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[摘要] 目的 体外制备供体特异性T细胞疫苗(T cell vaccine),探讨T细胞疫苗免疫(T cell vaccination)诱导异种胰岛细胞移植免疫耐受的作用。方法 腹腔注射链脲佐菌素诱导建立Balb/c小鼠Ⅰ型糖尿病模型,用酶消化法和密度梯度离心法无菌分离纯化Wistar大鼠胰岛细胞。制备Balb/c小鼠针对Wistar大鼠的T细胞疫苗:实验随机分为三组,G1:糖尿病小鼠对照组;G2:胰岛细胞移植组,糖尿病小鼠+胰岛细胞移植+1640培养液(对照组);G3:胰岛细胞移植加T细胞疫苗免疫组,糖尿病小鼠+胰岛细胞+T细胞疫苗(实验组),于d1,d7,d14,d21实验组Balb/c小鼠接种TCV(1×107/mL),对照组用1640替代。接种前和每次接种后第5d分别进行混合淋巴细胞反应。胰岛细胞移植:将Wistar大鼠的胰岛按1200～1500IEQ/kg经肝门静脉导入小鼠体内,移植后观测血糖和体重变化。结果 异种胰岛细胞移植可以诱导I型糖尿病小鼠血糖降至正常水平且维持一段时间,对照组小鼠移植后(5.12±1.36)d即发生排斥,实验组移植物存活时间达(20.25±3.45)d。结论 TCV能够有效延长异种胰岛移植物的存活时间,是一种潜在诱导异种胰岛移植免疫耐受的方法。
[关键词] T细胞疫苗;胰岛细胞;异种移植;免疫耐受
[中图分类号] R349.1 [文献标识码] A [文章编号] 1673-9701(2010)04-19-03
The Effects of T Cell Vaccination on Xenogeneic Islet Cell TransplantationHU Chunlei1ZHAO Zhenlin2
1.Shanxi Medical University,Taiyuan 030001,China;2.The Second Hospital of Shanxi Medical University,Taiyuan 030001,China
[Abstract] Objective To explore the the effects of T cell vaccination on xenogeneic islet cell transplantation. Methods Balb/c mice (H2-d) were used as recipients and Wistar rats were used as donors. TypeⅠdiabetes mice model were made by injecting streptozotocin intravenously. Type Ⅰ diabetes mice were randomLy divided into three groups,Group A:Diabetic mice with no transplantation and no TCV, Group B: Islet cell transplantation +RPMI1640 culture medium, Group C(experimental group): Islet cell transplantation+TCV.T cell vaccine was made by using attenuated spleen lymphocytes from Balb/c mice which were challenged with Wistar rat spleen lymphocytes in vitro and were stimulated with Con A. Balb/c mice as recipients were immunized with TCV on the time points of d1,d7,d14,d21 and;RPMI1640 culture medium was used in control group. One-way mixed lymphocyte reaction(MLR) was performed with effector of immunized Balb/c mice peripheric lymphocytes and stimulator of Wistar rat peripheric lymphocytes on the second day of T cell vaccination. Islet cells were isolated from Wistar rat pancreas using the protocol of enzyme digestion and density gradient centrifμgation. Islet cells were transplanted into immunized Balb/c mice at the density of 1200～1500IEQ/kg by the way of portal vein injection. Recipient"s blood glucose was monitored everyday and body weight was recorded weekly after transplantation. Results Xenogeneic islet cell transplantation can induce type I diabetic mice blood glucose to normal levels and remain for some time,the control group mice after transplantation(5.12±1.36) days, which happened rejection,the experimental group graft survival time of up to(20.25±3.45)days. Conclusion T cell vaccination can significantly prolong the survival time of xenogeneic pancreatic islet grafts;T cell vaccination is a potential approach to to induce xenogeneic islet transplant tolerance.
