论文Ⅰ 白介素-17调节致糖尿病性T细胞介导的NOD鼠1型糖尿病的发生 摘要 前言 白介素-17 (Interleukin-17，IL-17)，又称白介素-17A。主要表达于活化的CD4T淋巴细胞。IL-17A基因定位于人6p12,是一个由155氨基酸组成的糖蛋白二聚体构成，分子量约35kDa； IL-17受体(IL-17R)属于Ⅰ型跨膜蛋白，并广泛存在于各种组织中，通过转录因子NF-κB和JNK激酶途径活化。 IL-17是由初始CD4T淋巴细胞(naive CD4T cells)分化而来的T〓-17效应性细胞分泌的一种组织炎性细胞因子。IL-23可诱导初始CD4 T细胞发育成为T〓-17细胞。 目前已证实IL-17与风湿性关节炎、系统性红斑狼疮、哮喘、异体移植等自身免疫性疾病和炎性疾病有着密切的联系。T〓-17细胞分泌的IL-17主要通过刺激炎性因子和趋化因子的释放参与局部组织炎症反应。IL-17可调节大约60种编码炎症分子的基因，可以增强TNF-α诱导的炎性基因的表达。因此，目前普遍认为，IL-17是多种细胞的促炎性介质，与特异性炎症反应导致的粒细胞趋化作用有关,IL-17是CD4 T细胞参与组织炎症的重要途径之一。 1型糖尿病是一个慢性进行性的自身免疫性疾病，疾病的发展最终导致胰岛Beta(β)细胞破坏，胰岛素缺乏。非肥胖糖尿病(NOD)鼠的自发性糖尿病过程和胰腺病理改变与人类胰岛素依赖性糖尿病非常相似，具有显著的自发性糖尿病发生的倾向。在出生后第3-4周，NOD鼠胰岛内就可出现细胞浸润和进行性胰岛炎发生，于第12周，开始出现血糖升高等临床糖尿病症状。人们最初从NOD鼠胰岛炎的浸润性白细胞中分离出了BDC2.5 CD4 T细胞，这一细胞株具有特异性的识别胰岛细胞抗原和杀伤胰岛B细胞的能力，被称作致糖尿病性T细胞。 1993年Katz等利用NOD来源的BDC2.5 T细胞携带重组T细胞受体(TCR)α (Vα1)和β(Vβ4)基因，建立了BDC2.5/NOD TCR 转基因鼠(简称为BDC2.5/NOD鼠，BDC/NOD)。和NOD 鼠比较，BDC2.5/NOD 鼠提高了BDC2.5 T (BDC T)细胞在外周组织的表达，病程进展与NOD鼠相似。 在此基础上，Katz等把BDC2.5/NOD鼠和NOD.scid鼠交叉繁殖产生了BDC2.5/NOD.scid鼠，该鼠具有以下突出特点：(1)体内只含有CD4T淋巴细胞，且特异性表达BDC2.5 TCR，删除了其他的T、B细胞亚群。(2)加速胰岛炎和糖尿病的发生。出生后第14-16天即出现细胞浸润和胰岛炎，在第22-30天就表现为严重的胰岛炎并进展为糖尿病。出生后第33天，所有的BDC2.5/NOD.scid鼠就因糖尿病各种并发症而死亡。这一动物模型压缩了糖尿病发病进程，使研究者有更多的重复机会原位观察糖尿病发病全过程中炎细胞的浸润和胰岛细胞的凋亡的变化，为原位研究糖尿病提供了又一个极好的动物模型。 Katz实验表明，IL-17的表达与胰岛炎期和糖尿病前期的发生过程有着密切的关系。实验发现，在BDC2.5/NOD.scid出生后第10天，即胰岛炎和糖尿病前期，血浆IL-17显著升高，而在出生后第21天，即严重的胰岛炎和早期糖尿病期，血浆IL-17则显著下降。而在BDC2.5/NOD和NOD鼠，这一阶段血浆IL-17变化不明显。进一步研究给予外源性的IL-17抗体并不能显著阻止活化的BDC2.5 T细胞对NOD.scid鼠的转移性糖尿病的发生。通过IL-17基因敲除的小鼠证实IL-17对CD4 T细胞的增殖起着关键性作用。因此，调节BDC2.5T细胞自身的IL-17的表达，是否会影响BDC2.5T细胞的转移性糖尿病的发生和糖尿病的进程呢？ 为明确IL-17对1型糖尿病发病的影响，我们分别构建了携带IL-17和IL-17的siRNA (small interfering RNA,小分子干扰性RNA，siRNA IL-17在本文中被缩写为siIL-17)基因的MSCV-IRES-Thy1.1 (MIT)和pMND BANSHEE Thy1.1-U6 (pMND BANSHEE)逆转录病毒载体，采用分子克隆技术，产生目的病毒并转导入BDC2.5 T淋巴细胞，进一步观察IL-17对致糖尿病性T淋巴细胞的影响和对糖尿病发病的作用。 目的 ①研究逆转录病毒转染BDC T细胞能否稳定的表达和分泌IL-17； ②研究siIL-17能否在BDC T细胞的基因表达中抑制IL-17基因的表达。 ③观察IL-17转基因BDC T细胞体内能否诱导NOD.scid转移性糖尿病的发生，以及siIL-17能否通过BDC T细胞基因表达抑制IL-17的表达和体内抑制糖尿病的发生。 ④研究IL-17对1型糖尿病发病影响的机理。 研究方法 1、IL-17和siIL-17的病毒载体的构建： 借助于MSCV-IRES-Thy1.1逆转录病毒载体，通过基因重组的方法携带IL-17基因；以Thy1.1作为病毒载体上的目的基因的检验标志；以携带抗凋亡基因Bcl₂的重组MIT和空载体MIT为阳性对照；以空白转导作为阴性对照。 以pMND-BANSHEE逆转录病毒为载体，插入U6增强子和Thy1.1实验标志，构成了完整的逆转录病毒载体。siIL-17插入其多克隆位点，构建了siIL-17逆转录病毒载体。 2、Phoenix细胞生产病毒颗粒： Phoenix细胞培养和传代至对数生长期，在钙-磷转导试剂的作用下，加入目的病毒DNA，转染包装细胞，培养24小时即可收获携带目的基因的病毒上清。转染前后使用Thy1.1抗体和流式细胞仪鉴定Phoenix细胞。 3、BDC T细胞的活化： 以anti-mouse CD3和anti-mouse CD28体外共刺激脾细胞约24小时后，通过标记CD4和Vβ4抗体，流式细胞仪检测BDC T细胞的性质；标记CD69和CD62L抗体鉴定T淋巴细胞的活化率。 4、BDC T细胞的病毒感染和IL-17的表达： 目的病毒感染BDC T淋巴细胞。细胞培养后，流式细胞仪检测Thy1.1和Thy1.2抗体标记的双阳性T细胞的百分率并筛选出。ELISA检测双阳性T细胞培养液IL-17表达，确定转染效果。 5、转基因BDC T细胞的接种及效果： 通过目的病毒感染，大量生产转基因BDC T细胞，以抗体-磁珠筛选的方法，筛选出Thy1.1/Thy1.2双阳性细胞并注射NOD.scid鼠，以注射BDC T细胞和PBS作为阴性对照组，观察动物血糖变化。 6、ELISA方法检测NOD.scid小鼠血浆肿瘤坏死因子-alpha(TNF-α)、干扰素-gamma (IFN-γ)和白细胞介素-6 (IL-6)以及白细胞介素-4 (IL-4)，并送检胰腺进行病理检查，及流式细胞学检查脾脏和胰腺淋巴结。 结果 1、细胞克隆生产的重组逆转录病毒为分别携带目的基因IL-17和siIL-17的病毒DNA,测序和酶切结果和预期结果完全一致。 2、携带目的基因的病毒载体转染Phoenix包装细胞：抗体标记和流式细胞学鉴定转导前Phoenix细胞呈Thy1.1阴性，转导后，MIT空载体、重组Bcl₂-MIT质粒、重组IL-17质粒和siIL-17质粒的转导率分别是(96.1 ±4.2)%，(93.2±2.2)%， (93.5±3.2)%和(89.5±4.7)%，与空白对照组(0.4±0.1)%相比，均有显著性差异(P＜0.01)。 3、BDC T细胞的活化：分别用CD4/CD69和CD4/CD62L抗体标记活化前、后的脾细胞，与活化前比较，活化后BDC T细胞比例增加，活化前、后CD4⁺/CD69⁺细胞和CD4⁺/CD62L⁻细胞的比例分别由(3.4±1.5) %和(8.6±2.8 ) %提高至(33.7±6.9) %和(25.5±5.3) %,提示CD4⁺细胞处于较理想的被转染状态。 4、携带目的基因的逆转录病毒转染活化的T淋巴细胞：在Mock空白对照组、 MIT空载体组，Bcl₂阳性对照组、IL-17组和siIL-17组中，Thy1.1⁺/Thy1.2⁺细胞比例分别为(0.6±0.3) %，(7.2± 2.4) %，(6.8±2.6) %，(6.4±2.4) %和(4.6±1.8) %。 