Exogenous Htgf¦Â1 In Vitro Degenerative Disc Cells Expression

Objectives: 1) To provide experimental foundation for the clinical practice of gene therapy of disc degeneration diseases with transfecting adult degenerative disc cells cultured in vitro and determining the expressive product of hTGF-?1.Methods: 1) The plasmids PUC19- hTGF-?1 (supplied from ), contain full cDNA length of hTGF-?1 inserted between Hindâ…¢ and Xbaâ…  restriction sites, were first propagated in Escherichia coli JM109, then extracted and purified with the WizardTM Plus Milipre DNA purification kit (Promega) as recommended by the manufacturer. After plasmids PUC19-hTGF-?1 and plasmids PCI were prepared by double digestion with Hindâ…¢/Xbaâ… , the hTGF-?1 cDNA fragment was ligated with T4 ligase between the Hindâ…¢ and Xbaâ…  restriction sites in the linear plasmid PCI and PCI- hTGF-?1 was formed. The PCI- hTGF-?1, digested with Hindâ…¢/Xbaâ… , was found to contain the hTGF-?1 cDNA sequence by agarose gel electrophoresis and DNA sequence analysis.2) Cultured adult degenerative disc cells. 3) Transfected original degenerative disc cells in vitro with PCI-hTGF-?1 eukaryotic vector cultured in our laboratory and evaluated with control group. 4) Analyzed expressive product of the transfected cell with immunohistochemical staining method. 5) Used VIDAS Software analysis system to analyze the data quantitatively.Results: Immunohistochemical staining expose that: 1.The OD value of the positive cells production in the eukaryotic vector PCI-hTGF-?1 group is 3.49-3.56 times and 3.43-3.55 times of that in PCI group and untransfective group respectively in the original annulus fibrosus cells transfection;2. The OD value of the positive cells production in the eukaryotic vector PCI-hTGF-?1 group is 3.46-3.69 times and 3.33-3.63 times of that in PCI group and untransfective group respectively in the original nucleus pulposus cells transfection.Conclusions: Eukaryotic vector PCI-hTGF-?1 constructed in our laboratory could transfect adult degenerative disc cells cultured in vitro .The gene product of hTGF-?1 could be expressed .