以高粱无菌苗幼叶为试验材料,对颗粒状胚性愈伤组织的获得、细胞悬浮系的建立及影响悬浮细胞生长的主要因素进行了研究。结果表明:幼叶在MS(Murashige and Skoog)+2mg/L 2,4-D培养基上诱导出的愈伤组织,经2~3次继代筛选后获得了浅黄色...以高粱无菌苗幼叶为试验材料,对颗粒状胚性愈伤组织的获得、细胞悬浮系的建立及影响悬浮细胞生长的主要因素进行了研究。结果表明:幼叶在MS(Murashige and Skoog)+2mg/L 2,4-D培养基上诱导出的愈伤组织,经2~3次继代筛选后获得了浅黄色、颗粒状胚性愈伤组织;胚性愈伤组织接种于液体培养基中,于25±1℃、黑暗条件下,经45~60d的悬浮震荡培养建立了高质量的细胞悬浮系,细胞生长曲线呈"S"型,细胞密度可达6.45×10~5~5.08×10~6个/mL以上,活细胞率可达72.76%,近圆细胞率可达87.50%;培养基的基本成分、激素种类和水平对悬浮细胞生长状态有很大的影响,相比1/2MS培养基,MS培养基为适宜的培养基,2,4-D对保持细胞系的稳定增殖至关重要,适宜的浓度为1mg/L;培养液中加入0.5mg/L KT或6-BA会造成悬浮液褐化,影响悬浮细胞的生长;最适宜的细胞悬浮培养基为L2培养基(MS+1mg/L 2,4-D,30g/L蔗糖,pH5.8),震荡培养转速为110~120r/min,继代周期为7d,新旧培养基的接种比例为2∶1。展开更多
Studying the expression level of mRNA in living cells will offer tremendous opportunities for ad-vancement in cell biology research,disease diagnostics,and drug discovery.In this paper,a molecular beacon(MB)specific f...Studying the expression level of mRNA in living cells will offer tremendous opportunities for ad-vancement in cell biology research,disease diagnostics,and drug discovery.In this paper,a molecular beacon(MB)specific for the important tumor suppressor gene p21 has been designed and synthesized.The fluorescence signal was detected in real-time after the MB entered the cytoplasm of nasopharyn-geal carcinoma cells.After injecting the p21MB into nasopharyngeal carcinoma cell and p33-trans-fected nasopharyngeal carcinoma cell,the consistent increase of fluorescent signal intensity was de-tected in both cell lines,and maximum fluorescence intensity achieved in about 15 min.In about 4 min following microinjection,the fluorescence increasing rate was significantly different between these two cell lines,which indicate the different p21 mRNA expression levels.The results obtained in the real-time detection were also validated by RT-PCR.Analysis of the initial fluorescence increasing rate can effi-ciently reduce the side effect of enzyme and improve the accuracy in living cell mRNA detection.展开更多
基金the National Key Basic Research Program(Grant No.2002CB513110)the Key Technologies Research Development Program of China(Grant Nos.2003BA310A16and2005EP090026)+2 种基金International Technologies Collaboration Program of China(Grant No.2003DF000039)the National Natural Science Foun-dation of China(Grant Nos.90606003and20475015)Key Project of Hunan Province Technology Plan of China(Grant No.0399Y1006)
文摘Studying the expression level of mRNA in living cells will offer tremendous opportunities for ad-vancement in cell biology research,disease diagnostics,and drug discovery.In this paper,a molecular beacon(MB)specific for the important tumor suppressor gene p21 has been designed and synthesized.The fluorescence signal was detected in real-time after the MB entered the cytoplasm of nasopharyn-geal carcinoma cells.After injecting the p21MB into nasopharyngeal carcinoma cell and p33-trans-fected nasopharyngeal carcinoma cell,the consistent increase of fluorescent signal intensity was de-tected in both cell lines,and maximum fluorescence intensity achieved in about 15 min.In about 4 min following microinjection,the fluorescence increasing rate was significantly different between these two cell lines,which indicate the different p21 mRNA expression levels.The results obtained in the real-time detection were also validated by RT-PCR.Analysis of the initial fluorescence increasing rate can effi-ciently reduce the side effect of enzyme and improve the accuracy in living cell mRNA detection.