[Key words] T cell vaccine;Islet cell;Xenogeneic transplantation;Immune tolerance
异种移植为胰岛移植治疗I型糖尿病提供了充足的供体,但严重的排斥反应限制了这一方法的应用,应用常规的免疫抑制剂不能抑制异种免疫排斥反应,因此如何诱导免疫耐受成为一个重要课题。T细胞疫苗(TCV)是特异性抗原活化的反应性T细胞经过化学或物理方法处理灭活后制成的,是目前免疫耐受研究中较新的领域[1]。已有研究证明T细胞疫苗可以诱导同种异体移植免疫耐受[2-4]。本实验通过T细胞疫苗诱导受体对异种胰岛移植的免疫耐受状态,探讨TCV在诱导异种胰岛移植免疫耐受中的可行性。
1材料与方法
1.1动物
供者为Wistar雄性大鼠,180～200g,由山西医科大学生理教研室提供;受者为BALB/c(H-2d)雄性小鼠,20～25g;由中国医学科学院动物研究所提供。
1.2试剂及药品
胎牛血清(四季青公司);RPMI细胞培养基(HYCLONG);淋巴细胞分离液(TBD);丝裂霉素C(协和发酵工业株式);刀豆蛋白A(Sigma);链脲霉素(Sigma);ficoll400(sigma);胶原酶V(Sigma)。
1.3制备Wistar大鼠和Balb/c小鼠脾脏单个核淋巴细胞悬液
无菌取大鼠脾脏,研磨制成匀浆,Hanks液稀释,200目不锈钢网过滤,制成细胞悬液,10倍细胞压积的比例加入红细胞裂解液,充分吹打混匀后,Hanks液洗两次,轻轻加入到淋巴细胞分离液上层密度梯度离心,收集界面层细胞,即为单个核淋巴细胞,Hanks液洗涤两次,台盼兰拒染法鉴定活细胞数大于95%,RPMI1640培养液调细胞浓度至2×105/mL,丝裂霉素C(25μg/mL)37℃水浴45min灭活细胞,PBS洗涤3次,调细胞浓度至2×105/mL。同样方法制备小鼠脾淋巴细胞悬液,调细胞浓度至2×105/mL,分装于100mL的培养瓶内(5mL/瓶),不行丝裂霉素处理。
1.4制备T细胞疫苗
灭活大鼠脾淋巴细胞5mL接种于小鼠淋巴细胞培养瓶内,混合后加入ConA2.5μg/mL,37℃,95%O2,5%CO2孵箱中培养48h 后收集细胞,然后加入丝裂霉素(25μg/mL)处理灭活细胞(37℃水浴45min),RPMI1640培养液调细胞浓度至1×107/mL,制成供者特异性T淋巴细胞疫苗。
1.5小鼠Ⅰ型糖尿病模型的制备
将链脲霉素(STZ)用0.1mmol/l无菌枸橼酸-枸橼酸钠缓冲液配成2%溶液,调节PH4.5,滤菌器除菌。小鼠过夜禁食12h,按160mg/kg体重一次性腹腔注射STZ溶液,24h内随机测血糖≥16.5mmol/l,稳定一周即可视为造模成功,作为受体。
1.6胰岛细胞的获取和分离纯化
麻醉大鼠逆行注射胶原酶V10mL充盈胰腺组织,迅速完整取下胰腺组织,进一步去除脂肪和血凝块,加体积两倍的胶原酶V在37℃水浴锅内震荡消化15min,胎牛血清终止反应后100目不锈钢网过滤,Hanks液和1640液洗涤离心,用Ficoll400液不连续密度梯度离心纯化胰岛细胞后重悬于20mL完全PPMI1640培养液中,37℃,95%O2,5%CO2条件下培养24h。
1.7实验分组
T细胞疫苗免疫接种及异种胰岛细胞移植 将Ⅰ型糖尿病模型小鼠随机分为四组,每组8只,A组糖尿病小鼠,B组糖尿病小鼠+胰岛细胞+1640培养液(对照组),C组糖尿病小鼠+胰岛细胞+T细胞疫苗(实验组),胰岛移植前实验组注射TCV(1×107/mL、0.2mL/次、1次/周),连续3周,B组用1640液替代。第3次免疫后7d,B、C组经门静脉穿刺按1200～1500IEQ/kg导入大鼠胰岛细胞。
1.8单向混合淋巴细胞反应(MTT法)
接种前和每次接种后第5天进行单向混合淋巴细胞反应,Balb/c小鼠的脾细胞作为反应细胞,丝裂霉素处理的Wistar大鼠脾细胞作为刺激细胞,两者细胞浓度均为1×106/mL。取96孔板,每孔加入刺激细胞和反应细胞各100μl,每份标本设3个复孔,在孵育箱中培养72h,终止前4h每孔加入MTT10μl,培养结束后加入二甲基亚砜150μl,37℃下振荡20min,用酶标仪测每孔OD570值计算抑制率;抑制率=(1-实验组OD/对照组OD)×100。
1.9小鼠血糖和体重的检测
胰岛细胞经门静脉移植导入小鼠体内,每日相同时间点测空腹血糖,以非禁食血糖值≤10mmol/判定为移植物存活,移植有效;若连续2次血糖值≥11.1mmol/l为移植物排斥,以第一次的时间为排斥时间。每周相同时间点观察小鼠的体重变化。
1.10统计学分析
数据以均数±标准差(χ±s)表示,实验数据进行t检验、方差分析,检验水准α=0.05,P0.05),免疫后TCV组相同时间点同空白对照组比较,平均OD值有明显差异(P0.05)。见表2。
2.3移植组有效控制血糖天数的比较
t检验结果示:C组与B组比较(P [参考文献]
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(收稿日期:2009-10-23)
