| Objective:Use the rabbit bone marrow mesenchymal stem cells(rabBMSCs)for the study.By Solute LinkedCarrier 39A1(ZIP1)in vitro on rabBMSCs,research the possible mechanism of ZIP1 in vitro induced rabBMSCs differentiation into osteoblasts,to provide a vitro studies in clinical treatment of alcoholic avascular necrosis of the femoral head.Methods:(1)Establish culture system of rabBMSCs,using different concentrations of alcohol(0.00 mol / L,0.03 mol / L,0.09 mol / L,0.15 mol /L,0.21 mol / L)on rabBMSCs,using western-blotting to assay about the ALP?OCN?collagen I and Runx2 protein expression after 6 hours later.(2)Construction ZIP1 protein overexpress vector.After the full-length sequence with Gene Synthesis ZIP1 Gene,and use double enzyme digestion method digested it.After that link the Gene into pc DNA3.1(+)vector,Use of PCR products and Gene DNA sequencing when the vector constructed.The vector will be used as the next experiment if it accreditation.(3)To screening ZIP1 small interfer RNA(siRNA).Select rabbits ZIP1 Gene as target,then determine synthesis directly three pairs of effective ZIP1 siRNA(siRNA1,siRNA2,siRNA3)by the biotechnology company.Transfected it into rabBMSCs as 25 nM concentration.meanwhile setting normal culture as control group,and using RT PCR expression ZIP1 mRNA qualitative analysis after 36 hours.We selected the most efficient inhibition rate ZIP1 siRNA sequence to constructed ZIP1 siRNA vectors for further follow-upexperiments.(4)ZIP1 involvement of rabBMSCs osteogenic differentiation.The experiment was divided into three groups,A group of normal cell culture,group B is ZIP1 overexpressing,group C ZIP1 siRNA group;after 48 h cell culture to adopt western-blotting assay of ALP?OCN?collagen I protein expression.(5)To study the effect of ZIP1 protein on the apoptosis of rabBMSCs.The experiment was divided into four groups,the first group of normal cell culture,group 2 hypoxia and glucose deprivation in cell culture group,the third group was ZIP1 overexpression of hypoxia and glucose deprivation in cultured group,the fourth group was ZIP1 siRNA hypoxia and glucose deprivation in cultured group.After two hours in cell culture using the Tunel method and flow cytometry to detect cell apoptosis rate,WB detection expression of apoptosis-related Bax and Caspase6 protein.Results:(1)The relative protein content was detected by Western-blotting technology.The ALP were 0.66±0.03?0.58±0.06?0.51±0.02?0.4±0.02?0.38±0.01;The collagen I were 0.69±0.03?0.54±0.03?0.59±0.05?0.41±0.02?0.23±0.01;The OCN were 1.08±0.03? 1.0±0.05?0.93±0.02?0.68±0.03?0.63±0.01;The Runx2 were 1.08±0.03? 0.65±0.01?0.52±0.02?0.41±0.03 ? 0.35±0.02.(2)After the ZIP1 over expression vector was constructed,There is no miscellaneous peak or fold belt sequenced and identified with Universal primers,and the sequences were compared.(3)The relative mRNA content was detected by Real-time PCR technology.The ZIP1 mRNA were 1.00±0.32?0.41±0.18?1.96±0.08?0.11±0.07?(4)The relative protein content was detected by Western-blotting technology.The ALP were1.32±0.04? 2.67±0.19?0.8133±0.10;The collagen I were 1.72±0.09?2.68±0.17?1.54±0.17;The OCN were 1.45±0.15?2.08±0.11?0.87±0.06.(5)Tunel method to detect cell apoptosis rate were(0.66±0.18)% ?(13.19±1.8)%?(1.76±0.57)%?(24.43±6)%;(6)Flow cytometry to detect cell apoptosis rate were(0.56±0.02)% ?(1.29±0.05)% ?(0.77±0.04)% ?(2.53±0.02)%.(7)The relative protein content was detected by Western-blotting technology.The bax were 0.36±0.02?0.68±0.04?0.68±0.04?1.14±0.3;The caspase 6 were 0.30±0.05?0.63±0.02?0.38±0.02?0.95±0.03.Conclutions:Alcohol can inhibit the expression of ALP,OCN,collagen I and Runx2 in rabBMSCs,and the inhibition effect is more significant with the increase of concentration.ALP,OCN,collagen I and Runx2 can be one of a identify indicators as rabBMSCs osteogenic differentiation.ZIP1 protein could significantly increase the expression of ALP,OCN and collagen I in rabBMSCs,which could promote the osteogenic differentiation of rabBMSCs.ZIP1 protein can significantly inhibit the apoptosis of rabBMSCS.In summary,This study show that alcoholic avascular necrosis of the femoral head osteogenesis diminished in bone marrow,such may be due to the effect of alcohol inhibition of bone marrow mesenchymal stem cells osteogenic differentiation.Zip1 protein can accelerated osteogenic differentiation of rabBMSCs,and inhibit the apoptosis of rabBMSCS;So,Zip1 protein combined with rabBMSCs can be considered as a new kind of biomaterial in treating early alcoholic avascular necrosis of the femoral head. |
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