中 文 摘 要

目的：经实验研究探讨“补益阳明津气”方药益胃汤调控初老雌性大鼠生殖内分泌与延缓衰老的作用机理，与前期研究相结合以共同佐证中医传统经典理论“五七，阳明脉衰”在绝经前期生殖轴机能衰老中的意义和作用。为延缓绝经前期生殖轴机能衰老，促进绝经前期妇女健康，防治相关病症，提供一种新的研究与论治思路，并为研制相关方药提供药效学依据。

方法：4月龄~6月龄SD雌性大鼠为正常对照组；10月龄~12月龄，阴道细胞学表现动情期延长的SD雌性大鼠作为初老模型大鼠。模型动物随机分为：(1)益胃汤高剂量组、(2)益胃汤中剂量组、(3)益胃汤低剂量组、(4)己烯雌酚组、(5)模型对照组。各组实验动物灌药4周后，断头处死，取部分下丘脑、垂体及左侧卵巢组织固定切片，免疫组化法检测ER和FSHR表达、GnRH；取剩余部分下丘脑、垂体及右侧卵巢组织提取总RNA，用于RT-PCR检测下丘脑GnRHmRNA，下丘脑、垂体的P16mRNA及下丘脑、垂体、卵巢的ERmRNA、FSHRmRNA基因表达；另外取部分大脑组织制作脑组织匀浆，用于SOD和GSH-PX酶活力以及MDA含量的测定。

结果：与模型对照组比较，“补益阳明津气”方药益胃汤在对生殖轴内分泌调控方面：能增加下丘脑的GnRH及下丘脑、垂体、卵巢的ER和FSHR，并上调下丘脑的GnRHmRNA及下丘脑、垂体、卵巢的ERmRNA和FSHRmRNA的基因表达；仅高剂量组下丘脑和卵巢ERmRNA表达与正常对照组比较无统计学差异，其余均未达到正常对照组水平；衰老相关指标方面：减少初老雌性大鼠脑组织中升高的MDA含量，使其降低的SOD和GSH-PX酶活力升高，并可使降低的下丘脑、垂体P16mRNA基因表达升高。与正常对照组比较，高剂量组的SOD活力恢复到正常水平。

结论：“补益阳明津气”方药益胃汤能调节初老雌性大鼠与实验研究所选择的生殖内分泌与衰老相关检测指标，显示出相应的改善生殖激素内环境，上调脑组织抗氧化酶、下调过氧化物表达，调控衰老基因表达作用。提示益胃汤能够通过减轻自由基损伤，对抗大脑衰老；通过阻断细胞衰老进程，延缓下丘脑、垂体的衰老。从而改善生殖轴的内分泌，起到延缓生殖轴机能衰老的作用。

关键词：  补益阳明津气    初老雌性大鼠    生殖内分泌     延缓衰老    实验研究

Abstract

Objects:  Explore the functional mechanism of herbs for“tonifying Fluid and Qi of Yang Ming” on reproduction endocrine and delaying aging of the primary senile rat, to testify the importance of TCM traditional classic theory “Yang Ming Meridian Declines during Wu Qi period” in premenopause, in company with research in prophase. This research may offer a new study and differentiational thought to delay the aging of H-P-O-A during premenopause, improve premenopausal women health, prevent and treat relative diseases. It also can offer pharmaco- dynamics evidence for manufacturing relative effective formulas.

Methods:  4~6 months old SD female rats were set to be normal control group and 10~12 months old SD female rats whose oestrus elongation were set as primary senile female rat model. Primary senile female rats were devided into 5 groups randomly as following: 1. High dose Yiweitang group; 2. Middle dose Yiweitang group; 3. Low dose Yiweitang group; 4. Diethylstilbestrol group; 5. Model control group. After 4 weeks intervention, get the left ovarie, a part of hypothalami and pituitary gland from head-off rat, and observe expression of ER, FSHR and GnRH by immune histochemistry method . Then extract RNA from the rihgt ovarie and the rest hypothalami and pituitary gland, so as to measure the expression of GnRHmRNA、ERmRNA、FSHRmRNA and P16mRNA by RT-PCR. And the cerebrum were made to homogenate to examine SOD, GSH-PX and MDA.