5、转基因BDC T细胞的筛选和鉴定：以Thy1.1、Thy1.2和BDC2.5TCR抗体标记被筛选的细胞，并进行流式细胞学分析显示：①经Thy1.2和BDC2.5TCR抗体标记证实被筛选的细胞是BDC2.5 T细胞，②与筛选前对比，Thy1.1和Thy1.2抗体标记的双阳性IL-17转基因细胞从(5.1±2.4) %提高到(97.2±1.2) %， siRNA-IL-17转基因细胞从(4.6±1.6) %提高到(83.2±6.3) %。 6、转基因NOD/BDC T细胞IL-17的体外表达：IL-17转基因T淋巴细胞的IL-17水平显著升高，活化后可进一步促进IL-17的表达。siIL-17转基因T淋巴细胞可显著抑制IL-17的表达，IL-17的水平显著降低。 7、NOD.scid鼠血糖监测和组织学检查： 至观察期结束，注射IL-17转基因BDCT细胞的NOD.scid小鼠血糖显著升高，全部小鼠均发生糖尿病，而注射siIL-17细胞的NOD.scid小鼠、阴性对照组和BDC T细胞组无一例发病。胰腺组织学H&E染色发现注射IL-17转基因BDC T细胞的NOD.scid小鼠表现为严重的胰岛炎和胰岛结构破坏，模糊不清。而注射siIL-17的转基因NOD.scid小鼠和BDCT细胞组多表现为轻、中度胰岛炎，阴性对照组表现为正常胰岛。 8、NOD.scid鼠血浆IL-17、TNF-α、IFN-γ、IL-6和IL-4水平： 注射IL-17BDC T细胞的小鼠血浆IL-17、TNF-α IFN-γ和IL-6水平均显著高于siIL-17小鼠和对照组(P＜0.01)，而siIL-17小鼠各项血浆细胞因子水平与对照组无显著性差异(P＞0.05)。血浆IL-4水平在IL-17小鼠中未测出，在siIL-17小鼠和对照组中极低。未能检测出NOD.scid小鼠血浆各项细胞因子。 9、流式细胞学结果显示，在IL-17糖尿病发病组，脾脏和胰腺淋巴结BDCT增殖和聚集增多，树突细胞(DC)细胞显著增加。 结论 1、以逆转录病毒为载体携带IL-17和siIL-17基因转染BDC T细胞，可以体外获得显著高表达的IL-17 BDC T细胞和低表达IL-17 BDC T细胞，而siIL-17基因表达可以有效抑制BDC T细胞中IL-17的表达。 2、Phoenix包装细胞是一个安全有效的逆转录病毒“包装机”。 3、转移高表达IL-17的转基因BDC T淋巴细胞可诱发和加速NOD.scid鼠糖尿病的发生，表达siIL-17的BDC T细胞可通过抑制IL-17的表达抑制NOD.scid鼠糖尿病的发生。 4、IL-17可刺激自身的BDC T活化，导致淋巴细胞胰岛浸润，并通过促进DC活化和BDC T细胞释放TNF-α、IFN-γ, IL-6细胞因子和抑制IL-4细胞因子，最终破坏胰岛β细胞，诱发糖尿病。 5、DC细胞可以通过抗原递呈介导高表达IL-17的BDC T细胞活化增殖和胰岛β细胞损害。 关键词： 1型 糖尿病 白介素-17 转基因 siRNA NOD BDC2.5 小鼠 论文创新点： 首次建立了高表达IL-17和低/无表达BDC T细胞株，并研究IL-17与NOD鼠的关系，发现细胞因子和DC在转移性糖尿病中的作用。 论文Ⅱ 长期饮酒减少大鼠睾丸间质细胞PBR和StAR表达 前言 乙醇是目前滥用最广的成瘾物质，长期摄入乙醇可以对男性生殖系统产生多方面不利的影响。目前认为，乙醇是引起男性不育发病率逐年增高的重要原因之一。 乙醇损害男性生殖能力的机制目前还未阐明，可能与睾酮的合成、分泌的减少有关。睾酮是机体最重要雄激素之一，主要由睾丸间质细胞分泌，线粒体是睾酮的合成场所，胆固醇是睾酮的合成原料。睾酮的合成和分泌主要受下丘脑-垂体-性腺轴调节。多种激素如黄体生成素(LH)、人绒毛膜促性腺激素(hCG)、表皮生长因子(EGF)都可与睾丸间质细胞上相应的受体结合，通过各自的细胞内信号传递通路，最终可引起共同的生物学效应——胆固醇由胞浆进入线粒体，开始睾酮的线粒体合成过程，该过程被称为睾酮合成的共同通路。 大量的流行病学资料和动物实验证实，长期摄入乙醇可明显降低血清中的睾酮水平。乙醇对下丘脑-垂体-性腺轴活性的抑制被认为是乙醇抑制睾酮合成和分泌的可能机制之一，此外，乙醇还可直接作用于睾丸间质细胞，抑制睾酮的合成。 Murono和Khan等研究大鼠睾丸间质细胞发现，乙醇不仅可抑制LH刺激后的睾酮合成，在hCG和环磷酸腺苷(cAMP)刺激的睾酮合成也显著下降，同时基础状态下的睾酮合成也是降低的。从而推测，乙醇对睾丸间质细胞的直接作用，其作用环节很可能位于睾酮合成的共同通路上，即由胆固醇进入线粒体开始的睾酮合成的线粒体阶段。 外周型苯二氮卓类受体(PBR)和类固醇生成快速调节蛋白(StAR)都分布在细胞内线粒体外膜上，被公认为胆固醇进入线粒体的关键酶和控制阀。它们表达于肾上腺、卵巢和睾丸等重要的类固醇激素合成器官。研究显示，睾丸间质细胞(Leydig cells)是PBR在睾丸表达的唯一靶细胞，而StAR在间质细胞上也呈强阳性表达。实验表明，靶向损坏R2C间质细胞中的PBR基因，可直接阻止胆固醇转运进入线粒体和类固醇激素的合成，向该细胞转导PBR cDNA，又可恢复类固醇激素的合成；敲除StAR基因，小鼠的类固醇激素合成显著异常。因此， PBR和StAR在胆固醇合成类固醇激素中起着极其重要的作用。 West等借助劳光共振能量转移(Fluorescence resonance energy transfer, FRET)技术证实PBR和StAR在细胞线粒体膜上的相距不足100Å，在生理状态下，二者之间存在着能量转移，并推测StAR携带胆固醇并转移给PBR。因此，PBR和StAR共同参与胆固醇的转运和类固醇激素的合成，并且二者关系密切，相互协同。 文献显示，乙醇可影响肾脏的苯二氮卓类受体表达上调，增加肾脏PBR与其配体的结合；可引起嗅球细胞和人体血小板细胞中PBR表达的下调，并与乙醇的神经毒性发生和循环代谢有关；同样地，乙醇可快速诱导肾上腺细胞StAR的表达增加，并与孕酮、孕烯醇酮和皮质酮的变化相平行。Herman等[39]通过一次性给予大鼠胃内乙醇灌注，观察到睾丸Leydig细胞PBR和StAR表达在干预后10分钟开始下降，但48小时后又恢复到正常水平。而Calvo等通过乙醇喂养大鼠12周，发现PBR与肾脏配体的结合力升高，增加乙醇浓度，PBR和配体的结合力并不增加。上述研究显示乙醇对大鼠睾丸组织的PBR和StAR的影响趋势并不稳定。 基于上述研究现状，为了更深入的认识乙醇与PBR、StAR的关系和对睾酮的作用机理，本研究确定研究目的与方法如下： 目的 1、通过对大鼠睾丸组织染色，观察乙醇对大鼠睾丸组织的基本影响； 2、检测大鼠血清睾酮水平，了解乙醇对大鼠血清睾酮的抑制作用； 3、检测乙醇喂养大鼠睾丸StAR mRNA的表达水平；检测睾丸组织PBR和StAR的表达，了解乙醇对PBR和StAR在基因转录和蛋白质水平上的影响； 4、明确PBR和StAR在睾丸组织中的定位，观察定位组织蛋白表达情况。 研究方法 1、动物模型培养。为检测慢性乙醇对大鼠睾丸组织的影响，Wistar大鼠随机分成四组，每组给予不同剂量的乙醇胃管灌注，每天一次，共20周。麻醉后处死，迅速取睾丸置于液氮保存或4%多聚甲醛中固定、脱水、包埋，制成组织蜡块备用。 2、采用H&E染色，显示睾丸组织的乙醇影响状况。 3、采用罗氏全自动E170电化学发光仪测定大鼠血清睾酮水平。 4、采用RT-PCR检测StAR mRNA水平；采用免疫共沉淀和Western-blot检测PBR和StAR蛋白水平。 5、采用免疫组织化学技术检测PBR和StAR在睾丸组织中的定位和表达水平。 结果 1、大鼠睾丸病理学改变：H&E染色显示乙醇饲养大鼠睾丸组织结构的改变。和对照组相比较，曲精小管生精细胞层明显减少，管腔中精子数目明显减少，完整的精子减少，畸形和断裂的精子及鞭毛可见，提示慢性饮酒组精子生成减少，破坏增加，生精能力降低，生殖能力下降。 2、血清睾酮激素的测定：与对照组[(259.6±69.8)ng/dl]相比，各饮酒组大鼠睾丸血清睾酮含量明显下降。其中小剂量饮酒组血清睾酮含量下降67.2 % [(85.1±23.4) ng/dl，P＜0.01]；中剂量组下降 69.4 % [(79.6±23.8) ng/dl，P＜0.01]；大剂量组下降 90.0 % [(25.90±6.79) ng/dl, P＜0.01]。 