| Part One The role of islet beta- cell apoptosis in the initiation andprogression of diabetes's in NOD miceObjective: To observe the natural incidence of diabetes and islet beta-cell apoptosis rule in non-obese diabetic (NOD) mice and explore the mechanism of P cell apoptosis in the diabetes' s initiation and progress in NOD miceMethods: The apoptotic islet cells, insulitis and Fas/FasL expression were observed in neonatal period and at 4-, 6-, 12-, 30-week-old NOD mice respectively. Apoptotic islet P cells were counting with TUNEL in situ end- labeling method combined with avidin-biotin complex (sABC) immunohistochemical method. Insulitis severity was scored by hermatoxylin and eosin (H-E) staining, and Fas/FasL expression on islets evaluated with sABC. The islets were isolated by stationary digestion after pancreatic ductal injection of collagenase solution, and purified with discontinuous Ficoll solution centrifugation. Serum IL-4 and IFN- r were measured by ELISA. Relative levels of I-A g7, IFN- r, IL-4, TGF- p, IL-1 , and Fas mRNA transcription were semi- quantitated by RT- PCR in islets of NOD mice at 12- week of age. Results: 1. The diabetes incidence in NOD female mice was higher than that in NOD male mice(84.6% vs 10.0%, P<0.001).2. Neonatal wave of P cell apoptosis exists in NOD mice and BalB/C mice. Beta-cell apoptosis preceded insulitis in NOD mice. The apoptotic beta-cell rates were similar in male and female NOD mice at neonatal period being both higher than that in BalB/ C mice. Fas on islets expressed in neonatal NOD mice, and FasL expression was higher in NOD mice than that in BalB/C mice.3. The rates of apoptotic P cells in female NOD mice were higher than those in the males at the age of 12 weeks. Fas expression on islets in NOD female mice was higher than that in males. There was no differences of FasL expression between males and females.4. The peri-insulitis emerged at 4 weeks of age, and intra-insulitis at 6 weeks of age in NOD mice. The insulitis aggravated at 12-week-old. There was no differences of insulitis scores between males and females. The positive correlations were found between the apoptotic P cell rates and the insulitis scores in female NOD mice at 6 and 12 weeks of age, but no such correlations in male mice.5. Serum IL-4 level and intraislet IL-4 mRNA levels were elevated, and serum IFN-r, intraislet Ia(I-A g7), IFN-r, IL-1, Fas mRNA levels decreased significantly in male NOD mice compared to those in females at 12 weeks of age. There was no differences of TGF- P mRNA expression between male and female NOD mice.Conclusion: CD Islet p cell apoptosis preceded insulitis, and neonatal p cell apoptosis wave may initiate the autoimmune response to P cells in NOD mice. The high rate of apoptotic p cells in NOD mice may be related to Fas expression in islets. And higher FasL expression in islets of BalB/C mice may protect p cells from autoimmune damage. (2) Autoimmune diabetes proceeded in an age-linked and gender-dependent manner in NOD mice. The diabetes incidence in male NOD mice was lower than that in females. This gender-dependent protection was related to the pattern of cytokines produced by p-cell-reative T cells from Thl( IFN- r) to Th2 (IL-4) profile, thus reducing IL-1 and Fas expression and inhibiting the Fas-mediated p-cell apoptosis. The insulitis of male NOD mice is often non-destructive. (3) Apoptosis is the main mode of islet P cell death in NOD mice, and P cell apoptosis plays an important role in the initiation and progressionof autoimmune diabetes.
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