Results:  In modulating H-P-O-A aspect, compound Yiweitang which could tonify YangMing Fluid and Qi could up regulate GnRH and GnRHmRNA in hypothalami, also could up regulate ER、ERmRNA、FSHR and FSHRmRNA in ovary, hypothalami and pituitary gland. In delaying aging aspect, Yiweitang could decrease MDA , but increase SOD and GSH-PX in cerebrum,  up regulate P16mRNA in hypoth- alami and pituitary gland.

Conclusions:   The results showed that Yiweitang, which could tonify YangMing Fluid and Qi, could regulate the functional examination index of reproduction endocrine and delaying aging maker of primary senile rat which we selected to detect in this experiment.The result shows that it can ameliorat the internal hormone environment, up regulate antioxidase in cerebrum and P16mRNA in hypothalami and pituitary gland, down regulate peroxide in cerebrum. These effects may be the mechanism of Yiweitang delay the aging of  H-P-O-A.

Key Words: Tonify YangMing Fluid and Qi;  Primary senile female rat;  reproduction endocrine;  Delay aging;  Experiment research

目 次 页

中文摘要······················································································· 1

Abstract·························································································· 3

目次页··························································································· 5

英文缩略词表················································································ 8

照片目录······················································································· 9

引言······························································································· 11

实验研究······················································································· 14

1.实验研究材料············································································· 14

1.1研究对象·················································································· 14

1.2受试药物·················································································· 14

1.3主要试剂和药品······································································· 14

1.4主要仪器和设备······································································· 15

2.实验方法····················································································· 16

2.1模型的建立·············································································· 16

2.1.1初老大鼠模型········································································ 16

2.1.2正常对照大鼠········································································ 17

2.2分组及给药方法  ··································································· 17

2.3检测项目·················································································· 18

2.3.1生殖内分泌指标···································································· 18

2.3.2衰老相关指标········································································ 18

2.4组织标本的采集与处理方法···················································· 18

2.5检测方法·················································································· 19

2.5.1免疫组化··············································································· 19

2.5.2RT-PCR实验方法·································································· 20

2.5.3分光光度法··········································································· 24

2.6实验资料的统计与处理··························································· 29

3.实验结果····················································································· 30

3.1益胃汤对下丘脑GnRH及GnRHmRNA表达的影响·············· 30

3.2益胃汤对雌激素受体和卵泡刺激素受体的影响····················· 31

3.2.1益胃汤对下丘脑、垂体、卵巢ER及ERmRNA表达的影响·············· 32

3.2.2益胃汤对下丘脑、垂体、卵巢FSHR及FSHRmRNA表达的影响······ 37

3.3益胃汤对下丘脑、垂体p16mRNA表达的影响······················ 44

3.4益胃汤对脑组织SOD、GSH-PX、MDA的影响···················· 46

3.4.1益胃汤对脑组织SOD活性的影响········································ 46

3.4.2益胃汤对脑组织MDA含量的影响······································· 47

3.4.3益胃汤对脑组织GSH-PX活力的影响·································· 48

4.讨  论························································································· 49

4.1模型动物的选择······································································· 49

4.1.1实验动物的选择···································································· 49

4.1.2围绝经期模型动物的选择····················································· 49

4.1.3本研究实验动物模型的判定················································· 50

4.2补益阳明津气的立法依据和处方原则···································· 51

4.2.1祖国医学对 “阳明”的认识··················································· 51

4.2.2“阳明脉衰”与衰老的相关性·············································· 53

4.2.3未病先防，补益阳明，后天养先天····································· 54

4.2.4治疗上以益胃生津为法························································ 55

4.3益胃汤对生殖内分泌实验检测指标的影响····························· 57

4.3.1益胃汤对下丘脑GnRH及GnRHmRNA表达的影响············ 57

4.3.2益胃汤对雌激素受体和卵泡刺激素受体的影响·················· 58

4.4益胃汤对衰老相关指标的影响················································ 60

4.4.1益胃汤对P16mRNA表达的影响·········································· 60

4.4.2益胃汤对自由基的影响························································ 61

4.5益胃汤对生殖轴功能衰老的影响············································ 64

结语······························································································· 66

