【作者】 游方；

【导师】 黎烈荣；

【作者基本信息】 湖北中医药大学 ， 中医妇科学， 2014， 博士

【摘要】 目的：通过灌胃雷公藤多苷片建立大鼠卵巢功能低下模型，观察中药复方“调经助孕方”对卵巢功能低下模型大鼠生殖系统的影响，初步探讨调经助孕方对改善卵巢储备功能、提高卵泡质量及内膜容受性的机理；并验证本方治疗因卵巢储备功能下降，卵泡发育障碍所致不孕症的有效性，为后期调经助孕方的中药新药研究开发提供一定的科学依据。方法：除空白对照组(简称空白组)大鼠以生理盐水10000mg/(kg·d)灌胃外，其余大鼠采用雷公藤多苷片50mg/(kg·d)连续2周灌胃以复制肾虚型卵巢储备功能下降致卵泡发育障碍的生殖功能低下动物模型，并至灌胃第8天起行阴道脱落细胞学涂片观察动情周期变化情况以确定造模情况。将造模成功的大鼠随机分为模型对照组(简称模型组)、中西药联合组(简称联合组)、西药组及调经助孕方组(简称中药组)。后其中空白组和模型组大鼠以10000mg/(kg·d)生理盐水灌胃；西药组大鼠予以激素替代加促排卵治疗。具体方法为先在第一个性周期5天内行激素替代治疗，连续5日予以戊酸雌二醇片0.12mg/(kg·d)，至第4天起加用黄体酮胶囊18mg/(kg·d)，后在下一性周期内于大鼠动情前期连续2天灌胃枸橼酸氯米芬片4.5mg/(kg·d)以行促排卵治疗；中药组按成人等效剂量灌胃调经助孕中药复方颗粒5250mg/(kg·d)，连续10天；中西药联合组则参照中药组及西药组给药方法和计量，给予中西药联合用药10天。治疗后采用酶联免疫吸附法和化学发光免疫法分别检测血清促卵泡生成素、促黄体生成素、雌二醇、孕酮及抗苗勒氏管激素水平；利用组织病理学方法研究各组大鼠卵巢、子宫的形态及组织病理学改变，并统计各级卵泡及黄体计数；借助蛋白质印迹法观察卵巢内相关细胞因子：干细胞因子、生长分化因子-9及骨形态发生蛋白-15的蛋白表达水平变化；通过免疫组化法及末端脱氧核苷酸转移酶介导的dUTP缺口末端标记测定法分别观察卵巢细胞促凋亡基因Bax的表达情况及卵巢细胞凋亡率的变化；最后则运用实时荧光定量聚合酶链式反应法检测围着床期子宫内膜降钙素基因的表达情况以观察其容受性的变化。结果：1.对血清生殖激素的影响：模型组大鼠血清FSH、LH高于空白组，E2及AMH则较空白组低，均有统计学意义(P<0.01)。治疗后西药组和中药组大鼠血清各项生殖激素已恢复到正常水平，FSH、LH水平较模型组相比下降，E2及AMH水平则较模型组升高，差异均有统计学意义(P<0.01)，与空白组相比已不存在显著性差异(P>0.05)。2.对脏器指数的影响：造模后模型组大鼠卵巢及子宫指数均低于空白组，二者间的差异有统计学意义(P<0.05)。治疗后，西药组和中药组的卵巢、子宫指数上升，与空白组不存在明显差异(P>0.05)，而与模型组相比，则有显著性差异(P<0.05)。3.对卵巢、子宫显微结构的影响：HE切片光镜下可见空白组卵巢皮层中分布有许多原始卵泡，各级卵泡发育正常、卵泡形态大小各异，卵泡内卵母细胞大而圆，轮廓清晰，并见大量黄体；模型组卵巢中各级卵泡少见，卵泡直径小、卵泡内颗粒细胞层数较其余各组明显减少，有大量闭锁卵泡和白体，极少见黄体，髓质致密，血管较少；西药组和中药组卵巢内初级卵泡和次级卵泡发育较正常，可见卵泡内母细胞大而圆，轮廓清晰，可见黄体和闭锁细胞，髓质血管较丰富。另空白组和中药组子宫内膜上皮细胞排列整齐，腺体和基质丰富；模型组子宫内膜上皮细胞排列紊乱，腺体减少，基质稀疏水肿，大量嗜酸性粒细胞浸润；西药组子宫内膜上皮细胞排列尚整齐，腺体和基质较丰富，少量嗜酸性粒细胞浸润。4.对卵泡及黄体计数的影响：模型组大鼠卵巢内生长及成熟卵减少、黄体少见，闭锁卵泡增多，与空白组相比有极显著性差异(P<0.01)。经治疗后，西药组、中药组大鼠生长、成熟卵泡及黄体数均有明显增加，而闭锁卵泡减少，较模型组有很大改善(P<0.01)，与空白组间已无统计学差异(P>0.05)。其中，西药组生长卵泡数较空白组及中药组均明显增多(P<0.05)，而成熟卵泡、闭锁卵泡和黄体则与中药组无明显差异(P>0.05)。5.对卵巢内细胞因子的影响：模型组卵巢内细胞因子SCF、GDF-9及BMP-15的蛋白定量表达水平均较空白组有明显下降，有显著性差异(P<0.05)。而经治疗后均能明显上调大鼠卵巢内这三种细胞因子的表达，西药、中药组的细胞因子蛋白表达量同空白组相比，差异不存在统计学意义(P>0.05)。6.对卵巢细胞促凋亡基因Bax表达的影响：空白组大鼠卵巢细胞Bax基因阳性表达率较其余各组来说均处于明显低水平，其中与模型组及西药组间差异存在极显著性(P<0.01)；与中药组间则有显著差异(P<0.05)。经治疗后，西药组、中药组Bax基因阳性表达率较模型组有大幅下降，其二者同模型组间的差异均有统计学意义(P<0.01)，而其组间的差异则不没有统计学意义(P>0.05)。7.对卵巢细胞凋亡率的影响：空白组大鼠的卵巢细胞凋亡率在所有组别中为最低水平，与余各组间的差异均存在极显著性(P<0.01)。西药、中药组经治疗后，虽凋亡率较模型组有大幅下降(P<0.01)，组间没有显著性差异(P>0.05)，但仍与空白组存在极显著性差异(P<0.01)。8.对围着床期血清孕酮含量的影响：围着床期大鼠血清孕酮含量各组之间无显著性差异，空白组与西药组、中药组三组各组之间的差异均无统计学意义(P>0.05)。9.对围着床期子宫内膜CT mRNA表达的影响：西药组大鼠围着床期时内膜CT mRNA表达水平较空白组、联合组与中药组来说均处于低水平，其间的差异均存在极显著的统计学意义(P<0.01)，虽中药组的表达水平稍高于空白组及联合组，但其三者间的差异均没有显著性(P>0.05)。结论：徐升阳教授认为卵巢功能低下，卵泡发育障碍所致不孕症患者中医辨证主要属肾虚，其中合并黄体功能不全者或兼夹肝郁之证，并以此提出了“补肾以促卵泡发育，调肝以利黄体功能，健脾以益内膜容受性”的观点，自拟“调经助孕方”以补肾益精、调经种子之法来治疗因卵巢储备功能下降，卵泡发育障碍所致不孕症或作为IVF-ET的辅助治疗。本实验研究表明，调经助孕方能通过下丘脑-垂体-卵巢生殖轴以调节紊乱的生殖激素水平、修复模型大鼠子宫、卵巢组织损伤并改善其显微结构的病理改变；同时可促进模型大鼠卵泡及黄体发育，减少闭锁卵泡的生成，并提高卵巢内相关细胞因子的蛋白表达量来调节模型大鼠卵巢的自/旁(邻)分泌功能；还能抑制模型大鼠卵巢内卵泡颗粒细胞的凋亡、减缓卵泡闭锁以正向调节卵泡的正常发育；除此之外，调经助孕方还能上调围着床期时子宫内膜CTmRNA的表达来提高内膜的容受性以利于胚胎的着床。更多还原

