摘要
系统性红斑狼疮(SLE)是一种严重危害人类健康的自身免疫性疾病,其在我国人群的患病率接近千分之一,全国患病总人数高达100万。迄今为止,国内外尚无特异的有效治疗手段,现该病尚不能治愈。
在20世纪50年代,SLE的5年生存率仅为20%。近年来,由于免疫血清学检查的广泛应用,而且正确及时使用肾上腺皮质激素和免疫抑制剂,使得SLE的生存率大为改善。国外报道5年生存率90%,10年生存率80%。国内情况分别为98%及84%。免疫抑制剂中环磷酰胺(CTX)对狼疮肾炎的有效性已被多项临床研究证实,但其用量越大,效益越大,副作用也越大,其中包括感染、性腺抑制、及肿瘤。目前新的治疗方案不断出现,如新的免疫抑制剂、生物制剂、血浆置换、自体干细胞移植等,但上述治疗不仅疗效尚待长期临床考证,治疗费用少则数万元/年,多则数十万元/年,患者多难以承受。鉴于此,我们在治疗SLE的临床实践中,借鉴联合化疗治疗肿瘤的成功经验,根据细胞增殖动力学原理,将细胞周期非特异性药物(环磷酰胺CTX)与细胞周期特异性药物(长春新碱VCR)联合序贯间歇给药(每三周给药一次)治疗SLE,减少了CTX的用量,明显降低了不良反应,而且治疗效果显著。此方案价格低廉,使用方便。为了验证周期序贯联合VCR+CTX方案的疗效及协同机制,首先进行系统性红斑狼疮动物模型的实验研究。
动物模型选取慢性移植物抗宿主病狼疮样小鼠模型,该模型是一个良好的狼疮鼠肾炎模型。观测指标选取反映SLE肾脏及免疫系统病变的指标,包括SLE的肾脏生物指标—单核细胞趋化蛋白-1(MCP-1)、转化生长因子β1(TGF-β1)及导致SLE病情发生发展的一个重要环节—B淋巴细胞刺激因子及其受体系统(BAFF和BAFFR)。
[目的]为了从临床实践上升到理论认识的高度,本文探讨周期序贯联合免疫抑制剂(长春新碱、环磷酰胺)治疗系统性红斑狼疮动物模型的实验研究,以阐明序贯联合用药协同效应机制。
[方法]本研究分两部分进行:
第一部分系统性红斑狼疮动物模型的选择和建立
动物模型选取慢性移植物抗宿主病狼疮样小鼠模型,实验动物取6-8周龄雌性(C57BL/6J×DBA/2)F1杂交鼠及6-8周龄雌性DBA/2小鼠,体重(18-20)g。制作慢性移植物抗宿主病(cGVHD)狼疮样小鼠模型36只。造模前及造模6周时分别留取血尿检测尿蛋白定量、血肌酐、血抗双链DNA抗体并各处死解剖小鼠3只,剩余30只小鼠模型进入第二部分研究。
第二部分:VCR+CTX周期联合治疗SLE动物模型的实验研究
选取慢性移植物抗宿主病狼疮样小鼠模型30只,随机分五组:空白对照组、VCR间歇给药组、CTX间歇给药组、CTX隔日给药组及VCR+CTX联合间歇给药组。间歇给药为每隔三周(20天)给予一次,隔日给药为每隔一天给予一次。从诱导开始到全部处死动物,历时24周。
检测项目
1一般情况,包括体重、精神状态、存活。
2血液检测外周血白细胞、血清谷丙转氨酶、血清肌酐、血清双链DNA抗体、ELISA检测血TGF-β1、血MCP1、血BAFF。
3尿液检测24小时尿蛋白定量、尿TGF-β1、尿MCP1。
4肝脏病理肝脏病理切片(HE染色)
5肾脏病理肾脏病理切片(HE染色、Masson染色、PSAM染色、免疫荧光染色C3及IgG)免疫组织化学法定位测定TGF-β1、MCP1。
6半定量RT-PCR测定TGF-β1、MCP1的mRNA在肾脏的表达。
7脾脏病理免疫组化法定位测定BAFF、BAFFR。
8半定量RT-PCR测定BAFF、BAFFR的mRNA在脾脏的表达。
统计采用SPSS11.5统计软件,进行重复测量方差分析、相关分析、析因分析等。
[结果]
1造模情况及一般情况观察
①诱导造模后六周24小时尿蛋白定量达5.02±0.88mg,抗dsDNA抗体阳性,肾脏病理呈狼疮肾炎Ⅳ型表现,模型成功。不同治疗组治疗18周,最终对照组死亡4只,VCR间隔用药组死亡2只,余三组均存活。
②CTX隔日用药组对造模小鼠体重、血清ALT的影响较大,与其他四组相比有显著性差异(P＜0.05)。表现为小鼠体重不增,血清ALT明显升高及散在肝细胞空泡变性。各组对外周血白细胞计数改变无显著性差异(P＞0.05)。
2肾脏指标观察
①CTX隔日组、VCR+CTX联合组对造模小鼠24小时尿蛋白定量的改变最大,与其他三组相比有显著性差异(P＜0.01),对照组＜VCR间隔组＜CTX间隔组＜CTX隔日组≈VCR+CTX联合组。CTX隔日组、VCR+CTX联合组及CTX间隔组对造模小鼠血清肌酐、肾小球病变病理积分及抗dsDNA抗体滴度的减低较大,与对照组、VCR间隔组相比有显著性差异(P＜0.01)。
②CTX间隔治疗组、CTX隔日治疗组与VCR+CTX联合组三组对造模小鼠尿MCP-1值、尿TGF-β1值的降低幅度较大,与对照组、VCR间隔组相比有显著性差异(P＜0.01);而不同治疗方案对血清MCP-1、TGF-β1的改变无显著性差异(P＞0.05)。
③CTX隔日治疗组、VCR+CTX联合组造模小鼠肾脏MCP-1、TGF-β1表达在平均灰度及肾小球、肾小管中的阳性数及肾脏mRNA表达量最低,与对照组、VCR间隔组及CTX间隔组相比有显著性差异(P＜0.01)。
④造模小鼠24小时尿蛋白定量与血清肌酐、尿MCP-1、肾小球MCP-1的阳性细胞数、肾小管MCP-1阳性率的水平相关(P＜0.01)。
