脾肾双补法对衰老模型小鼠免疫功能的作用研究

【作者】 毛珺；

【导师】 陈刚；

【作者基本信息】 湖北中医学院 ， 中医基础理论， 2007， 硕士

【摘要】 目的本文通过用D-半乳糖背部注射建立衰老模型，用现代药理学和免疫学方法观察脾肾双补治法对实验小鼠脾脏、胸腺组织内SOD、MDA、IL-2含量、脾脏淋巴细胞CD28的表达，脾脏和胸腺细胞DNA氧化损伤的影响，探讨该治法对衰老的作用机制，为廷缓衰老和防治老年性疾病实践开拓了思路。方法2月龄昆明种小鼠60只，20±2g，随机分为6组：正常组、模型组、VitE组、脾肾双补法方治疗大剂量组(简称治疗大剂量组)、脾肾双补法方中剂量组(简称治疗中剂量组)，脾肾双补法方小剂量组(简称治疗小剂量组)。采用单细胞凝胶电泳法检测脾肾双补法对脾脏和胸腺细胞DNA氧化损伤修复的影响，取脾脏胸腺组织匀浆分别检测SOD、MDA含量，脾脏匀浆检测IL-2、CD28的表达。结果模型组彗星细胞尾长与正常组脾脏细胞相比具有显著性差异(P＜0.01)，VitE组和中药各组的彗星拖尾长度均较模型组减短，有显著性差异(P＜0.01)，与VitE组比较，大、中剂量组有显著性差异(P＜0.01)，与小剂量组无差异，与大剂量组比，中、小剂量组均有显著性差异(P＜0.01)。与正常组比较，模型组胸腺细胞尾长有显著性差异(P＜0.01)。与模型组比较，中、大剂量组均有显著性差异(P＜0.01)，VitE组和小剂量组有差异(P＜0.05)，与VitE组相比，大剂量组有显著性差异(P＜0.01)，中剂量组有差异(P＜0.05)，小剂量组无差异。与大剂量比较中、小剂量均有显著性差异(P＜0.01)。模型组脾脏组织SOD含量与各组相比均有显著性减少(P＜0.01)。与VitE组比较，大、中剂量组均有显著性差异(P＜0.01)，小剂量与VitE组无差异(P＞0.05)，与大剂量组比较，中剂量组有差异(P＜0.05)，小剂量组有显著性差异(P＜0.01)。与正常组比较，模型组胸腺组织SOD含量有显著性差异(P＜0.01)。与模型组相比，余胸腺各组均于之有显著性差异(P＜0.01)，与VitE组相比，大剂量有显著性差异(P＜0.01)，中剂量有差异(P＜0.05)，小剂量无差异。与大剂量组比较，小剂量组有差异(P＜0.05)，中剂量组无差异。模型组MDA含量明显高于各组脾脏中MDA的含量(P＜0.01)。与VitE组比较，中、大剂量有显著性差异(P＜0.01)，小剂量有差异(P＜0.05)，与大剂量组比较，小剂量有显著性差异(P＜0.01)，中剂量有差异(P＜0.05)。与正常组比较，模型组胸腺中MDA含量有显著性差异(P＜0.01)。模型组中MDA含量明显高于胸腺各组(P＜0.01)，与VitE组比较，中剂量有差异(P＜0.05)，大剂量有显著性差异(P＜0.01)，小剂量与VitE组无差异(P＞0.05)。与大剂量组比较，小剂量有显著性差异(P＜0.01)，中剂量无差异。正常组、大、中剂量组IL-2水平与模型组相比均有显著性提高(P＜0.01)，VitE组和小剂量与之有差异(P＜0.05)。与VitE组比较，大、中剂量均有差异(P＜0.05)，小剂量组无差异(P＞0.05)。与大剂量组比较，小剂量有显著性差异(P＜0.01)，中剂量无差异。CD28在正常组和大、中、小剂量组的表达与模型组相比均有显著性提高(P＜0.01)，VitE组与之无差异(P＞0.05)。与VitE组比较，大、中剂量组有显著性差异(P＜0.01)，小剂量有差异(P＜0.05)。与大剂量组比较，中、小剂量组无差异(P＞0.05)。结论本实验采用单细胞凝胶电泳技术，研究了脾肾双补法对D-半乳糖衰老模型小鼠脾脏和胸腺细胞的DNA氧化损伤的影响。发现脾肾双补法方可以减少彗星拖尾长度，增强DNA修复能力，使衰老时DNA修复的能力明显提高，脾肾双补法能有效的廷缓衰老。实验结果显示，脾肾双补法可以明显提高衰老模型小鼠中SOD的活性，提示可以通过增强SOD活力而提高对自由基清除的能力，减少自由基的损害而廷缓衰老，同时降低MDA的含量，降低过脂质过氧化的程度，延缓衰老。实验结果表明脾肾双补药治疗组可提高衰老机体IL-2的水平，可能是通过提高IL-2水平来发挥IL-2重要的免疫活性，从而调节免疫功能而延缓衰老。实验结果表明，衰老模型小鼠脾脏淋巴细胞CD28比例低于正常组，提示衰老可能使CD28分子对衰老小鼠淋巴细胞的重要活化作用下降，而中药各剂量组表达水平较衰老模型组增高，提示脾肾双补法可以提高衰老模型小鼠脾脏淋巴细胞CD28比例，作用机制可能是其提高了IL-2的水平刺激淋巴细胞增殖，从而增强了机体调节CD28分子的表达水平。更多还原

