UTI抑制骨肉瘤细胞侵袭及转移实验探究.docVIP

UTI抑制骨肉瘤细胞侵袭及转移实验探究 　 作者：张衣北,陈安民,郭风劲 【摘要】 目的 体外研究尿胰蛋白酶抑制剂（urinary trypsin inhibitor,UTI）抑制人骨肉瘤细胞系MG63细胞的侵袭与转移。 方法 在体外培养的MG63细胞培养基中加入不同浓度的UTI，对照组中加入50μl PBS缓冲液，用免疫组化、RTPCR和Western blot方法检测尿激酶型纤溶酶原激活物(urokinase type plasminogen activator，UPA)的表达情况。结果 免疫组化染色、RTPCR及Western blot检测发现随着UTI浓度的逐渐增加，UPA表达量逐渐减少，当UTI浓度增加到100nM时，抑制作用明显（Plt;0.05），并且呈剂量依赖性。结论 UTI不但从mRNA水平，而且从蛋白质水平抑制骨肉瘤细胞表达UPA，从而抑制骨肉瘤细胞侵袭与转移。 【关键词】 尿胰蛋白酶抑制剂；尿激酶型纤溶酶原激活物；骨肉瘤 Experimental Study on the Suppression of the Invasion and Metastasis of Osteosarcoma by UTI Department of Orthopedics,Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030,ChinaAbstract：Objective In order to investigate the effect of invasion and metastasis of human osteosarcoma MG63 by the urinary trypsin inhibitor(UTI) in vitro.Methods Different doses（10、50、100、300、500nM） of UTI were added into the human osteosarcoma MG63 medium and 50μl PBS was added into the control at the same time,then the expression of urokinase type plasminogen activator(UPA) was detected by immunohistochemistry,RTPCR and Western blot.Results The more concentration of UTI was transfected,the lesser UPA were expressed by the detections of immunohistochemistry and RTPCR and Western blot.When the concentration became 50nM,the suppression was obvious(Plt;0.05),and the suppression was in dosedependent manners.Conclusion The UTI can suppress efficiently the expression of protein UPA both at the mRNA level and at the protein level,then suppress the invasion and metastasis in osteosarcoma cells. Key words：Urinary trypsin inhibitor;Urokinase type plasminogen activator;Osteosarcoma 骨肉瘤是一种恶性度高和破坏性强的肿瘤，常在早期就发生侵袭与转移。在这种复杂且多步骤的侵袭与转移过程中，肿瘤细胞水解细胞外基质是一个非常关键的步骤，有一系列蛋白酶的参与[1]。在这些蛋白酶中，以尿激酶型纤溶酶原激活剂（urokinase type plasminogen activator，ＵＰＡ）尤为重要[2]。因此，如何减少肿瘤细胞产生UPA，对抑制肿瘤的侵袭与转移具有十分重要的意义。 1材料与方法 1.1主要实验材料与仪器 人骨肉瘤细胞系MG63由武汉大学生命科学院提供；SP试剂盒和AEC显色试剂盒购自武汉博士德生物技术有限公司；RTPCR试剂盒和Western blot检测试剂盒购自Fermentas公司，DMEM培养基及新生小牛血清购自美国GibcoBRL公司；UPA