王弘珺，李質馨，田洪艷，徐冶，劉忠平

NOD小鼠糖尿病早期Th1細胞與CD4+CD25+Treg細胞的變化

王弘珺，李質馨，田洪艷，徐冶，劉忠平

目的研究Th1細胞和CD4+CD25+Treg細胞在NOD小鼠糖尿病早期的變化，并評價其作用。方法選擇4周(A組)、8周(B組)和16周齡(C組)的雌性NOD小鼠，取脾、胸腺和胰腺組織，采用流式細胞術測定脾Th1和CD4+CD25+Treg細胞的比例，計算Th1/CD4+T、CD4+CD25+Treg/CD4+T和Th1/CD4+CD25+Treg的比值，再測定胸腺CD4–CD8–T、CD4+CD8+T、CD4–CD8+T和CD4+CD8–T細胞比例，計算CD25+Treg/CD4+CD8–T的比值。取胰腺組織，行HE染色和Foxp3免疫組化染色，觀察胰腺病理學變化。結果C組小鼠脾臟Th1細胞比例以及Th1/CD4+T和Th1/ CD4+CD25+Treg比值明顯高于A組和B組，但是A、B、C三組脾臟CD4+CD25+Treg細胞比例及CD4+CD25+Treg/CD4+T比值差異無統(tǒng)計學意義。三組間胸腺CD4–CD8–T、CD4+CD8+T、CD4–CD8+T和CD4+CD8–T細胞比例差異無統(tǒng)計學意義，但是B組和C組胸腺CD25+Treg/CD4+CD8–T比值明顯高于A組。HE染色結果顯示，B組和C組的胰島周圍可見淋巴細胞浸潤，但胰島周圍淋巴細胞浸潤部位免疫組化染色未見Foxp3陽性細胞。結論NOD小鼠糖尿病早期外周Th1細胞呈進行性增加，但CD4+CD25+Treg細胞相對缺乏，考慮與NOD小鼠糖尿病進展有關。

小鼠，近交NOD；Th1細胞；CD4+CD25+調節(jié)性T細胞；糖尿病，1型

1型糖尿病是由于T細胞進行性損傷胰島β 細胞引起的自身免疫性疾病[1]。NOD小鼠是1型糖尿病的動物模型，80%的雌性NOD小鼠在出生12周后發(fā)生自發(fā)性糖尿病。研究表明，隨年齡增加，NOD小鼠CD4+CD25+Treg細胞抑制功能明顯降低[2-5]。1型糖尿病患者IFN-γ分泌增加，Th1細胞活化增多[6]。但1型糖尿病發(fā)生是否與Th1和CD4+CD25+Treg細胞的失衡相關目前尚不完全清楚。據此，本研究選擇4、8、16周齡NOD小鼠，觀察Th1和CD4+CD25+Treg細胞的變化以及Th1/ CD4+CD25+Treg細胞平衡的改變，探討其與1型糖尿病發(fā)生的關系。

1 材料與方法

1.1 實驗動物及試劑 SPF級NOD雌性小鼠由上海斯萊克實驗動物有限責任公司提供，4周齡小鼠為A組，8周齡小鼠為B組，16周齡小鼠為C組，每組8只(4只用于流式細胞儀檢測，4只用于形態(tài)學染色)?？剐∈驝D4-Cy5抗體、抗CD25-PE抗體、抗IFNγ-PE抗體、抗CD8-PE抗體、抗CD25-FITC抗體(BD公司)，抗小鼠FcR單抗(自制)，兔抗小鼠Foxp3抗體(Santa Cruz公司)，免疫組化試劑盒(北京中杉金橋生物技術有限公司)，離子霉素(ionomycin)和佛波酯(PMA，Sigma公司)，蛋白轉運抑制劑GolgiStop、流式細胞儀(BD公司)。

1.2 方法

1.2.1 脾和胸腺流式細胞樣品的制備 小鼠麻醉后，分別取脾和胸腺，用毛玻璃片擠壓，過200目尼龍網制成單細胞懸液，用紅細胞裂解液(17mmol/ L Tris-HCl，140mmol/L NH4Cl，pH7.2)去除紅細胞，懸浮于PBS中，計數。

