【作者】 刘建民；

【导师】 王华；

【作者基本信息】 湖北中医学院 ， 针灸推拿学， 2009， 博士

【摘要】 目的:深入观察电针关元、足三里对D-半乳糖衰老模型大鼠的神经-内分泌-免疫网络相关物质的调节效应,探索针灸调控T细胞免疫延缓衰老的科学方法和作用机制。方法:立足中医和现代医学对于衰老的科学认识,制作D-半乳糖致衰老大鼠80只（雌雄各半）作为衰老模型,随机分组设衰老模型（Model）组×2、衰老模型电针（EA）组×2、衰老模型免疫抑制（ISU）组、衰老模型电针加免疫抑制（EA+ISU）组、衰老模型米非司酮（RU486）组、衰老模型米非司酮加电针（RU486+EA）组,每组各10只,另以3月龄大鼠作为正常青年对照（Normal）组10只（雌雄各半）×2。采用电针（HANS,LH202型电针仪,连续波,频率2Hz,强度为1mA,通电15分钟）关元、后三里治疗,每日1次,每周日休息1天,共治疗27天;采用MTT法检测T细胞增殖能力,双抗夹心ELISA法检测脾脏T细胞诱生IL-2和IL-2R水平,放射免疫法检测血清IL-6、ACTH、CORT含量,流式细胞术检测血清CD8⁺CD28⁺T细胞表达,免疫组织化学ABC法检测海马区IL-6R、下丘脑β-end和CRH免疫阳性物表达,荧光定量逆转录-聚合酶链反应（RT-PCR）法检测海马和胸腺细胞GRmRNA表达等,分析各组大鼠免疫系统和神经内分泌系统相关指标的变化及联系。为探讨电针强壮穴的“治未病”效应,借助环磷酰胺腹腔注射,检验电针强壮穴（关元、足三里）对衰老机体的免疫抗病能力影响;另借助糖皮质激素受体拮抗剂米非司酮（RU486）皮下注射,探讨海马对下丘脑-垂体-肾上腺（HPA）轴的负反馈调节在电针调节免疫效应中的作用。结果:1.Model组T细胞增殖能力、T细胞分泌IL-2及IL-2R水平、T细胞表面抗原CD28表达均明显低于Normal组（P<0.01）;电针强壮穴治疗后,EA组T细胞增殖及诱生IL-2和IL-2R的水平、T细胞表面抗原CD28的表达均提高（P<0.01）;电针对衰老模型大鼠T细胞增殖能力的调节与IL-2及其受体、CD8⁺CD28⁺T细胞密度呈正相关;2.ISU组衰老大鼠经免疫抑制剂腹腔注射后,与Model组比较T细胞增殖指数、T细胞诱生IL-2、IL-2R分泌、CD8⁺CD28⁺T细胞密度明显降低(P<0.01,P_IL-2<0.05);EA+ISU接受电针处置后,上述指标与未电针的ISU组比较却有显著性差异（P<0.01,）与Model组比较未见明显差异（P>0.05）;3.Model组外周血IL-6的分泌和海马区IL-6R表达,比Normal组明显升高（P<0.01）,电针强壮穴后,在提高衰老模型大鼠T细胞增殖能力同时,能降低外周血IL-6分泌和海马IL-6R表达水平（P<0.01）;4.下丘脑弓状核β-end免疫组织化学结果,β-end免疫阳性物主要分布在下丘脑弓状核,高倍镜下神经元和部分神经胶质细胞胞浆内可见棕褐色阳性颗粒,神经元轴突免疫反应物呈串珠状。光镜下Model组神经元胞体数比Normal组分布少,经电针强壮穴处理的EA组呈现增多趋势;光密度（OD）值分析表明,与Normal组比较,Model组明显降低（P<0.01）;经电针处置后的大鼠EA组与Model组比较,β-end阳性反应物OD值明显升高（P<0.05）,与Normal组比较无明显差异性（P>0.05）。5.下丘脑CRH免疫组织化学检测结果,CRH免疫阳性物主要分布在弓状核、室旁核,其中室旁核比较明显,高倍镜下可见CRH免疫阳性物呈棕褐色,分布于大细胞神经元的胞浆内,经D-半乳糖处理造模的Model组比组胞浆内CRH明显减少,而经过电针强壮穴处理的EA组胞浆内CRH呈明显上升趋势;CRH免疫阳性物OD值分析表明,Model组OD值较Normal组大鼠明显减少（P<0.05）,经电针强壮穴治疗后的EA组灰度值显著升高（P<0.05）,EA组与Normal组之间OD值比较无统计学差异（P>0.05）;6.与Normal组比较,Model组T细胞增殖能力降低,其血清ACTH和CORT含量却明显升高（P<0.01）,而经实验处理的EA组ACTH、CORT含量明显降低,与Model组组间比较有显著性差异（P<0.01）;电针对衰老模型大鼠T细胞增殖效应的调节与电针对HPA轴ACTH、CORT的分泌水平呈负相关,与CRH无明显相关性;7.海马GRmRNA表达完整,未被RNA酶降解;基因扩增产物只有单一峰值,扩增产物具有特异性;目的基因产物片断长度与设计长度相吻合;与Normal组比较,在Model组T细胞增殖能力降低、血清CORT水平明显升高同时,海马GRmRNA表达明显降低（P<0.01）;经电针强壮穴处理后,T细胞增殖能力升高、血清CORT水平降低,EA组与Model组比较有显著性差异（P<0.01）,海马GRmRNA表达也明显升高（P<0.01）;RU486组海马GRmRNA表达比Model组明显降低（P<0.01,或P<0.05）;与RU486组比较,EA+RU486组海马GRmRNA表达有所增加,但组间无显著性差异（P>0.05）;EA+RU486组GRmRNA的表达与EA组相比明显减少（P<0.01）。结论:通过本课题研究,我们认为,电针强壮穴调控T细胞免疫延缓衰老的作用机制是多环节、多途径的:1电针强壮穴能通过提高衰老模型大鼠T细胞增殖、T细胞诱生IL-2和IL-2R的水平、CD8⁺T细胞表面CD28的表达,调节T细胞免疫功能延缓衰老;2电针强壮穴能从T细胞增殖能力、T细胞分泌IL-2和IL-2R的水平以及CD8⁺T细胞表面抗原CD28表达等方面对抗环磷酰胺的免疫抑制作用,采用这种以治为防的策略,能增强老年个体免疫防御能力,电针强壮穴延缓衰老具有“治未病”效应;3电针强壮穴增强T细胞免疫功能,与电针对神经内分泌系统调节有关:（1）电针强壮穴能降低外周血IL-6分泌和海马IL-6R表达,并增加海马GRmRNA表达,进而增强海马对HPA轴的负反馈调节,降低体内高水平ACTH、CORT对T细胞的免疫抑制;（2）电针能提高下丘脑β-end表达,可能与海马功能恢复,增强对下丘脑β-end的良性调节、降低β-end向外周过多分泌造成对T细胞免疫功能的抑制效果。同时可能与提高β-end对外周ACTH和GC的负反馈调节能力有关;4.RU486能阻断电针调控海马对HPA轴的负反馈调节效应,并影响电针对T细胞免疫增殖的疗效,推测海马GR是实现海马对HPA的负反赖鹘诤投韵虑鹉粤夹缘鹘凇⒓潭訲细胞免疫产生调控作用的重要物质。综上所述,我们认为电针强壮穴调控T细胞免疫是通过神经-内分泌-免疫网络多个环节实现的,海马GR是实现神经内分系统对T细胞免疫产生调控作用的重要物质。血清IL-6,海马IL-6R,下丘脑β-end以及下丘脑-垂体-肾上腺（HPA）轴上CRH、ACTH和CORT参与了这个网络的具体调节过程。本课题在探讨针灸延缓衰老的机制中,从T细胞免疫入手,从神经-内分泌-免疫调节（NIM）网络综合探讨针灸调节免疫延缓衰老的作用机制,为阐明针灸作用机制提供科学的实验依据,在针灸调节免疫延缓衰老的同类报道中尚不多见;课题采用提前电针干预衰老模型大鼠,然后对大鼠进行免疫抑制刺激,探讨了“逆针刺”（提前针刺）对衰老机体免疫能力的调节影响,证实“逆针刺”强壮穴可以提高衰老机体的免疫抗病能力,验证了针灸治未病以治为防的策略的可行性和科学性。本课题未能观察β-end在外周血液的分泌情况,对实验结论的进一步证实造成了欠缺,有待今后在相关实验中继续完善。更多还原