【Abstract】 ObjectiveThis essay established senile model induced by injection of back D-gal to rats .Through the pharmacology and immunology research technique. This experiment is to studied the effect of "pi shen shuang bu " therapy on change of SOD ,MDA, IL-2, CD28 for analyzing the mechanism and action of this therapy for senility. The effects of PSSB therapy on oxygen-radical-generated DNA damage in senile rats spleen and chest gland cells .Discuss the effects of this therapy for delay senility. This will provide new prevention and cure for old age diseaseMethodsSixty normal rats,2-months in age,20±2g, randomly divided into six groups: normal control group, model group, high dosage PSSBP-treated group(high dosage group),middle dosage PSSBP-treated group(middle dosage group), low dosage PSSBP-treated group(low dosage group)and VitE medicine control group.Through with single cell gel eletrophoresis (comet assay) studied the effects of PSSB therapy on oxygen-radical-generated DNA damage in senile rats spleen and chest gland cells .Detection the content of SOD MDA in spleen , chest gland and level of IL-2,CD28 in spleen.ResultsThe rats′comet cell tail length between model group and spleen normal group had the remarkable difference (P<0.01) .Spleen cell in VitE medicine control group and every dosage PSSBP-treated group between the model group had the remarkable difference (P<0.01). Compared with VitE medicine control group, middle dosage group and high dosage group had the remarkable difference (P<0.01). Low dosage group did not have difference .Compared with high dosage group, middle dosage group and low dosage group had the obvious difference (P<0.01). The rats’ comet cell tail length between model group and chest gland normal group had the remarkable difference ( P<0.01 ) .The comet cell tail length in middle dosage group and high dosage group had the remarkable difference( P<0.01 ), VitE medicine control group and low dosage group had the difference ( P<0.05 ) , Compared with VitE medicine control group, chest gland cell in high dosage group had the remarkable difference( P<0.01 ), middle dosage group had the difference ( P<0.05 ) , low dosage group had no difference.Compared with high dosage group, low and middle dosage group had the remarkable difference( P<0.01 ).The content of SOD in spleen cell between model group and others group had the remarkable difference( P<0.01 ). Compared with VitE medicine control group, middle dosage group and high dosage group in spleen cell had the remarkable difference( P<0.01 ),low dosage group had no difference . Compared with high dosage group, spleen cell in middle dosage group had the difference ( P<0.05 ) , low dosage group had the remarkable difference( P<0.01 ). Compared with model group, chest gland cell in every group had the remarkable difference ( P<0.01 ).Compared with VitE medicine control group, chest gland cell in high dosage group had the remarkable difference( P<0.01 ), middle dosage group had the difference ( P<0.05 ) , low dosage group did not have difference. Compared with high dosage group, low dosage group had the remarkable difference( P<0.01 ), middle dosage group did not have obvious difference.The changes of MDA in model group was higher than the other groups in spleen cell obviously( P<0.01 ), compared with VitE medicine control group, middle dosage group and high dosage group had the remarkable difference( P<0.01 ), low dosage group had the difference ( P<0.05 ) , Compared with high dosage group, low dosage group had the remarkable difference( P<0.01 ) middle dosage group had the difference ( P<0.05 ). The changes of MDA in model was higher than the other groups in chest gland cell had the obvious difference ( P<0.01 ), compared with VitE medicine control group, middle dosage group had the difference (P<0.05) , high dosage group had the obvious difference ( P<0.01 ), low dosage group did not have obvious difference. Compared with high dosage group, low dosage group had the remarkable difference (P<0.01), middle dosage group did not have obvious difference.The level of IL-2 in model group, high and middle dosage group were obvious higher than model group( P<0.01 ), VitE medicine control group and low dosage group had the difference ( P<0.05 ). Compared with VitE medicine control group, high group and middle dosage group had the difference ( P<0.05 ) low dosage group did not have obvious difference. Compared with high dosage group, low dosage group had the remarkable difference (P<0.01), middle dosage group did not have obvious difference.The level of CD28 in model group, high and middle dosage group were obvious higher than model group( P<0.01 ),VitE medicine control group did not have obvious difference. Compared with VitE medicine control group, high group and middle dosage group had the obvious difference ( P<0.01 ), low dosage group had the difference ( P<0.05 ) Compared with high dosage group, middle and low group did not have obvious difference.ConclusionThis experiment through with single cell gel eletrophoresis (comet assay) studied the effects of PSSB therapy on oxygen-radical-generated DNA damage in senile rats spleen and chest gland cells. The result showed that the Chinese native medicine may reduce tail length of the comet cell and offered significant DNA protective effects. Obviously increase the DNA protective effects on senile process and delay senility.After the treatment ,the Chinese native medicine may increase the level of SOD in model group , The result showed that SOD may increase capability of eliminate Free Radical and delay senility. The Chinese native medicine may reduce the level of MDA and delay senility.After the treatment, The Chinese native medicine may increase the activity of IL-2 in senile organism. Through increase the IL-2′important immunity function and then delay senility.The experiment showed that the level of CD28 in model group was lower than normal group. The result may showed that senility reduce CD28′important immunity function in activity of T cells. The Chinese native medicine may increase the level of CD28.The mechanism of this may that CD28 have very important in regulating aged T cell function, and then delay senility.更多还原