Ⅱ型糖尿病獼猴部分靶器官中Th1/Th2型細胞因子表達分析

陳姍姍1?, 羅啟慧1?, 曾文2, 程安春1,3, 劉文濤1, 史良琴1, 陳正禮1,2*

( 1.四川農(nóng)業(yè)大學動物醫(yī)學院,動物疾病與人類健康中心四川省重點實驗室,四川 雅安625014;2.四川普萊美生物科技有限公司/

國家實驗獼猴種源基地,四川 雅安625014;3.四川農(nóng)業(yè)大學預防獸醫(yī)研究所,成都611130)

摘要檢測Ⅱ型糖尿病獼猴部分靶器官中Ⅰ型輔助T細胞(Th1)因子IL-2和IFN-γ以及Ⅱ型輔助T細胞(Th2)因子IL-4、IL-10的表達及分布變化情況,研究Th1/Th2型細胞因子在Ⅱ型糖尿病發(fā)病中的變化。?、蛐吞悄虿〖敖】但J猴胰腺、肝、腎和心臟,通過石蠟切片、常規(guī)染色觀察病理變化,同時采用免疫組織化學SABC法檢測各靶器官IL-2、IFN-γ、IL-4和IL-10的表達及分布情況。病理結果顯示：糖尿病獼猴肝血竇增寬伴中性粒細胞浸潤,腎、心臟和胰腺細胞呈不同程度腫脹、萎縮及壞死。免疫組織化學結果顯示：在腎中Th1型細胞因子IFN-γ表達水平高于健康組(P<0.01)并分布于近曲小管、遠曲小管及集合管。胰腺中IFN-γ表達水平與健康組相比差異無統(tǒng)計學意義(P>0.05),陽性產(chǎn)物分布于胰島部及外分泌部。胰腺及腎中Th1型細胞因子IL-2表達水平與健康組相比差異無統(tǒng)計學意義(P>0.05)。胰腺及腎中Th2型細胞因子IL-4表達水平顯著低于健康組(P<0.01),陽性產(chǎn)物分布于遠曲小管、胰腺胰島及外分泌部。在胰腺、腎、肝及心臟中,Th2型細胞因子IL-10表達水平顯著高于健康組(P<0.01),陽性產(chǎn)物分布于近曲小管、遠曲小管、胰腺胰島、胰腺外分泌部、心肌細胞及肝細胞的胞質中。Th1/Th2型細胞因子在Ⅱ型糖尿病(type 2 diabetes mellitus, T2DM)的發(fā)病過程中發(fā)生了顯著變化。

關鍵詞Th1/Th2; 細胞因子; Ⅱ型糖尿病; 獼猴

中圖分類號R 363.21; S 852.35文獻標志碼A

Expression of Th1/Th2 cytokines in partial target organs of type 2 diabetes mellitus rhesus monkey. Journal of ZhejiangUniversity(Agric. & LifeSci.), 2015,41(3)：302-308

Chen Shanshan1?, Luo Qihui1?, Zeng Wen2, Cheng Anchun1,3, Liu Wentao1, Shi Liangqin1, Chen Zhengli1,2*(1.KeyLaboratoryofAnimalDiseaseandHumanHealthofSichuanProvince,CollegeofVeterinaryMedicine,SichuanAgriculturalUniversity,Ya’an625014,Sichuan,China; 2.SichuanPrimedBiologicalTechnologyCo.,Ltd/NationalExperimentalMacaqueReproduceLaboratory,Ya’an625014,Sichuan,China; 3.InstituteofPreventiveVeterinaryMedicine,SichuanAgriculturalUniversity,Chengdu611130,China)

SummaryType 2 diabetes mellitus (T2DM) is a disease caused by carbohydrate metabolism disturbance, insulin resistance and reduction of insulin. Many studies confirmed that the disequilibrium of Th1/Th2 cells concerned with T2DM. IL-2 and IFN-γ mediate the growth of Th1 cells which induce cellular immunity. IL-4 and IL-10 are key mediators of Th2 cells which play an important role in humoral immunity. The principal goal of the present research is to detect the changes of Th1/Th2 cytokines in the pathogenesis of T2DM.

Five male healthy rhesus monkeys and five male T2DM rhesus monkeys induced by high-fat diets were used in the study. All rhesus monkeys had been checked without any infections such as bacteria and parasite before trials. After induced for 24 months, the fasting plasma glucose (FPG) mean value of all T2DM rhesus monkeys was higher than 7.0 mmol/L, and that of the control rhesus monkeys was below 6.11 mmol/L. Typical symptoms of type 2 diabetes mellitus were also observed in T2DM rhesus monkeys. The FPG met the WHO and ADA standards for type 2 diabetes mellitus.

