中 文 摘 要

目的：經(jīng)實驗研究探討“補益陽明津氣”方藥益胃湯調控初老雌性大鼠生殖內(nèi)分泌與延緩衰老的作用機理，與前期研究相結合以共同佐證中醫(yī)傳統(tǒng)經(jīng)典理論“五七，陽明脈衰”在絕經(jīng)前期生殖軸機能衰老中的意義和作用。為延緩絕經(jīng)前期生殖軸機能衰老，促進絕經(jīng)前期婦女健康，防治相關病癥，提供一種新的研究與論治思路，并為研制相關方藥提供藥效學依據(jù)。

方法：4月齡~6月齡SD雌性大鼠為正常對照組；10月齡~12月齡，陰道細胞學表現(xiàn)動情期延長的SD雌性大鼠作為初老模型大鼠。模型動物隨機分為：(1)益胃湯高劑量組、(2)益胃湯中劑量組、(3)益胃湯低劑量組、(4)己烯雌酚組、(5)模型對照組。各組實驗動物灌藥4周后，斷頭處死，取部分下丘腦、垂體及左側卵巢組織固定切片，免疫組化法檢測ER和FSHR表達、GnRH；取剩余部分下丘腦、垂體及右側卵巢組織提取總RNA，用于RT-PCR檢測下丘腦GnRHmRNA，下丘腦、垂體的P16mRNA及下丘腦、垂體、卵巢的ERmRNA、FSHRmRNA基因表達；另外取部分大腦組織制作腦組織勻漿，用于SOD和GSH-PX酶活力以及MDA含量的測定。

結果：與模型對照組比較，“補益陽明津氣”方藥益胃湯在對生殖軸內(nèi)分泌調控方面：能增加下丘腦的GnRH及下丘腦、垂體、卵巢的ER和FSHR，并上調下丘腦的GnRHmRNA及下丘腦、垂體、卵巢的ERmRNA和FSHRmRNA的基因表達；僅高劑量組下丘腦和卵巢ERmRNA表達與正常對照組比較無統(tǒng)計學差異，其余均未達到正常對照組水平；衰老相關指標方面：減少初老雌性大鼠腦組織中升高的MDA含量，使其降低的SOD和GSH-PX酶活力升高，并可使降低的下丘腦、垂體P16mRNA基因表達升高。與正常對照組比較，高劑量組的SOD活力恢復到正常水平。

結論：“補益陽明津氣”方藥益胃湯能調節(jié)初老雌性大鼠與實驗研究所選擇的生殖內(nèi)分泌與衰老相關檢測指標，顯示出相應的改善生殖激素內(nèi)環(huán)境，上調腦組織抗氧化酶、下調過氧化物表達，調控衰老基因表達作用。提示益胃湯能夠通過減輕自由基損傷，對抗大腦衰老；通過阻斷細胞衰老進程，延緩下丘腦、垂體的衰老。從而改善生殖軸的內(nèi)分泌，起到延緩生殖軸機能衰老的作用。

關鍵詞：  補益陽明津氣    初老雌性大鼠    生殖內(nèi)分泌     延緩衰老    實驗研究

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 頁

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

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

目次頁··························································································· 5

英文縮略詞表················································································ 8

照片目錄······················································································· 9

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

實驗研究······················································································· 14

1.實驗研究材料············································································· 14

1.1研究對象·················································································· 14

1.2受試藥物·················································································· 14

1.3主要試劑和藥品······································································· 14

1.4主要儀器和設備······································································· 15

2.實驗方法····················································································· 16

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

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

2.1.2正常對照大鼠········································································ 17

2.2分組及給藥方法  ··································································· 17

2.3檢測項目·················································································· 18

2.3.1生殖內(nèi)分泌指標···································································· 18

2.3.2衰老相關指標········································································ 18

2.4組織標本的采集與處理方法···················································· 18

2.5檢測方法·················································································· 19

2.5.1免疫組化··············································································· 19

2.5.2RT-PCR實驗方法·································································· 20

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

2.6實驗資料的統(tǒng)計與處理··························································· 29

3.實驗結果····················································································· 30

3.1益胃湯對下丘腦GnRH及GnRHmRNA表達的影響·············· 30

3.2益胃湯對雌激素受體和卵泡刺激素受體的影響····················· 31

3.2.1益胃湯對下丘腦、垂體、卵巢ER及ERmRNA表達的影響·············· 32

3.2.2益胃湯對下丘腦、垂體、卵巢FSHR及FSHRmRNA表達的影響······ 37

3.3益胃湯對下丘腦、垂體p16mRNA表達的影響······················ 44

3.4益胃湯對腦組織SOD、GSH-PX、MDA的影響···················· 46

3.4.1益胃湯對腦組織SOD活性的影響········································ 46

3.4.2益胃湯對腦組織MDA含量的影響······································· 47

3.4.3益胃湯對腦組織GSH-PX活力的影響·································· 48

4.討  論························································································· 49

4.1模型動物的選擇······································································· 49

4.1.1實驗動物的選擇···································································· 49

