[EXPERIMENTAL STUDY ON LENTIVIRUS-MEDIATED MULTI-GENES CO-TRANSFECTION IN BONE MARROW MESENCHYMAL STEM CELLS FOR TREATMENT OF KNEE OSTEOARTHRITIS IN CYNOMOLGUS MONKEY].

Objective: To observe the effect of lentivirus-mediated cyclooxygenase 2 (COX-2) and Aggrecanase-1 silencing and insulin-like growth factor 1 (IGF-1) in BMSCs after injecting into the knee joint cavity in cynomolgus monkeys with knee osteoarthritis (OA).
Methods: BMSCs were isolated from the bone marrow of 10 donors. The lentivirus vector expressing genes of COX-2, Aggrecanase-1, and IGF-1 were constructed, and transfected into the third generation human BMSCs at 40 multiplicity of infection (virus group); BMSCs transfected with lentivirus-empty vector served as blank-virus group. The growth status and number of BMSCs were observed under inverted phase contrast microscope, and normal BMSCs were used as normal control group. At 1 week after transfected, the mRNA expressions of COX-2, Aggrecanase-1, and IGF-1 were detected with RT-PCR. Nine 3-year-old cynomolgus monkeys were selected to establish the OA model according to Hulth modeling method, and were randomly divided into 3 groups ( n =3). At 6 weeks after remodeling, the right knee joint cavity was injected accordingly with 1 mL BMSCs (about 1×10 ⁷ cells) in virus group and blank-virus group, with 1 mL of normal saline in the blank control group; the left knee served as normal controls. The general condition was observed after injection; at 1, 4, and 6 weeks, the concentrations of prostaglandin E2 (PGE2), IL-1, Aggrecanase-1, and IGF-1 of double knee liquid were detected with ELISA; at 6 weeks, MRI, general observation, histology method, and immunohistochemistry method were used to detect the knee cartilage changes and the expressions of COX-2, Aggrecanase-1, and IGF-1 were measured with RT-PCR.
Results: No significant difference was found in cell morphology and growth curve between 2 groups after transfection. By RT-PCR, COX-2, and Aggrecanase-1 expressions were significantly reduced, IGF-1 expression was significantly increased in virus group when compared with normal control group and the blank-virus group ( P <0.05). All monkeys survived to the end of the experiment after injection. When compared with blank-virus group and blank control group, the concentrations of PGE2, Aggrecanase-1, and IL-1 significantly decreased and the concentration of IGF-1 significantly increased in the virus group ( P <0.05), but the indicators in 3 groups were significantly higher than those in the normal control group ( P <0.05). MRI showed that abnormal articular surface with high density could be found in virus group, blank-virus group, and blank control group, while the virus group had the minimum area. Gross observation and histological observation showed that the cartilage morphology of virus group, blank-virus group, and blank control group was accordance with early OA articular cartilage changes, but virus group was better than blank-virus group and blank control group in repair degree, whose improved Pineda score was significantly lower ( P <0.05). Immunohistochemical staining showed that the virus group had deeper dyeing with occasional brown particles and more chondrocytes than blank-virus group and blank control group. By RT-PCR, COX-2 and Aggrecanase-1 mRNA expressions of cartilage in virus group were significantly decreased, and IGF-1 expression was significantly increased when compared with blank control group and the blank-virus group ( P <0.05).
Conclusions: Lentivirus-mediated multi-genes co-transfection in BMSCs can inhibit the expressions of COX-2 mRNA and Aggrecanase-1 mRNA, and enhance the IGF-1 mRNA expression, which decreases the concentration of inflammatory factors, and protects the joint cartilage effectively.