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 GPFlabeled 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 (P lt;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 newlyformed 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. 

Citation： HU Yunsheng,MA Baoan,LI Wenhai,et al.. GREEN FLUORESCENT PROTEIN LABELING GENE TRANSFERRED INTO MESENCHYMALSTEM CELLS TO TRACE THEIR DIFFERENTIATION IN VIVO. Chinese Journal of Reparative and Reconstructive Surgery, 2007, 21(6): 633-637. doi: Copy