4.1.2圍絕經(jīng)期模型動物的選擇····················································· 49

4.1.3本研究實驗動物模型的判定················································· 50

4.2補益陽明津氣的立法依據(jù)和處方原則···································· 51

4.2.1祖國醫(yī)學對 “陽明”的認識··················································· 51

4.2.2“陽明脈衰”與衰老的相關性·············································· 53

4.2.3未病先防，補益陽明，后天養(yǎng)先天····································· 54

4.2.4治療上以益胃生津為法························································ 55

4.3益胃湯對生殖內(nèi)分泌實驗檢測指標的影響····························· 57

4.3.1益胃湯對下丘腦GnRH及GnRHmRNA表達的影響············ 57

4.3.2益胃湯對雌激素受體和卵泡刺激素受體的影響·················· 58

4.4益胃湯對衰老相關指標的影響················································ 60

4.4.1益胃湯對P16mRNA表達的影響·········································· 60

4.4.2益胃湯對自由基的影響························································ 61

4.5益胃湯對生殖軸功能衰老的影響············································ 64

結語······························································································· 66

1.本課題研究思路和創(chuàng)新點·························································· 66

1.1課題研究思路·········································································· 66

1.2實驗研究思路·········································································· 66

1.3實驗研究結論·········································································· 68

2.存在的問題和展望····································································· 68

2.1存在的問題·············································································· 68

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

參考文獻······················································································· 71

致謝······························································································· 75

附錄1：

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

附錄2：

綜述：··························································································· 83

在校期間公開發(fā)表的學術論文、專著及科研成果······················· 108

聲明······························································································ 109

英文縮略詞表

照 片 目 錄

照片1.正常對照組下丘腦GnRH染色·········································· 76

照片2.模型對照組下丘腦GnRH染色·········································· 76

照片3.益胃湯高劑量組下丘腦GnRH染色·································· 76

照片4.正常對照組卵巢ER染色··················································· 76

照片5.模型對照組卵巢ER染色··················································· 77

照片6.己烯雌酚組卵巢ER染色··················································· 77

照片7.益胃湯高劑量組卵巢ER染色··········································· 77

照片8.益胃湯低劑量組卵巢ER染色··········································· 77

照片9.正常對照組卵巢FSHR染色·············································· 78

照片10.模型對照組卵巢FSHR染色············································ 78

照片11.己烯雌酚組卵巢FSHR染色············································ 78

照片12.益胃湯高劑量組卵巢FSHR染色····································· 78

照片13.益胃湯低劑量組卵巢FSHR染色····································· 79

照片14.正常對照組下丘腦ER染色············································· 79

照片15.模型對照組下丘腦ER染色············································· 79

照片16.己烯雌酚組下丘腦ER染色············································· 79

照片17.益胃湯高劑量組下丘腦ER染色······································ 80

照片18.正常對照組下丘腦FSHR染色········································ 80

照片19.模型對照組下丘腦FSHR染色········································ 80

照片20.益胃湯高劑量組下丘腦FSHR染色································· 80

照片21.正常對照組垂體ER染色················································· 81

照片22.模型對照組垂體ER染色················································· 81

照片23.己烯雌酚組垂體ER染色················································· 81

照片24.益胃湯高劑量組垂體ER染色········································· 81

照片25.正常對照組垂體FSHR染色············································ 82

照片26.模型對照組垂體FSHR染色············································ 82

照片27.己烯雌酚組垂體FSHR染色············································ 82

照片28.益胃湯高劑量組垂體FSHR染色····································· 82

引    言

人口老齡化是世界性的問題，據(jù)WHO報道，全球老年人口將從1998年的5.8億增加至2050年的20億，所占總人口的比例由20%增加到35%。據(jù)我國2000年統(tǒng)計，全國60歲以上老年人口系數(shù)為10.46%，標志著我國人口的年齡結構已進入“老年型”^[1]。大城市老齡化的情況出現(xiàn)更早，情況更加突出^[2]。婦女約占人口的一半，目前我國婦女已有五分之一左右步入圍絕經(jīng)期。其間女性開始逐步出現(xiàn)的圍絕經(jīng)期癥狀，不同程度的影響了女性的健康和生活質量。世界各國醫(yī)學和預防醫(yī)學組織已經(jīng)日益重視老年人的醫(yī)療和預防保健問題。1994年開羅世界人口和發(fā)展會議之后，生殖健康的策略從以往多注重于生育期母兒健康方面，轉向圍絕經(jīng)期的生殖健康問題。正如Nakajima指出的：“我們的目的不單純是關心她們的疾病問題，還應該使她們以后能在更高的生活質量中度過”^[3]。隨著社會文明和經(jīng)濟的持續(xù)發(fā)展、生活水平的提高、科學技術與國際的接軌及性別平等的倡導，女性對健康水平的追求越來越高。因此，注重生殖健康，延緩生殖軸機能的自然衰老，提高圍絕經(jīng)期婦女的生活質量是醫(yī)學界嶄新的具有積極作用和重大價值的研究課題。

