Objective To construct a recombinant adenovirus vector pAdxsi-GFP-NELL1 that co-expressing green fluorescent protein (GFP) and homo sapiens NEL-l ike 1 (NELL1) protein (a protein bly expressed in neural tissue encoding epidermal growth factor l ike domain), to observe its expression by transfecting the recombinant adenovirus into rat bone marrow mesenchymal stem cells (BMSCs) so as to lay a foundation for further study on osteogenesis of NELL1 protein. Methods From pcDNA3.1-NELL1, NELL1 gene sequence was obtained, then NELL1 gene was subcloned into pShuttle-GFP-CMV (-)TEMP vector which was subsequently digested with enzyme and insterted into pAdxsi vector to package the recombinant adenovirus vector (pAdxsi-GFP-NELL1). After verified by enzyme cutting and gel electrophoresis, pAdxsi-GFPNELL1 was ampl ified in HEK293 cells and purified by CsCl2 gradient purification, titrated using 50% tissue culture infective dose (TCID50) assay. The rat BMSCs were cultured and identified by flow cytometry and directional induction, then were infected with adenoviruses (pAdxsi-GFP-NELL1 and pAdxsi-GFP). NELL1 expression was verified by RT-PCR and immunofluorescence; GFP gene expression was verified by the intensity of green fluorescence under fluorescence microscope. Cell counting kit-8 (CCK-8) was used for investigate the influence of vectors on the prol iferation of rat BMSCs. Results Recombinant adenoviral vector pAdxsi-GFP-NELL1, which encodes a fusion protein of human NELL1, was successfully constructed and ampl ified with titer of 1 × 1011 pfu/mL. The primary BMSCs were cultured and identified by flow cytometric analysis, osteogenic and adipogenic induction, then were used for adenoviral transfection efficiency and cell toxicity tests. An multipl icity of infection of 200 pfu/cell produced optimal effects in transfer efficiency without excessive cell death in vitro. Three days after transfection with 200 pfu/cell pAdxsi-GFP-NELL1 or pAdxsi-GFP, over 60% BMSCs showed green fluorescent by fluorescence microscopy. Imunofluorescence with NELL1 antibody also revealed high level expression of human NELL1 protein in red fluorescent in these GFP expressing cells. RT-PCR analysis confirmed that the exogenous expression of NELL1 upon transfection with pAdxsi-GFPNELL1 at 200 pfu/cell, whereas NELL1 remained undetectable in Ad-GFP-transfected rat BMSCs. The prol iferative property of primary rat BMSCs after adenoviral NELL1 transfection was assayed by CCK-8 in growth medium. Growth curve demonstratedno significant difference among BMSCs transfected with pAdxsi-GFP-NELL1, pAdxsi-GFP, and no treatment control at 7 days (P gt; 0.05). Conclusion Recombinant adenovirus vector pAdxsi-GFP-NELL1 can steady expressing both GFP and NELL1 protein after being transfected into rat BMSCs. It provides a useful tool to trace the expression of NELL1 and investigate its function in vitro and in vivo.
Objective To observe the tissue engineered bonefabricated with the cultured mesenchymal stem cells (MSCs) by the green fluorescent protein (GFP) gene transfer. Methods The recombinant Adeno-XTM-GFP expression vector was purified after being packed and proliferated by the HEK293 cells, and then it was used to infect the rabbit’s MSCs directly afer the virus titer was assayed. The cell morphological changes were observed under the inverted phase contrast microscope, and the expression of GFP was observed under the fluorescence microscope to confirm success of the labeling of MSCs.The GPFlabeled MSCs and the pure MSCs were cultured together in the conventional osteogenic supplements for 3 weeks, and then they were seeded onto the compound scaffold of the calcium phosphate cement (CPC) and the fibrin glue (FG) to form a new kind of the tissue engineered bone. It was implanted into the donator rabbit subcutaneously to be used as the experimental group; in contrast, the pure compound scaffold of the CPC-FG without any MSCs was implanted in the same rabbit as a control. The alkline phosphatase (ALP) activity assay was performed respectively at 1, 2 and 3 weeks after operation. GFP was observed under the laserconfocal microscope at 4 weeks after operation, and the formed new bone was harvested at 4 weeks and evaluated by the Masson staining, the immunohistochemistry staining of osteocalcin (OC) and collagen typeⅠ.Results The virus titer was 3×108pfu/ml after proliferation and purification. Expresstionof GFP was confirmed 96 h after MSCs were infected by the Adeno-XTM-GFP expression vector and the infection rate was proximally 50%-70%. In contrast to MSCs, division and proliferation of the GPF-labeled MSCs were not significantly different. The ALP activity in the experimental group (12.546±1.091, 16.567±0.659, 20.443±0.706) was significantly higher than that in the control group (0.453±0.113, 0.243±0.018, 0.308±0.056), respectively at 1, 2 and 3 weeks after operation (Plt;0.05). The tissue engineered bone formed at 4 weeks. There were newly-formed trabeculae around the pore of the compound scaffold, and theimmunohistochemistry staining of OC and collagen typeⅠ were positive. The laser confocal microscope revealed that the GFP-labeled cells existed in many newlyformed tissues，and the compound scaffold of CPC-FG was partly degraded. Conclusion The engineered bone is similar to the spongy bone and the composed cells originate from the cultured MSCs, all of which can be confirmed by the GFP gene transfer technique.