3、乙醇下调睾丸组织StAR mRNA表达水平：与对照组相比，慢性饮酒组睾丸组织mRNA表达均有不同程度的下降，其中小剂量饮酒组下降45.9% (P＜0.01),中剂量组和大剂量组分别下降60.2% (P＜0.01)和81.7%(P＜0.01)，提示乙醇抑制作用与睾丸组织StAR mRNA表达水平之间有剂量依赖性关系。 4、乙醇下调睾丸组织PBR和StAR蛋白表达水平：与对照组相比，PBR和StAR在小剂量饮酒组分别下降13.8% (P＞0.05)和34.5% (P＜0.01)，中剂量饮酒组下降20.9%(P＜0.05)和37.7%(P＜0.01),大剂量饮酒组下降50.4%(P＜0.01)和95.2% (P＜0.01)。结果显示长期慢性饮酒可在蛋白质水平抑制PBR和StAR的表达。PBR和StAR水平下降程度在大剂量饮酒组表现最为明显，在StAR中更加显著；相关分析显示PBR和StAR与血清睾酮呈中度相关(r=0.68，P＜0.01； r=0.74，P＜0.01，n=34)。提示在睾丸组织PBR和StAR表达与乙醇的量效关系上，乙醇对PBR和StAR的抑制作用随乙醇剂量增加而增加。 5、乙醇降低睾丸间质细胞PBR和StAR表达：免疫组织化学结果显示PBR和StAR主要表达在睾丸间质组织。定量分析睾丸间质中阳性细胞占间质组织面积百分比，与对照组相比，小、中、大剂量饮酒组PBR表达面积占间质组织面积百分比分别下降 33.3 % (P＜0.01)，37.7 % (P＜0.01)和 63.6 % (P＜0.01)，而在小、中、大剂量饮酒组StAR表达面积下降百分比分别是27.1 % (P＜0.01)、 51.8% (P＜0.01)和58.4% (P＜0.01)。相关分析显示PBR和StAR在睾丸间质组织中的表达与血清睾酮呈中度正相关(r=0.79，P＜0.01；r=0.71, P＜0.01, n=34)。 结论 1、长期慢性乙醇喂养，可降低大鼠血清睾酮的水平。 2、长期乙醇喂养可通过同时抑制大鼠睾丸组织间质细胞的PBR和StAR的表达，抑制睾丸组织睾酮的合成。 3、乙醇可通过抑制睾酮的合成或直接作用等其它途径影响睾丸组织的形态，抑制精子的发育和成熟，使精子数量和质量下降，使生育能力受损。 关键词 乙醇；受体，苯二氮卓；类固醇合成快速调节蛋白；大鼠， Wistar 论文创新点： 首次进行了长期慢性乙醇对大鼠睾丸间质细胞睾酮激素合成影响的在体研究，并发现乙醇对PBR和StAR的同时抑制的这一特性，有助于揭示长期慢性乙醇影响睾丸雄激素合成的发病机理。

 Part Ⅰ Interleukin-17 Modulates the Ability of Diabetogenic T cells to Mediate Type 1 Diabetes in NOD Mouse Perface Interleukin-17 (IL-17), also called IL-17A, is mainly expressed in activated CD4+ T cells. The gene location for IL-17 is 6p12 and is a homodimetric glycoprotein consisting of 155 amino acids of 35kDa molecular weight. The IL-17 receptor (IL-17R) is a type Ⅰ transmembrane protein, and its mRNA is distributed ubiquitously in various tissues, and its engagement activates both transcription factor NF-KB and kinase Jnk pathway. Now, it is known that IL-17 is a preinflammatory factor secreted by IL-17 effector CD4 cells (T〓-17 cells) which differentiated from naïve CD4 T cells. IL-23 is a key trigger for inducing the development of IL-17 T cells from naïve CD4 T cells. At present, it has been proved that the expression of IL-17 has closely linked to a growing list of autoimmune and inflammatory diseases such as rheumatoid arthritis, lupus, asthma and allograft rejection. IL-17 produced from T〓-17 cells mainly concerned to inflammation reaction in tissues by stimulation of inflammation factors and cytokines. Approximately 60 genes were upregulated by IL-17, many encoding proinflammatory molecules. IL-17 also increase TNF regulating the expression concerned to inflammatory genes. So, the published data have indicated that IL-17 is a proinflammatory mediator on multiple cells types in vitro and involved in specific inflammation processes leading to the mobilization of granulocytes. IL-17 is one of important pathways through which CD4 acts in inflammation in tissue. Type 1 Diabetes is a slow progressive autoimmune disease resulting in complete destruction of the insulin-production beta (β) cells in the pancreatic islets. As the pathogenic process and histopathological stages in nonbese diabetes (NOD) closely parallels of insulin-dependent diabetes mellitus (IDDM) in human, NOD mice are prone to develop diabetes spontaneously. The cellular infiltration into the pancreas that leads to insulitis and beta-cell destruction can begin as early as 3 to 4 weeks of age in the NOD mouse and up to 12 weeks of age, the animal may be overtly diabetic. The BDC2.5 CD4 T cells originally were isolated from islet-infiltrating leucocytes in NOD islets and have the ability to recognize specifically islet cells antigens and kill them. So, this kind of cells is called diabetogenic T cells. In 1993, Katz