【Abstract】 With the increasing problem of aging of the population all over the world,it gets more and more urgent and important to gain deep insight into the mechanism of aging and anti-aging in order to improve the life quality of the elders.Acupuncture study is one of the most characteristic and typical subjects in Traditional Chinese Medicine,and it has accumulated valuable experiences in human health care with its long history.The thought of "Preventive Treatment" which was originated from the HuangDi NeiJing（Huangdi’s Classic of Internal Medicine） embodies the core values of the adjustment of the current medical purpose and the change of medical pattern.The theoretical system of "Preventive Treatment",which includes the ideas of " preventive treatment before disease onset, preventing the disease to change,and preventing relapse after restoration",has provided an important theoretical foundation and specific means for the diagnoses and treatment of the diseases and health care in modern medicine.The principle of this study is the Treatment Determination Based on Syndrome Differentiation and the thought of "Preventive Treatment" in TCM.The subjects of this study are D-galactose-induced aging rats.The instrument of the study is to use electro-acupuncture on Important Tonifying Points such as Guanyuan（CV4）,Zusanli（ST36） as intervention means based on Prof.Wanghua’s "Shuang Gu Yi Tong"（Tonifying congenital and acquired essence,eliminating pathogen acupuncture therapy）.The study analyzed the effect and mechanism of acupuncture on adjusting immunity and preventing aging in the aspect of T cell-mediated immunity,which can provide a new scientific evidence for acupuncture to prevent aging.PurposeTo investigate the working mechanism of electro-acupuncture on Guanyuan and Zusanli for anti-aging with respect to Neuroendocrine-immuuno-modulation network.Methods:Based on the scientific understanding of aging in Traditional Chinese Medicine and Modern Medicine,in the present study,80 D-galactose-induced aging rats were taken as an aging model which was then divided into 8 groups:Aging Group（Model）×2,Aging Electro-acupuncture Group（EA）×2,Aging Immuno- suppressive Group （ISU）,Aging Electro-acupuncture plus Immunosuppressive Group （ISU+EA）,Aging mifepristone groups（RU486）,RU486 plus Aging Electric-acupuncture Group（RU486 + EA）,each having 10 rats.20 three-month old rats were taken as the Control Group（Normal）×2（5 males and 5 females）.The study used electro-acupuncture （HANS,LH202-type electric apparatus,continuous wave,frequency of 2Hz,intensity of 1mA,electricity for 15 minutes） on Guanyuan（CV4） and Zusanli（ST36） points,once a day,six days a week, and the treatment period was 27 days.The study also used the methods to observe target-related changes of neuroendocrine and the immune system of rats in each group,such as radioimmunoassay（radioimmunity） for testing the reproductivity of T cells,flow cytometry,enzyme-linked immunosorbent assay （ELISA） for inducible level of Interleukin-2（IL-2） and