免疫清零耐受诱导治疗1型糖尿病-NOD小鼠动物实验研究
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摘要
一研究背景
1型糖尿病(Type 1 Diabetes,TlDM)是一种T细胞介导为主的器官特异性的自身免疫性疾病(Autoimmune desease,AID),在病毒感染、理化因素等作用下,机体的免疫平衡状态被打破,对自身抗原的免疫耐受丧失,机体产生自身抗体和/或自身致敏淋巴细胞,导致胰腺β细胞进行性破坏,胰岛素分泌不足而引起一系列临床症状的疾病。
TlDM目前确切的发病机制尚不清楚,认为与自身免疫反应启动和自身免疫耐受的丧失有很大关系,研究发现CD4+CD25+调节性T(Treg)细胞在其中发挥重要功能,其表达水平与TlDM的发病有关,也与病情进展有关。Treg细胞具有免疫耐受和免疫抑制双重功能,正常表达下可以抑制自身免疫反应,维持机体内稳态的平衡。
近年来,对TlDM的临床治疗除胰岛素替代治疗外多采用免疫抑制治疗、造血干细胞移植(hematopoietic stem celltransplantation,HSCT)和/或联合胰岛移植,HSCT包括自体HSCT(autologous HSCT,Auto-HSCT)和异基因HSCT(allogeneic HSCT,Allo-HSCT)。免疫抑制疗法需长期甚至终生用药,使机体机会感染的危险增加,且往往仪能改善症状,停药后易反复,导致胰腺β细胞的进一步破坏;而Auto-HSCT虽耐受较好,移植后也大多可获得不同时间的稳定或缓解,但遗憾的是有部分患者移植后出现复发,移植过程中并发症也较多;Allo-HSCT虽能治愈,但因匹配移植源缺乏、价格昂贵及移植后的多种并发症,如常见的移植物抗宿主疾病(Graft verse host disease,GVHD),甚至继发的AID、感染等,这些因素限制了其临床应用。
研究发现Auto-HSCT后复发的原因可能有:1.病变处于干细胞水平;2.自身免疫性细胞克隆未完全清除或者再次回输体内;3.体细胞的变异抗原成分依然存在,新生免疫系统对其继续攻击。对于干细胞病变引起的TIDM我们必须用Allo-HSCT,而对干细胞病变之外的TlDM,我们期望通过对自身异反应细胞的克降清除,并继之通过诱导对自身抗原的重新耐受,达到防止疾病复发的目的。对于重新诱导机体免疫细胞对异己体细胞的耐受,我们可以从Allo-HSCT中借鉴经验,GVHD是最好的AID模型,在造血干细胞输入的同时我们给与环孢素A、骁悉等免疫调节剂可以诱导新生免疫系统对异己体细胞的良好耐受,而且不必终生服药;目前临床上的急性再生障碍性贫血(SAA)所使用的抗胸腺/淋巴细胞免疫球蛋白(ATG/ALG)+CSA的组合也使得70%的患者治愈,疗效较单用ATG或CSA明显提高,提示清除异常免疫与诱导耐受具有协同作用。上述事实说明环孢素A、骁悉等免疫调节剂可以诱导从干细胞分化来的初生免疫细胞对体细胞的重新耐受,重演胸腺的“阴性选择”。
TlDM是一种T细胞介导为主的AID,虽然其确切的发病机制尚未清楚,但可以肯定的是机体免疫系统丧失了对自身抗原的免疫耐受。所以我们认为对原有异反应细胞克隆彻底摧毁,重建正常免疫体系和诱导对自身抗原的重新耐受是本病治疗的关键,或许可以治愈TlDM。基于上述理论和事实的支持,所以我们提出了免疫清零联合诱导耐受的新模式治疗TlDM,对新发病的TlDM病人给与移植剂量的免疫抑制剂清除自身病态免疫系统,而不伴有造血干细胞的移植,继之用免疫调节药物诱导新生免疫系统对自身抗原的重新耐受,重演胸腺的“阴性选择”,达到治愈TlDM的目的。该治疗模式的一个重要环节是免疫抑制剂的选择:对机体免疫系统敏感而且特异,而对干细胞损伤较小的药物,以期取得更好疗效:1.特异性清除异常免疫系统而减少对干细胞的损伤,从而降低化疗的副损伤;2.由于不伴有自体细胞的回输避免了由自体异常克隆“污染”引起的二次复发;3.免疫清除后重新诱导免疫耐受,有望避免终生应用免疫抑制剂。
研究目的
对已发病的NOD小鼠行免疫清零联合耐受诱导治疗后观察其疗效,鉴于Treg细胞在TlDM发病和疾病进展中的重要性,本动物实验来观察该模式治疗后Treg细胞表达水平的变化,进一步探讨该治疗模式与Treg细胞的相关性,从而探索其可能的作用机制。
研究方法
1.实验动物
20只健康雌性8～10周龄NOD小鼠,南京大学模式动物研究所(许可证编号:SCXK(苏)2005-0002)提供,于南方医院实验动物巾心SPF级无菌环境实验室饲养(许可证编号:SCXK(粤)2006-0015),高脂高蛋白喂养,保持环境安静、清洁。
从20只小鼠中随机选择18只进入实验,平均分为3组,A组为空白对照组,B组为小剂量环磷酰胺(Low-dose Cyclophosphamide,LD-CTX)组,C组为移植剂量的环磷酰胺联合小剂量白舒非(HD—CTX+BU)组,并继之环孢素A(Ciclosporin A,USA)行耐受诱导。
实验过程中动物死亡或造模不成功,另随机取同样条件下的NOD小鼠相同的实验条件和步骤差额补齐。
2.试验路线设计
当NOD小鼠连续2次不同时间点血糖浓度≥11.3 mmol/L,即为模型成功进入实验,各组在处理前外周血白细胞、淋巴细胞,餐后2h血糖、血清C肽,动物的体重、周龄无显著性差异。
A组:新发病的NOD小鼠给与同B、C组等量的生理盐水腹腔注射,当血糖高于30.0mml/L给与皮下注射胰岛素控制,避免血糖过高导致动物死亡。
B组:新发病的NOD小鼠给LD—CTX 90mg/kg.d(相当于人用量10mg/kg.d)腹腔注射,每天1次,连续应用3天,给药期间及给药后监测血糖,当血糖高于30.0mml/L时,处理同A组。
C组:新发病的NOD小鼠给与HD-CTX联合BU腹腔注射给药,d1-3给与HD-CTX,d4-6给与小剂量BU,d7开始至死亡或实验结束给与CSA,给药期间及给药后监测血糖,当血糖高于30.0mml/L时,处理同A组。
给药方法:
CTX:d1-3给与360 mg/kg.d(相当于人用量40mg/kg.d),每目1次;
BU:d4-6给与7.2 mg/kg.次(相当于人用量0.8mg/kg/次),12小时一次,连续给药6次;
CSA:自d7始给与27mg/kg.d(相当于人用量3mg/kg.d),每天1次。
免疫清零:给药结束后(第7天)镜下计数外周血白细胞和淋巴细胞,目标:淋巴细胞计数趋近为零。
3.实验指标测定
