同浓度(10-7mol/l、10-8mol/l、10-9mol/l、10-10mol/l)干预24小时和1α,25-(oh)2d310-7mol/l干预不同时间(24h、48h及72h)。收集细胞培养上清液,elisa检测细胞培养上清中mcp-1的表达水平;提取各组细胞中总rna,rt-qpcr检测各样本mcp-1mrna的表达水平。结果:1.(1)血清25-(oh)d3测定结果:ln组血清25-(oh)d3水平显著低于正常对照组和nln组(p0.01),且血清维生素d缺乏的比例在ln组(71.2%)均显著高于正常对照组(23.3%)及nln组(41.3%)(x2=28.444,p0.01);(2)mcp-1mrna表达检测结果:nln组与ln组pbmc中mcp-1mrna的表达均高于正常对照组(p0.05);(3)24h尿mcp-1测定结果:ln组24h尿mcp-1水平均高于正常对照组和nln组(p0.05,p0.01);(4)相关性分析:nln组患者血清25-(oh)d3水平与pbmcmcp-1mrna表达呈负相关(r=-0.365,p0.01),ln组患者血清25-(oh)d3水平与24h尿mcp-1水平呈负相关(r=-0.437,p0.01)。2.(1)1α,25-(oh)2d3抑制lps诱导下hrmcsmcp-1的表达:lps可显著增加HRMCs MCP-1的表达水平(p0.01),而1α,25-(OH)2D3可显著降低经LPS干刺激MCP-1的表达(p0.01),其中在10-7mol/L干预24 h时对MCP-1表达的降低最为明显。(2)抗dsDNA抗体刺激HRMCs MCP-1的表达:163p.64、163p.77和452s.160均能增加HRMCs MCP-1 mRNA和蛋白的表达(p0.01),并且163p.77处理72h时较其他各处理组及其不同处理时间组作用更为显著(p0.01)。(3)不同浓度抗dsDNA抗体(163p.77)对HRMCs MCP-1表达的影响:163p.77 10μg/ml和15μg/ml浓度组刺激72h能显著增加细胞MCP-1的表达水平(p0.01)。(4)1α,25-(OH)2D3抑制163p.77作用下HRMCs MCP-1的表达:不同给药浓度的1α,25-(OH)2D3均可降低经163p.77刺激后MCP-1的表达水平(p0.05),且抑制强度在一定范围内随浓度及时间增加而逐渐增强。结论:1.SLE患者血清25-(OH)D3的缺乏及尿中MCP-1水平的增加可能与SLE肾脏损伤存在一定联系,并且维生素D可能对LN患者体内MCP-1表达存在调节作用。2.1α,25-(OH)2D3可显著抑制LPS刺激下HRMCs MCP-1的表达,且抑制强度在一定范围内随给药浓度及干预时间的增加而增强。3.抗dsDNA抗体可显著增加HRMCs MCP-1的表达,且可被1α,25-(OH)2D3抑制,并在一定范围内呈剂量-时间依赖性关系。4.1α,25-(OH)2D3可通过抑制MCP-1基因及蛋白的表达发挥抗炎作用。
[Abstract]:Objective: 1. to detect the level of 25- (OH) D3 in patients with systemic lupus erythematosus (systemic lupus erythematosus, SLE), the expression of -1 (monocyte chemotactic protein-1) in peripheral blood mononuclear cells (PBMC) and 24 hours of urine, and to analyze the correlation between the serum levels and the other two. The effect of active vitamin D on the expression of MCP-1 in patients with lupus nephritis (lupus nephritis, LN) was studied by.2. in human mesangial mesangial cells (human renal mesangial cells, HRMCs), with 1 alpha, 1 alpha (alpha hydroxyvitamin alpha). 2D3) for the intervention factors, the effect of anti dsDNA antibody on HRMCs and 1 alpha, 25- (OH) 2D3 on the expression of MCP-1 was observed. The possible mechanism of the development of LN disease was discussed. At the same time, the theoretical basis for the clinical improvement of LN from immunoregulation was provided. Methods: 1., 30 healthy persons, 80 nLN patients and 73 LN patients were collected, and the whole blood and 24 were small. The serum levels of 25- (OH) D3 were measured by electrochemiluminescence immunoassay (electrochemiluminescence immunoassay, ECLIA), and the expression of MCP-1mRNA in peripheral blood PBMC was detected by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR), and the enzyme linked immunosorbent assay (enzyme-linked immunosorbent as) was used. Say, ELISA) detected the level of MCP-1 in urine, and analyzed the expression level of MCP-1mRNA, the relationship between the level of MCP-1 in urine and the level of serum 25- (OH) D3 in the human kidney mesangial cells in vitro, and inoculated in the 6 orifice plate after the cell growth to about 80% fusion, and used the serum-free renal mesangial cell culture medium at 37, 5%co2 pre culture 6h, so that it was in synchronization. The following experiments were carried out in the following groups. (1) adding LPS to the final concentration of 0.1 u g/ml, adding or not adding different concentrations of 1 