Animals were scarified after ketamine anesthesia. Target organs (liver, kidney, heart and pancreas) were fixed in paraformaldehyde, embedded in paraffin and sliced. The pathological changes were studied by hematoxylin-eosin staining, and the expressions of IL-2, IFN-γ, IL-4 and IL-10 were observed by immunohistochemistry. Mean density was measured by IPP 6.0 (Image-pro plus 6.0) to evaluate their variation.

Neutrophil cell infiltration which means chronic inflammation was observed in hepatic sinusoid of T2DM rhesus monkeys, and hepatic sinusoid of T2DM was wider than healthy rhesus monkeys. Different degrees of cellular swell and atrophy as well as necrosis were found in kidney, heart and pancreas. All the tested organs in the study were damaged in the process of T2DM.

IL-2, IL-4 and IFN-γ were only expressed in kidney and pancreas, and IL-10 was expressed in all the four organs. IL-2 was only expressed in langerhans islet and kidney tubules, but IFN-γ, IL-4 and IL-10 were expressed in endocrine and exocrine pancreas, kidney tubules. IL-10 was also expressed in hepatic and myocardial cell. The expression of IFN-γ and IL-10 were significantly increased (P<0.01), but IL-4 was decreased oppositely (P<0.01). The expression of IL-2 in pancreas and kidney had no difference between T2DM and health rhesus monkeys (P>0.05).

In conclusion, IL-2, Th1 cytokines, has no significant change, but IFN-γ of Th1 cell is expressed higher in T2DM. Simultaneously, IL-4 and IL-10, Th2 cytokines, change oppositely. Inflammation exists in the process of T2DM. Difference distribution and level of Th1/Th2 cytokines between T2DM and health rhesus monkeys show that Th1/Th2 cell disequilibrium is key mediators in T2DM. According to the complex changes of cytokines of Th1/Th2 cells, it needs further researches to define the relationship of Th1/Th2 balance with T2DM.

Key wordsTh1/Th2; cytokines; type 2 diabetes mellitus; rhesus monkey

Ⅱ型糖尿病(type 2 diabetes mellitus, T2DM)是一種由環(huán)境、遺傳等多因素引起的,以胰島素分泌缺陷和胰島素抵抗為特征的代謝性綜合征。免疫功能紊亂是Ⅱ型糖尿病發(fā)病的重要原因,Marques-Vidal等[1]研究發(fā)現(xiàn),T2DM病人外周血液中部分細胞因子水平較高。大量研究[2-4]證實,T2DM動物及人外周血液中細胞因子水平發(fā)生了不同的變化。其中大部分細胞因子由Th1或Th2細胞分泌,而Th1/Th2平衡失調(diào)與許多疾病的發(fā)生密切相關.Ⅰ型糖尿病和糖尿病視網(wǎng)膜病均存在Th1/Th2向Th1亞群轉化的現(xiàn)象[5-7],提示Th1/Th2平衡失調(diào)可能是導致T2DM發(fā)生的重要因素。不少關于糖尿病及相關并發(fā)癥治療的報道,均觀察到Th1/Th2平衡失調(diào)有所緩解[6,8-11]。Th1細胞以分泌IL-2、IFN-γ為主,介導細胞免疫反應;Th2細胞以分泌IL-4、IL-10為主,介導體液免疫反應。未見細胞免疫及體液免疫在T2DM發(fā)病進程中的關系及T2DM發(fā)病過程中是否存在Th1/Th2平衡漂移的報道,因此T2DM是否存在Th1/Th2平衡漂移具有非常重要的研究價值。

目前,缺乏以獼猴T2DM模型為研究對象的相關報道。因此本實驗通過飼喂高脂飼料,建立川西亞種獼猴T2DM模型。該模型具有血糖規(guī)律、病理特征與在人類糖尿病患者中觀察到的臨床特征較為相似的優(yōu)勢,避免了藥物造模過程中毒副作用強、穩(wěn)定性差的問題,能真正很好地模擬人類糖尿病發(fā)展的長期緩慢進程及由此引起的全身機體變化的問題,科學性較強。在模型建立成功的基礎上,對T2DM動物部分靶器官組織病變,以及Th1細胞分泌的IL-2、IFN-γ,Th2細胞分泌的IL-4、IL-10表達及分布進行觀察,探討T2DM是否存在Th1/Th2平衡漂移,以期為該疾病的治療提供新思路。