et al generated a strain of mice carrying the rearranged TCRα (Vα1) and β (Vβ4) genes from a diabetogenic T cells isolated from a NOD mouse, which is called BDC2.5/NOD TCR transgenic mouse (also called BDC2.5/NOD). Compared to NOD, BDC2.5 T (BDC T) cells are increased in BDC2.5/NOD mice in the periphery, and approximately 95% of CD4 T and CD8 T cells in BDC2.5 mice displayed Vβ4. The process of disease development is similar to NOD. Based on these works, Katz et al reported that the transgene crossed from BDC2.5/NOD mice onto NOD.scid background, and produced a new strain, BDC2.5/NOD. scid with sudc distinguished characters as: (1) Only had CD4 T cells in this model and specifically express BDC2.5 TCR on the CD4 T cells. Other T cells subset and B cells had been deleted. (2) Hasten the development of insulitis and diabetes. BDC2.5/NOD.scid mice develop insulitis just from 14-16 days of age and become severe insulitis and diabetes by 22-30 days of age. All BDC2.5/NOD.scid mice died from diabetic complications on or before 33 days of age. The BDC2.5/NOD.scid mice had dramatically compressed the time of the development of diabetes and allowed us to determine by direct in situ cellular infiltration and apoptotic change of β cells. Katz's experiments suggest that the expression of IL-17 has a closely relationship with the development of insulitis and pre-diabetes. They observed the high level of plasma IL-17 as early as 10 days of life in BDC2.5/NOD.scid mice, concurrent with the onset of severe insulitis and per-diabetes. Whereas, at the time of the diabetes onset (21 days), the level of IL-17 plummet dramatically. Conversely, the levels of IL-17 tends to vary minimally in BDC/NOD and NOD mice. Moreover, treatment of NOD.scid recipient mice of activated BDC2.5 T cells with anti-IL-17 antibody did not prevent the onset of diabetes significantly. On the other hand, the study of IL-17 knockout mice proposed that IL-17 is critical for proliferation of CD4+ T cells. Therefore, whether the adjustment of IL-17 expression in BDC T cells themselves would affect the transfer diabetes from BDCT cells and the development of diabetes? To understand the influence of IL-17 on Type 1 diabetes, we have constructed MSCV-IRES-Thy1.1 (MIT) vector with IL-17 and pMND BANSHEE Thy1.1-U6 (pMND BANSHEE) vector with siRNA IL-17 (small interfering RNA of IL-17, abbreviated to siIL-17 here) and cloned to produce the target viruses for transduction BDC2.5 T cells. We hope to observe the endogenous regulation of IL-17 to the BDC2.5 T cells in the development of Type 1 diabetes animal models. Objectives (1)To explore and establish a stable expression system for IL-17 and si-17 in BDC T cells. (2)To test the inhibition of si-17 to IL-17 in BDC T cells. (3)To transfer the IL-17 transgenic BDC T cells to NOD.scid mice and identify the effect on inducing diabetes and suppressing as the expression of si-17 in BDC T in vivo. (4)To study the mechanism of IL-17 in the development of type 1 diabetes. Methods 1.The construction of retrovirus vector with IL-17 and siIL-17: IL-17 gene is linked to MSCV-IRES-Thy1.1 retrovirus vector by gene recombination with thy1.1 as marker of target gene. The MIT with anti-apoptosis gene of Bcl₂ and empty vector of MIT are as positive controls, and the blank control of Mock is as negative control. Both U6 promotor and Thy1.1 marker were inserted into the cloning site of pMND-BANSHEE to construct the integrated retrovirus vector for our experiment and the siIL-17 gene was inserted between the cloning sites to construct siIL-17 retrovirus vector. 