摘要
目的：不孕症是育龄妇女的多发病及疑难病,辅助生育技术(ART)为不孕症夫妇带来了希望。目前制约ART发展的主要瓶颈是子宫内膜容受性下降导致临床妊娠率持续在低水平徘徊。针对这一现状,我们在中医“肾主生殖”的经典理论指导下,从补肾活血法的角度出发指导临床用药。经过多年的临床实践,使中药改善子宫内膜容受性的临床疗效逐渐得到西医同道的认同。方法：由于伦理和实际操作等因素,在体研究子宫内膜容受性是很难实际开展的,本课题通过动物实验、细胞实验和临床试验,在客观评价补肾活血法改善子宫内膜容受性疗效的基础上,系统分析其内在分子作用机制,并从实验指导临床的角度出发进一步验证其临床治疗机制与途径。动物实验方面：通过复制大鼠超促排卵模型,以补肾活血方分别干预未孕及妊娠大鼠,分析子宫内膜形态、血管、子宫容受性分子标志物的改变；细胞实验方面：根据文献检索发现uNK细胞的数量受月经周期控制,在增殖期子宫内膜中很少,分泌早期(LH+3)开始大量增殖,分泌中晚期及早孕期间数量最多,可达蜕膜淋巴细胞总数的70%,uNK细胞所分泌的细胞因子、生长因子在螺旋动脉重构、胚泡着床及母胎界面免疫耐受中发挥着重要作用。另外,uNK细胞缺陷小鼠模型的胚胎丢失率高达64%,uNK细胞缺失或表型改变直接导致病理性妊娠发生甚至妊娠失败。由此本课题基于uNK细胞与子宫内膜上皮细胞以及间质／上皮细胞三维立体培养的旁分泌系统,应用基因芯片技术研究明确中药补肾活血方在此生理过程中的影响作用。临床试验方面：以东方医院生殖科IVF失败患者为研究对象,运用中药补肾活血方进行对症治疗,通过对比治疗前后患者的月经量、Salle评分、中医证候评分改善情况、超声评价指标(如：子宫内膜厚度、子宫内膜形态、子宫内膜及内膜下血流)及妊娠率评价临床疗效。结果：补肾活血方可改善未孕超促排卵大鼠模型子宫内膜MVD和LIF表达,其治疗机制可能是增加了子宫内膜的厚度、调节血管生成和调节子宫内膜容受性的相关因子；中药补肾活血方可改善超促排卵大鼠围种植期子宫内膜MVD及血管生成相关因子VEGF和OPN的表达,从而降低COH的负面作用,改善子宫内环境提高妊娠率。尽管Ang-2与胚胎血管生成有密切的关系,但此次试验我们并未发现Ang-2因子的表达与子宫内膜容受性的相关性。可能由于在妊娠早期(D3-D5天),Ang-2因子并不是早期胚胎血管生成所必须的相关因子,由此我们也推论胚胎种植前期与胚胎种植后期的血管生成参与因子有明显的不同与差异；我们实验首次验证了子宫内膜uNK细胞与上皮细胞的旁分泌关系,证实了uNK细胞对子宫上皮细胞的旁分泌作用可以促进uNK细胞自身的增殖,同时促进子宫内膜容受性而保证早期胚胎稳定着床、生长和发育的功能,但实验并不支持中药补肾活血方在此生理过程中产生相应的促进或抑制的作用；uNK细胞对子宫上皮细胞及子宫间质细胞／上皮细胞共培养体系的旁分泌作用可以促进uNK细胞自身的增殖,同时促进子宫内膜容受性而保证早期的妊娠稳定,正是由于uNK细胞的旁分泌作用,促进了uNK细胞的增殖,胚胎和滋养层细胞的迁移和子宫内膜的蜕膜化、血管生成化,以及子宫内膜微环境的免疫保护。而uNK+中药组较uNK组可明显上调CXCL9、CCL8、TAP1、CXCR2、VEGF-C、MHC class I polypeptide-related sequence A、 MHC class I polypeptide-related sequence B和ICAM1等重要因子的表达,从而达到提高胚胎移植率和子宫内膜容受性的治疗作用。临床试验部分运用中西结合,辨证与辨病相结合,借助西医检测手段,寻求病因,自拟补肾活血方,以IVF失败的不孕症患者为研究对象,通过治疗前后对比发现患者子宫内膜厚度、子宫内膜形态、子宫内膜及内膜下血流、妊娠率均有明显改善和提高。结论：补肾活血法可以改善子宫内膜容受性,其途径可能是通过uNK旁分泌系统。另外,补肾活血方可通过改善子宫内膜厚度、形态、内膜局部血流来提高肾虚血瘀型患者的症状,从而增高患者的IVF成功率。
Background:Infertility is the frequently-occurring disease and venereology women of childbearing age, assisted reproductive technology brought hope for infertile couples. At present the main bottleneck restricting the development of ART is to the receptivity of endometrium fall in clinical pregnancy rate hovering at a low level. For this situation, we in the traditional Chinese medicine under the guidance of the classical theory of "kidney main reproductive", therapy of traditional Chinese medicine to improve the clinical curative effect of endometrium and the receptivity of endometrium was accepted gradually. Methods:This topic through the animal experiment, cell experiments and clinical trials, the objective evaluation of kidney qi and improve curative effect and the receptivity of endometrium, on the basis of systematic analysis of its internal molecular mechanism and evaluate its clinical efficacy. By copying the rat super stimulate ovulation models to kidney invigorate the circulation of the intervention unfertilized and pregnancy rats respectively, analysis of endometrial morphology, blood vessels, and the receptivity of the uterus molecular markers of change; Cell experiments, based on uNK cells and endometrial cells paracrine system, application of gene chip technology research clear kidney invigorate the circulation of Chinese traditional medicine side effect in the physiological process. Results:Kidney and activating blood can improve the unfertilized super stimulate ovulation in rats model of endometrial MVD and expression of LIF; Kidney invigorate the circulation of the Chinese traditional medicine can adjust the MVD of the film inside the uterus of rats around the time of planting and angiogenesis factors related to the expression of VEGF and OPN; UNK cells of uterine epithelial cells and stromal cells uterus/epithelial cells co-culture system in paracrine effect can promote the proliferation of uNK cells themselves, at the same time promote the receptivity of endometrium and guarantee the stability of early pregnancy, and support Chinese medicine kidney invigorate the party in the physiological process. Conclusions:Chinese medicine kidney invigorate the endometrial receptive, perhaps by uNK paracrine effect to improving uterine microenvironment in angiogenesis.
引文
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