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

PartⅠThe construction of an eukaryotic expression voctor for pegfpN1-BDNFObjective To construct an eukaryotic expression vector pegfpNl-BDNF for genetically engineering cells in vitro.Methods Human brain-derived neurotrophic factor gene (hBDNF) was obtained by RT-PCR, and recombinant pegfpNl-BDNF plasmid was constructed by connecting the gene with pegfpNl plasmid. The recombinant plasmid was analyzed and identified by the EcoR I and BamH I restriction endonuclease after transformed, screened and amplified. Small amounts of recombinant plasmid was extracted and sequenced, then the correct recombinant pegfpNl-BDNF plasmid was massively extracted with QIAGEN-TIP100kit.Results To amplification of the hBDNF gene was performed by RT-PCR. Recombinant pegfpNl-BDNF plasmid was successfully constructed by connecting the hBDNF gene with pegfpN1 plasmid. The human BDNF gene encoding mature peptide was confirmed to be cloned by identified with restriction enzymes and sequenced.Conclusions The recombinant plasmid of pegfpNl-BDNF was constructed successfully, and it could be prepared for next experiment in vitro. Part II The establishment of BDNF-engineered bone marrow mesenchymal stem cells and the preliminary study of its electrophysiological character which differentiates into the neuronal-like cells in vitroObjective To investigate the differentiation of BDNF-engineered bone marrow mesenchymal stem cells(BMSCs) into neuronal-like cells in vitro, and detect its neuronal electrophysiologyical by patch clamp.Methods The BMSCs were obtained by density gradient centrifugation. After cultured for two or three passages, flow cytometry(FCM) analysis was used to detect markers CD34, CD44 and CD45. Transient transfction efficiency was detected 48h post-transfection by FCM, and stable DNA integration were selected by culturing with G418 of optimized concentration. Then BDNF-BMSCs were induced to neurnal-like cells under RA Expressed of NSE, GFAP and BDNF the three kinds cell were detected by immunocytochemistry.Comparion of the electrophysiologyical properties of induced BDNF-BMSCs and undifferentiated BMSCs were performed by whole-cell patch clamp.Results BMSCs population were isolated and expanded successful with distinct expression of CD34, CD45 and CD44 by 3.19%,4.42%, 96.82% respectively. Transient expressed Egfp was 10%-15% by electroporation. Stable expression cell lines of Egfp-BDNF engineered BMSCs, with 70-80% expression rate of Egfp by FCM. BDNF-BMSCs induced by RA, BDNF-BMSCs, BMSCs induced by RA, expression of NSE and GFAP were 45.87±5.82% and 21.92±2.29%,23.90±6.95% and 10.23±2.08%,4.81±1.71% and 1.93±1.34% respectively. The quickly activated like voltage-dependent Na+ channels of BDNF-BMSCs(7/15) induced by RA was examined by whole-cell patch clamp, it had statistical significance compared with the control(P<0.05).Conclusions BMSCs was separated and cultured in vitro easily. The high expressed BDNF of genetic engineering BMSCs were established by electroporation, and it was induced to neuronal-like cell who had neuronal electrophysiology.PartⅢComparison of the engineered BMSCs to the cochlear of normal hearing guinea pig with different path and observation of their safety for longObjective Choose the better path between scala tympani(ST) and modious(MO), and observe the safety of BMSCs transplant into the cochlea of the guinea pig for long.Methods The 40 guinea pigs of normal hearing serving as recipients were divided into two groups with Egfp-BMSCs:Ⅰa cochleostomy into the scala tympani(ST)(20 guinea pig); andⅡdirect access to modiolus (MO)(20 guinea pig). After Egfp-BMSCs was injected to the cochlea, auditory brainstem response audiometry(ABR) were recorded at day 7, day 28 and day 42 postoperative respectively at frequency 4kHz,8 kHz,16 kHz in the groups. Preoperative and postoperative ABR audibility thresholds were compared between and within group. In each group,5 guinea pigs were killed on each time point, and evaluated the distribution and survival of the transplanted cells in cochlear frozen section by fluorescence microscope. The another 10 guinea pig were killed in 6 monthes postoperatively, then the cochlear morphology were examined by paraffinsection.Results An identical significant elevated ARB thresholds were found in 7 days postoperatively at frequency 4kHz,8 kHz,16 kHz in groupⅠandⅡ, but the impairment was transient and recovered within 28 days. Most Egfp-BMSCs were survival in groupⅠandⅡin 7 days postoperatively, In groupⅠ, the distribution areas of Egfp-BMSCs were Scala tympani(ST), scala vestibule(SV), scala media(SM), no Egfp-BMSCs was examzined in MO. In GroupⅡ, the distribution area were MO, ST, SM, SV. As time goes by, the number of Egfp-BMSCs reduced. No tumor was found in gross specimen and no distinct structural alternation was found in the cochlea at paraffinsection in 6 monthes postoperation.Conclusions The ST injection was a better choose according to ABR. But the transplantation could be survivaled in the MO——the site of the SGN soma in MO injection, so it may be the most promising surgical approach if further refined to reduce the localized trauma associatedwith the procedure. The Egfp-BMSCs could be survival for 28-42 days. It had no oncogenicity for 6 monthes observation.Part IV The expression and differentiation of BDNF-engineered BMSCs transplanted into the cochlea of the deafened guinea pigs and its protection to the spiral ganglion neurons in vivoObjective Examination of BDNF-BMSCs survival and differentiation and its protection for the spiral ganglion neurons (SGNs) in the cochlea of deafened guinea pigs induced by aminoglycoside antibiotics.Methods 45 guinea pig deafened by amikacin intramuscular injection were randomly divided into three group, the transplantation was injected via scala tympani. The artificial perilymphatic fluid was injected in group A; differentiated BDNF-BMSCs in vitro was transplanted into the cochlea of the group B; group C with undifferentiated BDNF-BMSCs. The cochlea was respectively obtained and in paraffin-embedded on the day 7, day 28 and day 42 postoperatively. To evaluate and analyse the expression and transdifferentation of BDNF-BMSCs in the cochleas by immunohistochemisty including BDNF, NSE, GFAP antigens. The results were analysed in average optical density (AOD), and SGNs density was calculated in AOD.Results The NSE and GFAP expressive levels in experimental group (group B and C)was significant higher than that in the control group(A)(P<0.05) with the some result in the density of SGNs. However, the expression of NSE and GFAP were higher in group B than that in group C(P<0.05), with the some density of SGNs.Conclusions BDNF-BMSCs could be differentiated neuronal-like cell in the deafened cochlea. It has same protection for the SGNs whether differentiated or not.