1材料與方法

1.1材料

1.1.1實驗動物5只通過高脂飲食誘導建立的川西亞種獼猴T2DM模型,5只健康川西亞種獼猴(獼猴在實驗前檢疫合格,內(nèi)容包括：體檢,2次結核桿菌試驗，寄生蟲、沙門菌、致賀氏菌和B病毒檢查;獼猴單只飼喂,解剖前無外傷)均由四川農(nóng)業(yè)大學實驗動物工程技術中心/國家實驗獼猴種源基地提供[動物使用許可證為SCXK(川)：2013-105]。

T2DM川西亞種獼猴模型符合美國糖尿病學會(ADA)和世界衛(wèi)生組織(WHO)2006年糖尿病診斷標準：典型糖尿病癥狀(多尿、多飲和體質量下降),空腹血糖(fasting plasma glucose,FPG)≥7.0 mmol/L或餐后2 h血糖(2hPG)≥11.1 mmol/L,為糖尿病;7.77 mmol/L <2hPG<11.1 mmol/L時為糖耐量損傷(impaired glucose tolerance,IGT),6.11 mmol/L≤FPG<6.99 mmol/L時為空腹血糖損傷(impaired fasting glucose,IFG),FPG<6.11 mmol/L且2hPG<7.77 mmol/L,為正常,具體指標及空腹血糖變化情況(表1)參見本課題組已有文獻[12-13]報道。實驗中對動物的處置符合中華人民共和國科學技術部《關于善待實驗動物的指導性意見》的規(guī)定。

1.1.2儀器設備石蠟切片機(日本Leica公司),CH20BIMF200光學顯微鏡(日本Olympus公司),Nikon50i-BF生物數(shù)碼顯微鏡(日本Nikon光學株式會社),冰箱,恒溫培養(yǎng)箱等。

1.1.3主要試劑IL-2、IL-10兔抗人多克隆抗體,即用型SABC試劑盒(組成：5% BSA、生物素標志的山羊抗兔IgG和SABC),DAB顯色試劑盒,多聚賴氨酸均購自武漢博士德生物工程有限公司;IL-4、IFN-γ兔抗人多克隆抗體購自北京博奧森生物技術有限公司;0.01 mol/L pH 6.0檸檬酸緩沖液;0.02 mol/L pH 7.2磷酸鹽緩沖液(phosphate buffered saline,PBS)。

1.2方法

1.2.1實驗動物處理于高脂飼料誘導前和誘導后第2、3、5、7、10、11、12、15、16、18、19、20、22和24月，禁食16 h,從后肢股靜脈采血,分析空腹血糖(FPG)濃度。

高脂飲食誘導24個月后,用氯胺酮麻醉動物,經(jīng)頸動脈放血將動物處死并解剖。取出肝、胰腺、腎、心臟固定于4%多聚甲醛中24～48 h。

1.2.2石蠟切片制作及常規(guī)染色取材沖水24 h后用于常規(guī)石蠟組織包埋,玻片經(jīng)多聚賴氨酸處理后,連續(xù)切片,厚為5 μm,每個樣品切21張備用,并進行常規(guī)蘇木精-伊紅法(hematoxylin-eosin,HE)染色。

1.2.3免疫組織化學SABC法切片脫蠟至水后,用檸檬酸緩沖液微波抗原修復,4次,間隔6 min,PBS洗3次;3% H2O2室溫孵育50 min,蒸餾水洗3次,PBS洗1次;5% BSA,37 ℃孵育50 min;IL-2、IL-4、IL-10和IFN-γ兔多克隆抗體稀釋液(1∶200),4 ℃孵育過夜,室溫復溫30 min后,PBS洗4次;生物素化山羊抗兔IgG,37 ℃孵育40 min,PBS洗3次;SABC,37 ℃孵育20 min,PBS洗3次,蒸餾水洗1次;DAB藍色顯色液顯色,脫水、透明、封片。

2結果與分析

2.1空腹血糖濃度變化及主要病理變化

10只獼猴空腹血糖濃度變化詳見表1,可見經(jīng)過24個月高脂飲食誘導后,T2DM組獼猴空腹血糖平均水平已超過7.0 mmol/L,符合WHO和ADA公布的糖尿病判斷標準。

表1　高脂膳食誘導對川西亞種獼猴空腹血糖的影響

T2DM組肝、腎、胰腺及心臟發(fā)生了不同程度的炎性反應。肝：肝血竇增寬伴大量中性粒細胞浸潤,肝細胞腫脹,部分細胞核濃縮壞死。腎：腎小球萎縮,腎小管上皮細胞核濃縮,重度腫脹伴壞死脫落,部分區(qū)域可見大量上皮細胞管型。心臟：心肌細胞萎縮,間質增寬且成分增多。胰腺：胰島細胞中度到重度腫脹、空泡變性,局部細胞溶解壞死,外分泌部細胞核濃縮,細胞萎縮,腺管結構不清晰(圖1).