2.Production of virus by Phoenix cells: The phoenix cells were cultured and passaged to keep logarithmic growth. The target DNA was transfected into phoenix cells with aid of calcium-phosphate. After culture for 24hs, the virus supernatant was harvested. The phoenix cells were identified by Thy1.1 antibody and flow cytometry before and after transfection. 3.Activation of BDC T cells and virus transfection: The Spleen cells from BDC/NOD mice were stimulated by mouse CD3 and CD28 antibodies for about 24hs. BDC T cells marked CD4 & Vβ4 antibodies were checked by flow cytometry and the activation percentage of cells were identified by marked CD69 & CD62L antibodies. 4.The infection of BDC T cells and the expression of IL-17 BDC T cells were infected with target virus. After incubation, the infection percentage of T cells marked Thy1.1 & Thy1.2 antibodies was determined by flow cytometry and sorted them. The expression of IL-17 in the double positive cells was assayed by ELISA. 5.Transfer infected BDC T cells to NOD.scid mice: A large number of infected BDC T cells were produced and Thy1.1/Thy1.2 T cells were sorted by MACS Separation Columns. Each of NOD.scid mouse was injected the infected BDC T cells of and injection of BDC T cells as well as PBS injected as negative controls. The mice blood sugar was assayed. 6.The plasma tumor necrosis factor -alpha (TNF-α), interferon-gamma (IFN-γ), interleukin-6 (IL-6) and IL-4 were assay by ELISA and pancreas from NOD.scid were stained with hematoxylin and eosin (H&E). The spleen cells and PLN cells have been checked by Flow cytometry. Results 1.The recombinant retrovirus are the virus DNA with the target genes of IL-17 or siIL-17 respectively. The results from sequence and digestion are the same as the expected fragments of them. 2.Transfected phoenix cells by retrovirus with target genes: Identified the phoenix cells by specific antibodies and flow cytometry, They show Thy1.1 negative before transfection and the transfected percentage increase significantly in MIT, Bcl₂-MIT, IL-17 vector and siIL-17 vector of (96.1±4.2)%, (93.2±2.2)%, (93.5±3.2)% and (89.5±4.7)% respectively in the post-transfection, compared with Mock control (0.4±0.1) %. 3.Activate BDC T cells: Marked the spleen cells of pre- and post-activated with CD4/CD69 & CD4/CD62L antibodies. Compared to pre-activated, the percentage of post-activated BDC T cells increased from (3.4±1.5)% and (8.6±2.8)% to (33.7±6.9)% and (25.5±5.3)%, respectively. The results showed that the T cells were optimal for infection. 4.Infect BDC T cells by retrovirus with target genes: Marked the infected BDC T cells with Thy1.1/Thy 1.2 antibodies and flow, the percentage of infected BDC T cells in Mock (blank control), MIT(empty vector), Bcl₂ (positive control), IL-17 and siIL-17 were (0.6±0.3)%, (7.2±2.4)%, (6.8±2.6)%, (6.4±2.4)% and (4.6±1.8)%, respectively. 5.Identified the sorted transgenic BDC T cells: Marked the sorted cells by Thy1.1/Thy1.2/BDC2.5 TCR antibodies and flow cytometry, the results suggested that: (D the sorted cells marked by Thy1.1/Thy1.2/BDC2.5 TCR antibodies were BDC2.5 T cells. (2) Compared to pre-sorted, the percentage of Thy1.1/Thy 1.2 double positive IL-17 cells increased from (5.1±2.4)% to (97.2±1.2)%, and from (4.6±1.6)% to (83.2±6.3)% in double positive siIL-17 cells. 