中 文 摘 要

目的：經(jīng)實(shí)驗(yàn)研究探討“補(bǔ)益陽(yáng)明津氣”方藥益胃湯調(diào)控初老雌性大鼠生殖內(nèi)分泌與延緩衰老的作用機(jī)理，與前期研究相結(jié)合以共同佐證中醫(yī)傳統(tǒng)經(jīng)典理論“五七，陽(yáng)明脈衰”在絕經(jīng)前期生殖軸機(jī)能衰老中的意義和作用。為延緩絕經(jīng)前期生殖軸機(jī)能衰老，促進(jìn)絕經(jīng)前期婦女健康，防治相關(guān)病癥，提供一種新的研究與論治思路，并為研制相關(guān)方藥提供藥效學(xué)依據(jù)。

方法：4月齡~6月齡SD雌性大鼠為正常對(duì)照組；10月齡~12月齡，陰道細(xì)胞學(xué)表現(xiàn)動(dòng)情期延長(zhǎng)的SD雌性大鼠作為初老模型大鼠。模型動(dòng)物隨機(jī)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(duì)照組。各組實(shí)驗(yàn)動(dòng)物灌藥4周后，斷頭處死，取部分下丘腦、垂體及左側(cè)卵巢組織固定切片，免疫組化法檢測(cè)ER和FSHR表達(dá)、GnRH；取剩余部分下丘腦、垂體及右側(cè)卵巢組織提取總RNA，用于RT-PCR檢測(cè)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(dá)；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(cè)定。

結(jié)果：與模型對(duì)照組比較，“補(bǔ)益陽(yáng)明津氣”方藥益胃湯在對(duì)生殖軸內(nèi)分泌調(diào)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(diào)下丘腦的GnRHmRNA及下丘腦、垂體、卵巢的ERmRNA和FSHRmRNA的基因表達(dá)；僅高劑量組下丘腦和卵巢ERmRNA表達(dá)與正常對(duì)照組比較無(wú)統(tǒng)計(jì)學(xué)差異，其余均未達(dá)到正常對(duì)照組水平；衰老相關(guān)指標(biāo)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(dá)升高。與正常對(duì)照組比較，高劑量組的SOD活力恢復(fù)到正常水平。

結(jié)論：“補(bǔ)益陽(yáng)明津氣”方藥益胃湯能調(diào)節(jié)初老雌性大鼠與實(shí)驗(yàn)研究所選擇的生殖內(nèi)分泌與衰老相關(guān)檢測(cè)指標(biāo)，顯示出相應(yīng)的改善生殖激素內(nèi)環(huán)境，上調(diào)腦組織抗氧化酶、下調(diào)過(guò)氧化物表達(dá)，調(diào)控衰老基因表達(dá)作用。提示益胃湯能夠通過(guò)減輕自由基損傷，對(duì)抗大腦衰老；通過(guò)阻斷細(xì)胞衰老進(jìn)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(jī)能衰老的作用。

關(guān)鍵詞：  補(bǔ)益陽(yáng)明津氣    初老雌性大鼠    生殖內(nèi)分泌     延緩衰老    實(shí)驗(yàn)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(yè)

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

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

目次頁(yè)··························································································· 5

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

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

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

實(shí)驗(yàn)研究······················································································· 14

1.實(shí)驗(yàn)研究材料············································································· 14

1.1研究對(duì)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(shè)備······································································· 15

2.實(shí)驗(yàn)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(duì)照大鼠········································································ 17

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

2.3檢測(cè)項(xiàng)目·················································································· 18

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

2.3.2衰老相關(guān)指標(biāo)········································································ 18

2.4組織標(biāo)本的采集與處理方法···················································· 18

2.5檢測(cè)方法·················································································· 19

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

2.5.2RT-PCR實(shí)驗(yàn)方法·································································· 20

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

2.6實(shí)驗(yàn)資料的統(tǒng)計(jì)與處理··························································· 29

3.實(shí)驗(yàn)結(jié)果····················································································· 30

3.1益胃湯對(duì)下丘腦GnRH及GnRHmRNA表達(dá)的影響·············· 30

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

3.2.1益胃湯對(duì)下丘腦、垂體、卵巢ER及ERmRNA表達(dá)的影響·············· 32

3.2.2益胃湯對(duì)下丘腦、垂體、卵巢FSHR及FSHRmRNA表達(dá)的影響······ 37

3.3益胃湯對(duì)下丘腦、垂體p16mRNA表達(dá)的影響······················ 44

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

3.4.1益胃湯對(duì)腦組織SOD活性的影響········································ 46

3.4.2益胃湯對(duì)腦組織MDA含量的影響······································· 47

3.4.3益胃湯對(duì)腦組織GSH-PX活力的影響·································· 48

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

4.1模型動(dòng)物的選擇······································································· 49