3组小鼠发病初给药前(第1天)及治疗后2周取外周血测定餐后2h血清C肽浓度、外周血CD4+CD25+调节性T(Treg)细胞的表达水平;并在第1天、第7天镜下计数外周血白细胞、淋巴细胞比例;实验结束或动物死亡后立即取小鼠胰腺和脾脏,胰腺HE染色观察胰岛的破坏情况;用流式细胞仪测定脾脏Treg细胞表达水平。
数据统计学分析:
本实验纳入NOD小鼠18只,18只实验数据进入结果分析。
本实验数据应用SPSS13.0统计软件进行处理,结果用均数±标准差,治疗后2h-CP、外周血Treg细胞表达水平比较采用协方差分析(analysis ofcovariance,ANCOVA),脾脏Treg细胞表达水平的比较采用单因素方差分析(One-Way ANOVA),每组治疗前后指标的观察采用_酉己对样本t检验(Paired-Samples T Test),以p≤0.05为差异有统计学意义。
研究结果
1.实验动物观察
1.1小鼠发病初期异常活跃,饮水、进食增加、活动度较好。
1.2给药期间及给药后小鼠观察
A组饮水、进食、活动度及毛色等逐渐变差。自16天开始陆续死亡,最长生存时间19天,中位生存时间18天。
B组给与LD-CTX期间活动尚可,进食、饮水无明显变化,给药结束第2天开始小鼠毛色、进食、饮水都变差,15天始小鼠陆续死亡,最长生存时间19天,中位生存时间17天。
C组给与HD-CTX期间就已开始出现毛色变差,脱毛,弓背,消瘦,倦怠,活动明显减少,对刺激反应较差,进食、饮水量明显减少,给与CSA上述情况无明显改善,实验过程中观察到3例会阴部有血块,自16天开始陆续死亡,最长时间19天,中位生存时间18.5天。
1.3实验过程中无1例血糖超过30.0mmol/L。A组血糖波动,介于15-25mmol/L;B组给药后血糖持续丌高;C组血糖给药后血糖波动,但较前无明显升高,死前有2例测血糖偏低(1.9 mmol/L、2.1 mmol/L)。
2.第7天免疫清零效果:C组WBC:(0.20±0.07)G/L:LYM(0.09±0.07)G/L,基本达到免疫清零效果。
3.窄白对照组发病2周后指标观察
新发病的NOD小鼠观察2周后餐后2h血清C肽(2h-CP)、Treg细胞的表达水平、Treg/CD4+T都较发病初期偏低,但无显著统计学意义(p=0.144;P=0.221:P=0.430)。
4.新发病的NOD小鼠给与LD-CTX方法治疗2周后的疗效观察
4.1 LD-CTX治疗后发现2h-CP、Treg细胞的表达水平及Treg/CD4+T较治疗前显著降低(P=0.002;P=0.013;P=0.018)。
4.2 LD-CTX治疗2周后2h-CP与对照组相比无显著下降(p=0.158);Treg细胞的表达水平(1.20±0.39)与对照组(1.78±0.29)相比有显著降低(P=0.015);Treg/CD4+T无显著统计学差异(P=0.098)。
4.3 LD-CTX治疗后脾脏的Treg细胞表达水平与空白组相比无显著统计学差异(P=0.774);治疗后Treg/CD4+T%(7.38±3.61)较空白组(9.06±4.87)无显著下降(P=0.357)。
5.新发病的NOD小鼠给与HD-CTX+BU并CSA耐受诱导治疗2周后的疗效观察
5.1给与HD-CTX+BU并CSA耐受诱导治疗后2周发现2h-CP、Treg细胞的表达水平及Treg/CD4+T较治疗前显著增高(P=0.030;P=0.045;P=0.015)。
5.2治疗2周后2h-CP、Treg细胞的表达水平及Treg/CD4+T的比值较对照组明显增高(P=0.001;P=0.002;P=0.019),也较LD-CTX治疗组显著升高(P=0.000:P=0.000:P=0.011)。
5.3治疗后脾脏的Treg细胞表达水平及Treg/CD4+T细胞均较对照组有明显升高(P=000;P=0.005),也明显高于LD-CTX治疗组(P=000;P=0.001)。
结果显示:给与移植剂量的CTX联合小剂量BU并行CSA诱导后,小鼠餐后2h血清C肽分泌较给药前及空白组都显著升高,提示残存胰岛β细胞的功能较治疗前增强,胰腺B细胞的功能恢复。
6.胰腺HE染色胰岛观察
A、C两组腺泡基本完整,与B组相比胰岛破坏程度较轻,炎性细胞浸润较少,甚少出现胰岛的局部坏死。B组胰岛炎性细胞浸润较明显,胰岛破坏程度最重,取材肉眼观可见到胰腺糜烂。
研究结论
1.NOD小鼠是一个很好的TlDM实验动物模型,移植剂量的CTX联合小剂量BU基本达到免疫清零的效果。
2.新发病NOD小鼠LD-CTX治疗后2h-CP、Treg等表达较治疗前下降,治疗后也较空白组下降,胰腺组织病理中也观察到胰岛的炎性细胞浸润和破坏较其他组严重,认为LD-CTX对TlDM无治疗作用,反加速TlDM的进展,分析其可能的作用机制是:通过降低Treg细胞的表达来间接发挥自身免疫增强效应,可能是LD-CTX加速TlDM的原因之一。
3.新发病NOD小鼠HD-CTX+BU并CSA耐受诱导治疗后2h-CP、Treg细胞等表达较治疗前明显增加,治疗后也较空白组明显升高,认为给新发病的NOD小鼠HD-CTX+BU继之给与CSA诱导短期治疗效果好,可以延缓TlDM的进展。认为其可能的有效作用机制是:一移植剂量的化疗以“大扫荡”方式清除了病态异反应的克隆细胞,终止了自身免疫反应;二免疫调节剂CSA或许通过增加CD4+CD25+调节性T细胞的表达和活性,重新诱导了新生免疫系统对自身抗原的耐受。
Background
Type 1 Diabetes Mellitus(T1DM) is a organ specific autoimmune disease conducted by T lymphocyte.Immune system is broken under the influence of virus infection,physical or chemical factors and patient lose tolerance of auto-antigen, begin to produce auto-antibodies or auto-allergic lymphocytes which cause pancreaticβcell's progressing damage.Then insufficient insulin secretion causes a series of symptoms.
