完全弗氏佐剂抑制NOD鼠胰岛β细胞凋亡及其机制

Effect of complete Freund’s adjuvant on islet β cell apoptosis and its mechanism in non-obese diabetic mice

目的:探讨完全弗氏佐剂( complete Freund’s adjuvant, CFA)对非肥胖糖尿病( nonobese dia-betic, NOD)鼠胰岛β细胞凋亡及凋亡相关基因Fas,FasL和Bcl-x表达的影响。方法:将4周龄NOD雌鼠随机分为CFA组(n=5)和生理盐水( NS)对照组(n=5 ) ,给CFA组鼠后脚板注射50μLCFA,对照者鼠后脚板注射等量NS。监测血糖,若血糖连续2 d≥11.1 mmol /L即诊断为糖尿病。当NOD鼠发生糖尿病或至30周龄时,处死动物,取胰腺组织制成薄切片,HE染色观察胰岛炎,采用末端脱氧核苷酸转移酶介导的脱氧尿苷三磷酸缺口末端标记( TUNEL)及ABC免疫组织化学双标记染色观察并计数凋亡的胰岛β细胞,ABC免疫组织化学法染色观察并记数Fas,FasL和Bcl-x表达阳性细胞。结果:至NOD鼠30周龄时,CFA处理组鼠无1只发生糖尿病,对照组鼠有3只发生糖尿病;CFA处理组的胰岛炎积分低于NS对照组(1.820±0.962 vs. 3.020±1.040,P<0.05 ) ;CFA处理组胰岛β细胞凋亡率、Fas阳性细胞率、FasL阳性细胞率均低于NS对照组[ (10.2±2.8) % vs. (15.9±6.5) %,(54.9±14.5)% vs.(75.7±12.9) %,(20.3±10.4) % vs. (27.9±12.0) %,P<0.05] ,Bcl-x阳性细胞率高于NS对照组[ (74.9±10.7) % vs. (66.0±18.3) %,P<0.05]。结论:CFA能够减轻NOD鼠胰岛β细胞凋亡,其机制与减少促凋亡基因Fas和FasL表达及增加抑制凋亡基因Bcl-x表达有关。

Objective To investigate the effect of complete Freund＇ s adjuvant （ CFA ） on islet β cell apoptosis in preventing diabetes in non-obese diabetic （NOD） mice, and the influence on apoptotic related-gene expression. Methods Four-week-old female NOD mice were randomly divided into Group CFA （ n = 5 ） and Group saline （ NS ） control （ n = 5 ）. Mice in Group CFA were injected in the hind footpad with 50 μL CFA and mice in Group NS with 50 μL NS. Blood sugar was monitored and diabetes was diagnosed if blood sugar was higher than 11. 1 mmol/L for 2 continuous days in the NOD mice. The mice were sacrificed when diagnosed as diabetes or at 30 weeks of age. Pancreatic sections were made for： evaluation of insulitis severity with HE staining; counting of apoptotic β cells with the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling （TUNEL） method, and ABC immunohistochemical double labeling; counting of Fas, FasL and Bcl-x positive cells respectively with ABC immunohistochemical method. Results By 30 weeks of.age, none of the 5 CFA-treated mice, compared with 3 of the 5 control mice, had developed diabetes. The insulitis score was lower （1.820 ±0.962 vs. 3. 020 ±1.040, P〈0.05）,the rates of apoptotic β cells, Fas positive cells and FasL positive cells were lower [ （ 10.2 ± 2. 8 ） % vs. （15.9±6.5）%,（54.9±14.5）% vs. （75.7±12.9）%,（20.3±10.4）% vs. （27.9± 12.0）%, P 〈 0. 05）, and the Bcl-x positive cell rate was higher [（74. 9 ± 10. 7）% vs. （66.0 ± 18.3）%, P 〈0.05 ] in the CFA-treated group than those in the NS-treated group respectively. Conclusion CFA may inhibit β cell apoptosis in NOD mice by regulating Fas, FasL and Bcl-x expression on cells within islets.