| Normal human fibroblasts have been applied widely, which can be used as a cell model for many researchs, moreover, in the future which may be used as seed cell of tissue engineering for clinical application. Human skin fibroblasts have a finite proliferative life span in culture. After a defined number of divisions, they enter a phase known as "replicative senescence". Senescence cells are metabolically active but irreversibly growth arrested, and have a peculiar flat and enlarged morphology. Therefore, finite proliferative life span of human fibroblasts limits fibroblasts farther applied in scientific study and clinics. The reseach of fibroblasts with infinite proliferative life span will provide an immortalized cell model for medical research and clinical application.Telomere loss is thought to control entry into senescence. Human telomeres constitute the ends of the eukaryotic chromosomes, are formed by tandem repetitions of the hexamer TTAGGG bound to specific proteins. Telomeres allow cells to distinguishthe chromosome ends from double strand DNA breaks. The telomeric structure prevents the degradation or fusion of chromosome ends, and thus is essential for maintaining the integrity and stability of eukaryotic genomes. In most somatic cells, telomeres length is progressive shorter during each division. It has been proposed that when telomeres reach a critical length, they trigger the replicative block. Telomeres are synthesized by the specialized ribonucleoprotein enzyme telomerase. Telomerase is a ribonucleoprotein minimally consisting of an RNA subunit, containing the template for synthesis of telomeric DNA, and of a protein moiety with reverse transcriptase activity (hTERT). Telomerase is active in germline cells but negative in normal somatic cells. The restoration of telomerase activity allows human somatic cells to proliferate beyond the senescence limit. We introduced the human gene encoding for the enzyme catalytic subunit (hTERT) into human fibroblasts, and established a human fibroblast line. With hTERT gene inserted, hTERT-immortalized fibroblast cells expressed hTERT gene, and reconstituted telomerase activity. Cells extended life span with normal biological characteristic.Objective:To establish a human fibroblast cell line. We infect fibroblasts derived from normal human skin tissue with an hTERT containing retrovirus, and reconstitue telomerase activity of fibroblasts to extend the life span.Methods:1. Construction of retrovial vectors pLPCX- hTERT: The full hTERT cDNA fragment was subcloned into EcoR I site of the multicloning site of pLPCX Retrovial vector. The hTERT cDNA fragment was cloned in the 5'-3' orientation. Confirmation of the correct insert was determined by restriction digest with Xho I and sequencing.2. Primary isolation and culture of human fibroblasts: human fibroblasts was isolate by histotomy from the normal human prepuce. The fibroblasts was maintained in DMEM supplement with 15% bovine serum under an atmosphere of 5% CO2 at 37 .3.Production of retrovirus: The pLPCX- hTERT retrovial vectors was introduced into the packaging cell line PT67.After puromycin selection and cell cloning, cell clones producing high levels of recombinant virus were obtained. The viruses were used to infect fibroblasts directly.4.1nfect human fibroblasts: Fibroblasts were grown to 60% - 70% confluence.Approximately 2 105 fibroblasts were infect by retroviruses for 24 hours. After 48 hours, puromycin was added to the medium to a final concertration of 0.5 g/ml. Puromycin-resistant colonies were expanded, and the resulting stable clones were screened for the presence of hTERT insert by PCR.5.PCR assay the presence of hTERT insertion: The infected fibroblasts and parental control cells were harvested for PCR to detected the presence of hTERT insertion. Total genomic DNA was extracted from infectants and parental control cells using DNAeasy tissue Kit.Two primers were designed to detect the presence of hTERT insertion. The amplified product... |
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