1.本课题研究思路和创新点·························································· 66

1.1课题研究思路·········································································· 66

1.2实验研究思路·········································································· 66

1.3实验研究结论·········································································· 68

2.存在的问题和展望····································································· 68

2.1存在的问题·············································································· 68

2.2展望·························································································· 69

参考文献······················································································· 71

致谢······························································································· 75

附录1：

照片······························································································· 76

附录2：

综述：··························································································· 83

在校期间公开发表的学术论文、专著及科研成果······················· 108

声明······························································································ 109

英文缩略词表

照 片 目 录

照片1.正常对照组下丘脑GnRH染色·········································· 76

照片2.模型对照组下丘脑GnRH染色·········································· 76

照片3.益胃汤高剂量组下丘脑GnRH染色·································· 76

照片4.正常对照组卵巢ER染色··················································· 76

照片5.模型对照组卵巢ER染色··················································· 77

照片6.己烯雌酚组卵巢ER染色··················································· 77

照片7.益胃汤高剂量组卵巢ER染色··········································· 77

照片8.益胃汤低剂量组卵巢ER染色··········································· 77

照片9.正常对照组卵巢FSHR染色·············································· 78

照片10.模型对照组卵巢FSHR染色············································ 78

照片11.己烯雌酚组卵巢FSHR染色············································ 78

照片12.益胃汤高剂量组卵巢FSHR染色····································· 78

照片13.益胃汤低剂量组卵巢FSHR染色····································· 79

照片14.正常对照组下丘脑ER染色············································· 79

照片15.模型对照组下丘脑ER染色············································· 79

照片16.己烯雌酚组下丘脑ER染色············································· 79

照片17.益胃汤高剂量组下丘脑ER染色······································ 80

照片18.正常对照组下丘脑FSHR染色········································ 80

照片19.模型对照组下丘脑FSHR染色········································ 80

照片20.益胃汤高剂量组下丘脑FSHR染色································· 80

照片21.正常对照组垂体ER染色················································· 81

照片22.模型对照组垂体ER染色················································· 81

照片23.己烯雌酚组垂体ER染色················································· 81

照片24.益胃汤高剂量组垂体ER染色········································· 81

照片25.正常对照组垂体FSHR染色············································ 82

照片26.模型对照组垂体FSHR染色············································ 82

照片27.己烯雌酚组垂体FSHR染色············································ 82

照片28.益胃汤高剂量组垂体FSHR染色····································· 82

引    言

人口老龄化是世界性的问题，据WHO报道，全球老年人口将从1998年的5.8亿增加至2050年的20亿，所占总人口的比例由20%增加到35%。据我国2000年统计，全国60岁以上老年人口系数为10.46%，标志着我国人口的年龄结构已进入“老年型”^[1]。大城市老龄化的情况出现更早，情况更加突出^[2]。妇女约占人口的一半，目前我国妇女已有五分之一左右步入围绝经期。其间女性开始逐步出现的围绝经期症状，不同程度的影响了女性的健康和生活质量。世界各国医学和预防医学组织已经日益重视老年人的医疗和预防保健问题。1994年开罗世界人口和发展会议之后，生殖健康的策略从以往多注重于生育期母儿健康方面，转向围绝经期的生殖健康问题。正如Nakajima指出的：“我们的目的不单纯是关心她们的疾病问题，还应该使她们以后能在更高的生活质量中度过”^[3]。随着社会文明和经济的持续发展、生活水平的提高、科学技术与国际的接轨及性别平等的倡导，女性对健康水平的追求越来越高。因此，注重生殖健康，延缓生殖轴机能的自然衰老，提高围绝经期妇女的生活质量是医学界崭新的具有积极作用和重大价值的研究课题。

