本文選題：系統(tǒng)性紅斑狼瘡 + 狼瘡腎炎��； 參考：《川北醫(yī)學(xué)院》2017年碩士論文

【摘要】：目的:1.通過(guò)檢測(cè)系統(tǒng)性紅斑狼瘡(systemic lupus erythematosus,SLE)患者血清25-(OH)D3水平、外周血單個(gè)核細(xì)胞(PBMC)單核細(xì)胞趨化因子-1(monocyte chemotactic protein-1,MCP-1)mRNA表達(dá)及24小時(shí)尿MCP-1水平,并分析血清25-(OH)D3水平與其他兩者之間的相關(guān)性,探討活性維生素D對(duì)狼瘡腎炎(lupus nephritis,LN)患者M(jìn)CP-1表達(dá)水平的影響。2.以人腎系膜細(xì)胞(human renal mesangial cells,HRMCs)為研究對(duì)象,以脂多糖(lipopolysaccharide,LPS)及抗dsDNA抗體為刺激因素,以1α,25-二羥維生素D3(1α,25-dihydroxyvitamin D3,1α,25-(OH)2D3)為干預(yù)處理因素,觀察抗dsDNA抗體對(duì)HRMCs及1α,25-(OH)2D3對(duì)MCP-1表達(dá)水平的影響,探討LN疾病發(fā)生發(fā)展的可能機(jī)制,同時(shí)為從免疫調(diào)節(jié)方面提供臨床改善LN提供理論依據(jù)。方法:1.收集30例健康體檢者、80例nLN患者和73例LN患者血清、全血及24小時(shí)尿,通過(guò)電化學(xué)發(fā)光免疫分析法(electrochemiluminescence immunoassay,ECLIA)法測(cè)定各組血清25-(OH)D3水平,利用實(shí)時(shí)定量反轉(zhuǎn)錄-聚合酶鏈反應(yīng)(RT-qPCR)方法檢測(cè)各組外周血PBMC中MCP-1mRNA的表達(dá)水平,通過(guò)酶聯(lián)免疫吸附試驗(yàn)(enzyme-linked immunosorbent assay,ELISA)檢測(cè)各組尿液中MCP-1水平,并分析MCP-1mRNA的表達(dá)水平、尿液中MCP-1水平與血清25-(OH)D3水平的關(guān)系。2.體外培養(yǎng)人腎系膜細(xì)胞,接種于6孔板中待細(xì)胞生長(zhǎng)至80%左右融合后,用無(wú)血清腎系膜細(xì)胞培養(yǎng)基37℃、5%co2預(yù)培養(yǎng)6h,使其處于同步化狀態(tài)后進(jìn)行以下各組實(shí)驗(yàn)。(1)加入lps至終濃度0.1μg/ml,同時(shí)加或不加不同濃度1α,25-(oh)2d3(10-7mol/l、10-8mol/l、10-9mol/l、10-10mol/l)干預(yù)24小時(shí)和1α,25-(oh)2d310-7mol/l干預(yù)不同時(shí)間(3h、6h、12h、24h及48h)。(2)加入三種抗dsdna單克隆抗體(163p.64、163p.77及452s.160,各10μg/ml)組,分別刺激24小時(shí)、48小時(shí)及72小時(shí)。(3)加入163p.77不同濃度(1μg/ml、5μg/ml、10μg/ml、15μg/ml)組,刺激72小時(shí)。(4)加入163p.7710μg/ml,同時(shí)加或不加不同濃度1α,25-(oh)2d3不同濃度(10-7mol/l、10-8mol/l、10-9mol/l、10-10mol/l)干預(yù)24小時(shí)和1α,25-(oh)2d310-7mol/l干預(yù)不同時(shí)間(24h、48h及72h)。收集細(xì)胞培養(yǎng)上清液,elisa檢測(cè)細(xì)胞培養(yǎng)上清中mcp-1的表達(dá)水平;提取各組細(xì)胞中總rna,rt-qpcr檢測(cè)各樣本mcp-1mrna的表達(dá)水平。結(jié)果:1.