节点文献
补肾调周法在夫精人工授精技术中的应用及机制研究
【作者】 李楠；
【作者基本信息】 北京中医药大学 ， 中西医结合临床， 2012， 博士
【摘要】 目的1.评价补肾调周法干预行夫精人工授精(IUI)患者的临床疗效,观察补肾调周法对卵泡发育及子宫内膜容受性的影响,探讨其提高临床妊娠率的机理。2.研究育泡饮(补肾调周系列方组成之一)对体外培养大鼠卵巢颗粒细胞增殖、分泌、细胞周期的影响以及其对EGFR、CX43、P38MAPK蛋白表达的影响,从细胞及分子生物学水平探讨育泡饮促卵泡发育的作用机制,为验证育泡饮的有效性提供科学、客观的实验依据。方法临床研究1.将符合本研究入选标准的60例患者,随机分成两组,统一选用氯米芬促排卵方案,对照组(氯米芬组)34例,治疗组(补肾调周系列方联合氯米芬组)26例。观察两组患者治疗前后中医肾气虚证候变化；治疗后肾气虚证候积分与HCG注射日子宫内膜厚度、内膜血流参数以及卵泡周围血流、子宫动脉血流参数的相关性。2.比较两组患者HCG注射日子宫内膜厚度、类型,卵泡周围血流、子宫动脉血流、子宫内膜血流参数以及临床妊娠率。实验研究1.制备育泡饮水提醇沉液,过滤后4℃冰箱保存备用。2.育泡饮水提醇沉液作用于卵巢颗粒细胞药物浓度的确定：将1×10-7g/ml～1×10-3g/ml浓度的育泡饮水提醇沉液分别作用于体外培养大鼠卵巢颗粒细胞,培养24、48、72h,设立空白对照组。通过MTT法检测该药物对颗粒细胞增殖的影响,确定药物作用的量效时点以进行后期试验。3.育泡饮水提醇沉液对颗粒细胞分泌功能、细胞周期以及EGFR、CX43、P38MAPK蛋白表达的影响：颗粒细胞预培养48h后,随机分组,加入促卵泡激素FSH(50ng／m1)和不同剂量的育泡饮,并设立空白对照组,继续培养48h后,化学发光法检测颗粒细胞培养液中E2的分泌量,流式细胞术检测颗粒细胞周期的分布,Western-Blot法检测EGFR、CX43、P38MAPK的表达。结果1.临床观察表明,经补肾调周系列方治疗后,治疗组患者肾气虚症状改善情况优于对照组,组间比较有统计学意义(P<0.05)。两组患者中医证候疗效比较结果显示：治疗组显愈率为23.08%,总有效率为69.23%,对照组分别为11.76%和41.18%,治疗组显示出明显的疗效优势(P<0.05)；两组患者治疗后肾气虚证候积分与子宫内膜厚度、卵泡血流参数、子宫动脉及子宫内膜血流参数均存在相关性。其中与子宫内膜厚度呈负相关；与内膜血流PI、RI,子宫动脉血流RI及卵泡血流RI均成密切正相关。2.治疗组患者在IUI周期HCG注射日子宫内膜厚度明显增加,而卵泡周围血流PI、RI,子宫动脉PI、RI,子宫内膜血流PI、RI均明显降低,与对照组比较均具有显著性差异(P<0.05)；HCG注射日治疗组A型内膜占53.85%,B型占38.46%,而对照组分别为23.53%和29.41%,两组相比较,差异显著(P<0.05)；两组妊娠率比较结果显示,治疗组临床妊娠率为34.61%(9/26),周期妊娠率19.56%(9/46),对照组分别为11.76%(4/34)和6.06%(4/66),两组之间的差异有统计学意义(P<0.05)。3.育泡饮水提醇沉液在1×106g/ml～1×10-4g/ml药物浓度范围内,随着药物浓度的增加,培养时间的延长,其促进颗粒细胞增殖的能力逐渐增强,在培养48h时作用最佳。4.育泡饮水提醇沉液低、中、高剂量组均能促进颗粒细胞E2分泌,与空白组相比较有显著性差异(P<0.05),随着育泡饮药物浓度的增大,E2的分泌逐渐增加。与FSH组相比较,育泡饮低剂量组与其有显著性差异(P<0.05),而育泡饮中、高剂量组与FSH组促进E2分泌的作用无明显差异。5.经流式细胞仪检测,结果显示空白对照组颗粒细胞G0/G1期比例较育泡饮水提醇沉液低、中、高剂量组及FSH组明显增高,而S期比例及PI值则明显降低(P<0.05)。高剂量组与FSH组比较,G0/G1期细胞明显减少,S期细胞及PI值则明显增加(P<0.05)。而低剂量、中剂量组与FSH组比较,无差异(P>0.05)。6.育泡饮水提醇沉液呈剂量依赖性促进颗粒细胞EGFR表达。在低剂量作用下颗粒细胞EGFR表达开始增强,与空白对照组比较具有明显差异(P<0.05),高剂量组的促进作用最为显著(P<0.05)。与FSH组比较,低剂量组促颗粒细胞EGFR表达的能力明显降低(P<0.05)。而高剂量组与FSH的作用相比较无统计学差异(P>0.05)。7.与空白组相比较,育泡饮水提醇沉液低、中、高剂量组及FSH组CX43蛋白表达均显著升高(P<0.05),不同浓度的育泡饮水提醇沉液与FSH组比较,中、低剂量组CX43蛋白表达较之明显降低(P<0.05)。高剂量组CX43蛋白的表达与之无显著性差异(P>0.05)。提示随着育泡饮水提醇沉液浓度的增加,颗粒细胞CX43蛋白表达也随之增强,高浓度的育泡饮可以达到与FSH相同的效果。8.与空白组相比较,育泡饮水提醇沉液各剂量组及FSH组均能促进P38MAPK表达增加(P<0.05)。与FSH组比较,除低剂量组育泡饮P38MAPK表达明显降低(P<0.05),中、高剂量组P38MAPK表达与之无显著性差异(P>0.05)结论1.补肾调周法配合氯米芬,可明显提高IUI患者临床妊娠率。其机制可能与补肾调周系列方药改善卵泡周围血流,促进卵泡发育成熟；增加子宫内膜厚度、改变子宫内膜形态,降低子宫内膜及子宫动脉血流阻力,增加子宫动脉及子宫内膜血流灌注,增强子宫内膜容受性有关。2.颗粒细胞的增殖对于卵泡发育是必须的,育泡饮不仅能促进颗粒细胞的分泌,还能推动细胞周期进程,促进颗粒细胞增殖；上调CX43蛋白的表达,增强颗粒细胞的抗凋亡能力；调节EGFR的表达量,激活P38MAPK通路,促进卵母细胞成熟,通过以上多种途径共同作用,从而促进卵泡的发育与成熟。
【Abstract】 Objective1. To evaluate the treatment effect of kidney-reinforcing and menstrual cycle-regulation therapy in patients undergoing intrauterine insemination (IUI). To observe the effect of kidney-reinforcing and menstrual cycle-regulation therapy in follicle development and endometrial receptivity, and explore the mechanism of kidney-reinforcing and menstrual cycle-regulation therapy in improvement the clinical pregnancy rate.2. To research Yu Pao Decoction (a recipe of kidney-reinforcing and menstrual cycle-regulation) in rat’s ovarian GC proliferation, secretion, cell cycle and the protein expression of EGFR,CX43and P38MAPK.To explore the mechanism of it in promoting follicular development from the level of the cell and molecular biology in order to provide scientific and objective experimental evidences to verify the effectiveness of the recipe.MethodClinical research1.60sterile women according with the clinical research criteria were randomlydivided