【作者】 罗晓丽；

【导师】 杨侃； 孙明；

【作者基本信息】 中南大学 ， 内科学， 2009， 博士

【摘要】 第一章:不同剂量黄芪扶正汤对免疫抑制小鼠的溶血素和IL-2的影响目的:观察不同剂量的黄芪扶正汤对免疫抑制小鼠的免疫调节作用,寻找最佳的干预剂量。方法:取昆明种小鼠60只,随机分为6组,A组为空白对照组、B组为模型对照组、C组为9g生药/kg组、D组为18g生药/kg组、E组为36g生药/kg组、F组为72g生药/kg组,每组10只小鼠。除空白对照组外,其余小鼠在给药后第8、10、12天,腹腔注射氢化可的松25mg/kg/d,造出免疫抑制的动物模型。空白对照组和模型对照组予以生理盐水灌胃,其余组用不同剂量的黄芪扶正汤水煮液进行灌胃14天,检测小鼠血清溶血素的生成和IL-2的含量。结果:使用氢化可的松后,小鼠血清溶血素和IL-2的水平明显下降,与空白组比有统计学差异(P＜0.05)。药物组经黄芪扶正汤干预后,小鼠血清溶血素和IL-2水平在18g生药/kg的剂量开始升高,在36g生药/kg剂量时作用最强,差异有统计学意义(P＜0.05)。结论:1、使用氢化可的松造模成功。2、黄芪扶正汤的最佳干预剂量为36g生药/kg。第二章:黄芪扶正汤对免疫抑制小鼠的免疫调节作用目的:从不同方面观察黄芪扶正汤对免疫抑制小鼠的免疫调节作用。方法:取昆明种小鼠40只,随机分为4组,分别为空白组、模型组、黄芪扶正汤组和与玉屏风散组。除空白对照组外,其余小鼠在给药后第8、10、12天,腹腔注射氢化可的松25mg/kg/d,造出免疫抑制模型,空白对照组腹腔注射等量生理盐水。药物组小鼠灌胃给药14天,空白及模型对照组予等量生理盐水灌胃14天。给药后第15天,取小鼠血液和脾脏进行相关指标检测。采用TUNEL法检测小鼠脾细胞凋亡;免疫组化检测小鼠脾脏免疫细胞Bcl-2与Bax蛋白的表达;测定小鼠血清溶血素生成;ELISA法测血清中INF-γ、IL-4水平;流式细胞术测小鼠外周血T细胞亚群;RT-PCR和Realtime PCR方法测小鼠脾细胞INF-γ、IL-4、T-bet和GATA-3 mRNA表达。结果:1、模型组与空白组相比,脾细胞凋亡增多(P＜0.05),使用黄芪扶正汤和玉屏风散后凋亡减少,与模型组比有统计学意义,但组间也无差异。2、模型组与空白组相比,脾脏Bcl-2阳性细胞减少Bax阳性细胞增多(P＜0.05),经黄芪扶正汤和玉屏风散干预后,Bcl-2增加Bax减少(P＜0.05),但组间也无差异。3、模型组的血清溶血素低于空白组(P＜0.05),经黄芪扶正汤和玉屏风散干预后,溶血素较模型组明显升高(P＜0.05),但两组间差异无统计学意义。4、模型组与空白组相比,血清INF-γ下降、IL-4升高,差异有统计学意义,经黄芪扶正汤和玉屏风散干预后,INF-γ明显升高、IL-4下降(P＜0.05),且黄芪扶正汤比玉屏风散的效果更明显(P＜0.05)。5、模型组的CD3~+、CD4~+、CD8~+T细胞及CD4~+/CD8~+均明显低于空白组(P＜0.05),使用黄芪扶正汤或玉屏风散后上述指标均升高,差异有统计学意义,而黄芪扶正汤组比玉屏风散组升高更明显(P＜0.05)。6、RT-PCR和实时荧光定量PCR结果均显示,模型组与空白组对比,细胞因子INF-γmRNA表达下降、IL-4 mRNA表达增加,同时转录因子T-bet mRNA表达下降而GATA-3 mRNA表达升高,差异有统计学意义。黄芪扶正汤和玉屏风散均能升高INF-γ和T-bet的mRNA表达,降低IL-4和GATA-3的mRNA表达,使T-bet/GATA-3比值更接近正常,且黄芪扶正汤效果更显著,差异有统计学意义。。结论:1、黄芪扶正汤能提高免疫抑制小鼠的体液免疫功能,但与玉屏风散比无统计学差异。2、黄芪扶正汤能提高免疫抑制小鼠的细胞免疫功能,平衡Th1/Th2细胞因子的比例,效果优于玉屏风散。3、黄芪扶正汤调节Th1/Th2细胞因子比例的作用,可能部分源于对转录因子T-bet和GATA-3的mRNA表达的影响。第三章黄芪扶正汤对老年人免疫功能的影响目的:探讨黄芪扶正汤能否提高老年人的免疫和抗氧化功能。资料与方法:选择无严重疾病、能生活自理的老年人(年龄＞60岁)60例,男女各半。随机分为中药组和西药组,中药组服用黄芪扶正汤剂300ml每天分两次服用。西药组肌注卡介菌多糖核酸注射液(Polysaccharide nucleic acid fraction of BCG,BCG-PSN),商品名斯奇康针1mg/次,每周2次。两组均用药4周。干预前后分别抽取两组观察对象的静脉血标本,检测相关免疫学指标。检测方法为:使用ELISA法测血清中IgG、INF-γ和IL-4浓度,流式细胞术测人外周血T细胞亚群和NK细胞数量,黄嘌呤氧化酶法测人血清超氧化物歧化酶(SOD),比色法测人血清总抗氧化能力硫代巴比妥酸比色法测人血清微量丙二醛(MDA)。结果:1、斯奇康组研究对象失访率高于黄芪扶正汤组,差异有统计学意义。2、干预后,黄芪扶正汤组老年人血清IgG水平提高,与治疗前相比有统计学差异(P＜0.05)。但与斯奇康组对比无统计学差异。3、干预后,黄芪扶正汤组老年人血清INF-γ升高,IL-4下降,与治疗前相比均有统计学差异(P＜0.05)。但与斯奇康组对比无统计学差异。4、干预后,黄芪扶正汤组老年人全血CD4~+/CD8~+T细胞升高,与治疗前相比有统计学差异(P＜0.05)。而且治疗前后的差值高于斯奇康组,差异有统计学意义(P＜0.05)。5、干预后,黄芪扶正汤组全血CD56~+细胞数量升高,与治疗前相比有统计学差异(P＜0.05)。但与斯奇康组相比无差异。6、干预后,黄芪扶正汤组研究对象的血清SOD和总抗氧化能力水平较治疗前提高,差异有统计学意义(P＜0.05)。而阳性对照组(斯奇康组)与治疗前相比无明显改变(P＞0.05)。两组间治疗前后差值比较,黄芪扶正汤组显著高于斯奇康组,差异有统计学意义(P＜0.05)。7、干预后,黄芪扶正汤组研究对象的血清MDA水平较治疗降低,差异有统计学意义(P＜0.05)。而阳性对照组(斯奇康组)与治疗前相比血清MDA水平无明显改变(P＞0.05)。两组间治疗前后差值比较,黄芪扶正汤组显著高于斯奇康组,差异有统计学意义(P＜0.05)。结论:1、黄芪扶正汤能提高老年人的细胞免疫和体液免疫功能,增加外周血NK细胞数量,效果与斯奇康注射液无差异。2、黄芪扶正汤在调节Th1/Th2细胞因子的比例方面效果优于斯奇康注射液。3、黄芪扶正汤能改善氧化应激状态,这可能是其改善免疫衰老的机制之一。更多还原

