Studies Of Schwann Cells From Neonatal Rat Peripheral Nerves After Infected With A Deno-bdnf

The dissertation contents two parts:documentary research and experimental research.Documentary researchBy quantities of documentary data, several methods to culture Schwann cells(SC) have been systemically reviewed; the modern researching developments of the recombinant Adenoviruses(Ad) vectors and brain-derived neurotrophic factor (BDNF) have also been concluded and analyzed. By analyzing main issues in the past methods of culturing Schwann cells and gene transfection, we conceive to use Adenoviruses vectors to carry the neurotrophic factor gene into SCs, which can come true the stable and lasting expression of target gene.And we search for new way of gene therapy to treat peripheral nerve injury.Experimental researchPurpose:By the combination of several methods to provide highly-purified, good-state Schwann cells in a short time. Then using Adenoviruses vectors to carry the brain-derived neurotrophic factor gene into SCs to impore the feasibility of gene transfection and effectal pathway to treat peripheral nerve injury. Methods: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. At last these cells were identified by SABC immunohistochemistry methods with rabbit anti-S100 protein antibody. In order to explore the influence of BPE on the proliferation of SCs in vitro and get the optimal concentration of it, the second generation SCs were separated into five groups, four of which were added BPE with different concentration (50μg/ml,100μg/ml,150μg/ml,200μg/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-S100 protein antibody and the number of SCs and fibroblasts were counted respectively. Adenoviruses were amplified by infecting HEK293 cells respectively. When cytopathic effect (CPE) was evident, viruses were isolated through freeze-thaw method. With different adenovirusal quantities of different multiplicities of infection (MOI), human BDNF gene was transferred into the SC origined from Sprague-Dawley rat by recombinant adenovirus in vitro.RT-PCR and Western blot were used to detect the efficiency of infection 72 h after infection. Furthermore, SC was infected with adenovirusal quantity according to MOI, with which the efficiency of infection was highest. ELISA quantitative analysis was used to investigate the expression of BDNF in the culture supernatant of infected SC 3,6,9,12,15,18 and 21 d after infection. Results:The percentage of purified SCs was over 90% and in a good state. The statistical analyse of the results indicated that 100μg/ml was the optimal concentration of BPE for the SCs proliferation. The viral titer of Adeno-BDNF virus was 1.78×109 pfu/ml determined by plaque assay. SC was infected by recombinant adenovirus. 72 h after infection, the efficiency of infection was highest as MOI was 200. Furthermore, SC was infected with a denovirusal quantity as 200 of MOI. ELISA showed that the expression of BDNF in the culture supernatant of infected SC was higher than uninfected SC at each time point. Conclusion:The combination of enzymatic dissociation, differential adhesion methods, antimitotic treatment, bovine pituitary extract (BPE) and low-concentration-typsin passage method can achieve highly-purified, good-state Schwann cells in vitro. Bovine pituitary extract (BPE) with proper concentration can efficiently promote proliferation of SCs. High-titer Adenoviral vectors can be produced by using HEK293 cell lines. Through transcription and interpretation, the expression concentration of BDNF secreted by the SC, which infected by recombinant adenovirus with exogenous BDNF gene, was higher than uninfected SC, and could remain for a longer time. This constructed experiment foundation for Adenoviruses vectors to carry the brain-derived neurotrophic factor gene into SCs and explored a new pathway of gene therapy to treat peripheral nerve injury.