into two groups. They were given the same clomiphene treatment.34cases in the control group were treated with clomiphene alone, and26cases in the study group were treated with kidney-reinforcing and menstrual cycle-regulation decoction in combination with clomiphene. The syndrome points of the deficiency of kindey qi of both groups after treatment were observed, and the relevance of the syndrome points of deficiency of kindey qi and endometrial thickness, pulsatility index (PI) and resistant index (RI) of endometrial blood stream, follicle blood flow and uterine artery were discussed.2. It was evaluated between both groups including the thickness and types of endometrium, pulsatility index (PI) and resistant index (RI) of endometrial blood stream, follicle blood flow and uterine artery and clinical pregnancy rate.Experimental study1. Preparation of water extraction and alcohol precipitation of Yu Pao Decoction, after filtered and stored at4℃。2. Determination was made the drug level of Yu Pao Decoction:We treated rat’s ovarian GC with1×10-7g/ml～1×10-3g/ml of the decoction, and then respectively cultured for24h,48h,72h. At the same time, we set up a blank control group, through the MTT assaying different concentration of Yu Pao Decoction on GC growth and proliferation, determining the optimal dose-effort point to further experiments.3. Effect of Yu Pao Decoction on GC secretion, cell cycle, and the protein expression of EGFR, CX43and P38MAPK:GC was isolated from rat’s ovaries and cultured for48 hours, divided into five group; three dosage groups of Yu Pao Decoction, blank control group and FSH(50ng/ml) control group at random.,after cultured for48h, assayed the secretion quantity of E2by chemiluminescence, used flow cytometry to assess the cell cycle change of GC, respectively detected the protein expression of EGFR,CX43and P38MAPK by Western-Blot method.Results1. The results of clinical research showed that after treatment the symptoms of deficiency of kidney qi in the study group were significantly relieveded comparing with the control group, the difference was significant (P<0.05).The clinical curative effect comparison of both indicated that the apparent rate and total effect rate in the study group were both higher than in the control group(P<0.05). The apparent rate of the study group was23.08%,the total effective rate was69.23%, while the apparent rate of the control group was11.76%, the total effective rate was41.18%; There were correlations between deficiency of kidney qi and endometrial thickness, pulsatility index (PI) and resistant index (RI) of endometrial blood stream, follicle blood flow and uterine artery. Negative correlation was displayed in deficiency of kidney qi and endometrial thickness, positive correlation in deficiency of kidney qi and pulsatility index (PI) and resistant index (RI) of endometrial blood stream perfusion, RI of follicle blood flow and uterine artery.2. Yu Pao Decoction could obviously promote the growth of endometrium and decrease the PI and RI of follicle blood flow, uterine artery and endometrial blood stream on the day of HCG injection in the IUI cycle. Compared with the control group, the difference was significant (P<0.05); Type A endometrium accounted for53.85%, B was38.46%in the study group on the day of HCG injection. In the control group,Type of A and B endometrium separately accounted for23.53%and29.41%. The study results also revealed