6.IL-17 expression in transgenic BDC T cells in vivo: IL-17 levels in IL-17 transgenic BDC T cells increase significantly, and even more after activation. Conversely, the expression of IL-17 is inhibited in siIL-17 BDC T cells and IL-17 levels decrease. 7.Determined the blood sugar of NOD.scid mice and histopathology: The blood sugar from tails in NOD.scid mice are assayed at 4pm. By the end of observation (36 days), All of the NOD.scid mice (6 mice) injected IL-17 BDC T cells have diabetes and there is no diabetes in the NOD.scid mice of siIL-17 BDC T cells injected and negative control as well as BDC T cells groups (Thy1.1-/ Thy1.2+) to observe until the end of observation. Pancreases H&E stained showed that there were distinct severe insulitis and destruction of islets in NOD.scid mice injected IL-17 BDC T cells, and peri- or moderate insulitis in NOD.scid mice whether injected siIL-17 BDC T cells or BDC T cells, are normal in negative control group. 8.The plasma levels of TNF-α, IFN-γ, IL-6 and IL-4: The results show that the plasma levels of IL-17, TNF-α, IFN-γ and IL-6 in IL-17 mice are higher than those of in siIL-17 mice or in control (P＜0.01). Whereas, there are no difference in the plasma cytokines levels between the siIL-17 mice and control (P＞0.05). The plasma level of IL-4 in the IL-17 mice has not been detected and are very low in the siIL-17 mice and control. 9. Check the spleen cells and PLN cells by Flow cytometry: In the diabetes mice of injected IL-17 BDC T cells, the recruitment and proliferation of BDC T cells in spleen and PLN increase markably and the percentage of dendritic cells rise. Conclusions 1.The BDC T cells with high expression IL-17 can be obtained by infecting them with IL-17 retrovirus in vitro. The expression of siIL-17 in BDC T cells can efficiently suppress the expression of IL-17, namely BDC T cells of low expression. 2.Packaging system of phoenix cells is a safety and efficient "packaging machine" for retrovirus. 3.Transfer BDC T cells with high IL-17 expression can induce and speed up the development of diabetes in NOD.scid mice, and BDC T cells with si-17 expression can inhibit the development of diabetes in NOD.scid mice by suppression of IL-17 expression in BDC T cells. 4.IL-17 can activate the host DC and BDC T cells and result in the infiltration of lymphocytes to islets, and BDC T cells can destroy the islets β cells by release TNF-α、IFN-γ、IL-6 as well as inhabitation of IL-4 cytokines. 5.Dendritic cells (DC) in spleen and PLN may induce the activation and proliferation of BDC T cells with IL-17 high-expression, and moreover damage of β cells in islets. Key Words Type 1 Diabetes Interleukin 17 siRNA NOD BDC2.5 Mouse Part Ⅱ Chronic Ethanol Feeding Decrease PBR and StAR of Leydig Cells in Rat Perface The ethanol has been becomes broadly addicted substance for abuses at present. Excessive ethanol consumption may be among the causes of the various disadvantageous influence to the male reproductive system. Now, people believe that ethanol consumption is one of important factor which the rate of male infertility incidents rise year by year. Numerous epidemiological data and experiments confirmed that, excessive ethanol consumption can obviously reduce serum testosterone level. Ethanol could reportedly affect the activity of HPA axis, which is believed as the possible mechanism of ethanol. Moreover, ethanol can act to the Leydig cells directly and inhibit the production of testosterone. Recently, it was raised that PBR and StAR are functionally linked to leydig cells testosterone synthesis. Both