利用DDRT-PCR技术分析神经生长因子低亲和力受体诱导的细胞凋亡过程中基因表达的差异

转染了Ｐ７５ＮＧＦＲ的Ｒ２神经细胞系Ｒ２Ｌ１在去血清的培养时可以诱导细胞凋亡的发生．此凋亡可以被ＲＮＡ合成抑制剂放线菌素Ｄ和蛋白质合成抑制剂环己酰胺所抑制．利用ＤＤＲＴ－ＰＣＲ技术比较了去血清培养的发生凋亡的Ｒ２Ｌ１细胞与有血清培养的不发生凋亡的Ｒ２Ｌ１细胞以及去血清培养的不发生凋亡的Ｒ２Ｐ细胞基因表达的差异．克隆了数个特异或差异表达的短ｃＤＮＡ片段，经Ｎｏｒｔｈｅｒｎ杂交证实其中两个片段ＬＩＡＲＥＳＴ－１和ＬＩＡＲＥＳＴ－２表达量在凋亡细胞中显著高于不发生凋亡的细胞中，ＧｅｎＢａｎｋ检索表明此二片段为新的ｃＤＮＡ序列并给予登录号Ｕ４７３１５和Ｕ４７３１６．另有一个ｃＤＮＡ片段ＬＩＡＲＣＤ－３在凋亡细胞中受到了明显的抑制，经检索为一已知的与前强啡肽原上游调控区结合的ＤＮＡ结合蛋白ｃＤＮＡ编码区的一部分，首次被证实它与Ｐ７５ＮＧＦＲ诱导的神经细胞凋亡调控关联

Isolation and Identification of Differentially Expressed Short cDNA Fragments in Low affinity Neurotrophin Receptor(P 75NGFR ) induced

Apoptosis was observed in P 75NGFR expressing R2 cells (R2L1)after depriving serum from the culture medium,but not in PXT 1 transfected control R2 cells (R2P).Differential display technique was used to reveal the differentially expressed genes between normal control and apoptotic neural cells.Several short cDNA fragments were cloned and sequenced.These differentially expressed fragments were further confirmed by Northern blotting hybridization with their original total RNAs.The result showed that there were two of the short cDNA fragments LIAREST 1 and LIAREST 2 whose expression levels in apoptotic serum depriving cultured R2L1 cells were significantly higher than in normal serum containing cultured R2L1 cells and serum depriving cultured R2P cells.Sequence analysis showed that both of them contained a 3'poly A tail and a polyA tail adding signal in the 17 nucleotides upstream from 5' ending of polyA.Another short cDNA fragment LIARCD 3 was observed,whose expression level in apoptotic serum depriving cultured R2L1 cells was 63 4% and 62 3% lower than in serum containing cultured R2L1 cells and serum depriving cultured R2P cells,respectively.The full length of this cDNA fragment was cloned by Gu Jun of NIH in 1994 and is accessible with the accession number U08214.It encodes a protein(UreB 1)binding to an 8 nucleotide upstream regulation element(URE)for a translation initator element in the preprodynorphin promoter,and with a tyrosine kinase phosphorylation consensus.The role of these genes in apoptosis of neural cells in unknown at present.