that the clinical pregnancy rate and the cycle pregnancy rate of the study control were significantly higher than the control group (P<0.05). The clinical pregnancy rate and the cycle pregnancy rate of the study control were34.61%and19.56%. The control group was respectively11.76%and6.06%.3. Within the scope of1×10-6g/ml～1×10-4g/ml, the promoting proliferation capacity of Yu Pao Decoction gradually was enhanced increasing with the increase of the dosage and the culture-time. The best culture-time was48h.4. Each dosage group of Yu Pao Decoction could markedly increase secretion of E2compared with the blank control group (P<0.05), and the secretion quantity of E2gradually increased with the increase of the dosage. The difference was obvious between the low-dosage of Yu Pao Decoction and FSH control group (P<0.05). No significant difference of the secretion of E2was found between the medium、high-dosage group and FSH control group. 5. Though flow cytometry analysis operation, it showed that there was a remarkabe increase the in the ratio of GC Go/G1-phase and a obvious decrease in the ratio of S-phase and proliferation index compared with each dosage group of Yu Pao Decoction and FSH control group(P<0.05). The high-dosage group of Yu Pao Decoction could regulate up the ratio of S-phase and proliferation index. At the same time, regulate down the ratio of Go/G1-phase (P<0.05). There was no difference between the low、medium-dosage group of Yu Pao Decoction and FSH control group (P>0.05).6. EGFR protein expression gradually was increased with the increase of dosage of Yu Pao Decoction. The EGFR protein expression of each dosage group of Yu Pao Decoction was better than the blank control group (P<0.05).The high-dosage group of Yu Pao Decoction showed the most obvious effect (P<0.05), and the high-dosage group had the same effect with FSH control group(P>0.05). The EGFR protein expression of low-dosage group was significant lower than FSH control group (P<0.05).7. Three dosage groups of Yu Pao Decoction and FSH control group all could promote CX43expression. The CX43expression of low and medium-dosage group were lower than FSH control group (P<0.05). The high-dosage group could achieve the same effect with FSH group.8. Three dosage groups of Yu Pao Decoction and FSH control group all could promote P38MAPK expression. The difference was remarkable comparing with the blank control group (P<0.05). Compared with FHS control group, only decreased in low-dosage group, the P38MAPK expression in medium and high-dosage group had no difference (P>0.05).Conclusion1. Kidney-reinforcing and menstrual cycle-regulation therapy combined with clomiphene can significantly improve the clinical pregnancy rate of patients undergoing IUI.The possible mechanism of combined therapy is promoting oocyte maturation by benefiting follicle blood flow; enhancing endometrial receptivity by increasing endometrial thickness, changing the type of endometrium, reducing the blood flow resistance, increasing blood flow perfusion of uterine artery and endometrial blood stream.2. It is necessary to GC proliferation for follicle growth. Yu Pao Decoction can not only promote GC proliferation and secretion but also push cell cycle forward; as well as regulate up the CX43expression; enhance the ability of GC anti-apoptosis; adjust EGFR expression level; activate P38MAPK pathway,through above ways to achieve follicle growth and maturation.