NOD小鼠糖尿病早期Th1细胞与CD4+CD25+Treg细胞的变化

王弘珺，李质馨，田洪艳，徐冶，刘忠平

NOD小鼠糖尿病早期Th1细胞与CD4+CD25+Treg细胞的变化

王弘珺，李质馨，田洪艳，徐冶，刘忠平

目的研究Th1细胞和CD4+CD25+Treg细胞在NOD小鼠糖尿病早期的变化，并评价其作用。方法选择4周(A组)、8周(B组)和16周龄(C组)的雌性NOD小鼠，取脾、胸腺和胰腺组织，采用流式细胞术测定脾Th1和CD4+CD25+Treg细胞的比例，计算Th1/CD4+T、CD4+CD25+Treg/CD4+T和Th1/CD4+CD25+Treg的比值，再测定胸腺CD4–CD8–T、CD4+CD8+T、CD4–CD8+T和CD4+CD8–T细胞比例，计算CD25+Treg/CD4+CD8–T的比值。取胰腺组织，行HE染色和Foxp3免疫组化染色，观察胰腺病理学变化。结果C组小鼠脾脏Th1细胞比例以及Th1/CD4+T和Th1/ CD4+CD25+Treg比值明显高于A组和B组，但是A、B、C三组脾脏CD4+CD25+Treg细胞比例及CD4+CD25+Treg/CD4+T比值差异无统计学意义。三组间胸腺CD4–CD8–T、CD4+CD8+T、CD4–CD8+T和CD4+CD8–T细胞比例差异无统计学意义，但是B组和C组胸腺CD25+Treg/CD4+CD8–T比值明显高于A组。HE染色结果显示，B组和C组的胰岛周围可见淋巴细胞浸润，但胰岛周围淋巴细胞浸润部位免疫组化染色未见Foxp3阳性细胞。结论NOD小鼠糖尿病早期外周Th1细胞呈进行性增加，但CD4+CD25+Treg细胞相对缺乏，考虑与NOD小鼠糖尿病进展有关。

小鼠，近交NOD；Th1细胞；CD4+CD25+调节性T细胞；糖尿病，1型

1型糖尿病是由于T细胞进行性损伤胰岛β 细胞引起的自身免疫性疾病[1]。NOD小鼠是1型糖尿病的动物模型，80%的雌性NOD小鼠在出生12周后发生自发性糖尿病。研究表明，随年龄增加，NOD小鼠CD4+CD25+Treg细胞抑制功能明显降低[2-5]。1型糖尿病患者IFN-γ分泌增加，Th1细胞活化增多[6]。但1型糖尿病发生是否与Th1和CD4+CD25+Treg细胞的失衡相关目前尚不完全清楚。据此，本研究选择4、8、16周龄NOD小鼠，观察Th1和CD4+CD25+Treg细胞的变化以及Th1/ CD4+CD25+Treg细胞平衡的改变，探讨其与1型糖尿病发生的关系。

1 材料与方法

1.1 实验动物及试剂 SPF级NOD雌性小鼠由上海斯莱克实验动物有限责任公司提供，4周龄小鼠为A组，8周龄小鼠为B组，16周龄小鼠为C组，每组8只(4只用于流式细胞仪检测，4只用于形态学染色)。抗小鼠CD4-Cy5抗体、抗CD25-PE抗体、抗IFNγ-PE抗体、抗CD8-PE抗体、抗CD25-FITC抗体(BD公司)，抗小鼠FcR单抗(自制)，兔抗小鼠Foxp3抗体(Santa Cruz公司)，免疫组化试剂盒(北京中杉金桥生物技术有限公司)，离子霉素(ionomycin)和佛波酯(PMA，Sigma公司)，蛋白转运抑制剂GolgiStop、流式细胞仪(BD公司)。

1.2 方法

1.2.1 脾和胸腺流式细胞样品的制备 小鼠麻醉后，分别取脾和胸腺，用毛玻璃片挤压，过200目尼龙网制成单细胞悬液，用红细胞裂解液(17mmol/ L Tris-HCl，140mmol/L NH4Cl，pH7.2)去除红细胞，悬浮于PBS中，计数。

