羟氯喹抑制紫外线诱导的系统性红斑狼疮CD4+T细胞白细胞介素-10和干扰素-γ的表达

目的 研究紫外线对系统性红斑狼疮(SLE)CD4+T细胞因子的影响和羟氯喹的抑制作用.方法 选择SLE 30例,健康对照10名.磁珠分选SLE患者和健康人的CD4+T细胞,紫外线311 nm窄谱中波紫外线暴露,加入羟氯喹共培养,酶联免疫吸附试验(ELISA)检测培养上清白细胞介素(IL)-10和干扰素-γ的表达水平.采用t检验进行统计学分析.结果 SLE患者CD4+T细胞IL-10表达高于健康对照[(27±4)和(18±3) pg/ml,P=0.011];经45、100 mJ/cm2紫外线暴露后,SLE活动患者CD4+T细胞IL-10表达升高[(27±4)和(77±42) pg/ml,(40±18)和(77±42) pg/ml,P=0.022,P=0.048],经100 mJ/cm2紫外线暴露后,活动患者CD4+T细胞IL-10表达高于稳定患者[(77±42)和(24±4)pg/ml,P=0.029];羟氯喹降低SLE活动患者CD4+T细胞IL-10和干扰素-γ表达[(2.6±4.0)和(17.9±2.3)pg/ml,P=0.018,P=-0.017)];羟氯喹降低经45,100 mJ/cm2紫外线暴露后SLE活动患者T细胞IL-10表达[(40±18)和(22±6)pg/ml,(77±42)和(21±5) pg/ml,P=0.037,P=0.04];羟氯喹降低经100 mJ/cm2紫外线暴露的SLE活动和稳定患者T细胞干扰素-γ表达[(18±3)和(13±14) pg/ml,(19±7)和(12±5) pg/ml,P=0.013,P=0.049].结论 紫外线加重SLE患者体内Th1/Th2细胞因子的比例失衡;羟氯喹抑制了紫外线诱发SLE患者干扰素-γ和IL-10的表达.

Abstract：

Objective To explore the role of hydroxychloroquine (HCQ) in ultraviolet B (UVB)- induced expression of interleukin (IL)-10 and interferon (IFN)-γ from CD4+T cells in patients with systemic lupus erythematosus (SLE). Methods Thirty patients with SLE and 10 healthy controls were enrolled in the study. CD4+ T cells were isolated using magnetic beads from SLE patients and healthy controls. HCQ was added in culture media before and after irradiation with UVB 311 nm narrow band ultraviolet B (NB-UVB). The levels of IL-10 and IFN-γ in the supernatant were detected with enzyme-linked immunosorbent (ELISA). Comparisons between groups were performed by t-test. Results The level of IL-10 was higher in SLE patients [(27±4) pg/ml] than that in healthy controls [(18±3) pg/ml, P=0.011]. After exposure of CD4+T cells to UVB in 45 or 100 mJ/cm2 dosages, the level of IL-10 was increased significantly in patients with active disease (P=0.022, P=0.048). After exposure of CD4+T cells to UVB in 100 mJ/cm2 dosages, the levels of IL-10 was higher in patients with active disease [(77±42) pg/ml] than patients with stable disease [(24± 4) pg/ml, P=0.029]. When CD4+ T cell were cultured with HCQ, IL-10 and IFN-γ levels in patients with active disease [(2.6±4.0), (17.5±2.3) pg/ml] were decreased significantly (P=0.018, P=0.017). HCQ reversed UVB-induced IL-10 expression in active SLE patients after exposure of CD4+T cells to UVB in 45 or 100 mJ/cm2 dosages (P=0.037, P=0.04). HCQ also reversed UVB-induced IFN-7 expression in active SLE patients and stable SLE patients after exposure to CD4+T cells with UVB in 100 mJ/cm2 dosages (P=0.013, P= 0.049). Conclusion UVB can aggravate the imbalance of Th1 and Th2 cytokines. HCQ inhibits UVB-induced IL-10 and IFN-7 expression of CD4+T cells in patients with SLE, especially in patients with active disease.     