1.2.2 脾CD4+IFN-γ+T細胞的測定 取2×106個細胞加入50ng/ml PMA、500ng/ml Ionomycin和4μl/ml GolgiStop，培養(yǎng)4～6h，回收細胞。加入抗FcR單抗和抗CD4-Cy5抗體，4℃避光染色30min，抗IFNγ-PE抗體，染色過程按照BD說明書進行，流式細胞儀檢測。

1.2.3 脾CD4+CD25+Treg細胞的測定 取1×106個細胞，加入抗FcR單抗、抗CD4-Cy5抗體、抗CD25-PE抗體，4℃避光染色30min，流式細胞儀檢測。

1.2.4 胸腺CD4+CD8–CD25+T細胞的測定 取 1×106個細胞，加入抗FcR單抗、抗CD4-Cy5抗體、抗CD8-PE抗體、抗CD25-FITC抗體，4℃避光染色30min，流式細胞儀檢測。

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，常規(guī)脫水、透明，石蠟包埋，切片，HE染色，封片，光鏡下觀察。

1.2.6 胰腺Foxp3免疫組織化學染色 取胰腺行常規(guī)石蠟切片，梯度乙醇脫水，抗原修復；雙蒸水洗滌，3%H2O2孵育10min，PBS洗滌；山羊血清室溫封閉10min；加1:100稀釋兔抗小鼠Foxp3抗體，4℃過夜，PBS洗滌；加入生物素標記的山羊抗兔IgG抗體室溫孵育10min，PBS洗滌；加入鏈霉菌抗生物素蛋白-過氧化物酶室溫孵育10min，PBS洗滌；DAB顯色2min，脫水透明封片。陰性對照用PBS代替一抗。

1.3 統(tǒng)計學處理 采用SPSS 11.5統(tǒng)計軟件，結果用±s表示，組間比較采用單因素方差分析，進一步兩兩比較采用SNK-q檢驗，P＜0.05為差異有統(tǒng)計學意義。

2 結 果

2.1 脾流式細胞儀檢測結果 A、B、C組NOD小鼠脾Th1細胞和CD4+CD25+Treg細胞占總淋巴細胞的比例及Th1/CD4+T、CD4+CD25+Treg/CD4+T和Th1/CD4+CD25+Treg比值見表1。C組的Th1細胞比例、Th1/CD4+T比值和Th1/CD4+CD25+Treg比值明顯高于A組和B組，差異有統(tǒng)計學意義(P＜0.01)。但各組間CD4+CD25+Treg細胞比例和CD4+CD25+Treg/ CD4+T比值差異均無統(tǒng)計學意義(P＞0.05)。

表1 3組脾Th1細胞和CD4+CD25+Treg細胞的比較(±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組脾Th1細胞和CD4+CD25+Treg細胞的比較(±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 胸腺流式細胞儀檢測結果 A、B、C組NOD小鼠胸腺CD4–CD8–T細胞、CD4+CD8+T細胞、CD4–CD8+T細胞、CD4+CD8–T細胞比例及CD25+Treg/CD4+CD8–T比值見表2。其中CD4–CD8–T細胞、CD4+CD8+T細胞、CD4–CD8+T細胞和CD4+CD8–T細胞比例各組間差異均無統(tǒng)計學意義(P＞0.05)，但B組及C組CD25+Treg/CD4+CD8–T比值明顯高于A組，差異有統(tǒng)計學意義(P＜0.05或P＜0.01)。

表2 3組胸腺內不同表型T細胞的比較(±s, n=4)Tab.2 Comparison of thymus T cell phenotypes in three groups of mice (±s, n=4)

表2 3組胸腺內不同表型T細胞的比較(±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 胰腺組織學變化 HE染色結果顯示，A組NOD小鼠胰島組織結構正常，B組NOD小鼠部分胰島內可見少量淋巴細胞浸潤，C組NOD小鼠胰島內及其周圍胰腺組織內可見大量淋巴細胞浸潤(圖1)。

圖1 三組NOD小鼠胰腺組織學變化(HE ×200)Fig.1 Histological changes of pancreas in three groups of NOD mice (HE ×200)