【Abstract】 Objectives:Gastric administration of Tripterygium Glycoside (TG) Pill was given tothe rats to establish ovarian dysfunction model through which to observe theeffects TiaoJingZhuYun decoction on reproductive system of rat with ovariandysfunction and study futher the mechanism of this decoction in strengtheningovarian reserve, improving follicle qualilty and enhancing the endometriumreceptivity.Furth more, verified clinical curative effect on infertility caused byDecreasing Ovarian Reserve (DOR) and follicle development dysfunction canprovide a scientific evidence to some degree for the research in developingpatterned Chinese herbal products.Methods：The placebo group (P group) were perfused with physiological saline bydoes in10000mg/(kg·d), TG were administered to the rest for2consecutiveweeks to copy the model of low reproduction for follicle developmentdysfunction caused by DOR deemed as insufficiency of kidney. In the meantime, conduct viginal exfoliate cells test to observe the change in estrus so asto ensure whether the model was induced successfully. Next, the model-induced rats were randomized into model control group(M group), weartern medicine treated group (W group), Chinese herb treatedgroup (H group), combination group (C group). Then, P group and M groupwere perfused with physiological saline by does in10000mg/(kg·d). Whereas,conduct hormone replacement therapy (HRT) and ovarian stimulation in Wgroup. It comes specificly as below: implement HRT in1stestrus, perfuse ratswith Estradiol Valerate Tablete by does in0.12mg/(kg·d) for5sucessive daysbut add Progesterone Capsule by does in18mg/(kg·d) on4thday, thenadminister Clomifene Citrate Tablet to rats by does4.5mg/(kg·d) to conductovarian stimulation therapy for2consecutive days before the next proestrus.H group were ferfused with TiaoJingZhuYun complex priscription powder bydoes in5250mg/(kg·d) for10sucessive days. C group were treated withcombination of Chinese herb&Werstern drugs, and the does was the same asthe former2groups respectively.Adopting Enzyme Linked Immunosorbent Assay (ELISA) andChemiluminescence Immuno Assay (CLIA) after the therapy to detect thelevel of Follicle Stimulating Hormone(FSH)、 Luteinizing Hormone (LH)、Estradiol (E2)、 Progesterone (P) and Anti-Müellerian hormone (AMH)respectively. Taking advantage of Pathology to study the shape andPathological change of rats’ ovaries ang uteries, and make a statistics of thecount of follicles on each rank and corpus luteum. Observing the change onthe level of protein expressed of ovarian relevant cytokines by means ofWestern Blot. Watching the expression condition of ovarian cellspro-apoptotic gene Bax and the change on apoptosis rate throughImmunohistochemical Method and TdT-mediated Dutp Nick-End Labeling(TUNEL). Detecting the expression of endometrium Calcitonin inperi-implantation by Real-Time Quantitative Polymerase Chain Reaction(RT-PCR) to inspect the change of its receptivity comes to the final step. Results:1. Impact on the reproductive hormone in serum: FSH and LH of Mgroup were higher than that of P group; whereas, E2and AMH were lowercompared with P group, which maked a statistical significance (P<0.01). Thehormone level of W group and H group has recovered to the normalrespectively after the