⑤造模小鼠24小时尿蛋白定量与血清肌酐、尿TGFβ1、肾小球TGFβ1阳性细胞数、肾小管TGFβ1阳性率的水平相关(P＜0.01)。
3免疫系统指标观察
①CTX隔日组、VCR+CTX联合组对小鼠血清BAFF降低最大,与对照组有显著性差异(P＜0.01)。
②CTX隔日用药组、VCR+CTX联合组造模小鼠脾脏BAFF免疫组化表达的平均灰度及脾脏mRNA表达水平最低,与对照组相比有显著性差异(P＜0.01);不同治疗方案组造模小鼠脾脏BAFFR免疫组化表达的平均灰度及脾脏mRNA表达水平无显著性差异(P＞0.05)。
③血清BAFF与血清dsDNA滴度、脾脏BAFF表达的平均灰度相关(P＜0.05);脾脏BAFF表达的平均灰度与脾脏BAFFR表达的平均灰度不相关(P＞0.05)。
4 VCR与CTX的交互作用
①经析因设计的方差分析结果显示:VCR和CTX联合对造模小鼠24小时尿蛋白定量、血清Cr、尿MCP-1有明显协同作用(P＜0.05),对尿TGFβ1无交互作用(P=0.122)。
②经析因设计的方差分析结果显示:VCR和CTX联合对造模小鼠血清BAFF改变有协同作用(P=0.029＜0.05)、外周血白细胞减低无交互作用(P=0.085)。
[结论]
1慢性移植物抗宿主病狼疮样小鼠模型可出现狼疮肾炎Ⅳ型病理改变及MCP-1、TGFβ1、BAFF的表达排泌增加。
2序贯周期联合VCR和CTX联合治疗有交互作用,在降低24小时尿蛋白定量,血清肌酐,血清BAFF,减少肾脏MCP-1的表达和排泌等方面为协同作用,与CTX隔日用药组相当,优于VCR间隔治疗组、CTX间隔治疗组。
3 VCR+CTX联合间歇给药与单用药比较不良反应并没有增加,比较安全。CTX隔日给药组的不良反应明显大于其它各组,表现为体重不增、肝酶增高。
4 BAFF/BAFFR系统可能是VCR+CTX周期序贯联合治疗cGVHD狼疮样小鼠的一条途径。
Backgrounds
Systemic lupus erythematosus(SLE) is a kind of severity autoimmune disease.The prevalence of SLE is about 1/1000 in Chinese.To now,there are not effect therapeutic tool.
In 20 century,5 years' survival rate of SLE is 20%.For the past few years,survival rate of SLE has improved,for immune examination and to correctly use corticosteroids and immunodepressants.And now,5 years' survival rate of SLE is 90-98%,10 years' survival rate of SLE is 80-84%.It has been confirmed that cyclophosphamide(CTX) is effected to lupus nephritis(LN) by many clinical researches.But the more the total dosage of CTX,the more effect to LN,and the more side effect.CTX has many side effect about infections,sex gland inhibition,and tumor.Recently many new methods were discovered,such as new immunodepressants,biological agents,plasmapheresis,and stem-cellular transplant.But the effect of those methods had been not confirmed,and the cost of those methods was very expensive.So,for reference successfully combination therapy in leucemia,we first based on theory cell proliferation,sequential combined vincristine(VCR) with low dosage CTX intermittently to treat severe SLE.CTX is a cell cycle nonspecial stage agent,while VCR as an cell cycle special stage agent.And VCR was used to SLE thrombocytopenia.During the last decade,the new combination therapy has effective and safetive through we carried on clinicl practice.