Interleukin-2 receptor（IL-2R）,real time quantitative reverse transcription-polymerase chain reaction（RT-PCR）, radioimmunoassayfor the content of IL-6,ACTH,CORT in the serum, flow cytometry for the expression of CD8+CD28+T,ABC method of immunohistochemistry for expression of IL-6R,β-end and CRH, RT-PCR for the expression of GRmRNA,etc.In order to explore the efficiency of "preventive treatment theory", with the aid of intraperitoneal injection with cytoxan and the impact of the electro-acupuncture at important Tonifying Points on the capability of immune and anti-disease of the aging was examined in this study.In addition,the effect of hippocampus’ s negative feedback regulation under the adjustment of electric acupuncture in immune response on the hypothalamic-pituitary-adrenal（HPA） axis is investigated by injecting subcutaneously with glucocorticoid receptor,antagon,and Mifepristone（RU486）. Results:1.Thymus Cell（T Cell） indices,such as reproductivity,secretion level of Interleukin-2（IL-2） and Interleukin-2 receptor（IL-2R） and CD28 expression of surface antigen in Model Group,were found to be significantly lower than the Normal Group（P<0.01）.After electro-acupuncture treatment at Important tonifying points, Thymus Cell（T Cell） ’ s reproductivity,inducible level of Interleukin-2（IL-2） and Interleukin-2 receptor（IL-2R） and CD28 expression of surface antigen in EA Group were found to be significantly improved（P<0.01）.The adjustment of electric acupuncture to T Cell’ s reproductivity of EA Group was positively correlated to IL-2 and its receptor and the density of T cell （CD8+CD28+）.2.Compared with Model Group,T Cell’ s properties,such as proliferation index,inducible level of Interleukin-2（IL-2） and secretion level of Interleukin-2 receptor（IL-2R）,and T Cell’ s density（CD8+CD28+） in ISU Group after intraperitoneal injection with immunosuppressant decreased significantly(P<0.01,P_IL-2<0.05); while,the index levels mentioned above in EA+ISU Group after electro-acupuncture treatment had no significant differences compared with Model Group（P>0.05）,but had significant differences （P<0.01） compared with EA+ISU Group without electro-acupuncture treatment.3.IL-6 secretion of peripheral blood and IL-6R expression in the hippocampus area in Model Group were found to be significantly higher than that of the Normal Group（P <0.01）.Electro-acupuncture at Important tonifying points could improve the T Cell ’ s reproductivity in Model Group and at the same time reduced IL-6 secretion of peripheral blood and IL-6R expression levels in hippocampus area（P<0.01）4.The immunohistochemistry results ofβ-end immunohisto-chemistry of arcuate nucleus of the hypothalamus are as follows:β-end Immunoreactive material were mainly distributed in arcuate nucleus of the hypothalamus（nucleus arcuatus）,and it could be seen under the High-power microscope that tan Positive granules in the intracytoplasm of the nerve cell and part of the neuroepithelial cells,and immunoreactive objects of Axons of neurons presented the shape of a string of pearls.The number of neurons in Model Group under light