Experimental Studies On Rat Bone Marrow Stromal Cells Modified With Protein-tyrosine Kinase Receptor B Gene

Being disability of cell division, mature neurons can not proliferate to substitute lost cells after the injury of central nervous system(CNS). So, nerve repair is still a challenge for neuroscientists. Heretofore, transplantation of some materials including Schwann cells, olfactory tract, peripheral nerve and embryonic nerve tissues have been applied to repair injured spinal cord or brain. Although achievement has been procured, the deficient graft resource and disability to produce novel functional neurons limit further study and clinical application. Discovery of stem cells bring a new therapeutic strategy to repair nervous system after trauma.With self-renewal capacity and mutiple potential, study on stem cells is accordingly one of "foreland" fields in 21 century. Numerous experiments have confirmed that both embryonic stem cells and adult stem cells can different into neural stem cells, neurons, oligodendrocyte and astrocytes under special condition (in vivo or in vitro). Furthermore, transplantation with stem cells has unambiguous improvements on morphology and function of injured spinal cord and brain.Bone marrow stromal cells, special kind of stem cells, localized widely in bone marrow, were considered as one of the most promising cell resource for the repairing of CNS after injury due to abundant source and easy acquirement.How to enhance the activity of cultured BMSCs and its survival capability after transplantation effectively? How to induce specific differentiation of BMSCs into neurons and reduce its oncogenicity? All of these problems need to be resolved as early as possible.Brain-derived neurotrophic factor (BDNF) is a major member of neurotrophic factor family. During embryonic development and adult growth, BDNF can induce the neural precursor cells differentiate specifically into neurons and accelerate its survival. Moreover, the biological effect of BDNF was mediated by high-affinity membrane receptor-protein-tyrosine kinase receptor B (trkB).Based on these, the exogenous trkB gene was transfected into BMSCs in this study firstly. In order to provide definite basis for clinic experiments and applications, the growth characteristics and oncogenicity of BMSCs expressing trkB in long-term were examined and analyzed.Part one .- Construction of eukaryotic expression vector carrying brain-derived neurotrophic factor receptor trkB geneObjective To construct an eukaryotic expression vector carrying rat brain-derived neurotrophic factor receptor trkB gene.Methods: Primers were designed according to the sequence of rat trkB cDNA. The upstream primer was designed according to the 5' end sequence of trkB cDNA, added up EcoR I restrict endonuclease site, and the downstream primer upon the 3' end sequence, added up BamH I site as well. The reverse transcription and PCR were carried out in the same reaction system in which total RNA of rat brain tissue was used as as template. After RT-PCR, the product was analyzed by electrophoresis and then retrieved and purified. Via T-A cloning, the PCR product was ligated with pMD18-T cloning vector, and then transferred into E. coil JM 109. After screening onLB agar plate containing ampicillin/ X-gal/ IPTG, the positive colonies were picked out and amplified. After being extracted the pMD18-trkB recombinant plasmid was identified by PCR and restriction endonuclease analysis with EcoR I / BamH I . The recombinant plasmid of pMD18-trkB and pEGFP-C2 were cut by EcoR I and BamH I to get Insert DNA and Vector DNA . Then, Insert DNA and Vector DNA were combined and incubated with T4 ligase. In the follow step, the reaction product was transferred into E. coil DH5a and the bacteria were inoculated in the LB plate containing 30g/mL kanamycin. The positive colonies were picked out and amplified, and then extracted to get recombinant plasmids. The recombinant plasmid was analyzed by enzyme digestion. Moreover, gene sequence analysis was performed.Results: By using the primers we designed, the RT-PCR product was nearly 15OObp in length, which was in accordance with anticipate 1461bp. There was no DNA band amplified in the product of negative control group. From LB plate, the positive colonies which contain pMD 18-trkB were screened. The colonies were amplified and extracted to get recombinant plasmids. The plasmids were cut by restriction endonuclease EcoR I and BamH I , and then analyzed by gel electrophoresis. The positive recombinant plasmid was digested into 2600bp and 15OObp fragment, according with the length of trkB DNA and pMD18-Tvector respectively, but the false-positive only one linear band. Using the recombinant plasmid as template to perform PCR, we obtained the amplified product which was about 15OObp in length. The results suggested that cloning plasmids contained anticipated trkB gene. Screened by kanamycin, the positive colonies which