筛选转化生长因子β1刺激肝星状细胞差异表达基因的研究

A study on differentially expressed genes in hepatic stellate cells treated with transforming growth factor beta 1 using cDNA microarray technique

【摘要】目的筛选转化生长因子β1（TGFβ1）刺激大鼠肝星状细胞（HSC）的差异表达基因，以揭示TGFβ1介导肝纤维化的分子发病机制。方法分别用Trizol法抽提TGFβ1刺激的HSC及磷酸盐缓冲液刺激为对照的HSC总RNA，逆转录合成双链cDNA，制备掺入生物素标记的cDNA探针，与人基因表达谱芯片杂交，用Agilent扫描仪对芯片结果进行扫描，利用软件对差异表达基因进行生物信息学分析。结果从13824条目的基因中筛选出177条差异表达基因，其中123条基因表达上调，其中包括：结缔组织生长因子，微管蛋白ε1，V型胶原α2，连环蛋白δ2，钙粘蛋白6，2型，Smad3，丝裂源活化蛋白激酶4，生长因子受体结合蛋白7，丝裂原活化蛋白激酶相互作用／丝氨酸／苏氨酸激酶l等；54条基因表达下调，包括：肿瘤坏死因子受体相关因子4，干扰素调节因子7，干扰素诱生蛋白p78，骨形态发生蛋白7，基质gLa蛋白，人类丝氨酸蛋白酶抑制剂进化支B成员8，干扰素刺激基因2．0×10^4，死亡相关蛋白6，金属硫蛋白1H，超氧化物歧化酶2等；同时筛选到8个未知功能蛋白。结论应用基因表达谱芯片技术成功筛选了TGFβ1刺激HSC的差异表达基因，初步揭示了TGFβ1致肝纤维化的分子机制是诸多因素共同作用的结果，为进一步寻找新的基因治疗靶点奠定了基础。

Objectives To screen the differentially expressed genes in hepatic stellate cells （HSC） treated with transforming growth factor beta 1 （TGFβ1） by cDNA microarray technique, and to elucidate the molecular pathogenesis of liver fibrosis involving TGF β1. Methods Total RNA was extracted from HSC treated with TGF β1 and PBS by trizol and reverse-transcribed to double strand cDNA templates. Transcrip- tion of cDNA probe with biotin-labeling was performed, and then the obtained cDNA was hybridized with human cDNA microarray. The results were imaged by an Agilent scanner, and the differentially expressed genes were analyzed with bioinformatics software. Results One hundred seventy-seven differentially expressed genes were screened fi＇om 13824 targeting genes; 123 genes were up-regulated, including connective tissue growth factor, tubulin ε 1, collagen, type V, α 2, catenin δ 2, cadherin 6, type 2, Smad3, mitogen- activated protein kinase 4, growth factor receptor-bound protein 7 and MAP kinase-interacting serine/threo- nine kinase 1; 54 genes were down-regulated, including TNF receptor-associated factor 4, interferon regulatory factor 7, interferon inducible protein p78, bone morphogenetic protein 7, matrix gla protein, serine proteinase inhibitor, interferon stimulated gene 2.0- 104, death-associated protein 6, metallothionein 1H and superoxide dismutase 2; in addition, 8 genes with unknown functions were also found. Conclusion The differentially expressed genes in HSC treated with TGFβ1 were successfully screened by cDNA microarray technique. It revealed that the molecular pathogenesis of liver fibrosis involving TGF β1 was the result of coregulation by multiple factors. This information might be of help in searching for new targets in gene therapy.