microscope was less than that of the Normal Group,but it showed a trends to increase in EA Group after electro-acupuncture at Important tonifying points;OD value analysis showed that this number of Model group was significantly high compared with the Normal group,（P<0.01）;OD value ofβ-end positive reaction material in EA Group with electro-acupuncture treatment were significantly increased（P<0.05） compared with Model Group,while, there was no significant differences between that in EA Group and that in the Normal Group（P> 0.05）.5.The immunohistochemistry results of Hypothalamus orticotro-phin releasing hormone（CRH） are as follows:CRH immune was mainly distributed in the arcuate nucleus and periventricular nucleus. Especially in the periventricular nucleus CRH immunopositive material appeared to be in tan colour and distributed in the intracytoplasm of megacell nerve cell if a high-power microscope is used.The CRH in the intracytoplasm in D-galactose-induced Group reduced significantly compared with that in Nomal Group,while,CRH in EA Group with electro-acupuncture treatment tended to increase. OD value analysis showed that,the OD value of CRH immunopositive object was significantly reduced compared in the case of normal rats （P<0.05）;The value in EA Group with electro-acupuncture treatment was significantly increased（P <0.05）.There was no significant difference between the OD value of CRH immunoposi tive object in EA group and the Normal group（P>0.05）.6.Compared with the Nomal Group,T cell’s reproductivity in Model Group decreased,and the content of serum Adrenocorticotropic Hormone（ACTH） and corticosterone（CORT） were significantly increased（P<0.01）.The content of ACTH and CORT in EA Group was significantly decreased and it was significantly different from that in the Model Group（P<0.01）.The adjustment of electro-acupuncture to the effect of T Cell proliferation of rats in EA Group was negatively correlated to the secretion level of ACTH, CORT in HPA axis,and there was no any obvious relationship with CRH.7.The GRmRNA expression of Hippocampus were integrity,and had not degraded by RNA enzyme;gene amplification products only had one single peak and it had its own specificity;the length of fragment of the target gene product coincided with the designed length.Compared with the Normal Group,in Model Group T Cell reproductivity decreased,and serum CORT level was significantly increased.In the meanwhile,the GRmRNA expression of hippocampus were significantly decreased（P<0.01）;with the electro-acupuncture treatment,EA Group was significantly different from the Model Group（P< 0.01）,that is,T Cell reproductivity in EA Group increased,and serum CORT level decreased.In addition,the GRmRNA expression of hippocampus were significantly increased（P<0.01）;the GRmRNA expression of hippocampus were in RU486 Group were significantly decreased compared with the Model Group（P<0.01,or P<0.05）.the GRmRNA expression of hippocampus in EA+RU486 Group were increased compared with RU486 group,but there were no significant differences between these two groups（P>0.05）.GRmRNA expression in