筛选大鼠血管钙化消退差异表达基因及初步分析

Identification of differentially expressed genes in rats and preliminary analysis in regression of vascular calcification

目的:探讨大鼠在血管钙化消退过程中基因表达的变化。方法:选取6周龄SPF级雄性SD大鼠24只,随机分成3组(n=8),分别为对照组、钙化组和消退组。钙化组和消退组制作血管钙化模型(vitamin D3 plus nico-tine,VDN),对照组生理盐水和花生油灌胃。钙化组和对照组于实验第8周处死,消退组继续饲养至16周处死,测定各组大鼠动脉组织钙含量并作病理检查。采用抑制性消减杂交方法将消退组和钙化组大鼠血管cDNA作差减杂交,分离消退组较钙化组高表达或低表达基因的cDNA片段,建立消退组和钙化组的差异表达文库,扩增、鉴定文库并测序阳性克隆,BLAST比对测序序列。随机挑选4个基因进行RT-PCR验证和DNA条带半定量分析。结果:①血管组织钙含量测定显示钙化组((15.34±2.51)mg/g)较对照组((5.20±0.75)mg/g)血管组织钙含量明显升高(P<0.01);消退组((12.73±1.89)mg/g)较钙化组降低(P<0.05);②构建了血管钙化的差减文库,对所获阳性克隆进行测序和BLAST比对分析,获得28个表达上调基因和22个表达下调基因。RT-PCR验证示Prdx3,Ank2,Ror2,Abcc1等基因在消退组和钙化组间差异表达,消退组较钙化组表达增高,平均约为后者的1.7倍。结论:VDN模型诱导的大鼠血管钙化可以发生主动消退。钙化消退过程中焦磷酸合成相关基因、谷氨酸信号相关基因、还原及凋亡调节基因表达上调,同时见较多骨化相关基因及氧化活性等基因表达下调。钙化抑制基因的表达增多而钙化促进基因表达的下降可能是钙化发生主动消退的内源性机制。

Objective： To investigate the differentially expressed genes in rat in the process of regression of vascular calcification by using the suppression subtractive hybridization （SSH）. Methods： 24 SD male rots which aged 6 weeks and specific pathogen flee grade were selected and randomly divided into 3 groups（ n = 8） ：control group, calcification group and regression group respectively. Vascular calcification model （vitamin D3 plus nicotine, VDN） were made from rats in calcification group and regression group, and rats in control group were intragastric administered with normal saline and lavaged with peanut oil. Rats were bred for 8 weeks in calcification group and control group, while rats in regression group were fed for 16 weeks. All rats were killed to measure concentration of calcium in the arterial tissue and examine the patholo-gical lesion changes. Subtractive hybridization among vascular cDNA sequences from calcification group and regression group were established. The cDNA fragments which expressed higher or lower in regression group than those in calcification group were isolated. Differentially expressed genes with cDNA fragment were inserted into PMD18-T plasmid vector and transformed competent DH-5a, cDNA libraries of differentially expressed gene between calcification group and regression group were then coustmcted. Recombinant vectors were analyzed by colony PCR, positive genes were randomly selected for sequencing and analyzed by BLASr. 4 genes were randomly selected for RT-PCR certification combined with semi-quantitative analysis of DNA bands. Results： VDN model of rats were successfuUy constructed. Concentration of tissue calcium in calcification group（15.34 mg/g ± 2.51 mg/g） was significantly increased compared to that in control group （5.20 mg/g ± 0.75 mg/g, P 〈 0.001）, while in comparison with calcification group（ 15.34 mg/g ± 2.51 mg/g）, calcium in regression group was relatively lower （12.73 mg/g±1.89 mg/g, P 〈0.05）. 28 up-regulated genes and 22 down-regulated genes were gained through sequencing and BLAST analysis among positive clones. RT-PCR validation indicated that 4 genes such as prdx3 and Ank2 had increasedly expressed in regression group than those in calcification group, the average fold change was 1.7. Conclusion： Rat vascular calcification tissue had characteristic of active regression. Genes in relation to pyrophosphoric acid synthesis, glutamate signal peptides, anti-oxidant and ant-apoptesis were up-regulated, at the same time many genes related to ossification and oxidation activity were down-regulated in the process of calcification regression. Increased expression of calcification suppressor genes accompanying decreased expression of calcification promoting genes might be the intrinsic mechanisms which initiated the active regression of calcified tissues.