We chose murine chronic graft-versus-host disease(cGVHD) as a model for lupus nephritis.And we selected two biomarkers for lupus nephritis,MCP-1 and TGFβ1,and two immune markers for SLE,BAFF and BAFFR.
Objectives
From clinically to theoretically,to explore the effects and possible mechanisms of sequential combined VCR combined with low dosage CTX intermittently to treat severe SLE, by observing the changes of Urine protein,serum creatinine,HE staining of kidney, proinflammaton cytokines—MCP-1 and TGFβ1 expression in kidney,and BAFF and BAFFR expression in spleen.It is hoped that new promising combination therapy on SLE,the overall prognosis and outcome of SLE would be favorably improved accordingly.
Methods
The study was divided to two parts.
First part:to set up murine chronic graft-versus-host disease model for study.
experimental animal:6-8 week-age female(C57BL/6J×DBA/2) F1 murine and 6-8 week-age female DBA/2 routine.Weight(18-20)g.to create murine chronic graft-versus-host disease model was 36.
Second part:the effect and safety of VCR combined with low dosage CTX intermittently to treat murine cGVHD model.
After the murine cGVHD model was set up successfully,they were randomly divided into blank group,VCR pause group,CTX pause group,CTX every other day(EOD) group, VCR+CTX combination group.There are 24 weeks from set up model to sacrifice model.
Detection item
1 General state of health:weight,mental state,survival
2 Blood detection:peripheral blood leucocyte,ALT,Cr,anti-dsDNA,ELISA for MCP-1, TGFβ1,BAFF
3 Urine detection:24-hour urine protein quantitation,ELISA for MCP-1,TGFβ1
4 Liver:HE staining of liver
5 Kidney:HE,Masson,PSAM,IF staining of kidney,immunohistochemical method to MCP-1,TGFβ1 in kidney
6 RT-PCR to MCP-1,TGFβ1 mRNA
7 Spleen:immunohistochemical method to BAFF,BAFFR in spleen
8 RT-PCR to BAFF,BAFFR mRNA
Results
1 General state of health:
①After cGVHD models were set up 6 week,the models had 24-hour urine protein quantitation 5.02±0.88mg,anti-dsDNA positive,andⅣLN pathology.So cGVHD models were successful.②It exhibited a increase in weight,ALT,and liver pathology,statistical significance were found in CTX EOD group and other groups.(P＜0.05)
2 Kidney index
①It exhibited a decrease in 24-hour urine protein quantitation,statistical significance were found in CTX EOD group,VCR+CTX combination group and other groups.(P＜0.01) blank group＜VCR pause group＜CTX pause group＜CTX EOD group≈VCR+CTX combination group.
It exhibited a decrease in Cr,AI,anti-dsDNA,statistical significance were found in CTX EOD group,VCR+CTX combination group,CTX pause group and other groups.(P＜0.05)
②It exhibited a decrease in ELISA for urine MCP-1 and TGFβ1,statistical significance were found in CTX EOD group,CTX pause group,VCR+CTX combination group and other groups.(P＜0.01)
③MCP-1 and TGFβ1'expression in model kidney were reduced in CTX EOD group, VCR+CTX combination group and had statistical significance in CTX EOD group, VCR+CTX combination group with blank group,VCR pause group,CTX pause group.
④24-hour urine protein quantitation was closely correlated with blood Cr,urine MCP-1,and MCP-1 expression in tubular cells and glomcrulus cells.
⑤24-hour urine protein quantitation was closely correlated with blood Cr,urine TGFβ1,and TGFβ1 expression in tubular cells and glomcrulus cells.
3 Immune system index
①Blood BAFF were reduced in CTX EOD group,VCR+CTX combination group and had statistical significance in CTX EOD group,VCR+CTX combination group with blank group,VCR pause group,CTX pause group.
②BAFF expression in model spleen were reduced in CTX EOD group,VCR+CTX combination group and had statistical significance in CTX EOD group,VCR+CTX combination group with blank group,VCR pause group,CTX pause group.
③Blood BAFF was closely correlated with blood anti-dsDNA,BAFF expression in spleen.And BAFF expression in spleen was not correlated with BAFFR expression in spleen.
4 Interaction of VCR and CTX
①The result of ANOVA of factorial design show therapeutic alliance of VCR and CTX had interaction in 24-hour urine protein quantitation,blood Cr,and urine MCP-1(P＜0.05), and did not have interaction in urine TGFβ1(P=0.122).
②The result of ANOVA of factorial design show therapeutic alliance of VCR and CTX had interaction in blood BAFF(P＜0.05),and did not have interaction in blood WBC (P=0.085).
Conclusions
1 The murine cGVHD model hadⅣLN pathology and the increase in expression of MCP-1,TGFβ1,BAFF.
2 The combination of VCR and CTX had synergistic effect in decrease in 24-hour urine protein quantitation,Cr,and the expression of MCP-1,BAFF.