1.2.2 脾CD4+IFN-γ+T细胞的测定 取2×106个细胞加入50ng/ml PMA、500ng/ml Ionomycin和4μl/ml GolgiStop，培养4～6h，回收细胞。加入抗FcR单抗和抗CD4-Cy5抗体，4℃避光染色30min，抗IFNγ-PE抗体，染色过程按照BD说明书进行，流式细胞仪检测。

1.2.3 脾CD4+CD25+Treg细胞的测定 取1×106个细胞，加入抗FcR单抗、抗CD4-Cy5抗体、抗CD25-PE抗体，4℃避光染色30min，流式细胞仪检测。

1.2.4 胸腺CD4+CD8–CD25+T细胞的测定 取 1×106个细胞，加入抗FcR单抗、抗CD4-Cy5抗体、抗CD8-PE抗体、抗CD25-FITC抗体，4℃避光染色30min，流式细胞仪检测。

1.2.5 胰腺HE染色 小鼠麻醉后，取胰腺置于4%多聚甲醛中固定，常规脱水、透明，石蜡包埋，切片，HE染色，封片，光镜下观察。

1.2.6 胰腺Foxp3免疫组织化学染色 取胰腺行常规石蜡切片，梯度乙醇脱水，抗原修复；双蒸水洗涤，3%H2O2孵育10min，PBS洗涤；山羊血清室温封闭10min；加1:100稀释兔抗小鼠Foxp3抗体，4℃过夜，PBS洗涤；加入生物素标记的山羊抗兔IgG抗体室温孵育10min，PBS洗涤；加入链霉菌抗生物素蛋白-过氧化物酶室温孵育10min，PBS洗涤；DAB显色2min，脱水透明封片。阴性对照用PBS代替一抗。

1.3 统计学处理 采用SPSS 11.5统计软件，结果用±s表示，组间比较采用单因素方差分析，进一步两两比较采用SNK-q检验，P＜0.05为差异有统计学意义。

2 结 果

2.1 脾流式细胞仪检测结果 A、B、C组NOD小鼠脾Th1细胞和CD4+CD25+Treg细胞占总淋巴细胞的比例及Th1/CD4+T、CD4+CD25+Treg/CD4+T和Th1/CD4+CD25+Treg比值见表1。C组的Th1细胞比例、Th1/CD4+T比值和Th1/CD4+CD25+Treg比值明显高于A组和B组，差异有统计学意义(P＜0.01)。但各组间CD4+CD25+Treg细胞比例和CD4+CD25+Treg/ CD4+T比值差异均无统计学意义(P＞0.05)。

表1 3组脾Th1细胞和CD4+CD25+Treg细胞的比较(±s, n=4)Tab.1 Th1 cells and CD4+CD25+Treg cells in spleens in three groups of mice (±s, n=4)

表1 3组脾Th1细胞和CD4+CD25+Treg细胞的比较(±s, n=4)Tab.1 Th1 cells and CD4+CD25+Treg cells in spleens in three groups of mice (±s, n=4)

(1)P＜0.01 compared with group A; (2)P＜0.01 compared with group B

Group Th1(%) CD4+CD25+Treg(%) Th1/CD4+T CD4+CD25+Treg/CD4+T Th1/CD4+CD25+Treg A 0.920±0.028 1.843±0.012 2.260±0.136 7.673±0.544 0.499±0.015 B 1.420±0.104 1.747±0.069 2.970±0.212 6.960±0.161 0.818±0.081 C 2.947±0.219(1)(2) 1.630±0.166 6.370±0.368(1)(2) 6.710±0.626 1.828±0.165(1)(2)