The exact pathogenesis of T1DM is not thoroughly understand,we think that it has a close relationship with priming auto-immune response and loss of auto-immune tolerance.We found the expression levels of CD4+CD25+T regulatory cells play an important role in the incidence and progression of T1DM.T regulatory cell with immune tolerance and immune suppression can inhibit the immune response,to maintenance the balance homeostasis of the body.
Recent years,immune inhibited therapy is the mostly choose therapy for TIDM, as well as hematopoietic stem cell transplantation(HSCT) and/or combined pancreatic cell transplantation which includes autologous hematopoietic stem cell transplantation(auto-HSCT) and allogeneic hematopoietic stem cell transplantation (Allo-HSCT).Immuen inhibited therapy usually can just control the symptom and relapse after drug stopped;Although auto-HSCT has the advantage of better tolerance and patient has a period of stable and remission,but still some patients relapse after transplantation.Allo-HSCT can cure the disease,but it also has the disadvantages as expensive,limited resource,and post-transplantation complications such as GVHD,secondary AID which limit the clinical application of allo-HSCT.
Reasons of relapse after auto-HSCT probably are:pathological changes at stem cell level which will cause the reoccurrence after auto-immune system's rebuilding; Mutated immunologic cells were not eliminated thoroughly or reintroduced via auto stem cell transplantation cause the reoccurrence;Existence of mutated somatic cells (exp.Virus infected somatic cell) are attacked by newly generated immunologic cells will cause the relapse.Pathological changes at the stem cell level have to be cured by allogeneic stem cell transplantation.Beside that,we hope that clearing out auto-allergic clone and inducing tolerance of auto-antigen will prevent the relapse. Talking about feasibility of inducing tolerance of auto-antigen,we have something to learn from allo-HSCT,GVHD is a perfect model for AID.We give patients CsA, MMF right after stem cell infusion,this method will induce newly built immune system tolerate auto somatic cell,no need for life long drug administration.Presently, the combination of ATG\ALG plus CsA can cure 70%severe aplastic anemia patients,while single drug therapy doesn't have the same prognosis.Most of the 70%patients have immunologic disorder,this result can prove synergism of immunoablative and tolerance-inductive.All these above information suggest that immune modulate drugs as CsA,MMF have the ability to induce newly immunologic cells to tolerant auto-somatic cells,same as "negative choose" happens in thymus gland.
TIDM is a organ specific auto-immune disease,although its mechanism is not thoroughly understand,the one thing is sure that destroy the original auto-allergic immunologic cell,rebuild a normal immune system and induce a tolerance of auto-antigen is the key of treatment,it probably will cure TIDM.Based on this theory and facts,we propose to treat early stage TIDM with condition regime which is used on HSCT but without stem cell transfusion,then induce tolerance of auto somatic cell with immune modulate drugs,reenact the "negative choose" and cure the disease.The most important part of this treatment is the choice of immune inhibit drugs.We chose busulfan and cyclophosphomid,both drugs has less effect on stem cells and less sensitive to immune system.And we chose CsA for animal experiment based on the clinical experience we have on HSCT.The objection is to explore the feasibility of immnoablative and tolerance-inductive therapy,at the same time explore mechanism of TIDM and immune tolerance.
Objective
We treat the NOD mice by giving therapeutic regimen about Immunoablative and tolerance-inductive and observation its effect.We monitor the expression levels of CD4+CD25+T regulatory cells because the importance of Treg cells on T1DM,to explore the possible mechanism about tolerance-inductive.
Methods:
1,animal models:chose 18 NOD female mice randomly from 20,divided into 3 groups.Group A is control group,group B is lower dose CTX group,group C is conditioning dose CTX and BU group which will continue with CsA administration. During the experiment,if there is animal died or model failed,chose other NOD mouse to follow the same protocol.
2,experiment procedure design:If blood sugar value of NOD mouse is higher than 11.3 mmol/L twice consecutively,it will be included in experiment as animal model of TIDM.There is no significant difference of peripheral WBC,lymphocyte, blood sugar 2 hours after meal,serum C peptide and weight among groups before treatment.
Gourp A:early stage NOD mice were given saline injection,hypodermic insulin was given if the blood sugar value is higher than 30.0 mmol/L to prevent the animal die of high blood sugar.
Group B:early stage NOD mice were given LD-CTX intraperitoneal injection (mice dose 90mg/kg.d,equals to human dose 10 mg/kg),once daily,total 3 days, during the injection and after the treatment we monitored the blood sugar value,if the blood sugar value is higher than 30.0 mmol/L,treated them as group A.
Group C:early stage NOD mice were given intraperitoneal injection with HD-CTX and BU,day 1～3 CTX,day 4～6 BU.CTX 360 mg/kg.d(equals to human dose 40 mg/kg.d),once daily;Bu 7.2 mg/kg.d(equals human dose 0.8mg/kg/time), once every 12 hours,total 6 shots consecutively;followed with CsA since 7th day, dose 27 mg/kg.d(equals human dose 3 mg/kg.d),once daily.Monitor blood sugar value and when the value is higher than 30.0 mmol/L,same treatment as group A.
Immunoabrasive:run the peripheral blood cell count after 7 days after the injection.Objection:lymphocyte count is zero.
3,index:blood sugar value,serum C peptide concentration,peripheral CD4+CD25+ T lymphocyte's expression level were tested at the 1st and 14th day. WBC and lymphocyte count was tested at 1 st and 7th day.At the end of experiment of right after mice dead,took spleen and pancreas to observe the damage of pancreatic island,and test the spleen Treg cell's expression with flew cytometer.
Statistic analysis:
There are 18 NOD mice in our experiment,data of 18 mice was collected to run the statistic analysis.
Data of our experiment was analyzed with SPSS 13.0 software,results showed as means+standard deviation(X±SD),analysis of covariance was used to analyze the data of Treg expression and 2 hours of C peptide after meal in peripheral blood, one-way ANOVA was used to analyze the data of peripheral blood cell counts and Treg expression.Data before and after injection was analyzed with paired-samples t test,P≤0.05 was defined as having statistic meaning of significant difference.
Results:
1,observe animal model
1.1 before the eruption,all mice are very active,feed normally.
1.2 mice of group A gradually deteriorated.Feeding,activity and skin condition got worse gradually.Death had happened from day 16 to 19.
Mice of group B didn't have obvious change during the injection of LD-CTX.2 days after injection,feeding,activity and skin condition of them began to deteriorate.Death had happened from day 15 to 19.
Mice of group C experienced deteriorating of feeding,weight loosing, fatigue,activity and skin condition during the injection of HD-CTX,they also less responsive to stimulation,and the situation had no change after being given CsA injection.During the process,3 mice were found having blood clot around anus. Death had happened from day 16 to 19.
