【摘要】：目的:明確MTX周期聯(lián)合CTX是否通過JAK-STAT3通路抑制CIA小鼠脾臟Na?ve T細(xì)胞向Th17細(xì)胞分化。方法:本實驗以DBA1小鼠為研究對象,包括體內(nèi)實驗和體外實驗兩部分。體內(nèi)試驗:選取雄性DBA1小鼠56只,隨機(jī)分為正常對照組10只和CIA組46只。建立CIA模型后根據(jù)擬用藥不同隨機(jī)分為4組:CIA組、MTX組(1.5 mg/kg/3d)、CTX組(30 mg/kg/10d)和MTX+CTX組(聯(lián)合組,MTX:1.5 mg/kg/3d,CTX:30mg/kg/10d)。加強免疫3周后開始給藥,治療過程中連續(xù)監(jiān)測各組小鼠左踝關(guān)節(jié)腫脹度及關(guān)節(jié)炎指數(shù)d(arthritis index,AI)。給藥11周后處死小鼠,取雙膝、左踝關(guān)節(jié)包埋、切片,進(jìn)行HE染色,觀察病理改變。分選脾細(xì)胞中的Na?ve T細(xì)胞,經(jīng)細(xì)胞因子IL-6、TGF-β1、IL-1β、IL-23刺激于37℃5%CO2細(xì)胞培養(yǎng)箱中培養(yǎng)96小時,用流式細(xì)胞術(shù)檢測培養(yǎng)后Th17細(xì)胞所占比例,用RT-PCR檢測分化后Th17細(xì)胞中STAT3的表達(dá)水平。體外實驗:選取雄性的DBA1小鼠20只建立CIA模型,7周時處死,分選脾淋巴細(xì)胞中的Na?ve T細(xì)胞,分為空白對照組、抗CD3CD28激活組(激活組)、刺激Th17細(xì)胞定向分化組(Th17分化組)、CTX組、低濃度MTX組(MTX低組)、中濃度MTX組(MTX中組)、高濃度MTX組(MTX高組)、MTX低+CTX組(低聯(lián)合組),在37℃5%CO2細(xì)胞培養(yǎng)箱中培養(yǎng)96小時,用流式細(xì)胞術(shù)檢測培養(yǎng)后Th17細(xì)胞所占比例,用RT-PCR檢測分化后Th17細(xì)胞中STAT3的表達(dá)水平。結(jié)果:1.體內(nèi)實驗(1)治療前,各治療組小鼠關(guān)節(jié)腫脹度、AI與CIA組相比,差異無統(tǒng)計學(xué)意義;治療4周后,各治療組小鼠關(guān)節(jié)腫脹度、AI較CIA組改善明顯,差異有統(tǒng)計學(xué)意義,各治療組間差異無統(tǒng)計學(xué)意義;治療11周后,聯(lián)合組小鼠關(guān)節(jié)腫脹度、AI較CIA組、CTX組改善明顯,差異有統(tǒng)計學(xué)意義,聯(lián)合組較MTX組改善明顯,但差異無統(tǒng)計學(xué)意義;(2)治療11周后,各治療組Th17細(xì)胞所占百分較CIA組明顯降低,差異有統(tǒng)計學(xué)意義,聯(lián)合組Th17百分率較單用藥組降低明顯,但差異無統(tǒng)計學(xué)意義;(3)治療11周后,各治療組Th17細(xì)胞中STAT3 m RNA的表達(dá)均低于CIA組,差異有統(tǒng)計學(xué)意義,聯(lián)合組Th17細(xì)胞中STAT3 m RNA的表達(dá)低于單用藥組,但差異無統(tǒng)計學(xué)意義。2.體外實驗(1)體外培養(yǎng)96h后,Th17分化組Th17細(xì)胞所占百分比與空白組、MTX中組、MTX高組、低聯(lián)合組相比,差異有統(tǒng)計學(xué)意義;低聯(lián)合組與單用藥組相比,培養(yǎng)后Th17細(xì)胞所占百分比降低,差異無統(tǒng)計學(xué)意義;(2)體外培養(yǎng)96h后,Th17分化組Th17細(xì)胞中STAT3 m RNA的表達(dá)與空白組、MTX中組、MTX高組、低聯(lián)合組相比,差異有統(tǒng)計學(xué)意義;低聯(lián)合組與單用藥組相比,培養(yǎng)后Th17中STAT3 m RNA的表達(dá)降低,差異無統(tǒng)計學(xué)意義。結(jié)論:MTX周期聯(lián)合CTX可能通過JAK-STAT3通路影響CIA小鼠Th17細(xì)胞的分化。
[Abstract]:Aim: to determine whether MTX cycle combined with CTX inhibits the differentiation of Na?ve T cells into Th17 cells in the spleen of CIA mice via JAK-STAT3 pathway. Methods: DBA1 mice were studied in vivo and in vitro. In vivo test: 56 male DBA1 mice were randomly divided into normal control group (n = 10) and CIA group (n = 46). The CIA model was established and randomly divided into 4 groups according to the drug use. They were divided into 4 groups: 1. 5 mg/kg/3d group (1. 5 mg/kg/3d), CTX group (30 mg/kg/10d) and MTX CTX group (CTX: 1. 5 mg / kg / 3 d ~ (-1) CTX: 30 mg / kg / 10 d). Three weeks after immunization, the swelling degree of left ankle joint and arthritis index (d (arthritis index AI) were continuously monitored. After 11 weeks of administration, mice were killed, bilateral knees were taken, left ankle was embedded, sections were sliced, HE staining was performed and pathological changes were observed. Na?ve T cells were isolated from splenocytes and stimulated by IL-6 TGF- 尾 1 and IL-1 尾 -IL-23 in 37 鈩，

本文編號：2180459