| ObjectiveTo investigate the growth and the biocompatibility of hBDNF geneticallymodified rMSCs in C/GP hydrogel with NgR antibody.Methods1. Bone marrow cells were harvest from SD rat and cultured in culture platesusing adherent method. Inverted phase contrast microscope was used to observe themorphology of rMSCs. Flow cytometry was used to measure the surface antigenmarkers of rMSCs.2. Oxidized antibody was grafted into C/GP hydrogel to prepare C/GP-NgRantibody hydrogel. The level of NgR antibody was measureed by ELISA.3. rMSCs of third generation were transfected with type5replication defectadenovirus (Ad5) that carrying EGFP and hBDNF gene. The fluorescence expressionof EGFP in rMSCs were measureed by inverted fluorescence microscope.4. The co-cultivated experiment of rMSCs in hydrogel was divided into fourgroups: control group (rMSCs were seeded in C/GP hydrogel), hBDNF goup (hBDNFmodified rMSCs seeded in C/GP hydrogel), NgR graft group (rMSCs seeded inC/GP-NgR antibody hydrogel), and hBDNF+C/GP-NgR antibody group(hBDNFmodified rMSCs seeded in C/GP-NgR antibody hydrogel). Cells were cultured inculture plate. The growth of rMSCs was evaluated by acridine orange staining, theproliferative activity of rMSCs was detected by WST-1, the level of hBDNF mRNAwas measured by RT-PCR, and the protein level of hBDNF was detected by Westernblot.Results1. The rMSCs were successfully extracted, separated and cultured Five days afterculture; cell morphology is more uniform and spindle-shaped. After that, cellsproliferated rapidly, and reach confluence at7-10days. When cells were passaged to the third generation, all cells were spindle-shaped. The expression of CD34, CD45was negative in flow cytometry detection, while the expression of CD29, CD90waspositive, which conform to the phenotype of bone marrow mesenchymal stem cell.2. C/GP hydrogel can keep as clear homogeneous liquid solution for a long timeat room temperature and the pH is an average of7.2. When the temperature rose to37degree, it turned into transparent jelly-like gel in20minutes. After NgR antibody wasgrafted into C/GP hydrogel, the absorbing luminosity detected by ELISA were: liquidoxidized antibody group (0.669±0.235), solid oxidized antibody group (0.700±0.045),hydrogel only group (0.396±0.078). The expression of NgR antibody weresignificantly higher in first two groups compared to hydrogel only group (P <0.05);There was no significant difference between liquid and solid oxidized antibodygroups (P>0.05).3. Three days after transfected by Ad5-hBDNF-EGFP, the multiplicity ofinfection (MOI) increased multiply, the fluorescence expression of rMSCs enhanced.4.24hours after rMSCs was seeded in C/GP-NgR antibody hydrogels, cellsbegan to growth. The number of cells increased reached confluence in7days, whichgrew in shape of spindle or in shape of fiber. The nuclei of cells showed green inacridine orange staining, no orange condensation cell was detected, indicating that thecells were at active state.5. WST-1detection：rMSCs showed time-related growth pattern on the gel. Thenumber of cells increased significantly after3to5days of culture, then slow down atday7. The number of cells in hBDNF-rMSC+C/GP-NgR antibody group was muchhigher compared to control group, hBDNF goup, and NgR grafted group (P <0.05)after5days of culture.6. Real time-PCR results: The expression of hBDNF mRNA in control group isnegative. The expression of hBDNF mRNA in hBDNF goup and hBDNF-rMSC+C/GP-NgR antibody group were significantly increased when compared to controlgroup (P<0.01), and the expression of hBDNF mRNA was higher in hBDNF-rMSC+C/GP-NgR antibody group when compared to hBDNF group (P <0.01). Western blotshowed negative hBDNF protein expression in control group. The expression of hBDNF protein significantly increased in hBDNF group and hBDNF-rMSC+C/GP-NgR antibody group when compared to control group (P<0.01), and theexpression of hBDNF protein significantly increased in hBDNF-rMSC+C/GP-NgRantibody group when compared to hBDNF group (P <0.01).Conclusion1. hBDNF-rMSCs can survive and grow well in C/GP-NgR antibody hydrogel,and express hBDNF.2. The biocompatibility of C/GP-NgR antibody hydrogel is good for the growthof hBDNF-rMSCs that were seeded in. |