1.3 there was no blood sugar value higher than 30.0 mmol/L during the process, value range is 15 to 25 mmol/L.Mice of group B experienced consistent high blood sugar value after treatment.Mice of group C had volatile blood sugar value after treatment,but there was no significant difference compared with value before treatment.2 mice's blood sugar values were lower than normal before death.
2,Effect of immunoabrasive treatment at 7th day.WBC of group C: (0.20±0.07)G/L;LYM(0.09±0.07) G/L.Immunoablasive result was achieved.
3,After 2 weeks,the data of control group were collected.Mice have TIDM have lower C peptide,Treg expression and the ratio of Treg/CD4+ than the early stage of disease,but there is no statistic significant difference found.(P=0.144; P=0.221;P=0.430)
4,Effect of LD-CTX on early stage NOD mice.
4.1 2h-CP,expression of Treg and ratio of Treg/CD4+ of mice decreased significantly after being given LD-CTX injection(P=0.002;P=0.013;P=0.018).
4.2 2 weeks after LD-CTX injection,2h-CP of group B are lower than control group,but there is no statistic significance(p=0.158).Treg expression between two groups has significant difference(P=0.015).And the ratio of Treg/CD4+ has no difference(P=0.098).
4.3 there is no significant difference of Treg expression in spleen and ratio of Treg/CD4+ after LD-CTX injection(P=0.774;P=0.357),and the ratio of Treg/CD4+ of LD-CTX group(7.38±3.61) had slight decreasing compared with control group(9.06±4.87).
5.effect of HD-CTX+Bu followed with CsA treatment on early stage NOD mice.
5.1 after HD-CTX+Bu followed CsA treatment,2h-CP,Treg expression and ratio of Treg/CD4+ significantly increased compared with data before injection(P= 0.030;P=0.045;P=0.015)
5.2 after 2 weeks treatment,2h-CP,Treg expression and ratio of Treg/CD4+ of group C are significantly higher than that of control group(P=0.001; P=0.002;P=0.019) and LD-CTX(1.55±0.76) group(P=0.000;P=0.000;P=0.011).
5.3 Treg expression in spleen and ratio of Treg/CD4+ are also higher than control group significantly(P=0.000;P=0.005),and LD-CTX group(P=0.000; P=0.001).
6 pathologic examination of NOD mice's pancreas((hematoxylin and eosin stain)
The gland alveolus of group Aand B are nearly integrity,infiltrating of inflammatory cell and the necrosis of pancreas is fewer comparison with group B. We can find the erosion pancreas in group B.
Conclusion:
1,NOD mice is a very good TIDM animal model,conditioning dose of HD-CTX+Bu can achieve the immunoablative result.
2,2h-CP,Treg expression are lower after LD-CTX injection,and the value decreased after treatment compared with control group.It suggests that LD-CTX has no positive effect on TIDM,on the contrary,it might speed the development of disease.The possible mechanism is that LD-CTX decrease Treg expression and activity to achieve its immunologic intensified effect.
3,2h-CP,Treg expression are higher than before the HD-CTX +Bu followed by CsA treatment obviously,and higher than the control group obviously.It suggests that early stage NOD mice treated with immunoablasive and tolerance-inductive therapy have optimistic prognosis.TIDM progressing can be slowed.The probable mechanism is:first,conditioning dose of chemotherapy eliminated mutated cells clone;second,CsA modulate Treg's expression and activity by increasing CD4+CD25+ T cells,induce newly built immune system produce auto-tolerance to auto-antigen again.
1型糖尿病(Type 1 Diabetes,TlDM)是一种T细胞介导为主的器官特异性的自身免疫性疾病(Autoimmune desease,AID),在病毒感染、理化因素等作用下,机体的免疫平衡状态被打破,对自身抗原的免疫耐受丧失,机体产生自身抗体和/或自身致敏淋巴细胞,导致胰腺β细胞进行性破坏,胰岛素分泌不足而引起一系列临床症状的疾病。
TlDM目前确切的发病机制尚不清楚,认为与自身免疫反应启动和自身免疫耐受的丧失有很大关系,研究发现CD4+CD25+调节性T(Treg)细胞在其中发挥重要功能,其表达水平与TlDM的发病有关,也与病情进展有关。Treg细胞具有免疫耐受和免疫抑制双重功能,正常表达下可以抑制自身免疫反应,维持机体内稳态的平衡。
近年来,对TlDM的临床治疗除胰岛素替代治疗外多采用免疫抑制治疗、造血干细胞移植(hematopoietic stem celltransplantation,HSCT)和/或联合胰岛移植,HSCT包括自体HSCT(autologous HSCT,Auto-HSCT)和异基因HSCT(allogeneic HSCT,Allo-HSCT)。免疫抑制疗法需长期甚至终生用药,使机体机会感染的危险增加,且往往仪能改善症状,停药后易反复,导致胰腺β细胞的进一步破坏;而Auto-HSCT虽耐受较好,移植后也大多可获得不同时间的稳定或缓解,但遗憾的是有部分患者移植后出现复发,移植过程中并发症也较多;Allo-HSCT虽能治愈,但因匹配移植源缺乏、价格昂贵及移植后的多种并发症,如常见的移植物抗宿主疾病(Graft verse host disease,GVHD),甚至继发的AID、感染等,这些因素限制了其临床应用。