MRL/lpr狼疮小鼠CD4~+ CD25~+调节性T细胞和相关基因的研究

目的 通过研究MRL/lpr系统性红斑狼疮(SLE)小鼠(模型组)胸腺、脾脏中CD4+CD25+调节性T细胞(Tr)数量和相关基因的变化,探讨Tr在SLE发病中的作用.方法 采用间接免疫荧光法检测模型组和对照组血清中抗核抗体水平,流式细胞术检测胸腺、脾脏中Tr数量,RT-PCR检测Tr功能基因表达水平.结果 抗核抗体模型组均为阳性,而对照组均为阴性;胸腺、脾脏中Tr数量模型组与对照组无明显差别;Foxp3的表达水平在模型组胸腺中与对照组无明显差别,而在模型组脾脏中却低于对照组;CTLA-4的表达水平在模型组胸腺和脾脏均高于对照组.结论 Tr数量的变化在MRL/lpr小鼠的发病中不起关键作用,而Tr功能的变化,尤其在外周的功能缺陷可能是其发病的原因之一,至于Tr的抑制功能是否真正发生变化,还有待于进一步通过体外抑制功能的检测来确定.     

黄芪多糖联合间质干细胞治疗MRL/lpr狼疮鼠疗效研究

目的探讨黄芪多糖（APS）联合人骨髓间质干细胞（MSC）移植对MRL/lpr狼疮鼠的疗效及作用机制。方法 MRL/lpr鼠随机分为MSC＋APS治疗组、MSC治疗组、APS治疗组与生理盐水对照组。每2周测体质量,收集24h尿量检测尿蛋白。间接免疫荧光法检测抗核抗体（ANA）、抗ds-DNA抗体滴度,苦味酸法测血清肌酐,流式细胞仪检测T细胞内细胞因子。结果 32周时,MSC＋APS、MSC、APS治疗组及对照组病死率分别为0、20%、20%、40%;MSC＋APS组体质量高于其他3组;24、28、32周各治疗组24 h尿蛋白定量均低于对照组,MSC＋APS组低于MSC组和APS组;32周MSC＋APS组血清肌酐低于MSC组和对照组,而血红蛋白、白细胞计数则显著高于MSC组和APS组;MSC＋APS组和MSC组ANA滴度低于对照组,MSC＋APS组抗ds-DNA抗体滴度低于MSC组和对照组;与MSC组和对照组32周时比较,MSC＋APS组CD4＋T细胞和Th2亚群降低,CD8＋T细胞和Th1亚群升高。结论 APS联合人骨髓MSC移植治疗MRL/lpr鼠的疗效优于MSC单独治疗,调节Th1/Th2平衡可能是其机制之一。     

羟氯喹治疗系统性红斑狼疮合并妊娠24例临床研究

目的 评价羟氯喹治疗系统性红斑狼疮(SLE)患者合并妊娠的疗效与安全性.方法 回顾性分析2006年5月至2011年2月中日友好医院风湿免疫科收治的24例妊娠期间使用羟氯喹治疗SLE患者的疾病控制和新生儿安全情况,并对其子代进行随访.结果 24例患者中22例在妊娠期间持续应用羟氯喹治疗,21例患者SLE病情平稳,无复发.2例患者妊娠后停用羟氯喹,在妊娠中期出现SLE活动,增加糖皮质激素剂量并再度加用羟氯喹,SLE病情稳定至分娩和产后.3例患者诊断妊娠高血压,3例患者早产.副作用:2例患者在妊娠期加用羟氯喹后,分别出现一过性厌食和脱发,未予特殊治疗,症状大约持续3周后自行缓解.所有患者均未出现眼部不适及视野缺损症状.至目前为止,患者在我院眼科门诊定期复查视野及眼底均无异常发现.新生儿听力筛查和身高、体重均在正常范围内,未发现心肺异常及生长发育异常.结论 羟氯喹对SLE妊娠期妇女及胎儿具有良好的安全性,应在妊娠期间持续使用.     