therapy, FSH and LH decended but E2and AMH rised incomparision with M group, which counted a lot in statistic, on the contrast, thedifference with P group didn’t make any sense.2. Influence on the organ index: There was statistical significancebetween the P group and M group refer to the ovary and womb index whichwere lower in M group than in P group. But index in W group and H grouphas got up, not making a statistical significance (P>0.05) when compared withP group after the therapy.In contrast to M group, it did not (P<0.05).3. Effect on the microstructure of ovary and womb: a scale of primitivefollicle can be spotted in HE section by light microscope in ovarian cortex of Pgroup. The size and shape of follicle on every stage which developed normallyvaried. Oocytes were both large and round that were in distinct outline, andcorpus luteum in large amount can be caught.Follicles on each stage whichhad a short diameter were barely spotted in M group. Layers of granulosa cellsin follicle had reduced in comparision with other groups. There were also alarge sun of atrefic follicles and corpus albicans and infrequent corpus luteum.The medella of ovary were intensive and its blood vessels were fewer. Firstand second rank follicles in W group and H group developed in the normalway and the condition oocytes were the same as P group but atrefic folliclescan be seen and abundance in the blood vessels of medulla.Endometrial epithelial cells of P group and H group arranged orderly,glands and matrix were abundant.Whereas, the cells of M group were indisorder with decreasing glands and sparse papilledema matrix as well as massive infiltrating ensinophilic granulocyte. But the situation in W groupwere opposite.4. Impact on the count of follicle and corpus luteum: The developing andmature follicles has reduced with infrequent corpus luteum and increasingatrefic follicles in M group which maked a great statistical significance(P<0.01) compared with P group. After therapy, the developing and maturefollicles has increased obviously and atrefic follicles has reduced, improving alot in comparision with M group (P<0.01) and making any sense opposed to Pgroup (P>0.05). What was emphasize was that the developing follicles in Wgroup were up apparent than that in P group and H group (P<0.01), but thedistinction of mature follicles and atrefic follicles as well as corpus luteum canbe neglected (P>0.05).5. Influence on cytokines in ovary: The level of quantitative protein beingencoded of cytokines such as Stem Cell Growth Factor (SCF)、HumanGrowth Differentiation Factor-9(GDF-9) and Bone Morphogenetic Protein-15(BMP-15) has clearly decended opposed to P group, making a statisticalsignificance (P<0.05). Even though the expressin of three cytokines in Wgroup and H group has been evidential up-regulated, the distinct with P groupmake any sense (P>0.05).6. Effect on the expression of ovarian cell pro-apoptotic gene Bax: Thepositive expression ratio of ovarian cell pro-apoptotic gene Bax in P groupwere manifest lower than the rest, making a statistical significance with Mgroup and W group (P<0.01) but a bit difference compared with H group(P<0.05). The positive expression rate of pro-apoptotic gene Bax in W groupand H group were much lower than that in M group after the therapy. Thoughthe divergence between M group has statistical significance (P<0.01), thedifference between W group and H group can be ignored (P>0.05). 