关键词： 完全弗氏佐剂；胰岛炎；糖尿病；非肥胖糖尿病鼠；癌基因蛋白类；热休克蛋白类

【摘要】目的研究Bax蛋白和热休克蛋白(HSP)70在完全弗氏佐剂(CFA)预防NOD鼠胰岛炎和糖尿病中作用及意义。方法给4周龄雌性NOD鼠后脚板注射50μl CFA和生理盐水(NS组)，于30周龄或糖尿病发生时处死，胰腺组织置10%中性福尔马林固定后常规制成石蜡切片，HE染色后行光镜下胰岛炎评分，ABC免疫组化染色Bax蛋白和HSP70并光镜下计数。结果CFA组鼠胰岛内炎计数和胰岛炎评分均数均明显低于NS组鼠(P＜0.01)；CFA组鼠无1只发生糖尿病，NS组鼠中有3只(60%)发生糖尿病；CFA组鼠胰岛细胞Bax蛋白阳性率明显低于NS组鼠，而HSP70阳性率明显高于NS组鼠，均有极显著性差异(P＜0.01)；胰岛炎分值与Bax蛋白阳性率呈正相关(r=0.613)，与HSP70阳性率呈负相关(r=-0.913)。结论CFA能减轻NOD鼠胰岛炎发生程度和预防糖尿病发生，CFA能抑制胰岛细胞Bax蛋白表达和诱导胰岛细胞HSP70表达。

Prevention of insulitis and diabetes by complete Freund's adjuvant in NOD mice

YANG Zhulin,WU Hanwen,ZHOU Zhiguang,et al

Research Laboratory of Hepatobiliary Disease,The Second Affiliated Hospital,Hunan Medical University,Changsha,410011

【Abstract】ObjectiveTo study the effects and significance of Bax protein and heat shock protein 70 (HSP70) in prevention of insulitis and diabetes by complete Freund's adjuvant (CFA) in female NOD mice.Methods5 female NOD mice of CFA-treated group were injected into the pad of hind foot with 50μl CFA and 5 female NOD mice of control group with 50μl normal saline (NS) at 4 weeks of age. They were sacrificed at 30th week of age or when diabetes occurred. The pancreatic tissues were routinely processed and HE-stained and scoring of insulitis was done under optical microscopy. The Bax oncoprotein and HSP70 were stained with ABC immunohistochemical method and the counts of positive cells in islets were finished under high power field.ResultsThe incidence of in-insulitis and the average score of insulitis were significantly lower in CFA-treated mice than that in NS-treated mice (P＜0.01). None of CFA-treated mice and 3 out of 5 NS-treated mice developed diabetes. The positive rate of Bax oncoprotein in islet cells was significantly lower in CFA-treated mice than that in NS-treated mice, the positive rate of HSP70 in islet cells was significantly higher in CFA-treated mice than that in NS-treated mice. Positive correlation was found between the positive rate of Bax oncoprotein in islet cells and the score of insulitis (r=0.613), and negative correlation between positive rate of HSP70 in islet cells and the score of insulitis (r=-0.913) was also revealed.ConclusionCFA can reduce insulitis severity and decrease incidence of diabetes in NOD female mice. CFA is able to inhibit the expression of Bax oncoprotein and induce the expression of HSP70 in islet cells.

【Key words】Complete Freund's adjuvantInsulitisDiabetes mellitusNOD mouseOncoproteinsHeat shock proteins

(Chin J Endocrinol Metab, 1999,15:271-274)

NOD鼠是一种遗传性1型糖尿病的啮齿类动物模型，其发病机理为T细胞介导的胰岛β细胞进行性破坏和数量减少^〔1〕。完全弗氏佐剂(complete Freund's adjuvant, CFA)为一种免疫调节剂，近年发现CFA能通过免疫调节作用预防NOD鼠胰岛炎和糖尿病的发生，但CFA作用机理尚未完全清楚^〔2，3〕。Bax癌基因蛋白为促细胞凋亡蛋白^〔4〕；热休克蛋白70(heat shock protein 70, HSP70)为一种保护性蛋白，具有抑制细胞凋亡作用^〔5〕。本文应用免疫组化方法研究胰岛细胞Bax蛋白和HSP70的表达及意义。

材料和方法

一、材料

1.实验动物：NOD鼠从澳大利亚WEHI医学研究中心引进，由山西医科大学张志利教授提供。所有小鼠在标准饲养环境下喂养，自由饮净化水和全价营养颗粒饮食。本实验采用的NOD雌鼠群落在30周龄时糖尿病自然发病率为70%。