研究发现Auto-HSCT后复发的原因可能有:1.病变处于干细胞水平;2.自身免疫性细胞克隆未完全清除或者再次回输体内;3.体细胞的变异抗原成分依然存在,新生免疫系统对其继续攻击。对于干细胞病变引起的TIDM我们必须用Allo-HSCT,而对干细胞病变之外的TlDM,我们期望通过对自身异反应细胞的克降清除,并继之通过诱导对自身抗原的重新耐受,达到防止疾病复发的目的。对于重新诱导机体免疫细胞对异己体细胞的耐受,我们可以从Allo-HSCT中借鉴经验,GVHD是最好的AID模型,在造血干细胞输入的同时我们给与环孢素A、骁悉等免疫调节剂可以诱导新生免疫系统对异己体细胞的良好耐受,而且不必终生服药;目前临床上的急性再生障碍性贫血(SAA)所使用的抗胸腺/淋巴细胞免疫球蛋白(ATG/ALG)+CSA的组合也使得70%的患者治愈,疗效较单用ATG或CSA明显提高,提示清除异常免疫与诱导耐受具有协同作用。上述事实说明环孢素A、骁悉等免疫调节剂可以诱导从干细胞分化来的初生免疫细胞对体细胞的重新耐受,重演胸腺的“阴性选择”。
TlDM是一种T细胞介导为主的AID,虽然其确切的发病机制尚未清楚,但可以肯定的是机体免疫系统丧失了对自身抗原的免疫耐受。所以我们认为对原有异反应细胞克隆彻底摧毁,重建正常免疫体系和诱导对自身抗原的重新耐受是本病治疗的关键,或许可以治愈TlDM。基于上述理论和事实的支持,所以我们提出了免疫清零联合诱导耐受的新模式治疗TlDM,对新发病的TlDM病人给与移植剂量的免疫抑制剂清除自身病态免疫系统,而不伴有造血干细胞的移植,继之用免疫调节药物诱导新生免疫系统对自身抗原的重新耐受,重演胸腺的“阴性选择”,达到治愈TlDM的目的。该治疗模式的一个重要环节是免疫抑制剂的选择:对机体免疫系统敏感而且特异,而对干细胞损伤较小的药物,以期取得更好疗效:1.特异性清除异常免疫系统而减少对干细胞的损伤,从而降低化疗的副损伤;2.由于不伴有自体细胞的回输避免了由自体异常克隆“污染”引起的二次复发;3.免疫清除后重新诱导免疫耐受,有望避免终生应用免疫抑制剂。
研究目的
对已发病的NOD小鼠行免疫清零联合耐受诱导治疗后观察其疗效,鉴于Treg细胞在TlDM发病和疾病进展中的重要性,本动物实验来观察该模式治疗后Treg细胞表达水平的变化,进一步探讨该治疗模式与Treg细胞的相关性,从而探索其可能的作用机制。
研究方法
1.实验动物
20只健康雌性8～10周龄NOD小鼠,南京大学模式动物研究所(许可证编号:SCXK(苏)2005-0002)提供,于南方医院实验动物巾心SPF级无菌环境实验室饲养(许可证编号:SCXK(粤)2006-0015),高脂高蛋白喂养,保持环境安静、清洁。
从20只小鼠中随机选择18只进入实验,平均分为3组,A组为空白对照组,B组为小剂量环磷酰胺(Low-dose Cyclophosphamide,LD-CTX)组,C组为移植剂量的环磷酰胺联合小剂量白舒非(HD—CTX+BU)组,并继之环孢素A(Ciclosporin A,USA)行耐受诱导。
实验过程中动物死亡或造模不成功,另随机取同样条件下的NOD小鼠相同的实验条件和步骤差额补齐。
2.试验路线设计
当NOD小鼠连续2次不同时间点血糖浓度≥11.3 mmol/L,即为模型成功进入实验,各组在处理前外周血白细胞、淋巴细胞,餐后2h血糖、血清C肽,动物的体重、周龄无显著性差异。
A组:新发病的NOD小鼠给与同B、C组等量的生理盐水腹腔注射,当血糖高于30.0mml/L给与皮下注射胰岛素控制,避免血糖过高导致动物死亡。
B组:新发病的NOD小鼠给LD—CTX 90mg/kg.d(相当于人用量10mg/kg.d)腹腔注射,每天1次,连续应用3天,给药期间及给药后监测血糖,当血糖高于30.0mml/L时,处理同A组。
C组:新发病的NOD小鼠给与HD-CTX联合BU腹腔注射给药,d1-3给与HD-CTX,d4-6给与小剂量BU,d7开始至死亡或实验结束给与CSA,给药期间及给药后监测血糖,当血糖高于30.0mml/L时,处理同A组。
给药方法:
CTX:d1-3给与360 mg/kg.d(相当于人用量40mg/kg.d),每目1次;
BU:d4-6给与7.2 mg/kg.次(相当于人用量0.8mg/kg/次),12小时一次,连续给药6次;
CSA:自d7始给与27mg/kg.d(相当于人用量3mg/kg.d),每天1次。
免疫清零:给药结束后(第7天)镜下计数外周血白细胞和淋巴细胞,目标:淋巴细胞计数趋近为零。
3.实验指标测定
3组小鼠发病初给药前(第1天)及治疗后2周取外周血测定餐后2h血清C肽浓度、外周血CD4+CD25+调节性T(Treg)细胞的表达水平;并在第1天、第7天镜下计数外周血白细胞、淋巴细胞比例;实验结束或动物死亡后立即取小鼠胰腺和脾脏,胰腺HE染色观察胰岛的破坏情况;用流式细胞仪测定脾脏Treg细胞表达水平。
数据统计学分析:
本实验纳入NOD小鼠18只,18只实验数据进入结果分析。
本实验数据应用SPSS13.0统计软件进行处理,结果用均数±标准差,治疗后2h-CP、外周血Treg细胞表达水平比较采用协方差分析(analysis ofcovariance,ANCOVA),脾脏Treg细胞表达水平的比较采用单因素方差分析(One-Way ANOVA),每组治疗前后指标的观察采用_酉己对样本t检验(Paired-Samples T Test),以p≤0.05为差异有统计学意义。
研究结果
1.实验动物观察
1.1小鼠发病初期异常活跃,饮水、进食增加、活动度较好。
1.2给药期间及给药后小鼠观察
A组饮水、进食、活动度及毛色等逐渐变差。自16天开始陆续死亡,最长生存时间19天,中位生存时间18天。
B组给与LD-CTX期间活动尚可,进食、饮水无明显变化,给药结束第2天开始小鼠毛色、进食、饮水都变差,15天始小鼠陆续死亡,最长生存时间19天,中位生存时间17天。
C组给与HD-CTX期间就已开始出现毛色变差,脱毛,弓背,消瘦,倦怠,活动明显减少,对刺激反应较差,进食、饮水量明显减少,给与CSA上述情况无明显改善,实验过程中观察到3例会阴部有血块,自16天开始陆续死亡,最长时间19天,中位生存时间18.5天。
1.3实验过程中无1例血糖超过30.0mmol/L。A组血糖波动,介于15-25mmol/L;B组给药后血糖持续丌高;C组血糖给药后血糖波动,但较前无明显升高,死前有2例测血糖偏低(1.9 mmol/L、2.1 mmol/L)。
2.第7天免疫清零效果:C组WBC:(0.20±0.07)G/L:LYM(0.09±0.07)G/L,基本达到免疫清零效果。
3.窄白对照组发病2周后指标观察
新发病的NOD小鼠观察2周后餐后2h血清C肽(2h-CP)、Treg细胞的表达水平、Treg/CD4+T都较发病初期偏低,但无显著统计学意义(p=0.144;P=0.221:P=0.430)。
4.新发病的NOD小鼠给与LD-CTX方法治疗2周后的疗效观察
4.1 LD-CTX治疗后发现2h-CP、Treg细胞的表达水平及Treg/CD4+T较治疗前显著降低(P=0.002;P=0.013;P=0.018)。
4.2 LD-CTX治疗2周后2h-CP与对照组相比无显著下降(p=0.158);Treg细胞的表达水平(1.20±0.39)与对照组(1.78±0.29)相比有显著降低(P=0.015);Treg/CD4+T无显著统计学差异(P=0.098)。
4.3 LD-CTX治疗后脾脏的Treg细胞表达水平与空白组相比无显著统计学差异(P=0.774);治疗后Treg/CD4+T%(7.38±3.61)较空白组(9.06±4.87)无显著下降(P=0.357)。
5.新发病的NOD小鼠给与HD-CTX+BU并CSA耐受诱导治疗2周后的疗效观察
5.1给与HD-CTX+BU并CSA耐受诱导治疗后2周发现2h-CP、Treg细胞的表达水平及Treg/CD4+T较治疗前显著增高(P=0.030;P=0.045;P=0.015)。
5.2治疗2周后2h-CP、Treg细胞的表达水平及Treg/CD4+T的比值较对照组明显增高(P=0.001;P=0.002;P=0.019),也较LD-CTX治疗组显著升高(P=0.000:P=0.000:P=0.011)。
5.3治疗后脾脏的Treg细胞表达水平及Treg/CD4+T细胞均较对照组有明显升高(P=000;P=0.005),也明显高于LD-CTX治疗组(P=000;P=0.001)。
结果显示:给与移植剂量的CTX联合小剂量BU并行CSA诱导后,小鼠餐后2h血清C肽分泌较给药前及空白组都显著升高,提示残存胰岛β细胞的功能较治疗前增强,胰腺B细胞的功能恢复。
6.胰腺HE染色胰岛观察
A、C两组腺泡基本完整,与B组相比胰岛破坏程度较轻,炎性细胞浸润较少,甚少出现胰岛的局部坏死。B组胰岛炎性细胞浸润较明显,胰岛破坏程度最重,取材肉眼观可见到胰腺糜烂。
研究结论
1.NOD小鼠是一个很好的TlDM实验动物模型,移植剂量的CTX联合小剂量BU基本达到免疫清零的效果。
2.新发病NOD小鼠LD-CTX治疗后2h-CP、Treg等表达较治疗前下降,治疗后也较空白组下降,胰腺组织病理中也观察到胰岛的炎性细胞浸润和破坏较其他组严重,认为LD-CTX对TlDM无治疗作用,反加速TlDM的进展,分析其可能的作用机制是:通过降低Treg细胞的表达来间接发挥自身免疫增强效应,可能是LD-CTX加速TlDM的原因之一。
3.新发病NOD小鼠HD-CTX+BU并CSA耐受诱导治疗后2h-CP、Treg细胞等表达较治疗前明显增加,治疗后也较空白组明显升高,认为给新发病的NOD小鼠HD-CTX+BU继之给与CSA诱导短期治疗效果好,可以延缓TlDM的进展。认为其可能的有效作用机制是:一移植剂量的化疗以“大扫荡”方式清除了病态异反应的克隆细胞,终止了自身免疫反应;二免疫调节剂CSA或许通过增加CD4+CD25+调节性T细胞的表达和活性,重新诱导了新生免疫系统对自身抗原的耐受。