3 The adverse effect of VCR+CTX combination group was similar to VCR pause group and CTX pause group.
4 BAFF/BAFFR system may be a pathway that VCR+CTX combination group treat the murine cGVHD model.
引文
[1] Mok CC, Ho CT, Siu YP et al. Treatment of diffuse proliferative lupus glomerulonephritis: a comparison of two cyclophosphamide-containing regions. Am J Kidney Dis 2001;38:256-264.
[2] Gourley MF, Austin III HA, Scott D et al. Methylprednisolone and Cyclophosphamide, alone or in combination, in patients with lupus nephritis. A randomized, controls trail. Ann Intern Med 1996:125:549-557.
[3] Zhao Y, Zhou DB, Leng XM,Treatment of severe systemic autoimmune diseases with autologous peripheral blood stem cell transplantation. Zhonghua Yi Xue Za Zhi. 2004 Dec 17;84(24):2077-81.
[4] David I. Daikh and David Wofsy. Cutting Edge: Reversal of Murine Lupus Nephritis with CTLA4Ig and Cyclophosphamide. The Journal of Immunology, 2001, 166:2913-2916.
[5] Contreras G,Pardo V,Leclercq B et al.Sequential therapies for proliferative lupus nephritis.N Engl J Med 2004;350:971-980
[6] 李小平，崔恒，卵巢癌化疗的细胞动力学，实用妇产科杂志;2005，21(5);257-259.
[7] Haus E, Lakatua DJ, Swoyer J,et al; Chronobiology in hematology and immunology. Am J Anat. 1983 ,168(4):467-517.
[8] 张志义，孙燕。恶性肿瘤的化学治疗学。上海科技出版社，1981年，52
[1]Bergijk EC,Munaut C,Baelde JJ,et al.A histologic study of the extracellular matrix during the development of glomerulosclerosis in murine chronic graft-verse-host disease[J].Am J Pathol,1992,140(5):1147-1156.
[2]任文英,陈香美,邱全瑛,等。慢性移植物抗宿主病狼疮样小鼠模型的诱导。中国比较医学杂志,2004,14(4)215-220。
[3]董光富,叶任高,王俭勤,等。一种实用性狼疮样‘肾炎动物模型—鼠慢性移植物抗宿主病的制备。中国实验动物学报,2000,8(4)207-211。
[4]阳晓,叶任高,陈伟英。慢性移植物抗宿主病狼疮样肾炎小鼠模型的研制[J].中华肾脏病杂志,2000,16(5):277-281
[5]Yoshida S,Castles JJ,Gershwin ME.The pathogenesis of autoimmunity in the New Zealand mice.Semin Arthritis Rheum,1990,19(4):224-242
[6]Jevnikar AM,Grusby MJ,Glimcher LH.Prevention of nephritis in major histocompatibility complex class Ⅱ-deficient MRL-Ipr mice.J Exp Med,1994,179(4):1137-1143
[7]Wang B,Yamamoto Y,E1 Badri NS,et al.Effective treatment of autoimmune disease and progressive renal disease by bone marrow transplantation that establishes a stable mixed chimerism in BXSB recipient mice.Proc Natl Acad Sci USA,1996,96(6):3012-3016
[8]陈顺乐,陈嘉何。系统性红斑狼疮,上海科学技术出版社,2004,108-138
[9]Treurniet R A,Bergijk EC,Baelde J J,et al.Gender-related influences on the development of chronic graft-versus-host disease induced experimental lupus nephritis(J).Clin Exp Immunol,1993,91:442-448.
[10]Via CS,Shearer GM.T-cell interaction in autoimmunity:insights from a murine model of graft-verse-host disease[J].Immunol Today,1998,9:207-213
[11]Sutmuller M,Baelde HJ,Ouellette S,et al.T-cell receptor Vβ gene expression in expermental lupus nephritis[J].Immunol,1998,95:18-25
[12]Sutmuller M,Ekstijn GL,Ouellette S,et al.Non-MHC genes determine the development of lupus nephritis in H-2 identical mouse strains[J].Clin Exp Immunol,1996,106:265-272
[13]郭玲,吴厚生,胡新美。慢性移植物抗宿主反应诱导SLE样小鼠模型。上海医科大学学报,2000,27(4):264-266
[14]Jan A,Bruijn MD,Pancras CW,et al.Murine chronic graft-verse-host disease as a model for lupus nephritis.Am J Pathol,1988,130(3):639-642.
[1]苗明三.实验动物用量的确定及计算方法。见:苗明三主编。实验动物和动物实验技术,北京:中国中医药出版社,1997:146-145
[2]Dixon RH,Rosse WF.Platelet antibody in autoimmune thrombocytopenia.Br J Haematol.1975 Oct;31(2):129-134.