2.动物实验：10只4周龄NOD雌鼠随机分成CFA组和生理盐水组(NS组)，每组5只。CFA组给雌鼠后脚板注射50μl CFA，NS组注射等量NS。当发生糖尿病时或30周龄时处死小鼠，钝性分离全胰腺，置10%中性福尔马林液固定24～48小时，常规制成石蜡包埋切片，厚4μm。

3.主要试剂：CFA由美国生命技术公司进口，鼠抗人HSP70单抗(1/100)、兔抗人Bax蛋白多抗(1/100)、生物素标记羊抗鼠IgG或羊抗兔IgG(1/300)和ABC试剂盒(1/500)均购自Sigma公司，DAB-HCl为瑞士进口分装。

二、方法

1.胰岛炎评分：每例胰腺切2张不同切片(第一张切片后40μm再切第2张切片)，常规HE染色后光镜观察胰岛炎情况。参照Guytigco和Tisch评分标准^〔6，7〕，每例鼠胰2张不同切片在低倍光镜随机移动，随机找到胰岛后高倍镜观察胰岛炎情况并评分，共观察10个不同胰岛，所得值0分示正常胰岛，1～2分示胰岛周围炎，3～4分示胰岛内炎(图1，2)。

图1CFA组鼠，胰岛炎评分0分，属正常胰岛。HE染色，×100

Fig 1Zero score of insulitis in CFA mice. Normal islet. HE stain, ×100

图2NS组鼠，胰岛炎评分4分，属胰岛内炎。HE染色，×100

Fig 2Score 4 insulitis in NS mice. In-insulitis HE stain, ×100

2.糖尿病检测：从10周龄始观察NOD鼠是否有糖尿病症状出现，当出现多饮、多尿、消瘦等症状时，用断尾法取血，用血糖仪测血糖，如果连续2天血糖值＞11.1mmol/L时诊断为糖尿病。无糖尿病症状小鼠处死前均用上法测血糖值。

3.胰岛细胞Bax蛋白和HSP70蛋白阳性细胞计数：Bax蛋白和HSP70染色方法均为ABC免疫组化法，阳性细胞胞浆呈棕黄色，核呈兰色(复染苏木素)(图3，4)。两位观察者在低倍镜下随机移动切片观察到胰岛后转至高倍镜计数胰岛Bax蛋白和HSP70阳性细胞数及胰岛细胞总数，阳性细胞率=阳性细胞数/胰岛细胞总数，两位观察者在同一切片随机各计数5个胰岛的阳性细胞率，取其两者阳性细胞率均值，即为该例鼠胰阳性细胞率。用试剂盒阳性切片作为阳性对照，用0.01mol/L PBS(pH 7.5)替代一抗作为每次染色的阴性或替代对照。

图3胰岛细胞HSP70阳性细胞率为90.8%，胰岛炎评分均为0分，CFA组鼠。 ABC免疫组化法，×100

Fig 3Zero score of insulitis in CFA mice. Positive rate of Hsp70 in islet cells is 90.8%. ABC immunohistochemical method, ×100

图4胰岛细胞Bax蛋白阳性率为68.5%，胰岛炎评分4分，NS组鼠。 ABC免疫组化法，×100

Fig 4Score 4 insulitis in NS mice. Positive rate of Bax protein in islet cell is 62.5%. ABC immunohistochemical method, ×100

4.统计学处理：t检验、χ²检验或直接概率法，相关分析采用直线相关回归分析法。

结果

一、胰岛炎发生程度和糖尿病发生率

5只CFA组鼠4只发生胰岛周围炎(单个鼠胰胰岛炎均值≤2)和1只发生胰岛内炎(单个鼠胰胰岛炎均值≥2)，5只NS组鼠1只发生胰岛周围炎和4只发生胰岛内炎，CFA组鼠发生胰岛内炎鼠数明显低于NS组鼠，但无显著差异(P=0.103)。50个CFA组鼠胰岛中正常胰岛和胰岛周围炎数为36个(72%)，50个NS组鼠胰岛中正常胰岛和胰岛周围炎数为10个(20%)，两者存在极显著差异(P＜0.01)。CFA组鼠胰岛炎评分均数明显低于NS组鼠，有极显著差异(P＜0.01)。5只NS组鼠中有3只发生糖尿病，5只CFA组鼠中无1只发生糖尿病，NS组鼠糖尿病发生率高于CFA组鼠，但无显著差异(P=0.083)。

二、胰岛细胞Bax蛋白和HSP70表达

CFA组鼠胰岛细胞Bax蛋白阳性率明显低于NS组鼠(P＜0.01)，但HSP70阳性细胞率明<