Background
Type 1 Diabetes Mellitus(T1DM) is a organ specific autoimmune disease conducted by T lymphocyte.Immune system is broken under the influence of virus infection,physical or chemical factors and patient lose tolerance of auto-antigen, begin to produce auto-antibodies or auto-allergic lymphocytes which cause pancreaticβcell's progressing damage.Then insufficient insulin secretion causes a series of symptoms.
The exact pathogenesis of T1DM is not thoroughly understand,we think that it has a close relationship with priming auto-immune response and loss of auto-immune tolerance.We found the expression levels of CD4+CD25+T regulatory cells play an important role in the incidence and progression of T1DM.T regulatory cell with immune tolerance and immune suppression can inhibit the immune response,to maintenance the balance homeostasis of the body.
Recent years,immune inhibited therapy is the mostly choose therapy for TIDM, as well as hematopoietic stem cell transplantation(HSCT) and/or combined pancreatic cell transplantation which includes autologous hematopoietic stem cell transplantation(auto-HSCT) and allogeneic hematopoietic stem cell transplantation (Allo-HSCT).Immuen inhibited therapy usually can just control the symptom and relapse after drug stopped;Although auto-HSCT has the advantage of better tolerance and patient has a period of stable and remission,but still some patients relapse after transplantation.Allo-HSCT can cure the disease,but it also has the disadvantages as expensive,limited resource,and post-transplantation complications such as GVHD,secondary AID which limit the clinical application of allo-HSCT.
Reasons of relapse after auto-HSCT probably are:pathological changes at stem cell level which will cause the reoccurrence after auto-immune system's rebuilding; Mutated immunologic cells were not eliminated thoroughly or reintroduced via auto stem cell transplantation cause the reoccurrence;Existence of mutated somatic cells (exp.Virus infected somatic cell) are attacked by newly generated immunologic cells will cause the relapse.Pathological changes at the stem cell level have to be cured by allogeneic stem cell transplantation.Beside that,we hope that clearing out auto-allergic clone and inducing tolerance of auto-antigen will prevent the relapse. Talking about feasibility of inducing tolerance of auto-antigen,we have something to learn from allo-HSCT,GVHD is a perfect model for AID.We give patients CsA, MMF right after stem cell infusion,this method will induce newly built immune system tolerate auto somatic cell,no need for life long drug administration.Presently, the combination of ATG\ALG plus CsA can cure 70%severe aplastic anemia patients,while single drug therapy doesn't have the same prognosis.Most of the 70%patients have immunologic disorder,this result can prove synergism of immunoablative and tolerance-inductive.All these above information suggest that immune modulate drugs as CsA,MMF have the ability to induce newly immunologic cells to tolerant auto-somatic cells,same as "negative choose" happens in thymus gland.
TIDM is a organ specific auto-immune disease,although its mechanism is not thoroughly understand,the one thing is sure that destroy the original auto-allergic immunologic cell,rebuild a normal immune system and induce a tolerance of auto-antigen is the key of treatment,it probably will cure TIDM.Based on this theory and facts,we propose to treat early stage TIDM with condition regime which is used on HSCT but without stem cell transfusion,then induce tolerance of auto somatic cell with immune modulate drugs,reenact the "negative choose" and cure the disease.The most important part of this treatment is the choice of immune inhibit drugs.We chose busulfan and cyclophosphomid,both drugs has less effect on stem cells and less sensitive to immune system.And we chose CsA for animal experiment based on the clinical experience we have on HSCT.The objection is to explore the feasibility of immnoablative and tolerance-inductive therapy,at the same time explore mechanism of TIDM and immune tolerance.