2.2 胸腺流式细胞仪检测结果 A、B、C组NOD小鼠胸腺CD4–CD8–T细胞、CD4+CD8+T细胞、CD4–CD8+T细胞、CD4+CD8–T细胞比例及CD25+Treg/CD4+CD8–T比值见表2。其中CD4–CD8–T细胞、CD4+CD8+T细胞、CD4–CD8+T细胞和CD4+CD8–T细胞比例各组间差异均无统计学意义(P＞0.05)，但B组及C组CD25+Treg/CD4+CD8–T比值明显高于A组，差异有统计学意义(P＜0.05或P＜0.01)。

表2 3组胸腺内不同表型T细胞的比较(±s, n=4)Tab.2 Comparison of thymus T cell phenotypes in three groups of mice (±s, n=4)

表2 3组胸腺内不同表型T细胞的比较(±s, n=4)Tab.2 Comparison of thymus T cell phenotypes in three groups of mice (±s, n=4)

(1)P＜0.05, (2)P＜0.01 compared with group A; (3)P＜0.01 compared with group B

Group CD4–CD8–T(%) CD4+CD8+T(%) CD4–CD8+T(%) CD4+CD8–T(%) CD25+Treg/CD4+CD8–T A 2.900±0.340 79.33±1.220 4.717±0.570 13.03±0.463 2.360±0.187 B 2.457±0.107 82.47±0.696 3.473±0.127 11.60±0.473 4.430±0.162(1)2.677±0.075 80.17±0.745 3.427±0.168 13.73±0.664 5.090±0.280(2)(3)C

2.3 胰腺组织学变化 HE染色结果显示，A组NOD小鼠胰岛组织结构正常，B组NOD小鼠部分胰岛内可见少量淋巴细胞浸润，C组NOD小鼠胰岛内及其周围胰腺组织内可见大量淋巴细胞浸润(图1)。

图1 三组NOD小鼠胰腺组织学变化(HE ×200)Fig.1 Histological changes of pancreas in three groups of NOD mice (HE ×200)

2.4 胰腺Foxp3免疫组化染色结果 A组NOD小鼠胰岛周围未见淋巴细胞浸润；B组和C组NOD小鼠胰岛周围可见淋巴细胞浸润，但未见Foxp3阳性细胞(图2)。

图2 三组NOD小鼠胰腺Foxp3免疫组化(DAB ×400)Fig.2 Immunohistochemisty of Foxp3 in three groups of NOD mice (DAB ×400)

3 讨 论

自身免疫耐受失调和Th1细胞增多是引起NOD小鼠糖尿病的重要原因。Th1细胞分泌的炎性细胞因子如IFN-γ、TNF-α和IL-1等破坏胰岛β细胞引起胰岛β细胞凋亡[7-8]，使胰岛素分泌降低，从而出现糖尿病症状。研究表明，糖尿病NOD小鼠CD4+CD25+Treg细胞存在数目和功能缺陷，不能维持机体对自身抗原的免疫耐受，最终促进糖尿病的进展。

在本研究中，4周和8周龄的NOD小鼠脾Th1细胞比例已经明显增加，而脾CD4+CD25+Treg细胞无明显变化；16周龄的NOD小鼠脾Th1细胞比例升高至4周时的3倍以上，而CD4+CD25+Treg细胞比例则有降低趋势。提示在糖尿病早期，外周Th1细胞比例增加，而CD4+CD25+Treg细胞相对缺乏。

本研究结果显示，16周龄NOD小鼠脾CD4+CD25+Treg细胞比例虽然没有明显降低，但是Th1细胞比例已经接近CD4+CD25+Treg细胞的2倍。HE染色显示，部分胰岛周围可见明显的淋巴细胞浸润，但免疫组化染色在淋巴细胞浸润区域未见Foxp3+T细胞，表明NOD小鼠糖尿病早期胰岛周围缺乏CD4+CD25+Treg细胞。提示在糖尿病早期NOD小鼠外周CD4+CD25+Treg细胞相对缺乏，不能抑制Th1细胞的增殖，Th1细胞进行性损伤胰岛β细胞，从而导致糖尿病的发生。