are located on the outer membrane in mitochondria and plays a key role in the transport of cholesterol (substrate) from outer to inner membrane of mitochondria., known as the rate-limiting step in the steroidogenic stage of testosterone synthesis. Studies show that PBR was present exclusively in the interstitial Leydig cells and immuno- histochemical localization of StAR in leydig cells stained moderately to intensely. Series of experiments shown that targeted disruption of the PBR gene in R2C Leydig cells arrested cholesterol transport into mitochondria and steroid formation; transfection of the PBR-disrupted cells with a PBR cDNA rescued steroidogenesis. Whereas, StAR knockout mice exhibited multiple hormonal abnormalities. Therefore, both PBR and StAR play an very important role in steroidogenesis. Document shown that ethanol can affected PBR expression the kidney and increased binding of PBR with its ligand. Ethanol administration to rats for 30 days resulted in a significant decrease in the density of PBR in the olfactory bulb, and the reason may be concerned with neurotoxicity from ethanol. Similarly, experiments showed that ethanol rapidly induces the increased StAR expression and translocation in rat adrenal gland, and this StAR protein expression paralleled increases in plasma pregnenolone, progesterone and corticosterone levels. The experiments from Herman showed that the levels of StAR and of PBR were significantly decreased within 10 minutes of drug administration intragastrically, and by 48 hours, StAR, PBR levels had increased above control values. Whereas, Calvo reported the binding of ligand to PBR was increased in the kidneys of rats treated with ethanol for 12 weeks, but not to ethanol increased. So, above researches suggested that the trend of ethanol impacting on PBR and StAR be not clear. Based on above studies, it is necessary to deeply understand the relationship between ethanol and PBR & StAR, and mechanism of inhibition to testosterone. Therefore, we focus on the objective and methods as follow. Objectives 1.To observe the change of testes tissue by Hematoxylin and Eosin staining. 2.To understand the inhibition of ethanol to testosterone by assaying the rat serum testosterone level. 3.Todetermine the StAR mRNA expression levels by extraction of total RNA from testes tissues. Furthermore, to investigate the influence of ethanol on PBR and StAR protein western-blotting. 4.Todetermine the location and expression of PBR and StAR in testes by staining tissue slides with specific antibodies. Methods 1.Making ethanol-feeding Wistar rats model: To investigate the influence of chronic ethanol feeding on rats testes, Wister rats were randomly divided into four groups and administered intragastrically (i.g.) the different dose of ethanol to each group once a day for twenty weeks totally. After anesthetic executions, rapidly takes the testes into liquid nitrogen or fixed in the 30% formaldehyde, dehydrate and embed in paraffin. 2.Show the change of testes tissues affected by ethanol with Hematoxylin and Eosin staining. 3.To assay the serum testosterone level by Roche ECL170. 