【作者】 葛玲； 苏琦； 谭晖； 何洁； 黄卫国；

【Author】 Ge Ling Su Qi Tan Hui He Jie Huang Wei-Guo Cancer Research Institute, Nanhua University, Hengyang, Hunan, 421001, P.R.China

【机构】 南华大学肿瘤研究所；

【摘要】 目的:研究二烯丙基二硫(diallyl distalfide,DADS) 诱导人白血病HL-60细胞差异表达的基因,寻找与DADS抗白血病作用相关的基因,探讨DADS诱导人白血病HL-60细胞分化的分子机制。方法:采用抑制消减杂交技术,筛选 DADS处理HL-60细胞后差异表达的相关基因。经过消减杂交和巢式聚合酶链反应扩增,获得富集差异表达的cDNA片段(表达序列标签,EST),克隆并挑选经PCR鉴定含有插入片段的质粒测序,Genbank数据库同源性比较进行生物信息学分析。结果:细胞形态学观察发现,与对照组HL-60细胞相比较,DADS处理后HL-60细胞体积变小,胞浆丰富,细胞核变小,染色变淡。总RNA提取和mRNA纯化结果显示, 处理前后的HL-60细胞总RNA为28S和18S的清晰无拖尾条带,mRNA为清晰慧尾片状条带。Rsa I酶消化产物分析结果显示:未消化的ds cDNA电泳带为0．5-10kb清晰片状条带,消化后的cDNA为0．1-2kb清晰片状条带。接头连接效率检测结果表明:Rsa I酶消化产物cDNA与接头连接效率大干25％。巢式PER产物分析显示:消减组产物片段明显少于未消减组产物片段,且丰度较低,片段多在100- 600bp。消减效率的PCR鉴定表明,与未消减组PER产物相比,消减组PCR产物中G3PDH基因产物在28个循环之后才出现,且产物量明显减少,说明所构建的消减文库具有很高的消减效率。T／A克隆平板显示,平板白色菌落和蓝色菌落约为2:1。克隆产物的纯化和PER鉴定结果显示,纯化的阳性克隆质粒大于3kb,PCR产物多为200—300bp片段。随机挑取的89个白色阳性克隆(正向31个,反向58个),DNA 测序获得18个不同的cDNA序列。生物信息学分析,获得7 个差异表达基因,序列同源性为99％-100％。上调的有过氧化物酶(PRDX2)、CCR4-NOT转录复合物亚单位8(CNOT8)、聚谷氨酰胺结合蛋白变异体4(PQBP1)、核糖体蛋白S7 (RPS7)基因,下调的是核苷酸还原酶M2(hRRM2),钙离子／钙调蛋白依赖性蛋白激酶(CaMK)和非等位的IgE重链 (IGHV2-5)基因。分别与细胞代谢、免疫、抗氧化和耐药性形成有关。结论:SSH成功构建DADS诱导HL-60细胞的消减cDNA文库。DADS诱导HL-60细胞PRDX2,CNOT8, PQBP1,RPS7基因表达上调,RRM2,CaMK,IGHV2-5 基因表达下调。更多还原

【Abstract】 Objective: To screen and clone different expressed genes in human leukamia HL-60 cells. Methods: Different expressed genes in treated HL-60 cells by DADS were identified by using suppression subtractive hybridization (SSH), T/A cloning, sequenced and analyzed for homology in the Genbank database with BLAST. Results: Cellular size shrinked in HL-60 cells treated by DADS for 24h, as nuclear-to-cytoplasmic ratio was reduced, and coloring of nucleus weaked.Abstract of RNA: Total RNA Abstracted by TRLzol reagent was detected by agrose gel electrophoresis. 28S RNA and 18S RNA displayed as two bands without trail. mRNA was showed as a distinct smear strap.Enzymatic results of Rsa I: Undigested cDNA manifest as a distinct strap of 0.5-1.0kb ,digested cDNA manifest as a distinct strap of 0.1-2kb.Analysis of ligation: Efficiency of adaptor-ligation with digested cDNA exceed 25%.The nest PCR analysis of SSH: Fragments of subtracted products less than unsubtracted products, which size about 100-600 bp.PCR anallysis of subtraction efficiency: Using G3PDH 3’ primer and 5’ primer subtract product and unsubtract product were amplified, respectively. G3PDH product can be seen after 28 cycles, and the former’ s abundance less than the latter’ s. It demonstration that subtraction efficiency is very high. TV A clone: The ratio of white and blue clones is about 2.Pu-rified plasmid of positive clones exceed 3kb, Size of PCR products is about 200-300bp.Sequencing results showed that 89 clones were identified. After comparing with public database(Genbank/EMBL) and Expressed Sequence Tag(EST), 7 known genes were found in the two libaries. Data showed that those known genes represent a variety groups on the basis of their cellular functions and have 99-100% identities in sequences. In the forward substract-library, we found PRDX2, CNOT8, PQBP1 and RPS7 were up-regulated in HL-60 cells induced by DADS, while hRRM2, calcium/calmedulin-dependent protein kinase and Homo sapiens isolate C30 MK2 non-allergic IgE heavy chain were down-regulated in the reversed substract-library.This genes related to cell metabolism, immunification, antioxidation and drug resistance formation, respectively. Conclusion: The subtracted cDNA library constructed by SSH contains differently expression genes in human leukamia HL-60 cells treated by DADS .DADS up-regulated PRDX2, CNOT8, PQBP1 and RPS7 genes in HL-60 cells. DADS decline the expression of hRRM2, CaMk and IGHV2-5 in HL-60 cells.更多还原