基于TMEM16A基因敲除和MSX1基因重组构建输卵管妊娠小鼠模型的研究

Ectopic pregnancy is the most common cause of acute abdominal pain in gynecology, which is harmful to female reproductive health. About 98% of the ectopic pregnancies occur in the fallopian tubes. This may be caused by impaired embryonic transport in the fallopian tube and good tubal tolerance in human beings. However, the specific mechanism of ectopic pregnancy remains unclear. The main obstacle is the lack of effective experimental animal models. So far, tubal pregnancies of animals have never been reported. The MSX-Wnt-FGFs signaling pathway, which plays a key role in embryo implantation, regulates the intercellular dialogue between the basal layer and the functional layer of the endometrium. The mouse oviductal endometrium does not express the MSX gene and is in a non-acceptable state. Our previous study found that Ca2+-activated chloride channel TMEM16A down-regulates spontaneous contraction of human fallopian tube smooth muscle and epithelial ciliary beat frequency. The recombinant lentiviral vector plasmid integrates the gene of interest into the genome of the infected cell to achieve expression of the gene of interest in the cell or living tissue. Therefore, based on our existing research, we used the TMEM16A conditional knockout mouse to transfect the recombinant lentiviral vector plasmid expressing the MSX1 homeobox gene, which impairs the transportation of embryo and improves the receptivity of oviduct. We will try to construct the experimental animal model of ectopic pregnancy by this method.

异位妊娠是妇科常见的急腹症，对女性生殖健康危害大。约有98%发生于输卵管，其病因可能是胚胎输卵管内运输受阻以及良好的输卵管容受环境所致，然而其具体机制仍不明，主要障碍在于缺乏有效的实验动物模型，迄今尚未报道动物输卵管妊娠案例。MSX-Wnt-FGFs信号通路调控内膜基底层和功能层细胞间对话，对胚胎着床起关键作用，而小鼠输卵管内膜并不表达MSX基因而处于非容受态。我们前期研究发现Ca2+激活氯通道TMEM16A可下调人输卵管平滑肌自发性收缩以及上皮纤毛摆动。重组慢病毒载体质粒可将目的基因整合至所感染细胞的基因组，实现目的基因在细胞或活体组织中的表达。由此，本项目在我们已有研究的基础上利用输卵管TMEM16A条件性基因敲除小鼠，转染表达MSX1同源盒基因的重组慢病毒载体质粒，促使小鼠输卵管动力下降、内膜处于良好容受态从而构建异位妊娠实验动物模型。

宫外孕是妇产科的急腹症之一，尤其是输卵管妊娠。离子通道蛋白TMEM16A广泛分布于各种组织，在人类输卵管的平滑肌中也有分布。在本研究中，我们发现了TMEM16A在人类输卵管上皮细胞和平滑肌中的表达。值得注意的是，它在异位妊娠患者的输卵管中呈上调状态。此外，我们发现在异位妊娠中，甲基转移酶（METTL3, METTL14, WTAP）和m6A 结合蛋白（IGF2BP1, YTHDF1, YTHDF2）的表达减少，而脱甲基酶（FTO, ALKBH5）增加，并且TMEM16A的表达受到m6a甲基化水平的调节。通过对离体输卵管的检测，我们发现TMEM16A不仅调节输卵管的收缩，而且还改变了体外纤毛摆动的频率。此外，TMEM16A的激活或抑制都会导致胚胎滞留在输卵管中，并在体内出现异常的胚胎发育。我们的研究揭示了一个新的胚胎运输和发育的调控点，这可以为异位妊娠的诊断和治疗以及临床生殖研究提供支持。