【作者】 路文盛；

【导师】 张志利； 李昭瑛；

【作者基本信息】 山西医科大学 ， 内科学， 2003， 硕士

【摘要】 [目的] 本实验通过观察Ⅳ型磷酸二酯酶抑制剂Rolipram对非肥胖型糖尿病（NOD）小鼠Bcl-2、Bax、TGF-β₁、PDX-1在胰岛表达的影响以及对T淋巴细胞凋亡率、T淋巴细胞亚群分布、Th1／Th2细胞漂移的分析，旨在探讨Rolipram阻断NOD小鼠发生1型糖尿病的免疫机制。 [方法] 将90只体重相近（20g）的4周龄雌性NOD小鼠随机分为干预、对照及正常三组，每组30只。①干预组：腹腔注射Rolipram（8mg／kg），每日两次。②对照组注射等次等量的PBS。③正常组不施加任何处理。干预、对照两组小鼠均于实验第1和第14天各注射一次环磷酰胺（200mg／kg）以加速糖尿病的进程。实验第30天处死，观察发病率；HE染色观察胰岛炎；免疫组化检测Bcl-2，Bax，TGF-β1、PDX-1在胰岛的表达；凝胶电泳DNA梯度图谱定性分析及流式细胞仪定量测定T淋巴细胞的凋亡率；流式细胞仪检测胸腺、脾脏T淋巴细胞亚群的分布；逆转录PCR（RT-PCR）检测Th1细胞因子：IL-2，IFN-γ及TH2细胞因子：IL-4，IL-10 mRNA的转录。实验内容如图示： [结果] ①Rolipram干预组糖尿病发病率降低（26.7％vs 60.0％ x²=6.87p＜0.01） ②Rolipram干预组胰岛炎积分比PBS对照组显著降低（z=-1.987，P=0.047＜0.05） ③Rolipram干预组胰岛β细胞Bcl-2表达增加（0.6437±0.1277 vs 0.3543±0.0653 t=11.047 p＜0.01）而Bax表达减少（0.3263±0.0751山冠穿医才斗大学闷灭d匕学位论文vs 0.7890士0.0975t一20.583p<0.01），TGF一pl表达增加（0.7680士0.0645 vs0.3190士o.0320t二22.254p<0.01），PDx一1表达与Rolipram无相关性（0.3403士0.0409 vs 0.3539士0.03 16 t=l，ol7p=0.314）;而在T淋巴细胞Bel一2表达减少（0.3403士0.0809 vs 0.7533士o.ogs4t=一19.048p<0.01），Bax表达增加（0.6587士o.lllgvso.3553士o.o48lt=13.643p<0.01）④Rolipram干预组外周T淋巴细胞凋亡率增加（55.75%士5.38%vs 28.94%士3.26%、2.5一6，P=0.015）⑤Rolipram千预组脾脏T淋巴细胞亚群分布:CD3+无变化（43.7770士8.7707 vs 41.2500士7.lll8F=2.927op=0.218），CD4+增加（39.1413士6.8932vs34.40一7士4.s177F科.7397p一0.003），Cos+增加（一，5553士一4674 vs 8.5383士l.3673F=3.347P<0.01）;胸腺T淋巴细胞亚群分布:CD4+CDS一增加 （29.3807士0.9928 vs 17.5837士1.0816F=11.7970p<0.01），CD4+CDS+减少（ 73.9237士4.7985 vs 82.7147土4.3132F=一8.7910p<0.01），CD4一CDS+无变化（7.3050士0.9475 vs 7.2683士l，2778F=O.0367P=0.909），CD4一CDS-无变化（3.9153士0.7451 4.1337士o.7s95F=一0.2153P=0.255）⑥Rolipram干预组与PBS对照组相比ThZ细胞因子显著增加而Thl细胞因子明显减少:IL一4mRNA（0.8603士0.037Ivs0.2640士0.0155t’=40.445P<0.01），IL一10m刚A（0 .7309士0.0162 vs 0.33一0士0.0159p<0.05），IL一ZmRNA（0.4081士0.0178vs 0.9537士0‘0224P<0‘01），IFN一Y n1RNA（0.3707士0.0158 vs 0.5150士0.0363t=一23091 P<0.01）。⑦经相关分析发现胰岛炎积分与1型糖尿病发病率呈正相关 （rs=0 .451，P<o.05），与外周T淋巴细胞凋亡率呈负相关（rs二一0.472，P<0.ol），1型糖尿病发病率与外周T淋巴细胞凋亡率亦呈负相关（rs=一0.465，P<0 .05） l结论l①RoliPram能使CD4+Thl型细胞向cD4+ThZ型细胞漂移，从而减轻自身免疫反应。②Rolipram在胰腺组织能促进Bcl一2的表达抑制胰岛p细胞的凋亡;而在淋巴系统却能减少Bcl一2的表达以加速T淋巴细胞的凋亡从而减轻细胞免疫反应。③RoliPram能使中枢、外周T淋巴细胞亚群重新分布即:在中枢CD4+CDS一增加、CD4+CDS+减少、CD4一CDS+无变化、CD4一CDS一无变化;在外周CD4+增加、CDS+增加、CD3+无变化。更多还原

【Abstract】 [Objective] To study the immune mechanisms of rolipram on type 1 diabetes in non-obese diabetic mice by means of immunohistochemical method, DNA ladder analysis , flow cytometry quantitive analysis and reverse transcript PCR technology.[Methods] Ninety female 4 weeks NOD mice (20g) were randomly divided into three equal groups. Group1 mice received the intraperitoneal injection of rolipram (8mg/kg) twice every day. Group2 mice received the intraperitoneal injection of PBS equal quantity and times. Group3 mice were as a normal group. Group1 and Group2 experimental mice were administered the intraperitoneal injection of cyclophosphamide (200mg/kg) to accelerate the process of diabetes on day 1 and 14. All mice were killed on day 30 to observe the incidence of diabetes , the degree of insulitis and the expressions of Bcl-2 , Bax , TGF-β 1 and PDX-1 detected with immunohistochemical technology , the distribution of T lymphocytes in thymus and spleen detected by flow cytometry quantitive analysis and the apoptosis rate of T lymphocytes in spleen detected by DNA ladder qualitative analysis and by flow cytometry quantitive analysis. The production of IL-2,IFN-γ,IL-4, IL-10 mRNA was detected by reverse transcript PCR technology.[Results] Intraperitoneal injection of rolipram to NOD mice reduced the incidence of diabetes. The insulitis score and the severity of insulitis were lower in rolipram group than in PBS group. However, The ratio of Bc1-2/Bax, the apoptosis rate of T lymphocyte and the expression of TGF-β1 were higher in rolipram group than in PBS group. The expression of PDX-1 in pancreaticislets isn’t associated with rolipram. There are the shift from CD4+Th1 to CD4+Th2 cells in spleens in rolipram group. [Conclusions]:1.Rolipram can prevent NOD mice from developing into type 1 diabetes by increasing apoptosis of T lymphocyte or by shifting increasingly from CD4+Th1 to CD4+Th2 cells.2.Rolipram can decrease expression of Bc1-2 in lymphatic tissue , on the contrary, increase it in pancreatic tissue.3.Rolipram can increase CD4+CD8- and decrease CD4+CD8+ in thymus and increase CD4+ in spleen. However, CD4-CD8+, CD4-CD8-, CD8+ and CD3+ aren’t associated with Rolipram.4.Rolipram can increase expression of I1-4, I1-10 mRNA and decrease IFN-γ, I1-2 mRNA to protect pancreatic islets β cells.更多还原