【作者】 魏华；

【导师】 李小峰； 曾小峰；

【作者基本信息】 山西医科大学 ， 内科风湿病学， 2008， 博士

【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。更多还原

【Abstract】 BackgroundsSystemic 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.ObjectivesFrom 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. MethodsThe 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 item1 General state of health:weight,mental state,survival2 Blood detection:peripheral blood leucocyte,ALT,Cr,anti-dsDNA,ELISA for MCP-1, TGFβ1,BAFF3 Urine detection:24-hour urine protein quantitation,ELISA for MCP-1,TGFβ14 Liver:HE staining of liver5 Kidney:HE,Masson,PSAM,IF staining of kidney,immunohistochemical method to MCP-1,TGFβ1 in kidney6 RT-PCR to MCP-1,TGFβ1 mRNA7 Spleen:immunohistochemical method to BAFF,BAFFR in spleen8 RT-PCR to BAFF,BAFFR mRNAResults1 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 index①It 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 index①Blood 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 CTX①The 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).Conclusions1 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.更多还原