青蒿琥酯治疗MRL/lpr狼疮鼠的疗效及机制研究

目的 探讨青蒿琥酯对MRL/lpr狼疮鼠的疗效及作用机制.方法 MRL/lpr鼠随机分为青蒿琥酯治疗组、环磷酰胺(CTX)治疗组和对照组.16周龄时青蒿琥酯组给予青蒿琥酯125 mg ·kg-1·d-1治疗16周,CTX组给予CTX 100 mg/kg ×2 d腹腔注射.考马斯亮蓝法检测尿蛋白定量(24 h),间接免疫荧光法检测血清抗核抗体(ANA)、抗双链DNA(dsDNA)抗体滴度,过碘酸雪夫(PAS)染色观察病理改变,免疫荧光检测补体C3沉积,反转录-聚合酶链反应(RT-PCR)检测小鼠脾脏B细胞刺激因子(BAFF)mRNA的表达水平.结果 ①24、28、32周尿蛋白定量(24 h)青蒿琥酯组[(4.9±1.2),(5.2±1.0),(6.4±1.2)mg]显著低于对照组[(9.0±1.3),(9.3±0.6),(10.0±1.6)mg](均P＜0.01),28、32周尿蛋白定量(24h)青蒿琥酯组显著低于CTX组[(5.2±1.0)US(7.7±1.0),(6.4±1.2)vs(9.6±1.9)mg](P均＜<0.05).②32周龄时青蒿琥酯组体质量[(36.3±5.5)g]高于对照组[(32.8±30)g](P＜O.05),血清肌酐青蒿琥酯组[(15.9±2.4)μmol/L]显著低于对照组[(20.8±5.1)μmol/L](P＜0.05).③青蒿琥酯组和CTX组肾脏病理损伤较对照组减轻,肾脏内补体c3沉积较对照组减少.④青蒿琥酯组脾脏BAFF mRNA表达(0.81±0.05)和CTX组脾脏BAFF mRNA表达(0.74±0.13)均低于对照组(0.98±0.07)(P＜O.05).结论 青蒿琥酯治疗MRL/lpr狼疮鼠有效,可以改善肾脏病理损伤,降低尿蛋白,延长狼疮鼠生存期.抑制BAFF的产生可能是青蒿琥酯治疗MRL/lpr有效的的机制之一.     

脐带间充质干细胞移植治疗MRL/lpr狼疮鼠的疗效

目的 探讨脐带间充质干细胞(UC-MSCs)对MRL/lpr狼疮鼠的治疗作用.方法 24只18周龄雌性MRL/lpr小鼠,分为3组:UC-MSCs 1次治疗组(G1)、UC-MSCs 3次治疗组(G2)、对照组(G3).观察体质量,考马斯亮蓝法榆测尿蛋白定量(24 h),酶联免疫吸附法(ELISA)检测抗双链DNA(dsDNA)抗体水平,观察肾脏、肺病理改变.结果 ①尿蛋白定量(24 h)25周时G1组(2.3±1.9)mg和G2组(1.8±1.4)mg显著低于对照组(3.8±2.1)mg(P<0.05),27周时G1组(2.5±1.5)mg和G2组(1.9±1.2)mg也显著低于对照组(5.4±2.4)mg(P<0.01).②24周时治疗组体质量显著高于对照组(P<0.05),29周时血清肌酐G1组(7.2±3.2)μmol/L和G2组(6.2±2.8)μmol/L显著低于对照组(12.5±2.3)μmol/L(P<0.05).③移植1周时,抗dsDNA抗体滴度G1组(46±11)×102 U/ml和G2组(49±43)×102 U/ml显著低于对照组(99±42)×102 U/ml(P<0.05);29周时G2组(36±15)×102 U/ml显著低于对照组(68±32)×102 U/ml.④肾小球新月体形成率G1组(0.12±0.07)和G2组(0.08±0.02)显著低于对照组(0.20±0.06)(P<0.05),G2组较G1组显著减低(P<0.05).⑤间质性肺炎G1组和G2组较对照组减轻.结论 UC-MSCs对MRL/lpr狼疮鼠有显著疗效,安全且无排斥反应.     