2.2免疫組織化學染色結果

從圖2～5可以看出,免疫組織化學染色陽性反應呈藍黑色。IL-10在心肌、腎、肝、胰腺中有表達,IL-2、IL-4、IFN-γ只在腎和胰腺中有表達。與健康獼猴相比,T2DM模型獼猴胰腺中IL-4表達水平顯著降低(P<0.01),IL-10顯著升高(P<0.01),而IFN-γ表達差異無統(tǒng)計學意義(P>0.05),三者于胰島部及外分泌部均有表達。腎中,IL-4分布于遠曲小管且表達水平顯著降低(P<0.01),IL-10分布于近曲小管且表達水平顯著升高(P<0.01),而IFN-γ分布于近曲小管、遠曲小管及集合管且表達水平顯著增強(P<0.01)。與胰腺和腎表達水平相似,心臟及肝中IL-10的表達也顯著升高(P<0.01)。同時,IL-2在健康組和糖尿病組間差異無統(tǒng)計學意義(P>0.05),僅表達于胰腺胰島外周、腎近曲小管、遠曲小管及集合管。

圖1　糖尿病獼猴肝、腎、心和胰腺病理變化 Fig.1　Pathological changes of liver, kidney, heart and pancreas in T2DM rhesus monkey

**表示在0.01水平差異有高度統(tǒng)計學意義。 Double asterisk (**) indicate statistically highly significant difference at the 0.01 probability level。 圖2　IFN-γ、IL-2、IL-4和IL-10在Ⅱ型糖尿病獼猴靶器官陽性產(chǎn)物中的平均吸光度 Fig.2　Mean density of IFN-γ, IL-2, IL-4 and IL-10 positive staining in target organ of T2DM rhesus monkey

圖3　IFN-γ、IL-2、IL-4和IL-10在胰腺的表達分布 Fig.3　Expression and distribution of IFN-γ, IL-2, IL-4 and IL-10 in pancreas of T2DM rhesus monkey

圖4　IFN-γ、IL-2、IL-4和IL-10在腎的表達分布 Fig.4　Expression and distribution of IFN-γ, IL-2, IL-4 and IL-10 in kidney of T2DM rhesus monkey

圖5　IL-10在肝和心的表達分布 Fig.5　Expression and distribution of IL-10 in liver and heart of T2DM rhesus monkey

3討論

大量研究表明,Th1/Th2平衡失調(diào)與肥胖誘導的糖尿病及其并發(fā)癥發(fā)生關系密切,是許多疾病的重要致病因素[14]。T2DM的主要致病機制是胰島素抵抗與胰島素分泌受損,免疫功能低下是胰島素抵抗發(fā)生的關鍵因素。病理結果顯示T2DM靶器官發(fā)生了不同程度炎性反應,這與體內(nèi)細胞因子表達分布變化密切相關。同時,肝、心臟、腎及胰腺細胞腫脹壞死的現(xiàn)象證實它們是T2DM發(fā)病過程中受損的靶器官。

干細胞移植治療T2DM后,發(fā)現(xiàn)炎性細胞因子水平降低,且T2DM免疫功能缺陷得到了恢復[15]。用混合營養(yǎng)物質治療STZ小鼠糖尿病模型后,檢測到Th1細胞因子水平下降,而Th2細胞因子IL-10水平升高[16]。這些研究證明,Th1/Th2型細胞因子在糖尿病發(fā)病或治療過程中發(fā)生了顯著變化,與糖尿病關系密切。Cheng等[17]在使用富胍免疫抑制性寡核苷酸治療膳食誘導肥胖小鼠后,降低了胰島素抵抗反應,這是通過調(diào)節(jié)Th1/Th2平衡來達到的。因此,在T2DM發(fā)病過程中Th1/Th2型細胞因子的變化值得關注。本實驗結果發(fā)現(xiàn)肝、腎、心肌及胰腺細胞的胞質IFN-γ、IL-4、IL-10水平發(fā)生了變化。其中,IFN-γ表達升高與Mahmoud等[3]在對T2DM患者外周血液中T淋巴細胞因子的研究結果一致;同時,IL-10表達水平升高與Zhang等[18]對Ⅱ型糖尿病腎病患者血漿IL-10研究所得結果一致,Al-Shukaili等[19]對T2DM患者的血液學研究也得到了同樣的結果,但是該研究未發(fā)現(xiàn)T細胞亞群發(fā)生變化。

