ABSTRACT
Objective To construct the third-generation self-inactivating lentiviral vectors including BMP-2,BMP-4,BMP-6,BMP-7,BMP-9 and Wnt3a,then integrate them into the genome of mouse embryonic osteoblast cell,MC3T3-E1,and to explore the capability of osteogenic differentiation of individual bone morphogenetic control factor and the effective approaches to further improve the capability of osteogenic differentiation.Methods The plasmid vectors of gene expression including BMP-2,BMP-4,BMP-6,BMP-7,BMP-9 and Wnt3a were constructed,which were identified through enzyme cutting and further confirmed through sequencing.After packing pELNS-BMP-2,BMP-4,BMP-6,BMP-7,BMP-9 and Wnt3a,mouse embryonic osteoblast cell,MC3T3-E 1 was transfected,and the transfection efficiency was confirmed by GFP fluorescence imaging.The expression level of Runx2 mRNA and the transfection efficiency of individual bone morphogenetic control factor was detected by Real time PCR.Eight groups of MC3T3-E1 were dual-gene co-transfected,and the transfection efficiency was verified by GFP fluorescence imaging.ELISA was adopted to detect the expression level of BGP and ALP in MC3T3-E1culture supernatants;Real time PCR was adopted to detect the expression level of Runx2 mRNA;Western blot was adopted to detect the expression level of protein of BMP-2,BMP-4,BMP-6,BMP-7,BMP-9 and Wnt3a.Thus,the effectiveness of osteogenic differentiation of dual-gene co-transfection were evaluated.Results The recombination of lentiviruses,pELNS-BMP-2,pELNS-BMP-4,pELNS-BMP-6,pELNS-BMP-7,pELNS-BMP-9 and pELNS-Wnt3a were successfully constructed.MC3T3-E1 was successfully transfected.The expression levels of Runx2 mRNA were:BMP-2 ＞ BMP-4 ＞ BMP-9 ＞ BMP-7 ＞ Wnt3a ＞ BMP-6.Successful transfection of the dual-gene co-transfection of eight groups of MC3T3-E1 were verified by GFP fluorescence imaging.The expression level of Runx2 mRNA,the expression of BGP and ALP showed BMP-2 and BMP-7 co-transfection group was the most efficient in osteogenesis transfection.Western blot revealed thatthe protein expression of BMP-2,BMP-4,BMP-6,BMP-7,BMP-9 and Wnt3a increased after cotransfection of MC3T3-E1 by BMP-2 and BMP-7.Conclusion The third-generation lentiviral vector,pELNS can lead BMP-2,BMP-4,BMP-6,BMP-7,BMP-9 and Wnt3a into MC3T3-E1,mouse embryonic osteoblast cell,and stabilize its expression.Individual bone morphogenetic control factors can promote MC3T3-E1's differentiation to osteoblasts.The dual-gene co-transfection of BMP-2 and BMP-7 can effectively promote osteoblast conversion,which provides significant theoretical basis and technical support for remodeling of tissue engineering bone.
ABSTRACT
Objective To investigate the effect of depression symptoms on surgical outcome of posterior decompression among cervical spondylotic myelopathy (CSM) patients. Methods Between October 2006 and October 2011 in our hospital, lami?nectomy or laminoplasty was performed in 396 cases that were enrolled in the study. There were 132 males and 264 females with an average age of 60.2 years(ranged,39-84 years). All patients were divided into depression group and non?depressed group by the 21?item Beck Depression Inventory (BDI). There were no statistically significant differences between groups in age, sex, smok?ing status, duration of symptoms, and employment status (whether in the current working). The Japanese Orthopedic Association (JOA) scores, Neck disability index (NDI) and visual analogue scale (VAS) were compared after 1.5 months postoperatively. Re?sults All of 396 cases were followed up. The mean follow up duration was 32 months (range,24-50 months). There were no statistically significant differences in the CCI decline (7.1%±2.1% versus 6.8%±1.5%), expansion degree[(130.9±7.0) mm2 versus (150.8 ± 5.2) mm2] and the drift?back distance of the spinal cord [(5.7 ± 1.2) mm versus (6.2 ± 0.8) mm]. However, pa?tients with continuous depression showed poorer improvement than non?depressed patients in the surgery outcome: JOA im?proved (1.42±0.56 versus 6.76±3.12); NDI declined (7.31±2.18 versus 21.11±11.36); and VAS lightened (16.08±19.76 versus 23.85±20.79). Conclusion Depression contributed a dissatisfied surgery outcome after posterior decompression on functional re?covery, disability index and pain scores among patients of the cervical spondylotic myelopathy.