[3]Ahn YS,Harrington WJ,Seelman RC,Eytel CS.Vincristine therapy of idiopathic and secondary thrombocytopenias.N Engl J Med.1974 Aug 22;291(8):376-380.
[4]关永源,黄民。抗恶性肿瘤药的临床应用。见徐叔云主编:临床药理学第三版,北京,人民卫牛出版社,2004:447-460
[5]张志义,孙燕。恶性肿瘤的化学治疗学。上海科技出版社,1981年,52
[6]丁惠萍;抗肿瘤药物的血液系统毒性及防治。医药导报;2004,20(10):622-623。
[7]Mok CC,Ho CT,Siu YP et al.Treatment of diffuse proliferative lupus glomerulonephritis:a comparison of two cyclophosphamide-containing regions.Am J Kidney Dis 2001;38:256-264.
[8]bansal VK,Beto JA.Treatment of lupus nephritis:a meta-analysis of clinical trials.AM J Kidney Dis 1997;29:193-199.
[9]Boumpas DT,Austin Ⅲ HA,Vaughn EM et al.Controlled trial of pulse methylprednisolone versus two regimens of pulse cyclophosphamide in severe lupus nephritis.Lancet 1992;340:741-745.
[10]Gourley MF,Austin Ⅲ HA,Scott D et al.Methylprednisolone and cyclophosphamide,alone or in combination,in patients with lupus nephritis.A randomized,controls trail.Ann Intern Med 1996;125:549-557.
[11]Illei GG,Austin HA,Crane Met al.Combination therapy with pulse cyclophosphamide plus pulse methylprednisolone improves long-term renal outcome without adding toxicity in patients with lupus nephritis.Ann Intern Med 2001;135:248-257.
[12]Dooley MA,Hogan S,Jennette C,Falk R.Cyclophosphamide therapy for lupus nephritis:poor renal survival in black Americans.Glomerular Disease Collaborative Network.Kidney Int 1997;51:1188-1195.
[13]Schroeder JO,Euler HE,Loffler H.Synchronization of plasmapheresis and pulse cyclophosphmide in severe systemic lupus erythemaosus.Ann Intern Med 1987:107:344-346.
[14]Danieli MG,Palmieri C,Salvi A et al.Synchronised therapy and high-dose cyclophosphamide in proliferative lupus nepheitis.J Clin Apheresis 2002;17:72-77.
[15]Morrissey J.Angiotension and gene expression in the kidney[J].Am J kidney Dis,1998;31:171-179.
[16]Banas B,Luckow B,Moller M,et al.Chemokine and chemokine receptor expression in novel human mesangial cell line[J].J Am Soc Nephrol,1999;10(11):2314-2322.
[17]Gesualdo L,Grandaliano G.Monocyte recruitment in cryoglobulinemic membrano proliferative glomerulonephritis:A pathogenetic role for monocyte chemotatic peptide-1[J].Kidney International,1997;51:155-163.
[18]Oppenheim JJ,Murphy WJ,Chertox O.Prospects for cytokine and chemokine biotherapy[J].Clin Cancer Res,1997;3(12 pt2):2682-2686.
[19]Hasegawa H,Kohno M,Sasaki M,et al.Antagonist of monocyte chemoattractant protein-1ameliorates the initiation and progression of lupus nephritis and renal vasculitis in MRL/lpr mice.Arthritis Rheum,2003;48:2555-2566.
[20]Zhao Z,Burkly LC,Campbell S.TWEAK/Fn14 interactions are instrumental in the pathogenesis of nephritis in the chronic graft-versus-host model of systemic lupus erythematosus.J Immunol.2007 Dec 1;179(11):7949-7958.
[21]Schwartz N,Michaelson JS,Putterman C Lipocalin-2,TWEAK,and other cytokines as urinary biomarkers for lupus nephritis.Ann N Y Acad Sci.2007 Aug;1109:265-274.
[22]Tian S,Li J,Wang L,Liu To Urinary levels of RANTES and M-CSF are predictors of lupus nephritis flare.Inflamm Res.2007 Jul;56(7):304-310
[23]Li Y,Tucci M,Narain S.Urinary biomarkers in lupus nephritis.Autoimmun Rev.2006 Jul;5(6):383-388.
[24]Wada T,Yokoyama H,Su S,et al.Monitoring urinary levels of monocyte chemotactic and activation factor reflects disease activity of lupus nephritis.Kidney Int,1996;49:761-767.
[25]Openheim JJ,Murphy WJ,Chertox O.Prospects for cytokine and chemokine biotherapy.Clin Cancer Res,1997;3:2682-2686.
[26]刘冠贤,邓安国,方敬爱等。狼疮肾炎患者尿单核细胞趋化蛋白-1检测的临床意义。中华风湿病学杂志,2005;9(10),618-619。
[27]Biswas SK,Sodhi A.Tyrosine phosphorylation-mediated signal transduction in MCP-1-induced macrophage activation:role for receptor dimerization,focal adhesion protein complex and JAK/STAT pathway.Int Immunopharmacol,2002;2(8):1095-1107.