contain pEGFP-C2-trkB were picked out and amplified. Then the plasmids were extracted and analyzed by gel electrophoresis. The band of recombinant plasmid was behind the band of pEGFP-C2 vector which was extracted from the false-positive colonies. After the recombinant plasmid was cut by restriction endonuclease EcoR I andBamH I , two bands (about 5OOObp and 15OObp) were shown in gel electrophoresis. The result of gene sequence analysis suggested that the sequence of Insert DNA was in accordance with that of rat trkB gene in literature, and the target gene was in frame with the enhanced green fluorescent protein (GFP) coding sequences.Conclusion: In this study, the rat trkB gene was amplifies and the eukaryotic expression vector of pEGFP-C2-trkB was constructed successfully. Because trkB gene is in the same reading frame of GFP, we can monitor the expression of trkB protein in living cells and can also use this recombinant plasmid to transfer eukaryotic cells and trace them after transplant.Part two: Biologic characteristics of cultured rat bone marrow stromal cellsObjective: To establish the culture system of rat BMSCs in vitro and to explore its biologic characteristics.Methods: The nucleated cells in rat long bone marrow tissue were separated by gradient centrifugation on Lymophocyte Separating Liquid (density 1.077) and incubated randomly in the special BMSCs culture medium and DMEM/F12 respectively. The BMSCs were purified with discarding the suspended cells through exchanging medium. The morphology of cultured BMSCs was observed with inverted phase contrast microscope. Both cell counting and MTT were applied to plotted the cell growth curve. The cell cycle was examined with flow cytometry. The cell surface antigens, including the CD44, CD31, CD45, CD29, CD34 and flkl were detected to identify BMSCs with flow cytometry. The telomerase activity of BMSCs was also analyzed by using TRAP-ELISA.Results: Under microscope, the dissociated cells were suspended and round in shape with integral and clean outline. The attachment of bone marrow nucleated cells contained BMSCs started ahead of 24h and accomplished within 72h. Eliminatingsuspended cells, the attached cells were purified and displayed round in shape and reflected light well. The cell membrane was integral and cytoplasm were abundant. Furthermore, the nucleus could be shown indistinctly. During day 4 to day 7, the cell division and proliferation were observed. During day 10 to day 14, hearty proliferation and distinct information of cell clones were observed under microscope. The proliferated cells outgrew the cell clones and arranged like swirl, network or radialized shape. Cell culture till 20d, the BMSCs cultured in special medium differentiated gradually and the cell shape changed to shuttle, triangle or polygon. Round or similar round nucleuses with one to two nucleoli were shown in the cell central. Cells spread and connected each other with processes, displaying the morphological characteristics of cells of nervous system. The distinct morphological changes of cells in DMEM/F12 were not shown during the culture. The cell growth curve showed the cells enter into exponential phase during day 9 through day 11 after seeding. Till 15th day, eighty percent of cells were confused .Analysis of the cell cycle displayed that the values of Gl phase, S phase and G2 phase were 81.6%,9.5% and 8.8 % respectively , suggesting the cells held strong capability of self-renewal and self replication. The telomerase activity of BMSCs were positive and more slight than that of giloma cell line U251, which were performed as positive control. It suggested that cells were immature with low differentiation capability, but without immortalization of malignant tumor. Immuophenotye studies presented that the cultured BMSCs were CD34", CD45", CD31", flkr,CD29+ and CD44+, which were consistent with literatures.Conclusions: The results suggest that it is easy and feasible to dissociate and culture rat BMSCs, which were identified by using immuophenotye studies and had hearty activity, well purity and strong proliferation capability.Part three: Transfection of exogenous trkB into rat bone marrow stromal cellsObjective: To obtain the rat BMSCs expressing exogenous trkB gene stably and in long term and to evaluate the difference on transfection efficiency with electroporation and liposomes-mediated transfection method and their effects on cell activity.Methods: The eukaryotic expression vector pEGFP-C2-trkB was transfected into BMSCs with electroporation and liposomes-mediated transfection method randomly. And then, the survival and apoptosis of cells were detected by using flow cytometry. The transfection efficiency was calculated via expression of green fluorescent protein (GFP), which was detected with fluorescent microscope at 48h after transfection. After G418 screening, the trkB mRNA expression of positive BMSCs were analyzed with semi-quantitative RT-PCR. The