摘要
目的筛选和分析毛乳头细胞凝集性生长状态下差异表达基因。方法分别提取凝集性生长和非凝集性生长状态下毛乳头细胞中的总RNA,应用SMARTcDNA合成技术和抑制性消减杂交技术分离毛乳头细胞凝集性生长差异表达基因的cDNA片段,并构建消减cDNA文库,通过PCR筛选、cDNA斑点杂交验证、测序和同源性检索对差异表达基因进行分析。结果成功地构建了毛乳头细胞凝集性生长差异表达cDNA文库。通过对毛乳头细胞凝集性生长状态差异表达基因文库的筛选、克隆,证实毛乳头在体外凝集性生长状态下可以特异表达与细胞同源凝集、生长调控、分化发育及信号转导相关的基因,包括已知基因(如加帽蛋白、palladin,VEGF)、造血干细胞分化相关基因(HSPC011、HSPC016)及1条新基因。结论毛乳头细胞凝集性生长差异表达基因消减cDNA文库的构建,为进一步筛选和克隆毛乳头细胞凝集性生长相关基因奠定了基础。在凝集性生长状态下,多种基因可能协同作用,参与毛乳头细胞生长调控和功能发挥。
Objective To screen and analyze genes differentially expressed within dermal papillae cells(DPC)with aggregative behavior.Methods Total RNA was extracted from DPC with and without ag-gregative behavior,and double-stranded cDNA were synthesized by using SMART cDNA synthesis.The cD-NA fragments of differentially expressed genes in dermal papillae cells with aggregative behavior were isolat-ed by suppression subtractive hybridization,sequencing,and then subtracted library was set up.Positive clones were screened by PCR method and verified by cDNA dot blot and then analyzed through homologous retrieving.Results A subtractive cDNA library of DPC with aggregative behavior was successfully construct-ed.The results of screening and cloning of the library showed that DPC with aggregative behavior could ex-press genes related to homologous aggregation,regnlation of growth,differentiation and development,and sig-nal transduction proliferation and cycle control,which included known genes(capping protein,paladin,vas-cular endothelial growth factor),hematopoietic stem/progenitor cells(HSPC)related genes(HSPC011and HSPC016)and a new gene.Conclusions The construction of subtractive library of DPC lays solid founda-tion for screening and cloning new and specific genes related to aggregative behavior of DPC.Several genes may cooperatively involve in homologous aggregation,and regnlation of growth of DPC.Among these genes,capping protein and palladin may be closely related to aggregative behavior of DPC,and VEGF and HSPC re-lated clones may be responsible for the status of higher proliferation of DPC.
出处
《中华皮肤科杂志》
CAS
CSCD
北大核心
2003年第9期513-515,共3页
Chinese Journal of Dermatology
基金
国家自然科学基金资助课题(30200249)
