【作者】 施珊珊；

【导师】 江振友；

【作者基本信息】 暨南大学 ， 病原生物学， 2009， 硕士

【摘要】 目的研究海洋放线菌素X2的生物学活性,主要是抗肿瘤和抗病毒活性的研究。方法1.利用MTT法检测不同浓度的海洋放线菌素X2对正常和肿瘤细胞的抑制作用;FCM测定海洋放线菌素X2对HepG-2细胞周期、细胞凋亡的改变;经Hoechst 33258(8g/L)染色进行形态学观察。2.采用MTT法检测不同浓度的海洋放线菌素X2作用病毒后的抑制情况;采用RT-PCR和FCM分别测定CVB3诱导的ECV304细胞在海洋放线菌素X2作用前后不同时间点的ICAM-1和VCAM-1的mRNA和蛋白的表达水平;采用免疫细胞化学方法半定量分析CVB3诱导的ECV304细胞在海洋放线菌素X2作用前后不同时间点的NF-κB的活化情况。结果1、海洋放线菌素X2对多种肿瘤细胞都有抑制作用。在对HepG-2细胞增殖的研究中发现这种抑制作用具有时间-剂量依赖性,FCM检测发现HepG-2细胞细胞周期受到阻滞,停留于S期和G2/M期,细胞发生凋亡,荧光显微镜下观察发现HepG-2细胞中出现核固缩、凋亡小体。2、海洋放线菌素X2在病毒吸附前对H1N1、CVB3的SI分别为47.66和63.88。3.CVB3感染ECV304促进VCAM-1和的ICAM-1 mRNA表达,54h内表达均有增加,CVB3组、CVB3+X2组与对照组相比差异有显著性(P＜0.05).其中VCAM-1 mRNA表达在感染12h达到高峰,12h-24h表达量显著高于其他时间(P＜0.05);ICAM-1 mRNA表达在感染后显著增加(P＜0.05),24h-54h维持于高水平。海洋放线菌素X2干预后,分别于12-54h、24-54h降低VCAM-1和ICAM-1 mRNA的表达,与CVB3组相比差异有统计学意义(P＜0.05)。4.CVB3感染ECV304,细胞表达VCAM-1和ICAM-1蛋白在12h-54h显著高于对照组(P＜0.05)。海洋放线菌素X2干预后,分别于12h-24h、12h-54h降低VCAM-1和ICAM-1蛋白的表达,与CVB3组相比差异有统计学意义(P＜0.05)。5.免疫细胞化学方法和图像分析显示,ECV304经CVB3感染3h后NF-κB表达明显增强,活化入核细胞数增多,光密度值增加,与对照组相比较,差异有统计学意义(P＜0.05)。海洋放线菌素X2干预后,1h、3h时NF-κB的活化较CVB3组增强,而6h-24h时,NF-κB的活化受到明显抑制,表达下降。结论1.海洋放线菌素X2可抑制肿瘤细胞的增殖,均表现出时间-剂量依赖关系;阻滞HepG-2的细胞周期并可诱导其发生凋亡,形成凋亡小体。2.海洋放线菌素X2在病毒吸附前对H1N1、CVB3具有抗病毒作用,3.海洋放线菌素X2可能通过抑制NF-κB活化,下调CVB3诱导的ECV304细胞ICAM-1、VCAM-1的mRNA和蛋白表达。更多还原

【Abstract】 OBJECTIVEStudy on the biologic activity of marine actinomycin X2.The main research is about anti-tumor and anti-viral.METHODS1.The inhibition of marine actinomyein X2 on normal and tumor cells proliferation was detected by MTT method.Flow cytometer was used to detect the change of HepG-2 cell cycle、apoptosis and those disposed cells were stained with Hoechst 33258(8g/L).2.The inhibitory action of virus by use of different concentration marine actinomycin X2 by use MTT methods and study the effective of viral adsorption.Cytoplasmic RNA from ECV304 was extracted by the Trizol method and mRNA levels of ICAM-1, VCAM-1 were assayed by reverse transcript-polymerase chain reaction(RT-PCR) method respectively.Flow cytometer was used to detect the expression of ICAM-1, VCAM-1 on the infection ECV304 in different time courses respectively.Fluctuation of NF-κB was detected by semi-quantitative immunohistochemistry.RESULTS1.Marine actinomycin X2 has the inhibitory action to the tumor cells.FCM showed HepG-2 in G1 phase decreased,cells in S and G2/M phase increased,the cell cycle was arrested in S and G2/M phase.HepG-2 cell apoptosis with nuclear chromatin concentration and fragmentation as well as the formation of apoptotic bodies were observed.2.Marine actinomycin X2 can inhibit virus before absorption of CVB3、H1N1 into the normal cells and SI weree 63.88,47.66.3.The infection of CVB3 did promote the transcription of ICAM-1 and VCAM-1 in all 54h. The level of ICAM-1 and VCAM-1 mRNA in CVB3 group and CVB3+X2 group were higher than that of control group,and the differences had statistical significance(P＜0.05 ).The expression was increased in all 54h among which the VCAM-1 mRNA reached a peak at 12h point post-infection.There had expression of ICAM-1 mRNA in normal condition and increased after infection,especially between 24h-54h.In CVB3+X2 group at 12h-24h、12h-54h the expression of VCAM-1 and ICAM-1 mRNA was lower than CVB3 group(P＜0.05).4.At the same time,we observed the significance increased expression VCAM-1 and ICAM-1 protein on the surface of ECV304 by the way of FCM at 12h-54h.In CVB3+X2 group at 12h-24h、12h-54h the expression of VCAM-1 and ICAM-1 protein was lower than CVB3 group(P＜0.05).5.Our findings suggest that the level of NF-κB expression significantly increased in the CVB3 group aRer 3h.In CVB3+X2 group,at 1h、3h the level of NF-κB expression was increased,however,at 6h-24h the activation of NF-κB inhibited by X2.CONCLUSION1.Marine actinomycin X2 had significant inhibitory effect on tumor with dose and time-effect relationship.It arrested cell cycle、induced HepG-2 cells apoptosis and formed apoptosis bodies.4.The mechanism of anti-virus of Marine actinomycin X2 possibly arrested the absorption of virus.3.Marine actinomycin X2 decreased the expression of VCAM-1、ICAM-1 mRNA and protein maybe through inhibiting the activation of NF-κB.更多还原