Construction,Expression And Apoptosis Of Laryngeal Carcinoma Cell Line Hepa2 Induced By Tumor-specific Expression Of TRAIL Gene

TNF-related apoptosis -inducing ligand (TRAIL) was discovered as a member of TNF superfamily which can induce apoptosis . Because of its sequence homology to TNF and Apo-1 (FasL), TRAIL is called Apo-2L . TRAIL belongs to type â…¡ transmembrane protein and contains 281aa , of which 14aa in N-termini consists of its cytoplasm region , following 26aa makes its transmembrane region and a 241aa extracellular region . Extracellular region of TRAIL has protein-enzyme site and forms a soluble molecule upon cleavage . TRAIL have five receptors: death receptorsDR4, DR5 , decoy receptor DcR1, DcR2 and OPG which has also not death domain. In contrast to TNF and FasL , TRAIL receptor expression and distribution character suggest different apoptotic mechanism . TRAIL has important significance in regulating tumor occurrence and apoptosis , natural immunity ,virus infection , self-stabilization mechanism and so on . BecauseTRAIL can induce many tumor cell types apoptosis but protect normal cell types from apoptosis , so TRAIL is considered as a potential inducing-apoptosis factor . But some studies suggest TRAIL can also induce many normal cell types apoptosis ,such as human hepatocyte. We use IL-2 signal peptide to direct transcript protein of TRAIL cDNA functional sequence out of cells and induce tumor cells apoptosis .Tumor occurrence is directly related to gene changes , so tumor therapy studies in the level of gene can bring to human beings new hopes. Improvement of efficiency and specificity is always emphasised in gene therapy. In order to achieve tumor specific target, the specific promoters are often used to control the therapeutic gene expression by regulation of transcription . It has been already confirmed that the activity of human telomerase reverse transcriptase (hTERT) rise in approximately most human tumors whereas most nomal cells do not express the activity and it becomes a hotspot in transcriptional target of tumor gene therapy . We make use of expression difference of hTERT promoter activity between aryngeal carcinoma cells and nomal cells , regulate and control the expression of apoptotic gene by hTERT promoter, achieve specifical regulation and control of transcriptional level in tumor cells to avoid harmness in nomal cells .Objective: To construct TRAIL eukaryotic expression vector and explore targeted gene therapy of tumor by using the combination of TRAIL gene with the telomerase promoter.Methods: (1) TRAIL gene from the eukaryotic vector pRNDE2-l with interleukin 2 signal peptide was amplified by PCR and cloned into downstream of the vector pGL3-181h TERT promoter and confirmed by restriction endonucleases and was sequenced to form an eukaryotic vector...