Experimental Research On Co-cultivation Of BMSCs Transfected By Rabbit BDNF Gene Over-expressed Lentivirus Vector And Spinal Cord Neurons

Objective (s):To construct rabbit Brain Derived Neurotrophic Factor(BDNF) gene over-expressed lentivirus vector and transfect into bone marrow stromal stem cells(BMSCs).On co-cultivation of BMSCs transfected by rabbit BDNF gene over-expressed lentivirus vector and spinal cord neurons,we observed the expression of BDNF in BMSCs after transfection and its effect on the differentiations of BMSCs. Whether BMSCs transfected by rabbit BDNF gene over-expressed lentivirus vector played a protective role on spinal cord neurons in vitro experiment. To explore the experimental basis of BMSCs transfected by rabbit BDNF gene over-expressed lentivirus vector in vivo, to study the cell transplantation replacement therapy and gene therapy for the treatment of spinal cord diseases.Methods:1、Cell isolation, culture, identification of rabbit BMSCs(1) Primary culture and subculture of BMSCs;(2) Flow cytometry was used to identify BMSCs(CD29, CD34, CD45, CD90 staining);2、To construct rabbit BDNF gene over-expressed and inhibition lentivirus vector and transfect into BMSCs(1) RNA was extracted from the spinal cord of the Japanese big ear rabbit, the cDNA was synthesized, rabbit BDNF gene over-expressed and inhibition lentivirus vector were constructed and identified;(2) Using quantitative PCR and Western blot to screen the best plasmid of BDNF gene over-expressed and inhibition lentivirus vector;(3) Packaging of BDNF gene over-expressed and inhibition lentivirus vector,the recombinant plasmid was transfected into BMSCs by liposome method;(4) Expression of BDNF gene in BMSCs after transfection was confirmed with Western Blotting and RT-PCR;3、Co-cultivation of BMSCs transfected by rabbit BDNF gene over-expressed lentivirus vector and spinal cord neurons(1) The extraction, isolation and primary culture of rabbit spinal cord nerve cells;(2) Immunohistochemical method was used to identify the rabbit spinal cord neurons(NSE staining);(3) The co-cultivation cells were divided into four groups:group A:non-transfected BMSCs and spinal cord neurons;Group B:BMSCs transfected by rabbit BDNF gene over-expressed lentivirus vector and spinal cord neurons;Group C:BMSCs transfected by rabbit BDNF gene inhibition lentivirus vector and spinal cord neurons;Group D:BMSCs transfected by the empty lentivirus vector and spinal cord neurons.(4) Using Transwell double petri dishes to co-cultivate cells;(5) Immunohistochemical method was used to identify the neurogenic differentiations of BMSCs(Nestin, NSE staining);4. Results testSPSS 17.0 statistical software was used to analyze, all the data with mean ± standard deviation(x±s) said, the difference of data between groups were analyzed by independent sample t-test, the difference of data within group was analyzed by single factor analysis of variance, and P<0.05 had statistical significance.Results:1、Culture, identification of rabbit BMSCsDuring primary culture of BMSCs,adherent cell number gradually increased, the cell morphology changed for long fusiform, visible fibroblast scattered in the bottom of the culture bottle, colonys formed, the cells were arranged swirl around the center.The Cells fused to 80%can be subcultured.Flow cytometry was used to identify BMSCs when BMSCs spreaded to the third generation. BMSCs did’t express surface markers CD34 and CD45 of hematopoietic stem cell, but surface markers CD29 and CD90 were positive.2、Construction and identification of BDNF gene expression lentivirus vector,the best plasmid screeningConstructed lentivirus vectors were sequenced and compared with NCBI gene, all the genes of constructed lentivirus vectors can match with NCBI gene 100%. Enzyme digestion verified the expression of recombinant plasmid and PCR was performed to validate the inhibition recombinant plasmid,which confirmed that target gene had been inserted into the recombinant plasmid.Using QPCR and Western blot to screen the best plasmid,BDNF gene over-expressed lentivirus vector expressd more BDNF, BDNF gene inhibition lentivirus vector expressd less BDNF,the difference was statistically significant(p<0.01) compared with the control group.3、BDNF gene expression lentivirus vector were transfected into BMSCs,confirmed with Western Blotting and RT-PCRImmunohistochemical staining showed BDNF gene expression lentivirus vector were successfully transfected into BMSCs.Using RT-PCR and Western blot to confirm the expression of BDNF, BMSCs transfected by BDNF gene over-expressed lentivirus vector expressd more BDNF,BMSCs transfected by BDNF gene inhibition lentivirus vector expressd less BDNF,the difference was statistically significant(p<0.01) compared with the control group.4、Primary culture and identification of rabbit spinal cord nerve cellsDuring primary culture of spinal cord nerve cells, most of the cells adhered to the wall of the culture bottle,the cells body increased circular or elliptic,neurite surrounding the cells body stretched and extended to form monopolar, bipolar or multipolar neurite.Using immunohistochemical method to identify, the positive rate of NSE immunofluorescence staining of nerve cells was more than 86%after primary culture three days.5、Co-cultivation of BMSCs transfected by BDNF gene lentivirus vector and spinal cord neuronsIn Co-cultivation,using RT-PCR and Western blot to confirm the expression of BDNF,BDNF expressd more in group B,BDNF expressd less in group C,the difference was statistically significant(p<0.01) compared with the control group. Most of BMSCs differentiated into neurogenic cells in group B, cell morphological change of BMSCs was small in group A, C, D.Using immunohistochemical method to identify the neurogenic differentiations of BMSCs.Expression of NSE was about (89.49+1.25)%, Nestin was about (91.20+2.50)%in group B, Expression of NSE and Nestin was very little in group C, Expression of NSE and Nestin was very less in group A and D.The difference in group A,group B and group C were statistically significant(p<0.01).There were no significant differences in group A and,group D (P > 0.05).Conclusion(s):1、BMSCs can be isolation, culture, subcultured, amplification in vitro, flow cytometry was used to confirm the cells cultured were BMSCs.2、Rabbit BDNF gene over-expressed lentivirus vector was constructed and transfected into BMSCs successfully, which effectively enhanced the expression of BDNF gene and stably expressed BDNF protein.3、In co-cultivation of BMSCs transfected and spinal cord neurons, BMSCs transfected by rabbit BDNF gene over-expressed lentivirus vector expressed more BDNF, which accelerated the neurogenic differentiations of BMSCs,promoted the formation and growth of neurite, played a protective role on spinal cord neurons.