| Chondrogenic Differentiation of Mesenchymal StemCells Modified by Liposome Mediated Insulin-like Growth Factor-1 Gene TransfectionIntroductionArticular cartilage defect is a common injury. Since the self-repair ofcartilage is very weak, damage of cartilage will lead to necrosis and osteoarthritis: Insulin-like growth factor 1 (IGF-1) is one of the essential growth factors for development and self-steady-state regulation of cartilage. IGF-1 can stimulate the, division and multiplication of chondrocytes and promote synthesis of TypeⅡcollagen and proteoglycan so as to maintain the phenotype of chondrocytes, so it can be used as the effective drug for treating cartilage defect. Mesenchymal stem cells (MSCs) have the following characteristics: (1) They are easily obtained with stable sources (They are obtained from autologous and xenogenous adult bone marrow puncture), and they can also be obtained from adult fat and synovium; (2) They have strong capacity of passage and stable phenotype; (3) Multidirectional differentiation. They can be differentiated into cartilage, skeleton, nerve, muscle tendon and muscle tissues under the action of different induction factors in different microenvironments in vivo. They have received much attention in many fields, especially used as the seed cells for tissue engineering. Therefore, it is of great significance to use transgene and stem cell techniques to construct new types of seed Cells.Materials and Methods1. Experimental animals4 Japanese large-ear white rabbits, male and female, 1 month old, 0.5-1.0kg, provided by Experimental Animal Center of China Medical University.2. Main equipment and reagents(1) Main equipmentConstant temperature CO2 incubator (Thermo); superclean bench (Yue Tan double horizontal laminar flow type, Beijing Semiconductor Element Factory); inverted microscope (Olympus Model IX70, Olympus, Japan); high speed constant temperature refrigerated centrifuge (Sigma, 1K-15); vortex shaker (VORTEX-2GENE, USA); small type desk centrifuge (Sigma 1-13, USA); electrophoresis apparatus (BIO-RAD PowerPac2000, USA); vertical slab electrophoretic apparatus (BIO-RAD Mini-ProteinⅢ, USA); fluorescence microscope (Olympus AX70 type, Japan); semidry transfer system (BIO-RAD Semidry Transfer System, USA).(2) Main reagentsT4DNA ligase, DH5α, RNaseA, pcDNA3-GFP (Figure 1) and plasmid pcDNA 3.1-IGF-1 containing the total length of IGF-1 cDNA (Figure 2), presented by Professor Xu Zixiang (Jilin University); DMEM culture medium, purchased from Gibco Company (USA); standard fetal calf serum (FBS), purchased from Hyclone Company; trypsin (Sigma); Percoll separating medium (Phanrmcia); CD14 and CD45 antibodies (Santa Biotechnology Company Limited); CD29 antibody (Chemicon); CD44 antibody (BIOSOURCE, Camarillo, CA); transfection reagent LipofectamineTM 2000 (Invitrogen Company, USA); IGF-1 (Santa Bioteclmology Company Limited); polyvinylidene fluoride membrane (PVDF), filter paper, purchased from BIO-RAD Company; ECL (enhanced chemilluminent reagent) reagent kit, purchased from Pierce Company.3. Methods(1) Study of the isolation culture and biological characteristics of rabbit bone marrow-derived mesenchymal stem cells.①Bone marrow aspirate of Japan white rabbits was density gradient centrifuged and cultured in plastic culture bottles. ②Folw cytometry analysis of cell surface markers in MSCs.③The cells were examined by invert microscope and the growth curve was drawn.(2) Construction of expression vectors of human insulinlike growth factor-1 gene and green fluorescent protein with two multiclone sites.①Amplification and purification of plasmid DNA of pcDNA3. 1-IGF-1.②Identification ofplasmid DNA of pcDNA3.1-IGF-1.③Construction of recombinant pcGI.④Identification of recombinant pcGI.(3) The effect of insulinlike growth factor-1 gene transfer on the proliferation of rabbit bone marrow mesenchymal stem cells and its chondrocyte differentiation potential.①MSCs was transfected by IGF-1 gene and its identification.②MSCs was observed after being transfected and transfection efficiency was calculated.③Western blot analysis were used to measure collagenⅡand IGF-1 expressing in MSCs before and after being transfected by IGF-1 gene. SPSS10.0 statistical analysis software was used to conduct analysis of variance (ANOVA) on the calculated ratio.Results1. Isolation, culture and passage of MSCs(1) The cell suspension was inoculated into plastic culture flask. Most of the cells were round monocytes and uniformly distributed. On the 7th day of inoculation, the adherence and confluence rate of cells reached 80%-85%, and Some cells were confluent and grew in flaky and spiral form. The passaged cells grew fast and most of them adhered to the wall 12 hours after passage. They were long spindle cells with short latent period. After 7-10 days, the cells overgrew in the bottom of bottle, and the multiplication and growth were very fast. Different generations of cells had similar appearance and were all long spindle cells.(2) The fluorescence activated cell sorter (FACS) detection results showed that the expression of CD29 and CD44 was positive, while the expression of CD14 and CD45 was negative.(3) Growth curve showed proliferation ability of MSCs was similar at passage 1, passage 2 and passage 5.2. IGF-1 eukaryotic expression vector has been successfully constructed with GFP.(1) Amplification and purification of plasmid DNA of pcDNA3.1-IGF-1 were SUCCESS.(2) Enzyme digestion analysis of pcDNA3.1-IGF-1 proved that plasmid of pcDNA3.1-IGF-1 contained IGF-1 cDNA fragment.(3) Sequence data of IGF-1 cDNA fragment were in accordance with those reported in literature.(4) Enzyme digestion and agarose gel electrophoresis analysis revealed that pcGI vector contained correct GFP and IGF-1 cDNA.3. MSCs transfected with tGF-1 gene showed marked increase of IGF-1 expression and stable expression of collagenⅡ(1) Eukaryotic expression vector pcDNA3.1-IGF-1 plasmid was tranfected into MSCs with the help of lipofectamine. Green fluorescence was detected in successfully transfected MSCs with fluorescence microscopy.(2) Transfection efficiency was (30±2.5) % after 24 hours.(3) Western blot analysis showed that gray value of transfected group is higher than non-transfected group or transfected group by vector.Conclusion1. The combination of density gradient centrifugation and adherent culture is an effective method to isolate MSCs from bone marrow aspirate. MSCs has higher proliferation ability and can be used in tissue engineering.2. IGF-1 eukaryotic expression vector has been successfully constructed with GFP.3. MSCs transfected with IGF-1 gene showed marked increase of IGF-1 expression and stable expression of collagenⅡ.
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