【作者】 陈淑娇；

【导师】 李灿东；

【作者基本信息】 福建中医药大学 ， 中西医结合临床， 2013， 博士

【摘要】 本研究从临床和动物研究两个方面,通过研究围绝经期综合征(Perimenopausal Syndrome, PPS)的中医病理特征以及肝郁病理与雌激素(E2)、雌激素受体(Estrogen Receptor, ER)、单胺类神经递质表达关系；肝郁病理与Ca2+/cAMP信号通路功能基因表达关系,探讨PPS肝郁病理的生物学基础和分子学机制。目的(1)通过研究PPS症状、证素病理及兼杂特征,柴胡疏肝散治疗前后的PSS肝郁证患者肝郁积分、Kupperman法症状评分变化,肝郁病理分级与雌激素、神经递质关系,探讨PPS肝郁病理的部分生物学基础。(2)通过柴胡疏肝散干预PPS肝郁证大鼠,从E2、ER、神经递质变化及大鼠海马雌激素受体介导的Ca2+/cAMP信号通路探讨其分子机制。方法(1)临床研究方面收集PPS患者161例,以证素辨证的方法,分析PPS的症状分布频次、病位病性证素及其兼杂的特点。选取PPS患者中肝郁积分≥70分的患者100例,根据肝郁病理积分分为3级,予以4周的柴胡疏肝散干预,观察干预前后的肝郁病理积分、Kupperman法症状评分变化,肝郁病理分级与E2、神经递质5-羟色胺(5-HT),去甲肾上腺素(NE),多巴胺(DA)关系,探讨PPS肝郁证的生物学基础。(2)动物研究方面以3月龄成年大鼠为正常对照组(A组),13月龄自然衰老和慢性束缚法建立PPS肝郁证大鼠模型,分为围绝经期对照组(B组)、围绝经期对照组加中药组(C组)、PPS肝郁证加中药组(D组)和PPS肝郁证组(E组)等五组,药物干预3周后,予行为学观察,以实时定量PCR法检测海马区雌激素受体ERa, ERβ, GPR30(G protein-coupled receptor30) mRNA表达,用Fura-2/AM双波长荧光法测定海马细胞内游离钙浓度,用RT2Profiler PCR Arrays芯片检测海马Ca2+/cAMP通路上相关基因差异性表达,用ELISA法检测海马及血清雌激素及递质5-HT, NE,DA,血清钙调蛋白依赖性蛋白激酶Ⅱ (CaMKⅡ)表达。结果(1)临床研究部分1.PPS的症状分布频次前10位是阵发性烘热,汗出,急躁易怒,情绪抑郁或焦虑,月经紊乱,胸闷、失眠,喜太息,心悸,咽部异物感,各占91.93%,91.93%,85.09%,81.99%,78.26%,69.56%,67.70%,55.28%,52.17%,39.75%。2.PPS的病位证素涉及肝、肾、脾、心,主要是肝和肾,分别占91.93%和52.17%,与其它病位相比有显著性差异,P<0.01,肝的病位积分最高。3.PPS的病性证素主要涉及气滞、阴虚、气虚、阳虚、血虚,其中气滞所占的比例最高为90.1%,与其它病性所占比例相比有显著性差异,p<0.01,气滞的病性积分最高。4.PPS证素病位兼杂中,以肝肾最多见,其次为脾肾、肝脾,病性兼杂以气滞兼阴虚最为多见。5.临床干预前PPS患者肝郁病理0级0人,1级50人,2级34人,3级16人,干预后0级70人,1级22人,2级3人,3级5人,干预前后肝郁病理分级人数有显著性改变P<0.01。6.干预前后,Kupperman法症状评分有显著性改变,P<0.01。7.干预前后,肝郁病理分级与E2变化有显著相关关系,R=-0.497,P==0.001。8.干预前后,5-HT, NE, DA变化有显著性差异P<0.01。肝郁病理分级与神经递质变化有显著相关关系：与5-HT R=-0.417,P=001;与NER=-0.352P=0.001;与DAR=-0.609P=0.000。(2)动物研究部分1.E组与D组比,旷场实验水平得分和垂直得分有显著性差异,P<0.01。糖水嗜好实验,E组与D组比较,糖水饮用显著下降,P<0.01。E组与D组比较体重明显下降,P<0.01。2.海马区E组ERamRNA较B组减少,ERβ较B组升高但无显著性差异,GPR30mRNA组间没有显著性差异；E组ERamRNA/ERβmRNA比值较B组低,柴胡疏肝散干预后D组明显升高,P<0.01；E组E2较A组低,干预后D组升高,但无显著性差异；E组5-HT, DA较B组明显下降,NE, DA经干预后升高但无显著性差异,5-HT明显增高,P<0.01。血清中,E组5-HT较B组明显高,经干预后明显下降P<0.01；E组E2, DA, NE较B组低,经干预DA后上升明显,P<0.01,E2,NE有上升趋势,但无显著性差异P>0.05。3.E组海马Ca2+浓度显著升高,干预后Ca2+浓度下降P<0.01。E组CaMKII浓度较D组显著升高,干预后下降,P<0.01。4.PCR芯片显示,E组与B组比,在海马细胞cAMP通路上有6个功能基因(IL-2,NOS2, Th, Calm1, S100g和SSt)有差异性表达P<0.05或P<0.01,柴胡疏肝散干预后,E组与D组比较,SSt有差异性表达P<0.05。结论1.PPS的病位证素以肝为主,涉及肾、脾、心,病性证素以气滞为主,涉及阴虚、血虚、气虚,证素分布呈现多脏分布、虚实夹杂特点,肝郁是其主要病理特点,症状以阵发性烘热,汗出,急躁易怒,情绪抑郁或焦虑,月经紊乱,胸闷、失眠,喜太息,心悸,咽部异物感等为主。2.PPS患者干预前后Kupperman法症状评分及肝郁病理积分显著改善,肝郁病理分级与血清E2有显著性负性相关关系,与5-HT, NE, DA有显著性差异,呈负相关,说明肝郁是PPS主要病理,E2和神经递质改变是肝郁证的生物学基础。3.大鼠行为学实验和糖水嗜好实验、体重变化及血清雌激素变化等证明自然衰老和慢性束缚PPS肝郁证大鼠造模成功。4.PPS大鼠肝郁证与海马及周围血的E2神经递质的表达与有密切关系,与海马细胞的雌激素、雌激素受体ERamRNA/ERβmRNA比值表达有密切关系。5.PPS大鼠肝郁证与海马区Ca2+/CaMKII通路有密切关系,与海马Ca2+/cAMP通路调节神经传导的基因表达变化有关系,Ca2+/cAMP信号通路部分功能基因表达改变可能是PPS肝郁证的分子学机制。更多还原

【Abstract】 Perimenopausal syndrome (PPS) is a systematic disease occurred in perimenopausal women include imbalance in the spirit of the nemo-psychological, endocrine and metabolic, attributing to the change of the central and peripheral nerve neurotransmitter, vasomotor factor, cytokines free radicals. Liver-Qi stagnation plays an important role in perimenopausal syndrome, the priority of treatment is to soothe the liver and regulate the Qi and dispel the stagnation, such as Chaihu-Shugan-San(CSS), this study from the clinical and animal experiments explore the TCM pathological feature of perimenopausal syndrome and the relationship of Liver-Qi stagnation syndrome and strogen, estrogen receptors and monoamine neurotransmitters expression, to explore the relationship of gene expression in Ca2+/cAMP signaling pathway.ObjectiveTo explore the Liver-Qi