4.1.1實(shí)驗(yàn)動(dòng)物的選擇···································································· 49

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

4.1.3本研究實(shí)驗(yàn)動(dòng)物模型的判定················································· 50

4.2補(bǔ)益陽(yáng)明津氣的立法依據(jù)和處方原則···································· 51

4.2.1祖國(guó)醫(yī)學(xué)對(duì) “陽(yáng)明”的認(rèn)識(shí)··················································· 51

4.2.2“陽(yáng)明脈衰”與衰老的相關(guān)性·············································· 53

4.2.3未病先防，補(bǔ)益陽(yáng)明，后天養(yǎng)先天····································· 54

4.2.4治療上以益胃生津?yàn)榉?/SPAN>························································ 55

4.3益胃湯對(duì)生殖內(nèi)分泌實(shí)驗(yàn)檢測(cè)指標(biāo)的影響····························· 57

4.3.1益胃湯對(duì)下丘腦GnRH及GnRHmRNA表達(dá)的影響············ 57

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

4.4益胃湯對(duì)衰老相關(guān)指標(biāo)的影響················································ 60

4.4.1益胃湯對(duì)P16mRNA表達(dá)的影響·········································· 60

4.4.2益胃湯對(duì)自由基的影響························································ 61

4.5益胃湯對(duì)生殖軸功能衰老的影響············································ 64

結(jié)語(yǔ)······························································································· 66

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

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

1.2實(shí)驗(yàn)研究思路·········································································· 66

1.3實(shí)驗(yàn)研究結(jié)論·········································································· 68

2.存在的問(wèn)題和展望····································································· 68

2.1存在的問(wèn)題·············································································· 68

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

參考文獻(xiàn)······················································································· 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(xué)術(shù)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(duì)照組下丘腦GnRH染色·········································· 76

照片2.模型對(duì)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(duì)照組卵巢ER染色··················································· 76

照片5.模型對(duì)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(duì)照組卵巢FSHR染色·············································· 78

照片10.模型對(duì)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(duì)照組下丘腦ER染色············································· 79

照片15.模型對(duì)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(duì)照組下丘腦FSHR染色········································ 80

照片19.模型對(duì)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(duì)照組垂體ER染色················································· 81

照片22.模型對(duì)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(duì)照組垂體FSHR染色············································ 82

照片26.模型對(duì)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(wèn)題，據(jù)WHO報(bào)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?cè)丝诘谋壤?/SPAN>20%增加到35%。據(jù)我國(guó)2000年統(tǒng)計(jì)，全國(guó)60歲以上老年人口系數(shù)為10.46%，標(biāo)志著我國(guó)人口的年齡結(jié)構(gòu)已進(jìn)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(guó)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(zhì)量。世界各國(guó)醫(yī)學(xué)和預(yù)防醫(yī)學(xué)組織已經(jīng)日益重視老年人的醫(yī)療和預(yù)防保健問(wèn)題。1994年開羅世界人口和發(fā)展會(huì)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(zhuǎn)向圍絕經(jīng)期的生殖健康問(wèn)題。正如Nakajima指出的：“我們的目的不單純是關(guān)心她們的疾病問(wèn)題，還應(yīng)該使她們以后能在更高的生活質(zhì)量中度過(guò)”^[3]。隨著社會(huì)文明和經(jīng)濟(jì)的持續(xù)發(fā)展、生活水平的提高、科學(xué)技術(shù)與國(guó)際的接軌及性別平等的倡導(dǎo)，女性對(duì)健康水平的追求越來(lái)越高。因此，注重生殖健康，延緩生殖軸機(jī)能的自然衰老，提高圍絕經(jīng)期婦女的生活質(zhì)量是醫(yī)學(xué)界嶄新的具有積極作用和重大價(jià)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(shí)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(guò)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(shí)期，同時(shí)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(shí)期。圍絕經(jīng)期生殖軸機(jī)能的逐步衰老及其相應(yīng)病理性變化的發(fā)生，主要由絕經(jīng)前期機(jī)體的“內(nèi)因”所決定和導(dǎo)致。中醫(yī)典籍自《黃帝內(nèi)經(jīng)》伊始，即對(duì)女性生、長(zhǎng)、壯、老、已的全過(guò)程有了甚為明晰的分階段論述，其中《素問(wèn)·上古天真論》“女子……五七，陽(yáng)明脈衰……六七，三陽(yáng)脈衰于上”^[4]是對(duì)35歲~42歲年齡段女性，機(jī)體內(nèi)環(huán)境病理生理變化具代表性的經(jīng)典認(rèn)識(shí)。導(dǎo)師認(rèn)為《內(nèi)經(jīng)》所云“五七”、“六七”的“陽(yáng)明”、“三陽(yáng)脈衰”不僅可引起原文所述顏面、肌膚、毛發(fā)的改變，而且會(huì)因“沖脈隸于陽(yáng)明”的特定關(guān)系，導(dǎo)致月經(jīng)之本沖脈失于陽(yáng)明津氣資助而虛衰，引發(fā)女性生殖機(jī)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(wú)子也”，生殖機(jī)能的衰竭。故明代著名醫(yī)學(xué)家張景岳有“女為陰體，不足于陽(yáng)，故其衰也，自陽(yáng)明始”^[5]之說(shuō)。因而，在圍絕經(jīng)期之絕經(jīng)前期階段，注重“陽(yáng)明”進(jìn)而“三陽(yáng)”脈衰的中醫(yī)婦科學(xué)生理病理內(nèi)環(huán)境觀，應(yīng)用補(bǔ)益陽(yáng)明津氣之法，使陽(yáng)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(jī)能的逐步衰老，又可起到預(yù)防圍絕經(jīng)期相關(guān)疾病發(fā)生的作用，此即中醫(yī)學(xué)“必先伏其所主”的用意。

