本文關鍵詞： 銀屑病 紫草素 IL-23 IL-6 IL-17　出處：《中國醫(yī)科大學》2010年碩士論文　論文類型：學位論文

【摘要】： 前言 銀屑病的發(fā)病機制與近年新發(fā)現(xiàn)的CD4+T細胞亞群—Th17關系密切。目前研究顯示IL-23／Th17細胞通路是銀屑病發(fā)病機制中的重要環(huán)節(jié)之一。紫草系傳統(tǒng)中藥,具有涼血、活血、解毒、透疹之功效,治療銀屑病的療效已被臨床肯定。紫草素屬于萘醌類化合物,為紫草發(fā)揮藥理作用的主要成分,常被作為治療銀屑病的主要藥物,但其作用機制尚不清楚。為此,我們將探討紫草素對IL-23誘導銀屑病患者PBMCs產(chǎn)生IL-6、IL-17的影響。 材料和方法 一、研究對象 (一)病例組 銀屑病患者20例,均來自2009年10月至2010年3月期間中國醫(yī)科大學附屬第一醫(yī)院住院患者,其中男12例,女8例,年齡23～55歲,平均年齡40.9±10.9歲。 (二)對照組 健康志愿者11例,其中男5例,女6例,年齡27～50歲,平均年齡32.1±4.8歲�；颊呒爸驹刚呷虢M前均簽署知情同意書。 (三)入組標準 1、近三個月未用任何治療銀屑病及免疫抑制性藥物 2、無腫瘤及其他自身免疫性基礎疾病 3、近兩周無感染病史 4、疾病處于進展期 (四)排除標準 1、年齡60歲或20歲 2、近三個月應用治療銀屑病或免疫抑制性藥物 3、有腫瘤或自身免疫性疾病 4、近兩周有感染病史 5、疾病處于靜止期或消退期 二、試劑 重組人白介素23(rIL-23)購自BD/Pharminen公司。IL-23ELISA檢測試劑盒購自RD公司。IL-17ELISA檢測試劑盒購自CUSABIO BIOTECH Co.Ltd公司。IL-6ELISA檢測試劑盒購自武漢博士德公司。CCK-8購自日本同仁化學研究所。紫草素購自日本和光公司(WAKO)。環(huán)孢素A購自德國Merk公司。RPMI1640培養(yǎng)液和PBS緩沖液購自Hyclone公司。Ficoll液購自GE-healthcare bio-sciences AB公司。DMSO購自solarbio公司。 三、方法 (一)藥物處理 10mg紫草素溶于1mlDMSO,雙蒸水定容,細胞培養(yǎng)液中DMSO終濃度0.5%。100mg的環(huán)孢素溶于1mlDMSO,雙蒸水定容,細胞培養(yǎng)液中DMSO終濃度0.5%。 (二)ELISA法檢測血清IL-23的含量 取銀屑病患者及健康志愿者外周靜脈血,離心分離血清,操作步驟參照試劑盒的說明書進行。 (三)PBMCs的分離 將銀屑病患者外周靜脈血用Ficoll進行密度梯度離心,將獲得的PBMCs充分洗滌后,用RPMI1640培養(yǎng)液調(diào)整PBMCs的密度為6×108／L。 (四)細胞培養(yǎng) 將上述調(diào)整密度后的PBMCs細胞培養(yǎng)于48孔板中,每孔850μL,每個培養(yǎng)條件設3個復孔,在不同的刺激條件下置于37℃、50ML／L CO2孵箱中培養(yǎng)。 (五)細胞毒性分析 不同刺激條件下的PBMCs培養(yǎng)72h后收集細胞,將收集的各孔細胞計數(shù)后用100μLRPMI1640重懸,放入96孔板中,每孔均加入CCK-8(10μL),置于37℃、50ML／L CO2孵箱中培養(yǎng)4小時后,酶聯(lián)免疫檢測儀測OD(450nm)。按公式：細胞存活率(%)=[(As-Ab)／(Ac-Ab)]×100%計算細胞存活率。As：實驗孔(含細胞的培養(yǎng)基、CCK-8、紫草素),Ac：對照孔(含細胞的培養(yǎng)基、CCK-8、無紫草素),Ab空白孔(不含細胞和毒性物質(zhì)的培養(yǎng)基、CCK-8)。 (六)ELISA法檢測細胞培養(yǎng)上清液中IL-6、IL-17的含量 不同刺激條件下的PBMCs培養(yǎng)72h后收集細胞培養(yǎng)上清液,操作步驟參照試劑盒的說明書進行。 