stagnation syndrome’s TCM pathology and syndrome element mixed characteristics in PPS. To observe the relationship of Liver-Qi Stagnation score change and estrogen, neurotransmitters after treating with CSS in PSS Liver-Qi stagnation syndrome rats and to explore the biology basis of Liver-Qi stagnation in PPS.MethodsWe collected161patients of PPS to analyse the main locations and characteristic of the disease throught differentiation of syndrome elements method and syndrome differentiation to analyse main location and characteristic of the disease.100cases of PPS whose Liver-Qi Stagnation score exceed70are collected, after4weeks treatment with CSS, the relationship of Liver-Qi Stagnation score change and estrogen, neurotransmitters are observed. To observe the effective of CSS on PPS and Liver-Qi stagnation syndrome rat models, and explore the molecular mechanisms throught Ca2+/cAMP signaling pathway in hippocampal cell. In this experiments, twelve-week-old female rats (250-280g, n=12) are used as normal controls (Group A),52-week-old nature aging rat as PPS rat. A total of48nature aging rats (52-week-old,370-380g) are randomly assigned into four groups; PPS controls (Group B, n=12), PPS controls treate with CSS (Group C, n=12), PPS+Liver-Qi stagnation model treate with CSS (Group D, n=12), and PPS+Liver-Qi stagnation model (Group E, n=12). CSS is administered simultaneously when the Liver-Qi stagnation model being established. We selecte the sucrose consumption test and the open field test (OFT) for the Liver-Qi stagnation behavior test.We measure E2, neurotransmitters, CaMKII levels in serum and hippocampus by ELISA, Ca2+is measured by Fura-2/AM in hippocampus and estrogen receptors of hippocampal mRNA expression are measured by quantitative real-time PCR and Ca2+/cAMP signaling pathway by RT2Profiler PCR Arrays.ResultThe part of clinical Study1.The clinical symptoms distribution frequency of the top ten are hot flashes, sweating, irritability, depression or anxiety, menstrual disorders, chest distress, deep sigh, palpitations, globus hystericus, that is91.93%,91.93%,85.09%,81.99%,78.26%,69.56%,67.70%,55.28%,52.17%,39.75%.2.The locations of PPS are liver, kidney, spleen, heart, but mainly in the liver(91.93%) and kidney(52.17%), P<0.01.The score of liver locations is the highest.3. The nature syndrome elements include Qi stagnation and Yin deficiency, Yang deficiency, blood deficiency. Qi stagnation and Yin deficiency are in a high proportion, the ration is90.06%and71.43%。 The score of Qi stagnation is the highest.4. The mixed syndrome elements relationship of the locations mixed mainly are liver and kidney, spleen and kidney, liver and spleen, nature of mixed syndrome elements are Qi stagnation mixed Yin deficiency and Qi stagnation mixed blood deficiency.5.Before treatment, the cases of pathological Liver-Qi stagnation grade1is50, grade2is34, grade3is16, after treatment, the cases of pathological Liver-Qi stagnation grade0is70, grade1is22, grade2is3, grade3is5. The pathological classification changes significantly,P<0.01.6. Before and after treatment with CSS, the Kupperman score change significantly P <0.01.7. Before and after treatment with CSS, there is significant correlation relationship between Liver-Qi stagnation score and estrogen expression, r=-0.479P=0.001.8. Before and after treatment with CSS, there is significant correlation relationship between Liver-Qi stagnation score and neurotransmitters