【作者】 曹蕾；

【导师】 罗颂平；

【作者基本信息】 广州中医药大学 ， 中医妇科学， 2010， 博士

【摘要】 目的：探讨子宫内膜容受性与复发性流产的相关性及其影响因素,观察补肾健脾中药复方助孕丸对复发性流产患者子宫内膜容受性的作用,以研究其对复发性流产的防治机理。并通过建立肾虚内膜容受性不良大鼠模型,从子宫内膜形态学角度,研究助孕丸在改善子宫内膜容受性及其免疫环境方面的作用；建立肾虚内膜容受性不良大鼠妊娠模型,进一步探讨子宫内膜容受性及其免疫环境与自然流产的相关性,从中医“治未病”角度,研究助孕丸对复发性流产的防治机理。方法：1.助孕丸对脾肾亏虚型复发性流产患者内膜容受性及其免疫环境的影响：比较10例中医辨证为脾肾亏虚型复发性流产患者与10例正常生育妇女围着床期子宫内膜厚度及子宫内膜下螺旋动脉血流阻力指数(RI)、搏动指数(PI),比较同期血清E2、P、LIF、IL-2含量及外周血CD16+CD56+NK细胞含量。比较患者治疗前、治疗1月后、治疗2月后子宫内膜厚度及子宫内膜下螺旋动脉血流阻力指数(RI)、搏动指数(PI),治疗前及治疗2月后围着床期血清E2、P、LIF、IL-2含量及外周血CD16+CD56+NK细胞含量。2.助孕丸对肾虚大鼠内膜容受性、妊娠结局及其免疫环境的影响：以羟基脲建立肾虚大鼠模型,比较正常组、造模组、中药治疗组、中药治未病组大鼠动情期子宫内膜形态学及微细结构变化,子宫内膜及外周血各群NK细胞含量,血清LIF、IL-2、E2、P水平,子宫内膜LIF、IL-2表达。建立肾虚内膜容受不良妊娠大鼠模型,比较正常妊娠组大鼠、肾虚妊娠组大鼠、中药治疗肾虚妊娠组大鼠、肾虚妊娠后中药干预组大鼠、中药预防流产组大鼠妊娠率,各组妊娠大鼠蜕膜及外周血各型NK细胞亚群含量,蜕膜IL-2、LIF含量,血清IL-2、LIF、E2、P水平。结果：1.与正常组相比较,治疗组治疗前围着床期子宫内膜厚度明显减少(p<0.05),而子宫内膜下螺旋动脉血流阻力指数明显增高(p<0.05),血清E2含量及E2／P比值明显升高(p均<0.05)。两组间围着床期内膜下螺旋动脉搏动指数、血清P、LIF、IL-2含量、外周血CD56+CD16+NK细胞含量未见显著差异(p均>0.05)。2.与治疗前相比较,治疗组治疗1月后,围着床期子宫内膜下螺旋动脉阻力指数明显降低(p<0.05),治疗2月后,患者围着床期子宫内膜厚度明显增加(p<0.05)。治疗2月后患者围着床期血清P明显升高(p<0.05),E2／P比值、血清IL-2含量及外周血CD56+CD16+NK细胞含量有下降趋势,但未见统计学差异(p均>0.05)。未见治疗前后血清LIF变化的显著差异(p>0.05)。3.与正常大鼠相比较,使用羟基脲后,大鼠出现毛松、毛干枯、大便稀烂等表现,体重明显减轻(p<0.05),发情期子宫单位横截面上内膜腺体个数及腺体总面积均减少,但未见统计学差异(p均>0.05),内膜腺上皮细胞表面微绒毛分布稀疏,腺上皮细胞内见较多含空泡化线粒体,内质网数量较少,内质网上仅见少许散在核糖体附着。大鼠发情期内膜CD56+CD16-NK细胞含量明显减少(p<0.05),CD56+CD16-／CD56-CD16+比值有下降趋势,但未见统计学差异(p>0.05)；外周血CD56-CD16+NK细胞含量有上升趋势,而CD56+CD16-／CD56-CD16+比值亦可见下降趋势,但均未见统计学差异(p均>0.05)。发情期外周血E2、P含量未见显著差异,E2／P比值略有上升,但无显著性差异(p均>0.05)；外周血IL-2含量有所上升,内膜LIF表达明显减少(p<0.05)。各组大鼠内膜IL-2含量无差异(p>0.05)。各组大鼠血清LIF含量均极少,无统计意义。4.中药治疗后,大鼠毛松、毛干枯、大便稀烂、体重减轻表现有所改善,发情期子宫单位横截面上内膜腺体个数及腺体总面积较造模组均有所提高,但未见统计学差异(p均>0.05),内膜腺上皮细胞表面微绒毛分布较密集,细胞内可见个别线粒体空泡化,内质网上可见较密集核糖体附着,细胞内可见分泌颗粒。与模型组相比,大鼠发情期内膜CD56+CD16-NK细胞含量有所增加,内膜及外周血CD56+CD16-／CD56-CD16+比值均有所上升,但未见统计学差异(p>0.05)；发情期血清P含量明显提高(p<0.05),E2／P比值略有下降,但未见显著性差异(p>0.05)；血清IL-2含量有所上升,内膜LIF表达明显提高(p<0.05)。中药治未病组大鼠体重与正常大鼠无差异(p>0.05),发情期子宫单位横截面上内膜腺体个数及腺体总面积较模型组增加,与正常大鼠无显著差异(p>0.05),内膜腺上皮表面微绒毛分布密集,细胞内见较多线粒体,内部结构清晰、正常,个别线粒体内可见空泡,粗面内质网分布密集,上见核糖体附着密集,细胞内可见分泌颗粒。大鼠发情期外周血及内膜各群NK细胞含量及CD56+CD16-／CD56-CD16+比值均与正常大鼠无显著差异(p均>0.05)；发情期血清E2、P、IL-2含量均与正常组无显著差异(p均>0.05)；发情期内膜LIF表达较模型组明显增多(p<0.05),与正常大鼠无显著差异(p>0.05)。5.与正常妊娠大鼠相比较,肾虚大鼠妊娠后体重明显减轻(p<0.05),妊娠胚胎数减少,胚胎吸收率增加,但未见统计学差异(p>0.05)；外周血各群NK细胞含量与正常妊娠大鼠无显著差异(p>0.05),蜕膜CD56-CD16+NK细胞较正常组有较明显上升趋势,而CD56+CD16-／CD56-CD16+比值下降,但未见统计学差异(p>0.05)；血清E2、P、IL-2含量未见统计学差异(p>0.05),但P水平均较正常组及中药预防流产组偏低,而血清IL-2含量则偏高；蜕膜LIF表达仍明显减少(p<0.05),蜕膜IL-2表达无显著差异(p>0.05)。各组大鼠血清LIF含量均极少,无统计意义。6.中药孕前、孕后干预肾虚大鼠妊娠后体重较肾虚大鼠妊娠后体重增加,胚胎数明显增多(p<0.05),而胚胎吸收率降低；孕前、孕后中药预防流产大鼠体重较模型大鼠显著增加(p均<0.05),胚胎数增多,胚胎吸收率明显降低(p<0.05)。中药孕前、孕后干预肾虚大鼠外周血CD56-CD16+NK细胞含量均较模型组减少,中药预防流产组大鼠蜕膜CD56-CD16+NK细胞含量均较各组肾虚妊娠大鼠减少但未见统计学差异(p均>0.05)；中药孕前预防流产组大鼠血清IL-2含量较肾虚妊娠大鼠明显降低(p<0.05)；中药孕前治疗肾虚、孕后预防流产组大鼠蜕膜LIF含量较肾虚妊娠大鼠明显上升(p<0.05),与正常妊娠大鼠无差异(p>0.05)。结论：1.子宫内膜厚度、内膜下螺旋动脉血流阻力指数RI、E2／P比值可能是影响子宫内膜容受性的重要因素。助孕丸能够有效增加脾肾亏虚型RSA患者子宫内膜厚度,降低其内膜下螺旋动脉血流阻力指数,改善子宫内膜容受性。2.羟基脲450mg/kg/d×8d可造成肾虚证候之轻证,伴有子宫内膜腺上皮细胞线粒体、核糖体功能减退,细胞表面微绒毛数量减少,腺上皮细胞分泌功能与信号传导功能减退,内膜LIF含量减少。3.子宫内膜LIF可能是影响大鼠内膜容受性的关键因子之一,LIF高表达有助于子宫内膜容受性的形成。而外周血CD56-CD16+亚群NK细胞增多,内膜CD56+CD16-亚群NK细胞减少,NK细胞吞噬功能异常,可能会降低子宫内膜容受性。4.助孕丸具有调节肾虚模型大鼠内分泌-免疫功能紊乱的作用,调节改善NK细胞亚群含量,改善其功能,提高内膜LIF表达来改善子宫内膜容受性,提高妊娠成功率。5.正常大鼠孕前或孕后给予助孕3号方,亦可以通过维护、强化大鼠机体内分泌-免疫协调平衡功能,达到防止流产的目的。更多还原