[28]Panzer U,Thaiss F,Zahner G,et al.Monocyte chemoattractant protein-1 and osteopontin differentially regulate monocytes recruitment in experimental glomerulonephritis.Kidney Int 2001;59(5)1762-1769.
[29]Viedt C,Dechend R,Fei J,et al.MCP-1 induces inflammatory activation of human tubular epithelial cells:involvement of the transcription factors,nuclear factor-kappaB and activating protein-1.J Am Soc Nephrol,2002;13(6):1534-1547.
[30]王伟铭,姚建,石容.TGF-β1对人肾间质成纤维细胞PAI-1表达的影响.中华肾脏病杂志, 2000, 16:50-51
[31]Lise Wogensen, C. B. Nielsen, peter H. et al. Under control of the Ren-1C prmoter, Locally produced transforming growth factor-pi induces accumulation of glomerular extracellular matrix in transgenicmice. Diabetic, 1999; 48:182
[32]Wagrowska-Danilewicz M, Stasikowska O, Danilewicz M. Correlative insights into immunoexpression of monocyte chemoattractant protein-1, transforming growth factor beta-1 and CD68+ cells in lupus nephritis. Pol J Pathol. 2005; 56(3): 115-120.
[33] Chan RW, Lai FM, Li EK, Expression of chemokine and fibrosing factor messenger RNA in the urinary sediment of patients with lupus nephritis. Arthritis Rheum. 2004 Sep; 50(9):2882-2890.
[34]Isaka Y, Tsujie M, Ando Y, et al. Transforming growth factor-pi antisense oligodeoxynucleotides block interstitial fibrosis in unilateral ureteral obstruction. Kidney Int, 2000; 58:1882-1892
[35] Mackay F, Schneider P, Renner P, et al. BAFF and APRIL: a tutorial on B cell survival. Ann Rev Immunol,2003 ; 21:231-264.
[36] Mackay F, Mackay CR. The role of BAFF in B2cell maturation,T-cell activation and autoimmunity. Trends Immunol, 2002; 23:113-115.
[37] Mackay F, Browning JL. BAFF: a fundamental survival factor for B cells. Nat Rev Immunol, 2002,2 :465-475.
[38] Groom J, Kalled SL, Cutler AH, et al. Association of BAFF/BLyS overexpression an alter B cell differentiation wit h Sjogren's syndrome. J Clin Invest, 2002 ; 109 :59-68.
[39] Stohl W, Metyas S, Tan SM, et al. B lymphocyte stimulator overexpression in patients with systemic lupus erythematosus longitudinal observation. Art hritis Rheum, 2003 ; 48 :3475-3486.
[40]Szodoray P, Jellestad S, Teague MO, et al . Attenuated apoptosis of B cell activating factor expressing cells in primary Sjogren's syndrome. Lab Invest, 2003; 83 :357-365.
[41]Mukhopadhyay A et al. Identification and characterization of a novel cytokine, THANK, a TNF homologue that activates apoptosis, nuclear factor-kappa B, and c-Jun NH2-terminal kinase.J Biol Chem. 1999 Jun 4; 274(23): 15978-15981.
[42]Nardelli B et al. Synthesis and release of B-lymphocyte stimulator from myeloid cells.Blood. 2001 Jan 1; 97(1): 198-204.
[43] Shu HB et al. TALL-1 is a novel member of the TNF family that is down-regulated by mitogens.J Leukoc Biol. 1999 May; 65(5):680-683.
[44]Huard B et al. BAFF production by antigen-presenting cells provides T cell co-stimulation.Int Immunol. 2004 Mar; 16(3):467-475.
[45]Huard B et al. T cell costimulation by the TNF ligand BAFF.J Immunol. 2001 Dec 1; 167(11):6225-6231.
[46] Mackay F, Mackay CR. The role of BAFF in B-cell maturation, T-cell activation and autoimmunity. Trends Immunol. 2002 Mar; 23(3):113-115.
[47] Yan M et al. Identification of a novel receptor for B lymphocyte stimulator that is mutated in a mouse strain with severe B cell deficiency. Curr Biol. 2001 Oct 2; 11(19): 1547-1552.
[48] Thompson JS et al. BAFF-R, a newly identified TNF receptor that specifically interacts with BAFF. Science. 2001 Sep 14; 293(5537):2108-2111.
[49] Rolink AG, Melchers F. BAFF led B cells survive and thrive : roles of BAFF in B-cell development [J] . Curr Opin Immunol ,2002 ; 14 (2) :266-275.
[50] Batten M,Grovm J ,Cachero TG, et al . BAFF mediates survival of peripheral immature B lymphocytes [J] . J Exp Med ,2000 ; 192(10): 1453-1465.
