The Expression Of BDNF-engineered Mesenchymal Stem Cells And Its Protection To Spiral Ganglion Neurons Of Guinea Pigs In Vivo

Partâ… Establishment of BDNF-engineered mesenchymal stem cells and its expression in vitroObjective Cell replacement and gene therapy are attractive research giving new hope to cure sensorineural hearin loss.One of the key points for long-term expression of target gene in vivo is that what kind of vector to be selected.Bone marrow mesenchymal stem cells(MSCs) do possess easy-manipulation,differentiated multipotention,agility immigration,relatively immuno-privilege,which potentially enable their allogeneic therapeutic and autotransplantation.MSCs have gained rapidly increasing attention as a therapeutic tool for gene delivery.This part is aim to establish genetic engineering cell using brain-derived neurotrophic factor(BDNF) gene transected MSCs by means of non-virus vector,and explore its characteristics and expression in vitro,provide engineering cell population for subsequent research.Methods The MSCs were obtained from the tibias and femurs of the guinea pigs.After cultured for free passages,flow cytometry(FCM) analysis was used to detect markers CD44,CD34,and CD45.By comparison of two non-virus vectors as lipofectamine and electroporation,the better was choice to transfer pcDNA3.1(-)-BDNF to MSCs and stable DNA integration were selected by culturing with G418 of optimized concentration.Expressed BDNF were detected by immunohistochemistry 48h post-tranfection and after selection.The expression of BDNF also confirmed by RT-PCR following by electrophoresis.Results MSCs population were isolated and expanded successful with distinct expression of CD44,CD34 and CD45 by 94.65%,4.37%,7.81% respectively.Immunohistochemistry transient expressed BDNF was 5%by lipofectamine and 25%by electroporation.Stable expression cell lines of BDNF engineered MSCs,with 90%expression rate of BDNF by immunohistochemistry and expression of BDNF mRNA confirmed by RT-PCR, were successfully established by electroporation whereas failed by lipofectamine.Conclusions Genetic engineering cells using BDNF transected MSCs were established by electroporation whereas failed by lipofectamine,and the expressed BDNF was confirmed by immunohistochemistry and RT-PCR in vitro.Partâ…¡Transplantation of bone BDNF-engineered mesenchymal stem cells into the cochlea of normal guinea pigsObjective BDNF-engineered MSCs has the potential to treat an array of degenerative neurologic disorders including sensorineural hearing impairment. But the following questions must be identified in its practice.First is the functional and structural influence to recipients inner ear by cells transplantation;Second is the distribution and survival of grafted MSCs.Third is that how long can the BDNF-engineered MSCs maintain the expression of the target gene in vovo.By now there are no studies on transgened MSCs transplanted into the guinea pig cochleas.The main purpose of this prospective animal study was to reveal the questions as mentioned above, serving as control for the studies on deafened models.Methods the guinea pigs of normal hearing serving as recipients were performed an intraperilymphatic transplantation of different mixture with scala tympani injection.Groupâ… served as a negative control without any intervention,groupâ…¡were injected MSCs,and groupâ…¢injected BDNF-engineered MSCs,grafted cells were labeled by DAPI.Preoperative and postoperative ABR audibility thresholds were compared between and within group.Cochlear structure was evaluated by microscopic examination of paraffin sections cut through the cochlea of the recipient animals.The distribution and survival of transplanted cells was evaluated by fluorescence microscope.Expressed BDNF was detected by immunohistochemistry. Results An identical significant elevated ARB thresholds were found in 7 days postoperatively at frequency 4kHz,8 kHz,16 kHz in groupâ…¡andâ…¢, but the impairment was transcent and recovered within 28 days.NO distinct structural alternation was found in cochlea postoperatively.7 days postoperatively,most transplanted cells labeled by DAPI in groupâ…¢andâ…¢were found in the scala tympani and scala vestibule,even a small number located in the scala media and modiolus.28 days postoperatively,labeled cells still exist in perilymphatic space despite significantly decrement. BDNF-engineered MSCs had higher survivals in inner ears compare to bare MSCs.The positive immunohistochemical reaction was detected in groupâ…¢in 7 and 28 days postoperatively,differed from the other two groups which were negative.Conclusions The trancent hearing impairment was found when using MSCs transplantation with scala tympani injection,which could recover within 28 days.NO distinct structural alternation was found in cochlea postoperatively.The grafted cells can survive at least for 28 days in the cochlea and have the potential to migrate.BDNF transfection can elevated survivals of grafted MSCs.Thus,BDNF-engineered MSCs can be used as vehicles to deliver the BDNF gene into the cochlea with implications for the treatment of sensorineural deafness.Partâ…¢The expression of BDNF-engineered MSCs transplanted into the cochlea of the deafened guinea pigs and its protection to the spiral ganglion neurons in vovoObjective BDNF plays an important role in regulating the survival and differentiation of various neuronal populations including spiral ganglion neurons(SGNs).Based on these salutary effects of BDNF and the expression of BDNF-engineered MSCs in vitro and in normal cochlea,its expression level and efficacy in preventing the spiral ganglion neurons(SGNs) apoptosis in the deafened guinea pigs cochlea were evaluated in this part.Methods 24 guinea pig deafened by amikacin subcutaneous injection were randomly divided into two group,BDNF-engineered MSCs were transplanted into the cochlea of the experimental group with scala tympani injection,while the control group were injected artificial perilymphatic fluid.Total RNA were extracted for semiquantitatve real time RT- PCR,and TUNEL was used to identify the apoptotic neurons.Results The BDNF expressive level in experimental group was significant higher than in the control group,respectively,107 and 28 times higher at 7 days and 28 days postoperatively.The decreased apoptotic index of SGNs were detected in the experimental group compared to the control group.Conclusions BDNF-engineered MSCs can maintain high level expressed BDNF for at least 28 days after transplanted into deafened cochlea of the guinea pigs,and provide neurotrophic effect on SGNs by preventing its apoptosis.