【Abstract】 Objective:To investigate the correlation between endometrial receptivity and recurrent spontaneous abortion and immune factors related to endometrial receptivity. To study the mechanism of prevention and treatment of recurrent spontaneous abortion of Pill Zhuyun, by clinical observation of the improvement of endometrial receptivity of recurrent spontaneous abortion patients after taking the pill, and experimental study on endometrial morphological, endometrial immune microenvironment and pregnancy outcome, of rats with poor endometrial receptivity and the ones that have treatment with Pill Zhuyun.Methods:1. The effect of Pill Zhuyun on endometrial receptivity and endometrial immune microenvironment of recurrent spontaneous abortion patients with spleen and kidney deficiencyCompared the endometrial thickness and blood flow index of endometrial spiral arteries (RI, PI) between recurrent spontaneous abortion patients with spleen and kidney deficiency and the normal females with normal reproductive history in the mid-luteal, and the level of E2, P, LIF, IL-2 in blood serum, as well as the percentage of CD56+CD16+ NK cells in blood. Compared the endometrial thickness and blood flow index of endometrial spiral arteries (RI, PI) of the patients before treatment,1 month after treatment, and 2 months after treatment. Compared the level of E2, P, LIF, IL-2 in serum before treatment and 2 months after treatment, as well as the percentage of CD56+CD16+ NK cells in blood. 2. The influence of Pill Zhuyun on endometrial receptivity, pregnancy outcome and immune microenvironment of kidney deficiency rat modelWe used hydroxyurea to create kidney deficiency rat model. Compared the endometrial morphological and fine structure of endometrial, the percentage of different subsets of NK cells in endometrial and blood, the level of E2, P, LIF, IL-2 in serum, and the level of LIF, IL-2 in endometrial, of rats in estrus, among normal rats group, model without herbs group, model with herbal medicine group and normal rats with herbs group. Then we created kidney deficiency and poor endometrial receptivity pregnant rat model. Compared the abortion rate, the percentage of different subsets of NK cells in decidua and blood, the level of E2, P, LIF, IL-2 in serum, and the level of LIF, IL-2 in decidua, among normal pregnant rat group, model without herbs group, model with continued herbal medicine group, model with herbal medicine post-pregnancy group, and normal pregnant rat with herbs pre/post-pregnancy group.Result:1. The endometrial thickness was thinner, the blood flow index of endometrial spiral arteries (RI) was higher, and the level of E2 in serum and the ratio of E2/P were also higher in patients pre-treatment in mid-luteal, the differences were significant compared with the results of normal females with normal reproductive history in the mid-luteal (p<0.05).2. Treated with Pill Zhuyun a month later, the blood flow index of endometrial spiral arteries (RI) was significantly lower in patients in mid-luteal, compared with the same index in patients pre-treatment in mid-luteal (p<0.05). And after 2 months treatment, the endometrial thickness was significantly thicker and the level of P was higher in patients in mid-luteal, compared with the ones pre-treatment in mid-luteal (p<0.05). But the changes of the level of IL-2 and LIF in serum, the ratio of E2/P, the percentage of CD56+CD16+ NK cells in blood and the blood flow index of endometrial spiral arteries (PI) in patients after 2 months treatment, were not significant, compared with those of pre-treatment (p>0.05).3. The rats fed with hydroxyurea showed some