EA + RU486 Group were decreased significantly（P<0.01） compared with the EA Group. Conclusion: 1.Electro-acupuncture at Important tonifying points can improve proliferation of T cell,inducible level of IL-2 and IL-2R and T cell surface antigen expression of CD28.This implies that Electro-acupuncture at Important tonifying points can be used for anti-aging purpose through modifying immune function of T cell.2.Electro-acupuncture at Important tonifying points can be used to fight with Immuno-suppressive cytoxan through enhancing T Cell immune function in the aspects of T cell reproductivity,secretion level of IL-2 and IL-2R,and the T cell surface antigen expression of CD28,suggesting that this cure-to-prevent-disease strategy can enhance the immune defense ability of the elders,and electro-acupuncture at Important tonifying points on the old organism has the adjustment effect of "preventive treatment".3.Electro acupuncture at Important tonifying points enhancing T cell immune function is related to the adjustment of Electro acupuncture to the neuroendocrine system:（1） electro-acupuncture can reduce aging model rats’ IL-6R expression of hippocampus,and increased GRmRNA expression of hippocampus,and then enhance negative feedback regulation of the hippocampus in the HPA axis and reduce immune suppression of high-level ACTH and GC in serum.（2） electro-acupuncture can improve hypothalamic beta endorphin（β-end） expression and reduce the suppressive effect of T Cell immune function resulting from excessive secretion ofβ-end,which may be related to improving the negative feedback regulation capability ofβ-end on the peripheral high-level ACT and GC.4.RU486,to some extent,can block the electro-acupuncture on regulation of the hippocampus on the HPA axis negative feedback regulation,thus reducing the efficiency of electro-acupuncture on reproductivity of T cell.To sum up,electro-acupuncture on important tonifying points was proved to have the capability to improve the regulation of T cells immunological function and anti-aging,which was completed through Neuroendocrine-immuuno-modulation network,during the process, where serum IL-6,hippocampal IL-6R,hypothalamicβ-end,and the CRH,ACTH and CORT of hypothalamus-pituitary-adrenal（HPA） axis participated in the specific regulation of the network..The present study investigated the mechanism of electro-acupuncture in anti-aging purpose with reference to T cell-mediated immunity,Neuroendocrine-immuuno-modulation network and contributed to this research field with valuable experimental evidence.The general discussion from the Neuroendocrine-immuuno-modulation（NIM） on the network will have a certain degree of inspiration and innovative for finding out mechanism of acupuncture’ s immunological regulation.We stimulate the aging model in advance,which demonstrate the feasibility and science of the advance stimulate on important tonifying points can improve immuno-competence preventing disease.This study could not observe the secretion property ofβ-end in external blood.This should be recommended for the future study in this field.更多还原