LAT1对滋养层细胞侵袭的调节及相关机理研究

Invasion of trophoblast cells by hormones, cytokines, immune factors and other factors between the network adjustment, the new factors and mechanisms are constantly discovery in-depth. L-type amino acid transporter 1 (L-Type Amino Acid Transporter,-1, LAT1) is a key member of the L-type amino acid transporters and shows ability to regulate a variety of tumor invasion. At the same time, the expression of LAT1 was appeared in the whole pregnancy of mouse and the peak was showed in trophoblast cells on D8. Though the role of LAT1 in Invasion of trophoblast cells and the related mechanism need further research. In former sdudy, we first found LAT1 was expressed in specific pathologic state of trophoblast cell.In this study, we investigated the expression pattern in normal and choriocarcinoma trophoblast tissue and cell lines by immunohistochemistry, western blot, , and clarify LAT1 on trophoblast cell proliferation, invasion ability and its molecular mechanism by matrigel, invasion model, gelatin zymography . At last, we observe the effects of LAT1 on formation of the placenta in mouse as a model. We hope to clarify the role of LAT1 in trophoblast invasion and placenta formation in order to reveal the role in the process and elucidate the molecular mechanism of embryo implantation and prevent the occurrence of human miscarriage, premature birth, fetal growth retardation, obstetric diseases to provide a reliable theoretical basis.

滋养层细胞的侵袭受激素、细胞因子、免疫因子等多因素之间的网络调节，新的因子及机制正不断深入发现。L型氨基酸转运载体1（L-Type Amino Acid Transporter-1，LAT1）为L型氨基酸转运载体中主要成员，表现出调节多种肿瘤侵袭的能力。LAT1表达于小鼠整个妊娠阶段，于妊娠第8天滋养层细胞中呈现高表达。但LAT1对滋养层细胞侵袭调节及相关机制还有待深入研究。课题组前期首次发现LAT1在病理状态下的滋养层细胞特异表达。因此，本研究拟通过免疫组化，western blot、Matrigel侵袭模型、明胶酶谱等技术，首先调查LAT1在正常和绒癌滋养层组织及细胞系的表达规律，阐明LAT1对滋养层细胞侵袭能力的调节及其分子机理，随后以小鼠为体内研究模型，观察LAT1对胎盘形成的影响。以期为阐明胚胎着床的分子机理、防止人类流产、早产、胎儿发育迟缓等产科疾病的发生提供可靠的理论依据。

滋养层细胞的侵袭受激素、细胞因子、免疫因子等多因素之间的网络调节，新的因子及机制正不断深入发现。L型氨基酸转运载体1（L-Type Amino Acid Transporter-1，LAT1）为L型氨基酸转运载体中主要成员，表现出调节多种肿瘤侵袭的能力。本项目在分子、细胞、动物体内模型多个层面，首先调查LAT1在正常胎盘和先兆子痫绒毛组织及细胞系的表达规律，阐明LAT1对滋养层细胞侵袭能力的调节及其分子机理，随后以小鼠为体内研究模型，观察LAT1对胎盘形成以及蜕膜化进程的影响。研究发现，LAT1表达于妊娠第5、25周的流产组织，第40周足月胎盘组织及先兆子痫组织及细胞株HTR8-Svneo、JAR及JEG-3的细胞胞浆与胞膜，BCH及LAT1干扰质粒处理能显著抑制三株细胞增殖，促进细胞凋亡，干扰细胞周期分布，抑制细胞迁移、侵袭能力，而经过LAT1过表达质粒处理的细胞表现为增殖能力、迁移、侵袭能力上调，凋亡能力降低，LAT1对细胞株的体外功能可能与mTOR信号通路相关。在D4-8的小鼠子宫中，LAT1 mRNA和蛋白水平均高于非植入位点，LAT1蛋白主要定位于小鼠子宫的腔上皮、腺上皮，基质细胞和蜕膜细胞；高表达于D8子宫蜕膜区，在EPCs中亦呈现阳性表达。在D9-18天的小鼠胎盘中LAT1蛋白主要定位在母体蜕膜区细胞（Dec）、海绵滋养层细胞（Sp）以及迷路滋养层细胞（Lab）的胞质中。LAT1的特异性抑制剂BCH显著性抑制EPCs 的外延生长，表明LAT1表达于小鼠早期胎盘形成期的子宫中，促进EPCs的外延生长。体外培养小鼠子宫基质细胞并诱导蜕膜化，通过siRNA及 BCH处理下调LAT1的表达，可以体外抑制蜕膜化进程，伴随蜕膜化标志物PRL表达的降低；体内采取子宫角注射LAT1转运抑制剂BCH后抑制了小鼠体内蜕膜化进程，伴随蜕膜面积减少，胚胎体积减少。通过体内子宫角注射BCH抑制LAT1的转运活性导致胎盘形态学结构出现异常。综上结果提示我们， LAT1可能促进滋养层侵袭至母体蜕膜组织，在小鼠胎盘形成进程中可能扮演着重要的作用。该项目研究结果以期为阐明胚胎着床的分子机理、防止人类流产、早产、胎儿发育迟缓等产科疾病的发生提供可靠的理论依据。