圍絕經(jīng)期(perimenopause)是指自出現(xiàn)絕經(jīng)癥狀到停經(jīng)一年后的一段時間。其中從月經(jīng)周期出現(xiàn)明顯改變至絕經(jīng)前，稱絕經(jīng)過渡期(menopausal transition)。該期既是下丘腦-垂體-卵巢-子宮性生殖軸功能逐步衰老的時期，同時也是全身各器官和各系統(tǒng)發(fā)生一系列的生理性衰退，甚至是病理性變化的時期。圍絕經(jīng)期生殖軸機能的逐步衰老及其相應病理性變化的發(fā)生，主要由絕經(jīng)前期機體的“內(nèi)因”所決定和導致。中醫(yī)典籍自《黃帝內(nèi)經(jīng)》伊始，即對女性生、長、壯、老、已的全過程有了甚為明晰的分階段論述，其中《素問·上古天真論》“女子……五七，陽明脈衰……六七，三陽脈衰于上”^[4]是對35歲~42歲年齡段女性，機體內(nèi)環(huán)境病理生理變化具代表性的經(jīng)典認識。導師認為《內(nèi)經(jīng)》所云“五七”、“六七”的“陽明”、“三陽脈衰”不僅可引起原文所述顏面、肌膚、毛發(fā)的改變，而且會因“沖脈隸于陽明”的特定關系，導致月經(jīng)之本沖脈失于陽明津氣資助而虛衰，引發(fā)女性生殖機能的衰退。并因之逐漸形成“七七太沖脈衰少，天癸竭，地道不通，故形壞而無子也”，生殖機能的衰竭。故明代著名醫(yī)學家張景岳有“女為陰體，不足于陽，故其衰也，自陽明始”^[5]之說。因而，在圍絕經(jīng)期之絕經(jīng)前期階段，注重“陽明”進而“三陽”脈衰的中醫(yī)婦科學生理病理內(nèi)環(huán)境觀，應用補益陽明津氣之法，使陽明津氣充沛，“沖脈有所資助”，“先天得以充養(yǎng)”，既有助于延緩絕經(jīng)前期女性生殖軸機能的逐步衰老，又可起到預防圍絕經(jīng)期相關疾病發(fā)生的作用，此即中醫(yī)學“必先伏其所主”的用意。

衰老，是指在生命過程中，當生長發(fā)育達到成熟期以后，隨著年齡的增長，機體在形態(tài)結構與生理功能方面所呈現(xiàn)出的各種不利于自身的退行性變化，其中也包括絕經(jīng)前期女性生殖軸機能的衰老。絕經(jīng)前期時生殖功能減退出現(xiàn)較早，但在衰老征表現(xiàn)之前，衰老的機制早就發(fā)生了。部分學者認為，大腦是全身衰老的控制中心。腦細胞是不能進行有絲分裂的細胞，從出生到18歲左右，腦細胞數(shù)量變化不大。但自成年以來，腦細胞由于衰老死亡而逐漸減少，這是引起機體內(nèi)環(huán)境平衡失調和有關臟器功能低下的重要原因之一。1956年Harman提出了關于衰老機制的自由基學說，認為自由基及其誘導的氧化反應長期毒害可以導致機體衰老的發(fā)生，這一學說在現(xiàn)代衰老學中仍占有重要的地位。另外，內(nèi)分泌系統(tǒng)在維持動物機體內(nèi)環(huán)境穩(wěn)定和調節(jié)生長、發(fā)育與衰老過程中，具有重要作用。研究表明，下丘腦和垂體可能起衰老中心的作用，機體包括生殖軸在內(nèi)的眾多功能的衰退，都與下丘腦和垂體功能減弱，從而使機體控制內(nèi)環(huán)境平衡的能力下降相關^[6]。下丘腦-垂體軸隨年齡增長而發(fā)生的功能衰退可使其它內(nèi)分泌腺，包括性腺的功能都有所減退。并認為少數(shù)控制著機體全部生理功能的細胞（丘腦垂體軸）是不能為其它細胞所代替的，這些細胞受損是機體衰老的重要原因之一。

本實驗以中醫(yī)傳統(tǒng)理論為依據(jù)，通過對初老雌性模型大鼠（相類于絕經(jīng)前期）的研究，擬從調控生殖內(nèi)分泌和衰老相關指標兩方面入手，與前期“補益陽明津氣延緩初老雌性大鼠卵巢機能衰老的機理研究”及“補益陽明津氣方藥對雌性初老大鼠神經(jīng)免疫及生殖軸機能影響的實驗研究”相結合，共同驗證中醫(yī)學對絕經(jīng)前期“五七，陽明脈衰”“六七，三陽脈衰”特殊生理病理內(nèi)環(huán)境理論認識的科學性和實用性，并為延緩絕經(jīng)前期生殖軸機能衰老，提高圍絕經(jīng)期婦女生活質量、身體素質，預防和治療圍絕經(jīng)期相關疾病提示新的途徑與治療方藥。