| The cartilage injuries that caused by osteoarthritis or trauma is very common in the middle-aged and old crowds, which lead to high rate of mutilation and influence the quality of patients'lives seriously. Due to the limited selfrepair ability of articular cartilage, injuries to the joint cartilage will usually cause serious cataplasia of the articular cartilage .The repairing of articular cartilage injury is one of the problems in orthopedics, which is not solved finely today. Traditional methods have some disadvantages. For example, autologous osteocartilage, periosteum or perichondrium have limited source, and their harvesting may cause additional trauma. The immunologic rejection with allotransplantation has been a great concern that is hard to deal with. In recent years, the rise of tissue engineering has offered a new choice for the treatment of cartilage defect.The biggest challenge for the tissue engineering is the source of seed cells. Bone marrow mesenchymal stem cells (MSCs) are the subgroup of the non-hematopoiesis cells in the bone marrow, which have been focused highly for the past few years. They have strong ability of amplifying, and can differentiate to the osteoblast, chondrocyte, adipocyte, muscle cell, neurocyte and etc under some conditions. Bone marrow mesenchymal stem cells are to become the first-selected seed cells for the tissue engineering. But only 10- 20 ml of bone marrow can be taken suction in every adult, which contains a small amount of cells (about 1 MSC / 105 base support cells). Though a large amount of seed cells are demanded for the construction of tissue engineered cartilage. Therefore it is needed to largely amplify the MSCs in vitro to satisfy the demand. During the process of amplifying in vitro, the cell apoptosis occurs unavoidably. The cell apoptosis extently limits the application of seed cells.Using transgenic anti-apoptosis method is one good choice for amplying the seed cells in vitro. Bcl-xL is one important member of the Bcl-2 family which is the important molecule that regulates the cell apoptosis. Studies have shown that Bcl-xL can inhibit cell apoptosis caused by various kinds of inducing factors of cell apoptosis, such as the shortage of nutrition, radiant exposuring etc. Bcl-xL has better effects in inhibiting cell apoptosis than Bcl-2. Besides the function of inhibiting cell apoptosis, Bcl-xL also has certain ability in anti-inflammation. MSCs engineered by the Bcl-xL gene, which has both the anti-apoptosis and anti-inflammation ability, will have large prospects in the tissue engineering.Firstly, we designed the primers according to Bcl-xL gene order in GenBank and inducted the cut sites of XhoI and EcoRI in the up and down primers in the research. Then we amplified the aiming gene, utilized enzyme cut to enable the purpose gene insert eukaryotic expression vector pcDNA3, pEGFP-N2 correctly, identified by enzyme cut and gene sequencing. The results showed that the sequences and GenBank report were totally unanimous, which indicated that we succeed in structuring eukaryotic expression vector pcDNA3-Bcl-xL and pEGFP-N2-Bcl-xL.Secondly, we utilized the liposome to transfect eukaryotic expression vector pcDNA3-Bcl-xL and mock vector pcDNA3 to bone marrow mesenchymal stem cells. Using Western Blot and indirect immunofluorescence, we observed the expression of Bcl-xL protein.Further, we removed the serum in the culture medium and cultured the bone marrow mesenchymal stem cells for 24 hour after in the special culture medium. Using flow cytometry, we detected the cell apoptosis and observed the cell apoptosis rate in the experiment group was obviously lower than that in the control groups. These indicated that the expression of Bcl-xL had the function of inhibiting cell apoptosis which caused of lack of nutrition. Utilizing RT-PCR in mRNA level, we found the corresponding change in the expression of some related gene in bone marrow mesenchymal stem cells.Then we transfected the bone marrow mesenchymal stem cells with fluorescence eukaryotic expression pEGFP-N2-Bcl-xL. And the pEGFP-Bcl-xL-engineered MSCs suspended in rat tail collagen gel were injected into the knee synovial capsule three days after the surgery that created the cartilage defect. After two weeks, the frozen sections of the specimens were examined using a fluorescence microscope. The results confirmed that the injected cells were found in the neocartilage, which demonstrated that pEGFP-Bcl-xL-engineered MSCs could locate on the surface of the cartilage for tracing the gene engineered MSCs in vivo. MSCs were transfected with eukaryotic expression pxDNA3-Bcl-xL, mock vector pcDNA3. Bcl-xL engineered MSCs, mock vector engineered MSCs and non transfected MSCs suspended in rat tail collagen gel were injected into the knee synovial capsule respectively. In each group, four rabbits at six weeks and six rabbits at twelve weeks from the time of the injection were sacrificed. The entire knee was surgically removed, and the distal part of the femur was harvested, fixed in 10% paraformaldehyde, and then decalcified in 0.5 M EDTA (pH 7.4), dehydrated and embedded in paraffin. Serial sections cut longitudinally were stained with hematoxylin and eosin (H&E), Masson's trichome, Safranin O or toluidine blue. And we selected the Wakitani score for the histological scoring system. Our results at twelve weeks post injection in the Bcl-xL-MSC-treated group show definitive evidence of improved healing of cartilage histologically and morphologically when compared with the three control groups.In summary, we have confirmed that genetic modification of MSCs with the anti-apoptotic Bcl-xL gene resulted in a high survival rate in vitro and enhanced the survival of engrafted MSCs at the site of cartilage defect in the taxing in vivo environment. Genetically engineering Bcl-xL expression in cells using a non-viral vector could be an effective strategy for increasing cell survival after cell implantation while minimizing the potential risks.Intro-articular injection of anti-apoptotic Bcl-xL-engineered MSCs may provide a novel and effective approach in the treatment of cartilage defects. In our study, we chose rat tail collagen gel as the carrier for MSCs, which is permitted for use as a carrier for intro-articular injection by the SFDA of PR China. These results suggest that intra-articular injection of Bcl-xL-engineered MSCs is a potential non-invasive therapeutic method for effectively treating cartilage defects of the knee.
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