羟氯喹治疗系统性红斑狼疮的现状和展望

羟氯喹（hydroxychloroquine）是4-氨基喹诺酮类的抗疟药,也是一种独特的抗风湿病药物。自1951年Page等用米帕林治疗系统性红斑狼疮（systemic lupus erythematosus,SLE）获得成功并报道后,抗疟药在治疗SLE中的应用开始被人们认识,由于羟氯喹良好的安全性和可靠的疗效,正越来越广泛地应用于临床。     

UC-MSC移植对MRL/lpr鼠脾脏和淋巴CD+4/CD+25Foxp+3T细胞水平的影响

24只MRL/lpr鼠随机分为3组,一次移植组于18周龄时尾静脉输注脐带间充质干细胞(MSC)悬液0.5ml,三次移植组分别于18、19、20周龄时尾静脉输注脐带MSC悬液0.5 ml,对照组于18周龄时尾静脉输注生理盐水0.5 ml.MRL/lpr鼠输注脐带MSC后,用流失细胞仪检测脾脏和淋巴结CD4+CD25+Foxp3+ T细胞百分率.结果 :MRL/lpr鼠经一次或三次脐带MSC移植后脾CD4+ CD25+ Foxp3+ T细胞百分率均较对照组升高,但淋巴结CD4+ CD25+Foxp3+ T细胞百分率均较对照组降低.认为异种MSC移植可上调MRL/lpr CD4+ CD25+ Foxp3+ T细胞水平,CD4+ CD25+Foxp3+ T细胞水平上调可能是MSC移植治疗系统性红斑狼疮有效的机制之一.     

DNA甲基化转移酶在MRL/lpr狼疮鼠CD4+T淋巴细胞中的表达水平研究及功能分析

目的 研究DNA甲基化转移酶DNMT1、DNMT3A、DNMT3B在发病MRL/lor狼疮鼠脾脏CD4+T淋巴细胞的表达情况,并对其与免疫相关的甲基化敏感基因ITGAL、CD70的表达水平进行相关性分析.方法 CD4单抗标记的免疫磁珠分选小鼠脾脏CD4+T淋巴细胞,实时定量聚合酶链反应(real-time PCR)测定DNMTI、DNMT3A、DNMT3B和免疫相关的甲基化敏感基因ITGAL、CD70的相对表达量.结果 ①DNA甲基化转移酶DNMTI、DNMT3A、DNMT3B在发病MRL/lpr狼疮鼠CD4+T淋巴细胞中与健康对照BALB/c小鼠相比,表达水平均有降低趋势,其中DNMT3B差异有统计学意义(P<0.05);CD70在发病MRL/lpr狼疮鼠表达水平升高(P<0.01),ITGAL表达水平呈升高趋势,差异无统计学意义(P>0.05).②相关性分析显示:DNMT3B与CD70分子表达水平呈显著负相关(r=-0.769,P<0.01).结论 DNMT3B在发病MRL/lpr狼疮鼠CD4+T淋巴细胞中表达水平降低,可能通过影响免疫相关的甲基化敏感基因CD70的表达,造成CD4+T淋巴细胞功能异常,参与系统性红斑狼疮的发病.     