[1]Bansal VK,Beto JA.Treatment of lupus nephritis:a meta-analysis of clinical trials.AM J Kidney Dis 1997:29:193-199.
[2]Mok CC,Ho CT,Siu YP et al.Treatment of diffuse proliferative lupus glomerulonephritis:a comparison of two cyclophosphamide-containing regions.Am J Kidney Dis 2001;38:256-264.
[3]Houssiau FA.Cyclophosphamide in lupus nephritis.Lupus,2005,14:53-58.
[4]Boumpas DT,Austin Ⅲ HA,Vaughn EM et al.Controlled trial of pulse methylprednisolone versus two regimens of pulse cyclophosphamide in severe lupus nephritis.Lancet 1992:340:741-745.
[5]Gourley MF,Austin Ⅲ HA,Scott D et al.Methylprednisolone and cyclophosphamide,alone or in combination,in patients with lupus nephritis.A randomized,controls trail.Ann Intern Med 1996;125:549-557.
[6]Illei GG,Austin HA,Crane Met al.Combination therapy with pulse cyclophosphamide plus pulse methylprednisolone improves long-term renal outcome without adding toxicity in patients with lupus nephritis.Ann Intern Med 2001:135:248-257.
[7]Dooley MA,Hogan S,Jennette C,Falk R.Cyclophosphamide therapy for lupus nephritis:poor renal survival in black Americans.Glomerular Disease Collaborative Network.Kidney Int 1997;51:1188-1195.
[8]Guttuso T Jr,Roscoe J,Griggs J.Effect of gabapentin on nausea induced by chemotherapy in patients with breast cancer.Lancet 2003;361:1703-1705.
[9]Katsifis GE,Tzioufas AG,Vlachoyiannopoulos PG et al.Risk of myelotoxicity with intravenous cyclophosphamide in patients with systemic lupus erythematosus.Rheumatology(Oxford) 2002;41:780-786.
[10]Euler HH,Harten P,Zeuner RA et al.Recombinant human granulocyte colony stimulating factor in patients with systemic lupus erythemtosus associated neutropenia and refractory infections.J Rheumatol 1997:24:2153-2157.
[11]Pryor BD,Bologna SG,Kahl LE.Risk factors for serious infection during treatment with cyclophosphamide and high-dose corticosteroids for systemic lupus erythematosus.Arthritis Rheum 1996;39:1475-1482.
[12]Petri M.Infection in systemic lupus erythematosus.Rheumatic Dis Clinical N Am 1998;24:423-456.
[13]Petri M,Allbritton J.Antibiotic allergy in SLE:a case-control study.J Rheumatol 1992;19:265-269.
[14]杨岫岩,朱旬,梁柳琴。环磷酰胺治疗系统性红斑狼疮导致卵巢功能衰竭的相关因素 分析. 中华医学杂志 , 2005; 85: 690-692.
[15] Mok CC, Lau CS, Wong RWS. Risk factors for ovarian failure in patients with systemic lupus erythematosus receiving cyclophosphamide therapy. Arthritis Rheum 1998; 41: 831-837.
[16] Wang CL, Wang F, Bosco JJ. Ovarian failure in oral cyclophosphamide treatment for systemic lupus erythematosus. Lupus 1995; 4: 11-14.
[17] Ioannidis JP, Katsifis GE, Tzioufas AG et al. Predictors of sustained amenorrhea from pulsed intravenous cyclophosphamide in premenopausal women with systemic lupus erythematosus. J Rheumatol 2002; 29: 2129-2135.
[18] Huong du L, Amoura Z, Duhaut P et al. Risk of ovarian failure and fertility after intravenous cyclophosphamide. A study in 84 patients. J Rheumatol 2002; 29: 2571-2576.
[19] Mcdermott EM, Powell RJ. Incidence of ovarian failure in systemic lupus erythematosus after treatment with pulse cyclophosphamide. Ann Rheum Dis 1996; 55: 224-229.
[20] Dooley MA, Patterson CC, Hogan SL et al. ACR abstract, Arthritis Rheum 2000; 42: 107.
[21] McCune WJ, Somers EC, Ognenovski V,et al. Use of leuprolide acetate for ovarian protection during cyclophosphmide therapy of women with severe SLE: a case control study [abstract] Arthritis Rheum 2001; 44(suppl):s387.
[22] Masala A, Faedda R, Alagna S et al. Use of testosterone to prevent cyclophosphmide-induced azoospermia. Ann Intern Med 1997; 126: 292-295.
[23] Priftakis P, Bogdanovic G, Kokhaei P et al. BK virus quantification in urine samples of bone marrow transplanted patients is helpful for diagnosis of hemorrhagic cystitis, although wide individual variation exist. J Clin Virol 2003; 26: 71-77.
[24] Bedi A, Miller CB, Hanson JL et al. Association of BK virus with failure of prophylaxis against hemorrhagic cystitis following bone marrow transplantation. J Clin Oncol 1995; 13: 1103-1109.