从4周、8周到16周龄，NOD小鼠胸腺CD4–CD8–T、CD4+CD8+T、CD4+CD8–T和CD4–CD8+T细胞比例均未见明显变化。NOD小鼠胸腺CD4+CD8–T细胞中CD4+CD25+Treg细胞比例随年龄呈进行性增加，与正常小鼠无明显差异，说明NOD小鼠胸腺内CD4+CD25+Treg产生正常，外周CD4+CD25+Treg细胞的相对缺乏并非由于胸腺产生减少所引起。有研究表明，IL-2对维持CD4+CD25+Treg细胞稳定有重要作用，糖尿病早期外周CD4+CD25+Treg细胞减少可能与IL-2缺乏有关[9]。此外，NOD小鼠糖尿病中后期CD4+CD25+Treg细胞可能转化为效应性T细胞[10-11]。但导致16周龄NOD小鼠脾CD4+CD25+Treg细胞相对缺乏的原因尚需进一步研究。

总之，NOD小鼠糖尿病是由多因素引起的，而糖尿病早期Th1细胞进行性增加伴随CD4+CD25+Treg细胞数目和功能的相对缺陷与糖尿病进展密切相关。mTOR信号通路是控制Th1细胞和CD4+CD25+Treg细胞分化的关键[12-14]，调控该信号通路将在1型糖尿病治疗中起重要作用。

【参考文献】

[1] Santamaria P. The long and winding road to understanding and conquering type 1 diabetes[J]. Immunity, 2010, 32(4): 437-445.

[2] Bluestone JA, Tang Q, Sedwick CE. T regulatory cells in autoimmune diabetes: past challenges, future prospects[J]. J Clin Immunol, 2008, 28(6): 677-684.

[3] Feuerer M, Shen Y, Littman DR, et al. How punctual ablation of regulatory T cells unleashes an autoimmune lesion within the pancreatic islets[J]. Immunity, 2009, 31(4): 654-664.

[4] Zhang M, Xu SH, Mao XD, et al. Dynamic change of CD4+CD25+T cells in non-obese diabetic mice under natural condition[J]. Curr Immunol, 2008, 28(6): 479-482. [张梅, 徐书杭, 茅晓东, 等. NOD小鼠自然状态下CD4+CD25+T细胞的动态变化及意义[J]. 现代免疫学, 2008, 28(6): 479-482.]

[5] Li WP, Shen Y, Hu YQ, et al. Changes of function of CD4+CD25+T regulatory cells in NOD mice[J]. J Bengbu Med Coll, 2008, 33(6): 743-746. [李卫鹏, 申勇, 胡永全, 等.NOD小鼠CD4+CD25+调节性T细胞功能变化研究[J]. 蚌埠医学院学报, 2008, 33(6): 743-746.]

[6] Zhi DJ, Wang XC, Shen SX, et al. Immune responses of Th1/ Th2 in type 1 diabetic children[J]. Chin J Pediatr, 2001, 39(3): 148-150. [支涤静, 王晓川, 沈水仙, 等. 儿童1型糖尿病Th1/ Th2免疫应答状况研究[J]. 中华儿科杂志, 2001, 39(3): 148-150.]

[7] Cnop M, Welsh N, Jonas JC, et al. Mechanisms of pancreatic beta-cell death in type 1 and type 2 diabetes: many differences, few similarities[J]. Diabetes, 2005, 54(Suppl 2): S97-S107.

[8] Brusko TM, Wasserfall CH, Clare-Salzler MJ, et al. Functional defects and the influence of age on the frequency of CD4+CD25+T cells in type 1 diabetes[J]. Diabetes, 2005, 54(5): 1407-1414.

[9] Tang Q, Adams JY, Penaranda C, et al. Central role of defective interleukin-2 production in the triggering of islet autoimmune destruction[J]. Immunity, 2008, 28(5): 687-697.

[10] Zhou X, Bailey-Bucktrout SL, Jeker LT, et al. Instability of the transcription factor Foxp3 leads to the generation of pathogenic memory T cells in vivo[J]. Nat Immunol, 2009, 10(9): 1000-1007.

