昆明山海棠与类风湿关节炎滑膜细胞体外增殖和凋亡

背景:昆明山海棠应用于类风湿关节炎治疗已被证实有确切疗效,但对于其作用机制目前尚不明确.目的:验证昆明山海棠对类风湿关节炎滑膜巨噬样细胞和成纤维样细胞体外增殖活性及对其凋亡影响,分析其剂量效应关系.设计、时间及地点:细胞学体外分组对照实验,于2003-08/2007-06在汕头大学医学院第一附属医院中心实验室和南方医科大学细胞生物实验室完成.材料:类风湿关节炎6例及正常滑膜组织3例,来源于汕头大学医学院第一附属医院骨科和南方医院脊柱骨病科患者术中获得的标本.昆明山海棠制备水提取液,含生药0.667 g/mL..方法:收集获得的滑膜组织,通过消化法获得原代类风湿关节炎及正常滑膜细胞,第2-4代细胞用免疫磁珠法筛选滑膜CD68~+细胞和CD68~-细胞,在接种72 h后加入实验各组分别在培养液中加入最终体积分数分别为5,10,20 mL/L的昆明山海棠药液处理24 h和48 h.同时设立不加药物的阴性对照及不加细胞空白对照.主要观察指标:相差倒置显微镜、扫描电镜观察细胞形态,用MTT法测定细胞增殖情况,流式细胞仪检测和TUNEL.法检测细胞凋亡.结果:分选后滑膜CD68~-细胞为成纤维样细胞,滑膜CD68~+细胞为滑膜巨噬样细胞.施予干预因素后,各组有部分细胞体积缩小,胞膜皱缩,微绒毛和伪足减少或消失,部分细胞可见团块脱落形成凋亡小体.当昆明山海棠体积分数为2%时,各组细胞均有不同程度抑制,显示昆明山海棠有细胞毒性,对类风湿关节炎滑膜CD68~-、CD68~+细胞的抑制效应和诱导细胞效应强于同种正常滑膜细胞(P<0.01).在药物体积分数为1%时,对类风湿关节炎滑膜细胞有明显抑制效应(P<0.01).昆明山海棠对诱导类风湿关节炎滑膜细胞凋亡作用有明显时间依赖性和剂量依赖性,且凋亡率与相同条件下的抑制率表现出较好的直线相关关系(r=0.497,P<0.01).结论:昆明山海棠对于类风湿关节炎滑膜巨噬样细胞和滑膜成纤维样细胞有诱导凋亡和明显抑制异常增殖作用.在一定浓度下,昆明山海棠对于正常滑膜细胞毒性较小且对类风湿关节炎滑膜细胞的有较明显抑制异常增殖和诱导细胞凋亡的效应.

Effects of tripterygium hypoglaucum(Levl.)Hutch on in vitro proliferation and apoptosis of synoviocytes from patients with rheumatoid arthritis

BACKGROUND:Tripterygium hypoglaucum(Levl.)Hutch (THH) has been accurately confirmed a good clinical therapeutic effect for rheumatoid arthritis(RA).However.functionaI mechanism of THH remains unclearly.OBJECTIVE:To investigate the effect of THH on proliferation and apoptosis of macrophage-like synoviocytes and fibroblast-like synoviocytes from patients with RA and to explore its dose-effect relation.DESIGN.TIME AND SETTING:In vitro cytology grouping controlled observation was performed in the Center Laboratory of the First Clinical College of Shantou University and the Laboratory for Cellular Biology of Southern MedicaI University from August 2003 to June 2007.MATERIALS:Synovium was obtained from 6 patients with RA and 3 normaI synovial samples.All samples sourced from patients admitted in Department of Orthopaedics.the First Clinical College of Shantou University and in Spinal and Joint Surgery of Nanfang Hospital.The water extracts of THH contained 0.667 g/mL crude drug.METHODS:Synovial cells were isolated by digesting synovial tissue with collagenase.CD68+and CD68-synoviocytes were sorted from synovial cells by Dynabeads(magnetic celI sorting)from the 2~(nd)-4~(th) passages of synoviocyes.After 72 hours incubation,all groups of isolated synoviocytes were cultured in culture medium containing the flnaI concentrations of 0 mL/L,5 mL/L,1 0 mL/L and 20 mL/L of THH for 24 and 48 hours,respectively,and the common culture medium was sewed as the control. MAIN OUTCOME MEASURES:The morphology of synoviocytes were observed by inverted phase contrast microscope and studied by scanning electron microscopy(SEM).In vitro cell growth was assessed by the MTT assay,and synoviocytes apoptosis was evaluated using temlinaI deoxynucleotidyl transferase biotin-dUTP nick end labeling(TUNEL)assay and a flow cytometry.RESULTS:CD68~+ synoviocytes were appeared macrophage-like and CD68~- synoviocytes were exhibited fibroblast-like.After incubated with THH,some synoviocytes presented the volume of cells deflated,progressively destruction of cell membrane,microvillus or pseudopodium tended to be decreased even disappeared,and apoptosis bodies appeared.When incubated with 2% THH,proliferation inhibiUon and inducing apoptosis could be observed;it showed that THH had the cytotoxic effect on synoviocytes.The effect of proliferation inhibition and inducing apoptosis on CD68-and CD68+synoviocytes from RA patients was more significantly than that from normal arthritis (P<0.01).When incubated with 1%THH.proliferation inhibition and inducing apoptosis on synoviocytes from RA patients also could be found(P<0.01),but not found on normal synoviocytes(P>0.05).Compared with the negative controI group.proliferation inhibition was no significant difference On all groups with the low dose (≤5 mL/L) of THH(P>0.05).Furthermore,the effect of proliferation inhibition and inducing apoptosis on synoviocytes decreased in a dose-dependent and time-dependent manner.The inhibition rate of synoviocytes also could be found positively correlated with apoptosis rate aftat treatment of THH(r=0.497,P<0.01).CONCLUSlON:THH have cytotoxic effect on rheumatoid synoviocytes and normal synoviocytes.it can inhibit macrophage-like synoviocytes and fibroblast-like synoviocytes from RA on proliferation and induce them to apoptosis.In the some contraction of THH.proliferation Inhibition and inducing apoptosis can be occur on synoviocytes from RA but not on normal synoviocytes.