Effect On The Biological Properties Of Rabbit Bone Marrow Mwsenchymal Stem Cells And Osteoblasts After Silencing Annexin A1Expression

Background:The occurrence of steroid-induced avascular necrosis of femoral head (SANFH) is very common in clinical;however, the precise mechanism of its pathogenesis remains unknown.The present studies have shown that SANFH relates to the alter of proliferation and osteogenic differentiation capacity in bone marrow mesenchymal stem cells (BMSCs) and the increasing of apoptosis in osteoblasts. This experiment is part of the National Natural Science Foundation—the proteomics dynamic analysis of early steroid-induced avascular necrosis of femoral head.The previous results have demonstrated that the apoptosis of bone cells increased and annexin A1protein down-regulated in femoral head tissue of early SANFH animal model, which indicated that the downregulating expression of annexin A1protein is likely to lead to decrease the capacity of proliferation and osteogenic differentiation in BMSCs and increase the apoptosis of osteoblasts. Annexin A1(ANXA1) is a calcium-dependent phospholipid-binding multifunctional protein which involves in the regulating the differentiation and apoptosis of cells. However, relationships between expression the alters of Annexin A1gene and SANFH still remain unknown, and related reports have not been found at present.This study is divided into three parts.Part One CONSTRUCTION AND IDENTIFICATION OF SHRNA LENTIVIRAL VECTOR TARGETING RABBIT ANNEXIN A1GENEObjective:To construct a short hairpin RNA lentiviral vector targeting rabbit ANXA1gene, which lays a foundation for the following studies—to observe effects on the biological properties in rabbit bone marrow mesenchymal stem cells (BMSCs) and osteoblasts after silencing annexin A1expression and to further explore the mechanism of steroid-induced avascular necrosis of femoral head (SANFH)Methods:Targeting rabbit ANXA1gene sequences, a pair of complementary small hairpin RNA (shRNA) oligonucleotides were designed, synthesized, annealed and cloned into pGLV/H1GFP plasmid digested by BamHI and EcoRI to construct a vector named pGLV-shANXA1The recombinant plasmid was identified by PCR and DNA sequencing.293FT cells were co-transfected with pGLV-shANXA1, pGLV/helper-1,pGLV/helper-2and pGLV/helper-3to package into lentiviral vector named LV-shANXA1.The titer of virus was tested according to the expression level of GFP.Results:The PCR identification and DNA sequencing showed that the fragment and nucleotides were in accordance with the target sequence and the shRNA sequence was successfully inserted into the pGLV/H1/GFP vector. The titer of concentrated LV-shANXA1-764, LV-shANXA1-844, LV-shANXA1-901 and LV-shANXA1-NC were3.7×108TU/L,4.1×108TU/L,3.6×108TU/L and3.7×108TU/L, respectively.Conclusions:Lentiviral shRNA expression vectors targeting the ANXA1gene of rabbit are successfully constructed. Part Two INFUENCE ON THE CAPABILITY OF PROLIFERATION AND OSTEOGENIC DIFFERENTLATION IN RABBIT BONE MARROW MESENCHYMAL STEM OELLS AFTER SILENCING ANNEXIN A1GENE EXPRESSIONObjective:To study the influence on the capability of proliferation and osteogenic differentiation in rabbit bone marrow mesenchymal stem cells after silencing annexin A1gene expression.Methods:The whole bone marrow cells isolated from New Zealand white rabbits were cultured and purified in vitro, then flow cytometry was used to detected the purity quotient of bone marrow mesenchymal stem cells (BMSCs).Infection efficiency of BMSCs after infected with lentiviral vector LV-shANXA1-NC at different multiplicity of infection (MOI) values was detected to determined the optimum MOI value.BMSCs were infected by lentiviral vectors LV-shANXAl-764, LV-shANXA1-844and LV-shANXA1-901to down-regulate ANXA1gene expression, respectively. Real time PCR and Western blot were used to detect the silencing efficiency of lentiviral vectors targeting to ANXA1gene and to select the lentiviral vector with highest silencing efficiency.BMSCs cultured in vitro were divided into3groups:RNA interference group was cotransfected with lentiviral vector LV-shANXA1-901to down-regulate ANXA1gene expression; negative control group was cotransfected with lentiviral vectors carrying nonsense gene sequences (LV-shANXA1-NC); yet,there was no intervention in blank control group. MTT colorimetry was used to assessed the capacity of proliferation in BMSCs; the capacity of osteogenic differentiation in BMSCs was evaluated according to the results of alkaline phosphatase staining, alizarin red staining and alkaline phosphatase activity of the cells.Results:The positive rates of CD44, CD105and CD45in BMSCs after purification were97.2%,95.8%and2.5%, respectively.The optimum MOI value of LV-shANXA1infecting BMSCs was50, and the infection efficiency was (91.4±2.7)%at MOI of50. Both Real-time PCR and Western blot results showed that the highest silencing efficiency for BMSCs could be found in LV-shANXA1-901group, and the silencing efficiency reached above70%at MOI of50. After down-regulating the expression of ANXA1gene, significant growth inhibition could be found in BMSCs; the alkaline phosphatase positive rate and alkaline phosphatase activity decreased, and the alizarin red staining results showed negative reaction in the cells subsequently induced into osteoplasts.Conclusions:Lentiviral vector LV-shANXAl-901can effectively silences ANXA1gene expression in BMSCs, which reduces the capacity of proliferation and osteogenic differentiation in the cells. Part Three INFLUENCE ON THE APOPTOSIS OF RABBIT OSTEOPLASTS AFTER SILENCING ANNEXIN A1GENE EXPRESSIONObjective:To study the influence on the apoptosis of rabbit osteoblasts after silencing annexin A1gene expression.Methods:BMSCs cultured in vitro were induced into osteoblasts.Infection efficiency of osteoblasts after infected with lentiviral vector LV-shANXA1-NC at different MOI values was detected to determined the optimum MOI value.osteoblasts were infected with lentiviral vectors LV-shANXA1-901at the optimum MOI value to down-regulate ANXA1gene expression. Real time PCR and Western blot were used to detect the silencing efficiency of lentiviral vectors targeting to ANXA1gene and to select the lentiviral vector with highest silencing efficiency.Osteoblasts induced from BMSCs in vitro were divided into3groups:RNA interference group was cotransfected with lentiviral vectors carrying short hairpin RNA (LV-shANXA1) to down-regulate the expression of ANXA1gene; negative control group was cotransfected with lentiviral vectors carrying nonsense gene sequences (LV-shANXA1-NC); yet,there was no intervention in blank control group. The influence of down-regulating ANXA1expression on the apoptosis of osteoblasts was assessed according to caspase-3activity,flow cytometry and terminal dexynucleotidyl transferase(TdT)-mediated dUTP nick end labeling (Tunel) results.Results:The positive rate of alkaline phosphatase staining was (95.0±3.6)%and the alizarin red staining results showed strong positive reaction in BMSCs after induced into osteoblasts for14days.The optimum MOI value of LV-shANXA1infecting BMSCs was50, and the infection efficiency was (92.6±3.7)%at MOI of50.Both Real-time PCR and Western blot results showed that the silencing efficiency was above70%at MOI of50.After down-regulating the expression of ANXA1gene in osteoblasts, the caspase3activity and the apoptosis rate of osteoblasts increased in RNA interference group, which were higher than the blank control and negative control groups (P＜0.05)Conclusions:Lentiviral vector LV-shANXA1-901can effectively silences ANXA1gene expression in osteoblasts, which promotes the apoptosis of osteoblasts and leads to osteoblasts apoptosis of through activating caspase-3.