Objective
We treat the NOD mice by giving therapeutic regimen about Immunoablative and tolerance-inductive and observation its effect.We monitor the expression levels of CD4+CD25+T regulatory cells because the importance of Treg cells on T1DM,to explore the possible mechanism about tolerance-inductive.
Methods:
1,animal models:chose 18 NOD female mice randomly from 20,divided into 3 groups.Group A is control group,group B is lower dose CTX group,group C is conditioning dose CTX and BU group which will continue with CsA administration. During the experiment,if there is animal died or model failed,chose other NOD mouse to follow the same protocol.
2,experiment procedure design:If blood sugar value of NOD mouse is higher than 11.3 mmol/L twice consecutively,it will be included in experiment as animal model of TIDM.There is no significant difference of peripheral WBC,lymphocyte, blood sugar 2 hours after meal,serum C peptide and weight among groups before treatment.
Gourp A:early stage NOD mice were given saline injection,hypodermic insulin was given if the blood sugar value is higher than 30.0 mmol/L to prevent the animal die of high blood sugar.
Group B:early stage NOD mice were given LD-CTX intraperitoneal injection (mice dose 90mg/kg.d,equals to human dose 10 mg/kg),once daily,total 3 days, during the injection and after the treatment we monitored the blood sugar value,if the blood sugar value is higher than 30.0 mmol/L,treated them as group A.
Group C:early stage NOD mice were given intraperitoneal injection with HD-CTX and BU,day 1～3 CTX,day 4～6 BU.CTX 360 mg/kg.d(equals to human dose 40 mg/kg.d),once daily;Bu 7.2 mg/kg.d(equals human dose 0.8mg/kg/time), once every 12 hours,total 6 shots consecutively;followed with CsA since 7th day, dose 27 mg/kg.d(equals human dose 3 mg/kg.d),once daily.Monitor blood sugar value and when the value is higher than 30.0 mmol/L,same treatment as group A.
Immunoabrasive:run the peripheral blood cell count after 7 days after the injection.Objection:lymphocyte count is zero.
3,index:blood sugar value,serum C peptide concentration,peripheral CD4+CD25+ T lymphocyte's expression level were tested at the 1st and 14th day. WBC and lymphocyte count was tested at 1 st and 7th day.At the end of experiment of right after mice dead,took spleen and pancreas to observe the damage of pancreatic island,and test the spleen Treg cell's expression with flew cytometer.
Statistic analysis:
There are 18 NOD mice in our experiment,data of 18 mice was collected to run the statistic analysis.
Data of our experiment was analyzed with SPSS 13.0 software,results showed as means+standard deviation(X±SD),analysis of covariance was used to analyze the data of Treg expression and 2 hours of C peptide after meal in peripheral blood, one-way ANOVA was used to analyze the data of peripheral blood cell counts and Treg expression.Data before and after injection was analyzed with paired-samples t test,P≤0.05 was defined as having statistic meaning of significant difference.
Results:
1,observe animal model
1.1 before the eruption,all mice are very active,feed normally.
1.2 mice of group A gradually deteriorated.Feeding,activity and skin condition got worse gradually.Death had happened from day 16 to 19.
Mice of group B didn't have obvious change during the injection of LD-CTX.2 days after injection,feeding,activity and skin condition of them began to deteriorate.Death had happened from day 15 to 19.
Mice of group C experienced deteriorating of feeding,weight loosing, fatigue,activity and skin condition during the injection of HD-CTX,they also less responsive to stimulation,and the situation had no change after being given CsA injection.During the process,3 mice were found having blood clot around anus. Death had happened from day 16 to 19.
1.3 there was no blood sugar value higher than 30.0 mmol/L during the process, value range is 15 to 25 mmol/L.Mice of group B experienced consistent high blood sugar value after treatment.Mice of group C had volatile blood sugar value after treatment,but there was no significant difference compared with value before treatment.2 mice's blood sugar values were lower than normal before death.
2,Effect of immunoabrasive treatment at 7th day.WBC of group C: (0.20±0.07)G/L;LYM(0.09±0.07) G/L.Immunoablasive result was achieved.
3,After 2 weeks,the data of control group were collected.Mice have TIDM have lower C peptide,Treg expression and the ratio of Treg/CD4+ than the early stage of disease,but there is no statistic significant difference found.(P=0.144; P=0.221;P=0.430)
4,Effect of LD-CTX on early stage NOD mice.
4.1 2h-CP,expression of Treg and ratio of Treg/CD4+ of mice decreased significantly after being given LD-CTX injection(P=0.002;P=0.013;P=0.018).
4.2 2 weeks after LD-CTX injection,2h-CP of group B are lower than control group,but there is no statistic significance(p=0.158).Treg expression between two groups has significant difference(P=0.015).And the ratio of Treg/CD4+ has no difference(P=0.098).
4.3 there is no significant difference of Treg expression in spleen and ratio of Treg/CD4+ after LD-CTX injection(P=0.774;P=0.357),and the ratio of Treg/CD4+ of LD-CTX group(7.38±3.61) had slight decreasing compared with control group(9.06±4.87).
5.effect of HD-CTX+Bu followed with CsA treatment on early stage NOD mice.
5.1 after HD-CTX+Bu followed CsA treatment,2h-CP,Treg expression and ratio of Treg/CD4+ significantly increased compared with data before injection(P= 0.030;P=0.045;P=0.015)
5.2 after 2 weeks treatment,2h-CP,Treg expression and ratio of Treg/CD4+ of group C are significantly higher than that of control group(P=0.001; P=0.002;P=0.019) and LD-CTX(1.55±0.76) group(P=0.000;P=0.000;P=0.011).
5.3 Treg expression in spleen and ratio of Treg/CD4+ are also higher than control group significantly(P=0.000;P=0.005),and LD-CTX group(P=0.000; P=0.001).
6 pathologic examination of NOD mice's pancreas((hematoxylin and eosin stain)
The gland alveolus of group Aand B are nearly integrity,infiltrating of inflammatory cell and the necrosis of pancreas is fewer comparison with group B. We can find the erosion pancreas in group B.
Conclusion:
1,NOD mice is a very good TIDM animal model,conditioning dose of HD-CTX+Bu can achieve the immunoablative result.
2,2h-CP,Treg expression are lower after LD-CTX injection,and the value decreased after treatment compared with control group.It suggests that LD-CTX has no positive effect on TIDM,on the contrary,it might speed the development of disease.The possible mechanism is that LD-CTX decrease Treg expression and activity to achieve its immunologic intensified effect.
