紫草素抑制IL-23诱导银屑病患者外周血单个核细胞产生IL-6、IL-17的研究

发布时间：2018-02-24 13:02

  本文关键词： 银屑病 紫草素 IL-23 IL-6 IL-17　出处：《中国医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 前言 银屑病的发病机制与近年新发现的CD4+T细胞亚群—Th17关系密切。目前研究显示IL-23／Th17细胞通路是银屑病发病机制中的重要环节之一。紫草系传统中药,具有凉血、活血、解毒、透疹之功效,治疗银屑病的疗效已被临床肯定。紫草素属于萘醌类化合物,为紫草发挥药理作用的主要成分,常被作为治疗银屑病的主要药物,但其作用机制尚不清楚。为此,我们将探讨紫草素对IL-23诱导银屑病患者PBMCs产生IL-6、IL-17的影响。 材料和方法 一、研究对象 (一)病例组 银屑病患者20例,均来自2009年10月至2010年3月期间中国医科大学附属第一医院住院患者,其中男12例,女8例,年龄23～55岁,平均年龄40.9±10.9岁。 (二)对照组 健康志愿者11例,其中男5例,女6例,年龄27～50岁,平均年龄32.1±4.8岁。患者及志愿者入组前均签署知情同意书。 (三)入组标准 1、近三个月未用任何治疗银屑病及免疫抑制性药物 2、无肿瘤及其他自身免疫性基础疾病 3、近两周无感染病史 4、疾病处于进展期 (四)排除标准 1、年龄60岁或20岁 2、近三个月应用治疗银屑病或免疫抑制性药物 3、有肿瘤或自身免疫性疾病 4、近两周有感染病史 5、疾病处于静止期或消退期 二、试剂 重组人白介素23(rIL-23)购自BD/Pharminen公司。IL-23ELISA检测试剂盒购自RD公司。IL-17ELISA检测试剂盒购自CUSABIO BIOTECH Co.Ltd公司。IL-6ELISA检测试剂盒购自武汉博士德公司。CCK-8购自日本同仁化学研究所。紫草素购自日本和光公司(WAKO)。环孢素A购自德国Merk公司。RPMI1640培养液和PBS缓冲液购自Hyclone公司。Ficoll液购自GE-healthcare bio-sciences AB公司。DMSO购自solarbio公司。 三、方法 (一)药物处理 10mg紫草素溶于1mlDMSO,双蒸水定容,细胞培养液中DMSO终浓度0.5%。100mg的环孢素溶于1mlDMSO,双蒸水定容,细胞培养液中DMSO终浓度0.5%。 (二)ELISA法检测血清IL-23的含量 取银屑病患者及健康志愿者外周静脉血,离心分离血清,操作步骤参照试剂盒的说明书进行。 (三)PBMCs的分离 将银屑病患者外周静脉血用Ficoll进行密度梯度离心,将获得的PBMCs充分洗涤后,用RPMI1640培养液调整PBMCs的密度为6×108／L。 (四)细胞培养 将上述调整密度后的PBMCs细胞培养于48孔板中,每孔850μL,每个培养条件设3个复孔,在不同的刺激条件下置于37℃、50ML／L CO2孵箱中培养。 (五)细胞毒性分析 不同刺激条件下的PBMCs培养72h后收集细胞,将收集的各孔细胞计数后用100μLRPMI1640重悬,放入96孔板中,每孔均加入CCK-8(10μL),置于37℃、50ML／L CO2孵箱中培养4小时后,酶联免疫检测仪测OD(450nm)。按公式：细胞存活率(%)=[(As-Ab)／(Ac-Ab)]×100%计算细胞存活率。As：实验孔(含细胞的培养基、CCK-8、紫草素),Ac：对照孔(含细胞的培养基、CCK-8、无紫草素),Ab空白孔(不含细胞和毒性物质的培养基、CCK-8)。 (六)ELISA法检测细胞培养上清液中IL-6、IL-17的含量 不同刺激条件下的PBMCs培养72h后收集细胞培养上清液,操作步骤参照试剂盒的说明书进行。 结果 (一)病例组较对照组血清中IL-23的含量显著升高 与对照组相比,病例组血清中IL-23的含量显著升高。 (二)IL-23诱导银屑病患者PBMCs产生IL-17 10ng／ml、50ng／ml及100ng／ml IL-23与PBMCs共培养后,IL-23可诱导PBMCs产生IL-17。 (三)紫草素对银屑病患者PBMCs产生IL-17及细胞存活率的影响 ≤2ug/ml紫草素与PBMCs共培养后,可以剂量依赖的方式增加PBMCs产生IL-17。5 ug／ml、7.5ug/ml、10 ug／ml紫草素与PBMCs共培养后,产生IL-17的增加随紫草素剂量的增加而减少。12.5ug／ml、20ug／ml、30ug／ml紫草素与PBMCs共培养后,可以剂量依赖的方式减少IL-17的产生。与medium相比,≤12.5ug／ml紫草素与PBMCs共培养后,细胞存活率无明显下降,20ug／ml及30ug／ml紫草素与PBMCs共培养72h后,细胞存活率显著降低。 (四)紫草素对IL-23诱导银屑病患者PBMCs产生IL-17及细胞存活率的影响 PBMCs与IL-23共培养后,IL-23可诱导PBMCs产生IL-17,当加入7.5、10、12.5及20 ug／ml紫草素均可抑制IL-23诱导IL-17的产生。与medium相比,7.5ug/ml、10 ug／ml、12.5ug／ml紫草素与IL-23及PBMCs共培养后,细胞存活率无明显下降,20ug／ml紫草素与IL-23及PBMCs共培养72h后,细胞存活率显著降低。 (五)环孢素对IL-23诱导银屑病患者PBMCs产生IL-17及细胞存活率的影响 PBMCs与IL-23共培养后,IL-23可诱导PBMCs产生IL-17,加入7.5、12.5、20、30及50ug／ml环孢素均可抑制IL-23诱导IL-17的产生。与medium相比,7.5、12.5、20、30 ug／ml环孢素与IL-23及PBMCs共培养后,细胞存活率无明显下降,而50ug／ml环孢素与IL-23及PBMCs共培养72h后,细胞存活率显著降低。 (六)紫草素抑制IL-23诱导银屑病患者PBMCs产生IL-6、IL-17,及对细胞存活率的影响 PBMCs与IL-23共培养后,IL-23可诱导PBMCs产生IL-6及IL-17,当加入12.5ug／ml紫草素可抑制IL-23诱导IL-6及IL-17的产生。12.5ug／ml紫草素、IL-23与PBMCs共培养后,细胞存活率无明显下降。 (七)环孢素A(CsA)抑制IL-23诱导银屑病患者PBMCs产生IL-6、IL-17及对细胞存活率的影响 PBMCs与IL-23共培养后,IL-23可诱导PBMCs产生IL-6及IL-17,加入30ug／ml环孢素可抑制IL-23诱导IL-6及IL-17的产生。与medium相比,30ug／ml环孢素、IL-23与PBMCs共培养后,细胞存活率无明显下降。 结论 紫草素能抑制IL-23诱导银屑病患者PBMCs产生IL-6及IL-17。
[Abstract]:Preface
The pathogenesis of psoriasis associated with CD4+T cell subsets Th17 relationship discovered in recent years. The current study shows that IL-23 / Th17 pathway is one of the important links in the pathogenesis of psoriasis. Department of traditional Chinese medicine with shikonin, cooling blood, detoxification, blood circulation, efficacy Touzhen, curative effect in the treatment of psoriasis has been clinically confirmed. Shikonin belonging to the naphthoquinone compounds play a major component of pharmacological action for shikonin, often as the main drug in the treatment of psoriasis, but its mechanism is still unclear. Therefore, we will discuss shikonin induced by IL-23 on psoriasis patients PBMCs IL-6, IL-17.
Materials and methods
First, the research object
(1) case group
20 patients with psoriasis were from the First Affiliated Hospital of China Medical University from October 2009 to March 2010, including 12 males and 8 females, aged 23~55 years, with an average age of 40.9 + 10.9 years.
(two) control group
There were 11 healthy volunteers, of which 5 were male, 6 women, 27~50 years old, and the average age was 32.1 + 4.8 years. The patients and volunteers signed the informed consent before entering the group.
(three) standard of entry
1, no treatment for psoriasis and immunosuppressive drugs for nearly three months
2, no tumor and other autoimmune diseases
3, there was no history of infection for the last two weeks
4, the disease is in progress
(four) exclusion criteria
1, age 60 or 20
2, used for the treatment of psoriasis or immunosuppressive drugs for nearly three months
3, there is a tumor or autoimmune disease
4, the history of infection in the last two weeks
5, the disease is in a stationary period or a period of retreat.
