抑制性消减杂交法筛选大鼠血管钙化相关基因的研究

Screening for rat vascular calcification related genes using suppression subtractive hybridization

目的了解大鼠血管钙化过程中血管组织基因表达的改变。方法选取6周龄SPF级雄性SD大鼠24只，随机分成钙化组和对照组（各12只），钙化组大鼠制作血管钙化模型（VDN）。两组大鼠于实验第8周处死后测定动脉组织钙含量并作病理检查。提取两组大鼠主动脉中膜RNA，反转录成eDNA，采用抑制消减杂交法（SSH）分离钙化组较正常组高表达基因的cDNA片段，将差异片段与PMD18-T质粒载体连接起来，热转化法转化感受态菌DH-5α，建立钙化组与对照组之间的差异表达eDNA文库。菌落PCR法鉴定重组载体，测序阳性克隆，BLAST比对分析测序结果。随机对Nell 1等6个基因进行差异表达的RT—PCR验证。结果（1）病理检查示钙化组大鼠主动脉中膜扭曲并见明显钙盐沉积，对照组大鼠主动脉中膜无任何钙盐沉积。（2）血管组织钙测定示钙化组和对照组大鼠分别为（15．34±2．51）mg／g和（5．20±0．75）mg／g，钙化组大鼠血管组织钙含量显著高于对照组（P〈0．01）。（3）采用SSH方法成功构建血管钙化的差减文库，对正反双向差减文库进行菌落PCR鉴定后分别获得92个阳性克隆（正向文库）和18个阳性克隆（反向文库），插入片段的大小主要集中于150～400bp。对所获阳性克隆进行测序和BLAST比对分析，获得钙化相关43个上调基因和11个下调基因。RT—PCR验证示CytoP450、Nell1等5个基因在钙化组和对照组中表达变化较大，钙化组表达高于对照组，约为后者的1．71倍。结论成功构建血管钙化VDN模型。血管钙化过程中相关基因表达发生改变，一些骨化基因以及凋亡、氧化、炎性反应和细胞因子等相关基因表达上调，同时伴有少数可能抑制钙化的基因下调。

Objective To determine the differentially expressed genes in the development of vascular medium calcification in rats using the suppression subtractive hybridization （SSH）. Methods Twenty-four 6-week old SD rats of specific pathogen free grade were recruited and randomly allocated into calcified group （n=12） and control group （n=12）. Rats were made for vascular calcification model in calcified group （vitamin D3 plus nicotine, VDN）. All rats were sacrificed to measure concentration of calcium in the arterial tissue and examine the pathological lesion changes. RNA in rat aortic tuniea tissue was extracted and reverse transeripted into eDNA. eDNA fragments which highly expressed calcification were isolated in calcified group using the SSH. Differentially expressed genes with cDNA fragment were inserted into PMD18-T plasmid vector and transformed to competent DH-5α by means of heating transfer, cDNA libraries of differentially expressed gene between calcified group and control group were successfully constructed. Recombinant vectors were analyzed by colony PCR. Positive genes were randomly selected for sequencing and analyzed by BLAST. Six genes, for example, were randomly selected for RT-PCR certification. Results （1） The pathological examination results demonstrated that in calcified group there were obvious calcium diposits and media squirm in tunica media of rat aortic wall, while in control group no calcium diposit was found. （2） There was statistical significance in calcium concentration in vascular tissue between calcified group[ （15.34 ± 2.51）mg/g] and control group [（5.20± 0.75） mg/g ] （P〈0.01）. （3） Subtracted libraries in vascular calcification was successfully established. Ninety-two positive clones in positive library and 18 positive clones in reverse library were obtained after the colony PCR identification. The length of insertion fragments was concentrated between 150 bp and 400 bp. Calcification-related 43 up-regulated genes and 11 down-regulated genes were obtained through sequencing and BLAST analysis in positive clones. RT- PCR validation indicated that the expressions of 5 genes such as CytoP450 and Nelll had greater increase in calcified group than those in control group, the average fold change was 1.71. Conclusions Model of vascular calcification induced by vitamin D3 plus nicotine is successfully constructed. Related gene expression spectrum is changed in the process of vascular calcification. Some ossification genes and genes associated with apoptosis, oxidation, inflammation and cytokines are up-regulated. At the same time, some genes which possibly inhibit vascular calcification are down-regulated.