3,2h-CP,Treg expression are higher than before the HD-CTX +Bu followed by CsA treatment obviously,and higher than the control group obviously.It suggests that early stage NOD mice treated with immunoablasive and tolerance-inductive therapy have optimistic prognosis.TIDM progressing can be slowed.The probable mechanism is:first,conditioning dose of chemotherapy eliminated mutated cells clone;second,CsA modulate Treg's expression and activity by increasing CD4+CD25+ T cells,induce newly built immune system produce auto-tolerance to auto-antigen again.
引文
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6.Rinee Mukherjee,Pratibha Chaturvedi,Hui-Yu Qin CD4+CD25+ regulatory T cells generated in response to insulin B:9-23 peptide prevent adoptive transfer of diabetes by diabetogenic T cells[J].Journal of Autoimmunity,2001,21:221-237.
7.蒋铁建,周智广,苏恒等.胰岛素预防NOD鼠糖尿病的机制研究[J].中国糖尿病杂志,2007,15(12):750-52.
8.Brodsky RA.High-dose cyclophosphamide for aplastic anemia and autoimmunity[J].Curr Opin Oncol,2002,14:143-46.
9.Marmont AM.Intense immunosuppression and stem cell transplantation orrescue for severe systemic lupus erythematosus[J].Lupus,1999,8(4):256-59.
10.Brodsky RA,Petri M,Jones RJ.Systemic lupus erythematosus.Hematopoietic stem cell transplantation for MS:long-term remission after a high dose of cyclophosphamide 197 systemic lupus erythematosus[J].Rheum Dis Clin N Am,2002,26:166-173.
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12.Arnon K,Konstantin B,Wayne W H,et al.Cyclophosphamide modulates CD4+T cells into a T helper type 2 phenotype and reverses increased IFN-γ production of CDS+ T cells in secondary progressive multiple sclerosis[J].J Neuroimmunol,2004,146(122):189-98.
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14.Jonasova A,Neuwirtova R,Cermak J,et al.Cyclosporin A therapy in hypoplastic MDS patients and certain refractory anemias without hypoplastic bone marrow[J].Br J Haematol,1998,100(2):304-09.
15.Ferraccioli GF,Tomietto P,De Santis M.Rationale for T cell inhibition by cyclosporine A in major autoimmune diseases[J].Ann NY Acad Sci,2005,1051:658-65.
16.Notkins AL,Lernmark A.Autoimmune type 1 diabetes:resolved and unresolved issues[J].J ClinInvest,2001,108:1247-52.
17.Randolph DA,Fathman CG.Cd4(+)Cd25(+)regulatory T cells and their therapeutic potential[J].Annu Rev Med,2006,57:381-402.
18.Murphy T J,Choileain N N,Zang Y et al.CD4+CD25+regulatory Tcells control innate immune reactivity after injury[J].Immunol,2005,174(5):2957-63.
19.Yuko I,Masatoshi N,Chizuru T,et al.Cyclophosphamide decreases the number,percentage and the function of CD25+CD4+regulatory T cells,which suppress induction of contacthypersensitivity[J].J Dermatol Sci,2005,39(2):105-112.
20.Proietti E,Greco G,Garrone B,et al.Importance of cyclophosphamide-induced bystander effect on T cells for a successful tumor eradication in response to adoptive immunot herapy in mice[J].J Clin Invest,1998,101(2):429-41.
21.徐磊,王瑞,张卫星,等.生精冲剂对环磷酰胺致生精障碍小鼠的治疗作用[J].郑州大学学报:医学版,2008,43(2):267-70.
22.Proietti E,Greco G,Garrone B,et al.Importance of cyclophosphamide-induced bystander effect on T cells for a successful tumor eradication in response to adoptive immunot herapy in mice[J].J Clin Invest,1998,101(2):429-41.
23.李欣,崔永生,刘白楠.低剂量环磷酰胺对Lewis肺癌小鼠CD4+CD25+Treg 细胞功能的影响[J].吉林大学学报(医学版),2008,34(5):733-36.
24.Brode S et al Cyclophosphamide-Induced Type-1 Diabetes in the NOD Mouse Is Associated with a Reduction of CD4+CD25+Foxp3_Regulatory T Cellsl[J].The Journal of Immunology,2006,177:6603-12.
25.李薇,高素君,王冠军,等.环磷酰胺诱导的Fas(o|¨) FasL依赖的鼠胸腺细胞凋亡[J].免疫学杂志.2000,16(7):315.
26.施念玮,吴艺捷,王煜非,等.己酮可可碱对NOD小鼠1型糖尿病的免疫干预作用[J].上海医学,2005,28(10):877-82.
27.Ghiringheli F,Larmonier N,Schmitt E,et al.CD4+CD25+ regulatory T cells suppress tumor immunity but are sensitive to cyclophosphamide which allows immunotherapy of established tumors to be curative[J].Eur J Immunol,2004,34(2):336-44.
28.Christine Lut siak ME,Roshanak T,Smnani,et al.Inhibition of CD4+ CD25+ T regulatory cell function implicated in enhanced immune response by low-dose cyclophosphamide[J].Blood,2005,105(7):2862-68.
29.Arnon K,Konstantin B,Wayne W H,et al.Cyclophosphamide modulates CD4+T cells into a T helper type 2 phenotype and reverses increased IFN-γ production of CD8+ T cells in secondary progressive multiple sclerosis[J].J Neuroimmunol,2004,146(122):1892-1981.
30.Christe'le Ferry and Ge'rard Socie.Busulfan-cyclophosphamide versus total body irradiation-cyclophosphamide as preparative regimen before allogeneic hematopoietic stem celltransplantation for acute myeloid leukemia:What have we learned?[J].Experimental Hematology,2003,31:1182-86.
31.Remy Gb,Paul L,Mohamed E,et al.Hypersensitivity to insulin during rem issions in cyclo spo rine-reated IDDM patients[J].D iabetes care,1993;16(6):881.
32.Christie MR,Molvig J,Hawkes CJ,et al.IA-2 antibody-negative status predicts remission and recovery of C-peptide levels in type 1 diabetic pa-tients treated with cyclosporin[J].Diabetes Care,2002,25:1192-97.
33.杜美蓉,董琳,周雯慧,等.环孢素A诱导自然流产模型孕鼠脾脏CD4+CD25+Foxp3+的调节性T细胞扩增及外周母胎免疫耐受[J].中国免疫学杂志,2007,23:920-24.
34.雒王容,邱明才.环孢菌素A与自身免疫性糖尿病胰岛β细胞凋亡[J].国际内分泌代谢杂志,2007,27(2):101-103.
35.陈志红.1型糖尿病免疫学发病机制与免疫干预的研究玑状[J].J Appl C lin Pedia tr,2007,22(20):1523-25.
36.Zorn E.CD4 + CD25 + regulatory T cells in human hematopoietic cell transp lantation[J].Seminars in Cancer Biology,2006,16:150-59.
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