【作者】 曹俊岩；

【导师】 吴克明；

【作者基本信息】 成都中医药大学 ， 中医妇科学， 2011， 博士

【摘要】 目的：验证雷公藤多苷致大鼠卵泡发育障碍模型的中医证候属性,为中医药调节女性生殖轴的研究提供一个新的符合中医证候特点的病证结合动物模型。方法：将雌性育龄期SD大鼠行阴道脱落细胞学连续涂片,筛选出动情周期正常者为受试动物。随机留取8只为正常对照组,其余大鼠予雷公藤多苷50mg/(kg·d)灌胃。同时监测大鼠动情周期的变化,当全部大鼠均出现动情周期紊乱时,确定造模成功。将己造模大鼠随机分为5组,每组8只,分别为模型对照组、新加归肾丸组、圣愈汤组、加减逍遥散组、丹溪治湿痰方组。正常对照组与模型对照组每日生理盐水灌胃；各中药组分别以相应中药复方灌胃,每日1次,连续10天。各组均于末次灌胃24h后股动脉取血并处死动物,取出卵巢和子宫,称重法计算卵巢与子宫指数；放射免疫法检测血清E2、P和IGF－1水平；酶联免疫吸附法检测血清FSH、LH水平；免疫组化法检测卵巢FSHR、ERβ、IGF-1R、VEGF及Flk-1表达；将卵巢、子宫组织病理切片HE染色在光镜下观察分析卵巢、子宫组织细胞形态学改变以及计数卵巢中各级卵泡和黄体数。结果：(1)雷公藤多苷50mg/(kg·d)灌胃2周造模后,大鼠表现为一般情况差,动情周期紊乱。卵巢与子宫指数降低；卵巢中卵泡数及黄体数减少；卵巢门血管腔变小、血管壁增厚；血清IGF-1水平升高、E2与P水平明显降低；卵巢ERβ、VEGF及Flk-1表达减少、FSHR、IGF-1R表达增强。模型对照组以上指标与正常对照组相比差异均有统计学意义(p<0.05)。模型对照组血清中FSH与LH水平有升高趋势,但与正常对照组相比差异均无统计学意义(p>0.05)。(2)各中药复方组的FSH和LH水平与正常对照组、模型对照组比较,差异均无统计学意义(p>0.05)。新加归肾丸组的卵巢与子宫指数、卵巢中卵泡数及黄体数、卵巢门血管腔与血管壁厚度、血清E2、P与IGF-1水平及卵巢ERβ、VEGF、Flk-1、FSHR、IGF-1R表达与模型对照组比较,差异均有统计学意义(p<0.05)。各中药复方组中,以上各项指标以及子宫与卵巢组织形态学只有新加归肾丸组最接近于正常对照组。(3)相关性分析：卵巢指数与血清E2水平、卵巢VEGF及Flk-1的表达之间均呈正相关关系；血清E2水平与血清IGF-1水平、卵巢IGF-1R的表达呈负相关关系；血清E2水平与卵巢ERB及成熟卵泡数之间呈正相关关系。结果表明：ERβ、IGF-1、IGF-1R、VEGF及Flk-1的变化可反映卵泡发育及卵巢功能情况。结论：(1)雷公藤多苷50mg/(kg·d)连续灌胃2周可造成育龄期大鼠急性卵巢功能损伤和卵泡发育障碍状态。(2)补肾气益精血的新加归肾丸通过提高雌激素及其受体水平,能明显促进卵泡发育,提高生长卵泡数量；通过增加卵巢局部血供,能明显改善雷公藤多苷造成的子宫及卵巢组织形态损伤。(3)本研究通过多方比较“以药测证”在一定程度上验证了雷公藤多苷致大鼠卵泡发育障碍模型符合中医肾精气亏虚证候属性。更多还原

【Abstract】 OBJECTIVE:To discuss the TCM syndrome differentiation of follicular development disorder in rat model caused by GTW in order to have better animal models with required TCM syndrome characters for research in the effects of TCM treatment on female reproductive system.METHODS:The vaginal cytology smears of female reproductive SD rats were made, in order to select animals with normal estrous cycles as subjects.Eight of them were randomly selected as normal controls, and the others were all given intra-gastric administration with GTW 50mg/(kg·d) to make the required models,and to monitor the exchanges of estrous cycles of rats. The models were successfully made when all the animals became disordered. Model rats have been randomly divided into 5 groups, respectively, the control one, xinjiaguishen one, shengyutang one, jiajianxiaoyaosan one and danxizhishitan one, every one with 8 rats. The normal and control groups daily with normal saline; Xinjiaguishen, shengyutang, jiajianxiaoyaosan and danxizhishitan groups were all given intra-gastric administration with each prescription for 10 days. The rats were killed and taken femoral artery blood after 24 hours of intra-gastric administration, at the same time, to observe the general conditions, to calculate ovarian and uterine indexes; and to detect the serum levels of "FSH" and "LH" by means of ELISA; "E2"、"P" and "IGF-1" by means of RIA; and "FSHR", "ERβ" "IGF-1R", "VEGF" and "Flk-1" with IHCM. Pathological examinations were performed on their ovarian slides with HE stain to observe the histolytic morphological changes of ovarian and uterus cells, and the number of ovarian follicles and corpus luteum with optical microscope.RESULTS:1. The female reproductive SD rats can be posed to follicular development disorder by given intra-gastric administration with GTW 50mg/(kg·d) for two weeks. They displayed with general poor conditions, the disorders of estrous cycle, decreasing of ovarian and uterine indexes. GTW can reduce the number of ovarian follicles and corpus luteum, lumen of blood vessel of ovary hilum and thicken vascular wall, and raise serum IGF-1 level and FSHR and IGF-1R expressions of ovary tissue, and reduce serum E2 and P, and raise expression of ovary tissue in ERp, VEGF and Flk-1. Above indexes were different compared with normal group (P<0.05). GTW can raise FSH and LH levels. Above indexes were no difference (p>0.05).2. After the intervention of of using the compound Chinese herbal on DOS rats by GTW, serum FSH and LH levels of every compound Chinese herbal group were no difference compared with normal and model groups (p>0.05).The rest indexes of xinjiaguishen group were different compared with model group (P＜0.05). Above-mentioned indexes and HE dyeing of ovary and uterus of xinjiaguishenwan group were closesd to the normal group compared with others.3. Correlative analysis shows:there are positive-correlative relationships between ovarian index and E2, VEGF and Flk-1; positive-correlative relationships between E2 and ERβ, vesicula graafianae; negative-correlative relationships between E2 and IGF-1R, IGF-1R.The contents of IGF-1, IGF-1R, ERp,VEGF and Flk-1 can reflect conditions of follicular development.CONCLUSION:1. The tripterygium glycosides can cause acute injury of ovarian function and follicular development disorder in rats.2. Compound Herbal of xinjiaguishenwan with the functions to tonify Kidney Qi and nourish Blood and Essence can increase estrogen and ER levels and ovarian hemoperfusion, can significantly improve the damages of uterus and ovarian tissue of follicular development disorder rats by GTW, and effectively improve and enhance the ovarian function.3. The method of "syndrome identification with medicine" verifies the rat model with TCM syndrome of Kidney Deficiency in Qi and Essence.更多还原