[25] Bernatsky S, Ramsey-Goldman R, Boivin JF et al. Cancer in systemic lupus erythematosus(SLE): an international multi-centre cohort study [abstract] Arthritis Rheum 2003; 48(suppl):s696.
[26] Pryor B, Bologna S, Ernst C et al. Risk of malignancy in cyclophosphamide-treated SLE patients [abstract] Arthritis Rheum 1993; 36(suppl):s91.
[27] Bateman H, Yazici Y, Leff L et al. Increased cervical dysplasia in intravenous cyclophosphamide-treated patients with SLE: a preliminary study. Lupus 2000; 9: 542-544.
[28] Blumenfeld Z, Lorber M, Yoffe N. Systemic lupus erythematosus: predisposition for uterine cervical dysplasia. Lupus 1994; 3: 59-61.
[29] Lima FR, Guerra D, Sella EMC et al. Systemic lupus erythematosus and cervical intraepithelial neoplasia.Arthritis Rheum 1998;41(suppl):s66.
[30]顾颖莉,朱汉威。环磷酰胺心脏毒性作用的研究进展。上海第二医科大学学报,2004,24:772-774。
[31]Zhong S,Huang M,Yang X,Relationship of glutathione S-transferase genotypes with side-effects of pulsed cyclophosphamide therapy in patients with systemic lupus erythematosus.Br J Clin Pharmacol.2006 Oct;62(4):457-72.
[32]Tse KC,Tang CS,Lio WI,Lam MF,Chan TM.Quality of life comparison between corticosteroid-and-mycofenolate mofetil and corticosteroid-and-oral cyclophosphamide in the treatment of severe lupus nephritis.Lupus.2006;15(6):371-9.
[33]Ptera P,Manger B,Manger K et al.A pilot study of leflunomide in systemic lupus erythematosus.Arthritis Rheum.2000;43(suppl)241.
[34]Levy Y,Sherer Y,Ahmed Aet al.A study of 20 SLE patients with intravenous immunoglobulin-clinical and serologic response.Lupus,1999;8:705-712.
[35]Traynor AE,Schroeder J,Rosa RM.Treatment of severe SLE with high-dose chemotherapy and haemopoietic stem-cell transplantation phase Ⅰ study.Lancet 2000;356:701-707.
[36]邢莉民,王金铠,常英军等。自体外周造血干细胞移植治疗系统性红斑狼疮。中华风湿病学杂志2002;6:249-251。
[37]Zhao Y,Zhou DB,Leng XM,Treatment of severe systemic autoimmune diseases with autologous peripheral blood stem cell transplantation.Zhonghua Yi Xue Za Zhi.2004 Dec 17;84(24):2077-81.
[38]Schroeder JO,Euler HE,Loffier H.Synchronization of plasmapheresis and pulse cyclophosphmide in severe systemic lupus erythemaosus.Ann Intern Med 1987;107:344-346.
[39]Danieli MG,Palmieri C,Salvi A et al.Synchronised therapy and high-dose cyclophosphamide in proliferative lupus nepheitis.J Clin Apheresis 2002;17:72-77.
[40]D'Cruz D,Cuadrado MJ,Mujic F et al.Immunosuppressive therapy in lupus nephritis.Clin Exp Rheumatol 1997;15:275-282.
[41]Houssiau FA,Vasconcelos C,D'Cruz D et al.Immunosuppressive therapy in lupus nephritis:the Euro-Lupus Nephritis Trial,a randomized trial of low-doze versus high-dose intravenous cyclophosphamide.Arthritis Rheum 2002;46:2121-2131.
[42]刘湘源,徐宁,李胜光。环磷酰胺治疗系统性红斑狼疮不良反应的防治。中华风湿病学杂志2008,12(3),207-209。
[43]Mok CC,Ho CT,Chan KW et al.Outcome and prognostic indicators of diffuse proliferative lupus glomerulonephritis treated with sequential oral cyclophosphamide and azathioprine. Arthritis Rheum 2002; 46: 1003-1013.
[44] Brodsky RA, Petri M, Smith BD et al. Immunoblative high-dose cyclophosphmide without stem cell rescue for refractory, severe autoimmune disease. Ann Int Med 1998; 129:1031-1035.
[45] Santos GW, Owens AH Jr. A comparison of the effects of selected cyotoxic agents on allogeneic skin graft survival in rats. Bull Hopkins Hosp 1965; 116: 327-340.
[46] Zhang Q, Mayumi H, Umesue M et al. Fractionated dosing of cyclophosphamide for establishing long-lasting skin allograft survival, stable mixed chimerism, and intrathymic clonal deletion in mice primed with allogeneic spleen cells. Transplantation 1997; 63: 1667-1673.
[47] Petri M, Jones RI, Brodsky RA. High dose immunoblative cyclophosphamide without stem cell transplantation in systemic lupus erythematosus. Arthritis Rheum 2003; 48: 166-173.