[11] Zhou X, Bailey-Bucktrout S, Jeker LT, et al. Plasticity of CD4(+) FoxP3(+)T cells[J]. Curr Opin Immunol, 2009, 21(3):281-285.

[12] Delgoffe GM, Kole TP, Zheng Y, et al. The mTOR kinase differentially regulates effector and regulatory T cell lineage commitment[J]. Immunity, 2009, 30(6): 832-844.

[13] Liu G, Yang K, Burns S, et al. The S1P(1)-mTOR axis directs the reciprocal differentiation of T(H)1 and T(reg) cells[J]. Nat Immunol, 2010, 11(11): 1047-1056.

[14] Monti P, Scirpoli M, Maffi P, et al. Rapamycin monotherapy in patients with type 1 diabetes modifies CD4+CD25+FOXP3+regulatory T-cells[J]. Diabetes, 2008, 57(9): 2341-2347.

Changes in Th1 cells and CD4+CD25+Treg cells in non-obese diabetic mice at early stage of diabetes

WANG Hong-jun, LI Zhi-xin*, TIAN Hong-yan, XU Ye, LIU Zhong-ping
Department of Histology and Embryology, Jilin Medical College, Jilin City, Jilin Province 132013, China
*

, E-mail: lzx-62@163.com
This work was supported by the “Eleven Five-Year” Science and Technology Research Project of Jilin Provincial Education Department (2008401) and the Science and Technology Development Program of Jilin Province(20130101140JC)

ObjectiveTo investigate the changes in Th1 cells and CD4+CD25+Treg cells in non-obese diabetic (NOD) mice at early stage of diabetes, and to evaluate the significance of these changes.MethodsFour week- (group A), 8 week- (group B) and 16 week-old (group C) female NOD mice (8 each) were used in present study. The spleen, thymus and pancreas were harvested. Th1 and CD4+CD25+Treg cells in spleen were determined by flow cytometer, and the ratios of Th1/CD4+T, CD4+CD25+Treg/ CD4+T and Th1/CD4+CD25+Treg were calculated. Subsequently, CD4–CD8–T, CD4+CD8+T, CD4–CD8+T and CD4+CD8–T cells in thymus were determined by flow cytometer, and the ratio of CD25+Treg/CD4+CD8–T was calculated. The histopathological changes in pancreas were also evaluated by HE staining and immunohistochemistry staining.ResultsThe proportion of Th1 cells in spleen and the ratios of Th1/CD4+T and Th1/CD4+CD25+Treg were higher significantly in group C than in group A and B. However, no significant differences were found in the proportion of spleen CD4+CD25+Treg cells and the ratio of CD4+CD25+Treg/CD4+T among the three groups. Compared with group A, no obvious changes were found in thymus CD4–CD8–T, CD4+CD8+T, CD4–CD8+T and CD4+CD8–T cells in group B and C, but the ratio of thymus CD25+Treg/CD4+CD8–T increased significantly in group B and C. Lymphocytic infiltration was observed in pancreatic islets of group B and C as shown with HE staining, but Foxp3+T cells were not seen in pancreatic islets by immunohistochemistry.ConclusionTh1 cells are gradually increased at early stage of diabetes in NOD mice, but CD4+CD25+Treg cells are relatively default. These changes may play an important role in the progress of diabetes.

mice, inbred NOD; Th1 cells; CD4+CD25+Treg cells; diabetes mellitus, type 1

R392.11

A

0577-7502(2013)11-0888-04

10.11855/j.issn.0577-7402.2013.11.004

2013-04-24；

2013-08-18)

(责任编辑：张小利)

吉林省教育厅“十一五”科学技术研究项目(2008401)；吉林省科技发展计划项目(20130101140JC)

王弘珺，医学博士，副教授。主要从事免疫学方面的研究

132013 吉林省吉林市 吉林医药学院组织与胚胎学教研室(王弘珺、李质馨、田洪艳、徐冶、刘忠平)

李质馨，E-mail：lzx-62@163.com