丹参注射液处理动脉粥样硬化大鼠前后胸主动脉细胞相关基因表达的变化

Changes of expressions of genes related to thoracic aorta cells of atherosclerotic rats before and after salvia injection treatment

目的:观察经丹参注射液处理的动脉粥样硬化大鼠前后胸主动脉细胞与病理组未经处理的同类细胞基因表达的差异。方法:实验于2004-03/2005-02在泰山医学院生物化学与分子生物学研究室完成。18只Wistar大鼠随机分为2组:病理模型组和丹参注射液处理组,每组9只。两组均以50万U维生素D/kg体质量的总剂量分3d灌胃,每天喂高脂饲料15g/只,连续30d。同时丹参注射液处理组按1.5mL/(kg.d)体质量,腹腔注射丹参注射液15d。30d后应用DDRT-PCR技术分析丹参注射液处理动脉粥样硬化大鼠胸主动脉细胞基因表达的差异,并用反向Northern证实,计数统计处理后细胞表达上调和下调的基因数量。结果:纳入18只大鼠均进入结果分析。①大鼠胸主动脉mRNA的差异显示分析:大鼠胸主动脉细胞总RNA经DDRT-PCR分析,60对引物进行PCR反应,有32对引物组合中发现有差异片段,个别的引物组合扩增出2～3条差异带,共发现重复性较好的差异条带37条,其中4条在丹参注射液处理细胞中表达较强,其余在病理组细胞中特异表达或表达较强。②mRNA差别显示技术分离差异片段的克隆、序列分析及同源性比较:在29个阳性克隆中有21个克隆与已知基因序列高度同源,13个目前认为与动脉粥样硬化发病有关;;另外8个克隆为未知基因的序列。③病理组和丹参注射液处理组大鼠胸主动脉细胞mRNA的反向Northern分析:丹参注射液处理动脉粥样硬化大鼠前后胸主动脉细胞出现一些表达上调和下调的基因片段。上调基因有Apobec-1结合蛋白基因、ERP72、nitricoxidesynthase2和Gpx1;;下调的基因有胸腺素β4、FGFRI原癌基因伴随蛋白、ICAM-1、endothelin2、c-myc和TNF-α。结论:对29个阳性克隆中有21个克隆与已知基因序列高度同源,其中13个目前认为与动脉粥样硬化发病有关,将其各取约300ng的DNA产物,结果显示1、4、8和13号基因在丹参注射液处理组大鼠胸主动脉细胞后表达上调,2、3、6、7、9和12号表达下调,其余的诱导前后没有差异,可以认为是假阳性,总阳性率达76%。用DDRT-PCR方法证实经丹参注射液处理动脉粥样硬化大鼠前后胸主动脉细胞可发生一些基因表达水平的变化,引起血管内皮细胞发生病理变化减弱,阻碍动脉粥样硬化斑块的形成。

AIM： To study the difference of expressed gene in thoracic aorta cell of atherosclerotic rats before and after salvia injection treatment and those without salvia injection treatment. METHODS： This experiment was conducted in the Research Room of Biochemistry and Molecular Biology of Taishan Medical College from March 2004 to February 2005. Eighteen Wistar rats were divided randomly into two groups： pathological model group （n=9） and salvia injection treatment group （n=9）. All rats were administered with 500 000 U Vitamin D/ kg body mass for 3 days and 15 g/rat high fat feed for 30 continuous days. Rats in the salvia injection treatment group were treated with intraperitoneal injection of salvia injection （1.5 mL/kg per day body mass） for 15 days. After 30 days, the differential expressions of genes in thoracic aorta cell were analyzed with the differential-display reverse transcription-polymerase chain reaction （DDRT-PCR） in the atherosclerotic rat models with and without salvia injection treatment. The differential expressed cDNA was confirmed by reverse Northern blot. The numbers of up-regulated and down-regulated genes were counted. RESUILTS： All the 18 rats were analyzed in the experiment.①The resuits of DDRT-PCR showed that differential expression fragments were displayed in 32 pairs primer among 60 pairs primer. Two to three differential expression fragments were also found in several pair primer. Thirty-seven stable fragments were counted in this experiment. Among them, 4 fragments were up-regulated in salvia injection treatment group, and others were up-regulated in atherosclerosis pathological model group.②BLAST result of cloned differential expression fragments indicated that 21 fragments were homologous to known genes. Thereinto, 13 fragments were related to atherosclerosis, and other 8 fragments were unknown genes.③Reverse Northern results showed that up-regulated and down-regulated cDNA were isolated in thoracic aorta cell of atherosclerotic rat models with and without salvia injection treatment. The known up-regulated genes included Apo becl binding protein 1, ERP72, nitric oxide synthase 2 and Gpxl; The know down-regulated genes included thymosin 134, FGFRI oncogene,ICAM-1 ,endothelin2,c-myc and TNF-α. CONCLUSION： of the 29 positive clonings, 21 fragments were homologous to known genes, including 13 fragments related to atherosclerosis. Reverse Northern was conducted with 300 ng cDNA. The result showed that No.1,4,8 and 13 were up-regulated in salvia injection treatment group, and No. 2,3,6,7,9 and 12 were downregulated. The positive rate of DDRT-PCR was 76%. DDRT-PCR showed that salvia injection treatment could change the expression of some genes in thoracic aorta cell of atherosclerotic rat models and result in the low pathological change. Finally, the formation of atherosclerotic plaque was restrained.