脐带间质干细胞移植对MRL/lpr狼疮鼠间质性肺炎的影响

目的 探讨脐带间质干细胞(UC-MSCs)对MRL/lpr狼疮鼠间质性肺炎的治疗作用.方法 体外分离培养UC-MSCs,将24只18周龄雌性MRL/lpr小鼠采用随机数字表法分为1次治疗组、3次治疗组和对照组,1次治疗组和3次治疗组于第18周给予1×106第3代UC-MSCs尾静脉注射,3次治疗组在第19、20周时再分别重复1次;对照组给予0.5 ml生理盐水尾静脉注射.在第29周时处死小鼠,用HE染色观察各组小鼠的肺病理变化.结果 UC-MSCs移植能显著减轻MRL/lpr狼疮鼠的间质性肺炎.1次治疗组和3次治疗组的肺气管损害指数分别为1.40±0.24和1.02±0.29,明显低于对照组的1.95±0.35,差异有统计学意义(q值分别为0.551和0.937,均P<0.01);1次治疗组和3次治疗组的血管损害指数分别为1.20±0.18和1.08±0.16,明显低于对照组的1.56±0.32,差异有统计学意义(q值分别为0.360和0.479,P<0.05和P<0.01);1次治疗组和3次治疗组的肺间质中炎症细胞浸润程度分别为1.30±0.21和1.05±0.15,明显低于对照组的1.72±0.34,差异有统计学意义(q值分别为0.417和0.673,P<0.05和P<0.01).结论 UC-MSCs移植对MRL/lpr狼疮鼠的间质性肺炎具有治疗作用.     

The effect of hydroxychloroquine on lupus erythematosus-like skin lesions in MRL/lpr mice

Objectives: To evaluate the effect and safety of hydroxychloroquine (HCQ) on lupus erythematosus (LE)-like skin lesions in the MRL/lpr mouse, a model for systemic LE (SLE).

Methods: We divided the MRL/lpr mice into three groups that were given: (1) drinking water, (2) HCQ at a dose of 4?mg/kg/d, or (3) HCQ at a dose of 40?mg/kg/d. The HCQ was administered to examine the effect and safety of HCQ on skin lesions and the number of infiltrating cells including mast cells in the dermis.

Results: Six of 13 mice in the group given drinking water, 3 of 11 mice in the group administered low-dose HCQ (4?mg/kg/d), and 1 of 10 mice in the group administered high-dose HCQ (40?mg/kg/d) presented the skin lesions. The average number of mast cells was 81, 50, and 12 (magnification, ×100), the mortality rate was 24%, 8%, and 9% and the mean body weight gain was 4.6?g, 8.0?g and 5.1?g, respectively.

Conclusions: HCQ was demonstrated to decrease the appearance of LE-like lesions and the number of mast cells in the dermis. Furthermore, there were no obvious systemic adverse effects. This study provides evidence that suggests benefits in human patients.     

JAK/STAT1信号转导途径在MRL/lpr狼疮鼠不同器官中的活化研究

目的探讨Janus蛋白酪氨酸激酶(JAK)/信号转导和转录激活子1(STAT1)信号转导途径在MRL/lpr狼疮鼠肾脏、肺脏、脑等不同器官中的活化和作用。方法实验组是12周龄以上已经发病的MRL/lpr雌性小鼠．对照组是未发病的同龄MRL/lpr雌性小鼠。采用免疫组织化学方法研究肾脏中磷酸化STAT1的组织分布情况。采用Western blot方法研究STAT1磷酸化蛋白表达,采用SYBR greenⅠre- M-time定量聚合酶链反应(PCR)研究SOCS-1 mRNA的表达；并与肺脏、脑相对比。结果MRL/lpr狼疮鼠STAT1磷酸化蛋白在各器官均明显活化。肾脏和肺脏的STAT1磷酸化蛋白活化较脑组织明显；肾脏、肺脏和脑组织的SOCS-1基因的表达均升高．但肾脏的SOCS-1基因表达升高程度低于肺脏和脑组织。结论JAK/STAT1信号转导途径的异常可能参与和促进了系统性红斑狼疮(SLE)各器官病理损害的发生；狼疮肾炎的病理损害还可能与SOCS-1的负反馈调节作用降低有关。     

