【作者】 沈政；

【导师】 赵兴绪； 段恩奎；

【作者基本信息】 甘肃农业大学 ， 临床兽医学， 2004， 博士

【摘要】 一氧化氮(nitric oxide，NO ) 是一种无机自由基气体，作为一种特殊的生物传递信号分子，日益受到生命科学各领域的普遍重视。机体内的NO是通过三种一氧化氮合酶（nitric oxide synthase, NOS）合成。NOS在体内的分布极为广泛，几乎遍布机体的每一个系统。研究表明，哺乳动物雌性生殖过程中的重要环节，诸如卵泡的发育和成熟、胚胎的植入、妊娠的维持、分娩等许多生理过程中，NO发挥重要的调节作用。为探讨NO在哺乳动物胚胎植入过程中的信号通路及分子机制，本研究应用小鼠胚胎植入的体外模型，将常规方法获取的胚泡随机培养在含有不同处理药物的培养液中，在培养12、24和36 h后分别观察、统计胚泡黏附、扩展的情况，同时收集培养液，用明胶酶谱法分析其中MMP-2蛋白的变化，半定量RT-PCR分析各组胚泡中MMP-2 mRNA的变化。研究结果表明，500μM NOS抑制剂L-单甲基-精氨酸（L-NMMA）可以显著抑制小鼠胚泡的黏附、扩展及MMP-2的分泌（P<0.05），此抑制可以被同时加入的100μM NO供体S-亚硝基-乙酰青霉胺（SNAP）逆转（P﹥0.05）。提示NO对小鼠胚泡体外黏附、扩展及MMP-2的分泌具有重要的影响。此外，在使用NOS抑制剂500μM L-NMMA时，同时加入20μM的cGMP类似物8-Br-cGMP，也可以逆转这种抑制效果，胚胎的黏附和扩展与对照组相比差异不显著(P＞0.05)。结果进一步提示，NO对小鼠胚胎黏附、扩展及MMP-2的分泌影响可能是通过其下游分子cGMP通路进行，而且对MMP-2的作用可能是在基因水平进行调节。为了进一步了解NO调节胚胎植入过程的分子机理，将eNOS基因转入作为研究植入的模式细胞JAR当中，建立可控的内源表达NO的模型。应用脂质体介导转染技术将人内皮型一氧化氮合酶(eNOS)基因转入JAR细胞，获得转染阳性细胞。用RT-PCR和Western blot技术从基因及蛋白水平对表达产物进行鉴定，结果显示，阳性细胞有较高水平的mRNA转录和特异目的蛋白表达并通过免疫细胞化学方法获得证实，与对照组相比，转染eNOS基因的阳性JAR细胞<WP=6>高表达外源eNOS蛋白；但是一氧化氮合酶（NOS）活性及其催化产物NO并没有直接升高；但使用适当浓度的钙离子激动剂A23187处理则能增加NOS的活性。这些结果说明，在转染的JAR细胞中，eNOS已得到表达，但没有被直接激活。实验过程中，使用0.5μM A23187对转染阳性组（EA）与转染阴性组（MA）进行诱导，并与转染阴性非诱导组（M）及对照组（C）进行比较,差异均不显著(P>0.05)；使用2μM A23187进行诱导，EA组与MA组、M组和C组比较,差异显著(P<0.05)，但MA与M组及C相比NO增加,但统计分析差异并不显著;用8μM A23187进行诱导，EA组与ME组与C组相比，差异极显著(P<0.001)；而MA组在该剂量的A23187作用下，与E组及C组相比，NO产生差异显著(P<0.05)。这样用8μΜA23187诱导的转染组，建立一个产生内源NO的细胞模型。应用该转染细胞模型分别产生高、低浓度NO，进行细胞增生、迁移及MMP-2分泌实验；同时用外源NO供体SNAP分别以低浓度50μM（S1）和高浓度500μM（S2）分别处理JAR细胞，进行上述增生、迁移和MMP-2分泌实验。结果显示，EA组与M组相比，对细胞的迁移、增生及MMP-2分泌具有显著的抑制作用（P﹤0.05）；相反MA组与M组相比细胞的迁移、增生及MMP-2分泌具有显著的促进作用(P<0.05);而M组与C组无明显差异(P>0.05)。同时用外源NO供体50μM的SNAP处理细胞与C组相比，对细胞迁移、增生及MMP-2分泌具有显著的促进作用(P<0.05)；500μM SNAP处理细胞与C组相比则对迁移、增生及MMP-2分泌具有抑制效应（P﹤0.05）。应用免疫印迹分析，Akt/PKB Ser-473磷酸化状态均发生相应的变化，提示NO可能通过Akt/PKB信号通路影响MMP-2分泌及细胞增生、迁移。本研究首次系统地说明，在JAR细胞中,无论是内源产生NO或是外源NO,由于NO的浓度差异，造成NO对细胞迁移、增生和MMP-2分泌具有不同的作用，NO在低浓度时起促进作用，高浓度时起抑制作用；而调节过程可能是通过激活Akt/PKB信号通路而使Akt/PKB Ser-473磷酸化起作用。整合素连接激酶(Integrin-linked kinase,ILK)是新近发现的一种分子，该分子对细胞的生理功能和病理过程具有重要作用。本研究用野生型(WT-ILK)、酶灭<WP=7>活型(KD-ILK)及其空载体(Mock) 三种表达质粒转染JAR细胞，进而研究ILK对NO的调控作用。结果表明, 与M组相比，转染WT-ILK组细胞产生NO的量显著增加（P<0.05）；而转染KD-ILK组细胞产生的NO与M组相比却显著降低(P<0.05)，而转染空载体M组与对照组相比，无显著差异（P﹥0.05）。各实验组中NOS变化与NO相似，这些结果说明ILK通过激活NOS促进NO的产生。用MTT法测定各组细胞增生情况，发现WT-ILK转染组明显高于对照组（P<0.05）；而转染KD-ILK则低于对照组，差异显著（P<0.05）。同时用明胶酶谱方法分析MMP-2的变化，转染WT-ILK质粒的细胞MMP-2分泌与对照组比较，MMP-2分泌显著增加（P<0.05），而转染KD-ILK的细胞MMP-2分泌量与对照组相比，显著降低（P<0.05）。然后用免疫印记迹的方法分析eNOS及Akt/PKB Ser-473磷酸化的变化,与NO变化保持平行。本研究说明，ILK可以通过eNOS来产生NO,而对eNOS的调节可能是通过Akt/PKB信号?更多还原

