| Abstract: | 為瞭解氧化壓力及糖化蛋白質在糖尿病之淋巴細胞功能低下所扮演的角色，本研究進行二部分實驗。第一個部分將Wistar大鼠依體重隨機分為三組，分別經由尾靜脈注射STZ(65 mg/kg)、載劑或於STZ誘發糖尿病後第三天起每日注射一次長效胰島素(4 units/rat)。於注射後一、二、四及八週犧牲並收集血液及免疫組織測定GSH、TBARS及Vitamin E含量，並測定頸部淋巴細胞之複製功能同時於第八週時以DTH確認細胞免疫反應，然後分析淋巴細胞功能與淋巴結氧化還原狀態的關係。實驗的第二個部分是以取自正常Wistar大鼠之淋巴細胞，觀察糖化蛋白質對淋巴細胞功能之影響。在STZ誘發糖尿病的動物模型觀察發現：八週期間內T-淋巴細胞之活性雖有略低於控制組的趨勢但無統計學上的意義。然而CNBD誘發之DTH結果顯示糖尿病顯著造成細胞免疫反應低下。整體而言，GSH含量在不同免疫組織中並無一致的時程變化趨勢且在不同組動物間此含量大多無顯著的差異。TBARS含量在糖尿病組血液中有隨時間逐漸高於控制組的趨勢而於免疫組織之含量則隨糖尿病進行而降低但與控制組間大多無顯著的差異。而在vitamin E含量方面，則不論在血液或免疫組織中糖尿病組均有高於控制組的趨勢，但各組間也大多無顯著差異。由統計學分析發現淋巴細胞功能變化與氧化壓力變化之間無顯著相關性。在in vitro實驗方面發現：將BSA與葡萄糖共同培養形成AGE-BSA產物添加於由正常Wistar大鼠製備淋巴細胞培養液中，並以Con A(0-50 ug/mL)剌激時，T-淋巴細胞的複製會受AGE-BSA之抑制且此具有AGE-BSA濃度依賴性。若於AGE-BSA形成期間有精胺酸存在則可降低此抑制作用，且此抑制作用之降低與先前存在AGE-BSA培養液中之精胺酸濃度有關。總結本研究結果，免疫組織之氧化壓力與糖尿病淋巴細胞功能低下無關而白蛋白糖化產物則是造成淋巴細胞功能低下的一個原因。利用有抑制糖化蛋白形成作用之營養素精胺酸可逆轉高葡萄糖存在下白蛋白糖化引起的免疫抑制作用。
In order to investigate roles of oxidative stress and glycosylated protein on lymphocyte dysfunction in diabetes, two different experiments were carried out in the present study. In the first experiment, Wistar rats were randomly assigned to three different groups by weight. Rats of the three groups were injected (i.v.) with STZ (65 mg/kg) and with or without further treatment with Lente insulin (4 units/rat/day) three days after the injection (insulin group or diabetes group, respectively) or injected with vehicle (control group). One, 2, 4 and 8 weeks after the injection, rats were killed. Samples of blood, spleen, thymus and cervical lymph nodes were collected for the determination of GSH, TBARS and vitamin E content. Lymph node lymphocytes were also prepared for the determination of proliferation rate. Rats were determined for their delayed type hypersensitivity (DTH) response to CDNB at the end of 8 weeks after the injection. In the second experiment, cervical lymph node lymphocytes were prepared from normal Wistar rats. Effects of BSA-AGE deritives on the proliferation function of these cells were investigated. In the study wiwth ST2-DM model, we found that, throughout the investigation period, proliferation rates of lymphocytes from DM groups were slightly lower than that from the control groups without statistic significance. However, the DTH response in DM group were significantly lower than the control at 8 weeks after the injection. This result indicated decreased cellular immunity induced by diabetes. GSH contents in various immune tissues from diabetic rats were similar to that from the controls. TBARS or vitamin E contents in blood samples from DM group tend to be higher or lower than that from the control, respectively, without statistic significance. Statistic analysis indicated that there is no correlation between oxidative stress in lymph node and the function of lymph node lymphocytes. In the in vitro study, the present study incubated BSA with glucose in the absence or presence of various concentrations of arginine. The BSA-AGE products generated were dialyzed and added to the culture medium of lymphocytes isolated from normal rats and stimulated with Con A(0-50 ug/mL). It was found that the proliferation rate of these cells were significantly suppressed by the AGE-BSA products formed in the absence of arginine in a dose dependent manner. The presence of arginine during the formation of AGE-BSA could reverse the effect of AGE-BSA products on lymphocytes and this effect is arginine concentration dependent. In conclusion, the present study did not find correlation between oxidative stress in lymph node and the proliferation function of cells prepare from the nodes. However, the presence of glycosylated albumin product was found to inhibit the function of lymphocytes and this can be reversed by the addition of arginine during the formation of such products. |
