更年安怡片对更年期大鼠卵巢细胞凋亡及超微结构影响的实验研究

【作者】 江南；

【导师】 黎烈荣；

【作者基本信息】 湖北中医学院 ， 中医妇科学， 2003， 硕士

【摘要】 目的： 更年期是人体衰老过程中生理变化非常明显的阶段，是妇女由生育期向老年期过渡的时期。此时，由于卵巢功能逐渐衰退，雌激素水平明显下降，植物神经功能紊乱，患者出现腰膝酸软、潮热汗出、心悸不宁、烦躁易怒或抑郁等症状，统称为更年期综合征。部分进入更年期的妇女不仅出现上述症状，还有很明显的衰老的现象，如失眠健忘、四肢乏力、注意力不集中、性欲淡漠等。随着社会的发展，生活水平的提高，人们对自身健康和生活质量的要求越来越高，如何有效防治更年期综合征，并且能够延缓人体衰老成为医学研究的一大热点。中药复方更年安怡片的前期实验研究已经证明其具有调节植物神经功能，调节生殖内分泌，防治骨质疏松，升高去势大鼠靶细胞ER水平，上调免疫细胞ER的表达，改善免疫功能等作用。本课题将进一步探讨本方对更年期大鼠卵巢颗粒细胞凋亡及其相关的凋亡抑制基因Bcl-2和凋亡诱发基因Fas表达的影响，观察卵巢细胞的超微结构，研究该方对更年期大鼠卵巢颗粒细胞的作用，阐明其治疗更年期综合征和延缓人体衰老的机制，以期为临床运用提供新的理论和实验依据。 方法： 本实验以30只鼠龄为11～15月的自然更年期雌性SD大鼠为对象，体重250～350g，将其随机分为更年安怡片组、雌激素组、模型组，每组10只，另选用10只4～6月龄的青年大鼠作为青年组。治疗4周后，将所有大鼠颈椎脱臼法处死，取双侧卵巢，分别采用原位末端标记法(TUENL)检测卵巢颗粒细胞凋亡；免疫组化法检测颗粒细胞凋亡抑制基因Bcl-2和促进基因Fas的阳性表达结果；组织形态学检测用卵巢制成电镜超薄切片，通过透射电镜观察卵巢细胞超微结构。 结果： 1．模型组卵巢颗粒细胞凋亡阳性表达及Fas蛋白阳性表达明显高于青年组(P＜0.01)；Bcl-2蛋白阳性表达明显低于青年组(P＜0.01)； 2．经更年安怡片和雌激素治疗后，卵巢颗粒细胞凋亡阳性表达明显低于模型组(P＜0.05)；更年安怡片组和雌激素组之间比较无显著性差异(P＞0.05)； 3．更年安怡片和雌激素均能使凋亡抑制蛋白Bcl-2阳性表达较模型组显著升高(P＜0.05)；Fas蛋白阳性表达较模型组显著降低(P＜0.05)；更年安怡片组和雌激素组之间比较无显著性差异(P＞0.05)； 4．更年安怡片和雌激素都能使卵巢颗粒细胞内的线粒体、滑面内质网、胞质等有不同程度的改善。 结论： 上述结果表明：更年期大鼠卵巢颗粒细胞的凋亡阳性表达较青年期大鼠的阳性表达明显升高(P＜0.01)，其卵巢细胞内务细胞器明显衰退。更年安怡片能更年安怡片对更年期大鼠卵巢细胞凋亡及超微结构影响的实验研究够明显降低卵巢颗粒细胞的凋亡(P<0.05)，其治疗效果与西药雌激素比较无显著性差异(P>0.05)。实验还表明该方能够明显改善卵巢细胞超微结构，从而达到颗粒细胞增多，自身分泌更多雌激素的目的，体现了中医药治疗更年期综合征及延缓衰老的作用，为临床应用更年安怡片提供新的理论依据。更多还原

【Abstract】 Objective:The climacteric is the transitional period of a woman from bearing age to agedness. During this period, the body has very significant physical changes. Ovaries atrophy and dysfunction gradually. The excretion of estradiol (E2) decreased markedly and the vegetative nerve dysfunction gradually occurs. As a result, the patient may have many unhappy feelings such as hot, flashes, nervousness, depression, irritability, insomnia and so on, namely the Climacteric Syndrome. With the development of the society and the enhancing of life quality, the incidence of the disease take on a trend of increasing year after year. So how to prevent and cure Climacteric Syndrome effectively becomes a pop topic of medicine study. This report made a discussion of the effects of GNAT on the apoptosis and ultrastructure of ovary granulose cells of menopausal rat. It illustrated the possible mechanism of GNAT to cure the climacteric syndrome so as to supply the clinic application the basis of theory and experiment. Method:The study object was the female Sprague-Dawley (SD) menopausal rats of 11~15month.es old .The rats were divided randomly into 4 groups, i.e. GNAT groups, and estrogen groups, and menopausal contrast groups and with a normal groups (young rats of 4~6monthes old) as control. After 4 weeks, take the two ovaries each rat, the apoptosis expression was estimated by TUNEL, and Bcl-2 and Fas protein expression were estimated by immunohistochemical study. At the same time, we observe the ultrastructure of ovary granulose cells through electronic microscope. Result:1.The expression of ovary granulose cells apoptosis and Fas protein of the menopausal group increased evidently (P<0. 01), at the same time Bcl-2 protein expression decreased hugely than that of the young normal group (P<0. 01);2.After being treated with GNAT and estrogen, the expression of ovary granulose cells apoptosis decreased evidently than that of the menopausal group (P<0. 05); the differences of the above indexes between the two groups were not significant ( P>0. 05 ) ;3.After being treated with GNAT and estrogen, the expression of Fas protein decreased while Bcl-2 protein expression increased evidently than that of the menopausal group (P<0. 05); the differences of the above indexes between the two groups were not significant ( P>0. 05 ) ;4. GNAT and estrogen can improve the ultrastructure of ovary granulose cells. Conclusion:The results above indicate that GNAT can decrease the apoptosis in the ovary granulose cells, but also improve the ultrastructure of ovary granulose cells. Thismust be the probable action mechanism that GNAT to cure the climacteric syndrome. These evidences theoretically support the clinic application of GNAT.更多还原