7. Impact on the apoptosis rate of ovarian cell: The apoptosis rate ofovarian cell in P group was at the bottom among all the groups and thedivergence was much evidential compared to the other groups respectively(P<0.01). When the W group ang H group got treated even though theapoptosis rate has descended a lot in comparision with M group (P<0.01) anddifference made no sence between the two groups (P>0.05), there was stillstatistical significance (P<0.01) opposed to P group.8. Influence on the content of serum Progesterone in peri-implantation:There was no manifest differense among groups. Divergence between Pgroup、H group and W group can be neglected (P>0.05).9. Effected on the expression of endometrium CT mRNA inperi-implantation: The expression level of endometrium CT mRNA of Wgroup in peri-implantation was the lowest in comparision with P group、Cgroup and H group,making a great statistical significance (P<0.01). Specificly,the expression level of H group was a bit higer than that of P group and Cgroup, but the difference between the three groups maked no sense (P>0.05).Conclusions:Professor Shengyang Xu clings to the perspective that it is primary insufficiency of kidney Essence and Qi and partly hepatic stagnation whenmerged with luteal insufficiency based on syndrome differentiation thatcontributes to infertility as the result of DOR and follicle developmentdysfunction. So, he puts forwards the decoction of regulate menstruation forpregnancy promotion, a creative achievement from his perspective which isfocused on nourishing kidney to foster the development of follicle, smoothingliver to improve the function of corpus luteum, and enhancing theendometrium receptivity by strengthening the spleen as the therapy of this disease or supplementary for In Vitro Fertilization And Embryo Transfer(IVF-ET).This experimental research show that TiaoJingZhuYun decoction canregulate the disordered hormorne level, renovate model rats injured womb andovarian tissue as well as mitigate pathological change of microstructure bymeans of Hypothalamus-Pituitary-Ovary Axis. What’s more, it’s able to fosterthe development of follicle and corpus leteum, reduce the amount of atreficfollicle, accumulate the protein that relevant cytokines in ovarian encodes toregulate the function of model rats autocrine and paracrine as well asjustacrine, depress apoptosis of rats granulosa cells, slacken atrefic follicle toadjust it to cultivate in forward direction. Finally, TiaoJingZhuYun decoctioncan upgrade the expression of endometrium CT mRNA in peri-implantationfor enhancing the endometrium receptivity which can lay the foundation ofembryo implantation.更多还原