The Study About The RSKA /hIGF-Ⅰ Gene Mediated Delaying Atrophy Of Denervated Muscle

Insulin-like growth factor-I (IGF-1) is one of the most important regulate factors which can stimulate cell or tissue to grow and regenerate. With the development of gene transfection research, it is completely possible to introduce exogenous gene into target cells and get it expressed with high efficiency and to affect cells in some aspects. In the experiment, we constructed vector contained RSKA/hIGF-1 to delay skeletal muscle atrophy, which followed by determination of the gene expression in the C2C12 cells.0bjective:To construct a recombinant plasmid for expressing the IGF-I gene in C2C12 cells.Methods:The construct consists of rat skeletal a-actin promoter sequence, hIGF-1 coding sequence and human growth hormone 3'UTR region. The construct was initially cloned into a pBluescript II SK (+) after Synthesized, which contains the plasmid origin of replication and Ampicillin resistance gene. The sequence of Syntheticed hIGF-1 was confirmed by restriction analysis and DNA sequencing,then transfected into C2C12 cell.hIGF-1 gene was detected by RT-PCR and hIGF-1 by Western blot.Results : PBS-RSKA/hIGF-1 was transfected into C2C12 cells successfully .Specific bands could be tested in transfected cells by RT-PCR and Western blot.Conclusion:Recombinant plasmid PBS-RSKA/hIGF-1 constructed successfully and it can be effectively expressed after being transfected into C2C12 cells in vitro.Part two:The study about the RSKA / hIGF-I gene mediated by PEI delaying atrophy of denervated skeletal muscleOne of the most important reasons of the bad effect of the treatment to nerve injury is that skeletal muscle atrophy has taken place when it is dominated by nerve again, so the treatments to denervated muscle atrophy have aroused general interest of the clinicians and researchers. With the development of gene transfection research, it is completely possible to treat denervated muscle atrophy by gene transfection. We successfully constructed recombinant plasmid PBS-RSKA/hIGF-1, and transfected it into denervated gastrocnemius.0bjective: To examine the effects of the transfected of IGF-1 gene into denervated muscle.Methods: Seventy-two male SD rats randomly were divided into four groups.The sciatic nerve of the right leg of each rat was cut,and repaired at different time. Group A was injected sodium chloride into gastrocnemius when repaired the sciatic nerve at once; Group B was injected PBS-RSKA/hIGF-1 into gastrocnemius at the moment when nerves were cut and repaired, Group C was injected PBS-RSKA/hIGF-1 when nerves were cut, but nerves were repaired 2 weeks later; Group D was injected PBS-RSKA/hIGF-1 and repaired when the nerves were cut 2 weeks later. Equal amount of PBS-RSKA/hIGF-1 suspension or physiological saline was injected into the denervated gastrocnemius of the experimental groups or the control group respectively. 1,5,12 weeks later,nerve conduction velocity ,gastrocnemius muscle evoked potential and so on are examined in four groupsResults: The differences of nerve conduction velocity, gastrocnemius muscle evoked potential, tension of skeletal muscle among four groups are statistically significant. Conclusion: The transfection of RSKA / hIGF-I gene mediated by PEI in vivo can delay the atrophy of denervated muscle effectly.