2.4 胰腺Foxp3免疫組化染色結果 A組NOD小鼠胰島周圍未見淋巴細胞浸潤；B組和C組NOD小鼠胰島周圍可見淋巴細胞浸潤，但未見Foxp3陽性細胞(圖2)。

圖2 三組NOD小鼠胰腺Foxp3免疫組化(DAB ×400)Fig.2 Immunohistochemisty of Foxp3 in three groups of NOD mice (DAB ×400)

3 討 論

自身免疫耐受失調和Th1細胞增多是引起NOD小鼠糖尿病的重要原因。Th1細胞分泌的炎性細胞因子如IFN-γ、TNF-α和IL-1等破壞胰島β細胞引起胰島β細胞凋亡[7-8]，使胰島素分泌降低，從而出現(xiàn)糖尿病癥狀。研究表明，糖尿病NOD小鼠CD4+CD25+Treg細胞存在數目和功能缺陷，不能維持機體對自身抗原的免疫耐受，最終促進糖尿病的進展。

在本研究中，4周和8周齡的NOD小鼠脾Th1細胞比例已經明顯增加，而脾CD4+CD25+Treg細胞無明顯變化；16周齡的NOD小鼠脾Th1細胞比例升高至4周時的3倍以上，而CD4+CD25+Treg細胞比例則有降低趨勢。提示在糖尿病早期，外周Th1細胞比例增加，而CD4+CD25+Treg細胞相對缺乏。

本研究結果顯示，16周齡NOD小鼠脾CD4+CD25+Treg細胞比例雖然沒有明顯降低，但是Th1細胞比例已經接近CD4+CD25+Treg細胞的2倍。HE染色顯示，部分胰島周圍可見明顯的淋巴細胞浸潤，但免疫組化染色在淋巴細胞浸潤區(qū)域未見Foxp3+T細胞，表明NOD小鼠糖尿病早期胰島周圍缺乏CD4+CD25+Treg細胞。提示在糖尿病早期NOD小鼠外周CD4+CD25+Treg細胞相對缺乏，不能抑制Th1細胞的增殖，Th1細胞進行性損傷胰島β細胞，從而導致糖尿病的發(fā)生。

從4周、8周到16周齡，NOD小鼠胸腺CD4–CD8–T、CD4+CD8+T、CD4+CD8–T和CD4–CD8+T細胞比例均未見明顯變化。NOD小鼠胸腺CD4+CD8–T細胞中CD4+CD25+Treg細胞比例隨年齡呈進行性增加，與正常小鼠無明顯差異，說明NOD小鼠胸腺內CD4+CD25+Treg產生正常，外周CD4+CD25+Treg細胞的相對缺乏并非由于胸腺產生減少所引起。有研究表明，IL-2對維持CD4+CD25+Treg細胞穩(wěn)定有重要作用，糖尿病早期外周CD4+CD25+Treg細胞減少可能與IL-2缺乏有關[9]。此外，NOD小鼠糖尿病中后期CD4+CD25+Treg細胞可能轉化為效應性T細胞[10-11]。但導致16周齡NOD小鼠脾CD4+CD25+Treg細胞相對缺乏的原因尚需進一步研究。

總之，NOD小鼠糖尿病是由多因素引起的，而糖尿病早期Th1細胞進行性增加伴隨CD4+CD25+Treg細胞數目和功能的相對缺陷與糖尿病進展密切相關。mTOR信號通路是控制Th1細胞和CD4+CD25+Treg細胞分化的關鍵[12-14]，調控該信號通路將在1型糖尿病治療中起重要作用。

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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)

(責任編輯：張小利)

吉林省教育廳“十一五”科學技術研究項目(2008401)；吉林省科技發(fā)展計劃項目(20130101140JC)

王弘珺，醫(yī)學博士，副教授。主要從事免疫學方面的研究

132013 吉林省吉林市 吉林醫(yī)藥學院組織與胚胎學教研室(王弘珺、李質馨、田洪艷、徐冶、劉忠平)

李質馨，E-mail：lzx-62@163.com