基于HIF-1α/miR-210调节回路探讨化瘀消癥复方对低氧状态下输卵管妊娠滋养细胞自噬介导的血管生成与侵袭力影响

Tubal pregnancy is a common and frequently-occurring gynecological disease that influence the female reproductive health seriously. The treatment that dissolving stasis and eliminating mass had been proved effectively to treat the early stage of the disease, while the mechanism still has been exploring. The previous consequence of our reseach team has shown the Compound Prescription that Dissolving stasis and Eliminating mass could lower the proliferation and invasion, as well as upgrade the apoptosis of the tubal trophoblasts, which may be related to the effects of the PI3K/AKT/mTOR pathway to treat the tubal pregnancy. When the trophoblasts implant in the fallopian tube, the activity of the cells will be effected by the hypoxic condition. So，the differential expression of the HIF-1α/miR-210 on the villi tissue inside and outside of the uterus will be checked in this study. As the next step, the activity of the tubal trophoblasts will be contrasted when cultured by normoxia or hypoxic condition. MicroRNA-210 transfection model will be established, Real time PCR will be performed to detect the expression levels of target gene mRNA about the hypoxia-related index, the autophagy, the angiogenesis and the invasion of tubal trophoblasts. The impact on the above indicators mediated by different concentrations of the Compound Prescription that Dissolving stasis and Eliminating mass is the key point of the study, which will be the new scientific evidence of the Chinese medicine.

输卵管妊娠是严重影响女性生殖健康的常见病，化瘀消癥复方对早期输卵管妊娠有确切的疗效，但其作用机制仍处在不断的探索之中。研究团队前期通过实验研究证实化瘀消癥复方可下调输卵管妊娠滋养细胞的增殖与侵袭力，促进凋亡的发生，可能与PI3K/AKT/mTOR的通路效应相关，进而起到治疗输卵管妊娠的作用。滋养细胞着床于输卵管，局部的低氧状态影响其活性，故本项目拟以宫内与输卵管妊娠绒毛组织中HIF-1α/miR-210的差异表达为着眼点，在对比常氧或低氧状态下所培养的输卵管妊娠滋养细胞活性的前提下，建立miR-210沉默与过表达转染的细胞模型，采用Real time PCR的方法，对低氧相关、细胞自噬、血管生成与侵袭力等几个方面的目的基因进行检测，并探讨不同浓度化瘀消癥复方对上述指标的影响。为寻找中药作用靶点提供新的科学依据。

本项目的离体组织验证部分以宫内与输卵管妊娠绒毛为研究对象，结果发现输卵管妊娠绒毛中与低氧及自噬相关的蛋白表达较宫内妊娠绒毛上升，推测输卵管腔的低氧状态，通过影响HIF-1α表达，诱导并增强滋养细胞自噬，可能是导致输卵管妊娠破裂的原因之一。体外细胞验证部分以人绒毛滋养细胞系HTR-8/SVneo细胞为细胞载体，以化瘀消癥颗粒为研究对象，以Cocl2作为低氧的化学干预剂，以miR-210沉默和过表达的滋养细胞作为对照组，结果发现：①低氧状态可能通过增强滋养细胞中HIF-1α表达，影响VEGF、Beclin-1、LC-3、MMP-9、Timp-1、N-cadherin、uPA、PAI-1等蛋白表达，诱导细胞自噬并增强其血管生成能力、侵袭能力，加快上皮间质转化过程。②化瘀消癥颗粒在常氧状态及低氧状态均可下调滋养细胞中HIF-1α表达，影响上述的蛋白表达，抑制滋养细胞活性。③miR-210沉默的滋养细胞中，HIF-1α表达下降，细胞活性减弱；化瘀消癥颗粒高剂量组所逆转的低氧状态下滋养细胞中各蛋白表达的趋势，与miR-210沉默的滋养细胞中各蛋白表达的结果接近。④miR-210过表达的滋养细胞中，HIF-1α表达上调，细胞活性增强；化瘀消癥颗粒高剂量组逆转miR-210过表达滋养细胞中各蛋白表达，减弱miR210过表达后的细胞活性。综上，化瘀消癥颗粒可能发挥与miR-210沉默的细胞相似的生物学效应，通过下调HIF-1α表达，抑制“低氧状态下”与“miR-210过表达”的滋养细胞中自噬的发生，减少血管生成，削弱细胞侵袭力，影响上皮间质转化过程。因低氧状态更接近输卵管妊娠滋养细胞的生存环境，故化瘀消癥颗粒可能通过抑制细胞活性，减轻异位的滋养细胞对输卵管的浸润，降低输卵管妊娠破裂的风险，继而起到治疗输卵管妊娠的作用。