Preparation Of Recombinant Adenovirus Containing LacZ And Its Expression In The Rat Spinal Cord

Objective: The repair of the nerve injuries is still a tough problem puzzling the basic and clinical neuroscience. Because of the nerve's own structure and regeneration characters, repairing the broken nerve with surgery simply can not gain satisfactory result on the recovery of the long-term structure and function, especially for the recovery of the motor function. Among the various kinds of cure approach for the peripheral nerve injuries, the research progression of gene therapy gets the most attention. The gene therapy of peripheral nerve injuries means: by some methods or path, import the purpose gene into the human cell, and make it to produce a marked effect in vivo, so as to get the purpose of the gene therapy. It contains not only the protection effect for axoneuron, but also the promotion effect for the nerve repair. Among the total, the adenovirus vector has shown versatile superiority in empirical study of gene therapy on the regeneration of nerve, neuromuscular diseases and nerve tracing. The adenovirus vector is one of the most used viral vector currently, which is commonly used in expressing extrinsic protein. It is commendable carrier system for expressing recombinant antigen, possessing many advantages as follow: extensive host range, efficient gene transfer, relatively higher recombinant virus titre, comparatively large exogenous gene capacity, higher expressing level of mediated exogenous gene and never induce the genetic mutation of the host cell. Moreover its expressed foreign protein has the characteristics of native protein. All of these features make adenovirus the most important carrier. Recombinant adenovirus vector can not only infected mitotic phase cell, but also the nonmitotic phase cell, and the exogenous gene which it carried can long term exist and express stably. Therefore it is generally used to import exogenous gene into recipient cell, and known as a high performance gene transfection vector. Because of the high performance, diversity and security of cell transfection, Recombinant adenovirus vector bring about the hope on the gene therapy for the peripheral nerve injuries. We plan to build the recombinant plasmid containing LacZ and the virus taking along purpose gene LacZ, and expect furtherly transferring the vector from the injured peripheral verve to spinal cord, and obserbe its expression in targe neuron and the process marked by its product on the peripheral nerve, and master the expression law and characteristic of transgenes, Thus provides the basis of the therapy for peripheral nerve injuries with adenovirusmediated neurotrophic factors.Methods:1 Amplify the wrapped adenovirus vector plasmid containing LacZ in super competence to obtain enough plasmid. Collect the amplification plasmid and frozen at -20℃for use later.2 Cultivate the HEK-293 cell at 37℃in 5% CO2 cultivation box after its recovery. Change the cultivation liquid every 2 or 3 days. Observe the cell growth everyday, and transfer of culture is done when the cell density get close to 50%.3 Transfect the HEK-293 cell with the amplification plasmid in Liposome 2000 oligofectamine reagent box, wrap the recombinant adenovirus containing LacZ gene, and epurate the adenovirus through repeating freeze thawing between -70℃and 37℃.4 Infect the HEK-293 cell with the amplified virus in 96 hole board. Apparente CPE phenomenon on the 293 cell can be observed under micro. Determine the virus titre by 50% tissue culture infentive dosimetry.5 Import the well prepared adenovirus 2 ul to the lumbar intumescentia of the rats'spinal cord, and get the specimen 7 days later. Get the continuous transversally cut segment at the thickness of 40μm, and 4 hours dyeing is done with 0.1%X-gal staining solution at 37℃. Choose the slices which have positive reaction.Carry out the tabellae adhaesivus, neutral red counterstaining and mounting. Results:1 HEK-293 cell has a fusiform shape ,multi-feelers, little adherence strength, and prone to form cell monolayer. Cells contact with eath other by feelers.2 Three days after HEK-293 cell's transfection ,observe the cell blue-stain micro. after 20 min fixation in 4% paraform and 4 hours staining in 0.1% X-gal solution to detect the expression of LacZ gene. The experimental group have the positive result while the control group haven't found cell blue-stained.3 In the spinal cord transverse section, the blue-stained cell with LacZ gene positive expression always found at the cornu anterius medullae spinalis, and especially at the injection side. Mirky blue-stain makes the form of the masccline cell clear to be identified easily as cornu anterius medullae spinalis motoneuron and surrounding gliocyte.Conclusions:1 Maintaining the optimum growing conditions for the HEK-239 make for the transfection of plasmid and preparation of virus.2 Epurate the adenovirus through repeating freeze thawing after transfection, repeating inject to 293 cell is done and the cell died without other injection. This prove that the adenovirus gain stable expression in the cell, which open up the road for further experiment.3 Continuing expression of adenovirusmediated LacZ gene provide the basis of the therapy for peripheral nerve injuries with adenovirusmediated neurotrophic factors.