symptoms of kidney deficiency, such as withered fur, stool pulpy, and significant weight loss compared with the normal rats (p<0.05). The quantity of endometrial glands and endometrial glandular area both decreased in model rats, though the differences were not significant compared with the normal rats (p>0.05). Of the model rat in estrus, the distribution of microvilli at the surface of endometrial epithelial was sparse. There were more mitochondria with many vacuoles, the quantity of endoplasmic reticulum was reduced, and there was sporadic ribosomal attached to the surface of endoplasmic reticulum, compared with the normal rat in estrus. The percentage of CD56+CD16-NK cells in endometrial of model rats was significant lower (p<0.05), compared with the normal rats. The percentage of CD56-CD16+NK cells in blood was higher, and the ratios of CD56+CD16-/CD56-CD16+ both in blood and endometrial of model rats were lower, compared with the normal rats, but the differences were not significant (p>0.05). The differences of the level of E2 and P in serum between the normal rats and the model rats were not significant (p>0.05). Though the ratio of E2/P and the level of IL-2 in serum were higher in model rats compared with normal rats, the differences were not significant (p>0.05). The content of LIF in endometrial was significantly less in the model rats than that in the normal rats (p<0.05). There were no differences of the content of IL-2 in endometrial among all the groups (p>0.05). The level of LIF in serum was so little of all groups that there was no signification for statistics.4. The symptoms of kidney deficiency of model with herbal medicine group were improved. The quantity of endometrial glands and endometrial glandular area of model rats with herbal medicine both increased than the model rats, but the differences were not significant (p>0.05). Of the model with herbals medicine rat in estrus, the distribution of microvilli at the surface of endometrial epithelial was more dense. There were only a little vacuoles in mitochondria, there was more ribosomal densely attached to the surface of endoplasmic reticulum, and secretory granules could be saw in gland epithelial cells, compared with the model rat in estrus. The percentage of CD56+CD16-NK cells in endometrial and the ratio of CD56+CD16-/CD56CD16+ both in blood and endometrial of model rats with herbal medicine was higher, compared with the model rats without herbs, but the differences were not significant (p>0.05). The level of P in serum and LIF in endometrial of model with herbal medicine rats were both significantly higher than those of the model rats (p<0.05). The ratio of E2/P was a little lower and the level of IL-2 in serum was a little higher in model with herbal medicine rats than those in model rats, but the differences were not significant (p>0.05). The differences of above items between the normal rats with herbs and the normal rats without herbs were not significant (p>0.05). And the content of LIF in endometrial of the normal rats with herbs was significantly more than that of the model group (p<0.05).5. The weight of kidney deficiency pregnant model rats without herbs was significantly lighter than the normal pregnant rats (p<0.05). The quantity of embryos of the model rats was less and the abortion rate was higher of the model rats than those of normal pregnant rats, but the differences were not significant (p>0.05). There was no