围绝经期(perimenopause)是指自出现绝经症状到停经一年后的一段时间。其中从月经周期出现明显改变至绝经前，称绝经过渡期(menopausal transition)。该期既是下丘脑-垂体-卵巢-子宫性生殖轴功能逐步衰老的时期，同时也是全身各器官和各系统发生一系列的生理性衰退，甚至是病理性变化的时期。围绝经期生殖轴机能的逐步衰老及其相应病理性变化的发生，主要由绝经前期机体的“内因”所决定和导致。中医典籍自《黄帝内经》伊始，即对女性生、长、壮、老、已的全过程有了甚为明晰的分阶段论述，其中《素问·上古天真论》“女子……五七，阳明脉衰……六七，三阳脉衰于上”^[4]是对35岁~42岁年龄段女性，机体内环境病理生理变化具代表性的经典认识。导师认为《内经》所云“五七”、“六七”的“阳明”、“三阳脉衰”不仅可引起原文所述颜面、肌肤、毛发的改变，而且会因“冲脉隶于阳明”的特定关系，导致月经之本冲脉失于阳明津气资助而虚衰，引发女性生殖机能的衰退。并因之逐渐形成“七七太冲脉衰少，天癸竭，地道不通，故形坏而无子也”，生殖机能的衰竭。故明代著名医学家张景岳有“女为阴体，不足于阳，故其衰也，自阳明始”^[5]之说。因而，在围绝经期之绝经前期阶段，注重“阳明”进而“三阳”脉衰的中医妇科学生理病理内环境观，应用补益阳明津气之法，使阳明津气充沛，“冲脉有所资助”，“先天得以充养”，既有助于延缓绝经前期女性生殖轴机能的逐步衰老，又可起到预防围绝经期相关疾病发生的作用，此即中医学“必先伏其所主”的用意。

衰老，是指在生命过程中，当生长发育达到成熟期以后，随着年龄的增长，机体在形态结构与生理功能方面所呈现出的各种不利于自身的退行性变化，其中也包括绝经前期女性生殖轴机能的衰老。绝经前期时生殖功能减退出现较早，但在衰老征表现之前，衰老的机制早就发生了。部分学者认为，大脑是全身衰老的控制中心。脑细胞是不能进行有丝分裂的细胞，从出生到18岁左右，脑细胞数量变化不大。但自成年以来，脑细胞由于衰老死亡而逐渐减少，这是引起机体内环境平衡失调和有关脏器功能低下的重要原因之一。1956年Harman提出了关于衰老机制的自由基学说，认为自由基及其诱导的氧化反应长期毒害可以导致机体衰老的发生，这一学说在现代衰老学中仍占有重要的地位。另外，内分泌系统在维持动物机体内环境稳定和调节生长、发育与衰老过程中，具有重要作用。研究表明，下丘脑和垂体可能起衰老中心的作用，机体包括生殖轴在内的众多功能的衰退，都与下丘脑和垂体功能减弱，从而使机体控制内环境平衡的能力下降相关^[6]。下丘脑-垂体轴随年龄增长而发生的功能衰退可使其它内分泌腺，包括性腺的功能都有所减退。并认为少数控制着机体全部生理功能的细胞（丘脑垂体轴）是不能为其它细胞所代替的，这些细胞受损是机体衰老的重要原因之一。

本实验以中医传统理论为依据，通过对初老雌性模型大鼠（相类于绝经前期）的研究，拟从调控生殖内分泌和衰老相关指标两方面入手，与前期“补益阳明津气延缓初老雌性大鼠卵巢机能衰老的机理研究”及“补益阳明津气方药对雌性初老大鼠神经免疫及生殖轴机能影响的实验研究”相结合，共同验证中医学对绝经前期“五七，阳明脉衰”“六七，三阳脉衰”特殊生理病理内环境理论认识的科学性和实用性，并为延缓绝经前期生殖轴机能衰老，提高围绝经期妇女生活质量、身体素质，预防和治疗围绝经期相关疾病提示新的途径与治疗方药。