Two, reagents
Recombinant human interleukin 23 (rIL-23) was purchased from BD/Pharminen company.IL-23ELISA kit was purchased from RD company.IL-17ELISA kit was purchased from CUSABIO company BIOTECH Co.Ltd.IL-6ELISA kit was purchased from Wuhan boster company.CCK-8 was purchased from Japan on Tongren chemical. Shikonin purchased from Japan and the light company (WAKO). The purchase of cyclosporine A since the German company Merk.RPMI1640 medium and PBS buffer was purchased from Hyclone company.Ficoll GE-healthcare Bio-Sciences AB solution was purchased from.DMSO company was purchased from Solarbio company.
Three, method
(1) drug treatment
10mg shikonin dissolved in 1mlDMSO, volume of double distilled water, cell culture medium DMSO cyclosporine 0.5%.100mg final concentration dissolved in 1mlDMSO, volume of double distilled water, cell culture medium DMSO concentration 0.5%.
(two) the content of serum IL-23 was detected by ELISA
The peripheral venous blood of patients with psoriasis and healthy volunteers was centrifuged and serum was centrifuged. The procedures were carried out according to the instructions of the kit.
(three) separation of PBMCs
The peripheral venous blood of psoriasis patients was washed with Ficoll for density gradient centrifugation. After washing the obtained PBMCs, the density of PBMCs adjusted by RPMI1640 medium was 6 * 108 / L..
(four) cell culture
After adjusting the density, the PBMCs cells were cultured in 48 well plates, 850 L per hole, 3 holes in each culture condition, and incubated in 37 50ML, 50ML / CO2 incubator under different stimulation conditions.
(five) cytotoxicity analysis
Under the conditions of different stimulation PBMCs cultured 72h cells were collected after each hole cell counts collected after 100 LRPMI1640 suspension, in 96 Kong Banzhong, each hole was added to CCK-8 (10 L), at 37 DEG C, 50ML / L CO2 culture incubator after 4 hours, enzyme-linked immunosorbent assay test OD (450nm). According to the formula: the cell survival rate (%) n (As-Ab) / (Ac-Ab)] * 100% cell survival rate was calculated with.As: experimental hole (medium containing cells CCK-8, shikonin, Ac): the control hole (medium, containing CCK-8 cells, without shikonin). Ab blank hole (medium not containing cells and toxic substances, CCK-8).
(six) the content of IL-6 and IL-17 in the supernatant of cell culture by ELISA method
PBMCs was cultured for 72h under different stimulation conditions to collect cell culture supernatant. The operation procedure was carried out according to the instructions of the kit.
Result
(1) the content of IL-23 in the sera of the case group was significantly higher than that of the control group
Compared with the control group, the content of IL-23 in the sera of the case group increased significantly.
(two) IL-23 induced PBMCs in patients with psoriasis to produce IL-17
IL-23 can induce PBMCs production IL-17. after co culture of 10NG / ml, 50NG / ml and 100ng / ml IL-23 with PBMCs
(three) the effect of shikonin on IL-17 and cell survival rate of PBMCs in patients with psoriasis
2ug/ml shikonin after cultured with PBMCs, can dose dependently increased PBMCs IL-17.5 UG / ml, 7.5ug/ml, 10 UG / ml shikonin incubated with PBMCs, IL-17 increased with the increase of shikonin dose and the decrease of.12.5ug / ml, 20ug / ml, 30ug / ml and PBMCs shikonin after co culture, can dose dependently reduce the production of IL-17. Compared with medium, 12.5ug / ml shikonin after cultured with PBMCs, cell survival rate decreased significantly, 20ug / ml and 30ug / ml shikonin were co cultured with PBMCs 72h, the cell survival rate decreased significantly.
(four) the effect of IL-23 on IL-17 and cell survival rate induced by PBMCs in patients with psoriasis
PBMCs co cultured with IL-23, IL-23 can induce the production of PBMCs IL-17, when adding 7.5,10,12.5 and 20 UG / ml shikonin can inhibit IL-23 induced IL-17. Compared with medium, 7.5ug/ml, 10 UG / ml, 12.5ug / ml shikonin and IL-23 and PBMCs co culture, cell survival rate was no significant decline. 20ug / ml Shikonin and IL-23 and PBMCs were co cultured with 72h, cell survival rate decreased significantly.
(five) the effect of cyclosporin on PBMCs induced IL-17 and cell survival rate in patients with psoriasis induced by IL-23
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