significantly differences of the percentage of NK cells of all the subsets in blood among the groups (p>0.05), but the percentage of CD56×CD16+NK cells was higher and the ratio of CD56+CD16-/CD56-CD16+ was lower, in decidua of the model rats, than those of the normal pregnant rats, though the differences were not significant (p>0.05). The differences of level of E2, P, IL-2 in serum was not significant between the model rats and the normal pregnant rats (p>0.05), though the level of P and IL-2 in serum were higher in the model group. The content of LIF in deciduas of the model rats was significantly less than that of the normal rats (p<0.05), but the difference of the content of IL-2 in decidua between them was not significant (p>0.05). The level of LIF in serum was also so little of all groups that there was no signification for statistics.6. The weight and the quantity of embryos of the model rats with herbal medicine pre and post pregnancy increased and the abortion rate was lower than the model rats. The difference of the quantity of embryos was significant (p<0.05). The weight and the quantity of embryos of the normal rats with herbs pre and post pregnancy increased and the ratio of abortion was lower than the model rats, the difference of the weight and abortion rate were significant (p<0.05). The percentage of CD56-CD16+NK cells in blood of the model rats with herbal medicine pre and post pregnancy, and the percentage of CD56-CD16+NK cells in decidua of the normal pregnant rats with herbs, were both lower than those of all the kidney deficiency pregnant rats, but the differences were not significant (p>0.05). The differences of the level of IL-2 in serum between the normal pregnant rats with herbs pre-pregnancy and the model rats, the differences of content of LIF in decidua between the model rats with herbal medicine pre and post pregnancy and the model rats were both significant (p<0.05).Conclusion:1. The endometrial thickness, the blood flow index of endometrial spiral arteries (RI), and the ratio of E2/P could be the important factors that influence the endometrial receptivity. The pill Zhuyun could effectively improve these factors for recurrent spontaneous abortion patient with spleen and kidney deficiency.2. We could use hydroxyurea (450mg/kg/dX8d) to create kidney deficiency rat model in mild case, that showed such as dysfunction of mitochondria and ribosomal in endometrial epithelial cells, decrease of microvilli at the surface of endometrial epithelial cells, dysfunction of secretion and signal transduction of endometrial epithelial cells, and decrease of LIF in endometrial.3. LIF in endometrial could be one of the key factors that influence the endometrial receptivity of rats. More LIF in endometrial would be good for endometrial receptivity. The increase of CD56-CD16+NK cells in blood and the decrease of CD56+CD16-NK cells in endometrial could destroy the normal phagocytosis of NK cells, and then damage endometrial receptivity.4. Pill Zhuyun could effectively adjust the disorder of endocrine-immune system of kidney deficiency model rats, improve the function of NK cells, increase the content of LIF in endometrial, and then improve the endometrial receptivity and pregnancy success rate.5. Pill Zhuyun could maintain the balance of adjustment function of endocrine-immune system of the normal rats, and then prevent abortion of normal pregnant rats, by feeding them pre or post pregnancy.更多还原