IFN-γ可促進M1巨噬細胞分化,導致促炎性細胞因子產(chǎn)生,加重炎性反應。IL-4可誘導M2巨噬細胞分化,產(chǎn)生抗感染性細胞因子[20]。本研究結果發(fā)現(xiàn),在T2DM的發(fā)病過程中IL-4表達減弱,說明機體抗感染效應減弱,即炎性反應加強。同時,從各細胞因子在不同靶器官的分布差異可推斷出,細胞因子是通過血液運輸?shù)礁靼衅鞴賲⑴c炎性反應的。

眾所周知,Th1細胞以分泌IL-2、IFN-γ為主,Th2細胞以分泌IL-4、IL-10為主。從分泌變化的角度來看,Th1細胞分泌的IFN-γ增多,而IL-2不變,可表現(xiàn)為Th1細胞增多或功能增強。同樣,Th2細胞分泌的IL-10增多,而IL-4減少,與Th1 2個細胞因子變化相比較,可知Th1細胞亞群增殖強于Th2細胞。另外,IFN-γ與IL-4存在相互拮抗的關系,兩者平衡可影響Th1/Th2細胞亞群分化[21]。已知IFN-γ可促進Th1細胞增殖,也可抑制IL-4對B細胞分化的促進作用。同時,IL-4可抑制Th1細胞增殖來拮抗IFN-γ。本研究結果中T2DM組Th1細胞因子IFN-γ表達較健康組高,說明Th1細胞增殖作用加強;同時Th2細胞因子IL-4表達減弱,說明其促B細胞分化抑制Th1細胞增殖的作用減弱,從而可推斷出Th1細胞亞群的增殖強于Th2細胞。

通過本實驗可確定的是T2DM獼猴體內(nèi)Th1細胞亞群增殖加強,而IL-4的降低可能會抵消IL-10升高所代表的Th2細胞增殖現(xiàn)象,因此無法確定Th2細胞亞群的變化?？梢?，Th1/Th2亞群變化與T2DM的發(fā)生關系密切,但兩者因果關系需要進一步實驗證明。由于細胞因子間的作用太過復雜,僅通過細胞因子的變化來研究Th1/Th2平衡變化在T2DM發(fā)病過程中的作用還遠遠不夠,后續(xù)工作可考慮檢測CD4、CD8及CD3的比值來探討在T2DM發(fā)病過程中Th1/Th2平衡的變化方向。

總之，本實驗通過對T2DM獼猴部分靶器官中IFN-γ、IL-2、IL-10、IL-4的表達研究發(fā)現(xiàn),Th1/Th2型細胞因子表達水平發(fā)生了不同變化。Th1細胞因子IL-2表達水平無顯著變化,而IFN-γ表達水平升高；Th2細胞因子IL-4水平降低,而IL-10水平升高。Th1/Th2的顯著變化表明其相關因子與T2DM的發(fā)病進程密切相關。

參考文獻(References)：

[1]Marques-Vidal P, Schmid R, Bochud M,etal. Adipocytokines, hepatic and inflammatory biomarkers and incidence of type 2 diabetes. the CoLaus study.PloSONE, 2012,7(12):e51768.

[2]Costantini S, Capone F, Guerriero E,etal. Cytokinome profile of patients with type 2 diabetes and/or chronic hepatitis C infection.PloSONE, 2012,7(6):e39486.

[4]Badawi A, Klip A, Haddad P,etal. Type 2 diabetes mellitus and inflammation: Prospects for biomarkers of risk and nutritional intervention.Diabetes,MetabolicSyndromeandObesity:TargetsandTherapy， 2010,3:173-186.

[5]顧國浩,彭群新.Th1/Th2細胞的免疫功能變化及其意義.國外醫(yī)學:臨床生物化學與檢驗學分冊,2003,24(6):333-334.

