应用抑制性消减杂交技术筛选血小板衍生生长因子上调的大鼠肝星状细胞靶基因

Screening up-regulated genes in hepatic stellate cells treated with PDGF-BB using suppression subtractive hybridization technique

目的应用抑制性消减杂交（SSH）技术构建血小板衍生生长因子（PDGF）-BB刺激的大鼠HSC上调基因的cDNA消减文库,从分子生物学角度阐明PDGF在肝纤维化中的作用机制。方法以PDGF-BB刺激的HSC作为实验组,以未用PDGF-BB刺激的HSC作为对照组,分别收获细胞提取总mRNA,并逆转录为cDNA。经RsaⅠ酶切后将实验组cDNA分成两组,分别与两种不同的接头衔接,再与对照组cDNA进行两次消减杂交及两次抑制性PCR扩增。将产物与pGEM-Teasy载体连接,构建cDNA消减文库,并转化大肠杆菌进行文库扩增,随机挑选克隆经PCR扩增后进行测序及生物信息学分析。结果成功构建了PDGF-BB刺激HSC差异表达基因的cDNA消减文库。文库扩增后得到102个阳性克隆,经菌落PCR分析,得到93个200～1000 bp的插入片段。挑取含有插入片段的31个克隆进行测序,通过生物信息学分析获得13种已知基因序列,主要包括电压依赖性阴离子通道、热休克蛋白质47、Ras家族基因RAN等蛋白质基因。结论PDGF- BB作为最有效的促有丝分裂原在激活HSC过程中上调某些与细胞生长相关的蛋白质、参与细胞内代谢的蛋白质及分子伴侣蛋白质等基因的表达,这将为进一步阐明PDGF-BB刺激HSC介导肝纤维化的分子生物学机制提供理论依据。

Objectives To construct a subtractive cDNAs library of up-regulated genes in rat hepatic stellate cells （HSCs） stimulated with platelet-derived growth factor （PDGF）-BB by suppression subtractive hybridization （SSH） technique, to clone the up-regulated genes associated with its regulation effects, and to elucidate the mechanism of the molecular biology of hepatic fibrosis involved in PDGF-BB. Methods The mRNA was isolated from HSCs stimulated with PDGF-BB and controlled with identical cells untreated with PDGF-BB, and then the cDNAs were synthesized. The cDNAs were designated as tester and driver. After being digested by restriction enzyme Rsa I, small-sized cDNAs were obtained. Tester cDNA was then divided into two groups and ligated to the specific adaptor 1 and adaptor 2, individually. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, the amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain DH5 α. The cDNA was sequenced and analyzed in GenBank with Blast search after PCR. Results The subtractive cDNAs library of up-regulated genes in HSCs stimulated with PDGFBB was constructed successfully. The amplified library contained 102 positive clones. Colony PCR showed that 93 clones contained 200-1000 bp inserts. Sequence analysis was performed in 31 clones randomly, and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank; altogether 13 coding sequences were obtained, which were known ones. The genes mainly included voltage-dependent anion channel （VDAC）, heat shock protein 47 and RAN-member RAS oncogene family genes. Conclusion Acting as one of the most effective mitogens, PDGF-BB up-regulated some gene expressions during stimulation of the HSCs, including some cell growth associated proteins, some proteins participating in intracellular metabolism and some molecular chaperone proteins, This work brings some new clues for studying the molecular biological mechanism involved in the up-regulated genes in PDGF- BB transactivated HSCs in hepatic fibrosis.