亚砷酸对狼疮鼠自身抗体和白细胞介素-1012表达的影响

目的 探讨亚砷酸(ATO)对狼疮鼠自身抗体和白细胞介素(IL)-10、IL-12表达的影响.方法 MRL/lpr狼疮鼠随机分为ATO组、环磷酰胺(CTX)组和生理盐水(NS)组,给药2个月.用四色流式细胞术测脾脏CD3~+(T)细胞和CD3~+CD4~+(Th)细胞的百分率以及CD3~+CD4~+(Th)细胞内IL-10和IL-12的水平;用酶联免疫吸附试验(ELISA)法测血清中抗双链DNA(dsDNA)抗体、IL-10和IL-12的浓度.分别采用单因素方差分析、LSD检验,配对t检验进行统计学处理.结果 ①给药后ATO组小鼠血清的抗dsDNA抗体水平吸光度值(A)0.92±0.06,较给药前1.14±0.58明显下降(P<0.01);②ATO组CD3~+细胞和CD3~+CD4~+百分率[分别为(44±4)%和(20±4)%]均显著均低于NS组水平[分别为(59±5)%和(30±3)%](P<0.01);③ATO组的血清IL-12浓度(84±12)pg/ml较NS组水平(103±13)pg/ml显著降低(P=0.018);④ATO组Th细胞内IL-10和IL-12的表达水平(1.5±0.4)%和(2.43±0.42)%显著低于NS组(2.5±0.5)%和(3.24±0.40)%(P<0.01).结论 亚砷酸能显著降低MRL/lpr狼疮鼠血清抗dsDNA抗体的水平,抑制T细胞和Th细胞增生和活化功能,降低IL-12的血清水平和Th细胞的诱生水平,及能降低IL-10的Th细胞的诱生水平.     

Severer lupus erythematosus-like skin lesions in MRL/lpr mice with homozygous Kitwsh/wsh mutation

Objective: To clarify the roles of mast cells (MCs) on the pathogenesis of lupus erythematosus (LE)-like skin lesions on MRL/lpr mice.

Methods: MRL/lpr mice were mated with C57BL/6-Kit^wsh/wsh mice and the heterozygous F1 mice were 10 times backcrossed with the parental MRL/lpr to generate MRL/lpr-Kit^wsh/wsh mice. MC-deficient MRL/lpr-Kit^wsh/wsh mice were compared with MRL/lpr-Kit^+/+ and MRL/lpr-Kit^wsh/+ mice with intact MCs.

Results: MRL/lpr-Kit^wsh/wsh mice developed skin lesions without infiltrating MCs. As similar skin lesions on MRL/lpr-Kit^+/+ mice and MRL/lpr-Kit^wsh/+ mice contain comparable number of MCs, these mice were collectively analyzed as MRL/lpr mice with MCs. Compared with MRL/lpr mice with MCs, skin lesions developed earlier and showed consistently higher severity, with significantly higher mRNA expressions of many inflammatory cytokines in the dorsal skin on MRL/lpr mice without MCs. Furthermore, survival rate was significantly lower in MRL/lpr mice without MCs. The number of infiltrating MCs significantly increased in association with the severity of skin lesions in MRL/lpr mice with MCs.

Conclusions: These results demonstrated that MCs are infiltrated to suppress the progression of LE-like skin lesions in MRL/lpr mice.     

Quinacrine and hydroxychloroquine,a forgotten combination for patients with active systemic lupus erythematosus in Australasia?

In 1994, A 39‐year‐old Female Patient With Systemic Lupus Erythematosus (Sle) Was Diagnosed As Having Lupus‐induced Serositis. She Was Commenced On Hydroxychloroquine (Hcq) And Prednisone. Her Disease Kept Relapsing Whenever She Was Tailed Off Prednisone. In 1997, Quinacrine (Qn) Was Commenced, And Prednisone Was Gradually Stopped. Her Disease Has Remained In Remission On The Combination Of Hcq And Qn. In December 2000 She Ran Out Of Qn For A Week, And Within This Period She Started To Experience Fatigue And Polyarthralgia Again. Quinacrine Is Available From Compounding Pharmacies, And Is Relatively Cheap. The Combination Of Hcq And Qn In The Treatment Of Sle Should Be Considered More Often.