| After limited injury to the peripheral nervous system, Wallerian degeneration occurs and endogenous Schwann cells (SCs) are recruited to form a scaffold for regenerating axons to grow along, produce growth conducive extracellular matrix components, secrete neurotrophic factors and remyelinate regenerating axons in a phenotypical appropriate manner, which ultimately leads to reinnervation of the target and functional recovery. More and more attention has put on Schwann-cell-related studies. To attain Schwann cells (SCs) in vitro is the first step. The main problem of culturing Schwann cells is that how to eliminate fibroblasts and enrich SCs as much as possible. Several methods have been developed since the 1970s of last century, such as enzymatic dissociation, repeated explantation methods and antimitotic treatment. But the ever-deeper studies carried out by researchers nowadays need the combination of several methods to provide highly-purified, good-state Schwann cells in a short time.The enriched SCs after Wallerian degeneration are still insufficient because the secreted neurotrophic factors are not stable and degraded rapidly. Gene transfer has been used to provide a steady systemic supply of various neurotrophic factors under such circumstances. Various vectors have been exploited to carry thetarget gene into SCs. One of them is the recombinant Adenoviruses (Ad) vectors which are characterized by high titer, infecting both dividing and non-dividing cells and persisting as an episome in the nucleus of the transduced cells, therefore, much more importance has been attached to them.In this thesis, we used Adeno-X Tet-On System, which was integrated with the tetracycline-controllable expression system: tet-on system and carried LacZ gene, to infect the in-vitro-cultured Schwann cells which were produced through the comprehensive use of several culturing methods. After that, we observed the quantitative and qualitative expression of the LacZ gene product: -galactosidase ( -gal) under the inducement of doxycycline (DOX) , a derivative of tetracycline. From the whole experiment we explore the feasibility of promoting the functional recovery of injured peripheral nerve after infecting Schwann cells with the Adeno-X Tet-On System carrying therapeutic genes. Four parts are related during the experiment as follows:1. Culture Schwann cells in vitro. In order to get sufficient number of highly-purified, good-condition Schwann cells in a short time, six to seven-day-old Sprague-Dawley rats were sacrificed by decapitation and the sciatic nerves were removed aseptically to undergo such successive procedures to get Schwann cells as enzymatic dissociation and differential adhesion methods, Cytosine arabinoside and geneticin treatment to eliminate fibroblasts and bovine pituitary extract (BPE) to harvest SCs. And then low concentration typsin was used for passage. At last these cells were identified by SABC immunohistochemistry methods with rabbit anti-S100 protein antibody. The percentage of purified SCs was over 90% and in a good state.2. Enrich Schwann cells with BPE. In order to explore the influence of BPE on the proliferation of SCs in vitro and get theoptimal concentration of it, the second generation SCs were separated into five groups, four of which were added BPE with different concentration (50 g/mK 100 g/ml, 150 g/ml, 200 g/ml) and the last group without BPE as a control. On the second, fourth, sixth and eighth day that followed, one fourth of each group was dealt with SABC immunohistochemistry methods with rabbit anti-Si00 protein antibody and the number of SCs and fibroblasts were counted respectively. The statistical analyse of the results indicated that lOOug/ml was the optimal concentration of BPE for the SCs proliferation.3. Amplication of Adeno-X Tet-On System and viral titer assay. Two part of the system: Adeno-X Tet-On Virus Stock and Adeno-X TRE-pgal Virus Stock were amplified by infecting HEK293 cells respectively. When cytopathic effect (CPE) was evident, viruses were isolated throu... |
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