Effect of indianhedgehog gene transfection into rabbit bone marrow mesenchymal stem cells in promoting chondrogenic differentiation and inhibiting cartilage aging in rotary cell culture system

Abstract

?To investigate the effect of overexpressing the Indianhedgehog (IHH) gene on the chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells (BMSCs) in a simulated microgravity environment. ?The 2nd generation BMSCs from rabbit were divided into 2 groups: the rotary cell culture system (RCCS) group and conventional group. Each group was further divided into the IHH gene transfection group (RCCS 1 group and conventional 1 group), green fluorescent protein transfection group (RCCS 2 group and conventional 2 group), and blank control group (RCCS 3 group and conventional 3 group). RCCS group cells were induced to differentiate into chondrocytes under simulated microgravity environment; the conventional group cells were given routine culture and chondrogenic induction in 6 well plates. During differentiation induction, the ELISA method was used to detect IHH protein expression and alkaline phosphatase (ALP) activity, and quantitative real-time PCR to detect cartilage and cartilage hypertrophy related gene expressions, and Western blot to detect collagen type Ⅱ, agreecan (ANCN) protein expression; and methylene blue staining and Annexin V-cy3 immunofluorescence staining were used to observe cell slide. ?After transfection, obvious green fluorescence was observed in BMSCs under fluorescence microscopy in RCCS groups 1 and 2, the transfection efficiency was about 95%. The IHH protein levels of RCCS 1 group and conventional 1 group were significantly higher than those of RCCS 2, 3 groups and conventional 2, 3 groups (P<0.05); at each time point, ALP activity of conventional 1 group was significantly higher than that of conventional 2, 3 groups (P<0.05); ALP activity of RCCS 1 group was significantly higher than that of RCCS 2 and 3 groups only at 3 and 7 days (P<0.05). Conventional 1 group expressed high levels of cartilage-related genes, such as collagen type Ⅱ and ANCN at the early stage of differentiation induction, and expressed high levels of cartilage hypertrophy-related genes, such as collagen type X, ALP, and Annexin V at the late stage (P<0.05). RCCS 1 group expressed high levels of cartilage-related genes and low levels of cartilage hypertrophy-related genes at all stages. The expression of collagen type Ⅱ protein in conventional 1 group was significantly lower than that of conventional 2 and 3 groups at 21 days after induction (P<0.05); RCCS 1 group expressed high levels of collagen type Ⅱ and ANCN proteins at all stages (P<0.05). Methylene blue staining indicated conventional 1 group was stained lighter than conventional 2 and 3 groups at 21 days after induction; while at each time point RCCS 1 group was significantly deeper than RCCS 2 and 3 groups. Annexin V-cy3 immunofluorescence staining indicated the red fluorescence of conventional 1 group was stronger than that of conventional 2 and 3 groups at each time point. The expression of red fluorescence in each RCCS subgroup was weak and there was no significant difference between the subgroups. ?Under the simulated microgravity environment, transfection of IHH gene into BMSCs can effectively promote the generation of cartilage and inhibit cartilage aging and osteogenesis. Therefore, this technique is suitable for cartilage tissue engineering.