alpha, 25- (OH) 2D3 (10-7mol/l, 10-8mol/l, 10-9mol/l, 10-10mol/l) intervention 24 hours and 1 alpha, 25- (OH) intervention at different times. (2) add three kinds of anti monoclonal antibodies 160, each 10 g/ml) group, stimulated 24 hours, 48 hours and 72 hours respectively. (3) adding different concentrations of 163p.77 (1, 5, g/ml, 10, g/ml, 15 mu), stimulated 72 hours. (4) added 163p.7710 mu g/ml, plus or without the concentration of 1 alpha, 25- (OH) 2D3 concentration (10-7mol/l, 10-7mol/l, oh) interventions -7mol/l intervention at different time (24h, 48h and 72h). Collect cell culture supernatant, ELISA to detect the expression level of MCP-1 in cell culture supernatant; extract the total RNA in each cell and RT-qPCR to detect the expression level of MCP-1mRNA in each sample. Results: 1. (1) serum 25- (OH) D3 determination: the serum levels are significantly lower than that of the normal control group and the normal group. (P0.01) and the proportion of serum vitamin D deficiency in group ln (71.2%) was significantly higher than that in normal control group (23.3%) and NLN group (41.3%) (x2=28.444, P0.01); (2) MCP-1mRNA expression in group NLN and LN group PBMC MCP-1mRNA was higher than that in normal control group (P0.05); (3) Group and NLN group (P0.05, P0.01); (4) correlation analysis: the level of serum 25- (OH) D3 in NLN group was negatively correlated with pbmcmcp-1mrna expression (r=-0.365, P0.01). The expression level (P0.01), while 1 alpha, 25- (OH) 2D3 significantly reduced the expression of MCP-1 by LPS dry stimulation (P0.01), and the expression of MCP-1 was most obvious when 10-7mol/L intervention was 24 h. (2) the expression of anti dsDNA antibody stimulated HRMCs and proteins. 72h was more significant than other treatment groups and their different processing time groups (P0.01). (3) the effects of different concentrations of anti dsDNA antibodies (163p.77) on the expression of HRMCs MCP-1: 163p.77 10 mu g/ml and 15 micron concentration groups stimulated 72h to significantly increase the expression level of cell MCP-1 (P0.01). (4) 1 alpha The expression of 1 alpha, 25- (OH) 2D3 at different doses could reduce the expression level of MCP-1 (P0.05) after 163p.77 stimulation, and the inhibitory intensity increased gradually with the increase of concentration and time in a certain range. Conclusion: the deficiency of 25- (OH) D3 in serum of 1.SLE patients and the increase of MCP-1 water in urine may be associated with the damage of SLE kidney. 25- (OH) 2D3 could significantly inhibit the expression of HRMCs MCP-1 under the stimulus of LPS, and 25- (OH) 2D3 could significantly inhibit the expression of HRMCs MCP-1 under the stimulus of LPS, and the inhibitory intensity of 25- (OH) 2D3 can significantly increase the expression of.3. dsDNA antibodies in a certain range with the increase of drug concentration and intervention time, and can be inhibited by 1 alpha, and in a certain range, the expression of MCP-1 can be inhibited by a certain extent. A dose time dependent relationship between.4.1 and 25- (OH) 2D3 can play an anti-inflammatory role by inhibiting the expression of MCP-1 gene and protein.

【学位授予单位】：川北医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R593.242

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