結(jié)果 (一)病例組較對照組血清中IL-23的含量顯著升高 與對照組相比,病例組血清中IL-23的含量顯著升高。 (二)IL-23誘導銀屑病患者PBMCs產(chǎn)生IL-17 10ng／ml、50ng／ml及100ng／ml IL-23與PBMCs共培養(yǎng)后,IL-23可誘導PBMCs產(chǎn)生IL-17。 (三)紫草素對銀屑病患者PBMCs產(chǎn)生IL-17及細胞存活率的影響 ≤2ug/ml紫草素與PBMCs共培養(yǎng)后,可以劑量依賴的方式增加PBMCs產(chǎn)生IL-17。5 ug／ml、7.5ug/ml、10 ug／ml紫草素與PBMCs共培養(yǎng)后,產(chǎn)生IL-17的增加隨紫草素劑量的增加而減少。12.5ug／ml、20ug／ml、30ug／ml紫草素與PBMCs共培養(yǎng)后,可以劑量依賴的方式減少IL-17的產(chǎn)生。與medium相比,≤12.5ug／ml紫草素與PBMCs共培養(yǎng)后,細胞存活率無明顯下降,20ug／ml及30ug／ml紫草素與PBMCs共培養(yǎng)72h后,細胞存活率顯著降低。 (四)紫草素對IL-23誘導銀屑病患者PBMCs產(chǎn)生IL-17及細胞存活率的影響 PBMCs與IL-23共培養(yǎng)后,IL-23可誘導PBMCs產(chǎn)生IL-17,當加入7.5、10、12.5及20 ug／ml紫草素均可抑制IL-23誘導IL-17的產(chǎn)生。與medium相比,7.5ug/ml、10 ug／ml、12.5ug／ml紫草素與IL-23及PBMCs共培養(yǎng)后,細胞存活率無明顯下降,20ug／ml紫草素與IL-23及PBMCs共培養(yǎng)72h后,細胞存活率顯著降低。 (五)環(huán)孢素對IL-23誘導銀屑病患者PBMCs產(chǎn)生IL-17及細胞存活率的影響 PBMCs與IL-23共培養(yǎng)后,IL-23可誘導PBMCs產(chǎn)生IL-17,加入7.5、12.5、20、30及50ug／ml環(huán)孢素均可抑制IL-23誘導IL-17的產(chǎn)生。與medium相比,7.5、12.5、20、30 ug／ml環(huán)孢素與IL-23及PBMCs共培養(yǎng)后,細胞存活率無明顯下降,而50ug／ml環(huán)孢素與IL-23及PBMCs共培養(yǎng)72h后,細胞存活率顯著降低。 (六)紫草素抑制IL-23誘導銀屑病患者PBMCs產(chǎn)生IL-6、IL-17,及對細胞存活率的影響 PBMCs與IL-23共培養(yǎng)后,IL-23可誘導PBMCs產(chǎn)生IL-6及IL-17,當加入12.5ug／ml紫草素可抑制IL-23誘導IL-6及IL-17的產(chǎn)生。12.5ug／ml紫草素、IL-23與PBMCs共培養(yǎng)后,細胞存活率無明顯下降。 (七)環(huán)孢素A(CsA)抑制IL-23誘導銀屑病患者PBMCs產(chǎn)生IL-6、IL-17及對細胞存活率的影響 PBMCs與IL-23共培養(yǎng)后,IL-23可誘導PBMCs產(chǎn)生IL-6及IL-17,加入30ug／ml環(huán)孢素可抑制IL-23誘導IL-6及IL-17的產(chǎn)生。與medium相比,30ug／ml環(huán)孢素、IL-23與PBMCs共培養(yǎng)后,細胞存活率無明顯下降。 結(jié)論 紫草素能抑制IL-23誘導銀屑病患者PBMCs產(chǎn)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
PBMCs涓嶪L-23鍏卞煿鍏誨悗,IL-23鍙瀵糚BMCs浜х敓IL-17,鍔犲叆7.5,12.5,20,30鍙，

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