Gu G H, Peng Q X. Immune function changes and significance of Th1/Th2 cells.ForeignMedicalSciences:SectionofClinicalBiochemistryandLaboratoryMedicine, 2003,24(6):333-334. (in Chinese)

[6]Hassan G A, Sliem H A, Ellethy A T,etal. Role of immune system modulation in prevention of type 1 diabetes mellitus.IndianJournalofEndocrinologyandMetabolism, 2012,16(6):904-909.

[7]Liu J, Shi B, He S,etal. Changes to tear cytokines of type 2 diabetic patients with or without retinopathy.MolecularVision, 2010,16:2931-2938.

[8]Groen B, Links T P, Lefrandt J D,etal. Aberrant pregnancy adaptations in the peripheral immune response in type 1 diabetes: A rat model.PloSONE, 2013,8(6):e65490.

[5]李啟成.清末比附援引與罪刑法定存廢之爭——以《刑律草案簽注》為中心[J].中國社會科學，2013，(11):104-120.

[9]Zhang C L, Gui L, Xu Y J,etal. Preventive effects of andrographolide on the development of diabetes in autoimmune diabetic NOD mice by inducing immune tolerance.InternationalImmunopharmacology, 2013,16(4):451-456.

[10]Ezquer F, Ezquer M, Contador D,etal. The antidiabetic effect of mesenchymal stem cells is unrelated to their transdifferentiation potential but to their capability to restore Th1/Th2 balance and to modify the pancreatic microenvironment.StemCells, 2012,30(8):1664-1674.

[11]Chatzigeorgiou A, Harokopos V, Mylona-Karagianni C,etal. The pattern of inflammatory/anti-inflammatory cytokines and chemokines in type 1 diabetic patients over time.AnnalsofMedicine:Helsinki, 2010,42(6):426-438.

[12]曾文.恒河猴川西亞種生物學特性背景數(shù)據(jù)建立及在新藥評估中的應用研究.雅安，四川:四川農(nóng)業(yè)大學,2010.

Zeng W. Establishment of a biological background database of a subspecies of rhesus monkey (Macacamulattalasiotis) and its application in new drug evaluation. Ya’an, Sichuan: Sichuan Agriculture University, 2010. (in Chinese with English abstract)

[13]Gong L, Zeng W, Yang Z,etal. Comparison of the clinical manifestations of type 2 diabetes mellitus between rhesus monkey (Macacamulattalasiotis) and human being.Pancreas, 2013,42(3):537-542.

[14]姚金晶,陳宜濤.Th1/Th2平衡調(diào)節(jié)與疾病發(fā)生的研究進展.現(xiàn)代生物醫(yī)學進展,2009,9(13):2597-2600.

Yao J J, Chen Y T. Advances of regulation Th1/Th2 type cytokines balance in human disease。ProgressinModernBiomedicine, 2009,9(13):2597-2600. (in Chinese with English abstract)

[15]Zhao Y, Jiang Z, Zhao T,etal. Targeting insulin resistance in type 2 diabetes via immune modulation of cord blood-derived multipotent stem cells (CB-SCs) in stem cell educator therapy: Phase Ⅰ/Ⅱ clinical trial.BMCMedicine， 2013,11:160.

[16]Chang Y, Zhang G Z, Piao S L,etal. Protective effects of combined micronutrients on islet beta-cells of streptozotocin-induced diabetic mice.InternationalJournalforVitaminandNutritionResearch， 2009,79(2):104-116.

[17]Cheng X, Wang J, Xia N,etal. A guanidine-rich regulatory oligodeoxynucleotide improves type-2 diabetes in obese mice by blocking T-cell differentiation.EMBOMolecularMedicine， 2012,4(10):1112-1125.

[18]Zhang C, Xiao C, Wang P,etal. The alteration of Th1/Th2/Th17/Treg paradigm in patients with type 2 diabetes mellitus: Relationship with diabetic nephropathy.HumanImmunology， 2014,75(4):289-296.

[19]Al-Shukaili A, Al-Ghafri S, Al-Marhoobi S,etal. Analysis of inflammatory mediators in type 2 diabetes patients.InternationalJournalofEndocrinology， 2013,2013:976810.

[20]Winer S, Winer D A. The adaptive immune system as a fundamental regulator of adipose tissue inflammation and insulin resistance.ImmunologyandCellBiology， 2012,90(8):755-762.

[21]Pernis A, Gupta S, Gollob K J,etal. Lack of interferon gamma receptor beta chain and the prevention of interferon gamma signaling in TH1 cells.Science， 1995,269(5221):245-247.