| Objectives : 1.To observe the effect of changing the matrix Gla protein(MGP)gene expression on osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)of C57BL/6 mice,and to lay a foundation for a new pathogenesis of osteoporosis.2.To observe the effect of MGP adenovirus vector on lumbar spine in C57BL/6 mice.Methods: 1.The isolation,culture and identification of BMSCs from C57BL/6 mice;the primary BMSCs and BMSCs cell line of C57BL/6 mice were both divided into overexpression group which was transfected with the recombinant plasmid pIRES2-EGFP-MGP(OE)and pIRES2-EGFP(the negative control of overexpression group,OE-NC),and knockdown group was transfected with pKLO-EGFP-shRNA(KD)and pKLO-EGFP(the negative control of knockdown group,KD-NC);RT-PCR and Western blot were used to detect the expression of mRNA and protein of MGP;Proliferation of BMSCs was measured by CCK-8 assay,and ALP staining and assay was used to detect the differentiation,and the mineralization was detected by Alizarin Red S assay,quantitative analyzed by cetylpyridinium chloride.2.The C57BL/6 mice were divided into control group(0PFU)and intervention group(1×109PFU,3×109PFU,5×109PFU)by the different doses of MGP adenovirus vector injected into the mice,and each concentration has 3 mice,the lumbar spines were taken at the 7day,14 day and 21 day after injection,then RT-PCR and immunohistochemistry were used to detect the expression of mRNA and protein of MGP in the lumbar spine,and the microstructure of the third lumbar vertebrae was examined by Micro-CT.Results: 1.Effects of MGP on osteogenic differentiation of BMSCs:(1)The expression levels of MGP mRNA: in the primary BMSCs,the OE group was 1.43 times higher than that in the OE-NC group,and in the KD group,it was0.57 times of the KD-NC group,and in the BMSCs cell line they were 1.15 timesand 0.61 times higher than those of the control group(P <0.05);(2)The expression of MGP protein in the primary BMSCs OE group was 1.36 times higher than that in the control group,0.30 times in the KD group,and in the BMSCs cell line,1.16 times and 0.26 times of that of the corresponding control groups(P <0.05);(3)The results of CCK-8 assay showed that in the two kinds of cells,both the OE group and the OE-NC group showed an increasing trend,and the OE group was faster than the control group,at 24 and 48 hour there were no significant differences in the cell growth trend,but at 72 and 96 hour the cell proliferation was significantly increased(P<0.01),while that of the KD group was slightly lower than that of the control group,the primary cells were inhibited at 72 hours,the cell lines were significantly inhibited at 48 and 72 hours(P<0.01);(4)ALP staining showed in the OE group the differentiation ability of the two cells was increased,the ALP activity of the primary cells was about 2.18 times of that of the control group,and the cell lines were 3 times of the control group,on the contrary,the differentiation ability of the KD group was inhibited,the activity of ALP in the primary cells was about 0.26 times of that of the control group,and the cell lines was 0.2 times(P<0.01);(5)The Alizarin red S staining showed that both the two kinds of cells,the OE group formed more mineralized nodules,and the mineralization capacity was increased,on the contrary,knockdown MGP decreased mineralized level(P<0.05,P<0.01).2.Effects of MGP adenovirus on bone tissue:(1)RT-PCR: In the 0PFU dose group,the MGP mRNA expression of14 days and 21 days were both about 2 times higher than the 7 days(P<0.05),and the expression level of MGP mRNA in the 1×109PFU dose group was higher than that of 21 days at 7 days and 14 days(P<0.05),the expression level of MGP mRNA in the 3×109PFU dose group of 7 days was about 4.3 times higher than that in the blank control group(P<0.05),in the 5×109PFU dose group of 14 days it was 2.32 times higher than that in the control group(P<0.05,P<0.01);(2)HE staining showed that the bone structure of lumbar vertebrae were normal in mice,but on the 1×109PFU 7 days and 14 days,5×109PFU 7 days and14 days the density of osteoblasts was significantly higher than the respective control groups;Immunohistochemical staining showed that in the 1×109PFU and 3×109PFUgroup the positive cells were more than 0PFU,in the 1×109PFU group on the 7th day and the 14 th day,the lumbar positive cells were more than the 21 th day,in the 5×109PFU group,only on the 7th day the positive cells were increased(P<0.05,P<0.01);(3)Micro-CT detection of lumbar morphology: Lumbar structure is complete,trabecular bone is clear,comparison of parameters between the two groups: the BMD of the intervention group increased,is about 1.39 times of the control group(P<0.01),BV/TV was 1.22 times than that in the control group(P<0.05),but there was no significant difference between Tb.N,Tb.Th and Tb.Sp at the two groups(P>0.05).Conclusions: 1.MGP can induce the proliferation of bone marrow mesenchymal stem cells(BMSCs),and promote their differentiation and mineralization,thereby promoting the bone formation;2.The expression of MGP adenovirus vector in C57 BL / 6 mice was better at about 14 days of titer of 5×109PFU,and the MGP on the mouse lumbar spine may have a protective effect. |
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