【作者】 黎刚；

【导师】 王应雄；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2004， 博士

【摘要】 白血病抑制因子（leukemia inhibitory factor, LIF）是一种多功能的细胞因子,在不同的细胞和组织中具有多种生物学活性，近年来发现它在生殖中发挥着重要的作用。LIF启动靶细胞发挥其生物学功能是通过其受体系统实现的。LIF首先与膜受体gp190连接, 而后再同gp130（IL-6信号转导子）连接构成异源性二聚体,启动下游信号传导发挥其生物学效应。1998年发现人体液中存在LIF可溶性受体, soluble gp190（sgp190）,但目前对sgp190具体的生物活性的研究尚未见报道。由于LIF在诸多生命活动中的重要作用，研究sgp190的生物活性具有非常重要的意义。 本研究对sgp190在酵母Pichia pastoris中实现了重组sgp190分泌表达。利用基因重组技术，将编码人可溶性gp190（LIF受体α亚基gp190胞外区）cDNA克隆到毕赤酵母Pichia pastoris分泌表达载体pPIC9K,构建了重组表达质粒pPIC9K－sgp190。原生质体法转染Pichia pastoris GS115菌株，经过G418筛选，得到了高效分泌表达sgp190蛋白的毕赤酵母菌株GS115/pPIC9K-sgp190，sgp190蛋白占摇瓶培养表达上清中总蛋白质的27%。经初步纯化，对表达产物的性质鉴定表明，其分子量约125 kD,具有免疫活性。获得的sgp190蛋白为体内外深入研究其生物学活性奠定了基础。 <WP=10> 本研究第二部分以体外培养滋养层细胞为载体，从sgp190对滋养层细胞增殖、分化，激素分泌，IL-10表达，与侵袭力有关的MMP-9和TIMP-1分泌水平及对Jak1-STAT3信号通路中STAT-3磷酸化水平的影响等几个方面对sgp190活性进行了研究。结果显示，30 ng/ml sgp190基本能完全抑制10 ng/ml LIF对滋养层细胞诱导分化作用。LIF能够促进细胞滋养层细胞向合体滋养层细胞分化（P < 0.05），而同时施加sgp190与LIF组对细胞分化基本无影响（P > 0.05）。细胞增殖（MTT实验）表明，sgp190与LIF不影响滋养层细胞的增殖（P > 0.05）。在滋养层细胞体外培养48h前，LIF促进HCG的分泌，而此后，LIF抑制HCG的分泌，sgp190则能抑制LIF的促进或抑制作用。滋养层细胞的侵袭能力与MMPs－TIMPs系统的活性有关，我们通过分析LIF与sgp190对MMP-9 和TIMP-1 蛋白表达水平的影响来研究LIF及其可溶性受体sgp190在MMPs－TIMPs系统中扮演的角色。结果显示，LIF抑制MMP-9 与TIMP-1 蛋白表达（P < 0.05），而sgp190促进MMP-9的表达（P < 0.05），但对TIMP-1 蛋白表达无影响，sgp190可抑制LIF的作用。 细胞滋养层分泌的Th1/Th2 细胞因子对妊娠的维持起着重要的作用，对妊娠中期滋养层细胞施加LIF和/或sgp190刺激，用ELISA法检测Th2 型细胞因子IL-10 蛋白，结果显示，LIF刺激IL-10 蛋白表达，sgp190无此效应，但sgp190可阻断LIF的作用。 对sgp190作用于LIF的机制研究表明，LIF刺激gp130与gp190 mRNA与蛋白表达，sgp190对gp130与gp190 mRNA与蛋白表达无影响，而sgp190＋LIF处理组与空白对照组无差异，说明sgp190可能通过结合<WP=11>LIF，使LIF游离浓度减少，对细胞的刺激降低，从而导致LIF的膜受体gp130与gp190表达减弱，以此抑制LIF的作用。 LIF传导信号的主要通路是通过STAT3磷酸化作用。在信号通路方面，LIF与JAK-STAT（signal transducer and activator of transcripition, STAT）途径的关系明确，LIF作用于受体之后可以激活JAK1-STAT3途径。结果提示LIF使STAT-3磷酸化水平升高，sgp190能够抑制LIF刺激磷酸化水平升高的作用。此外，sgp190也能单独抑制STAT-3磷酸化。LIF在胚泡着床窗口期出现爆发性表达，其在胚泡植入中的重要性已得到公认。由于sgp190可能对人体整个循环中LIF表达具有调节作用，因此，在第三部分我们对正常能育妇女和原因不明不孕患者植入窗口期（D19-21）血浆中sgp190与LIF进行测定，以探索sgp190在胚泡植入中可能起的生物学作用。结果显示原因不明不孕患者在植入窗口期（D19-21）LIF表达较正常能育者低(P<0.01)，而sgp190表达略高，进一步说明sgp190在LIF发挥效应中具有调节作用。第二、三部分研究结果表明：sgp190对局部或整体LIF的活性起负性调节作用，可能是LIF生物活性的一个调节子。通过抑制LIF活性而在滋养层细胞的增殖、分化、侵袭特性等方面具有调节作用，与胚泡植入、妊娠建立与维持相关。其作用机制可能是通过与LIF结合，使局部或整体的LIF有效浓度下降，通过反馈机制，LIF的膜受体gp130与gp190表达减少，下游信号通路活动减弱，从而使LIF的效应受到抑制。更多还原

