广枣总黄酮体外抗CVB_3病毒活性  

Anti-Coxsackievirus B_3 effects of Total Flavones of Fructus choerospondiatis in vitro

作　　者：刘小玲[1] 梁彬 张勇[1] 刘小雷[2] 

机构地区：[1]内蒙古自治区医院,内蒙古呼和浩特010018 [2]内蒙古医学院药学院,内蒙古呼和浩特010059

出　　处：《中国医院药学杂志》2007年第12期1637-1642,共6页Chinese Journal of Hospital Pharmacy

基　　金：国家教育部"春晖计划"项目(编号:Z2004-2-15001);内蒙古自治区自然科学基金资助项目(编号:200408020908)

摘　　要：目的:研究广枣总黄酮(TFFC)体外抗柯萨奇B组3型病毒(CVB3)作用及其作用机制,探究其作用靶点,为TFFC进一步开发利用奠定基础。方法:采用差速贴壁法培养原代乳鼠心肌细胞,镜下观察心肌细胞搏动次数,使用α-actin免疫组化法鉴定培养心肌细胞的纯度;在细胞水平测定阳性药盐酸胍以及TFFC的药物毒性;以CVB3感染心肌细胞建立病毒性心肌炎细胞模型;MTT染色分析TFFC对病毒感染后的Hela细胞及心肌细胞是否有保护作用;病毒繁殖抑制实验比较不同分组间TCID50的差别以验证TFFC在Hela细胞及心肌细胞上的抗病毒活性;测定各实验组心肌细胞培养上清液中心肌酶LDH、CK-MB的变化;测定各实验组心肌细胞培养上清液中肿瘤坏死因子TNF-α含量的变化;运用逆转录聚合酶链式反应测定心肌细胞内病毒相关基因的表达。结果:经过在Hela细胞及心肌细胞水平的初筛,确定TFFC具有保护细胞免受CVB3病毒侵袭的作用。病毒繁殖抑制实验显示TFFC的高、中剂量组TCID50与病毒对照组TCID50相差一个以上对数值;TFFC能使心肌酶(LDH、CK-MB)的释放较病毒组显著降低(P<0.05),还可抑制被病毒感染的心肌细胞TNF-α的分泌;同时,TFFC还可明显抑制CVB3相关基因c-Myc、TNF-α、Fas的表达。结论:通过建立病毒性心肌炎细胞模型,证实TFFC在体外细胞培养物上具有抗CVB3病毒活性作用。OBJECTIVE A cell-based assay was established to research effect of Total Flavones of Fructus choerospondiatis （TFFC） on coxsackieviruses group B3（CVB3）-induced viral myocardiam and its action mechanism, which have facilitated its further development in clinical trials. METHODS Cell-based viral myocardial model was established by using CVB3 to infect cardiomyocytes. Cardiomyocytes derived from neonatal mice were purified by differential adhesion method and characterized by immunohistochemical staining method. Their rhythmical beat were observed, too. Protection effects of TFFC on virus-infected Hela cells and cardiomyocytes were detected by MTT assays. At the same time,its anti-virus activity was verified by comparing difference of TCID50, between various groups. In addition, changes of activities of LDH and CK-MB in cell-flee releastes were measured by LDH kit and CK-MB kit, respectively. At last, the supernatants of cultured fluid in each experimental groups were analyzed to investigate levels of TNF-α by usage of the corresponding kits. RT-PCR assays were used to determine expression of CVB3-associcated genes in cardiomyocytes. RESULTS The results of primary screening based on CVB3-induced viral myocardial model have desmonstrated that TFFC could protect Hela cells and cardiomyocytes from CVB3. TFFC at high and middle dosages can significantly inhibit viral reproduction, decrease release of LDH and CK-MB and suppress serection of TNF- from cardiomyocytes infected by CVB3. At the same time, TFFC could significantly inhibit expression of c-Myc.TNF-α,Fas genes. CONCLUSION The outcomes of cell-based assays have identified the anti-CVB3 activity of TFFC in vitro.

关 键 词：广枣总黄酮 CVB3病毒 心肌酶 

分 类 号：R96[医药卫生—药理学]