expression,(5-HT, R=-0.417,P=0.001;NE, R=-0.352P=0.001;DA, R=-0.609P=0.000)Part II The part of Basical Study1. The open field test and the sucrose consumption test, the dynamic changes of BW. The BW of group E decrease sharply, while that of group D decreased slowly. We found the sucrose intake in group E are significantly lower than that in group B, p<0.01), whereas the CSS treatment ameliorate the sucrose intake (group D vs. group E, p<0.01).2. CSS administration effects on hippocampal ERa, ERβ, or GPR30mRNA expression in aging rats or Liver-Qi stagnation group rats.No significant difference is observed in ERβ mRNA expression between the four groups, thereby indicating that CSS administration does not affect the ERβ mRNA expression in groups B and C or groups D and E. Similarly, GPR30mRNA expression does not differ significantly between treatment groups. The ERa/ERβ ratio in group E is markedly decrease compared with the other groups, p<0.01. CSS administration increase ERa/ERβ ratio.E2levels in A group rats were significantly higher than those in the group B. No significant difference is observed between the four aging rat groups, indicating that CSS does not affect E2levels in aging rats. Hippocampal5-HT, NE, DA levels in A group are significantly higher than those aging group, after CSS treatment NE, DA levels increase. The change of serum NE and DA are the same as of hippocampus except5-HT.3. The E group hippocampal Ca2+expression increase sharply than D group, P<0.01. The change of CaMKII expression is the same.4. PCR Arrays indicate there are6functional gene namely S100g, Th, NOS2, IL-2, Calml and SSt expression change between E group and B group (P<0.05or P<0.01), after CSS administration only the SSt functional gene change P<0.05.Conclusion1. The main clinical symptoms of PSS is Liver-Qi stagnation symptoms.The main locations of PPS are liver. The characteristic of syndrome elements in PPS is multiple organ involvement, mixed excessiveness and deficiency. Liver-Qi stagnation is the main pathology. The main clinical symptoms of the PSS are hot flashes, sweating, irritability, depression or anxiety, menstrual disorders, chest distress, deep sigh, palpitations, globus hystericus.2. There are significant differences in Liver-Qi Stagnation score and Kupperman score before and after treatment. There is significant difference between Liver-Qi Sagnation score and estrogen before and after treatment. There are significant negative correlation between the pathology grade and estrogen. There are significant differences between Liver-Qi stagnation score and neurotransmitters5-HT, NE, DA before and after treatment. There are significant negative correlation between the pathology grade and neurotransmitters5-HT, NE, DA.3. The open field test and the sucrose consumption test, the dynamic changes of BW and E2content in serum and hippocampus verify the PPS+Liver-Qi Stagnation syndrome rats model is good.4. It indicates that there is close relation between ERa/ERβ ratio and Liver-Qi Stagnation syndrome in PPS rats. There is close relation between neurotransmitters and Liver-Qi stagnation syndrome in PPS.5. There is close relation between Liver-Qi stagnation syndrome and Ca2+/CaMKII signal pathway in PPS. There is close relation between genes relating to neurotransmitter in Ca2+/cAMP signaling pathway and Liver-Qi stagnation syndrome, maybe it was the molecular mechanisms of Liver-Qi stagnation syndrome of PPS.更多还原