【Abstract】 Nitric oxide (NO) is produced by a group of enzymes named nitric oxide synthases (NOS), which has three isoforms: endothelial (eNOS), neural (nNOS) and inducible (iNOS). Nitric oxide is a multifunctional messenger molecule and is produced through the oxidation of L-arginine to L-citrulline by the enzyme NO synthase. NO acts as an important intracellular messenger in a variety of systems including reproduction. In mammals, NO is known to be a major paracrine mediator and an important regulator in female reproductive processes, such as ovulation, implantation, pregnancy maintenance, labor and delivery. NO donors may contribute to a reduction of fetal and maternal perinatal morbidity and mortality. NO donors and NOS inhibitors may provide novel, effective, safe and inexpensive drugs to regulate and steer various functions in female reproductive life.In the present study, mouse blastocysts were cultured in the medium containing different treatments according to the experimental design. The implantation capacity of blastocysts was evaluated by calculating the percentage of embryos with attachment or outgrowth after being cultured for 12 h, 24 h and 36 h. MMP-2 mRNA was detected by RT-PCR and MMP-2 protein was detected by gelatin zymography. Inhibition of blastocyst attachment and outgrowth was observed in embryo cultured with NOS inhibitor NG-monomethyl- L-arginine (L-NMMA). Both NO donor S-nitroso-N-acetylpenicillamine (SNAP) and cGMP analogue 8-Br-cGMP blocked this inhibition. The expression and secretion of MMP-2 in the blastocysts were suppressed by L-NMMA and this suppression was also reversed by SNAP or 8-Br-cGMP. These results indicated that NO/cGMP signaling pathway induced embryo implantation by increasing <WP=9>the expression of MMP-2. Based on the above results, the next experiment was designed to elucidate the detailed molecule mechanism of NO in embryo implantation, using JAR cells, a well propagated human first trimester extravillous trophoblast cell line in vitro. The first study was designed to determine the enzyme activity of eNOS in the transfected JAR cell. The results showed that mRNA of eNOS in the transfected JAR cell was significantly increased compared to untreated cells. Immunocytochemical studies revealed a strong green perinuclear immuno -fluorescence in eNOS transfected JAR cell. NOS activity and NO release did not change in transfectants. In this study, three independent methods demonstrated that transfection resulted in over-expression of eNOS in JAR cell, but the enzyme was not active until being treated with calcium ionophore A23187.The effect of endogenous NO on the proliferation, migration and matrix metalloproteinase-2 (MMP-2) production in the JAR cells was investigated by transfected nitric oxide synthase (eNOS) gene. The transfected JAR cells treated with different levels of calcium ionophore A23187 produced different concentrations of endogenous NO. The results indicated that eNOS gene transfer significantly inhibited the proliferation, migration and MMP-2 production in JAR cells when A23187 was at the dosage of 8μM. In contrast, NO at a low concentration produced by the mock cells treated with 8μM A23187 had opposite results in the same model. Furthermore, these effects may be regulated via Akt/protein kinase B (Akt/PKB), a well-known important mediator of cell survival signaling pathways. Exogenous NO donor SNAP (50μM and 500μM ) had the similar effect in this experiment. The current results indicated that different concentrations of NO, regardless of endogenesis or exogenesis, had different effects on the proliferation and migration of the embryos and MMP-2 production, which were regulated by Akt/PKB pathways. These results implied that the dual effects may depend on the NO concentration. Integrin-linked kinase (ILK) is an intracellular serine/threonine protein kinase that interacts with the cytoplasmic domains of integrins and numerous <WP=10>cytoskeleton-associated proteins. ILK has been shown to be involved in the regulation of a number of integrin-mediated pr更多还原