Biological Effects Of Lentiviral-mediated Survivin、TGFβ₃、TIMP-1Co-transfection In Human Degenerative Nucleus Pulposus

Objective: To construct Survivin、TGFβ3、TIMP-1three-gene lentiviral vector and to achieve stable transfection of the three gene in human degenerative intervertebral disc nucleus pulposus through one single transfection. By the ways of prohibiting apoptosis of intervertebral disc nucleus pulposus, enhancing synthesis of extracellular matrix(ECM) and inhibiting its catabolism to revere or slow down the degeneration of intervertebral disc.Methods:Constructing Survivin、TGFβ3、TIMP-1three-gene lentiviral vector and transfecting human degenerative intervertebral disc nucleus pulposus by using Survivin、 TGFβ3and TIMP-1lentiviral vector. Applying Real-Time PCR technique to detect mRNA expression of the three gene, aggrecan and collagen II. Appling Western blot to detect protein level of aggrecan and collagen II. Appling caspase-3Kit to detect activity of caspase-3protein. Appling Annexin V-PI to detect the rate of apoptosis.Result: Survivin、TGFB3and TIMP-1over expressed respectively after stable transfection and its biological expressions can last to P3generation or more. Real-time PCR shows no significant difference between the experiment group and the control group in mRNA expression. There is also no significant difference in Western blot result, capsase activity and apoptosis rate.Conclusion:The Co-transfection is stable which is mediated by Survivin、TGFB3and TIMP-1lentiviral vector and the three gene expressions can last for a long period of time and function biological effect. But, in vitro culture, by transfecting Survivin、TGFB3and TIMP-1three-gene lentiviral vector into the intervertebral disc nucleus pulposus cell can not reduce the apoptosis rate and enhance the protein level of aggrecan and collagen II. How Survivi、TGFβ3and TIMP-1three-gene lentiviral vector would function still need to be find out in vivo culture.