衰老，是指在生命過(guò)程中，當(dāng)生長(zhǎng)發(fā)育達(dá)到成熟期以后，隨著年齡的增長(zhǎng)，機(jī)體在形態(tài)結(jié)構(gòu)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(jī)能的衰老。絕經(jīng)前期時(shí)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(jī)制早就發(fā)生了。部分學(xué)者認(rèn)為，大腦是全身衰老的控制中心。腦細(xì)胞是不能進(jìn)行有絲分裂的細(xì)胞，從出生到18歲左右，腦細(xì)胞數(shù)量變化不大。但自成年以來(lái)，腦細(xì)胞由于衰老死亡而逐漸減少，這是引起機(jī)體內(nèi)環(huán)境平衡失調(diào)和有關(guān)臟器功能低下的重要原因之一。1956年Harman提出了關(guān)于衰老機(jī)制的自由基學(xué)說(shuō)，認(rèn)為自由基及其誘導(dǎo)的氧化反應(yīng)長(zhǎng)期毒害可以導(dǎo)致機(jī)體衰老的發(fā)生，這一學(xué)說(shuō)在現(xiàn)代衰老學(xué)中仍占有重要的地位。另外，內(nèi)分泌系統(tǒng)在維持動(dòng)物機(jī)體內(nèi)環(huán)境穩(wěn)定和調(diào)節(jié)生長(zhǎng)、發(fā)育與衰老過(guò)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(jī)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(jī)體控制內(nèi)環(huán)境平衡的能力下降相關(guān)^[6]。下丘腦-垂體軸隨年齡增長(zhǎng)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(rèn)為少數(shù)控制著機(jī)體全部生理功能的細(xì)胞（丘腦垂體軸）是不能為其它細(xì)胞所代替的，這些細(xì)胞受損是機(jī)體衰老的重要原因之一。

本實(shí)驗(yàn)以中醫(yī)傳統(tǒng)理論為依據(jù)，通過(guò)對(duì)初老雌性模型大鼠（相類于絕經(jīng)前期）的研究，擬從調(diào)控生殖內(nèi)分泌和衰老相關(guān)指標(biāo)兩方面入手，與前期“補(bǔ)益陽(yáng)明津氣延緩初老雌性大鼠卵巢機(jī)能衰老的機(jī)理研究”及“補(bǔ)益陽(yáng)明津氣方藥對(duì)雌性初老大鼠神經(jīng)免疫及生殖軸機(jī)能影響的實(shí)驗(yàn)研究”相結(jié)合，共同驗(yàn)證中醫(yī)學(xué)對(duì)絕經(jīng)前期“五七，陽(yáng)明脈衰”“六七，三陽(yáng)脈衰”特殊生理病理內(nèi)環(huán)境理論認(rèn)識(shí)的科學(xué)性和實(shí)用性，并為延緩絕經(jīng)前期生殖軸機(jī)能衰老，提高圍絕經(jīng)期婦女生活質(zhì)量、身體素質(zhì)，預(yù)防和治療圍絕經(jīng)期相關(guān)疾病提示新的途徑與治療方藥。