Kruppel样因子4在人类原始生殖细胞特化调控中的作用机制研究

As the precursors of spermatozoa or oocytes, human primordial germ cells (PGCs) serve as the foundation for totipotency. However, owing to inaccessibility of early human germ line sample in vivo and ethical limitations, most of the mechanisms underlying human germ cell development are still unclear. Thus, generation of appropriate research models in vitro could provide better understanding for the mechanisms of human germ line development. Kruppel-like factor 4 (KLF4) is a zinc-finger transcription factor with specific genome binding sites. KLF4 is involved in the regulation of cell proliferation, differentiation, embryonic development by binding to different target genes. Our previous research showed that the expression level of KLF4 was increased during the in vitro PGC induction from embryonic stem cells (ESCs). In contrast, we found that the overexpression of KLF4 in ESCs resulted in decreased differentiation efficiency. These results suggest that KLF4 may play an important role in the process of human PGC specification. In this study, we aim to discover the location of KLF4 in human PGCs during early embryo development. Moreover, we try to elucidate the function of KLF4 and the related mechanisms in PGC specification process by an in vitro ESC differentiation model with CRISPR-Cas9 technology, ChIP-sequencing and luciferase reporter system. This research would help us to know more about the biological functions of KLF4 and also the regulation of human PGC development, which is very meaningful for the exploration on mechanisms of human infertility and basic research in reproductive biology.

原始生殖细胞(primordial germ cell, PGC)是精子和卵子的来源，人类PGC发育调控研究进展缓慢，大部分机制尚未阐明，建立合适的体外研究模型有助于探究人类生殖细胞的特化机制。Kruppel样因子4（KLF4）是一种结合位点特异的锌指蛋白转录因子，我们的前期研究发现其在人类PGC体外特化过程中表达上调，而在胚胎干细胞中过表达KLF4降低了其向PGC分化的效率，可见KLF4在人类PGC特化过程中发挥重要的调控作用。因此，我们拟探究KLF4在人类PGC中的表达与定位，并利用胚胎干细胞体外分化技术模拟生殖细胞体内特化过程，通过CRISPR-Cas9介导的基因表达调控技术，探究KLF4在人类PGC特化过程中的功能，并进一步利用ChIP-seq技术及荧光素酶报告系统探究KLF4调控人类PGC特化的分子机制。本研究有助于完善对人类PGC特化机制和生殖细胞发育异常相关不育症机制的认识。

原始生殖细胞(primordial germ cells, PGCs)是精子和卵子的来源，人类早期生殖细胞发育调控研究进展缓慢，大部分机制尚未阐明，建立合适的体外研究模型有助于探究人类生殖细胞的特化机制。Krüppel样因子4（Krüppel-like factor 4, KLF4）是一种结合位点特异的锌指蛋白转录因子，我们的前期研究发现其在人类PGCs体外特化过程中表达上调，而在人胚胎干细胞（human embryonic stem cells, hESCs）中过表达KLF4降低了其向PGCs分化的效率，可见KLF4在人类PGCs特化过程中可能发挥重要的调控作用。本课题通过CRISPR-Cas9技术成功构建KLF4敲除的hESCs，并利用体外分化技术探究KLF4对人类PGCs体外诱导分化的影响，然而结果显示KLF4敲除的hESCs体外向PGCs诱导的效率与野生型细胞无明显差异。相反，我们的前期研究已证实过表达KLF4显著抑制hESCs向PGCs诱导的效率，这可能与KLF4介导的原始多能性有关。基于此，我们推测KLF4是PGCs特化的下游基因，并非PGCs特化所必须，KLF4在PGCs特化后的激活可能为进一步分化做准备。因此，我们体外诱导hESCs向早期精原干细胞（spermatogonia stem cells, SSCs）分化，结果证实敲除KLF4后，hESCs体外向早期SSCs分化的效率明显下降；为探究机制，我们对KLF4敲除前后体外诱导分化所得的早期SSCs进行转录组测序，发现KLF4敲除后Nodal基因的表达受到抑制，NODAL蛋白水平表达及其下游分子Smad2/3磷酸化水平均下降。综上，本课题发现KLF4可能通过NODAL信号调控人类早期精原干细胞的分化，为揭示人类生殖细胞的早期发育机制奠定基础并值得后续的深入研究。