4.To explore the influence of ethanol on PBR and StAR in testes of rats, RT-PCR was employed for determination of StAR mRNA and Western-blot was used for assaying the protein of PBR and StAR. 5s To determine the location and expression of PBR and StAR in testes by immunohistochemistry Results 1.The pathological change in testes: The morphology and structure of testes tissue were shown by Hematoxylin and Eosin (H&E) staining. Compared to the control, seminiferous tubular wall of testes became thin and the layer of germ cells reduced significantly. Moreover, the number of spermatozoon in the seminiferous tubes decreased, and the broken and misshapen spermatozoon's flagella were frequently observed and no integrated spermatozoon produced. These change strongly suggested that ethanol can decrease spermatozoon production, destroy spermatozoon development and damage the function of spermatogenic cells, even the ability of male reproduction. 2.Assay the serum testosterone level: As Compared to the control [(259.62±69.80) ng/dl ], the serum testosterone level in each ethanol-feeding group decreased significantly, the percent of decline in the small-, middle- and large-dose ethanol feeding groups were 67.2% [(85.1±23.4) ng/dl, P＜0.01], 69.4% [(79.6±23.8) ng/dl, P＜0.01] and 90.0 % [(25.9±6.8) ng/dl, P＜0.01] respectively. 3.Ethanol down-regulated the mRNA expression of StAR in testes: The StAR mRNA level in each ethanol-feeding group decreased with different extent. The descending percent in the small-,middle- and large-dose ethanol feeding group were 45.9% (P＜0.01), 60.2 % (P＜0.01) and 81.7 % (P＜0.01) respectively, compared to the control, and suggested that there was a dose-dependent relationship between ethanol and the inhibition of StAR mRNA expression. 4.Ethanol down-regulated the expression of PBR and StAR protein level: Compared to the control, the descending percentage of PBR and StAR in small-dose ethanol feeding group were 13.8% (P＞0.05) 和 34.5% (P＜0.01) respectively, the descending percentage of PBR and StAR were 20.9% (P＜0.05 ) 和 37.7% (P＜0.01) respectively in the middle-dose group, and the descending percent of PBR and StAR in large-dose group were 50.4% (P＜0.01) 和 95.2% (P＜0.01) respectively. The descending degree in the large-dose group was the most significant in three groups, and even in the StAR large-dose group. Correlation analysis showed that there were middle correlation between ethanol and PBR or StAR (r=0.68, P＜0.01; r=0.74, P＜0.01 , n=34) , suggesting that the suppression of ethanol to PBR and StAR depend on the dosage of ethanol. 5.Decrease the expression of PBR and StAR in Leydig cells: The results of immunohistochemistry suggest that PBR and StAR expressed mainly in the leydig cells of interstitial testes. Quantitative analysis has been taken with the square percentage of positive cell in the total interstitial tissues. Compared to control, the square percentage of PBR and StAR expression in low-, middle-, high-dose ethanol groups reduced to 33.3% (P＜0.01) ,37.7% (P＜0.01) ,63.6% (P＜0.01) and 27.1 % (P＜0.01 ),51.8 %(P＜0.01) ,58.4 %(P＜0.01)respectively. Correlation analysis show that there were medium positive correlation between ethanol and PBR or StAR (r=0.79, P＜0.01; r=0.71, P＜0.01, n=34) respectively. Conclusions 1.The serum testosterone level decreased in the chronic ethanol-feeding rats. 2.The expression of PBR and StAR in testes can be suppressed by ethanol feeding rats for 20 weeks and this may be one of the mechanismto responsible for ethanol inhibition to testosterone. 3.Ethanol can inhibit the testosterone biosynthesis, affect tissue morphology in testes, decredse the number and the quality of spermatozoon, even damage the ability of reproduction by direct or other indirect pathways. Key words Ethanol Receptor Benzodiazepine Steroidogenic acute regulatory protein (StAR) Rats Wistar