【Abstract】 Chapter 1 Enhancement of immunity in the Immunosuppressive Mice by Different Dosage of Huangqi Fuzheng TangObjective:To study if Huangqi Fuzheng Tang could enhance the immunity in the immunosuppressive mice and find out the best dosage.Methods:Sixty male Kunming mice were divided into 6 groups: normal control group,model group,9g/kg,18g/kg,36g/kg and 72g/kg Huangqi Fuzheng Tang treatment groups.Set up the immunosuppressive animal model by using hydrocortisone.Different dosages of Huangqi Fuzheng Tang apozema were given to the treatment groups by intragastric administration once daily for 14 days.The normal control group and the model group received the same volume of normal saline.The contents of serum hemolysin and IL-2 of the mice were measured.Results:After giving hydrocortisone,compared to that of the normal control group,the serum hemolysin and IL-2 of the mice were decreased obviousely(P＜0.05).Huangqi Fuzheng Tang could elevate serum hemolysin and IL-2 when the dosages were 18g/kg,36g/kg and 72g/kg.The effect of 36g/kg was the strongest,the disparity had statistical significance.Conclusion:1.The immunosuppressive mice model was set up successfully by giving hydrocortisone intraperitoneal injection.2.The most effective dosage of Huangqi Fuzheng Tang was 36g/kg. Chapter 2 Enhancement of Immunity in the Immunosuppressive Mice by Huangqi Fuzheng TangObjective:To study the enhancement of immunity in the immunosuppressive mice by using Huangqi Fuzheng Tang.Methods:Forty mice were divided into 4 groups:normal control group,model group,Huangqi Fuzheng Tang(HFT) group and Yupingfeng San(YPF) group.Set up the immunosuppressive animal model by giving hydrocortisone intraperitoneal injection.Huangqi Fuzheng Tang and Yupingfeng San apozema were given to the mice of the treatment groups by intragastric administration once daily for 14 days.The normal control group and the model group received the same volume of normal saline. After 14 days the apoptosis of the spleen cells were assayed by using TUNEL staining.Bcl-2 and Bax proteins in the spleen were assayed by immunohistochemistry.The contents of hemolysin,IL-2、INF-γin the mice serum and the expression of CD3~+、CD4~+、CD8~+ in the peripheral blood lymphocytes were measured.RT-PCR and Realtime PCR were used to evaluate the mRNA of INF-γ、IL-4、T-bet and GATA-3 in the spleen cells.Results:1.Compared to that of the normal control group,the apoptosis of the spleen cells in mice of the model group was increased significantly(P＜0.05).After treatment,the apoptosis of the spleen cells decreased significantly(P＜0.05),but there was no difference between HFT group and YPF group(P＞0.05).2.The mice of the model group had less Bcl-2 positive cells and more Bax positive cells in the spleen than that of the normal group mice(P＜0.05).After treatment,Bcl-2 positive cells increased and Bax positive cells decreased in the mice of HFT group and YPF group(P＜0.05),but there was no statistical difference between the two groups.3.The serum hemolysin in the mice of the model group were lower than that of the normal group mice(P＜0.05).After treatment,the serum hemolysin elevated obviously in the mice of the HFT group and YPF group,but there was no statistical difference between the two groups.4.Compared to that of the normal control group,the model group had a lower level of serum INF-γ,but a higher level of serum IL-4(P＜0.05).Huangqi Fuzheng Tang could obviously elevate INF-γ,and descent IL-4,acted more effective than Yupingfeng San(P＜0.05).5.Huangqi Fuzheng Tang could elevate the ratio of CD4~+/CD8~+T cells,and there was statistical difference between Huangqi Fuzheng Tang and Yupingfeng San(P＜0.05).6.Both the RT-PCR and Realtime PCR revealed the Huangqi Fuzheng