完全弗氏佐剂预防NOD鼠糖尿病的机制及其与细胞因子Th表达和β细胞凋亡的关系

CFA preventing diabetes in NOD mice correlates with Thcytokine shifts and islet β cell apoptosis

目的 探讨完全弗氏佐剂 (CFA)预防非肥胖糖尿病 (NOD)小鼠胰岛炎与糖尿病的作用机制。 方法 42只NOD雌鼠随机分为CFA处理组 (n =2 1)和磷酸盐缓冲液 (PBS)对照组 (n =2 1) ,3周龄时分别于后脚板一次性注射 50 μlCFA和PBS。于 12周龄时观察胰岛炎的程度、胰岛Fas和FasL的表达、β细胞凋亡情况以及测定血清白细胞介素 4(IL 4)、γ干扰素 (Ifn γ)水平和胰岛内IL 4、Ifn γ、白细胞介素 1β(IL 1β)、FasmRNA的表达水平。 结果 12周龄时CFA组胰岛炎积分和β细胞凋亡率均显著低于PBS组 (P均 <0 .0 5)。Fas只在PBS组胰岛内表达 ,而FasL在CFA和PBS组均有相似表达。 12周龄CFA组血清IL 4水平较PBS组增高而Ifn γ水平降低 (P均 <0 .0 5) ;RT PCR结果显示 ,CFA组胰岛内IL 4mRNA转录水平较对照组显著增高 (P <0 0 1) ,而Ifn γ、IL 1β、FasmRNA转录水平则明显降低 [P均 <0 .0 1(Ifn γ ,IL 1β) ,P <0 .0 5(Fas) ]。 3 0周龄时CFA组糖尿病发病率 (9.1% ,1/ 11)比PBS组 (81.8% ,9/ 11)显著降低 (P =0 .0 0 1)。 结论 CFA预防NOD鼠胰岛炎及糖尿病可能与胰岛自身反应T细胞由Th1向Th2转型 ,从而抑制Fas介导的

ObjectiveTo explore the effects and significan ce of complete Freund adjuvant (CFA)-induced Th cytokines expression and β cell apoptosis on prevention of insulitis and diabetes in female Non-obese diabetic (NOD) mice.MethodsForty-two female NOD mice were randomly divided into CFA group (n=21) and phosphate buffered saline(PBS) group (n =21). in CFA-treated mice 50 μl CFA were injected into the pad of hind fo ot and in control group 50 μl PBS injected at 3 weeks of age. Insulitis sever ity were scored by routine HE staining. Fas and Fas ligand(FasL) expression on i slets of Langerhans were examined by immuhistochemical techniques. Apoptotic β cells were counted with TUNEL in situ end-labeling method combined with sABC im muhistochemical method. Serum Th2 cytokine (IL-4) and Th1 cytokine( Ifn-γ) w ere measured by ELISA and the RT-PCR product semi-quantitated relative express ion levels of IL-4, Ifn-γ, IL-1β and Fas mRNA transcription of isolated p ure pancreatic islets at the age of 12 weeks.ResultsThe insulitis scores and apoptotic β cell rates were both significantly lower in CF A group than those in PBS group(insulitis scores: 0.7±0.4 VS 1.7±0.2, β cell rates: 0.061±0.038 vs 0.322±0.051, P<0.01)at 12 weeks of age. Fas expres sed only on islets of the PBS treated mice and did not express on the islets of CFA-treated mice, whereas FasL expressed on islets of both group. Serum IL-4 l evel was elevated and Ifn-γ level was declined in CFA-treated mice while comp ared with control group[IL-4 (85±6 VS 59±7)pg/ml, Ifn-γ (41±5 VS 64±18)p g/ml, P<0.05]. RT-PCR demonstrated intraislets IL-4 mRNA transcription le vel increased and Ifn-γ?IL-1β?Fas mRNA level decreased significantly in CF A group while compared with control group [IL-4 , P<0.01; Ifn-γ, P< 0.01; IL-1β,P=0.001; Fas,P<0.05]. Diabetes incidence of CFA group and PBS group were 9.1% (1/11) VS 81.8% (9/11) at the age of 30 weeks (P=0.01). ConclusionThese results show that CFA prevention of insuliti s and diabetes in female NOD mice is correlated with skewing the pattern of cytokines produced by β-cell autoreative T cells from Th1 (Ifn-r) to a Th2 (I L-4) profile, thus inhibiting the Fas-mediated β-cell apoptosis .