【Abstract】 Leukemia inhibitory factor(LIF) is a multifunctional cytokine, which has been shown to have a wide variety of actions in many different cell types and tissues, including its ability to inhibit HIV-1 replication in vivo and in vitro , preserve the totipotentiality of embryonic stem cells, regulate nerve differentiation, stimulate acute phase protein synthesis by hepatocytes, inhibit lipid accumulation in adipocytes, stimulate the function of osteoblasts and enchance the formation of platelets. In addition, its critical role in female reproduction system have also been reported in recently years, since female mice with LIF-deficient are unresponsive to their viable blastocysts, resulting in implantation failure.LIF exerts its biological actions must through its receptor system,which consists of two distinct membrane-bound glycoproteins, a 190 kD specific LIF-binding receptor α-subunit, gp190, and a 130 kD affinity converter, gp130. LIF binds first to gp190 with low affinity and then to gp130 to form a high affinity functional receptor complex leading to activation of <WP=13>down-stream signal transduction pathways. In addition to the cell membrane-anchored forms of LIF receptor, it has been reported that naturally occurring soluble forms of these receptor molecules are present in biological fluids and may act as natural inhibitors of LIF activity. A soluble form of the mouse gp190 with a molecular weight(Mr) of approximately 90 000-150 000 occurs at high levels in normal mouse serum and is elevated dramatically during pregnancy. It increased sharply around day 8 of pregnancy, just after several days of the transient burst of LIF expression during the implantation process, and peaked at day 12, then dropped to relatively low levels at day 21. An approximately 20-fold increase in the level of transcript encoding soluble LIFR was observed between normal and day 12 pregnant mice. The pattern correlated with the changing levels of soluble mLIFR protein that were detected in the serum.To study the role of the soluble receptor and the mechanism of its production , Tomida et al isolated mouse cDNA for the transmembrane LIFR,and found that an inserted nucleotide sequence introduced a stop codon before the LIFR transmembrane domain, so the mRNA encoded the soluble LIFR α-subunit,sgp190.The recombinant msgp190 containing the extracelluar domain of gp190 by genetic engineering have the same activity with the naturally occurring soluble form of gp190,which further confirmed that sgp190 may be generated by expression of alternatively spliced mRNA of gp190,and the whole or part of gp190 extracellular domain mRNA could <WP=14>encode sgp190 with the identical stimulatory/inhibitory properties of the naturally occurring soluble gp190.Despite the high levels of soluble gp190 appeared in mouse serum, its analogue was not detected in human serum until Zhang et al reported that a soluble form of gp190 was also detected in normal human urine and plasma in 1998. It can inhibit STAT-3 phosphorylation induced by LIF in M1 myeloid cells, which is similar to mouse soluble gp190 function in vitro. But its levels is lower than that in mice serum.This suggested that the soluble human gp190(shgp190) is also produced in the human body and may play some physiological and pathological roles.However,its function remains to be investigated. So we cloned cDNA encoding soluble human gp190, and expressed it in the methylotropic yeast Pichia pastoris. The expression supernatant of GS115/pPIC9K-sgp190 was analysized by SDS-PAGE and western blotting . The results showed that the protein band migrated at MW around 125kD could be specifically recognized by polyclonal antibodies against human sgp190, which indicated the MW of expression protein is about 125kD and have good antigenicity.However,the MW have dramatically difference from its expected MW. We supposed that the higher MW than that estimated from the amino acid sequenced are likely be due to posttranslational modification, including glycosylation.By purification ,we obtaine更多还原