localization, distribution and expression of trkB protein of BMSCs prelabeled PE were examined further with confocal microscopy.Results: The survival percentages of trancfected BMSCs with electroporation and liposomes-mediated transfection method were 53.2±2.3 % and 74.6±3.7 % respectively. Whereas, the survival percentage of untransfected BMSCs was 97.0±3.5 %. Significant differences were found between them (P<0.05) .After transfection, about 20.5±l.l %of survival cells transfected with electroporation displayed apoptosis in early period. As to other groups, the volumes were 7.6±0.6%( liposomes-mediated method group) and 1.6±0.1 % (control group) . To both trancfection methods, the expression of GFP was observed at 12h and came to peak at 48h. At this point, the transfeciton efficiency in electroporation group (41.7±1.3%) was higher than that in liposomes-mediated group ( 17.3±0.8%) with statistical difference. Cell culture till 48h, 500ng/L G418 was added into medium. Five days later, all of the untransfecedBMSCs came to death and some of BMSCs contained exogenous pEGFP-C2-trkB and pEGFP-C2 were fall off from the culture bottle and dead. The concentration of G418 was changed to 300ng/L at 5th day and its effect was maintained over 20 days. The expression of trkB in pEGFP-C2-trkB BMSCs was higher significantly than those in pEGFP-C2 BMSCs and untrasfected cells. Under confocal microscope, the pEGFP-C2-trkB BMSCs displayed red fluorescence, which was concentrated on cell membrane. On the contrary, no red fluorescence was found in the pEGFP-C2 BMSCs and untransfected BMSCs.Conclusions: The results suggest that the exogenous trkB had been successfully transfected into rat BMSCs through two techniques above-mentioned. The stable expression of exogenous trkB, which were validated at mRNA and protein levels, provide credible basis for further study. Compare with liposomes-mediated transfection method, the electroporation was of advantage to transfection efficiency and disadvantages to cell survival in early period resulting to the prolongation of attachment time. In this study, we prefer selecting electroporation.Part four: Biological characteristic changes of rat bone marrow stromal cells after transfection with trkB geneObjective: To investigate the effects of trkB and BDNF on the activity and tumorigenic abilities of BMSCs in vitro.Methods: After transfection with the eukaryotic expression plasmids of pEGFP-C2-trkB and pEGFP-C2 respectively, G418-resistant clones were isolated, and then cultured in DMEM/F12 containing 5u.g/mL BDNF. Using cells without transfection as control, growth curve of each group was plotted with cell counting and MTT respectively. These cells were also seeded in soft agar medium containing 5ng/mL BDNF and glioma U251 cells were used as control. Two weeks later, theclone forming efficiency was calculated. Semi-quantitative RT-PCR was carried out to evaluate the expression of c-myc mRNA in BMSCs of each group.Results: The exponential phase of proliferation of cells tansfected with pEGFP-C2-trkB began 7 days later after being seeded, compared to cells tansfected with pEGFP-C2 or without transfection 9-11 days later. The proliferation incubation period of BMSCs tansfected with pEGFP-C2-trkB was shorter than that of other BMSCs. But the difference of proliferation ratio was not significant. The clone(containing 20 cells or more) forming rates were: BMSCs tansfected with pEGFP-C2-trkB 0.2347 ± 0.0389 %( F+s), BMSCs tansfected with pEGFP-C2 0.6751+0.0506%, BMSCs without transfection 0.7023 + 0.0467%, and glioma U251 cells 7.3381+0.6813%. The capacity of colony formation of BMSCs transfected with pEGFP-C2-trkB was inferior to that of other groups (P< 0.05). The relative expression level of c-myc mRNA were: BMSCs tansfected with pEGFP-C2-trkB 0.0385±0.0073, BMSCs tansfected with pEGFP-C2 0.1329±0.0233, BMSCs without transfection 0.1314±0.0430, and glioma U251 cells 1.3482±0.4866. Compared with other groups, the relative expression level of c-myc mRNA decreased markedly (P< 0.05).Conclusion: After transfection with pEGFP-C2-trkB and followed treatment of BDNF, the activity of BMSCs can increase, and the tumorigenic abilities be depressed on the contrary.In this study, a eukaryotic expression vector carrying trkB gene was constructed and rat BMSCs were cultured and identified. And then the trkB gene was transfected into BMSCs with two different transfection methods. The positive cells were harvested which could express the protein of trkB in long term. Finally, the activity and tumorigenic abilities were evaluated. So, it provide academic and experimentbasis for BMSCs clinical applying to transplantation and regeneration of CNS after injury.There are still lots of work to do for us to understand the migration, survival, differentiation of the BMSCs transfected with trkB gene and their effects to regeneration of CNS after transplantation.Much to be learned. Much to be gained.