Tang could enhance the mRNA expression of INF-γ,and T-bet, but reduce the mRNA expression of IL-4 and GATA-3.And there was statistical difference between Huangqi Fuzheng Tang and Yupingfeng San.Conclusion:1.Huangqi Fuzheng Tang could enhance the humoral immunity,but there was no statistical difference between the two.2.Huangqi Fuzheng Tang could enhance the cellular immunity, balance the ratio of Th1/Th2 cytokines,and acted more effectively than Yupingfeng San in some aspects.3.Huangqi Fuzheng Tang regulated the Th1/Th2 cytokines might through regulating the transcriptional factor T-bet and GATA-3. Chapter 3 Immunomodulatory and Anti-oxidative Activity in the Elderly by Huangqi Fuzheng TangObjective:To investigate whether taking Huangqi Fuzheng Tang apozema daily could enhance immunity and have anti-oxidative effect in the elderly.Methods:Sixty elderly(age＞60 years) volunteers participated in this program.They were divided randomly into two groups:Huangqi Fuzheng Tang(HFT) group and the Polysaccharide nucleic acid fraction of BCG(BCG-PSN) group.Volunteers in the HFT group took Huangqi Fuzheng Tang apozema 300ml/day,and the BCG-PSN group received BCG-PSN injection 1mg i.m twice a week.The treatment continued for 4weeks.Before and after the treatment the immunological parameters of the subjects’ blood were examined.The parameters included serum IgG、INF-γand IL-4,expression of CD3~+、CD4~+、CD8~+、CD56~+in the peripheral blood lymphocytes.Serum concentration of SOD,total anti-oxidative capability and MDA were also measured.Results:1.The rate of dropping out was higher in the BCG-PSN group than that of the HFT group(P＜0.05).2.After the treatment,subjects’serum IgG elevated significantly in the HFT group than that before treatment(P＜0.05).But there were no statistical differences between the two groups.3.After the treatment,subjects’serum INF-γincreased and IL-4 decreased significantly in the HFT group than that before treatment(P＜0.05).But there were no statistical differences between the two groups.4.After the treatment,the ratio of CD4~+/CD8~+T cells of the elderly was higher than that before treatment(P＜0.05).And in HFT group that increased more obviously than that of BCG-PSN group(P＜0.05).5.Huangqi Fuzhegn Tang could elevate the CD56~+ cells in the blood, statistically different from before the intervention.But there was no statistical difference between Huangqi Fuzheng Tang and BCG-PSN.6.The levels of serum SOD and the total anti-oxidative capability in the HFT group increased significantly than that before the therapy(P＜0.05).But in the BCG-PSN group the levels of SOD and the total anti-oxidative capability were not changed before or after the treatment(P＞0.05).7.The subjects’serum MDA were lower than that before in the HFT group(P＜0.05).But in the BCG-PSN group there were no differences before or after the treatment(P＞0.05).Conclusion:1.Huangqi Fuzheng Tang could enhance the cellular immunity and the humoral immunity in the elderly,increase the amount of NK cells.2.Huangqi Fuzheng Tang could balance the ratio of Th1/Th2 cytokines,and had a better effect than BCG-PSN.3.Huangqi Fuzhegn Tang had anti-oxidative effect and improved the oxidative stress,that might be one of the anti-immunosenescence mechanisms.更多还原