Objective To investigate the function of bone-marrow mesenehymal stem eells(BMSCs) differ-entiating into neuron-like cells in vitro after transfected by human brain-derived neurotrophie factor (BDNF) gene. Methods Human BDNF genes were cloned and recombinant pcDNA3. 1(-)-BDNF plasmids were construc-ted. BMSCs from five guinea pigs were isolated and cultured while their morphologies were observed by microscope. The surface antigen was detected by flowcytometry. BDNF genes were transfected into BMSCs with electroporation, and the transfected BMSCs were induced by ratinoie acid(RA)after bolted by Geneticin-418 (G418), then the dif-ferentiated BMSCs were identified by immunocytochemistry. Results The culture ceils demonstroted the typical mor-phology and surface antigen of BMSCs. The transfected cells expressed neuron- specific enolase(NSE), Nestin and glial fi-brillary acid protein(GFAP) also secreted BDNF. Conclusion BMSCs transfected by BDNF genes can differentiate into neu-ron- like cells in vitro, electroporation can enhame the transfection efficiency, RA can promote cell induction.

BACKGROUND:Gene transfection of cells includes virus and non-virus vector.As virus vector has some issues,such as safety and immunological rejection,the present study explored lipofectamine and electroporation transfection methods.OBJECTIVE:To establish genetic engineering cells using human brain-derived neurotrophic factor(hBDNF)gene transfected bone marrow mesenchymal stem cells(BMMSCs)by lipofectamine or electroporation,and explore its characteristics and expression in vitro.METHODS:Lipofectamine method:The BMMSCs were obtained from the tibias and femurs of the guinea pigs.The third passage BMMSCs were cultured with plasmid-lipofectamine mixture for 6 hours,followed by fetal bovine medium for 48 hours.Immunohistochemistry was performed for transient expression.G418 was added after 48 hours.Electroporation method:BMMSCs were trypsinized and resuspended with serum-free medium.Cell suspension was added into electrotransformation pool,and plasmid was added.The electrotransformation pool was moved between electrodes.After transfection for 48 hours,gene transient expression was detected.G418 was added after 48 hours.Brain-derived neurotrophic factor expression was detected by immunohistochemistry and RT-PCR.RESULTS AND CONCLUSION:Immunohistochemistry showed that BDNF transient expression was 5.80% by lipofectamine and 24.29% by electroporation.Cells almost died at 14 days following lipofectamine transfection.Stable expression cell lines of BDNF engineered BMMSC were successfully established by electroporation,with 90% expressive rate by immunohistochemistry and expression of BDNF mRNA by RT-PCR.Genetic engineering cells using BDNF transected BMMSC were established by electroporation whereas failed by lipofectamine,and the expressed BDNF was confirmed by immunohistochemistry and RT-PCR in vitro.

Objective To investigate the function of bone-marrow mesenchymal stem cells(BMSCs) differentiating into neuron-like cells in vitro after transfected by human brain-derived neurotrophic factor (BDNF) gene.Methods Human BDNF genes were cloned and recombinant pcDNA3.1(-)-BDNF plasmids were constructed. BMSCs from five guinea pigs were isolated and cultured while their morphologies were observed by microscope. The surface antigen was detected by flowcytometry. BDNF genes were transfected into BMSCs with electroporation,and the transfected BMSCs were induced by ratinoic acid(RA)after bolted by Geneticin-418(G418),then the differentiated BMSCs were identified by immunocytochemistry. Results The culture cells demonstroted the typical morphology and surface antigen of BMSCs. The transfected cells expressed neuron-specific enolase(NSE),Nestin and glial fibrillary acid protein(GFAP) also secreted BDNF.Conclusion BMSCs transfected by BDNF genes can differentiate into neuron-like cells in vitro,electroporation can enhame the transfection efficiency,RA can promote cell induction.

OBJECTIVE: To study the incidence and locations of facial nerve dehiscence (FND) in mastoidectomy for the patients with cholesteatoma and chronic otitis media, and to determine its relevance as pre-operative prediction. METHOD: Three hundred and fifteen ears (217 ears with cholesteatoma and 98 with chronic otitis media) undergoing mastoidectomy with or without tympanoplasties were selected for retrospective study, in which the incidence and locations of FND was studied, and the relevance for FND were analyzed by univariate test following by multivariate stepwise logistic regression. RESULT: The presence of FND was 22.9% of total surgical procedures and the locations of FND were 93.1% in the tympanic segment, which was significantly higher than in the mastoid segment. The factors as otogenic facial paralysis, pathologic style (cholesteatoma or chronic otitis media) and lateral semicircular canal (LSC) fistula were related to FND, while others factors as sex, age, revision operations, preoperative complications, dural exposure, sigmoid sinus exposure were not risk factors for FND. CONCLUSION The incidence of FND was 22.9% in this study, the most common location for FND was in the tympanic segment, therefore, the facial nerves should be especially taken care in mastoidectomy for patients with presence of otogenic facial paralysis, cholesteatoma and LSC fistula.
Adolescent ; Adult ; Aged ; Child ; Cholesteatoma, Middle Ear ; surgery ; Chronic Disease ; Facial Nerve Injuries ; epidemiology ; Female ; Humans ; Incidence ; Male ; Mastoid ; surgery ; Middle Aged ; Otitis Media ; surgery ; Retrospective Studies ; Young Adult