The function analysis of the apoptosis of pancreatic β-cells in autoimmune (Type I)diabetes using microdissection methods

显微切割方法分析自身免疫性（I型）糖尿病胰腺β细胞凋亡的功能

基本信息

批准号：
10670439
负责人：
ANZAI Keizo
金额：
$ 2.05万
依托单位：
Fukuoka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670439/
关键词：
Type I diabetes NOD mice Apoptosis

项目摘要

We produced frozen sections from the pancreas of non-obese diabetic (NOD) mice. which used as model animal of autoimmune diabetes (type I). The apoptotic cells were detected within islets of non-diabetic NOD mice by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) methods (Topics of the internal medicine advance, l998).The micodissection methods using micromanupulator was adopted in order to analyze mechanism of pathogenesis of the apoptosis, and either pancreatic β cells or infiltrating lymphocytes around and within islets from pancreas section were cut off and were collected. and mRNA was extracted.In addition, We analyzed gene expression in either islets or lymphocytes, for example IL-4. IFN-γ. FasL, Fas. perforin, granzyme. TNF-receptor family, iNOS and Bcl-2 family associated with apoptosis. by RT-PCR methods. By arranging result at present the contribution preparation is being carried out.We measured the serum soluble Fas and FasL in the human NK leukemia patient by ELISA methods (British J Haematology. l998). The NOD mice is also being examined the serum soluble Fas and FasL used the similar measuring method.In order to clarify the difference in population of infiltrated lymphocytes and in the ratio of CD40. CD40L, CD28, CD86 positive cells between diabetic NOD mice and non-diabetic NOD mice (40 wk-old and over), islets with insulitis were immunohistochemically examined for subsets of infiltrated lymphocytes. We found no difference in between the two groups.In addition, in order to clarify the difference in cytokine and TCR repertoire between B cell deficient NOD mice or B cell deficient streptozocin induced diabetic AKR/J mice and control littermates with B cells. we studied the cytokine and TCR repertoireby infiltrated T cells into islets in both groups by RT-PCR from islets (International Immunology, in press).

我们从作为自身免疫性糖尿病（I 型）模型动物的非肥胖糖尿病 (NOD) 小鼠的胰腺中制备了冷冻切片，通过末端脱氧核苷酸转移酶介导的 dUTP 在非糖尿病 NOD 小鼠的胰岛中检测到凋亡细胞。切口末端标记（TUNEL）方法（内科进展专题，l998）。采用显微操作器的微解剖方法，以便为了分析细胞凋亡的发病机制，从胰腺切片中切下胰岛周围和胰岛内的胰腺β细胞或浸润淋巴细胞并提取mRNA。此外，我们分析了胰岛或淋巴细胞中的基因表达。例如，通过 RT-PCR 方法检测 IFN-γ、Fas、TNF 受体家族、iNOS 和 Bcl-2 家族。目前整理结果正在进行准备工作。我们用ELISA方法测定了人NK白血病患者的血清可溶性Fas和FasL(British J Haematology.1998)，还检测了NOD小鼠的血清可溶性Fas和FasL。采用类似的测量方法，以明确浸润淋巴细胞数量和CD40L、CD28、CD86阳性比例的差异。糖尿病 NOD 小鼠和非糖尿病 NOD 小鼠（40 周龄及以上）之间的细胞，对患有胰岛炎的胰岛细胞进行免疫组织化学检查，以了解两组之间的浸润淋巴细胞亚群。我们研究了 B 细胞缺陷型 NOD 小鼠或 B 细胞缺陷型链佐星诱导的糖尿病 AKR/J 小鼠与 B 细胞对照同窝小鼠之间细胞因子和 TCR 的差异。通过从胰岛进行 RT-PCR，将 T 细胞浸润到两组胰岛中，研究了细胞因子和 TCR 库（国际免疫学，出版中）。