(1)血清25-(oh)d3測(cè)定結(jié)果:ln組血清25-(oh)d3水平顯著低于正常對(duì)照組和nln組(p0.01),且血清維生素d缺乏的比例在ln組(71.2%)均顯著高于正常對(duì)照組(23.3%)及nln組(41.3%)(x2=28.444,p0.01);(2)mcp-1mrna表達(dá)檢測(cè)結(jié)果:nln組與ln組pbmc中mcp-1mrna的表達(dá)均高于正常對(duì)照組(p0.05);(3)24h尿mcp-1測(cè)定結(jié)果:ln組24h尿mcp-1水平均高于正常對(duì)照組和nln組(p0.05,p0.01);(4)相關(guān)性分析:nln組患者血清25-(oh)d3水平與pbmcmcp-1mrna表達(dá)呈負(fù)相關(guān)(r=-0.365,p0.01),ln組患者血清25-(oh)d3水平與24h尿mcp-1水平呈負(fù)相關(guān)(r=-0.437,p0.01)。2.(1)1α,25-(oh)2d3抑制lps誘導(dǎo)下hrmcsmcp-1的表達(dá):lps可顯著增加HRMCs MCP-1的表達(dá)水平(p0.01),而1α,25-(OH)2D3可顯著降低經(jīng)LPS干刺激MCP-1的表達(dá)(p0.01),其中在10-7mol/L干預(yù)24 h時(shí)對(duì)MCP-1表達(dá)的降低最為明顯。(2)抗dsDNA抗體刺激HRMCs MCP-1的表達(dá):163p.64、163p.77和452s.160均能增加HRMCs MCP-1 mRNA和蛋白的表達(dá)(p0.01),并且163p.77處理72h時(shí)較其他各處理組及其不同處理時(shí)間組作用更為顯著(p0.01)。(3)不同濃度抗dsDNA抗體(163p.77)對(duì)HRMCs MCP-1表達(dá)的影響:163p.77 10μg/ml和15μg/ml濃度組刺激72h能顯著增加細(xì)胞MCP-1的表達(dá)水平(p0.01)。(4)1α,25-(OH)2D3抑制163p.77作用下HRMCs MCP-1的表達(dá):不同給藥濃度的1α,25-(OH)2D3均可降低經(jīng)163p.77刺激后MCP-1的表達(dá)水平(p0.05),且抑制強(qiáng)度在一定范圍內(nèi)隨濃度及時(shí)間增加而逐漸增強(qiáng)。結(jié)論:1.SLE患者血清25-(OH)D3的缺乏及尿中MCP-1水平的增加可能與SLE腎臟損傷存在一定聯(lián)系,并且維生素D可能對(duì)LN患者體內(nèi)MCP-1表達(dá)存在調(diào)節(jié)作用。2.1α,25-(OH)2D3可顯著抑制LPS刺激下HRMCs MCP-1的表達(dá),且抑制強(qiáng)度在一定范圍內(nèi)隨給藥濃度及干預(yù)時(shí)間的增加而增強(qiáng)。3.抗dsDNA抗體可顯著增加HRMCs MCP-1的表達(dá),且可被1α,25-(OH)2D3抑制,并在一定范圍內(nèi)呈劑量-時(shí)間依賴性關(guān)系。4.1α,25-(OH)2D3可通過(guò)抑制MCP-1基因及蛋白的表達(dá)發(fā)揮抗炎作用。
[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.

【學(xué)位授予單位】：川北醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R593.242

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相關(guān)期刊論文 前1條

1 王茜;潘海峰;李文先;葉冬青;;IgG型抗dsDNA抗體及其亞型與狼瘡腎炎臨床和病理關(guān)系探討[J];中華疾病控制雜志;2008年05期

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本文編號(hào)：1783479